CHAP TER 2 
Hematopoietic System
V. E. O. (Ted) Valli  •  Matti Kiupel  •  Dorothee Bienzle (with R. Darren Wood)
BONE MARROW 103
General considerations 103
Sample procurement and processing 107
Disorders of leukocytes 109
Disorders of erythrocytes 112
Disorders of platelets 128
Hematopoietic neoplasia 129
Other hematopoietic conditions 136
LYMPHOID ORGANS 138
Developmental diseases of the lymphoid system 139
Immunodeficiency 139
THYMUS 141
Structure and function of the normal thymus 141
Developmental diseases of the thymus 144
Thymic involution and atrophy 144
Thymic hemorrhages/hematoma 147
Inflammatory diseases of the thymus 148
Hyperplastic and neoplastic diseases of the thymus 150
Thymic hyperplasia 150
Thymic cysts 151
Thymic neoplasms 151
SPLEEN AND HEMOLYMPH NODES 158
Structure and function of the normal spleen 158
Developmental diseases of the spleen 162
Degenerative diseases of the spleen 163
Rupture of the spleen 165
Volvulus of the spleen 167
Splenomegaly and splenic nodules 167
Circulatory diseases of the spleen 169
Specific infections of lymphoid tissues 171
Anthrax 171
Leishmaniasis 174
Theileriosis 176
Tick-borne fever 178
Classical swine fever 178
African swine fever 181
Inflammatory diseases of the spleen 182
Tularemia 184
Histoplasmosis 186
Melioidosis 188
Hyperplastic diseases of the spleen 189
Splenic cysts 189
Lymphoid hyperplasia 189
Nodular hyperplasia 189
Fibrohistiocytic nodules 190
Hematopoietic alterations 191
Neoplastic diseases of the spleen 191
Jembrana disease 195
LYMPH NODES 196
Structure and function of normal lymph nodes 196
Developmental diseases of lymph nodes 198
102
Degenerative diseases of lymph nodes 198
Hyperplasia of lymph nodes 200
Inflammatory diseases of lymph nodes 202
Caseous lymphadenitis 204
Streptococcal adenitis in swine 208
Streptococcal adenitis in dogs 209
Pseudotuberculosis 209
Porcine circovirus-associated diseases 210
Parasitic infestations of lymph nodes 212
Neoplastic metastatic diseases of lymph nodes 212
Lymphoid neoplasms 213
Clinical features of lymphomas 214
Diagnosis of lymphomas 214
Classification of lymphomas 215
HISTIOCYTIC PROLIFERATIVE DISEASES 243
Canine cutaneous histiocytoma 243
Canine cutaneous Langerhans cell histiocytosis 245
Feline pulmonary Langerhans cell histiocytosis 246
Canine reactive histiocytosis 247
Cutaneous reactive histiocytosis 247
Systemic reactive histiocytosis 249
Histiocytic sarcoma 250
Feline progressive histiocytosis 253
Hemophagocytic histiocytic sarcoma 254
DISORDERS OF HEMOSTASIS 255
Platelet plug formation 255
Adhesion 256
Aggregation 256
Platelet disorders 257
Evaluation of platelet function 257
Thrombocytopenia 258
von Willebrand disease 258
Intrinsic disorders of platelet function 259
Acquired platelet disorders 260
Thrombin formation 260
Initiation 260
Amplification of thrombin generation 261
Fibrin formation 262
Laboratory evaluation of thrombin and fibrin formation 262
Disorders of thrombin formation 263
Inherited disorders 263
Acquired disorders 264
Disorders of fibrin formation 265
Regulation of coagulation: endogenous anticoagulants 265
Inhibitor disorders 266
Fibrinolysis 266
Endothelium-mediated fibrinolysis 266
Plasma-mediated fibrinolysis 266
Systemic inflammation and neoplasia: dysregulation
of hemostasis 267
 Bone Marrow 103

ACKNOWLEDGMENTS of the marrow space of bones to concentration in marrow

We gratefully acknowledge the contributions to past editions of
this chapter by Drs. Ken Jubb, Peter Kennedy, Nigel Palmer, Bruce
Parry, and Patricia Gentry. We thank the image archive of Michi-
gan State University for use of its images.
BONE MARROW
General considerations
The hematopoietic system is broadly divided into myeloid and
lymphoid tissues. The myeloid tissues consist of bone marrow
and the blood cells derived from it (e.g., red blood cells, white
blood cells, and platelets), whereas primary lymphoid tissues
include the lymph nodes, spleen, thymus, and circulating lym-
phocytes. Hematopoiesis refers to the generation of blood
cells, which in mammals proceeds from the mesonephric
region in the embryo to the liver in the fetus, and then to bone
marrow prior to birth. During maturation from neonate to
adult, hematopoietic activity contracts from occupying most
space of axial bones (skull, vertebrae, ribs, sternum, pelvis) and
the proximal regions of the humerus and femur. In healthy
adult animals, this active bone marrow appears grossly red and
is composed of a dynamic composite of hematopoietic and
adipose tissue. Marrow in the distal appendicular bones con-
sists predominantly of adipocytes and appears white-yellow.
The relative proportion of hematopoietic and adipose tissue
in marrow is highly variable, reflecting age-related increases
in the proportion of fat and the relative demand for blood
cells. Although adult marrow contains few lymphocytes, cells
of both myeloid and lymphoid tissues are derived from
common precursor cells in bone marrow. Such hematopoietic
stem cells (HSC) have self-renewing and pluripotent differ-
entiating ability, and give rise to all blood and lymphoid tissue
cells (Fig. 2-1).
Bone marrow comprises 2-5% of body weight in adult
animals, and hematopoietic tissue is identified grossly as red
marrow amidst the labyrinth of boney trabeculae in flat bones
and the proximal parts of long bones. A layer of endosteal cells

Hematopoietic
stem cell

Multipotent
Lymphoid Myeloid
precursor
progenitor progenitor

T-cell B-cell
thymus lymphoid CFU-GM CFU-Meg-E
tissue

Mitotic
Committed activity
precursor

Monoblast Myeloblast Mega- Erythro-

karyoblast blast

Terminally
committed
precursor
Pro- Neutrophil Eosinophil Basophil Mega- Basophilic
monocyte metamyelocyte karyocyte rubricyte
Distinct cell
morphology
Mature
blood cells

Monocyte Neutrophil Eosinophil Basophil Platelets Erythrocyte

Figure 2-1  Production of blood cells proceeds in an orderly and highly regulated manner from
hematopoietic stem cells through multiple precursor stages to mature blood cells. CFU, colony-
forming unit; GM, granulocyte-macrophage; Meg-E, megakaryocyte-erythrocyte.
104 CHAPTER 2  •  Hematopoietic System
covering cortical and trabecular bone surfaces lines the marrow
cavity containing hematopoietic cells. Endosteal cells are mul-
tipotent mesenchymal progenitor cells with ability to give rise
to osteoblasts, osteocytes, and osteoclasts; all are cells that
are commonly identified at endosteal surfaces. Together with
specialized endothelial cells, macrophages and nerve cells,
endosteal cells comprise a HSC niche or microenvironment.
Trabecular bone homeostasis is interconnected with hemato-
poiesis, and altered hematopoiesis may be associated with
changes in density or diameter of the boney trabeculae. Bone
marrow has a vascular supply through nutrient arteries that
enter through cortical bone, branch to run parallel to the long
axis of bone, and give rise to arterioles directed toward the
marrow periphery. Anastomosis with venous sinuses leads to
venules that drain into a central longitudinal vein, which in
turn feeds into nutrient veins. The sinusoidal network in bone
marrow is extensive, but most sinusoids are very small and
inconspicuous, and may be entirely inapparent in biopsy sec-
tions. Bone marrow lacks lymphatic vessels. Other compo-
nents of the hematopoietic microenvironment are nerve cells,
adventitial barrier cells, adipocytes, macrophages, and mesen-
chymal cells.
Mature blood cells are derived from precursor cells through
orderly and hierarchical maturation under the precise influ-
ence of transcription factors and lineage-specific growth
factors. HSC and committed precursor cells lack distinct mor-
phologic features, and are defined by functional assays such as
in vitro colony formation or expression of specific antigens.
Committed precursor cells are morphologically recognizable as
rubriblasts, myeloblasts, or megakaryoblasts. Precursor cells
without distinct morphologic features are infrequent in bone
marrow, and are rarely enumerated for diagnostic purposes.
However, during times of hematopoietic stress, HSC may be
mobilized from bone marrow, travel via the circulation to
tissues outside the bone marrow, and establish extramedullary
foci of hematopoiesis. Healthy animals, particularly dogs and
cats, frequently have hematopoietic activity in the red pulp of
the spleen, and virtually any other tissue, but most commonly
liver and lymph nodes, may acquire hematopoietic activity in
stress or disease states.
Hematopoiesis is a highly dynamic process, given the short
life-span of blood cells. Neutrophils circulate for 8-10 hours
after release from bone marrow, platelets for 10-14 days, and
erythrocytes for 70 (cats) to 150 days (cows). Hence it is
common to observe mitotic activity in bone marrow samples
from healthy animals, which may be markedly increased
during sustained inflammation, or in response to blood loss or
premature destruction of blood cells. Chronic blood loss or
inflammation in an otherwise healthy animal may increase the
proportion of marrow hematopoietic relative to adipose tissue
from ~50% to >90% within a few weeks. Further prolonged
increased demand for blood cells results in progressive conver-
sion of adipose tissue first in distal trabecular bone regions of
the metaphysis, and then in areas lacking trabecular bone
toward the diaphysis. Such expanded hematopoiesis depends
on formation of new HSC niches through interaction with
endosteal cells, and is grossly apparent as a red line along the
endosteal surface of long bones. High proliferative capacity
also renders hematopoietic tissue uniquely susceptible to
injury from cytotoxic drugs, toxins, and infectious agents. The
ratio of hematopoietic to adipose tissue in red marrow is
80-90% to 10-20% in healthy animals <1 year of age (Fig. 2-2).
With progressive age, the proportion of marrow space taken
Bone Marrow
up by adipocytes increases, and in healthy geriatric animals,
hematopoietic tissue may comprise only 10% of red marrow.
Hematopoiesis within the microenvironment of the bone
marrow takes place in the interstitium, and maturing cells
acquire properties that allow them to transit across a special-
ized blood–bone marrow barrier. This barrier consists of a
single layer of endothelium and a discontinuous basement
membrane with adventitial cells, which together regulate
cell transition and release into the blood stream. Erythropoi-
esis occurs in discrete clusters, whereas granulopoiesis is
more dispersed as individual cells throughout the interstitium.
Megakaryocytes are typically located adjacent to sinusoids.
The term “myeloid” refers to all hematopoietic cells in bone
marrow exclusive of lymphocytes, but is often also applied
to cells of only the neutrophil lineage or to spinal cord tissue.
Therefore specific terminology such as “granulocytic” and
“erythrocytic” should be used to describe cells in the marrow.
Rubriblasts are the first recognizable erythrocyte precursor.
Rubriblasts are medium-sized round cells with darkly staining
cytoplasm, a small Golgi zone, high nuclear-to-cytoplasmic
ratio, and one or several nucleoli. With maturation, rubriblasts
progress to rubricytes that progressively synthesize cytoplas-
mic hemoglobin, which imparts initially a purple cytoplasmic
hue and eventually bright orange-pink staining to metarubri-
cytes, immature anucleated reticulocytes, and mature anucle-
ated erythrocytes (Fig. 2-3). Concurrent with homogeneous
cytoplasmic hemoglobin synthesis, rubricyte nuclei progres-
sively condense and decrease in size. Late-stage rubricytes have
small, round, and intensely dark-staining nuclei that are
expulsed prior to transition across the blood–bone marrow
barrier. Rubriblasts and different stages of maturing rubricytes
may comprise clusters surrounding macrophages (“nurse
cells”) that provide trophic factors and iron for synthesis of
hemoglobin. Rubricyte maturation follows a continuum with
mitotic capability throughout, and reliably identifying indi-
vidual cell stages is challenging. Thus, for practical purposes,
synchronous erythropoiesis is characterized by low number of
rubriblasts and immature large rubricytes with round, cen-
trally located nuclei and a moderate amount of blue cyto-
plasm, and high number of late-stage small rubricytes with
increasingly hemoglobinized cytoplasm. Immature (polychro-
matophilic) erythrocytes are difficult to distinguish from mature
erythrocytes in hematoxylin and eosin (H&E)-stained sec-
tions, but are readily identified on cytology preparations.
Maturation from rubriblast to reticulocyte takes ~6 days
under homeostatic conditions, but may be accelerated relative
to the degree of hypoxic stress. Rubricytes are not normally
released from bone marrow, and their presence in blood in the
absence of regenerative anemia suggests altered blood–bone
marrow barrier function, abnormal splenic erythropoiesis, or
lead toxicity. Specific causes of altered blood–bone marrow
barrier function include injury caused by hyperthermia or
chemotherapy, and disruption resulting from granulomatous
inflammation, hematopoietic neoplasia, myelofibrosis, or met-
astatic neoplasia.
Myeloblasts are the precursors of neutrophils, eosinophils, and
basophils. They are larger than rubriblasts, have pale-staining
and abundant cytoplasm, and have one large nucleolus, or,
more commonly, several small nucleoli. Myeloblasts are con-
centrated adjacent to trabeculae (Fig. 2-4) and generate
differentiating granulocytes of progressive maturity toward
the sinusoids in the center of the marrow cavity. Recogniz­
able sequential stages of granulocyte differentiation are
 Bone Marrow 105

A B

C D

E F
Figure 2-2  Bone marrow biopsy sections from a healthy young dog. A. Bone marrow is comprised
of ~70% hematopoietic and 30% adipose tissue. B. Myeloblasts are concentrated adjacent to tra-
beculae, erythrocytes are inconspicuous, and hemosiderin is not yet apparent given the age of the
dog. C. Periodic acid–Schiff stain highlights megakaryocyte and granulocyte cytoplasm. D. CD31
immunohistochemistry identifies small collapsed sinusoids that are difficult to appreciate on H&E
stains. E. Silver impregnation for reticulin is negative in normal bone marrow. F. Masson trichrome
stains erythrocytes and granulocyte and megakaryocyte nuclei red. Extracellular collagen is absent.
106 CHAPTER 2  •  Hematopoietic System Bone Marrow

# *

* *
*

A
Figure 2-3  Bone marrow aspirate with erythroid hyperplasia.
Rubriblast has dark basophilic cytoplasm (#). Promyelocytes have
slightly granular and light blue cytoplasm (*). Metarubricytes
(arrows) are frequent.

promyelocytes with round nuclei, single or multiple nucleoli

and faint cytoplasmic primary granules; myelocytes that are
smaller and have specific cytoplasmic granules identifying
them as committed to neutrophil, eosinophil or basophil dif-
ferentiation; metamyelocytes with indented nuclei; and granu-
locytes with band-shaped and segmented nuclei. Monocytes
are derived from a granulomonocytic precursor cell shared
with granulocytes. Three morphologic stages are recognized:
monoblasts that have round to oval nuclei, one or several
nucleoli, and pale blue cytoplasm; promonocytes that lack
nucleoli and have more angular nuclei; and monocytes with B
butterfly- or horseshoe-shaped nuclei, pale blue cytoplasm,
and frequent cytoplasmic vacuoles. Ultrastructurally, mono-
cytes have scant electron-dense cytoplasmic granules. Fine
pink granules may be apparent in cytology preparations in
monocytes from animals with inflammatory disease. Matura-
tion of monoblast to monocyte requires 1-2 days. Monocytes
and their precursors are very infrequently observed in bone
marrow.
Megakaryocytes are giant cells with multilobulated nuclei
that release cytoplasmic fragments as platelets. A bipotential
megakaryocyte-erythroid progenitor gives rise to megakaryo-
blasts, which further differentiate to megakaryocytes under the
influence of thrombopoietin. Megakaryoblasts are larger than
rubriblasts or myeloblasts, have 1 or 2 nuclei, intensely dark
basophilic cytoplasm, and an irregular cytoplasmic membrane
with frequent small protrusions. Megakaryoblasts are unique C
relative to other cells by undergoing endomitosis without
cytoplasmic separation during differentiation to megakaryo- Figure 2-4  Bone marrow from a dog with chronic inflammation
cytes. This process results in polyploid giant cells containing up and leukocytosis. A. Granulocytes mature from myeloblast/
to 32 nuclei. During maturation, megakaryocytes form an promyelocyte (*) with a large round nucleus and basophilic cyto-
extensive cytoplasmic demarcation membrane system and plasm, to myelocyte (arrows) with faint cytoplasmic granules, to
cytoplasmic granules, which are then transferred to platelet metamyelocytes (arrowheads), band, and segmented neutrophils.
progeny. In general, megakaryocytes with low ploidy have B. Myeloblasts are concentrated in paratrabecular regions
high replicative potential and limited platelet production, (bracket). C. Maturing granulocytes and rubricytes are randomly
whereas high ploidy corresponds to low replicative potential interspersed in small groups.
and abundant platelet production. Mature megakaryocytes
reside proximal to sinusoids, and release platelets from cyto-
plasmic extensions into the sinusoids. In health, individual or
clustered hematopoietic cells and adipocytes are distributed
 Bone Marrow
Table • 2-1  poietic cells. Hematologic manifestations of such conditions
Histologic evaluation of bone marrow
Magnification Features of normal bone marrow
Low power Intact nonfragmented trabeculae with ≥3
intertrabecular spaces free of artifact
Randomly distributed hematopoietic and
adipose cells
Inconspicuous sinusoids and lack of
extravasated red blood cells
Cellularity 10% (old age) to 90% (young
age)
Randomly distributed megakaryocytes (2-5
per intertrabecular space) of different
maturational stages
Iron stores, except in young animals and
cats
Lack of aggregated lymphocytes, plasma
cells, or histiocytes
High power Granulocytic to erythrocytic (G : E) cell
ratio ~1 : 3 (large animals) to ~3 : 1
(small animals)
Differentiated granulocytes
(metamyelocytes, band, and segmented
neutrophils) and rubricytes outnumber
myeloblasts and rubriblasts by >5 : 1
Undifferentiated blast cells, apoptotic or
mitotic cells each comprise <5% of all
cells
randomly throughout the interstitium of the bone marrow,
with maturing granulocytes and rubricytes in similar propor-
tions throughout. Individual megakaryocytes should also be
randomly distributed and encompass a range of maturational
stages and sizes. Clustering of megakaryocytes is indicative of
altered extracellular matrix or hematopoiesis. Other nonhe-
matopoietic cells that may be routinely observed in marrow
include plasma cells, neurons, and stromal cells.
Bone marrow needs to be evaluated systematically and inter-
preted in light of peripheral blood findings. Each cell line has to
be identified and assessed for synchronous maturation reflected
by a low proportion of immature cells and high proportion of
differentiated rubricytes and granulocytes (Table 2-1). The
ratio of granulocytes to rubricytes normally ranges from 1 : 3
in large animals to 3 : 1 in small animals. Blast and mitotic cells
should be rare, and iron should be apparent in all species
except cats. Hematopoietic cells should be closely apposed to
each other, denoting high cell density. Hemorrhage, as indi-
cated by extravasated erythrocytes, should be absent, but bone
marrow biopsies frequently contain artifactual hemorrhage.
Diseases of hematopoietic tissue have a propensity to also
involve lymphoid tissues, for instance, neoplasms of hemato-
poietic cells preferentially spread to lymph nodes, spleen, and
liver, whereas neoplasms of lymphoid tissue frequently also
involve bone marrow. Conditions that alter bone marrow
architecture, such as myelofibrosis, granulomatous inflam­
mation, or metastatic cancer, can disrupt the blood–bone
107
marrow barrier and interfere with orderly release of hemato-
are presence of immature precursor cells, such as rubricytes
and myelocytes, in blood. Concurrence of nonregenerative
anemia with circulating immature leukocytes and rubricytes
has been termed leukoerythroblastic anemia, and reflects
severely disturbed hematopoiesis in myeloid neoplasia or
myelofibrosis.
Myeloid metaplasia is replacement of an extramedullary
mature tissue type by mature hematopoietic tissue, sometimes
including osteocytes, osteoid, and endosteal cells. Myeloid
metaplasia has been described in mixed mammary carcinoma
of dogs. The condition “myelofibrosis with myeloid metapla-
sia” occurs in people, and has also been termed “agnogenic
myeloid metaplasia” and “idiopathic myelofibrosis.” In people,
this is a clonal myeloproliferative neoplasm with proliferation
of nonmalignant bone marrow stromal cells caused by factors
produced by neoplastic hematopoietic cells. Affected people
have massive splenomegaly, neoplastic cytosis (such as
increased erythrocytes in polycythemia vera or increased
platelets in essential thrombocythemia), and cytopenia of at
least one cell line. In essence, in this condition the hematopoi-
etic neoplasm is translocated to spleen because of filling of
marrow cavities with fibrous tissue. All of the above terms
have also been variably used in veterinary medicine; however,
lack of well-documented clinical cases and inability to accurately
distinguish clonal from nonclonal hematopoiesis and fibroplasia
have precluded clear understanding of pathogenesis. Extensive
splenic hematopoiesis is observed in animals with malignant
(myeloid neoplasia) and nonmalignant (myelofibrosis) bone
marrow failure. If such splenic hematopoiesis consists pre-
dominantly of a single cell line with asynchronous maturation,
and there is concurrent lymphoid atrophy and organomegaly,
myeloid neoplasia is likely and should prompt comprehensive
hematologic and bone marrow assessment in the animal. If
splenic hematopoiesis involves multiple cell lines, there is
preservation of white and red pulp organization, and there is
lack of organomegaly, benign extramedullary hematopoiesis sec-
ondary to prolonged high demand for blood cells or nonma-
lignant bone marrow failure should be considered.
Iron for hemoglobin synthesis is stored bound to proteins as
hemosiderin in marrow macrophages of all domestic animals
except cats, which typically lack iron in bone marrow during
health, but have iron stores in spleen and liver. Iron stores are
absent in neonates, and at 6-12 months of age become appar-
ent in bone marrow samples.
Sample procurement and processing
Comprehensive assessment of hematopoietic tissue includes
cytologic preparations for identification and enumeration of
individual cell stages and infectious agents, and sections of fixed
bone marrow tissue to determine the relative proportions of
hematopoietic and adipose tissue, changes in tissue architec-
ture, and presence of myelofibrosis, metastasis, or inflamma-
tion. Cell stage identification is challenging on histopathology
because of subtle changes in cytoplasmic features that distin-
guish immature monocytes from granulocytes, and that char-
acterize different maturational stages of leukocytes and
rubricytes. Therefore in vivo investigation of hematopoietic
disease should include assessment of both types of sample. Bone
marrow core biopsies should be obtained prior to aspiration
of bone marrow for smear preparation to maximize tissue
108 CHAPTER 2  •  Hematopoietic System Bone Marrow

architectural preservation. To collect bone marrow core

samples of adequate size and quality requires special trephine
needles and substantial technical expertise. Suitable sites for
core biopsy are the ilium, proximal femur, and proximal
humerus in small animals, and tuber coxae and sternum in
large animals. Given the small size of some dogs and of cats,
biopsies <1 cm in length are often submitted, which do not
yield an adequately representative sample, particularly if arti-
fact from tissue handling is also present. In general, an artifact-
free biopsy 1.5 cm in length prior to fixation will yield at least
3 intact intertrabecular spaces, which is the minimum required
for representative assessment. Cores should be appropriately
fixed in buffered formalin before demineralization and sec-
tioning. Given the thin nature of trabecular bone, demineral-
ization for 10-15 minutes is adequate for bone marrow
biopsies. Central metaphyseal samples collected at autopsy A
often lack trabecular bone and do not require demineraliza-
tion. To maximize identification of individual cells, bone
marrow cores should be sectioned at 2-3 µm thickness. Bone
marrow aspirates should be obtained from accessible flat
bone sites, such as ilium or sternebrae, or if these sites are
deemed unsuitable, from the proximal humerus or femur.
Preparation of high-quality thin bone marrow smears requires
special effort to minimize sample clotting and hemodilution,
to concentrate particles of loosely adherent hematopoietic
cells on the glass slide, and to avoid drying artifact.
Grossly, normal bone marrow fat is firm and opaque, and
processing dissolves fat to leave empty adipocytes on histol-
ogy. In serous atrophy from malnutrition or malassimilation,
there is gelatinous transformation of bone marrow fat to
glycosaminoglycan, which has a translucent and soft gross
appearance. Histologically, adipocytes are atrophied, and with
hemotoxylin and eosin (H&E) staining, amorphous pink- B
staining glycosaminoglycan matrix is apparent. This matrix
material stains with acidic Alcian blue (Fig. 2-5). Serous
atrophy of fat reflects advanced cachexia. Hematopoiesis
is frequently impaired in animals with such severe cachexia,
and can result in hypocellular bone marrow and blood
cytopenias.
Hematopoietic tissue is preserved relatively well postmor-
tem, possibly because of the physical barrier to infectious
agents imposed by cortical bone. This allows reasonable esti-
mates of cellularity and some estimates of cell composition
after death, but cell identification is limited. Postmortem bone
marrow should be sampled from grossly red (active) marrow,
and sections should be prepared as for biopsies. Only samples
obtained within ~30 minutes after death yield acceptable
cytologic preparations of bone marrow. Associated with death,
mature granulocytes appear to frequently transit from perisi- C
nusoidal locations into the vasculature; therefore a relative
paucity of mature neutrophils is a common finding in post- Figure 2-5  Bone marrow from a horse with serous atrophy
mortem samples and does not necessarily indicate antemor- of marrow fat caused by starvation. A. Cytologic preparations
tem leukopenia. Most bone marrow sections are adequately have a ground-glass background appearance and drying artifact.
assessed with routine H&E staining, but special stains may be B. There is hematopoietic tissue and serous atrophy. C. The gelati-
applied to estimate iron stores (Prussian blue), and to assess nous material stains with Alcian blue (pH 2.5).
presence of reticulin fibers (Gomori silver impregnation) or
collagen (Masson trichrome). Iron may leach during acidic
demineralization; therefore assessment of iron stores may be
underestimated, and cytologic preparations yield more accu- detection of clonally rearranged lymphocyte antigen receptor
rate estimates. Additional techniques useful for diagnosis and genes or clonality in nonlymphoid cells, and immunohisto-
characterization of hematopoietic neoplasms are automated chemistry. These assays have proven value for identification
hematology analysis of marrow aspirate suspensions, flow and characterization of lymphoid cells, but are at the devel-
cytometric analysis of cell antigen expression, PCR-based opmental stage for nonlymphoid hematopoietic cells.
 Bone Marrow
Further reading
Beeler-Marfisi J, et al. Gelatinous marrow transformation and hemato-
poietic atrophy in a miniature horse stallion. Vet Pathol 2011;
48:451-455.
Keller SM, Moore PF. A novel clonality assay for the assessment of
canine T cell proliferations. Vet Immunol Immunopathol 2012;145:
410-419.
Tan E, et al. Automated analysis of bone marrow aspirates from dogs
with haematological disorders. J Comp Pathol 2014;151:67-79.
Tattoli L, et al. Postmortem bone marrow analysis in forensic science:
study of 73 cases and review of the literature. Forensic Sci Int
2014;234:72-78.
Travlos G. Histopathology of bone marrow. Toxicol Pathol 2006;
34:566-598.
Disorders of leukocytes
Blood cells are maintained in clearly defined limits during
health, and are easily counted in automated hematology ana-
lyzers. Therefore numerical changes such as leukocytosis and
leukopenia are readily identified, and many associated condi-
tions are well characterized. Leukocytosis is most commonly
of a reactive nature in response to inflammation, infection, or
increased concentration of systemic mediators, such as gluco-
corticoids or epinephrine. Healthy adult dogs, cats, and horses
normally have more circulating neutrophils than lymphocytes,
whereas lymphocytes outnumber neutrophils in healthy adult
ruminants. The number of neutrophils in blood reflects a
dynamic equilibrium of cell release from bone marrow, release
of cells from storage pools such as splenic and hepatic sinu-
soids, entry from marginating into circulating pools in larger
blood vessels, and extravasation followed by cell death. During
homeostasis, few neutrophils persist in extravascular sites;
therefore numbers in circulation directly reflect bone marrow
production.
Neutrophil production is increased in response to cyto-
kines and growth factors elaborated during inflammation and
infection. The major pathway involves increased production
of granulocyte colony-stimulating factor (G-CSF) and
granulocyte-macrophage colony-stimulating factors (GM-
CSFs) through stimulation of bone marrow stromal cells and
T lymphocytes by interleukin-1 (IL-1) and tumor necrosis
factor. If tissue demand for neutrophils exceeds storage pools,
immature band neutrophils, metamyelocytes, or more imma-
ture forms are released from bone marrow and constitute a
blood left shift. The degree of left shift in blood is proportional
to the acuteness and magnitude of inflammation. For example,
acute pancreatitis in dogs is commonly accompanied by a
moderate blood neutrophilia and a left shift. Immune hemo-
lytic anemia with intravascular destruction of red blood cells
is frequently associated with marked neutrophilia and a
marked left shift, presumably because of extensive intravas-
cular complement activation. Other factors that increase
blood neutrophil concentration are glucocorticoids, which are
thought to decrease neutrophil extravasation, and catechol-
amines and vigorous exercise, which are thought to decrease
neutrophil margination. Effects of catecholamines on leuko-
cytes are particularly evident in young animals, and will also
result in lymphocytosis. In general, neutrophilia from increased
glucocorticoids or catecholamines remains below twice the
upper reference limit, and may include a mild left shift. Other
causes of persistent and marked neutrophilia are prolonged
109
exposure to estrogens, pyometra, extensive tissue necrosis,
ectopic production of G-CSF by nonhematopoietic cancers
such as carcinoma or leiomyosarcoma (paraneoplastic neutro-
philia), leukocyte-adhesion deficiency (LAD), and neutro-
philic leukemia. LAD in cattle and dogs is caused by an
autosomal recessive mutation, resulting in lack of expression
of the integrin subunit CD18. Without expression of CD18,
functional integrins composed of CD11/CD18 heterodimers
are not formed, and neutrophils are unable to adhere to endo-
thelial cells, extravasate, and migrate toward a chemotactic
stimulus. Affected animals have extreme blood neutrophilia
but lack tissue neutrophils at sites of inflammation or infec-
tion, and die because of recurrent infections.
Toxic changes in neutrophils consist of cytoplasmic clear
spaces from organelle dispersion, cytoplasmic basophilia
because of increased RNA concentration, and cytoplasmic
basophilic aggregates (Döhle bodies) composed of RNA and
endoplasmic reticulum. Toxic changes in neutrophils reflect
accelerated or stress granulopoiesis. Bacterial infections, par-
ticularly those involving production of bacterial leukotoxins,
are associated with the most pronounced toxic changes in
neutrophils, but intense noninfectious inflammation such as
pancreatitis can also cause marked neutrophil toxic changes.
An example of a bacterial infection causing severe toxic
changes in neutrophils is Salmonella enteritis in horses.
Neutropenia indicates that demand for neutrophils exceeds
release from bone marrow or storage pools. Neutrophils are
the first line of defense against infection, and prolonged or
severe neutropenia renders animals highly susceptible to
sepsis and death. Acute overwhelming inflammation from
bacterial infection is the most common cause of neutropenia,
with salmonellosis in horses as a classic example. Cattle have
more limited storage and mobilization of bone marrow neu-
trophils than nonruminants, and are particularly prone to
become neutropenic during bacterial infections such as acute
mastitis, metritis, or pneumonia. Other causes of neutropenia
are inadequate production as a result of hematopoietic
progenitor cell injury from drugs or infection by viruses,
premature destruction of neutrophils by immune-mediated
mechanisms, or myelophthisis resulting from leukemia, meta-
static cancer, or myelofibrosis. Parvovirus in dogs and cats
infects dividing cells in the intestinal epithelium and bone
marrow, resulting in cell death or lack of normal proliferation.
Neutropenia is an early effect, and concurrent with a compro-
mised epithelial barrier, permits systemic invasion of intestinal
bacteria in a host unable to mount an effective inflammatory
response. Affected animals are at risk of fatal secondary infec-
tions. Many retroviruses may cause neutropenia, either
through direct infection of hematopoietic precursor cells
(feline leukemia virus) or through indirect mechanisms (feline
immunodeficiency virus [FIV]). Bovine viral diarrhea virus
(BVDV) infects hematopoietic precursor cells, and infection
manifests with neutropenia and thrombocytopenia in cattle.
Immune-mediated neutropenia in neonatal animals caused
by maternal immune response to polymorphic paternal or
unknown antigens occurs in piglets, calves, foals, kittens, and
puppies. Antibodies transferred either in utero or postnatally
via colostrum bind to antigens on neutrophils (and often also
erythrocytes and platelets) and induce either direct cell lysis
or removal of antibody-coated cells by splenic macrophages.
In foals, concurrent neutropenia, thrombocytopenia, and
ulcerative dermatitis have been described, suggesting concur-
rent immune reactivity with antigens on hematopoietic and
 109.e1

Further reading
Auler P, et al. Myeloid metaplasia in canine mixed mammary tumors:
occurrence and characterization. Vet Q 2011;31:173-177.
Mochizuki H, et al. Demonstration of the cell clonality in canine hema-
topoietic tumors by X-chromosome inactivation pattern analysis.
Vet Pathol 2015;52:61-69.
Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic
stem cells. Nature 2014;505:327-334.
Tefferi A. The forgotten myeloproliferative disorder: myeloid metapla-
sia. Oncologist 2003;8:225-231.
Wilkins BS. Pitfalls in bone marrow pathology: avoiding errors in
bone marrow trephine biopsy diagnosis. J Clin Pathol 2011;64:
380-386.
Wilkins BS, Clark DM. Making the most of bone marrow trephine
biopsy. Histopathol 2009;55:631-640.
110 CHAPTER 2  •  Hematopoietic System
epithelial cells. In cattle, an adjuvanted inactivated BVDV
vaccine administered to breeding age cows in Europe and
New Zealand caused a widespread condition termed “bovine
neonatal pancytopenia” in offspring. Approximately 50% of
affected calves died because of massive internal and external
hemorrhage. Affected animals also had an increased incidence
of infectious diseases. Detailed analysis of hematologic find-
ings showed that affected calves had predominantly thrombo-
cytopenia and neutropenia, and became anemic as a result of
hemorrhage. Allo-immunization against polymorphic antigens
incorporated into the vaccine from in vitro producer cells was
considered to be the cause of this neonatal immune-mediated
cytopenia.
Several genetic causes of congenital neutropenia have been
identified. Trapped neutrophil syndrome occurs in Border
Collies across the world, and is caused by an autosomal reces-
sive deletion in the gene coding for vesical protein sorting 13B
(VPS13B). The carrier rate of the mutation is 4-8%, and
affected dogs have neutropenia, slender extremity bones, poly-
arthritis, and microencephaly. The cause of the neutropenia is
unclear, but is likely related to impaired generation and trans-
port of neutrophil granules. Canine cyclic hematopoiesis occurs
in Collie-like dogs and is the result of an autosomal recessive
insertion into the gene coding for an adapter protein subunit
important in trafficking of vesicles and proteins, such as neu-
trophil elastase. Concentration of all blood cells, not just neu-
trophils, fluctuates in this condition, and affected animals also
have limited melanin pigmentation of hair follicles resulting
in a “gray” phenotype.
Abnormally large granules in neutrophils and other cells
occur in animals with Chediak-Higashi syndrome (CHS).
CHS is caused by mutations in the lysosomal trafficking regu-
lator LYST gene that result in reduced or absent formation,
migration, and exocytosis of lysosomes. Mutations that pro-
foundly reduce the LYST protein correlate with a more severe
clinical phenotype than those that generate a truncated
protein. Abnormal granule formation may be observed in
many cell types. In neutrophils, lack of transcellular movement
and extrusion of abnormally large lysosomes impairs
phagosome-lysosome fusion and microbial killing. Affected
animals generally have normal neutrophil counts but reduced
innate immunity. Granules of cytotoxic and natural killer lym-
phocytes also are not extruded properly; platelets have
reduced dense granules, impairing aggregation and resulting in
a bleeding diathesis; and animals with CHS have partial or
complete albinism from reduced transfer of melanin-
containing granules. The condition occurs in cattle, cats, mink,
humans, mice, and other species. CHS has been described in
Hereford, Brangus, and Japanese black cattle that have partial
oculocutaneous albinism and leukocyte and platelet defects.
CHS is the cause of the blue smoke fur color variant in Persian
cats and the light blue-grey fur color in Aleutian mink. Affected
mink have normal neutrophil and platelet number, giant neu-
trophil granules, and reduced platelet aggregation. All mink
are susceptible to infection by a parvovirus that results in
neuropathy, hyperglobulinemia, anemia, and progressive
weight loss. Mink with the Aleutian trait have particularly
severe and fatal disease, and it has been speculated that the
cause is defective innate immune function as a result of CHS.
Pelger-Huët anomaly (PHA) is caused by a genetic muta-
tion that in heterozygotes manifests with band-shaped or
bilobed instead of segmented granulocyte nuclei. In affected
humans, the lamin B receptor (LBR) that stabilizes nuclear
Bone Marrow
membrane interactions with heterochromatin, and contrib-
utes to nuclear segmentation, is mutated. Identical morpho-
logic changes in granulocytes of animals suggest that mutation
is LBR is also the cause of PHA in animals, but the actual
genetic defect remains to be mapped. In PHA, neutrophil and
eosinophil nuclei are hyposegmented but have condensed
clumped chromatin like normal mature granulocytes. PHA is
usually an incidental finding on a complete blood count
(CBC), given that the hyposegmented granulocytes are not
functionally impaired. PHA has been reported in multiple dog
breeds, a few cats, and 2 Arabian horses, and is inherited as a
dominant or co-dominant trait. Homozygous mutation of the
LBR gene in people results in more severely hyposegmented
nuclei, skeletal abnormalities, and frequent fatality, and a
similar phenotype occurs in homozygous cats. Absence of
toxic changes in neutrophils and clinical signs of infection
distinguish PHA from a severe left shift. Acquired PHA, also
called pseudo-PHA, is also characterized by hyposegmented
granulocyte nuclei, but in the absence of bacterial or fungal
infection. Acquired PHA has been described in association
with retroviral infection, chemotherapy, myeloid neoplasia,
and myelodysplastic syndrome. Thorough clinical history,
repeat CBCs, and hematologic evaluation of related animals
will distinguish the different entities.
Overall, genetic causes of neutropenia and abnormal neu-
trophil morphology or function are rare in animals, but serve
as useful models of corresponding human conditions.
Some agents infect neutrophils and cause profound neu-
trophilia. Hepatozoon is a large genus of hemogregarine para-
sites for which vertebrates serve as the intermediate host and
invertebrates as the definitive host. Hepatozoon gamonts infect
leukocytes in mammals and birds, and erythrocytes in reptiles.
Dogs infected with H. canis or H. americanum have marked
neutrophilia. Hepatozoon parasites have a life cycle distinct
from other vector-borne protozoa because they are acquired
by vertebrates through ingestion of an invertebrate host such
as a tick containing oocysts, rather than being transmitted via
saliva by a tick during a bite.
Hepatozoon canis is prevalent in Africa, Asia, southern
Europe, and South and North America, and causes predomi-
nantly infection of hemolymphoid organs. H. americanum
occurs in dogs in the southern and central United States, and
causes myositis and severe lameness. Several ticks, including
Rhipicephalus sanguineus in Europe and Amblyomma ovale in
South America, transmit H. canis. Sporozoites ingested with
the tick migrate through the intestinal wall of the dog, invade
mononuclear cells and disseminate via blood or lymph to
hemolymphatic organs, liver, kidney, and lungs. Merogony
with formation of micromerozoites and macromerozoites
occurs in host tissues, and mature meronts released from
tissues invade neutrophils and monocytes to develop into
gamonts that can be observed on blood smears. The tick
Amblyomma maculatum transmits H. americanum, but it
appears that H. americanum can also be acquired by predation
of other infected mammalian hosts and through intrauterine
transmission.
Infection of dogs with H. canis can result in subclinical
illness or severe life-threatening cachexia, lethargy, and
anemia. A mild subclinical infection is most common and
associated with <5% of leukocytes on blood films containing
H. canis gamonts and mild anemia. Severe illness with marked
parasitemia and neutrophilia occurs in immunocompromised
or debilitated dogs, or with concurrent other infections. Other
 Bone Marrow
abnormalities in H. canis infection are polyclonal hyperglobu-
linemia, and increased creatine kinase and alkaline phospha-
tase activity. Periostitis is uncommon. Diagnosis is usually
based on observing gamonts on blood films, or meronts in
tissue biopsies.
Infection with H. americanum results in more severe
disease and is fatal after several months unless treated. Target
organs of H. americanum are skeletal and cardiac muscle
where infected macrophages form cysts composed of zoites
surrounded by layers of glycosaminoglycan. Merogony fol-
lowed by rupture of the cyst and release of merozoites causes
intense myositis. Massive neutrophilia of up to 200 × 109/L is
observed in H. americanum infection, although the number of
infected neutrophils on blood smears is typically <0.1%.
Hence diagnosis may require biopsy of affected muscle for
demonstration of cysts. Affected dogs have fever, muscle pain
and atrophy, mucopurulent ocular discharge, and bone pain.
Chronic infection with persistent hyperglobulinemia may
result in renal amyloidosis, glomerulonephritis, and uveitis.
Untreated infection with H. americanum results in debilitation
and death.
Hepatozoonosis has been reported in cats from several
countries with H. canis infection, but neither the species of
Hepatozoon nor the definitive host is identified. Infection was
associated with meronts in myocardium and skeletal muscle,
and low levels of parasitemia.
Another vector-borne agent of leukocytes is Anaplasma
phagocytophilum, which is the current name of the former
Ehrlichia equi, Ehrlichia phagocytophila, and human granulo-
cytic ehrlichiosis agent. Anaplasma phagocytophilum is a widely
distributed, obligate intracellular, gram-negative bacterium
transmitted by various ixodid ticks. The bacterium infects and
survives in neutrophils by interfering with phagocytosis, oxida-
tive burst, motility, and endothelial adhesion. Groups of aggre-
gated bacteria in endosomes of neutrophils may be observed
during acute infection and are called morulae. Infection can
variably cause fever, leukopenia, thrombocytopenia, edema,
petechiation, and reluctance to move. Infection in domestic
and nondomestic ruminants in Europe with A. phagocytophi-
lum is called tick-borne fever. Equine granulocytic anaplasmosis
is associated with fever, neutropenia, thrombocytopenia,
ataxia, and icterus. Experimentally infected dogs have tran-
sient high fever, thrombocytopenia, and leukopenia. In immu-
nocompetent animals, infection with A. phagocytophilum is
self-limiting and induces robust antibody responses. Infection
is diagnosed by observing morulae on blood films, rising anti-
body titers, and PCR detection of bacterial nucleic acids.
Canine monocytotropic ehrlichiosis is caused by Ehrlichia
canis, which is transmitted by the brown dog tick Rhipicepha-
lus sanguineus. Infection of dogs, and rarely cats, occurs world-
wide. German Shepherd dogs are suggested to be particularly
susceptible to infection. Acute infection is similar to that with
A. phagocytophilum, except that ophthalmic lesions are more
common and consist of anterior uveitis, chorioretinitis, papill-
edema, and retinal hemorrhage, and thrombocytopenia is
moderate to severe. Infection progresses from an acute phase
to a subclinical phase, and in some cases to a chronic phase.
Dogs in the chronic phase of monocytotropic ehrlichiosis are
severely ill and have marked weight loss. Morulae may be
observed in monocytes on blood films during the acute phase.
In dogs with advanced disease, marked lymphocytosis com-
posed of large granular lymphocytes has been observed, which
is an immune, rather than a neoplastic, response.
111
Other vector-borne rickettsial infections of domestic
animals, such as salmon poisoning disease caused by Neorick-
ettsia helminthoeca and equine neorickettsiosis (Potomac horse
fever, also called equine monocytic ehrlichiosis), caused by
Neorickettsia risticii, are covered in later sections of this
chapter and other chapters.
Lymphopenia is most often the result of redistribution of
lymphocytes to lymphoid organs rather than an absolute
decrease in body lymphocytes. Causes for such altered distri-
bution include increased endogenous and exogenous gluco-
corticoids, and acute bacterial or viral infection. Absolute
lymphopenia may result from intestinal loss of lymphatic fluid
in lymphangiectasia or severe enteropathy, immunosuppres-
sion, chemotherapy, and radiation. Rare causes of absolute
lymphopenia are congenital severe combined immunodefi-
ciency caused by impaired DNA repair in Arabian horses, or
lack of cytokine signals in various dog breeds as a result of
mutation in the cytokine receptor subunit shared by IL-2,
IL-4, IL-7, IL-9, IL-15, and IL-21. Of note, blood lymphocyte
concentration is generally higher in healthy young than adult
animals; hence lymphopenia in young animals should prompt
assessment of immune competency.
A common cause of lymphocytosis is endogenous catechol-
amine release. Hypoadrenocorticism in dogs, and infections
with bacterial and protozoal agents, such as Ehrlichia, Ana-
plasma, Theileria, and Babesia, may manifest with lymphocy-
tosis. Retroviruses such as feline leukemia virus (FeLV), equine
infectious anemia virus (EIAV), FIV, and bovine leukemia
virus (BLV) establish permanent infections that may result in
transient lymphocytosis. Lymphocyte concentration is gener-
ally lower in BLV-seropositive than seronegative healthy cows,
but ~30% of infected cows develop persistent non-neoplastic
B-cell lymphocytosis. Neoplastic lymphocytosis occurs in leu-
kemic lymphoma or leukemia.
Causes of monocytosis are chronic infections, excess glu-
cocorticoids, histiocytic proliferative diseases, and myelopro-
liferative neoplasia.
Eosinophilia can be seen in animals with extensive tissue
parasite migration, allergic responses, or as a paraneoplastic
condition. Blood eosinophil transit can be very brief as
reflected by blood concentrations that are normal or only
slightly increased despite substantial tissue eosinophil accu-
mulations. This is considered to result from prolonged tissue
eosinophil survival in the presence of cytokines such as IL-5.
Massive blood eosinophilia and tissue eosinophil infiltrates are
observed in cats, and distinction between reactive and neo-
plastic proliferation may be difficult in these cases.
Basophilia is rarely observed in animals, and is associated
with parasitic and neoplastic conditions such as heartworm
infection, or mast cell and myeloproliferative neoplasms.
Further reading
Allen KE, et al. Hepatozoon spp infections in the United States. Vet
Clin North Am Small Anim Pract 2011;41:1221-1238.
Allison RW, Little SE. Diagnosis of rickettsial diseases in dogs and cats.
Vet Clin Pathol 2013;42:127-144.
Anistoroaei R, et al. A frameshift mutation in the LYST gene is
responsible for the Aleutian color and the associated Chediak-
Higashi syndrome in American mink. Anim Genet 2013;44:
178-183.
Baneth G. Perspectives on canine and feline hepatozoonosis. Vet Para-
sitol 2011;181:3-11.
 111.e1

Further reading
Aroch I, et al. Clinical, biochemical, and hematological characteristics,
disease prevalence, and prognosis of dogs presenting with neutro-
phil cytoplasmic toxicity. J Vet Intern Med 2005;19:64-73.
Baird JD, Arroyo LG. Historical aspects of Potomac horse fever in
Ontario (1924-2010). Can Vet J 2013;54:565-572.
Dallap Schaer BL, et al. Identification of predictors of Salmonella shed-
ding in adult horses presented for acute colic. J Vet Intern Med
2012;26:1177-1185.
de Caprariis D, et al. Evolution of clinical, haematological and bio-
chemical findings in young dogs naturally infected by vector-borne
pathogens. Vet Microbiol 2011;149:206-212.
Deshuillers P, et al. Pelger-Huët anomaly in a cat. Vet Clin Pathol
2014;43-337-341.
Dotta L, et al. Clinical, laboratory and molecular signs of immunodefi-
ciency in patients with partial oculo-cutaneous albinism. Orphanet
J Rare Dis 2013;8:168.
Harrus S, Waner T. Diagnosis of canine monocytotropic ehrlichiosis
(Ehrlichia canis): an overview. 2011;187:292-296.
Meng R, et al. Neutrophil elastase-processing defect in cyclic hemato-
poietic dogs. Exp Hematol 2010;38:104-115.
Perkins GA, et al. Ulcerative dermatitis, thrombocytopenia, and neu-
tropenia in neonatal foals. J Vet Intern Med 2005;19:211-216.
Petterino C, et al. Paraneoplastic leukocytosis in a dog with a renal
carcinoma. Vet Clin Pathol 2011;40:89-94.
Shearman JR, Wilton AN. A canine model of Cohen syndrome: trapped
neutrophil syndrome. BMC Genomics 2011;12:258.
112 CHAPTER 2  •  Hematopoietic System
Brovik L, et al. Pelger-Huët anomaly and a mild skeletal phenotype
secondary to mutations in LBR. Am J Med Genet A 2013;
161A:2066-2073.
Carrade DD, et al. Canine granulocytic anaplasmosis: a review. J Vet
Intern Med 2009;23:1129-1141.
Demasius W, et al. Bovine neonatal pancytopenia (BNP): novel insights
into the incidence, vaccination-associated epidemiological factors
and a potential genetic predisposition for clinical and subclinical
cases. Res Vet Sci 2014;96:537-542.
Gleich S, Hartmann K. Hematology and serum biochemistry of feline
immunodeficiency virus-infected and feline leukemia virus-infected
cats. J Vet Intern Med 2009;23:552-558.
Mason SL, et al. Presentation and management of trapped neutrophil
syndrome (TNS) in UK border collies. J Small Anim Pract 2014;
55:57-60.
Nagahata H. Bovine leukocyte adhesion deficiency (BLAD): a review. J
Vet Med Sci 2004;66:1475-1482.
Reissmann M, Ludwig A. Pleiotropic effects of coat colour-associated
mutations in humans, mice and other mammals. Semin Cell Dev
Biol 2013;24:576-586.
Swenson CL, et al. Impact of bovine leukemia virus infection on neu-
trophil and lymphocyte concentrations in dairy cattle. J Am Vet Med
Assoc 2013;243:131-135.
Zimmerman KL, et al. Leukocyte adhesion deficiency type I in a mixed-
breed dog. J Vet Diagn Invest 2013;25:291-296.
Disorders of erythrocytes
Anemia is a reduction in the red blood cell (RBC) mass and
therefore in oxygen-carrying capacity. Causes of anemia are
reduced erythrocyte production, premature destruction, and
blood loss (Table 2-2). The degree of anemia is generally cat-
egorized as mild, moderate, or marked if the hematocrit is
within 13 , within 2 3 , or > 2 3 below the lower reference interval,
respectively. Anemia is further characterized as regenerative if
there is an increased concentration of polychromatophilic
RBC or reticulocytes in blood and an increased proportion of
rubricytes in bone marrow, or as nonregenerative if production
of RBCs is not increased. Mechanistically, there are 3 main
categories of anemia: blood loss, hemolytic, and decreased
production.
Blood loss anemia
Acute blood loss anemia is usually the result of trauma. Most
external blood loss is readily identifiable, whereas internal
blood loss may be obscured. In both instances, there is a rapid
shift of water from the interstitial fluid compartment into the
vasculature to restore blood volume, which results in hemo-
dilution and a lowered hematocrit and protein concentration.
Reduced oxygen delivery stimulates erythropoietin produc-
tion in the kidney, which in turn increases proliferation of
committed erythroid progenitor cells in bone marrow. Newly
released erythrocytes appear in blood in 3-5 days, depending
on the intensity of the erythropoietic stimulus and the capac-
ity of the host to respond. Horses release very few reticulocytes
from bone marrow in regenerative anemia; these few reticulo-
cytes may be detectable with automated analyzers that scru-
tinize large numbers of cells, but they are typically not
identified on blood smear review. However, newly released
erythrocytes in horses and other species are larger than mature
erythrocytes and have lower hemoglobin concentration, trans-
lating to higher mean cell volume (MCV), and lower mean
cell hemoglobin concentration (MCHC). Therefore regenera-
Bone Marrow
tive anemia is usually macrocytic and hypochromic. Hemorrhage
into tissues results in recycling of the iron in hemoglobin, and
therefore a more robust regenerative response than hemor-
rhage into the gastrointestinal tract or other external sites with
loss of iron.
Chronic blood loss leads to iron deficiency. Dietary iron
deficiency is the most common human nutritional disorder in
the world. Diets for domestic animals are generally iron
replete, therefore iron deficiency in adult animals is rare and
virtually always the result of whole-body blood loss. Iron
metabolism is tightly regulated: dietary iron is absorbed by
duodenal enterocytes, and transferred from enterocytes to
plasma through interaction with ferriportin and oxidative
enzymes. In plasma, iron is bound to transferrin and trans-
ported to rubricytes and macrophages for incorporation into
hemoglobin and storage, respectively. Free iron is highly toxic;
therefore iron is always bound to proteins such as ferritin, and
blood ferritin concentration is a good indicator of body iron
stores. Intracellular partially degraded ferritin aggregates into
hemosiderin granules. The iron in hemosiderin reacts with
potassium ferrocyanide and turns blue-black, which is the
Prussian blue stain reaction. Multiple additional proteins regu-
late iron transport within and across cells. Hepcidin is a central
regulator of iron metabolism. Increased hepcidin inhibits iron
transfer from enterocytes into plasma, and iron release from
macrophages, leading to loss of iron with enterocyte sloughing
and iron retention in macrophages. In iron-replete states, hep-
cidin concentration is high, which limits iron absorption and
iron mobilization from macrophage stores. In iron-deficient
states, hepcidin concentration is low, which enhances iron
transfer from enterocytes. However, hepcidin is also increased
in chronic inflammation and disease, which inhibits enterocyte
iron transfer and iron mobilization from storage in macro-
phages, leading to the conundrum of abundant stainable
iron in bone marrow and spleen despite a functional iron-
limited anemia. Anemia of chronic inflammation and disease
is most common in dogs, but is also seen in other domestic
species.
Iron deficiency resulting from blood loss develops gradu-
ally, usually weeks to months. With modern hematology ana-
lyzers, changes in erythrocytes caused by iron-limited
erythropoiesis can be detected long before there is iron defi-
ciency characterized by lack of stainable iron in bone marrow
and spleen, and reduced blood iron and ferritin concentration.
Neonatal animals have very limited iron stores, and milk is
low in iron, resulting in physiologic iron deficiency during the
first few months of life. By 4-6 months of age, bone marrow
and/or spleen iron stores become apparent, and persistent lack
of stainable iron and anemia are abnormal. The most common
cause of anemia in juvenile animals is blood loss caused by gas-
trointestinal parasites, such as strongyles in horses, Haemonchus
contortus and Trichostrongylus spp. in ruminants, and hook-
worms and ascarids in dogs and cats. Heavy ectoparasite infes-
tation can also cause iron deficiency anemia. Piglets in intensive
production systems are born severely iron deficient and gener-
ate iron stores very slowly; therefore, despite routine paren-
teral iron supplementation, piglets remain highly susceptible
to debilitating or fatal anemia from blood loss. In adult pet
dogs and cats, iron deficiency anemia is most often secondary
to a bleeding gastrointestinal neoplasm. Internal bleeding and
epistaxis associated with vascular erosion in guttural pouch
mycosis may cause anemia, and occasionally iron deficiency
anemia, in horses. Bleeding into the esophagus, stomach, and
 Bone Marrow 113

Table • 2-2 
Classification of anemia by mechanism
Mechanism Hematologic and clinical findings Examples, affected breeds

Blood Loss
  Acute Low to normal hematocrit, low to normal plasma Trauma with external blood loss
protein concentration, normal MCV
  Chronic Low hematocrit, normal plasma protein concentration, Gastrointestinal or urogenital bleeding
low to normal MCV
Hemolysis
  Immune- Marked anemia, spherocytosis, agglutination, marked Primary (autoimmune), drug-induced, transfusion
mediated jaundice, splenomegaly, hemoglobinuria reaction, infectious agents, paraneoplastic
  Infectious Mild to marked anemia, spherocytosis, mild or absent Babesiosis, anaplasmosis, cytauxzoonosis, hemotropic
jaundice, mild or absent splenomegaly mycoplasmosis, equine infectious anemia
  Mechanical Mild to moderate anemia, poikilocytosis, RBC Endocardiosis, endocarditis, vasculitis, intravascular
trauma fragmentation, mild or absent jaundice coagulation, burns
  Toxic injury Mild to moderate anemia, poikilocytosis, Heinz bodies, Oxidant injury, clostridial sepsis, leptospirosis,
mild or absent jaundice hyperglycemia, snake venom
  Hereditary Pyruvate kinase (PK) deficiency—dog: mild to moderate Various small to medium-sized dog breeds; Somali,
hemolytic anemia, persistent reticulocytosis, Abyssinian, and domestic shorthair cats
splenomegaly, develop myelofibrosis and
osteosclerosis; cat: mild or absent anemia, persistent
reticulocytosis
Phosphofructokinase (PFK) deficiency: mild to moderate Spaniel-type dogs, Whippets, mixed-breed dogs
anemia worsened by exercise, persistent
reticulocytosis
Glucose-6-phosphate dehydrogenase (G6PD) deficiency: American Saddlebred
episodic mild to moderate anemia, jaundice
Erythrocyte flavin adenine dinucleotide (FAD) deficiency: Spanish Mustang, Kentucky Mountain Saddle Horse
eccentrocytosis, pyknocytosis, methemoglobinemia,
normal hematocrit
Cytochrome b5 reductase deficiency: normal Multiple dog breeds, domestic shorthair cats
hematocrit, methemoglobinemia, cyanosis
Stomatocytosis: mild to moderate anemia Standard Schnauzer, Alaskan Malamute, Patrijshond
Spherocytosis: mild to moderate anemia Golden Retriever
Elliptocytosis: normal hematocrit Various dog breeds

Decreased Production
Chronic inflammation with iron sequestration: Cancer, polyarthritis, enteritis, etc.; common in dogs;
normocytic normochromic or microcytic hypochromic uncommon in other species
anemia
Iron deficiency: macrocytic or normocytic hypochromic All species; most common in dogs
anemia progressing to microcytic hypochromic
anemia
Erythropoietin deficiency: normochromic normocytic Chronic kidney disease; all species
anemia
Cobalamin deficiency: mild to moderate anemia, Giant Schnauzer, Australian Shepherd, Border Collie,
leukopenia, megaloblastic change in hematopoietic Shar Pei, Beagle
cells
Immune or toxin-mediated progenitor cell injury: Pure red cell aplasia (lack of RBC production);
moderate to severe anemia +/− other cytopenia aplastic anemia (lack of production of all
hematopoietic cells); all species
Primary hematopoietic neoplasm: acute myeloid or All species and ages
lymphoid leukemia, myelodysplasia,
myeloproliferative neoplasm; marked cytopenia of
one or several cell lineages
Metastatic hematopoietic neoplasm: mild cytopenia Myeloma, carcinoma, mast cell tumor
MCV, mean cell volume; RBC, red blood cell.
114 CHAPTER 2  •  Hematopoietic System
Figure 2-6  Blood smear from a dog with iron deficiency anemia.
Note severely hypochromic erythrocytes, acanthocytes (arrow-
heads), and rare polychromatophilic cells (arrows).
proximal small intestine allows partial digestion and reabsorp-
tion of heme and globins, and therefore more gradual develop-
ment of iron deficiency than blood loss into the distal small
or large intestine. Hemoglobin in digesta increases protein
absorption and catabolism, and therefore blood urea concen-
tration. Morphologically, erythrocytes in iron deficiency anemia
are hypochromic and microcytic, fragmented erythrocytes are
common, and there is often a thrombocytosis composed of
microcytic platelets (Fig. 2-6).
Further reading
Lipinski P, et al. Benefits and risks of iron supplementation in anemic
neonatal pigs. Am J Pathol 2010;177:1233-1243.
Schaefer DMW, Stokol T. The utility of reticulocyte indices in distin-
guishing iron deficiency anemia from anemia of inflammatory
disease, portosystemic shunting, and breed-associated microcytosis
in dogs. Vet Clin Pathol 2015;44:109-119.
Hemolytic anemia
Hemolysis of erythrocytes can be intravascular or extravascu-
lar, or both. Immune-mediated hemolysis is most common in
animals, and involves premature removal of antibody-coated
erythrocytes by splenic macrophages. The antibodies may be
directed against antigens on erythrocytes recognized as foreign
as a result of changes in conformation, exposure of hidden
epitopes, or other reasons, or may be directed against infec-
tious agents or drugs absorbed onto, or inducing altered
expression of, membrane components. In dogs with immuno-
hemolytic anemia, the life-span of antibody-coated erythro-
cytes is usually reduced from 120 days to <10 days, resulting
in intense erythropoietic stress. Partial phagocytosis of
antibody-coated erythrocyte membranes by splenic macro-
phages changes the erythrocyte shape from discoid to spher-
oid, with morphologically identifiable spherocytes on blood
smears (Fig. 2-7). Spherocytes have reduced deformability in
splenic red pulp sinusoids, leading to phagocytic removal.
Other mechanisms likely also contribute to immunohemo-
lytic anemia. Apoptosis of erythrocytes has been termed
Bone Marrow
eryptosis, and is characterized by cell shrinkage, extracellular
exposure of phosphatidylserine, membrane blebbing, and
intracellular protease activation. In people, eryptosis is consid-
ered to be a major contributor to anemia of toxic injury, fever,
metabolic derangement, and erythrocyte infection. Intermedi-
aries of the eryptotic pathway have been identified in eryth-
rocyte membranes of dogs with immunohemolytic anemia,
but detailed understanding of this process in the pathogenesis
of anemia is lacking.
Intravascular hemolysis results from activation of com­
plement, and is characterized by presence of erythrocyte
ghosts on blood smears, free plasma hemoglobin, and hemo-
globinuria and hemosiderosis in renal tubular cells from reab-
sorbed filtered hemoglobin. Free plasma hemoglobin is highly
oxidative, and rapidly bound to haptoglobin. Hemoglobin-
haptoglobin complexes have reduced glomerular filtration and
are scavenged by tissue macrophages expressing the CD163
receptor. Once the capacity of haptoglobin to bind free hemo-
globin is exceeded, hemoglobinemia, methemoglobinemia,
and hemoglobinuria ensue. Myoglobin does not have a carrier
protein and is filtered rapidly across the glomerulus; therefore
discolored plasma is the result of free hemoglobin and not
myoglobin. However, both urinary hemoglobin and myoglo-
bin result in grossly indistinguishable pigmenturia.
Immunohemolytic anemia manifests differently in cats than
in dogs. Onset in cats is often <2 years of age, concurrent
neutropenia and thrombocytopenia are common, evidence of
cell regeneration in blood or bone marrow may be absent, and
prolonged or even lifelong immune suppression is required to
normalize blood cell counts. Thus the conditions in cats may
be more appropriately termed immune-mediated cytopenia
(IMC). Although IMC appears increasingly frequent in cats,
pathogenic mechanisms and detailed epidemiologic and clini-
cal characterization remain to be determined.
All erythrocyte parasitic infections have the potential to
cause anemia by multiple mechanisms, including direct eryth-
rocyte lysis, immune-mediated recognition and removal,
shortened life-span caused by oxidative stress, and blood loss
resulting from endothelial disruption or inadequate platelet
function.
Infectious agents of erythrocytes induce predominantly extra-
vascular hemolysis that manifests with hyperbilirubinemia,
jaundice, anemia, and splenomegaly. Depending on the type
of organism, anemia and jaundice may be subtle, such as in
cats infected with Mycoplasma haemofelis and horses with
piroplasmosis, or profound, as in cattle infected with Ana-
plasma marginale, or cats infected with Cytauxzoon felis.
Severity of illness is often variable and influenced by the
genetic background in the host, age of the host at the time of
infection, preexisting immunity, and concurrent other illness.
Erythrocyte infections that are either widespread or economi-
cally important are described in detail later.
Equine infectious anemia.  Species Equine infectious
anemia virus (EIAV) is a member of the Lentivirinae subfam-
ily of retroviruses that infects horses, mules, and donkeys.
Infection occurs worldwide and is lifelong. Diagnosis of EIAV
infection by detection of antibodies (by the agar gel immuno-
diffusion “Coggins test,” or now by enzyme-linked immuno-
sorbent assay [ELISA]) has aided in establishing control
measures in many countries. The virus is transmitted by
various flies, and transmission is mechanical not biological, and
may also occur accidentally by contaminated needles, syringes,
and tattoo equipment, or experimentally by parenteral
 114.e1

Further reading
Powers JM, Buchanan GR. Diagnosis and management of iron defi-
ciency anemia. Hematol Oncol Clin North Am 2014;28:729-745.
 Bone Marrow 115

A B

C D
Figure 2-7  Samples from a dog with immunohemolytic anemia and intravascular hemolysis.
A. There are numerous ghost cells (arrows), spherocytes (arrowheads), and polychromatophilic
erythrocytes in the blood smear. The nucleated cell is a rubricyte. B, C. The bone marrow aspirate
and section show marked rubricyte hyperplasia but orderly maturation. D. Renal tubules contain
heme pigments reabsorbed from urinary filtrate.

administration of blood or virus. Infected horses are the sole
source of viral transmission.
Infection causes acute severe illness that may be fatal, then
cycles of illness characterized by thrombocytopenia and
anemia, and eventual asymptomatic infection. However, even
horses without clinical signs of illness remain a reservoir of
infectious virus. The cyclic nature of infection corresponds to
an intense immune response that drives selection of viral
escape mutants in a manner akin to immunity to the human
immunodeficiency virus in humans.
EIAV infects cells of the monocyte-macrophage system, and the
mechanism for thrombocytopenia and anemia is thought to
be a combination of premature removal of platelets and eryth-
rocytes coated with immune complexes, and direct infection
of megakaryocytes. Hemolysis is predominantly intravascular,
and during chronic infections, the bone marrow becomes
hypoplastic with plasma cell hyperplasia. Acute disease is
characterized by pyrexia and marked depression, with
anorexia, jaundice, weight loss, and pitting and dependent
edema. There are petechial hemorrhages on the ventral surface
of the tongue but also on ocular and vulvar mucosae that
reflect thrombocytopenia. Mild icterus develops after a short
febrile period and is accompanied by pallor of mild to marked
degree. The febrile periods may progress to death in less than
a week, or regress and recur at irregular intervals. Rarely, an
acute episode is followed by many years of an asymptomatic
carrier state.
During acute illness anemia is severe and sufficient to cause
death, with hemoglobin of 25-50 g/L, and hematocrits of
0.08-0.15 L/L. There is consistent thrombocytopenia during
febrile periods, with resulting petechiae. Anemia is initially
regenerative as indicated by anisocytosis and macrocytosis,
and later becomes nonregenerative. There may also be leuko-
penia as a result of neutropenia and lymphopenia, and at
least a relative monocytosis. Monocytes may contain ingested
erythrocytes (sideroleukocytes) within 2-3 days of a febrile
episode. Grossly, horses that die or are euthanized have edema
of the ventral abdominal wall and in the suspensory ligaments
of the viscera. The spleen is enlarged, turgid, and fleshy with
capsular hemorrhages; a bulging but not oozing cut surface;
and inapparent lymphoid follicles. The liver is enlarged,
dark, and turgid, with a fine lobular pattern and focal capsular
hemorrhages. Petechial hemorrhages are present on the
renal capsules and in the perirenal tissues. On cut surface,
116 CHAPTER 2  •  Hematopoietic System
there are multiple fine hemorrhages throughout the cortex
and medulla. The most significant gross lesions occur in bone
marrow, where the degree of reddening is in direct proportion
to the duration of the disease. In acute cases, the conversion
of fat to hematopoietic tissue in the femoral marrow occurs
first in proximal cancellous and then subendosteal diaphyseal
and distal cancellous areas. The red and yellow areas are ini-
tially firm and opaque, often with focal areas of hemorrhagic
infarction. In chronic cases, the red conversion may include
all of the medullary marrow and is alternately pink and trans-
lucent in areas of serous atrophy of fat, and cyanotic where
congested sinuses have dilated as adjacent hematopoietic
areas atrophy.
Microscopic lesions occur in most tissues, but are most promi-
nent in the heart, lungs, liver, spleen, kidney, bone marrow, and
lymph nodes. Their severity varies with chronicity, and they are
described here in the fully developed state. The myocardium
has fiber atrophy in chronic cases, and interstitial edema in
acute cases, with perivascular lymphocytic aggregations that
irregularly permeate the surrounding interstitium. There is
mild pulmonary alveolar thickening and an overall appearance
of hypercellularity. Occasionally, hemosiderin-bearing macro-
phages are found in alveolar walls and are likely intravascular.
The liver presents a spectrum of changes, which varies from
mild periportal lymphocytic infiltrates to atrophic cords with
sinusoidal dilation, Kupffer cell hyperplasia, broad loosely
arranged periportal lymphoid infiltrates, and increased inter-
stitial connective tissue. As the disease progresses, hepatic
hemosiderosis increases, largely in Kupffer cells. Generalized
endothelial prominence, along with sinusoidal Kupffer cells
and infiltrates, gives an overall picture of hypercellularity. In
subacute cases, there is periacinar fatty vacuolation, and in
acute cases hemorrhage and necrosis. In animals in which the
disease has been quiescent for some months, hepatocytes
appear normal and lymphoplasmacytic infiltrates subside but
sinusoidal hemosiderin-bearing macrophages remain as evi-
dence of previous hemolysis.
Splenic follicles are variably enlarged but hypocellular,
often with a “bull’s-eye” appearance resulting from a sharp
distinction between the cells of the follicular center and the
mantle and marginal layers, and a sharp transition to conges-
tion in the surrounding sinusoids. In acute cases, the spleen is
congested and, being largely composed of unsupported red
cells, fractures on sectioning. Lymph nodes are edematous and
have medullary hemosiderosis with persistence of follicles and
thin moth-eaten paracortical areas. In chronic disease, there is
sclerosis and lymphoid atrophy. Renal lesions in acute disease
are largely hemorrhagic with some glomeruli obscured by
erythrocytes and fibrin. Lymphocytic infiltrates may be intense
and separate tubules in an irregular manner. There is some
degree of epithelial atrophy and mild pigmentation that
appears to be both bilirubin and hemosiderin.
Bone marrow in acute disease is 60% or more cellular, and
cells are densely packed. Megakaryocytes are not increased,
which suggests that the thrombocytopenia is at least partially
the result of ineffective production. With chronicity and
repeated febrile periods, there is a progressive reduction of fat
cells until the marrow is completely converted to hematopoi-
esis. At the same time, cellular packing decreases and sinusoi-
dal expansion occurs so that marrow that appears red grossly
is nevertheless histologically hypocellular. Stromal cells are
increased, endothelial nuclei are prominent, and there is
diffuse plasmacytosis.
Bone Marrow
Figure 2-8  Blood smear from a cow with severely hypochromic
erythrocytes and numerous round purple Anaplasma marginale
organisms. There is one large lymphocyte and several platelets.
Further reading
Cook RF, et al. Equine infectious anemia and equine infectious anemia
virus in 2013: a review. Vet Microbiol 2013;167:181-204.
Anaplasmosis.  Bovine anaplasmosis is a caused by infec-
tion of erythrocytes by Anaplasma marginale and extravascu-
lar removal of infected erythrocytes leading to anemia. Three
species infect large animals in the genus Anaplasma, family
Ehrlichiaceae, order Rickettsiales: A. marginale and A. centrale
infect cattle; A. ovis infects sheep and goats. These obligate
intracellular bacteria are spherical or oval, from 0.3-1.0 µm in
diameter, and situated as membrane-bound bodies (initial
bodies) near the erythrocyte margin (A. marginale and A. ovis)
or in the center of the cell (A. centrale). A. centrale rarely
causes disease in cattle (Fig. 2-8).
Anaplasma marginale is transmitted biologically by ixodid
ticks such as Dermacentor and Rhipicephalus, referring to
development of the bacterium in the tick. A. marginale is
transmitted mechanically by bloodsucking flies of the genera
Tabanus and Stomoxys, several mosquito species, and blood-
contaminated hypodermic needles and instruments used for
dehorning, castration, and ear tagging. There is geographic
variation in the type of vector that transmits A. marginale, and
some strains are neither infective for, nor transmitted by, ticks.
Anaplasmosis has worldwide distribution and poses a major
constraint on cattle production. In North America, bovine
anaplasmosis is enzootic throughout the southern, western,
and midwestern United States, and sporadic outbreaks occur
in most other states and in several Canadian provinces. Non-
domestic ruminants in contact with domestic cattle may
provide a source of infected ticks.
Naive cattle become ill ~20-40 days after infection, and
within 5-10 days thereafter, organisms are detectable on blood
smears. The numbers of organisms increase rapidly during the
acute phase, and greater than 70% of erythrocytes may be
parasitized. Infected cells are removed by the monocyte-
macrophage system, resulting in mild to marked anemia and
jaundice. Intravascular hemolysis is minimal, and therefore
animals have neither hemoglobinemia nor hemoglobinuria.
 116.e1

Further reading
Issel CJ, et al. A perspective on equine infectious anemia with an
emphasis on vector transmission and genetic analysis. Vet Microbiol
1998;17:251-286.
Payne SL, Fuller FJ. Virulence determinants of equine infectious anemia
virus. Curr HIV Res 2010;8:66-72.
 Bone Marrow
However, marked bilirubinemia results in bilirubinuria, which
gives an intense yellow-brown color to urine. Hemoglobin
concentration may be as low as 30-40 g/L, and hematocrit
0.10-0.15 L/L, and there typically is a leukocytosis because of
neutrophilia. About 1 week after onset of parasitemia, eryth-
rocyte regeneration appears on blood smears. Clinical illness
consists of fever, weight loss, reduced milk production, abor-
tion, lethargy, and death, in particular in animals older than 2
years. Mortality rates of 50-60% are observed in naive adult
animals first infected with A. marginale, although this varies
by bacterial strain, geographic region, and nutritional status of
cattle. High-producing dairy cows appear most susceptible to
severe illness and high mortality from acute anaplasmosis.
Animals resist movement, have a rapid heart rate and increased
respiratory rate, and may have incoordination. Cattle that
survive become persistently immune carriers with cyclical
low-level parasitemia and do not develop clinical illness upon
reinfection.
Anaplasmosis, distinct from babesiosis, does not cause
intravascular erythrocyte lysis, and therefore no hemoglobin-
uria. Recycling of iron and globins by splenic and hepatic
macrophages is considered to be the main reason for more
effective erythrocyte regeneration in anaplasmosis relative to
babesiosis, in which hemoglobin is lost externally via the
urinary system. Erythrocytes are the only target cell of A.
marginale, and are frequently sphered in acute disease, likely
because of removal of parasitized erythrocyte membrane com-
ponents by splenic macrophages. Identifying organisms on
blood smears is a useful diagnostic test in acute infection, but
in chronically infected carrier animals, very few erythrocytes
contain organisms, and other tests such as ELISA or PCR must
be applied.
There are no pathognomonic gross lesions of anaplasmosis.
There is pallor of all tissues and mild to marked icterus, with
relatively good body condition in those animals dying acutely,
and cachexia in chronic cases. The lungs are pale and discol-
ored and may have bullous emphysema if there has been
severe terminal dyspnea. There are frequently ecchymotic
hemorrhages on the epicardium, and the heart is flabby and
dilated. The liver is pale and icteric, and the gallbladder is
usually distended. The spleen is enlarged, turgid, and con-
gested in acute cases, and firm, dark red, and fleshy in chronic
ones. The enteric tract is unremarkable, and the bladder may
contain deeply bilirubin-stained urine. The marrow hemato-
poietic space is variably expanded, depending on the stage
of the disease, and there may be serous atrophy with
chronicity.
Anaplasma centrale produces a natural infection of cattle,
but is also used, being a mild pathogen, as an immunizing
agent against A. marginale in some areas of endemic infection.
A. centrale infection usually produces a mild disease, although
sometimes it can be severe, with fever and anemia but no
icterus. Immunity to A. centrale does not protect against infec-
tion by all strains of A. marginale, and coinfections have been
reported. A. centrale also causes a persistent infection.
Anaplasmosis in sheep and goats is caused by A. ovis. A.
ovis is highly prevalent in sheep in many Eurasian and south-
ern European countries, but rarely associated with overt clini-
cal disease unless animals have concurrent other infections,
debilitation, or poor nutrition. However, infection of goats
more often results in anemia and clinical disease, and there
are reports of infection of humans in Cyprus with an Ana-
plasma organism genetically similar to A. ovis via tick bite.
117
Although cattle are not susceptible to A. ovis, sheep and goats
develop a latent infection if inoculated with A. marginale. The
economic impact of widespread ovine anaplasmosis on sheep
production has not been fully determined.
Further reading
Kocan KM, et al. The natural history of Anaplasma marginale. Vet
Parasitol 2010;167:95-107.
Renneker S, et al. Can Anaplasma ovis in small ruminants be neglected
any longer? Transbound Emerg Dis 2013;60(Suppl. 2):105-112.
Babesiosis.  The apicomplexan protozoan Babesia parasit-
izes erythrocytes of a wide range of mammals, including humans.
Similar to Anaplasma, Babesia infects and replicates exclu-
sively in erythrocytes. The number of Babesia spp. recognized
has increased dramatically over the past decade, given applica-
tion of molecular tools, and morphologically similar organisms
may be genetically distinct. Classification of Babesia is not
reviewed here, but rather the pathogenesis and pathology of
natural infections. Babesia has high host specificity, and is
transmitted by ticks. During the tick bite, sporozoites are
injected with tick saliva into the host and directly infect eryth-
rocytes. Sporozoites in erythrocytes divide asexually into 2 or
4 pear-shaped merozoites (“piroplasm”) that are then capable
of infecting new erythrocytes (Fig. 2-9). Some merozoites
develop into extracellular gametocytes, which if ingested by
ticks replicate in their gut. Some Babesia spp. have transovarial
transmission in the tick, implying potentially prolonged sur-
vival in ticks without feeding on vertebrate hosts. Transmission
by biting insects or contaminated instruments appears to be
less common than for Anaplasma, which may be because
of preferential location of erythrocytes in capillaries rather
than veins.
The severity of the disease produced in the mammalian
host depends more on the strain and species of infecting
Babesia than on the number of organisms inoculated. Patho-
genic effects are, in most infections, related directly to lysis of
red cells by emerging parasites, but other mechanisms, including
sludging of infected red cells, hemoglobinuric nephrosis, and
release of vasoactive peptides, contribute to the signs and are
Figure 2-9  Blood smear from a cow with Babesia bigemina
infection.
 117.e1

Further reading
Stuen S, Longbottom D. Treatment and control of chlamydial and
rickettsial infections in sheep and goats. Vet Clin North Am Food
Anim Pract 2011;27:213-233.
118 CHAPTER 2  •  Hematopoietic System
responsible for death. The severity of anemia and illness is
enhanced by splenectomy, which is used commonly in patho-
genetic studies. Splenectomy is a risk factor for human babe-
siosis, and likely also for some veterinary infections. B. bovis
merozoites are smaller than those of B. bigemina.
“Tick fever” in cattle is caused by one or several of A.
marginale, B. bovis, and B. bigemina. Babesia bovis causes the
most severe disease. Bos indicus cattle breeds are relatively more
resistant to the effects of this parasite than Bos taurus, which
has led to the suggestion that the organism evolved in these
breeds. Young cattle have pronounced resistance to severe
infection. Colostral antibodies transiently protect young
calves, and infection of young animals induces long-term
immunity. Hence, in endemic areas, there often is a high level
of herd immunity, and outbreaks result from interruption in
exposure to ticks or introduction of naive animals. For these
reasons, the disease is mostly seen in adult cattle recently
introduced from tick-free areas, or in areas of greatly fluctuat-
ing tick populations.
Infection by B. bovis causes severe febrile illness, which
begins ~2 weeks after exposure to ticks. The animal may
become extremely ill before severe anemia, parasitemia, or
hemoglobinuria are apparent, and sometimes the clinical
picture is dominated by neurologic signs such as seizures,
hyperesthesia, and paralysis, likely associated with the ten-
dency for parasitized red cells to sludge in cerebral capillaries.
Animals progress to weakness, fever, hemoglobinuria, and
anemia; the latter is more severe in animals surviving more
than a week. Causes of death are shock and respiratory dis-
tress. The parasite is never numerous in circulating erythro-
cytes, rarely being seen in >5% of circulating erythrocytes.
Parasitized erythrocytes are more likely to be found in smears
of blood prepared from cut skin capillaries than in routine
blood samples from large veins. Recovery is usually fairly
rapid in animals that survive the acute phase, although chronic
ill thrift has been described. Photosensitization may occur
in the convalescent phase, probably as a result of reduced
excretion of phylloerythrin in animals with severe
hyperbilirubinemia.
The most typical postmortem findings are those to be expected
of an acute intravascular hemolytic crisis. There is anemia, vari-
ably severe jaundice and hemoglobinuria, and the kidneys are
deep red-brown throughout as a result of hemoglobin staining
and intense congestion of capillaries. There is capillary conges-
tion of most organs; the spleen is grossly swollen, soft, and
dark; and the liver is acutely congested and may be heavily
stained with bile pigment. The gallbladder is distended with
thick, dark, viscous bile.
In some acute cases, the lungs are congested and edema-
tous, and the larger airways contain stable foam. Recent hem-
orrhages are common in the thoracic serosal membranes. The
most characteristic macroscopic feature of B. bovis infections is
striking, uniform congestion of the gray matter throughout the
brain, imparting to it a dramatic, deep pink color (the cerebral
flush), which contrasts strongly with the white matter; the
latter is often stained faint yellow by unconjugated bilirubin.
In some peracute cases, there is minimal hemoglobinuria or
jaundice, but the brain still has characteristic capillary conges-
tion, and imprint preparations yield many intraerythrocytic
organisms.
Histologically, there is thickening and congestion of pulmo-
nary capillaries with hemosiderin in intravascular macro-
phages. Kidneys and liver have the histologic lesions to be
Bone Marrow
expected of an acute hemolytic disease. There is variably
severe hemoglobinuric nephrosis with severe congestion, focal
hemorrhage and hemosiderin in tubular epithelium, and
diffuse vacuolar degeneration with interstitial mixed mono-
nuclear cell reaction. There is hepatic periacinar congestion
with uninfected red cells, and vacuolar degeneration in
midzone and periacinar hepatocytes, accompanied by cana-
licular cholestasis. There is hemosiderin accumulation in both
hepatocytes and Kupffer cells, and the latter contain both
infected and noninfected red cells. Lymphocytes and plasma
cells accumulate in portal areas and around central veins.
Parasitized erythrocytes may be seen in vessels in all tissues, but
they are particularly common in interstitial capillaries in the
kidney, in the gray matter of the brain, in the heart, and espe-
cially in skeletal muscle; in these locations, nearly every eryth-
rocyte packed into the distended capillaries appears to contain
1-2 parasites. The organisms are faint blue in routine sections,
and are best demonstrated in imprint preparations of fresh tissue.
In sections, Giemsa stain demonstrates them as small paired
or single spherical bodies with denser staining at one pole. In
well-fixed fresh brain of acute cases, there may be edema of
the neuropil around the clogged capillaries.
Animals that die with peracute disease have necrosis of
lymphocytes in germinal centers of node and spleen, although
there may be some degree of recovery in animals surviving a
week, and general depletion of lymphocytes in those dying
after protracted illness. Characteristic changes in the medul-
lary region of nodes include sinus histiocytosis with extensive
erythrophagocytosis. The histology of the spleen is often
unhelpful, the organ being so suffused with intracorpuscular
and extracorpuscular hemoglobin that evidence of erythro-
phagocytosis is obscured. There is little extramedullary hema-
topoiesis in liver or spleen. The bone marrow has hyperplasia
of immature rubricytes and, in animals that survive the acute
stage, there is strong reticulocytosis and slow recovery of
anemia. There is increased hemosiderin in bone marrow, sug-
gesting that marrow is also a site of erythrolysis.
Babesiosis results in metabolic disease more complex than a
simple syndrome of intravascular hemolysis. Severely affected
animals may die before significant anemia has set in, and in
these cattle, the intense visceral congestion and pulmonary
edema suggest that death may be at least partly the result of
circulatory shock. This has been supported by studies that
have shown that the parasite is the source of proteases that
activate plasma kallikrein, which, as well as being a hypoten-
sive agent, may activate bradykinin, another potent vasodila-
tory agent. Rather unexpectedly, metabolic alkalosis occurs in
B. bovis infections, whereas acidosis is the rule in severe B.
canis infections. The result of these profound metabolic upsets is
a syndrome of circulatory failure, likely because of extensive
plugging of microvasculature by sequestered red cells. The
apparent anemia is therefore caused in considerable measure
by the vasodilatory effects of kallikrein and shift of red cells
away from large veins. Although plasmin activation occurs, the
effects on the coagulation system are unlikely to be a signifi-
cant factor in the pathogenesis because hemorrhage is not a
major part of the morbid picture.
The actual mechanism of penetration of red cells by the mero-
zoites of Babesia is unclear. Parasites remodel the erythrocyte
surface with their variable erythrocyte surface antigen (vesa)
proteins, which changes the membrane mechanical and adhe-
sive properties. The organism causes complement activation
by the alternative pathway, and by C3b achieves adherence
 Bone Marrow
and later invasion. The mechanism of hemolysis is also obscure.
Immune mechanisms do not appear to play a significant role
in the early stages of the disease, but they may be a factor in
delayed recovery. Osmotic fragility is markedly increased in
nonparasitized cells. Generation of intravascular fibrin may
contribute to capillary erythrocyte sludging.
Babesia bigemina infection may be transmitted by the
same tick as B. bovis, and both parasites may cause losses in
the same geographic area. B. bigemina, however, usually causes
much less severe illness, although parasitized erythrocytes cir-
culate early, sometimes before clinical signs are apparent, and
in much greater numbers than in B. bovis infection. B. bigemina
also causes erythrocyte destruction, but this appears to be
directly proportional to the level of parasitemia, unlike B. bovis
infection. There are few of the vasoactive and erythrocyte
adherence effects, thus profound shock-like signs are usually
absent. Anemia in severe cases may be marked before the
animal becomes ill and, as in any case of severe anemia, death
may occur suddenly as a result of myocardial failure. Fever is
not usually as severe as in B. bovis infection.
The pathology of B. bigemina and B. bovis infections is
similar, except that in B. bigemina infection there is little capil-
lary congestion of the viscera and none of the cerebral gray
matter impact, the presence or absence of the cerebral flush being
the most reliable gross feature for differentiating the 2 infections.
Pulmonary edema may be more often seen in fatal cases of B.
bigemina infections, probably because the greater severity of
anemia causes terminal left ventricular failure.
Babesia divergens and B. major are more or less restricted
to northern and western Europe and Asiatic Russia and, for
the most part, are of relatively minor importance to the cattle
industries there, although significant outbreaks of disease and
death may occur. Although B. divergens is small and resembles
B. bovis in size, and B. major is as large as B. bigemina, the
pathology and pathogenesis of the disease produced by these
organisms is apparently very similar to that produced by B.
bigemina. Thus there is intravascular hemolysis with little or
no capillary agglutination of erythrocytes, and death results
primarily from severe anemia. Spontaneous splenic rupture has
been reported in B. major infections, a complication rarely, if
ever, reported in other bovine babesial infections.
Differential diagnosis of bovine babesial infections in coun-
tries where more than one species of Babesia is present is impor-
tant and at times difficult, and may be complicated by dual
infections and by the additional possibility of Anaplasma
infection. B. bovis and B. bigemina may be distinguished from
one another in well-prepared blood smears, but erythrocytes
parasitized by B. bovis are rare in jugular blood, or even in
blood from deep skin punctures. They are best demonstrated
in smears of blood expressed from a superficial skin scrape;
this is most conveniently obtained from the tail-tip in the live
animal. B. bigemina–containing erythrocytes, on the other
hand, are quite numerous in circulating blood while clinical
signs are severe. The morphology of the parasites will usually
be insufficiently preserved in postmortem material to allow
distinction, but the preference of B. bovis for capillaries of
organs such as kidney, heart, and brain will, in most cases, serve
to distinguish the infections. It must be remembered that
Sarcocystis spp. may be present in smears of myocardial blood.
Serologic tests are available for antemortem diagnosis, and PCR
tests for postmortem samples. It is necessary to demonstrate
a very heavy parasite burden in cerebral capillaries if brain
smears are to be relied on as evidence of active B. bovis
119
infection, as most clinically normal carriers of this organism
have some parasitized erythrocytes in this site.
In the last 2 decades, it has been recognized that canine
babesiosis is caused by different organisms transmitted by spe-
cific vectors and has highly variable pathogenesis. Large babe-
sial organisms in dogs include B. canis, B. rossi, B. vogeli, and an
as yet unnamed Babesia in North America. B. canis occurs
mostly in southern and central Europe, is transmitted by Der-
macentor reticulatus and causes disease of variable severity
consisting of fever, hemolytic anemia, and jaundice in adult
dogs, and less severe disease in young dogs. B. rossi is restricted
to sub-Saharan Africa, is transmitted by Haemaphysalis ellip-
tica, and causes severe peracute and acute illness with 10-20%
mortality. Clinical disease results from severe hemolytic
anemia, acid-base derangement, hypoxia, and a systemic
inflammatory response. Death is attributed to multiple organ
failure as manifested by hepatopathy and hypoglycemia, acute
kidney failure, cerebral ischemia, and respiratory distress. Con-
trary to most babesial infections, B. rossi affects young dogs as
severely as adult dogs. B. vogeli occurs in tropical and sub-
tropical regions of most continents, including North America,
and is transmitted by the brown dog tick Rhipicephalus san-
guineus. Disease associated with B. vogeli infection is less
severe than that of other large babesial organisms in dogs, and
may be subclinical in immunocompetent animals. However,
young dogs are also more likely to be clinically ill with hemo-
lytic anemia and thrombocytopenia. The unnamed large
babesial organisms in dogs were identified in splenectomized
or immunosuppressed animals in North America and Britain,
and were also associated with severe anemia and thrombocy-
topenia. Large babesial organisms in dogs are morphologically
indistinguishable.
Small Babesia of dogs were until recently thought to all
be B. gibsoni, but are now recognized to belong to at least 5
different taxa: B. gibsoni (sensu stricto), B. conradae, a B.
microti–like organism, Theileria annae, and an unnamed Thei-
leria spp. The number of organisms in erythrocytes is often
high, but disease associated with small babesial organisms in
dogs is usually mild. Anemia results mostly from extravascular
erythrocyte removal and not intravascular hemolysis.
In dogs with pathogenic babesiosis at postmortem, there
is staining of tissues with both bilirubin and hemoglobin.
The kidneys are dark brown, and there is splenomegaly and
copious thick bile in the gallbladder. In addition, there is evi-
dence of vascular injury in the form of hemorrhages and
edema, which may be severe in the lung (eFig. 2-1). Parasit-
ized erythrocytes may be found plugging capillaries in smears
and sections of cerebral cortex, reminiscent of B. bovis infec-
tion, but the phenomenon is never as severe as in that disease.
Disseminated intravascular coagulation is a consistent occur-
rence in severe B. rossi and B. canis infection, and is presum-
ably related to, and is likely to aggravate, the hemolysis and
the vascular damage. Microthrombi can be demonstrated in
many tissues.
Piroplasmosis in horses is caused by Theileria equi and
Babesia caballi. T. equi was previously classified as B. equi, but
is molecularly distinct from other Babesia spp. and was reclas-
sified. Also, during initial infection, T. equi sporozoites invade
lymphocytes, monocytes, and macrophages, and develop
schizonts and then merozoites in those cells. Merozoites are
released to invade erythrocytes. This property is more like
other Theileria spp. than Babesia spp. Infection with either
organism is widespread, and their distinct ixodid ticks are also
 119.e1

B
eFigure 2-1  Babesiosis in a dog. A. Babesia canis can be detected
as single or paired, piriform organisms within erythrocytes in a
vessel within the stomach. B. Centrilobular congestion and hepa-
tocellular degeneration and extramedullary hematopoiesis. (Cour-
tesy E.P. Lane.)
120 CHAPTER 2  •  Hematopoietic System Bone Marrow

widely distributed. North America is generally considered free
of equine piroplasmosis, although sporadic outbreaks have
occurred in Florida and Texas. T. equi infection is considered
to be more widespread and causes more severe disease. Clini-
cal disease in acute infection with either organism is similar,
and consists of fever, lethargy, petechiation, ventral and peri-
orbital edema, thrombocytopenia, jaundice, hemoglobinuria,
splenomegaly, and intravascular hemolysis leading to anemia.
T. equi are small piroplasms, and 1-5% of erythrocytes may
contain organisms during acute infection, whereas B. caballi
are large piroplasms, and <1% of erythrocytes are infected.
Mortality is low in immunocompetent horses, but experimen-
tal infection of foals with severe combined immunodeficiency
results invariably in fulminant infection and death. Following
acute infection, most horses become persistently infected
clinically inapparent carriers resistant to illness from subse-
quent exposure. Such horses constitute a source of infection
for naive animals. It has been suggested that some horses clear
B. caballi infection over time without treatment. Immunity to
one organism does not cross-protect against infection by the
other piroplasm.
Babesial infection of cats is uncommon worldwide except
in southern Africa. The small B. felis piroplasm causing infec-
tion in those areas induces severe hemolytic anemia. Several
other small and large piroplasms have been reported in domes-
tic and nondomestic cats, but neither disease association nor
classification is well established.
Babesial infections of other species are for the most part
mild or clinically inapparent. The course of these diseases in
general follows the pattern described for B. bigemina infection,
in that there is quite severe parasitemia, anemia caused by
intravascular hemolysis, with hemoglobinuria in acute cases.
At autopsy, there is splenomegaly and variably severe jaundice
and, in some cases (notably B. trautmanni infection in pigs),
edema, and petechiae. An organism resembling B. divergens
may infect white-tailed deer, and mice are susceptible to
numerous Babesia organisms.
the tick vector, in addition to clades of distinct Theileria and
Cytauxzoon organisms.
Cytauxzoon felis is naturally transmitted by ixodid ticks,
and can be transmitted experimentally by fresh and cryopre-
served blood or tissue homogenates from infected animals.
Infection of domestic cats results in acute febrile illness and
death in ~19-21 days. Clinically ill cats have fever, depression,
pallor, icterus, dark urine, and occasionally dyspnea. Hematol-
ogy is characterized by nonregenerative anemia and frequently
neutropenia and thrombocytopenia. Lack of erythrocyte regen-
eration and neutropenia distinguish C. felis from hemotropic
Mycoplasma infection. Babesia felis piroplasms are indistin-
guishable from C. felis, but infection with the former is not
usually characterized by leukopenia (Fig. 2-10). Cytauxzoon
organisms in erythrocytes are variable in shape, but most
common are signet ring forms with a thickened nuclear area
at one point of the ring. The level of parasitemia is low, with
piroplasms in 1-4% of peripheral blood erythrocytes, but in
terminal stages of illness; infected macrophages may be in
blood samples from larger veins and therefore on blood films.
Macrophages containing schizonts can be readily identified in
imprints from virtually any organ at postmortem, in particular
lung and spleen. In the early hemolytic stages of the disease,
the urine is highly concentrated, acidic with high levels of

Further reading
Gohil S, et al. Bovine babesiosis in the 21st century: advances in
biology and functional genomics. Int J Parasitol 2013;43:
125-132. A
Penzhorn BL. Why is Southern African canine babesiosis so virulent?
An evolutionary perspective. Parasit Vectors 2011;4:51.
Schnittger L, et al. Babesia: a world emerging. Infect Genet Evol
2012;12:1788-1809.
Wise LN, et al. Equine piroplasmosis. Vet Clin North Am Equine Pract
2014;30:677-693.

Cytauxzoonosis.  Cytauxzoonosis is caused by a protozoal

piroplasm with an erythrocytic and tissue macrophage stage.
Cytauxzoon spp. infect wild ungulate species in Africa, includ-
ing the kudu, eland, and giraffe, as well as domestic and wild
felids in North America. Babesia, Theileria, and Cytauxzoon
are closely related non–pigment-forming piroplasms that
infect erythrocytes. Theileria also forms schizonts in lympho-
cytes, and Cytauxzoon forms schizonts in intravascular and B
subendothelial macrophages. These life-cycle features corre-
spond to recent molecular taxonomic characterization, which Figure 2-10  Acute cytauxzoonosis in a cat with numerous mero-
suggests that there are multiple clades of different Babesia, in zoites in erythrocytes (A), and a giant schizont in bone marrow
part characterized by presence or absence of development in (B). (Courtesy J. Tarigo.)
 120.e1

Further reading
Johnsson NN, et al. Productivity and health effects of anaplasmosis and
babesiosis on Bos indicus cattle and their crosses, and the effects
of differing intensity of tick control in Australia. Vet Parasitol
2008;155:1-9.
Short MA, et al. Outbreak of equine piroplasmosis in Florida. J Am Vet
Med Assoc 2012;240:588-595.
 Bone Marrow
protein and large amounts of bile and blood, including intact
red cells and bilirubin crystals. Consistent with a rapidly fatal
course of disease, animals at postmortem are generally in good
body condition with adequate adipose tissue. There is exten-
sive vascular obstruction from schizont-containing macro-
phages, leading to congested edematous lungs, reddened
lymph nodes, petechial and ecchymotic hemorrhages, and
pleural, pericardial, and peritoneal effusion.
North American bobcats are considered to be a reservoir
of C. felis, and generally do not become ill after experimental
tick infection. Inoculation of cats with blood from parasitemic
bobcats induces parasitemia in cats, but not severe illness. This
is thought to be because transferred piroplasms are unable to
undergo schizogony in recipient cats without a developmental
stage in ticks. Other nondomestic cats also harbor C. felis
without apparent clinical illness.
The host-pathogen relationship between C. felis and
domestic cats appears to evolve. Recently, infection in domes-
tic cats has been identified over an expanded range of the
southern and midwestern United States, and domestic cats
have been recognized as parasitemic over several years but
healthy. Anecdotally, cats that have survived C. felis infection
may be resistant to subsequent challenge, and immunosup-
pressive treatment may increase parasitemia. These findings
suggest that a more benign host-pathogen relationship of C.
felis and domestic cats may be emerging.
Further reading
Meinkoth JH, Kocan AA. Feline cytauxzoonosis. Vet Clin North Am
Small Anim Pract 2005;35:89-101.
Rizzi TE, et al. Prevalence of Cytauxzoon felis infection in healthy cats
from enzootic areas in Arkansas, Missouri, and Oklahoma. Parasit
Vectors 2015;8:13.
Trypanosomiasis.  Trypanosomes are insect-transmitted
protozoa, uniform in type but varying in morphologic details.
Some species are morphologically stable, whereas others are
polymorphic, and the differentiation of species by morphol-
ogy is challenging (Fig. 2-11).
Figure 2-11  Trypanosoma vivax trypomastigote in blood smear
from a cow.
121
The trypanosomes important to human and animal health
are transmitted by insects that carry the metacyclic stages in
either their oral or anal tracts. Thus, Trypanosoma cruzi, the
cause of Chagas disease in humans and animals in South
America, is known as a stercorarian parasite because it is
spread by passage in the feces of the reduviid insects (Triato-
minae subfamily); after the insects bite (often around the
mouth or eye), they defecate, and the trypanosomes gain entry
to the host through the bite wound, often assisted by the host
scratching. The important African trypanosomes of animals
(T. congolense, T. vivax, T. brucei brucei) and of humans
(T. brucei rhodesiense and T. brucei gambiense) are called sali-
varian parasites because of their transmission through the
salivary glands of the tsetse fly. Other biting and sucking
insects, such as tabanids, stable flies or fleas, and vampire bats
may transmit trypanosomes mechanically. Infection of the
definitive host, the tsetse fly, is probably lifelong.
Trypanosoma equiperdum, the cause of dourine, does not
require a vector host but is transmissible by direct contact
with mucous membranes. It is described in Vol. 3, Female
genital system.
Not all species of Trypanosoma of mammals are pathogenic,
and among the common nonpathogenic species of veterinary
interest are T. melophagium, ubiquitous in sheep and trans-
mitted by the ked, Melophagus ovinus; T. theileri, which is
widespread in cattle and transmitted by several types of biting
flies, including Tabanidae; T. theodori, which occurs in goats
in the Middle East, transmitted by the hippoboscid fly, Lipo-
ptena caprina, and probably synonymous with T. melophagium;
and T. lewisi of rats, which is of much interest as a convenient
subject for research on the genus. T. rangeli of South Ameri-
can cats, dogs and humans is also apparently nonpathogenic
to the vertebrate hosts but differs from the above in that it
can be transmitted by inoculation or contamination, the
vector being the triatomine Rhodnius prolixus. The species of
trypanosomes listed above, although widespread in their hosts,
are ordinarily sparse in blood and seldom observed unless
specifically looked for in thick smears, after splenectomy, or
by culture. Occasionally, however, T. melophagium and T. thei-
leri are found in very large numbers in the blood of cattle and
sheep suffering from a primary and usually an immunosup-
pressive disease. It is not clear whether T. theileri may become
pathogenic in the compromised host; however, its increase in
number in association with other diseases suggests that it may
be of secondary importance.
In endemic areas, the incidence of infection by pathogenic
species varies depending on such factors as the availability of
mammalian reservoirs, the density of vector tsetse flies, and
the systems of husbandry adopted, but, as a rule, domestic
animals and trypanosomes cannot coexist. Trypanosomiasis pro-
hibits domestication of livestock in perhaps 1 4 of the total land
surface of Africa. Knowledge of host-parasite relationships is
not fully understood, neither for the vector nor the mamma-
lian hosts. Within a species of the organism, local strains may
be distinct in their basic and predominant antigens, but within
the host in the course of chronic infections, a series of geneti-
cally regulated antigenic variants appears in succession so that
the animal exhausts itself without clearing the infection. Suc-
cessive waves of parasite and antibody dominance account for
the characteristic recrudescences of the infection and for the
known difficulty in producing an effective vaccine.
The pathogenic trypanosomes differ widely in virulence and
are apparently nonpathogenic in mammalian reservoir hosts.
122 CHAPTER 2  •  Hematopoietic System
Most of them are parasites of a variety of domestic and wild
animals, in some of which they produce fulminating disease,
in some chronic disease, and in others mild or unapparent
infections. Strains within a species of the parasite also differ
greatly in virulence, some strains producing rapidly fatal infec-
tions, some producing chronic infections with premunition,
and some allowing complete recovery and acquired immunity.
Much of the knowledge of trypanosomiasis has been gained
by studies of T. brucei, which conveniently infects dogs, rabbits,
rats, and mice, and poses little problem of dissemination in
developed countries, where it has served as a research model.
In terms of the overall problem, T. brucei is not nearly as
important as T. vivax and T. congolense and the pathogenic
trypanosomes of humans. Of the latter organisms, only T.
congolense is studied with any frequency outside their natural
habitats because the other trypanosomes are thought to con-
stitute too great a hazard of dissemination, even under condi-
tions of laboratory restraint. Most of the research on T. vivax
and T. congolense has been directed at defining the disease in
British breeds of cattle. Efforts are now being made to under-
stand the host-parasite relationship in indigenous species of
animals, such as the Thomson’s gazelle, wildebeest, Cape
buffalo, and eland, which are heavily exposed and apparently
carry the organisms, but rarely, if ever, have clinical disease.
The components of pathogenicity of trypanosomes are largely
unknown. Their effects vary with their tissue tropism. The
edema and interstitial reactions caused by T. brucei are the
result of the fact that this parasite resides in perivascular
tissues, in contrast to the more serious pathogens of humans
and animals, which are obligate intravascular parasites. In
many respects, bovine trypanosomiasis is similar clinically and
pathogenetically to equine infectious anemia in that infection
is followed by an asymptomatic period until antibody devel-
ops. There is then anemia and generalized compensatory
hyperplasia of the mononuclear phagocyte system, which ulti-
mately results in cachexia and hematopoietic and lymphoid
exhaustion.
Bovine trypanosomiasis.  In cattle, parasitemia occurs a
week after inoculation with 105 or more organisms that have
been passed in rats and column separated (see Fig. 2-11). By
the second week of infection, there is a sharp drop in the red
cell count and hemoglobin levels, accompanied by an ery-
throid shift and macrocytosis, anemia being the predominant
aspect of the disease. There is concurrent thrombocytopenia of
moderate degree and hypocomplementemia. Fibrinogen is
reduced to about half of normal levels, and there is irregular
appearance of fibrin degradation products as animals go
through febrile periods. Kinetically, the erythrocyte life-span
is reduced to a half or less of normal, and iron utilization is
initially rapidly increased, but slows down with cachexia and
inhibition of the erythroid response because of inflammatory
changes. The platelet life-span is not shortened in infections
with T. congolense, although it may be with T. vivax. In the
former, the truncation appears to result from proliferation of
marrow megakaryocytes that are impaired in their maturation
and produce fewer platelets that survive normally, thus con-
stituting an ineffective response. The disease tends to stabilize
at ~4-6 weeks in well-fed animals under laboratory conditions,
and to proceed to death from secondary causes in animals that
are forced to forage for feed as well as maintain their immune
defenses.
Red cells are destroyed by erythrophagocytosis as a result
of adsorbing toxic and metabolic products of the trypano-
Bone Marrow
somes, and as a product of direct injury from parasite-derived
neuraminidases, such as trans-sialidases. There may be competi-
tion among precursor cells for erythroid and granulocytic differ-
entiation, which may explain the clinical finding that animals
with severe anemia are more susceptible to secondary infection
by bacteria and viruses. A dilutional component to the anemia
has been suggested, but no increase in blood volume has been
detected. Calves infected during the first week of life have less
severe hematologic disease than calves infected at 6 months
or older. Some of these animals become cachectic and die,
although it appears that infection at an early age results in a
more tolerant host-parasite relationship that is less injurious
to the host. T. vivax and T. congolense differ in that T. vivax
tends to circulate relatively freely and uniformly in the vascu-
lar system, and, in this respect, the level of parasitemia gives
a reasonable estimate of the total parasitic burden. In contrast,
T. congolense “homes” to the microvasculature of the brain and
skeletal muscle, and the paucity of organisms in the peripheral
blood makes diagnosis and estimates of total burden difficult.
It appears that the tissue tropism of T. congolense is a response
to developing immunity and is antibody mediated. It is not
known why brain and muscle are selectively parasitized, but
the phenomenon may be related to trypanosomal energy
requirements, because the endothelial cells in these areas tend
to have more mitochondria than in other tissues.
Trypanosoma vivax is more virulent than T. congolense.
Whereas both cause anemia and cachexia, infection with T.
congolense regularly results in chronic disease, and in a high
percentage of animals infected with T. vivax, there is sudden
death from salmonellosis or other infections. Infected animals
are moderately stunted in growth, although this effect is less
apparent on body weight than on dressed weight (the part of
the carcass that is edible). This disparity is the result of the
fact that infected animals pool fluid in the intestinal tract and
become pot-bellied while failing to deposit muscle and fat.
When viewed from behind, infected animals have poorly
covered bony prominences, poor “spring of rib,” and a deep
and pendulous abdomen. The skin is scurfy and rough, par-
ticularly about the head, ears, and hindquarters. There is
chronic low-grade pyrexia with intermittent temperature
increases, and periodic watery diarrhea with passage of poorly
digested ingesta of normal color. There is irregular oculonasal
discharge, but no loss of appetite, and the animals continue to
eat. There is mild mucosal pallor without petechial hemor-
rhages, and the urine may be dark but never blood tinged. The
heart and respiratory rates are not remarkably altered under
laboratory conditions but are increased in nonspecific response
to anemia under field conditions, and animals may show open-
mouth breathing on forced exercise. In the absence of second-
ary infection, there is little or no depression or abnormal
behavior despite high levels of cerebral parasitism.
Anemia is of moderate degree, with hemoglobin generally
between 50 and 80 g/L, is initially macrocytic and normo-
chromic, and later becomes normochromic, normocytic, and
poorly responsive. In the well-developed disease, there are
always some hypochromic red cells and at least mild poikilo-
cytosis. In the early stages of infection, the radio-iron uptake
time is reduced from a normal of ~3 hours to about half that
figure but in chronic disease is only mildly reduced. There is
increased osmotic fragility of red cells without spherocytosis.
The platelets are 100-200 × 109/L and are often lower during
intercurrent infection. The leukocyte count is reduced to
about half of normal (5-6 × 109/L) as a result of an absolute
 Bone Marrow
reduction in both neutrophils and lymphocytes. Changes are
less severe in neonatally infected animals. Biochemical changes
consist of a reduction in total protein, largely because of
reduced albumin, but there are no consistent changes in trans-
aminases or bilirubin. There is a consistent reduction in total
serum lipids, cholesterol, and triglyceride, and erythrocyte
phospholipid levels are consistently elevated, which may indi-
cate that there is an acquired injury to red cell membranes
that contributes to shortened life-span. Aspirated marrow is
hypercellular with an erythroid shift that drops the
granulocytic:erythrocytic ratio to 0.4 or lower in animals
infected with T. congolense. There is a less marked erythroid
response in T. vivax infection.
There are no pathognomonic gross lesions of trypanosomiasis.
Chronically affected animals are cachectic and have a rough
haircoat, and there is increased clear fluid in body cavities.
There is generalized lymph node enlargement to 2-4 times
normal, and the hemal nodes become prominent in subcuta-
neous areas and in association with the para-aortic and pelvic
nodes, where they may reach 1 cm in diameter or larger. The
lungs are heavy and have increased density on palpation, and
may show intercurrent cranioventral bronchopneumonia. The
heart is generally flabby, with serous atrophy of pericardial fat,
and there may be white foci 1-2 mm in diameter on the epi-
cardium and on the cut surface. The liver and kidneys are
symmetrically enlarged and constitute a greater than normal
percentage of body weight. The liver is unusually firm and not
easily penetrated by digital pressure, and there is a fine lobular
pattern visible through the capsule and on cut surface. Renal
medullary fat has serous atrophy. There are fine focal, raised,
reddened areas 1-2 mm in diameter throughout both layers
of the omentum. The enteric tract is unremarkable, except
that the small intestine is atonic and contains an excessive
amount of normal-appearing fluid content. The most remark-
able changes occur in lymph nodes, which have generalized
medullary pigmentation, and in bone marrow, where there is
a regular increase in hematopoietic areas, which may occupy
all of the cancellous and diaphyseal fatty areas in animals that
have been infected 6 months or more. The spleen is uniformly
enlarged and bulges, but does not ooze blood, on cut surface.
The Malpighian corpuscles are generally visible grossly.
Microscopically, there is a generalized increase in septal
width in the lungs and accumulation of intravascular
hemosiderin-bearing macrophages. There are multifocal areas
of fiber atrophy with sclerosis and lymphoplasmacytic prolif-
eration in the myocardium, most prominently in those animals
that have succumbed to the disease. The bone marrow goes
through a cycle of changes similar to those in equine infec-
tious anemia. There is early conversion of fatty to hematopoi-
etic marrow with high cell density, erythroid shift, and
plasmacytosis, followed by a reduction in cellular packing and
dilation of sinusoids, and ultimately by reduction in hemato-
poiesis and serous atrophy of the remaining tissue. The rubri-
cytes are increased in number and are relatively synchronous
in maturation with numerous mature cells, suggesting late
asynchrony associated with poor availability of iron. There is
a variable degree of hemosiderosis, depending on the duration
of the disease, and erythrophagocytosis may be observed in
the marrow. Marrow granulocyte reserves are reduced, and, as
a result, there is mild early asynchrony, although it is likely
that with the expanded volume of hematopoietic marrow
there is a normal volume of granulopoietic tissue. Megakaryo-
cytes are increased in number to about twice the normal
123
level, and the nuclei are increased in diameter, but the cyto-
plasmic volume is reduced, indicating ineffective thrombopoi-
esis. There is a mild generalized increase in endothelial and
true reticular cells in bone marrow, but myelofibrosis is not
severe. Trypanosomes may be found free and phagocytosed in
small vessels throughout the body, but are most commonly
observed in the liver and cerebral cortex. Hepatic changes
are constant, and consist of atrophy of hepatocellular cords
with sinusoidal dilation and periportal lymphoplasmacytic
proliferation. There is increased interstitial stroma, without
change in lobular organization, and generalized Kupffer cell
hyperplasia.
Changes in the lymphoid tissue are remarkable and constant
in all areas of the body. There is first follicular hyperplasia, with
a competent response and the formation of large and densely
cellular germinal centers. These changes are accompanied by
variable paracortical hyperplasia, and the paracortex has a
moth-eaten appearance because of the many large lympho-
blasts and tingible body macrophages. At this time, the lymph
nodes are physically enlarged, and there is thinning of the
capsules with focal colonization of perinodal fat and a histo-
logically compressed peripheral sinus. In chronic disease, the
nodes remain enlarged, but this is due, at least in part, to a
marked increase in capsular and intranodal stroma with
numerous collagenous septa extending from the medulla to
the capsule. The medullary follicles remain, but their cellular-
ity is reduced, and there is marked atrophy of paracortical
areas. Concurrently, there is sinus histiocytosis and at least
some degree of hemosiderosis without medullary cord hyper-
plasia. The changes are thus those of continuing stimulation with
an initial competent response followed by hyperplasia, then
atrophy and sclerosis.
Concurrent with the changes in lymph nodes, there is
remarkable atrophy of the thymus, with the reduction most
prominently affecting cortical regions. With the loss of cortical
volume, there is a reduction in cell density and an increase in
size of nuclei in cortical thymocytes. The splenic lymphoid
changes reflect those in other areas of the body, with atrophy
of periarteriolar cuffs and the formation of large hypocellular
follicles, occasionally with “target” ringing of mantle cell and
marginal zone layers. The number of follicles is not increased.
The most marked splenic change is sinus hyperplasia charac-
terized by a marked increase in fixed cells in the Billroth cord
(splenic cord, red pulp cord) areas, which contain within their
interstices many macrophages and plasma cells, with increased
hemosiderin and an irregular appearance of extramedullary
thrombopoiesis and erythropoiesis. Renal changes consist of a
moderate generalized increase in interstitial connective tissue
with mild epithelial atrophy and the appearance of large lym-
phoid cuffs around arterioles at the corticomedullary junction.
Glomeruli are regularly increased in diameter and in cellular-
ity and occasionally contain hemosiderin-laden macrophages.
There are focal adhesions between the visceral and parietal
layers of Bowman’s capsule, and a mild epithelioid appearance
to the central areas of the tuft. The nuclei of the juxtaglo-
merular cells are unusually prominent, and there is mild pig-
mentation by both iron and bilirubin in the proximal
epithelium. The overall changes in glomeruli are compatible
with membranoproliferative glomerulonephritis.
Subtle changes are consistently present in skeletal muscu-
lature and consist of an overall hypercellularity with a mild
reduction in fiber diameter and increased perivascular lym-
phocytes. The reddened foci in the omentum consist of focal
124 CHAPTER 2  •  Hematopoietic System
venous ectasia with mild interstitial sclerosis and perivascular
lymphoplasmacytic reaction.
Trypanosomiasis in other species.  Trypanosoma brucei
rhodesiense and T. b. gambiense are human pathogens causing
African sleeping sickness and are not important in domestic
animals, although T. gambiense has produced meningoenceph-
alitis in experimental goats and cats. These trypanosomes are
morphologically indistinguishable from each other and from
T. brucei, from which they are presumed to have evolved.
Trypanosoma brucei brucei is a cause of nagana in most
domestic species in Africa; humans are refractory to infec­
tion. Transmission is by Glossina spp. or mechanical. Equids,
small ruminants, camels, and dogs are very susceptible; the
disease is more chronic in cattle; and pigs may recover. Inco-
ordination and spinal paralysis are reported in horses and dogs.
Dogs may also develop parasitism and inflammation of the
anterior segments of the eye. The neurologic signs are undoubt-
edly the result of invasion of cerebrospinal fluid and to inflam-
mation of meninges, brain, and cord. Diagnosis depends on
demonstration of the organism in blood in acute or relapsing
febrile cases, or otherwise in lymph nodes by direct smear,
inoculation into the very susceptible laboratory mouse, or
cultivation.
Trypanosoma evansi was originally shown to be the cause
of surra in horses and camels, but it is pathogenic for most
domestic species and distributed in North Africa, Asia, and
Central and South America. Transmission is mechanical,
chiefly by Tabanidae, but also by other blood-sucking flies, and
apparently by vampire bats in the Americas. The disease in
horses and dogs is severe, and probably uniformly fatal in the
absence of adequate treatment. Cattle are mildly affected and
act as reservoirs, although acute disease may occur in suscep-
tible cattle introduced to endemic areas.
Trypanosoma equinum occurs in South America as the
cause of mal de caderas of horses, a disease that resembles
surra. The parasite is transmitted mechanically by tabanids. It
is probably a stable variant of T. evansi.
Trypanosoma suis is a West African species transmitted
by tsetse flies. It produces a nagana-like disease in pigs but is
apparently nonpathogenic for other domestic animals.
Trypanosoma simiae was originally isolated from African
monkeys. Although its pathogenicity for pigs is unpredictable,
it is ordinarily highly virulent and causes death in a few days.
Pathogenicity for other domestic species is insignificant. Trans-
mission is cyclical in tsetse flies. Trypanosomes of the species
T. congolense, T. dimorphon, T. vivax, and T. uniforme are causes
of nagana in Africa. Most domestic species are susceptible to
infection, although dogs are resistant to T. vivax. There is
considerable variation in the pathogenicity of different strains,
especially of T. congolense.
Trypanosoma cruzi is the cause of American trypanoso-
miasis (Chagas disease), which is an uncommon illness of
children, although infection of human adults and animals is
apparently common. The reservoir hosts are chiefly wild
species, but cats, dogs, and pigs can be infected and act as
reservoirs. Sheep and goats are susceptible to experimental
infections. This trypanosome is of zoologic interest because,
both in the tissues of the vertebrate host and in the alimentary
canal of the vector, it forms developmental phases resembling
the other genera Leptomonas, Crithidia, and Leishmania of the
family Trypanosomatidae.
Whereas some of the manifestations of T. cruzi infection
may be attributable to parasitemia with the trypanosomal
Bone Marrow
form, the disease is characterized by invasion of mesodermal
tissues, and the growth therein of the leishmanial forms of the
organism, which show a preference for cardiac and skeletal
muscle. It has been suggested that, because the cysts are focal
and the interstitial reaction may be widespread and diffuse,
there may be an autoimmune component to the myocarditis
associated with release of fiber proteins associated with para-
sitic injury.
Further reading
Guegan F, et al. Erythrophagocytosis of desialylated red blood cells is
responsible for anaemia during Trypanosoma vivax infection. Cell
Microbiol 2013;15:1285-1303.
Omotainse SO, Anosa VO. Comparative histopathology of the lymph
nodes, spleen, liver and kidney in experimental ovine trypanosomo-
sis. Onderstepoort J Vet Res 2009;76:377-383.
Van den Bossche P, et al. A changing environment and the epidemiol-
ogy of tsetse-transmitted livestock trypanosomiasis. Trends Parasitol
2010;26:236-243.
Hemotropic mycoplasmas.  Hemotropic mycoplasmas, also
called hemoplasmas, are cell wall–less unculturable bacteria
that attach to erythrocyte membranes and cause variably
severe anemia. The organisms were previously known as
Eperythrozoon or Haemobartonella spp., but based on nucleic
acid sequences, they are most similar to Mycoplasma, and
have all been renamed as such. All hemotropic mycoplasmas
are considered to be vector transmitted, although the specific
vector has been identified in only a few cases. Based on
sequence analysis, multiple mycoplasmal agents are identified
in blood in most species, but association with disease is infre-
quent. Mycoplasmas are at or below the limit of resolution of
light microscopy, and identification of organisms on blood
films is an insensitive method of diagnosis (Fig. 2-12).
In cats, there are currently 3 hemoplasmas: M. haemofelis,
M. turicensis, and M. haemominutum. Only M. haemofelis is
consistently associated with anemia, and can be identified by
light microscopy as rings or short chains of perimembranous
organisms. M. turicensis and M. haemominutum are only identi-
fied by PCR. Cats that survive acute infection with M. hae-
mofelis mount an immune response coincident with increase
in hematocrit and disappearance of organisms from blood
smears. It is unclear whether cats subsequently clear infection
or become subclinical carriers. Dogs are also host to multiple
hemoplasmas, but only M. haemocanis has been associated
with anemia, and only in splenectomized or immunosup-
pressed dogs. Cattle are host to M. wenyonii and M. hemobos.
M. wenyonii has been associated with anemia, edema, lymph-
adenopathy, and fever in dairy cattle. The pathogenicity of M.
hemobos is unclear. Similarly, infection of sheep with M. ovis
only rarely causes hemolytic anemia. M. haemolamae is preva-
lent in llamas and alpacas, and acute infection or infection in
crias or stressed adults may cause anemia with a large number
of erythrocyte organisms (see Fig. 2-12).
Acute infection with M. suis in young pigs results in illness
characterized by fever, hemolytic anemia, and hypoglycemia.
Older pigs may be infected, but clinical disease is usually
not apparent. As for other erythrocytic infectious agents, M.
suis-induced anemia is multifactorial, and physical damage
leading to premature splenic removal, induction of immune
responses to erythrocyte and parasite components, eryptosis,
 124.e1

Further reading
Anosa VO, et al. The haematology of Trypanosoma congolense infec-
tion in cattle. I. Sequential cytomorphological changes in the blood
and bone marrow of Boran cattle. Comp Haematol Int
1997;7:14-22.
Batista JS, et al. Highly debilitating natural Trypanosoma vivax infec-
tions in Brazilian calves: epidemiology, pathology, and probable
transplacental transmission. Parasitol Res 2012;110:73-80.
Forsberg CM, et al. The pathogenesis of Trypanosoma congolense
infection in calves. IV. The kinetics of blood coagulation. Vet Pathol
1979;16:229-242.
Gutierrez C, et al. Trypanosomosis in goats. Ann N Y Acad Sci
2006;1081:300-310.
Magona JW, et al. A comparative study on the clinical, parasitological
and molecular diagnosis of bovine trypanosomosis in Uganda.
Onderstepoort J Vet Res 2003;70:213-218.
 Bone Marrow
A A
B B
Figure 2-12  A. Mycoplasma haemofelis are minuscule epicel-
lular erythrocyte bacteria associated with regenerative anemia.
B. M. haemolamae can cause massive parasitemia in young or
debilitated camelids.
and endothelial activation from attachment of infected cells
all likely contribute to disease.
Rangelia vitalii is a tick-transmitted piroplasmic proto-
zoon that infects endothelial cells and hematopoietic cells of
dogs in South America. Infection is frequently fatal in domes-
tic dogs, and the most prominent clinical feature is bleeding
from pinnae. An unusual feature of R. vitalii infection is pre-
dominant targeting of endothelial rather than hematopoietic
cells. As a result, organisms are present in low number or
transiently in erythrocytes or leukocytes, although infected
dogs have immunohemolytic anemia with splenomegaly and
jaundice. Thrombocytopenia is variable, and not considered
to be the primary cause of bleeding. Organisms are most
readily identified in capillaries of lymph node and other tissues
(Fig. 2-13).
Further reading
França RT, et al. Canine rangeliosis due to Rangelia vitalii: from first
report in Brazil in 1910 to current day—a review. Ticks Tick Borne
Dis 2014;5:466-474.
Hoelzle LE, et al. Pathobiology of Mycoplasma suis. Vet J 2014;
202:20-25.
125
10 m
Figure 2-13  Rangelia vitalii cytoplasmic zoites in bone marrow
cell (A) and in an endothelial cell (B) from a dog. (Courtesy
A.P. Loretti.)
Sykes JE. Feline hemotropic mycoplasmas. Vet Clin North Am Small
Anim Pract 2010;40:1157-1170.
Mechanical, physical, and chemical causes of hemolytic
anemia.  Hemolytic anemia from mechanical trauma is caused
by physical damage to erythrocytes in vessels where turbulent
blood flow and pressure gradients exert shear forces. This
may be observed in animals with disseminated intravascular
coagulation (DIC), vasculitis, endocarditis, endocardiosis,
hemangiosarcoma, and burn wounds. DIC is characterized
by microangiopathy with luminal narrowing from fibrin
strands and platelet aggregates, which generates fragmented
erythrocytes (schistocytes) that are removed prematurely by
splenic macrophages. Anemia caused by mechanical trauma is
generally only mild, and jaundice is absent or mild. Both
intravascular and extravascular hemolysis induce erythropoi-
etic stress.
Many substances have the ability to cause oxidative injury.
Hemoglobin, normally maintained in a reduced state by eryth-
rocyte enzymes, is an abundant target for substances with
oxidative potential. Oxidized hemoglobin undergoes confor-
mational change and forms membrane-bound precipitates
 125.e1

Further reading
dos Santos AP, et al. Complete genome sequence of Mycoplasma
wenyonii strain Massachusetts. J Bacteriol 2012;194:5458-5459.
126 CHAPTER 2  •  Hematopoietic System
Figure 2-14  Blood smear from a cat with Heinz body hemolytic
anemia as a result of marked hyperglycemia. Note numerous
Heinz bodies (arrows) and erythrocyte ghosts (arrowheads).
called Heinz bodies (Fig. 2-14). Erythrocytes containing Heinz
bodies have reduced deformability and are removed by splenic
macrophages. The process of Heinz body removal may yield
deformed cells called “bite cells.” Oxidative substances associ-
ated with Heinz body hemolytic anemia in animals are red
maple leaves (horses), acetaminophen, Brassica and onion
ingestion, copper, zinc, propofol (cats), hyperglycemia, vitamin
K1 and K3, and others. The oxidative effect may cause minor
Heinz body formation without anemia (for example, repeated
administration of propofol in cats) or intravascular and extra-
vascular hemolysis with severe anemia (red maple leaf and
copper toxicity). Multiple factors such as excess dietary con-
centration, changes in intracellular transporters, decreased
excretion or imbalance with other trace elements contribute
to massive hepatocyte copper accumulation in sheep. Sudden
release of hepatocyte copper as a result of hepatic hypoxia,
parasitism, or stress increases blood concentration; free copper
ions enter erythrocytes and oxidize hemoglobin, leading to a
frequently fatal hemolytic crisis.
Certain bacterial hemolysins can directly lyse erythrocyte
membranes through phospholipase C or lecithinase activity
(Clostridium haemolyticum, C. novyi type D, C. perfringens), or
induce erythrocyte membrane changes resulting in immuno-
hemolytic anemia (C. perfringens type A in horses). Leptospira
spp. infection in cattle, lambs, and pigs may induce hemolysis
and marked jaundice, although the mechanism remains
unclear. Direct hemolysins have not been identified in Lepto-
spira, and the mechanism of hemolysis is assumed to be the
result of vasculopathy. Different types of snake venom contain
enzymes, including phospholipases A2 and metalloprotein-
ases, with potential to lyse erythrocytes and digest tissue to
induce hemorrhage, respectively. In most instances, hemolytic
anemia caused by venom is among the less severe outcomes
of snakebites.
Inherited erythrocyte enzyme deficiencies causing hemo-
lytic anemia are uncommon autosomal recessive conditions
that are informative regarding their normal enzymatic func-
tion and the effect of chronic hemolysis-stimulated erythro-
poiesis (see Table 2-2). Pyruvate kinase (PK) deficiency
diminishes generation of adenosine triphosphate (ATP) in
erythrocytes, which shortens their life-span and results in
Bone Marrow
anemia. Affected dogs are young, have macrocytic hypochro-
mic highly regenerative anemia, and develop hemosiderosis,
hemochromatosis, and eventual liver fibrosis, as well as myelo-
fibrosis and osteosclerosis from excessive iron absorption
resulting in iron overload. PK deficiency in cats is rarely associ-
ated with clinical disease, and anemia is only mild and associ-
ated with reticulocytosis. Phosphofructokinase (PFK) deficiency
impairs glycolysis in erythrocytes and myocytes, resulting in
persistent but compensated hemolytic anemia. Hemolytic
crises occur after vigorous exercise or in alkalemia because
PFK-deficiency erythrocytes have increased alkaline fragility.
Affected dogs develop hepatic hemosiderosis but not myelo-
fibrosis. Cytochrome b5 reductase deficiency causes methemo-
globinemia. The condition is usually clinically silent and
identified incidentally in dogs or cats with cyanotic mucus
membranes and brown discolored venous blood. Other eryth-
rocyte enzyme deficiencies have no or only limited clinical
consequence (see Table 2-2).
Hereditary abnormalities in erythrocyte membrane con-
stituents, such as spectrin and cationic transporters, are associ-
ated with elliptocytosis, spherocytosis, or stomatocytosis in
dogs. Although these shape changes are very noticeable on
microscopic review of blood films, they are usually incidental
findings and cause only mild hemolytic anemia compensated
for by increased reticulocyte production.
Hemolytic anemia is virtually always associated with
increased reticulocytes in blood and an increased proportion
of rubricytes in the bone marrow. Erythropoiesis may be
increased more than 10-fold in animals with chronic hemo-
lytic anemia and adequate nutrition and iron stores. Many
causes of hemolytic anemia also induce a systemic inflamma-
tory response, and therefore increased granulopoiesis and
overall bone marrow hypercellularity. Splenomegaly and
hepatomegaly are prominent features of extravascular hemo-
lysis, and less prominent in intravascular hemolysis. The
degree of hyperbilirubinemia and jaundice reflect the rate of
heme breakdown, and also liver capacity to conjugate and
excrete bilirubin. Dogs with immunohemolytic anemia are at
increased risk of venous and arterial thrombosis, and particu-
larly pulmonary thromboembolism. Induction of tissue factor
and abundant anionic phospholipid exposed on fragmented
cell membranes are thought to activate the coagulation
cascade.
Further reading
Alward A, et al. Red maple (Acer rubrum) leaf toxicosis in horses: a
retrospective study of 32 cases. J Vet Intern Med 2006;
20:1197-1201.
Goddard A, et al. Clinicopathologic abnormalities associated with
snake envenomation in domestic animals. Vet Clin Pathol
2011;40:282-292.
Owen JL, Harvey JH. Hemolytic anemia in dogs and cats due to eryth-
rocyte enzyme deficiencies. Vet Clin North Am Sm Anim
2012;42:73-84.
Anemia of decreased production
The most common causes of inadequate erythrocyte produc-
tion are chronic disease with persistent systemic inflamma-
tion, chronic kidney disease, and iron deficiency. Anemia
resulting from decreased erythrocyte production is nonregen-
erative, as indicated by lack of reticulocytosis and reduced
 126.e1

Further reading
Auza NJ, et al. Diagnosis and treatment of copper toxicosis in rumi-
nants. J Am Vet Med Assoc 1999;214:1624-1628.
Harvey JH. Pathogenesis, laboratory diagnosis, and clinical implications
of erythrocyte enzyme deficiencies in dogs, cats, and horses. Vet
Clin Pathol 2006;35:144-156.
 Bone Marrow 127

proportion of erythrocyte precursors in bone marrow. Changes

in erythrocyte and reticulocyte indices on the hemogram may
indicate the cause of anemia.
Anemia of inflammatory disease (AID) is very common
among hospitalized dogs, and less common in hospitalized
other species. AID is associated with a wide range of chronic
conditions that have an inflammatory component, such as
dermatitis, arthritis, enteritis, and microbial infections. Animals
with cancer also often have AID, although inflammation may
not be obvious. The predominant mechanism leading to AID is
thought to be increased production of inflammatory mediators,
such as IL-6, that stimulate hepatic production of hepcidin.
In turn, hepcidin reduces transfer of iron from enterocytes
into plasma and inhibits iron export from macrophages to
erythroid precursors in bone marrow. As a result, despite
abundant hemosiderin in bone marrow and splenic macro- A
phages, erythroid precursors are starved of iron for synthesis
of hemoglobin, and anemia ensues. Most AID is normocytic
and normochromic, but in some cases, microcytosis may be
present. Other factors thought to contribute to AID are
reduced erythropoietin production, reduced precursor cell
erythropoietin sensitivity, and reduced erythrocyte life-span.
AID is distinguished from true iron deficiency by presence of
ample hemosiderin in tissues, and normal to high serum fer-
ritin concentration.
Chronic renal disease is very common in older cats, and
virtually all affected cats have anemia. Anemia also occurs in
other species with chronic kidney disease, and is a major cause
of morbidity in people with kidney disease and hemodialysis.
Causes are reduced erythropoietin production by renal inter-
stitial cells and altered iron use, but altered sensing of oxygen
tension, reduced erythrocyte life-span, and blunted erythro-
poietin responsiveness are also likely factors. Exogenous sup- B
plementation with erythropoiesis-stimulating agents has
Figure 2-15  A, B. Bone marrow sections from a cat with FeLV
ameliorated anemia in cats, but therapeutic efficacy and ideal
infection, myelofibrosis, and lymphoid aggregates. Also note
hematocrit and iron concentration are ill defined. Morphologi-
coarse hemosiderin, not normally found in cat bone marrow.
cally, bone marrow in animals with anemia of chronic kidney
disease has reduced erythropoiesis despite abundant tissue
iron stores. Beagles. Lack of this receptor impairs intestinal absorption and
Iron deficiency anemia, as discussed previously, is physio- renal tubular reabsorption of intrinsic factor/cobalamin com-
logic in neonatal animals, and pathologic in adult animals. The plexes and other proteins. Cobalamin and folic acid are
cause is almost always chronic blood loss. Iron-limited eryth- required for synthesis of thymidine in DNA. In affected dogs,
ropoiesis is initially responsive with bone marrow erythroid defective DNA synthesis manifests with normochromic nor-
hyperplasia, and blood reticulocytosis, macrocytosis, and mocytic anemia, leukopenia, megaloblastic change in hema-
hypochromasia. With progressive iron depletion, microcytic hypo- topoietic and intestinal cells, and metarubricytosis. Dogs, in a
chromic erythrocytes become predominant in blood. In animals difference from people, do not have macrocytosis. Decreased
with anemia caused by iron deficiency, bone marrow and serum folate concentration, thought to result from lack of
splenic hemosiderin stores are absent or sparse. Cats normally absorption in primary gastrointestinal disease or from cobala-
do not store hemosiderin in bone marrow, but iron deficiency min deficiency, has been identified in Golden Retriever and
is nevertheless characterized by microcytic hypochromic Boxer dogs. It is unknown whether affected dogs also have
anemia, and low serum iron and ferritin concentration. Hemo- anemia.
siderin, sometimes very coarsely aggregated, may be seen in Pure red cell aplasia (lack of bone marrow erythroid
bone marrow of cats with myelofibrosis, feline leukemia precursors) and aplastic anemia (lack of all hematopoietic
virus (FeLV) infection, and myeloproliferative neoplasms precursor cells) result from cytotoxic viral infections, and
(Fig. 2-15). immune- or toxin-mediated injury to progenitor cells. In
Other causes of anemia from reduced erythrocyte produc- young animals, parvovirus or pestivirus infection are possible
tion are portosystemic shunting, cobalamin deficiency, and pure etiologies, whereas in adult animals, idiosyncratic drug reac-
red cell aplasia. The anemia of portosystemic shunting is tions or autoimmune destruction are more likely etiologies. In
usually microcytic and normochromic, and thought to result most cases, a definitive cause cannot be identified. The pres-
from reduced hepatic production of proteins involved in iron ence of lymphocytes and plasma cells in bone marrow sections
transport. Cobalamin (vitamin B12) deficiency from nonfunc- otherwise devoid of hematopoietic cells, and improvement of
tional cubam receptors has been described in Giant Schnauzers, cytopenia in response to immune suppression, supports an
Australian Shepherd dogs, Border Collies, Shar Peis, and immune-mediated etiology (Fig. 2-16) in some cases. Drugs
128 CHAPTER 2  •  Hematopoietic System
A B
C D
Figure 2-16  Bone marrow biopsy sections from an older dog with aplastic anemia. A, B. The bone
marrow is profoundly hypocellular, sinusoids are prominent, and there are lymphocytes and plasma
cells. C. CD3-positive lymphocytes around blood vessels. D. Occasional CD79a-positive B
lymphocytes.
with known potential to cause hematopoietic precursor cell
injury are chloramphenicol, phenylbutazone, trimethoprim-
sulfamethoxazole, and cephalosporins. Prolonged high estro-
gen concentration, either from endogenous production by
Sertoli cell tumors in dogs, granulosa cell tumors in horses and
other species, adrenal tumors in ferrets, or from exogenous
administration, may cause precursor cell injury and bone
marrow hypoplasia or aplasia. Bracken fern (Pteridium aquili-
num) ingestion causes hematuria (enzootic bovine hematuria)
and bone marrow suppression in ruminants caused by ptaqui-
loside toxin. Antiproliferative therapy with chemotherapeutic
agents or irradiation also affects bone marrow. Neutropenia is
the main manifestation of toxicity, and to a lesser degree
thrombocytopenia, reflecting their 8-hour and 10-day life-
spans, respectively. Overall, hematopoietic stem cells are
thought to be highly resistant to fatal injury, and with suffi-
cient time, protection from opportunistic infections and cell
replacement through transfusion, there can be recovery of
normal hematopoiesis in many instances.
Erythrocytosis is most often the result of dehydration, and
may be very severe in animals with extreme external loss of
free water or internal water sequestration. Neonatal bovine
diarrhea, parvoviral enteritis, and equine salmonellosis are
conditions typically associated with severe hemoconcentra-
Bone Marrow
tion. Physiologic erythrocytosis results from hypoxic stimula-
tion of erythropoietin production in animals with chronic
cardiac or pulmonary disease. Paraneoplastic erythrocytosis is
the result of ectopic production of erythropoietin by malig-
nant tumors, most often carcinomas. Lymphoid tumors, and
specifically renal lymphoma in dogs, have also been shown to
produce erythropoietin and induce erythrocytosis.
Further reading
Fyfe JC, et al. Selective intestinal cobalamin malabsorption with pro-
teinuria (Imerslund-Gräsbeck syndrome) in juvenile Beagles. J Vet
Intern Med 2014;28:356-362.
Weiss DJ. Bone marrow pathology in dogs and cats with non-
regenerative immune-mediated haemolytic anaemia and pure red
cell aplasia. J Comp Pathol 2008;138:46-53.
Disorders of platelets
Platelets are produced as cytoplasmic fragments of bone
marrow megakaryocytes and function in primary hemostasis,
inflammation, and repair (see also later section, Disorders of
hemostasis). Platelet number on the complete blood count
(CBC) is the most widely used indicator of adequate platelets,
 128.e1

Further reading
Aroch I, et al. Peripheral nucleated red blood cells as a prognostic
indicator in heatstroke in dogs. J Vet Intern Med 2009;23:
544-551.
Chalhoub S, et al. Anemia of renal disease: what it is, what to do and
what’s new. J Fel Med Surg 2011;13:629-640.
Cortinovis C, Caloni F. Epidemiology of intoxication of domestic animals
by plants in Europe. Vet J 2013;197:163-168.
Dandrieux JR, et al. Canine breed predispositions for marked hypoco-
balaminaemia or decreased folate concentration assessed by a labo-
ratory survey. J Comp Pathol 2013;54:143-148.
Durno AS, et al. Polycythemia and inappropriate erythropoietin con-
centrations in two dogs with renal T-cell lymphoma. J Am Anim
Hosp Assoc 2011;47:122-128.
Seguro AC, Andrade L. Pathophysiology of leptospirosis. Shock
2013;39:17-23.
Shmukler BE, et al. Cation-leak stomatocytosis in Standard Schnauzers
does not cosegregate with coding mutations in the RhAG, SLC4A1,
or GLUT1 genes associated with human disease. Blood Cells Mol
Dis 2012;48:219-225.
Waldner CL, Blakley B. Evaluating micronutrient concentrations in liver
samples from abortions, stillbirths, and neonatal and postnatal
losses in beef calves. J Vet Diagn Invest 2014;26:376-389.
 Bone Marrow 129

but as for erythrocytes, platelet volume is variable in health,
higher in recently released platelets, and lower in iron defi-
ciency and immune-mediated destruction. Therefore platelet-
crit (analogous to hematocrit) is actually a more meaningful
indicator of total platelet mass, and its use will become more
widespread because modern automated hematology analyzers
determine plateletcrit, platelet number, and platelet volume.
Thrombocytopenia results from increased consumption, pre-
mature destruction, or decreased production. Increased con-
sumption occurs in conditions such as hemorrhage, enteritis,
anticoagulant toxicity, pancreatitis, necrosis, endotoxemia,
envenomation, and vasculitis, and usually causes mild throm-
bocytopenia. Many hemotropic infectious agents can infect all
blood cells, including platelets; Anaplasma platys specifically
targets platelets. Bovine viral diarrhea virus (BVDV) has
tropism for megakaryocytes, and either direct toxicity or
immune-mediated injury render thrombocytopenia a common
feature of BVDV infection. FeLV infection of megakaryocytes
may also cause thrombocytopenia.
Immune-mediated thrombocytopenia (IMT) is a relatively
common primary autoimmune disease in dogs. The pathogen-
esis involves premature removal of antibody-coated platelets
by splenic macrophages. The nature of antigens targeted by
antibodies is poorly characterized. Platelet concentration and
plateletcrit in IMT are usually very low, and megakaryocytes
in bone marrow sections are markedly increased. Concurrent
immune destruction of erythrocytes and platelets (Evans syn-
drome) also occurs in dogs. IMT in cats is also most commonly
of an autoimmune nature, and often part of a generalized
antihematopoietic cell immune response, leading to multiple
cytopenias. Treatment with immune suppression appears to
be more effective in dogs than cats. IMT in horses is more
commonly associated with the use of drugs such as penicillin,
cephalosporins, and sulfonamides, which are presumed to act
as a hapten for induction of antiplatelet antibody responses.
Decreased platelet production is most often the result of
myeloproliferative or infiltrative bone marrow diseases, such
as leukemia or myeloma. Regeneration of platelets can be
monitored in a similar manner as for erythrocytes by detecting
increased mean platelet volume (MPV).
Thrombocytosis is a frequent finding in dogs with a variety
of illnesses, including inflammation and cancer. Increased
platelet number does not consistently correlate with increased
plateletcrit, and clinical associations of thrombocytosis have
not clearly been identified.
Further reading
Fielding CL, et al. Rattlesnake envenomation in horses: 58 cases (1992-
2009). J Am Vet Med Assoc 2011;238:631-635.
Schwartz D, et al. Platelet volume and plateletcrit in dogs with pre-
sumed primary immune-mediated thrombocytopenia. J Vet Intern
Med 2014;28:1575-1579.
Hematopoietic neoplasia
Integration of cellular, genetic, molecular, and immunologic
features of primary hematopoietic neoplasms with prognosis
over the past decade has established 3 main categories in
people: acute myeloid leukemia (AML), myeloproliferative neo-
plasm (MPN, formerly called “chronic leukemia”), and myelo-
dysplastic syndrome (MDS). Despite limited knowledge about
genetic and molecular features, most myeloid neoplasms in
animals can be grouped into one of these broad categories,
based on careful morphologic and immunochemical charac-
terization (Table 2-3).
Acute myeloid leukemia.  Animals with AML are usually
acutely but nonspecifically ill and have profound cytopenia in
one or several cell lines. Undifferentiated blast cells may be
absent in peripheral blood or may comprise a massive leuko-
cytosis. By definition, either bone marrow or blood, or both,
contain >20% blast cells for a diagnosis of AML. Blast cells are
large cells with round to oval nuclei, 1 or 2 nucleoli, and
basophilic cytoplasm. Blast cells lack differentiated features
such as granules that would allow classification into lineage.
Identification and enumeration of blast cells is best accom-
plished on cytology; determining the extent of myelophthisis
and presence of myelofibrosis or myelonecrosis requires
histopathology. Thus accurate diagnosis and classification of
AML requires, at minimum, a CBC, bone marrow cytology,
and histopathology. Bone marrow films should be differen-
tially counted (500 nucleated cells) to determine the propor-
tion of blast cells, the proportion of differentiated granulocytic
and erythrocytic cells, and the granulocytic:erythrocytic (G : E)

Table • 2-3 
Categories of myeloid neoplasms
Category Definition Subcategory

Acute myeloid leukemia
(AML)
Myeloproliferative neoplasms
(MPN, old term = chronic
leukemia)
Myelodysplastic syndrome
(MDS)
Cytopenia and ≥20% blast
cells in blood or bone
marrow
Cytosis of mature appearing
cells in blood, hypercellular
bone marrow, <5% blasts
Nonregenerative cytopenia,
dysplasia, 5-20% bone
marrow blast cells,
+/− myelofibrosis
According to cell morphology and/or immunophenotypic
features of cells: acute undifferentiated leukemia (AUL);
AML with neutrophilic differentiation; AML with
granulomonocytic differentiation; AML with
megakaryoblastic differentiation; etc.
According to cell morphology: polycythemia vera (PV); essential
thrombocythemia (ET); chronic neutrophilic leukemia (CNL);
chronic monocytic leukemia (CMoL); mastocytosis; etc.
According to most pronounced cytopenia, termed: refractory
anemia with excess blasts (RAEB); refractory neutropenia
with excess blasts (RNEB); refractory thrombocytopenia with
excess blasts (RTEB)
 129.e1

Further reading
O’Marra SK, et al. Treatment and predictors of outcome in dogs with
immune-mediated thrombocytopenia. J Am Vet Med Assoc 2011;
238:346-352.
130 CHAPTER 2  •  Hematopoietic System Bone Marrow

ratio. Concurrent assessment of CBC and bone marrow cytol-
ogy is essential for interpreting bone marrow histopathology,
and at times serial CBCs and repeated bone marrow biopsies
are required to arrive at a definitive diagnosis. Recovery from
hematopoietic stem cell injury, parvovirus, and acute feline
immunodeficiency virus (FIV) infection can cause transient
cytopenia and excess bone marrow blast cells, which needs to
be distinguished from AML by repeated CBCs.
AML is a highly heterogeneous cancer. In some cases, the
entire bone marrow consists of blast cells, and animals have
severe cytopenia; in other cases, blast cells encompass only
20-30% of bone marrow cells, with partial differentiation into
granulocytes or rubricytes (Figs. 2-17, 2-18). There usually is
lack or severe decrease in rubricytes, granulocytes, or mega-
karyocytes. Although animals are presented with acute illness,
retrospectively, cytopenia has often existed for weeks or
months before diagnosis of AML. Bone marrow cellularity in
AML is also highly variable and can range from 10-100%.
Investigations in people have shown that the molecular lesions
in AML are heterogeneous and mirror the variable clinical and
morphologic features of this cancer. Some understanding of
the molecular basis of AML in dogs is emerging with muta-
tions identified in the oncogenes RAS, FLT3, and C-KIT, trans-
location of BCR to ABL, copy number changes in PTEN and
BRCA1, and large deletions. Clonality in AML and other
hemolymphatic tumors has been identified in dog tissues with
a PCR assay targeting polymorphic regions of the androgen
receptor gene located on the X chromosome. Usefulness of
this assay is currently limited because ~50% of female dogs
normally lack heterozygosity at this gene locus.
Practically, once a diagnosis of AML has been derived by
the above criteria, the cancer should be subcategorized based
on morphology (Figs. 2-19, 2-20; see Table 2-3). In cases of
acute undifferentiated leukemia, flow cytometric analysis of
fresh aspirated bone marrow cells may be useful to rule out
lymphoid cell type, and to classify blast cells as monocytic
(CD11c/CD14/major histocompatibility complex II [MHC
II]), granulomonocytic (CD4/CD11b/CD11c), or megakaryo-
cytic (CD9/CD41/CD61). Detection of CD34 is consistent
with acute leukemia, but does not differentiate between acute
myeloid or acute lymphoid leukemia. Specialized laboratories
performing flow cytometry generally apply a panel of 10-20
antibodies and derive interpretation about blast cells from
detecting presence or absence of a combination of antigens.

A B

C D
Figure 2-17  Acute myeloid leukemia with neutrophilic differentiation in a dog. A. The blood
smear shows anemia, neutropenia, thrombocytopenia, and numerous blast cells. B. Bone marrow
aspirate has lack of rubricytes and segmented neutrophils, and increased immature granulocytes.
C, D. There were green masses throughout the mesentery and in kidney. (Courtesy R. Fraser.)
 Bone Marrow 131

E F
Figure 2-17, cont’d  E, F. Bone marrow sections have a predominance of blast cells with single
or multiple nucleoli, and reduced metamyelocytes and band neutrophils.

A B

C D
Figure 2-18  Samples from a dog with acute myeloid leukemia with rubricytic differentiation.
A. Bone marrow aspirate is devoid of granulocytes; blast cells are sparse in this field. B. The pro-
portion of blast cells is >20%, there is differentiation to rubricytes, granulocytes are rare. C. The
spleen is enlarged, mottled, and firm. D. There are clusters of rubricytes in many tissues; shown
here is a pulmonary arteriole.
132 CHAPTER 2  •  Hematopoietic System
A A
B B
Figure 2-19  Acute myeloid leukemia with neutrophilic differen-
tiation, in a dog. A, B. Blast cells comprised 25%, but differentia-
tion to band and segmented neutrophils is apparent.
IHC is useful to differentiate acute T-lymphocyte (CD3 posi-
tive) or B-lymphocyte (CD79a or CD20 positive) leukemia
from AML, but few additional antibodies specific for leuko-
cyte antigens are reactive with formalin-fixed tissue. Antibody
to canine CD18 reactive with formalin-fixed tissue labels all
leukocytes, but granulocytic and monocytic cells stain more
intensely than lymphocytes. Some antibodies to canine MHC
II are also reactive with formalin-fixed tissue, and label mono-
cytic cells and macrophages (Fig. 2-21). Infidelity in antigen
expression on neoplastic leukocytes is relatively common in
human leukemia, and has also been described for leukemic
cells of dogs. Hence interpretation of antigen expression
should always be based on absence or presence of multiple
antigens, and not only a single antigen.
All subcategories of AML (see Table 2-3) have been
reported in dogs and most other domestic animals. Granulo-
monocytic and megakaryoblastic AML appear to be more
common than neutrophilic or other subcategories in dogs,
whereas erythroblastic AML in association with FeLV infec-
tion was most common in cats. However, overall data on
prevalence of well-characterized AML cases in animals are
sparse, and much remains to be learned about this cancer. As
might be expected for a morphologically heterogeneous
Bone Marrow
Figure 2-20  Undifferentiated acute myeloid leukemia, in a dog.
A, B. Blast cells comprise >80% of cells, and mitotic figures are
frequent. At low power, individual rubricytes and granulocytes are
apparent, but megakaryocytes are absent.
cancer, response to therapy and prognosis are also very vari-
able. Although many cases of AML have life-threatening
thrombocytopenia or neutropenia at diagnosis, and the animal
dies within days despite therapy, survival in dogs of a year and
longer has also been reported. Organomegaly caused by leu-
kemic infiltrates is usually subtle, and in decreasing order of
frequency involves spleen, liver, lymph nodes, and any other
tissue. Several subcategories of AML have been reported in
horses, and clinical presentation usually consisted of hemor-
rhage and thrombocytopenia.
Acute lymphoid leukemia (ALL) arises from large precur-
sor lymphocytes in bone marrow. Morphologically, lympho-
blasts and myeloblasts are indistinguishable, but in most cases,
ALL cells express markers such as CD3, CD4, CD5, CD8,
CD20, CD21, or CD79a to indicate lymphoid origin. Dogs
with ALL are more likely to have severe neutropenia and
thrombocytopenia than dogs with AML. B-ALL is more com-
monly reported than T-ALL in dogs, but it is unknown whether
there are differences in natural history for either subcategory
(Fig. 2-22). The mean age of dogs with ALL is higher (8.0
years) than that of dogs with AML (6.3 years). Canine mul-
ticentric stage V leukemic lymphoma may be challenging to
distinguish from ALL with enlarged lymph nodes. Usually,
 Bone Marrow 133

A A

B B

C C
Figure 2-21  Acute myeloid leukemia with monocytic differentia- Figure 2-22  B-lymphocyte acute lymphoid leukemia, in a dog.
tion in a dog. A. Blood smear shows pancytopenia with occasional The dog had mild anemia and no morphologically abnormal cells
monocytes. B. Bone marrow section has predominance of large on the blood smear. A. Bone marrow aspirate shows a uniform
blast cells with a single nucleolus. C. Immunohistochemistry for population of hyperchromatic blast cells and many lysed cells
major histocompatibility complex II expression labels approxi- (arrowheads). B. Bone marrow section is >80% cellular, and mega-
mately half of the blast cells. karyocytes and hemosiderin are rare. C. Cells have indistinct
cytoplasm, uniformly round nuclei, and prominent central
nucleoli.

dogs with stage V lymphoma have greater tissue tumor burden
than those with ALL, and lymph nodes are more massively
enlarged. Also, expression of CD34 on leukemic cells is very
strongly suggestive of ALL rather than stage V lymphoma. In
horses, ALL is more commonly reported than AML, whereas
in dogs and cats AML is slightly more commonly reported
than ALL. Infection of cats with certain strains of FeLV results
in relatively frequent AML, most commonly of erythroblastic
type (erythremic myelosis), but monoblastic AML and ALL
have also been described.
134 CHAPTER 2  •  Hematopoietic System Bone Marrow

Further reading
Comazzi S, et al. Acute megakaryoblastic leukemia in dogs: a report
of three cases and review of the literature. J Am Anim Hosp Assoc
2010;46:327-335.
Comazzi S, et al. Immunophenotype predicts survival time in dogs with
chronic lymphocytic leukemia. J Vet Intern Med 2011;25:
100-106.
Juopperi TA, et al. Prognostic markers for myeloid neoplasms: a com-
parative review of the literature and goals for future investigation.
Vet Pathol 2011;48:182-197.
Novacco M, et al. Prognostic factors in canine acute leukaemias: a
retrospective study. Vet Comp Oncol 2015 Jan 26. [Epub ahead of
print].
Tan E, et al. Automated analysis of bone marrow aspirates from
dogs with haematological disorders. J Comp Pathol 2014;151:
A
67-79.
Usher SG, et al. RAS, FLT3, and C-KIT mutations in immunophenotyped
canine leukemias. Exp Hematol 2009;37:65-77.
Vernau W, Moore PF. An immunophenotypic study of canine leukemias
and preliminary assessment of clonality by polymerase chain reac-
tion. Vet Immunol Immunopathol 1999;69:145-164.
Willmann M, et al. Chemotherapy in canine acute megakaryoblastic
leukemia: a case report and review of the literature. In Vivo
2009;23:911-918.

Myeloproliferative neoplasms.  Myeloproliferative neo-

plasms (MPN) are the former chronic leukemias, also previ-
ously called myeloproliferative diseases. The terminology was
changed in the most recent World Health Organization clas-
sification of human myeloid neoplasms, to make clear that
these are neoplasms, although their natural progression is B
usually very gradual. The hallmarks of MPN are accumulation
Figure 2-23  Myeloproliferative neoplasm, chronic neutrophilic
of a large number of relatively normal-appearing cells in blood,
leukemia in a dog. A, B. The dog had mild anemia and thrombo-
bone marrow, spleen, and eventually other organs, and mild or
cytopenia, and a neutrophil count of 109 × 109/L. The bone
absent cytopenia. Splenomegaly may be massive, and reflects a
marrow consists predominantly of differentiated segmented and
large tumor burden. Bone marrow biopsy is usually not
band neutrophils, with ~5% blast cells. Rubricytes and mega-
needed for diagnosis, but, if performed, bone marrow is
karyocytes are much reduced. The dog had an increasing neutro-
highly cellular, with numerous terminally differentiated cells
phil count over 18 months prior to presentation for fatigue and
(Figs. 2-23, 2-24).
poor appetite.
MPN are rare in animals but are readily diagnosed and
categorized (see Table 2-3). Typically, a large number of
mature erythrocytes, granulocytes, monocytes, platelets, or tions. MPN may eventually progress to “blast transformation”
mast cells are discovered incidentally on the CBC of animals with appearance of immature or blast cells in circulation and
with no or vague illness. Retrospectively, hematocrit or leuko- progressive cytopenia. Chronic myelogenous leukemia (CML)
cyte concentration in affected animals often have increased is a distinct MPN entity in humans characterized by a recipro-
gradually over more than a year, and the diagnosis is estab- cal translocation resulting in expression of a constitutively
lished relatively late in the life of the neoplasm. Hematocrit active tyrosine kinase. Translocation of equivalent regions has
in polycythemia vera may be twice normal, and associated been identified in several dogs with MPN.
hyperviscosity can cause retinal or brain ischemic events man-
ifesting with seizures or other neurologic abnormalities. Simi-
larly, animals with chronic neutrophilic leukemia often have Further reading
neutrophil concentrations exceeding 10 times the upper refer- Culver S, et al. Molecular characterization of canine BCR-ABL-positive
ence interval, and in essential thrombocythemia, platelet con- chronic myelomonocytic leukemia before and after chemotherapy.
centration and plateletcrit exceed twice the upper reference Vet Clin Pathol 2013;42:314-322.
interval. The molecular pathogenesis of MPN in people Vardiman JW, et al. The 2008 revision of the World Health Organiza-
involves mutations that constitutively activate kinases involved tion (WHO) classification of myeloid neoplasms and acute leukemia:
in growth factor receptor signaling. Hence in some respects, rationale and important changes. Blood 2009;114:937-951.
MPN may be considered accumulations of relatively normal cells
unable to perceive signals to stop growth. All hematopoietic cells
in MPN share key molecular lesions, but predominant pheno- Myelodysplastic syndrome.  Myelodysplastic syndrome
type is determined by cell lineage-specific additional muta- (MDS) is defined as a condition with persistent nonregenerative
 134.e1

Further reading
Adam F, et al. Clinical pathological and epidemiological assessment of
morphologically and immunologically confirmed canine leukaemia.
Vet Comp Oncol 2009;7:181-195.
Figueiredo JF, et al. Acute myeloblastic leukemia with associated
BCR-ABL translocation in a dog. Vet Clin Pathol 2012;41:
362-368.
Fischer C, et al. Erythroleukemia in a retrovirus-negative cat. J Am Vet
Med Assoc 2012;240:294-297.
Giantin M, et al. Evaluation of tyrosine-kinase receptor c-KIT (c-KIT)
mutations, mRNA and protein expression in canine leukemia: might
c-KIT represent a therapeutic target? Vet Immunol Immunopathol
2013;152:325-332.
Mochizuki H, et al. Demonstration of the cell clonality in canine hema-
topoietic tumors by X-chromosome inactivation pattern analysis.
Vet Pathol 2015;52:61-69.
Valentini F, et al. Use of CD9 and CD61 for the characterization of
AML-M7 by flow cytometry in a dog. Vet Comp Oncol
2011;10:312-318.
Wilkerson MJ, et al. Lineage differentiation of canine lymphoma/
leukemias and aberrant expression of CD molecules. Vet Immunol
Immunopathol 2005;106:179-196.
134.e2

Further reading
Cruz-Cardona JA, et al. BCR-ABL translocation in a dog with chronic
monocytic leukemia. Vet Clin Pathol 2011;40:40-47.
Perez M, et al. Partial cytogenetic response with toceranib and pred-
nisone treatment in a young dog with chronic monocytic leukemia.
Anticancer Drugs 2013;24:109-1103.
 Bone Marrow 135

A A

B B
Figure 2-24  Myeloproliferative neoplasm, mastocytosis in a dog.
The dog had mild anemia and a low number of mast cells on
routine blood smear review. A. Bone marrow shows rubricytes,
granulocytes, megakaryocytes, and a moderate number of round
granular cells. B. Toluidine blue staining identifies numerous mast
cells.

cytopenia, dysplasia of blood or bone marrow cells, and presence

of 5-20% blast cells in bone marrow. MDS may be more
common than AML in specialty small animal practice, but
unlike in people, causes and prognosis of MDS in animals are
ill defined. Hematopoietic cells in primary MDS of people
have clonal molecular lesions, which is likely also the case in
at least a proportion of primary MDS in animals. Primary
MDS has not been described in large animals, which may
reflect lack of investigation. Cytopenia, in most cases anemia,
is usually severe in MDS. Dysplasia can manifest as asynchro-
nous cytoplasmic and nuclear maturation or binucleation in
rubricytes, neutrophil hypersegmentation or gigantism, plate-
let gigantism, and other morphologic abnormalities (Figs.
2-25, 2-26). Despite severe nonregenerative cytopenia, the
bone marrow is usually hypercellular and has an excess pro-
portion of blast cells. Some dogs with MDS progress to AML,
sometimes within weeks of diagnosis, but morphologic char-
acteristics to identify those individuals are lacking. Other dogs
with MDS either remain static, become transfusion dependent
if they have anemia, or progress to develop additional cytope-
nias and bone marrow failure. Myelofibrosis is a common feature
C
Figure 2-25  Myelodysplastic syndrome, severe anemia and
thrombocytopenia in a dog. A. The blood smear has giant neutro-
phils with condensed poorly segmented nuclei. B. Similar atypical
neutrophils on bone marrow cytology. C. The bone marrow is
hypercellular but lacks megakaryocytes and adequate rubricytes.
Blast cells comprise 18%.
of MDS in dogs, and presumed to be the result of excess pro-
duction of growth factors, such as transforming growth factor
(TGF)-β or platelet-derived growth factor (PDGF) in a bone
marrow with disturbed cytokine environment. In people,
MPN with MDS is recognized as a separate category of
136 CHAPTER 2  •  Hematopoietic System
A Gomori), and should be absent in normal bone marrow (Fig.
B may induce severe MF, anemia, and bone marrow lymphoid
Figure 2-26  Acute myeloid leukemia with neutrophilic differen-
tiation and dysplasia in a dog. A, B. Blast cells comprise 41% of
cells, and there is severe rubricytic hypoplasia. Neutrophils have
abnormal nuclear segmentation (arrows).
myeloid tumors. Clear diagnostic features for such a category
are lacking for animals.
Cytopenia and dysmyelopoiesis morphologically indistinguish-
able from primary MDS may result from idiosyncratic responses
to drugs such as phenobarbital; treatment with chemothera-
peutic drugs; altered hematopoiesis in immune-mediated
hematologic disease; precursor cell infection with vector-
borne pathogens such as Anaplasma platys, Leishmania infan-
tum, Babesia vogeli, and Hepatozoon canis; and bracken fern
myelotoxicity. It is essential to distinguish primary from sec-
ondary MDS because discontinuation of particular drugs or
toxin exposure or treatment of immune-mediated or infec-
tious disease may resolve dysmyelopoiesis. A thorough clinical
history and repeated hematologic assessment can differentiate
these etiologies.
Further reading
De Tommasi AS, et al. Evaluation of blood and bone marrow in selected
canine vector-borne diseases. Parasit Vectors 2014;7:534-541.
Jacobs G, et al. Neutropenia and thrombocytopenia in three dogs
treated with anticonvulsants. J Am Vet Med Assoc 1998;212:
681-684.
Bone Marrow
Vardiman J. The classification of MDS: from FAB to WHO and beyond.
Leuk Res 2012;36:1453-1458.
Weiss DJ. Recognition and classification of dysmyelopoiesis in the dog:
a review. J Vet Intern Med 2005;9:147-154.
Other hematopoietic conditions
Myelofibrosis.  Myelofibrosis (MF) is excessive production of
collagen by bone marrow fibroblasts causing cytopenia and even-
tual bone marrow failure. MF is suspected in animals with
cytopenia (usually anemia), deformed tear-drop shaped or
oval erythrocytes on blood smears, and inability to aspirate
bone marrow. Thus diagnosis of MF requires assessment of a
core biopsy. Deposition of fine type 3 collagen fibrils can be
detected with silver impregnation stains (Warthin-Starry or
2-27). With progressive MF, coarse type 1 collagen fibers
appear that can be highlighted with trichrome stains. In
people, primary MF is a clonal disorder diagnosed by compos-
ite assessment of clinical, laboratory, and molecular features.
Secondary MF is a not a clonal disorder, and results from
chronic hyperstimulated hematopoiesis in conditions such as
chronic hemolytic anemia, response to bone marrow micro-
environment injury, and cytokine dysregulation. Many people
with MPN eventually develop MF.
Clear diagnostic criteria and clonality assays to distinguish
primary from secondary MF are lacking for animals. MF in
dogs is most often attributed to bone marrow injury and
cytokine dysregulation, or identified as part of MDS. A pro-
portion of dogs with MF respond to immune suppression and
antifibrotic drugs, whereas some dogs with MF and MDS
progress to AML. Infection of cats with some strains of FeLV
aggregates, which is an irreversible and progressive lesion.
Chronic lymphocytic leukemia.  Chronic lymphocytic leuke-
mia (CLL) is the most common leukemia in dogs. Approxi-
mately 70% are of T-cell type, arise in the splenic red pulp,
and have minimal bone marrow involvement. The other 30%
are of B-cell type, arise in bone marrow, are frequently associ-
ated with a monoclonal gammopathy, and cause gradual
myelophthisis. CLL should be suspected in middle-aged to
older dogs with lymphocytosis of small- to medium-sized
cells. Lymphocytosis may be mild or extreme. Immunopheno-
typing distinguishes neoplastic from reactive polyclonal lym-
phocytosis of inflammatory conditions. CLL is an indolent
leukemia, and survival of more than 1 year after diagnosis is
common for both T- and B-cell types. In a small proportion
of dogs, CLL cells do not express typical T- or B-cell antigens,
and these dogs have shorter survival. Diagnosis of CLL may
be incidental during CBC for another indication; thus the
duration of CLL prior to diagnosis may be highly variable and
affect data concerning survival.
CLL occurs in older cats, and is also an indolent leukemia.
The T-cell type is much more common than the B-cell type,
organomegaly is uncommon, and mild anemia, likely resulting
from myelophthisis, is common. Mean survival with chloram-
bucil treatment was >1 year. CLL in old horses is a rare condi-
tion, and both B-cell and T-cell types have been reported.
Advanced small cell lymphoma with leukemia may be dif-
ficult to distinguish from advanced CLL with involvement of
lymph nodes. Clinical history and historical laboratory data
may distinguish the conditions that, at an advanced disease
state, have similar prognosis and therapy.
 136.e1

Further reading
Perez-Alenza MD, et al. Clinico-pathological findings in cattle exposed
to chronic bracken fern toxicity. N Z Vet J 2006;54:185-192.
Weiss DJ, Aird B. Cytologic evaluation of primary and secondary myelo-
dysplastic syndromes in the dog. 2001;32:67-75.
 Bone Marrow
A teinuria. Bleeding diatheses, including epistaxis, gingival bleed-
B proteins detected in serum or urine. Lytic bone lesions are
C by identifying morphologically abnormal and highly frequent
Figure 2-27  Myelofibrosis in a dog. A. The dog had anemia,
and teardrop-shaped (arrowhead) and oval (arrows) erythrocytes
in the blood smear. B, C. Bone marrow was patchy with fibrous
tissue and areas of hematopoietic cells.
Mastocytosis.  Bone marrow involvement in mast cell neo-
plasia of dogs is most often the result of metastatic spread of
high-grade solid mast cell tumors to multiple hemolymphatic
sites. Mastocythemia in cats is rare, and in most instances
reflects release of cells from visceral (usually splenic) or cuta-
neous mast cell tumors. Primary bone marrow mastocytosis is
137
a rare MPN in dogs and cats not associated with solid mast
cell tumors. Animals have gradually progressive myelophthisis
(see Fig. 2-24).
Multiple myeloma.  Multiple myeloma (MM) is a malignant
tumor of plasma cells that occurs in older animals. In dogs and
horses, the tumor is most often located in the medullary cavity
of bones with active bone marrow, and frequently at multiple
sites. Animals with MM are usually presented with a history
of lameness or paresis secondary to spinal cord compression,
or the neoplasm may be suspected from the radiographic
evidence of multiple foci of bone lysis. Other clinical signs are
usually nonspecific and include lethargy, weakness, and
anorexia. Hypercalcemia may occur in the course of disease
as a result of bone lysis, and polyuria, polydipsia, and kidney
disease occur secondary to hypercalcemia and light-chain pro-
ing, retinal hemorrhage, and, less frequently, melena or
hematuria, are common. In cats, malignant plasma cell tumors
are more often located at extramedullary sites, either in
abdominal organs or skin. In most MM, the neoplastic cells
secrete a clonal immunoglobulin (also called paraprotein or M
protein) that is detectable by serum electrophoresis as a mono-
clonal protein. Free light chains readily cross the glomerulus;
therefore, in many cases, urine protein electrophoresis is a
sensitive means of identifying clonal immunoglobulin light
chains (Bence Jones proteins) that may not accumulate in
serum.
Diagnostic criteria for MM in people are applicable to dogs,
and require presence of neoplastic cells in bone marrow plus either
serum or urine monoclonal proteins or osteolytic lesions. In cats,
diagnostic criteria for MM are presence of a plasma cell tumor,
either in abdominal organs or bone marrow, plus monoclonal
uncommon in cats. Cutaneous plasma cell tumors in cats may
also be associated with monoclonal gammopathy, and have a
better prognosis than those involving abdominal organs. The
diagnosis of MM is usually made by fine-needle aspiration or
bone marrow core biopsy from an area of bone lysis. Immu-
noglobulin G (IgG) followed by IgA gammopathies are most
common.
Nonsecretory MM has been reported, and differentiation
from plasmacytoid lymphoma is based on the location of
MM in the bone medullary cavity causing bone lysis. Cellular
features of MM are similar to those of plasma cells, and
can include binucleation or multinucleation and moderate
anisokaryosis and anisocytosis. Some infectious agents such
as Ehrlichia and Leishmania may induce clonal immunoglobu-
lin production, which needs to be differentiated from MM
plasma cells, and with serologic assays. Plasma cell features
in bone marrow supportive of a diagnosis of MM are propor-
tion >5% of nucleated cells, occurrence in clusters, and abnor-
mal morphology, such as binucleation or multinucleation,
anisocytosis, and anisokaryosis. Clustering of bone marrow
plasma cells is best identified on histopathology, and in
some cases proximity to osteoclasts indicates bone lysis
(Fig. 2-28).
Circulating nonhematopoietic neoplastic cells.  Cancers
that metastasize via the blood stream frequently shed low
numbers of cells that may be detectable in blood with highly
sensitive techniques such as flow cytometry. Sufficient
numbers to be apparent on routine review of blood smears
may occur with disseminated histiocytic sarcoma, high-grade
138 CHAPTER 2  •  Hematopoietic System
A
B animals, the bone marrow and the thymus as well as the liver
C organized tissues that include the tonsils and Peyer’s patches
Figure 2-28  Multiple myeloma in a dog. A. Plasma cells in bone
marrow aspirate are morphologically normal but very numerous.
B. Bone marrow biopsy shows uneven cell density at low magni-
fication. C. At high magnification, plasma cells predominate in
dense areas.
mast cell tumors, and widely metastatic carcinomas. In most
instances, prominent neoplastic cells on blood or bone marrow
films reflect bone marrow metastases and advanced cancer.
Morphology is generally sufficient for distinction from neo-
plastic leukocytes.
Lymphoid Organs
Further reading
Campbell MW, et al. Chronic lymphocytic leukaemia in the cat: 18
cases (2000-2010). Vet Comp Oncol 2013;11:256-264.
Comazzi S, et al. Immunophenotype predicts survival time in dogs with
chronic lymphocytic leukemia. J Vet Intern Med 2011;25:
100-106.
Giraudel JM, et al. Monoclonal gammopathies in the dog: a retrospec-
tive study of 18 cases (1986-1999) and literature review. J Am Anim
Hosp Assoc 2002;38:135-147.
Mellor PJ, et al. Myeloma-related disorders in cats commonly present
as extramedullary neoplasms in contrast to myeloma in human
patients: 24 cases with clinical follow-up. J Vet Intern Med
2006;20:1376-1783.
Tefferi A. Primary myelofibrosis: 2014 update on diagnosis, risk-
stratification, and management. Am J Hematol 2014;89:915-925.
LYMPHOID ORGANS
The lymphatic system consists of lymphatic organs and a
conducting network of lymphatic vessels that carry the circu-
lating lymph directly toward the heart. Because the lymphatic
vessels are a major component of the circulatory system, they
have been discussed in the chapter on cardiovascular diseases.
The bone marrow, lymph nodes, spleen, thymus are classified
in the Nomina Anatomica Veterinaria as lymphoid organs.
However, these organs are also part of the immune system,
which is more complex and includes a number of other organs
and tissues. Central or primary lymphoid organs provide lym-
phoid precursor cells that circulate and take up residence in
peripheral or secondary lymphoid organs. In most domestic
are considered primary lymphoid organs. Peripheral lymphoid
organs include the spleen and the lymph nodes, which are
responsible for production of antibodies and cell-mediated
immune responses. Furthermore, there are numerous types
of mucosa-associated lymphoid tissues (MALTs) that repre-
sent local collections of lymphoid tissue that underlie the
submucosa, such as the gastrointestinal tract–associated lym-
phoid tissue (GALT) or the bronchus-associated lymphoid
tissue (BALT). Other such locations of lymphoid tissues at
various sites of the body include the conjunctival-associated
lymphoid tissue (CALT); the nasal-associated lymphoid tissue
(NALT); the larynx-associated lymphoid tissue (LALT); the
duct-associated lymphoid tissues (DALT) of different salivary
glands, including mammary glands; and the skin-associated
lymphoid tissue (SALT). MALT can be further divided into
and diffuse lymphoid cells in epithelial layers and their under-
lying connective tissue that are not organized into a structure
such as the SALT. The Peyer’s patches are a major site of
B- and T-cell production after thymic involution, and can
therefore be classified as primary and secondary lymphoid
organs.
In other chapters, pathologic changes have been character-
ized based on primary or secondary disease processes affecting
a particular organ system. This is extremely difficult for the
lymphoid organs because they play a central role in many
immune-mediated disease processes that manifest in nonlym-
phoid organs. In most disease processes, the lymphoid organs
have a vital immunologic function; however, the pathophysi-
ologic processes of the lymphoid cells do not necessarily
 138.e1

Further reading
Amati M, et al. Carcinocythaemia (carcinoma cell leukaemia) in a dog:
an acute leukaemia-like picture due to metastatic carcinoma. J
Small Anim Pract 2012;53:476-479.
Blackwood L, et al. European consensus document on mast cell
tumours in dogs and cats. Vet Comp Oncol 2012;10:e1-e29.
Cian F, et al. Leukemic small cell lymphoma or chronic lymphocytic
leukemia in a horse. Vet Clin Pathol 2014;42:301-306.
Geigy C, et al. Multiple myeloma in a dog with multiple concurrent
infectious diseases and persistent polyclonal gammopathy. Vet Clin
Pathol 2013;42:47-54.
Hikasa Y, et al. Connective tissue-type mast cell leukemia in a dog. J
Vet Med Sci 2000;62:187-190.
Piviani M, et al. Significance of mastocytemia in cats. Vet Clin Pathol
2012;42:4-10.
Ramos-Vara JA, et al. Immunohistochemical detection of multiple
myeloma 1/interferon regulatory factor 4 (MUM1/IRF-4) in canine
plasmacytoma: comparison with CD79a and CD20. Vet Pathol
2007;44:875-884.
Weiss DJ. Bone marrow pathology in dogs and cats with non-
regenerative immune-mediated haemolytic anaemia and pure red
cell aplasia. J Comp Pathol 2008;138:46-53.
Workman HC, Vernau W. Chronic lymphocytic leukemia in dogs and
cats: the veterinary perspective. Vet Clin North Am Small Anim Pract
2003;33:1379-1399.
 Lymphoid Organs
manifest as changes in the lymphoid organs themselves. For
those diseases, the reader is referred to the appropriate chapter
with the manifestation of the immunologic function within
that organ. Furthermore, the bone marrow is also the major
component of the myeloid tissues and has therefore been
discussed previously. However, the division of myeloid and
lymphoid organs is arbitrary given that some disorders of
erythrocytes are caused by antibody formation and could also
be classified as a disease of lymphocytes. The liver, together
with the yolk sac, plays a major part as the primary site of
blood cell formation during early ontogeny and may be,
together with bone marrow, the primary site of maturation or
production of B lymphocytes in mammals. This is evidenced
by the large number of islets of hematopoietic cells within the
parenchyma of the fetal liver. Similarly, the yolk sac may also
represent a site of production of immature hematopoietic
cells. Regardless, detailed discussions of the liver, the yolk sac,
and the MALT can be found in the appropriate chapters. This
section will only discuss disease processes that manifest within
the thymus, spleen, and lymph nodes.
Developmental diseases of
the lymphoid system
Immunodeficiency
Immunodeficiency syndromes are characterized by an
increased risk of the host becoming infected by various patho-
gens, including less virulent or even commensal organisms or
uncommon pathogens, and to be less responsive to standard
antimicrobial therapies. These syndromes are a reflection of
the immune system’s inability to protect the host against
infectious disease or the development of neoplastic diseases.
In general, antibody deficiencies are associated with bacterial
infections, and cell-mediated deficiencies are associated with
protozoal, fungal, viral, or obligate intracellular bacterial infec-
tions. The vast majority of immunodeficiencies are caused by
an acquired impairment of the immune system that can be
caused by malnutrition, aging, neoplastic or infectious dis-
eases, or chronic diseases. Such immunodeficiencies are also
referred to as secondary immunodeficiencies. In contrast, primary
immunodeficiencies are caused by genetic or congenital defects.
Most of these deficiencies manifest themselves early in life,
and the affected animal is often clinically described as “poor
doing” or failing to thrive. However, in some animals, primary
immunodeficiencies may manifest clinically only later in life.
The type of infection in affected animals often indicates which
component of the immune system may be defective. This
chapter will only discuss primary immunodeficiencies character-
ized by an absence or defect in a class or subclass of lymphocytes
derived either from bone marrow or thymus. Primary immuno-
deficiencies caused by defects of phagocytic cells, or of com-
plement, or secondary immunodeficiencies, or the failure to
passively transfer immunoglobulin from dam to offspring are
not included in this chapter. Primary defects in adaptive
immune response can be partial or combined, affecting either
only T cells or B cells or both. Such conditions usually lead to
severe combined immunodeficiency.
Immunoglobulin deficiencies.  These primary immunode-
ficiencies are characterized by an immunoglobulin deficiency
that can affect all 3 types of major immunoglobulins (agam-
maglobulinemia) or appear as selective deficiencies of indi-
vidual classes of immunoglobulins (selective immunoglobulin
deficiency).
139
Agammaglobulinemia is characterized by lack of all types
of major immunoglobulins and an absence of mature B cells
and plasma cells. It occurs in male foals, and affected horse
breeds include Thoroughbreds, Quarter Horses, and Standard-
breds. Equine agammaglobulinemia resembles Bruton con-
genital X-linked agammaglobulinemia of human infants in
both expression and progression. In humans, there is a muta-
tion in the BTK gene encoding Bruton tyrosine kinase, which
is closely related to the Src kinase family. This kinase is
required for B-cell maturation, and B-cell development is
arrested in the pre-B stage. Histologically, there is an absence
of plasma cells and germinal centers in lymph nodes, Peyer’s
patches, and tonsils, but the thymus is normal.
Selective deficiencies of immunoglobulins can be observed
in any class of immunoglobulins, but selective IgM and IgA
deficiencies have been reported in dogs and horses. These
diseases are characterized by serum levels of the affected
immunoglobulin being at least 2 standard deviations below
normal values, but B-cell counts are normal. Clinical signs
commonly manifest after degradation of maternal antibodies.
An IgM deficiency occurs in foals of Quarter Horse and Arabian
breeding, and both sexes are affected. IgM deficiencies
are very rare in humans. In affected foals, immunoglobulin
levels vary from undetectable to 10% of normal, and the
lymphocyte counts are normal, with normal proportions of
B- and T-derived classes. These animals have neutrophilic leu-
kocytosis without anemia and are presented for examination
at 4-8 months of age with febrile disease involving the respira-
tory tract. Most animals die as a result of septicemia or pneu-
monia before 10 months of age. The few surviving horses will
have recurrent respiratory infections, and most die before
reaching full adulthood. In dogs, an IgA deficiency has been
reported in Beagles, German Shepherd dogs, Irish Setters, and
Shar Peis. IgA deficiencies are the most common deficiency in
humans in the United States. They are usually asymptomatic,
but can be associated with autoantibodies to IgA, recurrent
respiratory diseases, allergies, and autoimmune diseases. In
dogs, affected animals may have disease manifestation in the
skin, gastrointestinal tract, or respiratory tract. Atopy is
common. The number of lymphocytes, and plasma cells in
affected dogs appear normal, suggesting an inability to produce
or secrete IgA.
Severe combined immunodeficiency.  Severe combined
immunodeficiency (SCID) describes a group of diseases that
are characterized by defects in both humoral and cellular immu-
nity. In most cases, both B- and T-cell lineages are affected,
and animals with SCID have lymphopenia, nonresponsiveness
of lymphocytes to antigen stimuli, and minimal levels of
immunoglobulins or responses to immunization. Although
numerous defects have been shown to cause SCID in humans,
and mouse models of SCID have been developed, in domestic
animals, SCID has only been described in foals, primarily of
the Arabian breed, and Basset hounds, Cardigan Welsh Corgi,
and Jack Russell Terrier dogs.
SCID occurs in ~2% of Arabian and Arabian-crossbred foals,
but the incidence of phenotypically normal heterozygotes will
be much higher. Both sexes are affected, and the disease is the
result of an autosomal recessive trait expressed as an absence of
both B- and T-lymphocyte functions. The genetic defect is a
spontaneous frame-shift mutation of the PRKDC gene encod-
ing the catalytic subunit of DNA-dependent protein kinase
(DNA-PKcs) located on chromosome 9. The mutation causes
a complete loss of function of DNA-PKcs, which is required
140 CHAPTER 2  •  Hematopoietic System
for formation of functional V regions during recombination of
immunoglobulin heavy-chain and T-cell receptor genes. This
results in an inability to produce functional T and B cells.
Furthermore, DNA-PKs have an important function in DNA
repair, and affected horses have been shown to have defects
in their DNA repair mechanism. Homozygous horses rarely
live beyond 5 months of age; heterozygous horses have a
higher risk of developing equine sarcoids in the skin. Whether
a single allele mutation in PRKDC predisposes such horses to
the development of neoplastic disease is unknown as is the
potentially synergistic effect of the mutation and bovine papil-
lomavirus 1 and 2 infection, which has been associated with
the development of equine sarcoids.
Foals with SCID are normal at birth, but at ~10 days of
age develop a range of diseases, including pneumonia and
diarrhea, that are commonly associated with equine adenovi-
ruses, Cryptosporidium parvum, and Pneumocystis carinii infec-
tions. Foals commonly develop bilateral nasal discharge that
becomes sufficiently profuse to impair suckling. There is pro-
gressive weight loss and intermittent pyrexia with coughing,
depression, and rough haircoat. Intractable respiratory disease
is the most common clinical sign, but diarrhea and swollen joints
are also observed. In spite of intensive therapy, affected foals
die at 3-5 months of age.
Hematologically, the animals are profoundly lymphopenic,
and it is suggested that counts of <1 × 109/L are diagnostic for
the disease. Lymphopenia is present from birth and is constant
throughout life. Because IgM is synthesized by the normal
equine fetus, absence of IgM from serum of foals that have not
suckled supports a diagnosis of SCID. In animals from 1-100
days of age, the serum IgM and IgA levels are low and remain
between 0.2 and 0.6 g/L. The IgG, which is maternally
derived, progressively drops from 8.0 g/L shortly after birth
to 2.0 g/L in foals that approach 100 days of age. There is
usually mild anemia that increases in severity with resistant
and repeated infections, but is often masked by dehydration.
Leukocyte counts are highly variable, being primarily depen-
dent on the numbers of neutrophils. Reduced lymphocyte
levels in blood are usually more than compensated by neutro-
philia, so that total leukocyte counts are usually in the normal
range.
The characteristic lesions in foals with SCID are bilateral
cranioventral bronchopneumonia in association with hypoplasia
of all lymphoid tissue, including thymus, lymph nodes, and spleen.
The thymus is identified only as a thin mediastinal raphe
cranial to the heart. The bone marrow is unusually reactive
for the age of the animal, and hematopoiesis persists in cancel-
lous bone and partially occupies the central femoral cavities.
Terminally, areas of marrow that appear red grossly may be
hypoplastic with dilated sinusoids. Histologically, the lesions
in the lung are typical of a suppurative bronchopneumonia,
but basophilic adenoviral inclusion bodies are usually present
in the epithelial nuclei of bronchi and bronchioles as well as
other organs. The primary SCID lesion is marked lymphoid
hypoplasia of all lymphoid organs. The spleen has no lymphoid
follicles, the small arterioles are devoid of lymphocytic cuffs,
and there are no plasma cells. The lack of connective tissue
stroma in follicular sites helps to differentiate this hypoplastic
lesion from atrophy. Some spleens have increased extramedul-
lary hematopoiesis. The lymph nodes consist of a stromal
framework that is essentially an unoccupied superstructure.
The capsule is delicate, with the peripheral sinus often heavily
colonized by macrophages and granulocytes. Some lymph
Lymphoid Organs
nodes, particularly from the mediastinal area, may have corti-
cal microabscesses. Follicles are absent, and the cortex consists
of a reticular framework in which there are small foci of lym-
phocytes around small arterioles. Postcapillary venules have
high vesicular endothelium and are usually well demonstrated
because of the lack of perivascular and intramural lympho-
cytes. Medullary cords are collapsed, and medullary sinuses
are dilated and contain neutrophils and macrophages. If
thymus can be identified, it consists of small lobules of 1-2 mm
diameter surrounded by normal fat. The lobules have normal
stromal capsulation and consist almost entirely of medullary
structures with central Hassall’s corpuscles and a vesicular
reticular network with a very low lymphocyte population. The
presence of the Hassall’s corpuscles is worthy of note because,
besides identifying the tissue as thymus, their presence also
indicates that there has been successful embryologic migration
of the epithelial duct system. In human infants, the corpuscles
are present only in SCID patients with an enzyme deficiency
of adenosine deaminase (ADA), whereas in X-linked SCID
the thymus only contains lobules of undifferentiated epithelial
cells resembling fetal thymus.
An X-linked SCID has been reported in Basset Hounds and
Cardigan Welsh Corgi dogs that is characterized by defects in
the gene encoding the gamma chain that is common to the
receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and
IL-21. Affected dogs therefore cannot complete the signal
transduction pathways of any of these interleukins. Different
mutations in the gamma chain of the IL-2 receptor are also
the most common mutations encountered in SCIDs in
humans. Similarly, different mutations have been detected in
Basset Hounds (4 base-pair deletion in exon 1) and Cardigan
Welsh Corgi dogs (single base-pair insertion in exon 4), and
the location of the mutation within target genes influences
the spectrum of diseases observed in affected animals. Because
the mechanisms of gene rearrangement are not impaired,
affected dogs have lymphocytes that express mature T- and
B-cell markers. Therefore this variant of SCID is characterized
by only moderate lymphopenia, with normal percentages of
circulating B lymphocytes and low to normal percentages of
mature, but nonfunctional, T cells. There is a decrease in cir-
culating CD8-positive cells, and the ratio of CD4 to CD8 cells
is ~15 : 1 compared to 2 : 1 in normal dogs. Affected dogs also
have normal serum level concentrations of IgM. In contrast,
serum IgG and IgA concentrations are significantly reduced or
not detectable, respectively. Only antibodies of the IgM class
are produced, which is consistent with an inability to class-
switch to IgG and IgA. Affected puppies fail to produce anti-
bodies in response to immune stimuli, including vaccination
and are highly susceptible to bacterial and viral infections.
They rarely live more than 3-4 months. As with SCID in
horses, lymphoid organs are severely hypoplastic, and tonsil,
thymus, Peyer’s patches, or lymph nodes are often grossly
undetectable. The histologic changes are similar to those
described in horses. The small lobules of the thymus have few
to no lymphocytes, and there is no corticomedullary demarca-
tion. The ratio of CD8 to CD4 lymphocytes has markedly
shifted toward CD8-positive cells. Similar to human X-linked
SCID, Hassall’s corpuscles are not detectable.
An autosomal recessive SCID as a result of a mutation in
DNA-PKcs has also been described in Jack Russell Terriers
(Fig. 2-29). Both the mechanisms and the pathology, including
adenoviral hepatitis, are similar to reports in Arabian foals. A
single litter of Rottweiler puppies was suspected to have SCID
 Thymus
A median survival times of 7 months. This syndrome is similar
B Don-van’t Slot HP, van der Kolk JH. Severe combined immunodeficiency
Figure 2-29  Severe combined immunodeficiency (SCID) in a
dog. A. Thymic hypoplasia characterized by small lobules that
have few to no lymphocytes and no corticomedullary demarca-
tion. B. Immunohistochemistry for CD3 highlights the loss of T
cells.
based on few T cells and few follicles with B cells in lymphoid
tissues and abnormally low IgA levels. The mechanisms of this
syndrome were not investigated.
T-cell immunodeficiency.  Rare T-cell immunodeficiencies
have been reported in dogs and cattle. In Weimaraners, a
growth hormone deficiency has been reported that was associ-
ated with constant severe bacterial infections and poor growth
rates. Affected puppies not only had decreased levels of
growth hormones, but also low T-cell counts. The thymus was
small, and there was loss of corticomedullary delineation
because of the absence of T cells. Affected puppies had normal
leukocyte counts and gammaglobulins and were able to
produce antibody in response to bacterial challenges. The
underlying mechanisms are unknown. Treatment with bovine
growth hormone was beneficial. In humans, mutations in the
STAT5b gene have been described causing a combined phe-
notype of growth hormone insensitivity and immunodefi-
ciency because of T-cell lymphopenia. Clinical findings
included congenital growth hormone deficiency that mani-
fested as persistently low growth rate, severely delayed bone
age, and postnatal growth failure as well as recurrent infec-
tions of the skin, severe chronic lung disease, and multiple
141
episodes of herpetic keratitis because of T-cell immunodefi-
ciency. Growth hormone receptors and IL-2 receptors share
STAT5 proteins for their signaling pathways, which would
explain the dual deficiency. Whether similar mutations exist
in Weimaraners is unknown.
In Bull Terrier dogs, an autosomal recessive acrodermatitis has
been associated with an ultimately fatal syndrome character-
ized by growth retardation, progressive acrodermatitis, chronic
pyoderma, diarrhea, pneumonia, and abnormal behavior.
Affected puppies had decreased plasma zinc concentrations
and a reduced lymphocyte response to antigenic stimulation.
Clinically, they had neutrophilia, parakeratosis, hyperkeratosis,
superficial bacterial infections of the skin, bronchopneumonia,
and dilation of the cerebral ventricles. There was a severe
decrease of T cells in all lymphoid organs. Affected dogs had
to acrodermatitis enteropathica in humans that is associated
with thymic atrophy and a selective decline of CD4-positive
T helper cells.
A similar autosomal recessive disease, called lethal trait
A46, has been described in Friesian and Shorthorn cattle. The
disease is characterized by a reduced capacity to absorb zinc
through the intestinal tract, causing anorexia, diarrhea, alope-
cia, and hyperkeratosis. Furthermore, affected cattle have a
reduced lymphocyte response to antigenic stimulation,
reduced levels of CD4-positive T cells, and a relative decrease
of B cells.
Further reading
Bell TG, et al. Autosomal recessive severe combined immunodeficiency
of Jack Russell terriers. J Vet Diagn Invest 2002;14:194-204.
disease (SCID) in the Arabian horse. Tidschr Diergeneeskd
2000;125:577-581.
Flaminio MJ, et al. Common variable immunodeficiency in horses is
characterized by B cell depletion in primary and secondary lymphoid
tissues. J Clin Immunol 2009;29:107-116.
Perryman LE. Molecular pathology of severe combined immunodefi-
ciency in mice, horses, and dogs. Vet Pathol 2004;41:95-100.
Tallmadge RL, et al. Expression of essential B cell development genes
in horses with common variable immunodeficiency. Mol Immunol
2012;51:169-176.
Verfuurden B, et al. Severe combined immunodeficiency in Frisian
Water Dogs caused by a RAG1 mutation. Genes Immun
2011;12:310-313.
THYMUS
Structure and function of the normal thymus
The thymus is a composite organ of epithelial and lymphoid
tissues, both of which have distinct developmental origins. The
thymic anlage develops from the third paired branchial (pha-
ryngeal) pouch of the foregut endoderm. Endodermal diver-
ticula arise bilaterally from the middle series of the branchial
pouches. Following outgrowths of the epithelial component to
the cranial mediastinal area, the ducts connecting the diver-
ticula to the pharynx soon disappear, setting the thymus anlage
free. While extending laterally and backward, the diverticula
grow into the surrounding mesoderm and neural crest–derived
mesenchyme of the branchial arch and give rise to the capsule,
trabeculae, and blood vessels. The diverticula lose their lumina,
and their lower ends enlarge and migrate caudally toward the
 141.e1

Further reading
Crisman MV, Scarratt WK. Immunodeficiency in horses. Vet Clin North
Am Equine Pract 2008;24:299-310.
Felsburg PJ, et al. Canine X-linked severe combined immunodeficiency.
Vet Immunol Immunopathol 1999;69:127-135.
Fox-Clipsham LY, et al. Population screening of endangered horse
breeds for the foal immunodeficiency syndrome mutation. Vet Rec
2011;169:655.
Hutchison JM, et al. Immunodeficiency syndrome associated with
wasting and opportunistic infection in juvenile llamas: 12 cases
(1988-1990). J Am Vet Med Assoc 1992;201:1070-1076.
Meek K, et al. SCID dogs: similar transplant potential but distinct intra-
uterine growth defects and premature replicative senescence with
SCID mice. J Immunol 2009;183:2529-2536.
Moroff SD, et al. IgA deficiency in Shar-Pei dogs. Vet Immunol Immu-
nopathol 1986;13:181-188.
Nolte T, et al. Oxazolone and ethanol induce colitis in non-obese
diabetic-severe combined immunodeficiency interleukin-2R (null)
mice engrafted with human peripheral blood mononuclear cells.
Clin Exp Immunol 2013;172:349-362.
Roth JA, et al. Thymic abnormalities and growth hormone deficiency
in dogs. Am J Vet Res 1980;41:1256-1262.
Tallmadge RL, et al. Fell Pony syndrome: characterization of develop-
mental hematopoiesis failure and associated gene expression pro-
files. Clin Vaccine Immunol 2012;19:1054-1064.
Taylor SD, et al. Protective effects of broadly neutralizing immuno-
globulin against homologous and heterologous equine infectious
anemia virus in horses with severe combined immunodeficiency.
J Virol 2011;85:6814-6818.
142 CHAPTER 2  •  Hematopoietic System
thorax and become the thoracic lobes. The upper ends either
atrophy or persist as cervical lobes. In ruminants and pigs, both
large cervical lobes and thoracic lobes exist, whereas the cervi-
cal lobes vary in size in cats, are small in horses, and absent in
dogs. The thoracic lobe develops in all domestic animals in the
ventral portion of the cranial mediastinum, except in rumi-
nants, where it can be found dorsally.
There are 2 streams of epithelial migration, with that from
the uppermost part of the pharyngeal pouch forming the
thymic duct epithelium and later the Hassall’s (thymic) cor-
puscles, and that from the lower part of the pouch forming
the reticular epithelial component of the cortex and medulla of
the adult thymic lobule. Embryologically, the reticular epithe-
lial component first invades the mediastinal interstitium with
irregular, solid, rosette-like buds that enlarge to form the pri-
mordial lobules. This reticular epithelium forms loose cuffs
around small vessels that persist in adult life and become
obvious following lymphoid atrophy. The second component
migrates as a system of branching ducts that ramify in the
interlobular stroma and penetrate the lobules to form a central
cord that broadly communicates between the lobules of a
single lobe. In early development, this tubular system has a
basement membrane and layered epithelium that becomes
solid and cystic and loses the outer membrane in the mature
thymic medullary corpuscle. In terms of induction, it appears
that these medullary ducts are essential to colonization of the
thymic anlage by thymocytes (T lymphocytes at different
stages of development), and are the source of trophic thymic
hormones. The colonizing lymphocytes are derived from
Thymic cortical
epithelial cell Trabeculae
Thymocyte Capsule
Subcapsular
epithelium
Cortex
Medulla
Macrophage Hassall’s
Thymic medullary
(bone marrow origin) corpuscle
epithelial cell
Figure 2-30  Normal thymus in a dog. The illustration on the left depicts the thymic structure
and cell populations; the image on the right parallels the microscopic appearance of these
structures.
Thymus
blood islands of the primordial yolk sac. A further cell type,
which resembles striated muscle fibers, is of indefinite origin, but
has importance in the pathogenesis of myasthenia gravis.
These relationships are pertinent to the histologic interpreta-
tion of the thymus in pathologic states; as in dysplasia, atrophy,
regeneration, and hyperplasia, these components recapitulate
their embryologic relationships.
In most species, the thymus reaches maximal development
at about the time of puberty. It can become hyperplastic,
and in calves given repeated injections of endotoxin can
extend from the rami of the mandibles to the base of the
heart. Normally, the thymus regresses in adult life, and this
regression may be accelerated during severe or chronic illness,
but it never entirely disappears. Even a very small organ
weighing <10 g can be immunologically potent in autoim-
mune dysfunction.
The thymus differs from the spleen and lymph nodes in a
number of important aspects: There are no lymphoid follicles
and B lymphocytes in the normal thymus; there are no affer-
ent lymphatics, and the cortex is composed of small, densely
packed T-cell precursors and T lymphocytes at various stages
of proliferation and differentiation as well as apoptotic cells;
the medulla does not contain sinusoids, but instead represents
a mesenchymal-endothelial reticular network that contains
Hassall’s corpuscles and large numbers of lymphocytes (Fig.
2-30, eFig. 2-2). The thymic epithelial cells are best high-
lighted by immunohistochemistry for cytokeratins; pancyto-
keratin antibodies MNF116 or AE1/AE3 are most commonly
used to detect the different subpopulations of epithelial cells
Corticomedullary
junction
 142.e1

eFigure 2-2  Normal thymus in a cat.

 Thymus
and to visualize their multiple delicate interconnecting cyto-
plasmic processes. The lymphoid component is best character-
ized immunohistochemically by CD3. Cortical thymocytes
(immature T lymphocytes) will have cytoplasmic expression,
whereas medullary thymocytes are usually positive for both
membranous and cytoplasmic CD3. CD1a is only available
for frozen sections, and antibodies against terminal deoxynu-
cleotidyl transferase (TdT) or CD99 have only been estab-
lished in humans for cortical thymocytes. There is also an
appreciable number of CD20-positive B lymphocytes in the
medulla that may be the cells from which mediastinal large
B-cell lymphomas arise. These cells commonly synthesize IgG,
IgM, and IgD.
Thymic cortical lymphocytes are small cells with nuclei
slightly larger than erythrocytes, little internal nuclear detail,
and minimal cytoplasm. Despite their small size and lack of
nucleoli, there is intense lymphopoiesis in the peripheral
thymic cortex and continual migration of cells to the periph-
ery from this area. Most (99%) of this proliferation is ineffec-
tive and balanced by a high rate of cell death, apparently in a
process of immunologic selection. A complete turnover of
cells is believed to occur within 3-4 days. In normal states,
cortical lymphocytes are closely packed so that the cortex
appears uniformly dark histologically. In conditions of stress
and atrophy, cortical epithelial cells and phagocytes become
prominent, and these larger cells with ingested nuclear debris
give the cortex a moth-eaten appearance.
Thymic medullary lymphocytes are larger than their corti-
cal precursors and have a more vesicular nucleus with small
nucleoli and more cytoplasm. They appear less tightly packed
than the cortical cells and, with the higher density of epithelial
cells and the greater variability in nuclear size and shape, cause
the medullary areas to appear less dense histologically, giving
the thymus its characteristic corticomedullary differentiation. In
many disease states where the animal is faring poorly, this
corticomedullary boundary is indistinct and blurred by dilu-
tion of the cortical cells and an increase in macrophages in
both regions. In normal states, the squamous ductal epithe-
lium is obvious in central medullary areas, but the reticular
epithelium of the cortex and medulla is not apparent in the
background of cells and only becomes apparent following
atrophy of the lymphocyte population.
Physiologically, prothymocytes from bone marrow colonize the
outer cortex of the thymus, where they proliferate and are selected
and trained by the cortical epithelium. In this region, the epi-
thelial cells form large membrane-lined cavities called caveo-
lae in which the marrow-derived lymphocytes divide and
undergo phenotypic development. On release from the
marrow, prothymocytes have the ability to home selectively
to the thymus, but they lack helper, suppressor, or killer func-
tions. In contrast, peripheral T lymphocytes are characterized
phenotypically by CD4 (cluster derived, helper) or CD8 (sup-
pressor) expression, functionally by the major histocompati-
bility complex (MHC) determinants they recognize, and by
their collective inability to react against normal self-antigens.
The naive T cells arriving in the thymus from the bone marrow
express CD34 and CD7 and are pluripotent in their ability to
become types of T cells as well as dendritic or natural killer
(NK) cells. They undergo a sequential pattern of antigen
expression as a function of orderly gene rearrangements
encoding the T-cell receptor (TCR), which requires the activ-
ity of the recombinase genes (RAG-1 and RAG-2). The earli-
est T-cell development occurs in the outer cortex with the
143
expression of CD2 and CD44, and such cells are termed
“triple-negative” as they do not yet express CD3, CD4, or
CD8. Most T cells develop a TCR heterodimer composed of
α and β chains, and a much smaller proportion develop the
γδ TCR configuration. This rearrangement process brings vari-
able (V), diversity (D), and joining (J) gene segments together.
The numerous combinations among the V, D, and J segments,
together with non–template-encoded nucleotides (N) by ter-
minal deoxynucleotidyl transferase, for instance, V-N-D-N-J,
create the large repertoire of T-cell types within the αβ and
γδ T-cell lineages. Although intrathymic dendritic cells were
originally thought to derive from progenitors with lymphoid
potential, it has recently been shown that thymic dendritic
cells are also derived from myeloid-restricted progenitors.
Besides becoming CD3-positive with this level of maturation,
the young thymocytes expressing the αβ form of TCR also
express CD4 and CD8 molecules and are known as double-
positive cells during the time they express both molecules.
Those cells that recognize antigen in association with class II
MHC gain additional CD4 expression and lose CD8. Simi-
larly, those cells that recognize antigen in association with class
I MHC persist as CD8-positive cells. A much smaller popula-
tion of γδ T cells form a cytotoxic T-cell population as mature
cells. Thymocytes that do not pass the applied selection crite-
ria usually die through apoptosis.
Prothymocyte development is believed to occur within the
caveolae of the large epithelial “nurse cells.” Nurse cells tend to
express either MHC II, primarily in the cells of the outer
cortex and dendritic cells of the inner cortex, or, in association
with the more flattened reticular epithelium of the medulla,
expressing both type I and II determinants. This close apposi-
tion of epithelium and young lymphocytes permits the trans-
mission of inductive signals for proliferation and development,
or for death of the great majority of the cells produced. The
thymic nurse cells themselves produce interleukin-1 (IL-1),
which drives lymphoproliferation, and in the course of matu-
ration, the T cells acquire receptor and production capability
for IL-2, as well as other reactive lymphokines.
The progeny of this system are the recognition and regulation
specialists of the adult immune system. They identify antigens
presented to them by other cells as having either the MHC I
or II determinants, and determine the presence or absence
of foreign antigen, which is handled by killing or by stimula-
tion of antibody development. T-cell development involves
the rearrangement of the T-cell receptor genes analogous
to the same process by which the B cells gain antibody diver-
sity. Whereas B cells recognize antigen on the basis of molecular
shape, T-cell recognition is on the basis of peptides. Because
antigen is presented to the T cell by antigen-processing
phages and dendritic cells, denaturation of antigen is of no
consequence because it is the amino-acid sequence that is
detected as foreign. In nonhematopoietic cells, internal antigen
is displayed within a groove in the MHC I molecule on cell
membranes so that viral infection or tumor antigen are avail-
able for recognition by T lymphocytes. This process of cell
recognition is accomplished by intracellular transporter pumps
that display all of the proteins (self or foreign) produced
within the cell on the cell surface. The diversity of T-cell rec-
ognition appears to be a function of receptor diversity and
permits survival of cells with acceptable recognition of self
to avoid “holes” in antigen detection. Thus most NK cells
recognize targets without MHC restriction, whereas the
CD8+ cytotoxic cells with appropriate antibody mediate
144 CHAPTER 2  •  Hematopoietic System
antibody-dependent cytotoxicity (ADCC). Cytotoxic T cells
and NK cells can induce apoptosis in adjacent cells by releasing
cytotoxic granules called perforins or granzymes, both of which
act in a Ca2+-dependent manner.
Cells exiting the thymus have not only managerial recep-
tors but also “homing” receptors to guide them to appropriate
areas in the peripheral nodes and Peyer’s patches. Only 1-3%
of thymocytes bear homing receptors, all of which are in the
cortex and all of which are phenotypically mature. Thus it
appears that the homing direction is added last, with most of
the cortical cells homed to nodes and likely medullary cells homed
to Peyer’s patches.
It appears that the thymic epithelium, besides making
trophic hormone for the immune system, also makes the pep-
tides oxytocin and vasopressin in common with the pituitary,
thus indicating a mechanism for the interaction of the nervous,
endocrine, and immune systems.
In terms of ontogeny, lymphocytes first appear in the
thymus of the fetal lamb at day 43, nodes at day 45, spleen
at day 54, and Peyer’s patches at day 65. These relationships
are altered by fetal infection (see Vol. 3, Female genital
system). In the dog, the thymic epithelial anlage is present by
a gestational age of 23 days, lymphopoiesis begins by day 33,
and corticomedullary differentiation with formation of Has-
sall’s corpuscles is first evident at ~38 days.
Further reading
Cejalvo T, et al. Ephrin-B-dependent thymic epithelial cell-thymocyte
interactions are necessary for correct differentiation and thymus
histology organization: relevance for thymic cortex development. J
Immunol 2013;190:2670-2681.
Cheng G, et al. IL-2R signaling is essential for functional maturation of
regulatory T-cells during thymic development. J Immunol
2013;190:1567-1575.
Coquet JM, et al. Thymic epithelial cells use macroautophagy to turn
inside out for CD4 T cell tolerance. J Exp Med 2013;210:
715-728.
Farley AM, et al. Dynamics of thymus organization and colonization in
early human development. Development 2013;140:2015-2026.
Developmental diseases of the thymus
Thymic hypoplasia is commonly observed in a number of
immunodeficiencies that affect T cells. Nude mice have a
specific defect that results in developmental arrest of the
thymus and a defective cell-mediated immune response. In
humans, a condition called DiGeorge syndrome is caused by an
embryologic development defect of the third and fourth pha-
ryngeal pouches. The defect is caused by a deletion mutation
of a gene located on chromosome 22q11 and results in low
number of T cells in blood and lymphoid organs. Affected
individuals are predisposed to viral and fungal infections.
Although rare forms of right persistent aortic arch have been
associated with mutations in the DiGeorge syndrome critical
region of the canine chromosome 26, there have been no
reports of thymic hypoplasia caused by a DiGeorge-like syn-
drome in domestic animals.
In domestic animals, thymic hypoplasia most commonly
occurs as part of severe combined immunodeficiencies in foals or
certain breeds of dogs. In affected animals, the thymus is small
and consists of 1-2 mm diameter lobules that are almost
entirely depleted of lymphocytes, and thymic cortex is
Thymus
replaced by adipocytes. The only recognizable structures are
the thymic medulla and central Hassall’s corpuscles. In
X-linked SCID, Hassall’s corpuscles are not detectable.
A case of hypotrichosis and concurrent thymic hypoplasia
has been reported in a single litter of Birman kittens that all
died within the first 13 weeks of life. The condition is believed
to be autosomal recessive inherited.
Further reading
Casal ML, et al. Congenital hypotrichosis with thymic aplasia in nine
Birman kittens. J Am Anim Hosp Assoc 1994;30:600-602.
Philipp U, et al. A rare form of persistent right aorta arch in linkage
disequilibrium with the DiGeorge critical region on CFA26 in
German Pinschers. J Hered 2011;102(Suppl. 1):S68-S73.
Thymic involution and atrophy
The reduction of the size of the thymus is caused by alteration
of its cellular density and/or cellular composition. A physio-
logic, age-associated decrease of cellularity is called thymic
involution, whereas induced shrinkage of thymic lobules
caused by inadequate nutrition, intoxications, infectious
agents, lack of antigenic stimuli, medical therapy, and so on,
is referred to as thymic atrophy. Thymic involution is part of
the normal process of ageing that is gradual and irreversible. It
normally begins at sexual maturity and may be slowed by
dietary restriction or gonadectomy or can be accelerated by
toxic insult. The morphologic change of thymic involution
reflects its functional change from lymphocyte production to
recirculation.
Atrophy of the thymus is commonly observed in cachectic
animals and has been associated with vitamin B6, fatty acid,
or zinc deficiencies, all of which cause immunosuppression.
The recognition that a number of environmental and dietary
contaminants may accelerate immune deterioration has
resulted in the development of testing protocols specifically
designed to assess adverse effects of this nature. In a general
sense, thymic atrophy can be used as an index of disease severity
and duration in young animals, in which the organ would
normally be much larger.
The thymus is the most sensitive of the lymphoid tissues
to fluctuations in sex hormone levels, and changes in adreno-
cortical hormone levels or growth hormone levels. Elevated
levels of glucocorticosteroids as induced by stress can result in
significantly reduced thymic weight. Apoptosis of cortical thymic
T cells with secondary digestion by macrophages occurs
within hours of treatment with corticosteroids. However,
thymic atrophy secondary to stress-induced glucocorticoste-
roid levels is reversible upon removal of the stressor.
A further form of thymic atrophy occurs as a result of
exposure to a variety of environmental contaminants, with the
most serious effects being those caused by exposure to various
forms of dioxin and various congeners of the polychlorinated
and polybrominated biphenyls (PCB, PBB). Heavy metal ions
such as lead or mercury have direct effects on cell membranes,
organelles, or cell enzymes, causing apoptosis of cortical thy-
mocytes. Mycotoxins such as fumonisins B1 and B2 produced
by fungi of the genus Fusarium or aflatoxins can cause severe
lymphocytolysis of the thymic cortex.
The thymus is also highly sensitive to a wide variety of
immunotoxicants. Many physical, chemical, or other agents,
including anticancer drugs or radiation treatment, can cause
 144.e1

Further reading
Ardavin C, et al. Thymic dendritic cells and T cells develop simultane-
ously in the thymus from a common precursor population. Nature
1993;362:761-763.
Cowan JE, et al. the thymic medulla is required for Foxp3+ regulatory
but not conventional CD4+ thymocyte development. J Exp Med
2013;210:675-681.
LeFrancois L, Puddington L. The role of the thymus in intestinal intraepi-
thelial T-cell development. Ann NY Acad Sci 1996;778:36-46.
Pezzano M, et al. Questionable thymic nurse cell. Microbiol Mol Biol
Rev 2001;65:390-403.
Suster S, Rosai J. Histology of the normal thymus. Am J Surg Pathol
1990;14:284-303.
Wilson CB. The ontogeny of T lymphocyte maturation and function.
J Pediatr 1991;118:S4-S9.
 Thymus
Figure 2-31  Thymic atrophy in a dog. Infection with canine
distemper virus caused multilobular acute hemorrhage. Histologi-
cally, there was marked depletion of lymphocytes.
immune dysfunction resulting in immunosuppression evident
in marked thymic atrophy. Because the generation of T cells
in the thymus is especially important in the early stages of life,
immune system toxicants often have profound effects in even
low doses during prenatal and postnatal periods. Immunotox-
icity most commonly manifest as immunosuppression that
causes T-cell apoptosis and manifests as a decrease in relative
thymic weight in young animals. Very high doses of immuno-
toxicants can also cause degeneration of epithelial cells and
proliferation of glandular epithelial structures.
Many viruses, including canine parvovirus (CPV-2) and
canine distemper virus (CDV) in dogs (Fig. 2-31), feline pan-
leukopenia virus (FPV), feline leukemia virus (FeLV) and
feline immunodeficiency virus (FIV) in cats, equid herpesvirus
(EHV-1) in horses, classical swine fever virus (CSF) and
porcine reproductive and respiratory syndrome virus (PRRSV)
in pigs, and bovine viral diarrhea virus (BVDV) in cattle can
cause severe thymic atrophy.
Cats that succumb to feline parvoviral infection (panleuko-
penia) have marked thymic atrophy to the extent that the
organ may not be identifiable grossly, and can only be exam-
ined histologically by systematically sampling the cranial
mediastinal tissues. Microscopically, the thymus is character-
ized by collapsed lobules with angular facets to the lobes that
become flattened and triangular. The changes are uniform in
each lobule and consist of complete thymic atrophy with only
a very narrow rim of small lymphocytes 1-2 cells thick beneath
the capsule. Hassall’s corpuscles are prominent, and the
lobules in effect consist of a condensation of medullary tissue
and a prominent epithelial/stromal background that contains
a few large lymphoblasts (Fig. 2-32). Small lymphocytes
typical of cortical cells are virtually absent from the medullary
areas.
Cats infected with FeLV suffer thymic cortical atrophy of
variable degree, and a reduction in functional capability of
neutrophils that may persist for a year after viremia is cleared.
The p15E envelope protein appears to be the major factor in
impaired T-cell function, apparently by inhibition of IL-2 pro-
duction and responses. Interferon-γ production is inhibited,
and NK-cell cytolytic function is impaired, but binding to
target cells is unaffected. Circulating immune complexes con-
145
Figure 2-32  Thymic atrophy in a cat. Infection with feline pan-
leukopenia virus caused lobular collapse, and only a narrow rim
of lymphocytes remains beneath the capsule. Hassall’s corpuscles
are prominent, and there is condensation of medullary tissue.
tribute to the impairment of defenses, and some improvement
in condition can be achieved by their removal. There is an
age-related response to infection, with cats infected at <8
weeks of age surviving ~3 months, and cats 4 months of age
at infection surviving about a year. There is an early stage of
lymphoid hyperplasia accompanied by weight loss, and fol-
lowed by lymphoid depletion, lymphopenia, reduced lympho-
cyte response to lectins, hypogammaglobulinemia, ulcerative
stomatitis, diarrhea with necrosis of crypt epithelium, and
death. Thymic atrophy occurs in all cats that die, and termi-
nally there is marrow hypoplasia.
The lesions associated with FIV are largely those of immune
activation and secondary invaders, and the most specific
changes are in tests of immune cell number and function.
Neonatal cats infected with FIV will develop chronic thymic
atrophy and degeneration that is associated with decreasing
productive viral infection and low-level viremia. However,
fetal cats infected with FIV develop acute thymic atrophy at
birth associated with peak viremia. The thymus will partially
regenerate ~6 weeks postpartum because of decrease of pro-
ductive infection, and thymic size and architecture, including
cell numbers, will normalize. Infected cats maintained in a
clean environment survive indefinitely, but have reduced
numbers of CD4 lymphocytes and reversal of the CD4/CD8
ratio, suggesting specific infection of the helper cell popula-
tion. Lymphocyte response to lectins is reduced, and there is
reduced IL-2 production by cells from infected cats. The
disease process is likely accelerated when FeLV and FIV
coexist, which occurs in ~1% of sick cats. Both viruses may
be present in cats with lymphoma.
In foals aborted because of equid herpesvirus 1 infection, the
thymus is grossly normal or of reduced size with edema. There
is lack of distinction between cortical and medullary areas
given the loss of cortical cells. Cytologically, there is extensive
lymphocytolysis in the subcapsular areas, with pyknotic
nuclear debris persisting in these areas. The necrotic lesions
tend to be patchy, but there is an overall reduction in cell
density, with edema of the lobules and separation of lympho-
cytes by clear fluid (eFig. 2-3). Hassall’s corpuscles may be
surrounded by relatively clear spaces, and viral inclusion
 145.e1

eFigure 2-3  Thymic necrosis in a horse. Focally extensive necro-

sis caused by equine herpesvirus.
146 CHAPTER 2  •  Hematopoietic System Thymus

A
Figure 2-33  Thymic necrosis in a horse. Equine herpesvirus
caused extensive necrosis of lymphocytes. Pyknotic nuclear debris
persists in the multiple necrotic foci.

bodies are often present in medullary epithelial cells. There is,

in addition, a mild increase in monocytes and granulocytes in
the interlobular connective tissue. The presence of nuclear
debris in the outer cortices indicates acute lympholysis (Fig.
2-33). Foals that survive 1-2 days may remove the cellular
debris, and the cortices are then characterized by hypocellu-
larity alone.
Regardless of the mechanism, the end result of thymic involu-
tion or atrophy is loss of cortical lymphocytes and lobular shrink-
age, and the histologic changes are similar for both pathways.
Microscopically, there is apoptosis of cortical thymocytes that B
leads to cortical T-cell depletion and thinning of the thymic
cortex (Fig. 2-34). The process is accompanied by an increase
of tingible body macrophages. Perivascular spaces at the cor-
ticomedullary junction become prominent, and there is accu-
mulation of perivascular B cells and plasma cells that may
form lymphoid follicles with prominent germinal centers.
During the later stages of either disease process, the interlobu-
lar septa become more prominent, adipocytes infiltrate, and
ultimately the medulla has a higher cellular density than the
cortex. There is some degree of epithelial cell proliferation
occurring during that process that can lead to various degrees
of epithelial cell pleomorphism and architectural rearrange-
ment. Epithelial cords, tubules or even cysts may form during
that process.
It is basically impossible to differentiate thymic involution from
atrophy histologically, and the age of the animal and clinical C
presentation should be considered when making the diagnosis. It
is important to recognize that complete involution does not Figure 2-34  Thymic involution in a cat. A. Apoptosis of cortical
occur in any species, and remnants of thymic epithelial cells thymocytes leads to thinning of the thymic cortex, and there is
can usually be found embedded in some stroma in older loss of corticomedullary demarcation. Interlobular septa become
animals (Fig. 2-35). This may explain why thymomas can be more prominent. B. Immunohistochemistry (IHC) for CD3 high-
primarily found in dogs or cats older than 10 years. lights the loss of T cells. C. There is some degree of epithelial cell
Rarely, dystrophic mineralization secondary to renal or para- proliferation and architectural rearrangement that can be appreci-
thyroid disease and focal areas of fibrosis (Fig. 2-36) secondary ated with IHC for pancytokeratin.
to localized inflammation have been reported.

Further reading
Day MJ. Review of thymic pathology in 30 cats and 36 dogs. J Small
Anim Pract 1997;38:393-403.
 146.e1

Further reading
Chu I, et al. Subchronic toxicity of 3,3′,4,4′,5-pentachlorobiphenyl in
the rat. I. Clinical, biochemical, hematological and histopathological
changes. Fundam Appl Toxicol 1994;22:457-468.
Keenan KP, et al. The effects of overfeeding and moderate dietary
restriction on Sprague-Dawley rat survival, pathology, carcinogenic-
ity and the toxicity of pharmaceutical agents. Exp Toxic Pathol
1996;48:2-3.
Little S, et al. Feline leukemia virus and feline immunodeficiency virus
in Canada: recommendations for testing and management. Can
Vet J 2011;52:849-855.
National Toxicology Program. Mirex. Rep Carcinog 2011;12:273-275.
Orandle MS, et al. Selective thymocyte depletion and immunoglobulin
coating in the thymus of cats infected with feline immunodeficiency
virus. AIDS Res Human Retrovir 1997;13:611-620.
146.e2

A
eFigure 2-5  Thymic hemorrhage, in a dog. Extensive hemor-
rhage expands the thymic capsule and has largely replaced cortex
and medulla.

B
eFigure 2-4  Thymic hematoma in a dog. A. A well-circumscribed
single hematoma in the thymus. B. Idiopathic thymic hemorrhage
resulting in hemothorax in a dog. (Courtesy D.J. Patrick.)
 Thymus
Figure 2-35  Thymic remnant in a dog. Complete thymic involu-
tion does not occur, and thymic remnants can usually be found
embedded in some mediastinal stroma in older animals.
Figure 2-36  Thymic fibrosis in a dog. Focal areas of fibrosis occur
most commonly secondary to localized inflammation.
Kolenda-Roberts HM, et al. Immunopathogenesis of feline immunode-
ficiency virus infection in the fetal and neonatal cat. Front Biosci
2007;12:3668-3682.
Maranghi F, et al. Dietary exposure of juvenile female mice to polyha-
logenated seafood contaminants (HCBD,BDE-47,PCB-153, TCDD):
comparative assessment of effects in potential target tissues. Food
Chem Toxicol 2013;56:443-449.
Pathak S, Kundu R. Short-term PCB (Aroclor 1254) toxicity on few
phosphatases in mice brain. Dose Response 2013;11:1-8.
Pearse G. Histopathology of the thymus. Toxicol Pathol 2006;34:
515-547.
Thymic hemorrhages/hematoma
Severe thymic hemorrhage or thymic hematoma occurs most
commonly in young dogs that are presented with acute, severe
mediastinal hemorrhage, and they often die suddenly because
of hypovolemic shock. Affected dogs commonly exhibit
marked pallor in multiple organs consistent with anemia as a
result of massive blood loss into the thymus and thorax.
Grossly, the thymus and adjacent connective tissue are
147
Figure 2-37  Thymic hematoma in a dog. The thymus contains a
single, large, well-demarcated area of hemorrhage.
Figure 2-38  Idiopathic thymic hemorrhage in a dog. Extensive
hemorrhage expands the thymic capsule and interlobular septa,
and also extends into the thymic cortex and medulla.
extended by a large hematoma (Fig. 2-37), and severe hemor-
rhages are commonly found in the thoracic cavity causing
compressive atelectasis of the lungs (eFig. 2-4). Microscopi-
cally, there is extensive hemorrhage expanding the thymic
capsule and interlobular septa and also extending into the
thymic cortex and medulla and adjacent connective tissue
(Fig. 2-38, eFig. 2-5). Within hemorrhagic areas, there is a
mixture of fibrin, edema fluid, and inflammatory cells, pre-
dominantly neutrophils and macrophages. The affected
thymic parenchyma commonly undergoes lymphoid necrosis
(Fig. 2-39).
There is a wide range of causes for thymic hemorrhage,
including thoracic trauma, overstretching of the neck, rupture
of dissecting aortic aneurysms, bleeding thymic neoplasms, or
intoxication with anticoagulant rodenticides (Fig. 2-40). In
addition, a spontaneous idiopathic form has also been reported.
The suggested underlying cause for idiopathic thymic hemor-
rhages is thymic involution causing the thymus to become
fragile. Under these conditions, thin-walled thymic vessels
148 CHAPTER 2  •  Hematopoietic System
Figure 2-39  Idiopathic thymic hemorrhage in a dog. Hemor-
rhagic areas commonly contain foci of fibrin accumulation
admixed with variable numbers of inflammatory cells and necrotic
lymphocytes.
Figure 2-40  Thymic hematoma in a dog. Intoxication with anti-
coagulant rodenticides is one of the most common causes of
thymic hemorrhages/hematomas. (Courtesy K.G. Thompson.)
would lack support from the adjacent, lymphocyte-depleted
parenchyma and would be predisposed to hemorrhage in cases
of sudden increase of blood pressure or microtrauma. A single
case was reported in a puppy following transportation in an
unpressurized cargo chamber of an airplane during a multi-
hour flight. Idiopathic thymic hemorrhages are diagnosed in
young dogs following exclusion of other causes and morpho-
logic evidence of thymic involution, including depletion of
cortical lymphocytes and more pronounced Hassall’s corpus-
cles within an adipose tissue framework. Anticoagulant roden-
ticide poisoning must be excluded as the primary differential,
because it may cause localized fatal thymic hemorrhages
similar to those observed in the idiopathic form. However,
poisoned dogs may also have abnormalities in the intrinsic,
extrinsic, and common coagulation systems, including pro-
longed activated partial thromboplastin time, prolonged pro-
thrombin time, and prolonged activated clotting time. Stomach
content and liver should be collected to screen for anticoagu-
lants in cases of thymic hemorrhage.
Thymus
Further reading
Coolman BR, et al. Severe idiopathic thymic hemorrhage in two lit-
termate dogs. J Am Vet Med Assoc 1994;205:1152-1153.
Liggett AD, et al. Thymic hematoma in juvenile dogs associated with
anticoagulant rodenticide toxicosis. J Vet Diagn Invest 2002;
14:416-419.
Inflammatory diseases of the thymus
Inflammation of the thymus, thymitis, is a rather uncommon
lesion. Many infectious diseases, specifically viral diseases,
attack lymphoid cells, causing destruction of the lymphoid
component of the thymus. Viral infections most commonly
result in atrophy rather than an exudative or cellular inflam-
matory response, and atrophy can be the result of lysis of
lymphocytes or net emigration. Marked lymphocytolysis with
severe atrophy occurs in foals aborted because of infection
with equid herpesvirus 1, in cats with feline panleukopenia
virus, or in pigs with chronic classical swine fever virus infec-
tion, as discussed previously. In calves with epizootic bovine
abortion and dogs with salmon poisoning (rickettsiosis), there
is both lymphocytolysis and infiltration with inflammatory
cells. Bacteremia can cause thymitis, but suppurative inflam-
mation is uncommon, and abscesses have rarely been observed
(Fig. 2-41).
Epizootic bovine abortion, also known as foothill abortion,
is a tick-transmitted reproductive disease of pregnant cattle
that graze on foothill pastures in California and adjacent
states. The disease has recently been associated with a unique
16S deltaproteobacterial rDNA gene sequence that was detected
in 90% of thymus tissues from aborted fetuses. Intracytoplas-
mic bacterial rods were detected in thymuses of aborted
calves by using silver stains and immunohistochemistry by
using hyperimmune serum from infected cattle. Thymic
lesions develop late in the course of the disease and are char-
acterized by decreased size of thymic lobules. Histologically,
there is loss of cortical T cells and infiltration of the medullary
region with macrophages. The interlobular septa of the thymus
are distended with fibrin, hemorrhage, and inflammatory
cells, predominantly macrophages and fewer lymphocytes
(Fig. 2-42).
Neorickettsia helminthoeca (salmon poisoning disease) is
a North American rickettsial disease that occurs mainly in the
northwestern United States and western Canada and is trans-
mitted by the trematode Nanophyetus salmincola. The trema-
tode harbors the rickettsiae throughout its life-cycle stages.
The miracidia (ciliated larvae) develop from eggs in water and
enter snails (Oxytrema silicula) as the intermediate host. Free-
swimming larvae (cercariae) enter fish, the second intermedi-
ate host, mainly of the family Salmonidae. In salmon or trout,
metacercariae localize largely to the kidney, but can be found
in the eyes, liver, intestinal wall, musculature, and fins. When
an infected fish is ingested by the definitive host, adult trema-
todes attach and penetrate the intestinal mucosa of such fish-
eating mammals, including dogs. The flukes apparently cause
minimal damage or clinical signs to dogs, which shed fluke
eggs within about a week after ingestion of infected salmon
and continue to shed for ~8 months. The rickettsiae are inocu-
lated into the intestinal mucosa by the fluke via an unknown
mechanism. Dogs are apparently susceptible to the infection
at all ages, with the disease characterized by lethargy and
pyrexia with splenomegaly, generalized lymphadenopathy,
 148.e1

Further reading
Sheafor SE, Couto CG. Anticoagulant rodenticide toxicity in 21 dogs.
J Am Anim Hosp Assoc 1999;35:38-46.
van der Linde-Sipman JS, van Dijk JE. Hematomas in the thymus in
dogs. Vet Pathol 1987;24:59-61.
 Thymus
B Figure 2-42  Epizootic bovine abortion (foothill abortion) in a
Figure 2-41  Suppurative thymitis in a horse. A. Bacteremia can
cause suppurative thymitis, but grossly visible inflammation is
uncommon and abscesses are rarely observed. B. Histologically,
there is localized vasculitis with associated lymphocytolysis and
infiltration with inflammatory cells.
vomiting, and diarrhea. Following initial replication of the
rickettsiae in villar epithelial cells, the organisms are dissemi-
nated by histiocytic cells to the lymph nodes, spleen, tonsils,
thymus, liver, lungs, and brain. Dogs succumbing to infection
with salmon poisoning have marked enlargement of spleen
and lymph nodes with focal hemorrhages in the thymus
and pale often swollen liver with an exaggeration of the
lobular pattern visible through the capsule. In the thymus,
infection with N. helminthoeca causes depletion of lymphocytes,
foci of necrosis with neutrophilic infiltrates, and increased numbers
of histiocytes in the cortex and medulla (Fig. 2-43). Affected
lymph nodes are characterized by massive lympholysis and
loss of germinal centers and generalized cortical depletion of
cells. In animals that survive 10-12 days, there is prominent
node enlargement because of diffuse paracortical hyperplasia
with a prominent “starry-sky” appearance, but without the
development of germinal centers (eFig. 2-6). Splenic lesions
are characterized by follicular lympholysis in the acute phases
of the disease, as well as loss of cells in the periarteriolar
lymphoid sheaths. There is marked congestion of sinus areas
with numerous macrophages containing ingested debris as
well as the neorickettsial organisms. Hepatic lesions consist of
isolated foci of necrosis with macrophage reaction. The
149
calf. A. Thymic lesions develop late in the course of disease and
are characterized by decreased size of thymic lobules and pete-
chiae. B. Histologically, there is necrosis of cortical thymocytes,
and interlobular septa are distended with fibrin and inflammatory
cells. (Courtesy M.L. Anderson.)
gram-negative, 0.3-2.0 µm, pleomorphic organisms are located
exclusively within macrophages. They appear blue-purple with
blood stains, and blue with Macchiavello stain. At the time
dogs are showing acute signs of salmon poisoning, there are
usually large numbers of operculated fluke eggs in fecal
smears. Mortality rate is very high, and approaches 100% in
untreated animals. Organisms are present in virtually all
lymph nodes.
Salmon poisoning needs to be distinguished from a related
disease known as Elokomin fluke fever, named after the river
in Washington State where the disease was first recognized.
There are antigenic differences between the agent of salmon
poisoning and that of Elokomin fever (Neorickettsia elokomi-
nica), which were initially distinguished on the basis of the
specificities of the antibody reaction in surviving animals
applied in fluorescent antibody staining of the infectious
agents. The lesions in both diseases are similar.
Further reading
Anderson ML, et al. Histochemical and immunohistochemical evidence
of a bacterium associated with lesions of epizootic bovine abortion.
J Vet Diagn Invest 2006;18:76-80.
 149.e1

B
eFigure 2-6  Neorickettsia helminthoeca (salmon poisoning
disease) in a dog. A. N. helminthoeca causes increased numbers of
histiocytes in the cortex. B. Lower magnification of the lymph
node shows loss of lymphoid follicles and infiltrating histiocytes
in the cortex. (Courtesy R.J. Bildfell.)
150 CHAPTER 2  •  Hematopoietic System
A
B
Figure 2-43  Neorickettsia helminthoeca (salmon poisoning
disease) in a dog. A. N. helminthoeca causes cortical depletion of
lymphocytes and increased numbers of histiocytes in the cortex
and medulla. (Courtesy R.J. Bildfell.) B. The gram-negative,
0.3-2.0 µm, pleomorphic organisms are located exclusively within
macrophages and appear blue-purple with Wright stain. (Cour-
tesy J.L. Johns.)
Headley SA, et al. Neorickettsia helminthoeca and salmon poisoning
disease: a review. Vet J 2011;187:165-173.
Sykes JE, et al. Salmon poisoning disease in dogs: 29 cases. J Vet Intern
Med 2010;24:504-513.
Hyperplastic and neoplastic diseases
of the thymus
Thymic hyperplasia
Hyperplasia of the thymus is an often misleading term because
the size of the thymus varies widely and a larger thymus in a
young animal may simply represent physiologic variation.
Regardless, diffuse enlargement or large thymuses are com-
monly referred to as diffuse hyperplasia (Fig. 2-44). This
process may occur in any species, but is most commonly
observed in calves, rabbits, and birds that have been repeatedly
immunized. Grossly, the glands may fill the cranial mediasti-
num and extend up the neck in calves. The capsules remain
thin and delicate and the lobulation is distinct. The ratio of
cortical and medullary components is similar to that of a
Thymus
Figure 2-44  Diffuse thymic hyperplasia in a horse. Diffuse
enlargement or large thymuses are commonly referred to as
diffuse hyperplasia, but may simply represent a physiologic
variation.
Figure 2-45  Thymic follicular hyperplasia in a dog. Thymic fol-
licular hyperplasia indicates chronic inflammation or another
immunologic response, for instance, immune hemolytic anemia
or systemic lupus erythematosus.
normally sized thymus. Thymic enlargement is usually
observed as an incidental finding on autopsy, and no clinical
signs have been reported.
In most cases, thymic hyperplasia consists of lymphoid hyper-
plasia characterized by the development of B-cell germinal centers
and therefore referred to as follicular lymphoid hyperplasia. A
much less common form of lymphoid hyperplasia is called
focal lymphoid hyperplasia.
Follicular hyperplasia is the result of typical B-cell germi-
nal centers forming, usually at the corticomedullary junction
(Fig. 2-45). Follicular hyperplasia most commonly appears in
animals >6 months of age. The presence of hyperplastic lym-
phoid follicles in the thymus is independent of the size of the
actual thymus, and may be concurrent with thymic involution
or atrophy. In most cases, thymic follicular hyperplasia indi-
cates chronic inflammation or another immunologic response.
Follicular development has been reported in dogs with
 Thymus
immune hemolytic anemia and systemic lupus erythematosus,
but the frequency is not documented. Follicular hyperplasia
may also be found in association with thymoma, suggesting
some period of immune dysfunction preceding development
of the tumor. In humans, thymic follicular hyperplasia is
strongly associated with autoimmune disease, particularly
myasthenia gravis, and 75% of patients with myasthenia gravis
develop thymic follicular hyperplasia. Histologically, germinal
centers in the thymus may be surrounded by a thin layer of
epithelial cells and physiologically can be considered to be
outside of the thymus and do not indicate immune
dysfunction.
Focal hyperplasia is an unusual and incidental finding in
older animals, and is most often of the nonimmune type. It is
an appropriate term for lesions characterized by proliferation
of cortical lymphocytes in one or more lobules of the gland,
usually with compression of surrounding, often atrophic,
lobules, suggesting an attempt at regeneration. The lesions are
usually lightly encapsulated, often with some laminar infiltra-
tion of the perithymic tissues. Single epithelial cells are usually
scattered through a relatively diffuse area of lymphoid prolif-
eration. Focal hyperplasia of the thymic cortex is visible on
architectural or low-power examination by the presence of
increased numbers of large tingible body macrophages impart-
ing a “starry-sky” appearance, similar to that seen in a reactive
germinal center.
Epithelial hyperplasia is seen most frequently in the
thymus of rats, especially females, but has rarely been observed
in dogs. Treatment with diethylstilbestrol has caused similar
lesions in mice. In rats, the hyperplastic change is age associ-
ated and can be focal or diffuse, and is characterized by loosely
defined aggregates of cells arising at the corticomedullary
junction (Fig. 2-46). These aggregates are recognizable on
low-power examination by increased density because of a
greater proportion of small cells than the surrounding thymic
medulla. Epithelial cells are columnar to cuboidal and often
form cords or tubules. Tubular epithelial cells are commonly
ciliated and contain secretory material. Hassall’s corpuscles
appear not to be present within epithelial hyperplastic foci.
The significance of epithelial hyperplasia is unclear, but the
Figure 2-46  Thymic epithelial hyperplasia in a dog. Proliferating
epithelial cells are columnar to cuboidal and often form cords or
tubules.
151
lesion does not appear to undergo neoplastic transformation.
No clinical signs have been reported, and the lesion is primarily
important because of its resemblance to thymomas. In thymic
hyperplasia, there is always normal separation of cortex and
medulla, whereas thymomas have a diffuse architecture and
loss of corticomedullary delineation.
Pseudoepitheliomatous hyperplasia has rarely been
reported within thymic cysts in young dogs as a regenerative
response to thymic injury or inflammation. The hyperplastic
lesions are characterized by 1-8 layers of plump to slightly
attenuated polygonal squamous epithelial cells lining thymic
cysts. Scattered lymphocytes and Hassall’s corpuscles are com-
monly admixed within the squamous epithelial lining. The
squamous epithelial cells have indistinct cell borders; variable
amounts of wispy, pale eosinophilic cytoplasm; and vesicular
nuclei with peripheralized chromatin. Mitotic figures are
absent. The pleomorphic presentation of thymic pseudoepi-
theliomatous hyperplasia is similar to some variants of
thymoma; however, the association with thymic cysts and the
early onset of the lesion help distinguish the 2 entities.
Rarely, ectopic thymic tissue has been reported in some
animals.
Thymic cysts
Thymic cysts can occur in both the developing and mature
thymus, or persist during involution in the cranial mediasti-
num in all domestic species. Congenital thymic cysts are usually
thin walled and unilocular, whereas acquired thymic cysts are
often thick walled and multilocular. Cysts are lined by ciliated
columnar to cuboidal epithelial cells, indicating their origin
from the remnants of the branchial arch epithelium (eFig.
2-7). There are commonly histologic differences of the epi-
thelium within an individual cyst. Areas of ciliated epithelium
interchange with pseudostratified epithelium (Fig. 2-47).
Some epithelial cells may have elongated ovoid nuclei, whereas
others have narrower, indented nuclei. Although most cases
are incidental findings, if the cysts become large, they may
cause dyspnea. In other cases, thymic cysts may be formed
secondary to compression and distortion of the normal thymic
parenchyma by a neoplastic process.
Thymic neoplasms
Primary neoplasms of the thymus are uncommon. The most
commonly observed neoplasms include thymic epithelial
tumors (thymomas and thymic carcinomas) and thymic lym-
phomas. Rare neoplasms include thymic germ cell tumors.
The distinction between thymomas with benign cellular fea-
tures from thymic carcinomas is often straightforward;
however, accurate diagnosis and subclassification of thymomas
with benign cellular features can be difficult. Most thymic
epithelial neoplasms have a favorable long-term prognosis follow-
ing surgical removal, but a small subset will recur or metastasize.
Furthermore, like other thymic diseases, thymic epithelial
neoplasms increase the risk of having one or more autoim-
mune disorders, most notably myasthenia gravis. Thymomas
in the cat may be associated with a type of chronic exfoliative
dermatitis. The successful management of autoimmune disease
remains one of the biggest challenges, especially in dogs.
Unfortunately, there is little information available that would
allow predicting the likelihood of occurrence of paraneoplas-
tic syndromes, such as myasthenia gravis or erythema multi-
forme, based on the observed thymic epithelial neoplasm
subtype.
 151.e1

eFigure 2-7  Thymic cyst in a dog. The cyst is lined by ciliated

columnar and attenuated epithelial cells.
152 CHAPTER 2  •  Hematopoietic System Thymus

A B
Figure 2-47  Thymic cyst in a dog. A. Cysts are lined by ciliated columnar to cuboidal epithelial
cells indicating their origin from the remnants of the branchial arch epithelium. B. Immunohisto-
chemistry for pancytokeratin is useful for identifying the epithelial lining.

Table • 2-4 
The WHO classification of thymic epithelial neoplasms as applied for use in animals
Histologic subtype Histologic criteria

Type A thymoma Neoplastic thymic epithelial cells are spindle/oval shaped, lack nuclear atypia, and are accompanied
by few, if any, non-neoplastic lymphocytes.
Micronodular thymoma Neoplastic spindle-shaped epithelial cells form nodules that are separated by large aggregates of
predominantly B cells. Immunohistochemistry for pancytokeratin highlights the distinct separation
of the 2 cell components. This thymoma is probably a subtype of type A thymoma.
Type AB thymoma Neoplastic epithelial cells have cellular features of type A thymoma and are admixed with foci of
non-neoplastic lymphocytes. The segregation of such foci can be sharp or indistinct, and a wide
range exists in the relative amount of the 2 components.
Type B1 thymoma Neoplastic epithelial cells closely resemble the normal thymus with an appearance almost
indistinguishable from thymic cortex with some areas resembling thymic medulla.
Type B2 thymoma Neoplastic epithelial cells are plump with vesicular nuclei and distinct nucleoli and are scattered
among a heavy population of non-neoplastic lymphocytes. Perivascular spaces are common and
prominent.
Type B3 thymoma Neoplastic epithelial cells are round or polygonal and exhibit no or only mild atypia. They are
admixed with a minor component of non-neoplastic lymphocytes, which results in a sheet-like
growth of neoplastic epithelial cells.
Thymic carcinoma Neoplastic epithelial cells have malignant cellular features and are named based on their
differentiation status. They lack immature lymphocytes, and lymphocytes within the tumor tend
to be mature and are usually admixed with plasma cells.
Combined thymoma Benign thymic tumors that demonstrate a combination of thymoma subtypes.
Thymoma (type X) with anaplasia Subgroup of tumors with morphologic features between a thymoma and thymic carcinoma.
WHO, World Health Organization.

Thymic epithelial tumors are uncommon cranial mediastinal
neoplasms that are derived from the thymic epithelial components
of ectodermal origin with the potential to differentiate toward
either a cortical or medullary phenotype. They are highly vari-
able histologically, making their diagnosis and classification
difficult. In human medicine, different histologic classifica-
tions have been proposed for thymic epithelial tumors to
better correlate their histology with clinical behavior, but
the 2004 World Health Organization (WHO) classification
scheme for thymic epithelial tumors is most widely used. This
classification has also been shown to be applicable to thymic
epithelial tumors in dogs (Table 2-4). The morphologic basis for
this classification is the cell morphology of the neoplastic epithelial
cells and the relative proportion of non-neoplastic lymphocytes. It
is rare for thymic epithelial tumors to be purely epithelial, and
the association of lymphocytes with epithelium, even in meta-
static sites, suggests that the latter retains some lymphoid
inductive capacity, notwithstanding the malignant state. The
2004 WHO classification system for thymic epithelial tumors
identifies encapsulated or invasive thymomas and thymic
 Thymus
Figure 2-48  Thymoma in a dog. The vast majority of thymomas
in dogs or cats are heavily encapsulated.
Figure 2-49  Thymoma in a dog. Large, well-circumscribed mass
in the cranial mediastinum with multifocal hemorrhages.
carcinomas. Because invasive thymomas can only be distin-
guished from encapsulated thymomas when the entire neo-
plasm is available for histologic examination, this distinction
is rarely applicable in veterinary medicine. Furthermore, the
vast majority of thymomas in dogs or cats tend to be slow
growing, heavily encapsulated, and rarely metastasize (Fig.
2-48). Dogs with surgically excised thymomas have a favor-
able long-term prognosis for both invasive and noninvasive
thymomas. Given their rarity and their generally favorable
postsurgery outcome, thymic epithelial neoplasms have not been
extensively studied in most animal species and are commonly only
classified as thymomas or thymic carcinomas, with the latter
being exceedingly rare. In dogs, cats, horses, and occasionally
cattle, thymic epithelial neoplasms most commonly occur
with clinical signs of respiratory and/or cardiovascular impair-
ment, whereas they are usually seen as incidental findings at
autopsy in sheep and goats.
Thymomas.  Thymomas occur in dogs and cats 10-12 years
of age (Fig. 2-49, eFig. 2-8). They have historically been clas-
sified on the basis of cellular composition and the type and
degree of atypia of the epithelium as lymphocytic, epithelial, or
mixed. A clear cell thymoma variant has been described in a
153
Figure 2-50  Compressed thymic remnant in a dog. Remnant of
normal thymus has been compressed to the periphery of the
thymic capsule by the expanding thymoma.
Figure 2-51  Thymoma in a goat. Large mediastinal masses in
female, adult goats, most likely represent thymomas. (Courtesy
K.G. Thompson.)
single dog. Regardless of their histologic appearance, thymo-
mas tend to be localized to one lobe, or, more commonly, a
remnant of benign thymus may be found compressed to the
periphery of the thymic capsule (Fig. 2-50). In the cat, thy-
momas may occasionally occur as an ectopic mass in the
mid-cervical area and are often focally cystic. Goats, especially
female goats, have a high likelihood of developing thymomas
at 7-8 years of age (Fig. 2-51). Most caprine thymomas are
completely contained within the thoracic cavity and vary
in size from 3-8 cm in diameter. They are encapsulated,
often cystic, and may have large areas of ischemic infarction
(Fig. 2-52).
A more accurate classification of thymomas is based on
2 major histologic thymoma phenotypes that demonstrate
 153.e1

B
eFigure 2-8  Thymoma in a dog (A) and a sheep (B). A. The
thymoma appears as an expansile mass in the mediastinum.
B. Cross section of a large well-circumscribed thymoma with
multifocal areas of cyst formation.
154 CHAPTER 2  •  Hematopoietic System
Figure 2-52  Thymoma in a goat. The thymoma is encapsulated,
cystic, and areas of neoplasm have undergone ischemic infarction.
(Courtesy K.G. Thompson.)
benign cellular features. Type A thymomas are composed of
spindle-shaped cells, and type B thymomas are composed of epi-
thelioid cells. Admixtures of spindle-shaped cells and epitheli-
oid cells are designated as type AB thymomas. Type B thymomas
are further subdivided into type B1, B2, or B3 thymomas. The
relative ratio of the epithelial cell population compared to
non-neoplastic lymphocytes is the major distinguishing feature
between the different type B thymomas. A major diagnostic
difficulty involves the recognition of epithelium in the lym-
phocytic type, which distinguishes it from thymic lymphoma.
Because some thymomas may have marked intratumoral het-
erogeneity, they are also classified as combined thymomas
with a percentage of each component noted. Occasionally,
cystic changes may become so prominent that the neoplasm
might be misdiagnosed as a thymic cyst. Rare clear cell or
plasma cell–rich variants have been observed in dogs.
The spindled epithelium in type A thymomas is usually
arranged in storiform, fascicular, or pericytomatous pattern
(Fig. 2-53). These cells have uniform elongated nuclei with
dense chromatin and inconspicuous nucleoli. Mitoses are
absent. Rare signet ring cells and pseudorosettes are observed.
In many cases, there is focal formation of microcystic or glan-
dular spaces that may even contain secretory material. These
cystic structures are branchial duct remnants and have a lining
that varies from squamous to columnar and ciliated, and may
change abruptly from one form to the other. They most likely
do not represent true glandular structures, but are formed by
stromal edema. Lymphocytes are usually rare to absent and
represent a more mature medullary lineage. This subtype rep-
resents ~10% of canine thymomas.
Micronodular thymoma is probably an unusual variant of
type A thymoma and exceedingly rare (Fig. 2-54). Most com-
monly, features of this type of thymoma can be observed in
some lobules of type A thymomas. It is characterized by mul-
tiple nodules composed of epithelial spindle cells with similar
morphology to what has been described with type A thymoma.
These nodular structures are separated by an abundance of
lymphocytes that may form follicular centers. Many lympho-
cytes are actually B cells admixed with a few T cells. Immu-
nohistochemistry for pancytokeratin helps to visualize the
total separation between the epithelial and lymphoid
Thymus
A
B
Figure 2-53  Type A thymoma in a dog. A. Neoplastic cells are
spindle-shaped, lack nuclear atypia, and are accompanied by a few
non-neoplastic lymphocytes. B. Neoplastic spindle cells are posi-
tive for pancytokeratin with immunohistochemistry.
aggregates. There are no epithelial cells in the lymphoid com-
ponent, whereas lymphocytes still infiltrate the epithelial
component.
Type B thymomas are the most common subtype of canine
thymomas, representing ~70% of all cases, with types B1
(24%) and B2 (36%) being most common. They are also fre-
quently seen in cattle, sheep and goat, and rarely in horses.
Grossly, they appear as irregularly shaped masses 5-15 cm in
diameter, with irregular rounded projections, and most often
firm fibrous encapsulation. Type B1 thymomas represent the
greatest diagnostic challenge because they most closely resem-
ble the normal thymus and have the most dense lymphocyte
population that may be misdiagnosed as a lymphoid neoplasm
(Fig. 2-55). Type B1 thymomas appear as lobulated masses
that resemble an exaggeration of normal thymic structure and
are separated by fibrous septa. Among the most characteristic
features are randomly distributed foci of pale-staining medul-
lary differentiation, which may be misdiagnosed as pale-
staining proliferation centers in chronic B-cell lymphocytic
leukemia on low magnification, and the background of epi-
thelial cells with large ovoid nuclei and small nucleoli that can
be easily recognized on high magnification. Epithelial cells do
not form aggregates. Scattered poorly developed Hassall’s
 Thymus 155

A A

B B
Figure 2-54  Micronodular thymoma in a dog. A. Multiple
nodules composed of neoplastic epithelial spindle cells separated
by an abundance of lymphocytes. B. Immunohistochemistry for
pancytokeratin shows the epithelial character of the proliferating
spindle cells.

corpuscles are present singly and in cornified clusters and help

distinguish these neoplasms as thymomas. The infiltrating thy-
mocytes are mostly immature T cells. Immunohistochemistry
for cytokeratin will highlight the neoplastic epithelial cells.
Cytologically, type B1 thymoma is difficult to distinguish from
lymphoma on fine-needle aspirates and relies on the recogni-
tion of epithelial cells with large, pale nuclei and abundant
cytoplasm throughout the material obtained. If epithelial cells
are not readily apparent in the aspirate, immunocytochemistry C
for cytokeratin might prove helpful.
Type B2 thymomas also have prominent lobulation. Figure 2-55  Type B1 thymoma in a dog. A. Type B1 thymomas
Neoplastic epithelial cells are plump with vesicular nuclei closely resemble the normal thymic cortex, and dense populations
and distinct nucleoli and are scattered singly or as clusters of small lymphocytes can mask the large neoplastic epithelial cells
among a heavy population of non-neoplastic lymphocytes in the background. B. In foci of pale-staining medullary differen-
(Fig. 2-56). In contrast to type B1 thymomas, epithelial cells tiation, lymphocytes are less densely packed. C. Immunohisto-
are more easily recognized and stand out more easily from the chemistry for pancytokeratin highlights neoplastic epithelial cells
lymphocytic background. Although immature non-neoplastic in the background.
lymphocytes are abundant, they are less prominent than in
type B1 thymomas. Lymphoid follicles with germinal centers particularly myasthenia gravis and less often polymyositis.
may be observed. Prominent germinal centers with plasma Perivascular spaces are common and prominent. These peri-
cells present in their vicinity and along the fibrous septa have vascular spaces are characterized by cuffs of palisading epithe-
especially been reported in dogs and cats, where such follicular lial cells around small veins that create a clear space resembling
activity has been associated with autoimmune disease, a lymphatic. Immunohistochemical staining for cytokeratins
156 CHAPTER 2  •  Hematopoietic System
A
B
Figure 2-56  Type B2 thymoma in a dog. A. Epithelial cells form
discrete groups surrounded by lymphocytes and are easily recog-
nizable. B. Clear cells can be the dominant neoplastic cell type in
thymomas, and these cells are rich in glycogen.
will highlight the palisading epithelial cells and the striking
resemblance to perivascular spaces in the normal thymus.
Type B3 thymomas are characterized by lobules of round
or polygonal epithelial cells that exhibit no or only mild atypia
and separated by thick fibrous septa (Fig. 2-57). They are
admixed with a minor component of non-neoplastic imma-
ture T lymphocytes, which results in a sheet-like growth of
neoplastic epithelial cells. Palisading of epithelial cells around
small veins or fibrous septa is prominent, but perivascular
spaces tend to be narrow or may undergo hyalinization. The
epithelial cells have oval to irregular nuclei with inconspicu-
ous nucleoli and ample eosinophilic cytoplasm that often
shows some perinuclear clearing. Some cells have a squamous
appearance. Few mitoses can be found. It is not uncommon
to find a transition from B2 to B3 thymomas in the same
neoplasm.
Type AB thymomas represent ~15% of canine thymomas.
This type of thymoma is seen most frequently in the goat and
sheep as space-occupying lesions. They tend to appear as well-
formed lobules composed of the lymphocyte-poor type A
thymoma component and the lymphocyte-rich type B
thymoma component (Fig. 2-58). The percentage of each
component will vary among neoplasms and even within the
same neoplasm, with one component being predominant in
Thymus
Figure 2-57  Type B3 thymoma in a dog. Neoplastic epithelial
cells are round to polygonal and admixed with only small numbers
of non-neoplastic lymphocytes.
Figure 2-58  Type AB thymoma in a dog. Sharply demarcated
areas of type A thymoma on the right and type B thymoma on
the left.
one area but not the other. Both components can appear as
histologically distinct areas or intermixed with each other. In
the latter form, the spindled epithelial cells of the type A
component commonly form septum-like structures that sur-
round the type B component. These tumors often have firm
encapsulation with regular, but coarse septation, but with
minimal stroma in the pseudolobular areas. Cystic spaces are
frequently present as part of the type A component and
are not seen in pure type B thymomas. Lymphocytes in the
type B component commonly have smaller nuclei with
indistinct nucleoli than usually observed in B1 or B2 thymo-
mas. Hassall’s corpuscles are rarely present in the type B
component.
Malignant thymomas are classified as thymic carcinomas
(Fig. 2-59). Thymic carcinomas are further subdivided into
additional subtypes based on the presence of histologic
features that are not found in the normal thymus, for
instance, squamous cell carcinoma, clear cell carcinoma, and
lymphoepithelioma-like carcinoma. All of these entities are
rare; squamous cell carcinomas are the most commonly
 Thymus
Figure 2-59  Thymic carcinoma in a dog. Anaplastic malignant
epithelial cells are arranged in tubulopapillary structures.
observed thymic carcinoma in dogs. These neoplasms are
lobulated, have ample hyaline stroma, and neoplastic cells
have pleomorphic, vesicular nuclei. Mitoses are common, and
a high mitotic index (>10 mitoses/high-power field [HPF])
carries a worse prognosis. Islands of neoplastic cells undergo
squamous differentiation with various degrees of keratiniza-
tion, and are surrounded by desmoplastic stroma that is com-
monly infiltrated by plasma cells and mature lymphocytes.
Thymic carcinomas are highly invasive and have to be dif-
ferentiated from pulmonary squamous cell carcinomas that
invade the mediastinum. Accurate diagnosis is relevant because
pulmonary carcinomas carry a worse prognosis. In humans,
thymic carcinomas are commonly positive for KIT; a similar
expression profile has not been described in domestic animals.
Thymoma (type X) with anaplasia is the suggested diag-
nostic term for a very uncommon group of thymomas that
demonstrate morphologic features that do not fit into either
the thymoma or thymic carcinoma categories.
Myasthenia gravis is an autoimmune disorder of humans,
dogs, and cats, characterized by progressive muscle weakness
and reduced exercise tolerance. Specific areas of involvement
include the facial and extraocular muscles with the develop-
ment of megaesophagus associated with difficult swallowing,
regurgitation, and aspiration pneumonia. It can be congenital
or acquired. Only acquired myasthenia gravis commonly
occurs secondary to thymic abnormalities, especially thymoma
in dogs and thymic follicular hyperplasia in humans. All of the
physiologic features of the acquired myasthenia gravis can be
accounted for by the reduction in functional acetylcholine recep-
tors of neuromuscular junctions (see Vol. 1, Muscle and tendon).
The disease arises as an immune-mediated phenomenon,
whereby an antibody reaction is developed against the thymic
myoid cells that express intact acetylcholine receptors. The myoid
cells tend to be associated with Hassall’s corpuscles and the
germinal centers, which resemble those of antigen-activated
lymph nodes, and diffuse areas of B-cell proliferation that are
centered on the medullary areas with sparing of the cortex,
which is often reduced in volume. It has been hypothesized
that thymic epithelial cells present epitopes from the isolated
acetylcholine receptor subunits, which they express and auto-
immunize helper T cells. These helper T cells induce an early
antibody response directed against the rare thymic myoid
157
Figure 2-60  Thymic lymphoma in a dog. A large, homogeneous,
pale mass expands the cranial mediastinum.
cells. The autoantibodies diversify in the resulting germinal
centers and recognize native acetylcholine receptors. Myasthe-
nia gravis is primarily caused by the attachment of antibodies to
skeletal muscle acetylcholine receptors of the postsynaptic mem-
brane, leading to decreased of functional receptors. In addition,
antibodies against titin and ryanodine receptors have been
shown to be associated with more severe disease, an older
onset, or the presence of a thymoma in humans and dogs.
Although the physiologic role of titin is unknown, ryanodine
receptors act as calcium-release channels in skeletal muscle
and are therefore important for excitation/contraction cou-
pling in skeletal muscle. In a thymoma model in mice, even
in the absence of acetylcholine receptor antibodies, antibodies
against ryanodine receptors caused severe skeletal muscle
weakness. Recent studies in humans strongly suggest that
myasthenia gravis may develop after viral infections, leading to
interferon I overexpression, together with the activation of
innate immunity pathways in thymoma-associated myasthe-
nia gravis. This hypothesis is supported by detection of neu-
tralizing antibodies against interferon I in thymoma-associated
myasthenia gravis patients, but not in patients with thymomas
without muscular disease or those with normal thymuses.
However, no specific viral etiology has been described. Surgi-
cal removal of the neoplastic or hyperplastic thymus has been
reported to restore muscle function.
Thymic lymphomas.  In general, lymphoid tumors of the
cranial mediastinum in cats and cattle, and less commonly dogs,
are most commonly precursor T-lymphoblastic lymphomas (see
details of the histologic and immunohistochemical features
under lymphomas) that can be easily misdiagnosed as
lymphocyte-rich thymomas (eFig. 2-9). A less common type
of thymic lymphoma that has rarely been observed in dogs is
a large B-cell lymphoma (Fig. 2-60). Because these neoplasms
have rarely been reported, there is limited data on whether
they truly resemble mediastinal B-cell lymphoma in humans
or are actually diffuse large B-cell lymphomas arising in the
mediastinal lymph nodes.
In the cat, mediastinal lymphomas can reach a large size,
with dorsocaudal displacement of the lungs before they are
presented for clinical examination (Fig. 2-61). There is a wide
age distribution of feline thymic lymphoma, with 90% occur-
ring relatively evenly between 1 and 10 years of age.
 157.e1

B
eFigure 2-9  Thymic lymphoma in a dog (A) and a pig (B). A. A
large, homogeneous, pale mass has displaced the lungs caudally.
(Courtesy K.G. Thompson.) B. Thymoma and mediastinal lym-
phoma are often difficult to differentiate grossly.
158 CHAPTER 2  •  Hematopoietic System
A tic germ cells seen in seminomas, and are large, discrete, rounded,
B 515-547.
Figure 2-61  Thymic lymphoma in a cat. A. The thymic lym-
phoma has displaced the lungs caudodorsally. There are mul­
tifocal areas of necrosis and petechiae throughout the mass.
B. Precursor T-cell lymphoblastic lymphoma characterized by
diffuse, dense infiltrates of monomorphic, intermediate-sized lym-
phoid cells with oval or folded convoluted nuclei with dispersed
chromatin and indistinct nucleoli.
Involvement of the lung, even by direct extension, is unusual,
although there may be infiltration into the atria and through
the pericardium into the base of the heart and around the
origins of the great vessels. Infiltration beneath the sternal
pleura is consistently present. Metastases may be widespread,
but they are rare and small compared to the mediastinal neo-
plasm, suggesting that these lesions spread by local expansion
until late in the disease.
Thymic lymphoma in cattle characteristically occurs in
yearlings of the beef breeds, and is not associated with infec-
tion with bovine leukemia virus (BLV). Typically, there is
swelling, sometimes massive, at the base of the neck, and
brisket edema. Bloating and dysphagia associated with esopha-
geal compression are common. The animals are seldom leu-
kemic, although in advanced cases there are usually neoplastic
lymphocytes in the peripheral blood without changes in abso-
lute numbers of cells, and neoplastic cells may be identified
in sternal marrow aspirates. The thymic lymphoma may
extend from the rami of the mandibles to the base of the
heart, with a constriction at the entrance to the thoracic cavity,
and may weigh 20 kg or more. They are lightly encapsulated
Spleen and Hemolymph Nodes
and infiltrate the surrounding tissues, often with smooth,
rounded, shiny implants appearing on the pulmonary and
parietal pleura. There is compression of the lungs by tumor
and excess pleural fluid. The lungs themselves may be focally
invaded but are rarely extensively involved. Invasion of the
pericardial sac produces pericardial effusion that contains neo-
plastic lymphocytes in large numbers. On cut surface, the
tumors are gray with irregular yellow areas of infarction that
have hyperemic borders, and they tend to have coarse col-
lagenous septation.
Thymic germ cell tumors.  Rarely, primary germ cell
tumors, resembling seminomas, occur in the thymus in the
absence of a primary neoplasm in the testicle. However, the
possibility of a previously removed testicular neoplasm should
always be excluded. The neoplastic cells closely resemble neoplas-
or polygonal. The cytoplasm is eosinophilic or palely baso-
philic and scanty, finely granular, and encloses a relatively
large, central, oval, or rounded nucleus with usually a single
prominent nucleolus. Rare karyomegaly and binucleated cells
occur. Areas of necrosis and foci of lymphocytes are typically
present. Necrotic foci cause a characteristic feature of thymic
seminomas that manifests as variable numbers of round clear
spaces throughout the neoplasm, which give the lesion a moth-
eaten appearance. Neoplastic cells are immunohistochemi-
cally positive for Oct4 and negative for CD45 and CD18.
Further reading
Lara-Garcia A, et al. Cervical thymoma originating in ectopic thymic
tissue in a cat. Vet Clin Pathol 2008;37:397-402.
Pearse G. Histopathology of the thymus. Toxicol Pathol 2006;34:
Ponce F, et al. A morphological study of 608 cases of canine malignant
lymphoma in France with a focus on comparative similarities
between canine and human lymphoma morphology. Vet Pathol
2010;47:414-433.
Rickman BH, Gurfield N. Thymic cystic degeneration, pseudoepithelio-
matous hyperplasia, and hemorrhage in a dog with brodifacoum
toxicosis. Vet Pathol 2009;46:449-452.
Robat CS, et al. Clinical features, treatment options, and outcome in
dogs with thymoma: 116 cases (1999-2010). J Am Vet Med Assoc
2013;243:1448-1454.
Suster S, Moran CA. Histologic classification of thymoma: the World
Health Organization and beyond. Hematol Oncol Clin North Am
2008;22:381-392.
Tepper LC, et al. Diagnosis of erythema multiforme associated with
thymoma in a dog and treated with thymectomy. J Am Anim Hosp
Assoc 2011;47:19-25.
Tillman H, et al. Application and clinical relevance of the WHO histo-
logical classification system to canine thymomas. Vet Pathol
2009;46:1043.
Zitz JC, et al. Results of excision of thymoma in cats and dogs: 20 cases
(1984-2005). J Am Vet Med Assoc 2008;232:1186-1192.
SPLEEN AND HEMOLYMPH NODES
Structure and function of the normal spleen
The spleen is a peripheral lymphoid organ that, together with
the hemal nodes, is also part of the blood circulatory system,
and functions as an effective filter of blood through a sinusoidal
system.
 158.e1

Further reading
Hadlow WJ. High prevalence of thymoma in the dairy goat. Report of
seventeen cases. Vet Pathol 1978;15:153-169.
Oksanen A. Fine structure of epithelial thymus cysts in dogs. Acta
Pathol Microbiol Scand [A] 1977;85:470-480.
Rottenberg S, et al. Thymoma-associated exfoliative dermatitis in cats.
Vet Pathol 2004;41:429-433.
Shelton GD, et al. Titin and ryanodine receptor autoantibodies in dogs
with thymoma and late-onset myasthenia gravis. Vet Immunol
Immunopathol 2001;78:97-105.
Willcox N, et al. Autoimmunizing mechanisms in thymoma and thymus.
Ann N Y Acad Sci 2008;1132:163-173.
 Spleen and Hemolymph Nodes
The spleen develops between the serosal membranes that
are part of the dorsal mesogastrium that connects the stomach
to the cranial abdominal wall and from which the greater
omentum develops. The spleen is a highly elastic organ of
purple-gray color that tends to be darker in herbivores and
redder in carnivores. There are significant differences in shape,
structure, attachment, and function of the spleen among
domestic animals, but all spleens are flattened, elongated
organs. The spleen is located primarily in the intrathoracic
portion of the abdominal cavity in the left cranial hypogastric
region. It is attached to the greater curvature of the stomach
by the gastrosplenic ligament, except in ruminants, where it
closely adheres to the dorsolateral aspect of the rumen. The
position of the spleen depends in carnivores and, to a lesser
degree in horses, on the amount of food in the stomach. Espe-
cially in dogs, large amounts of food content can displace the
spleen far caudally. Each spleen has a parietal or diaphrag-
matic surface and a visceral surface, a cranial and a sharp
caudal border, as well as a proximal and distal end. In carni-
vores, the spleen is dumbbell shaped with a wider distal end,
whereas in pigs it remains uniformly strap-like. In cows, the
spleen has the shape of an elongated oval, whereas in sheep
or goats it is more triangular or quadrangular, respectively. In
horses, the spleen is falciform with a broad dorsal end and a
pointed ventral end. On the visceral surface is the splenic hilus,
which is characterized by insertion of the greater omentum
and the splenic arteries and veins as well as the beginning of
the gastrosplenic ligament. In ruminants, the hilus appears as
a small grove only while the spleen is fused through connec-
tive tissue with the rumen at a serosa-free region. In rumi-
nants, the spleen is also anchored through the phrenicosplenic
ligament that connects the dorsal portion of the spleen to the
diaphragm. In horses, the gastrophrenic ligament becomes
the phrenicosplenic and splenorenal ligaments and connects
the spleen to the left kidney and the transverse colon.
On macroscopic examination, 3 components of the splenic
structure are recognizable: the splenic capsule and connective
tissue trabeculae, the red pulp, and the white pulp. A strong
capsule surrounds the spleen of all domestic animals, and the
trabeculae extend from the capsule internally. The chambers
between the trabeculae are filled with the soft splenic paren-
chyma, the splenic pulp. This pulp is composed of darker red,
blood-rich areas—the red pulp—that surround lighter-colored
areas that are devoid of erythrocytes—the white pulp.
Except for the serosal-free area in ruminants, the spleen is
covered by the peritoneum that continues onto the splenic
ligaments. The internal coat of the capsule, also called the
elastic coat, is formed by fibrous connective tissue with a high
percentage of elastic fibers, and in contrast to humans, up to
80% of this coat is composed of smooth muscle fibers. In
ruminants and pigs, a large percentage of smooth muscle fibers
can also be found in the red pulp. In carnivores, the internal
capsule is formed by a single layer, whereas in ruminants and
horses, 2 layers can be identified. The trabeculae extend from
the capsule into the splenic parenchyma and form, together
with the reticulum, the supporting stroma. The high percent-
age of elastic fibers combined with the capsular and trabecular
musculature allows for tremendous expansion of the spleen
in domestic animals and enables the spleen to store blood.
Spleens with such storage capacity are classified as storage
spleens, and horses and carnivores have the highest capacities
of domestic animals. Approximately 13 of the blood volume
can be stored in the spleen of dogs, and half of the circulating
159
blood volume can accumulate in the spleen of horses. Pigs and
ruminants have less capacity. Spleens that have limited storage
capacity are classified as defense spleens. Such spleens have
fewer elastic fibers and smooth muscles in their trabeculae and
capsule, and examples include the spleens of humans and
rabbits. The autonomic nervous system regulates the ability of
storage spleens to actively contract through innervation by
vagal and sympathetic fibers and release of catecholamines.
The spleen has no cortex or medulla, but the parenchyma
is composed of 2 distinct compartments: the white pulp and
red pulp (Fig. 2-62). The white pulp represents macrophages,
antigen-presenting cells, and B and T lymphocytes in the peri-
arteriolar lymphoid sheaths (PALS), the splenic follicles (Mal-
pighi bodies), and the marginal zone. The red pulp is composed
of a meshwork of reticular fibers, reticular cells, and associated
monocytes/macrophages, and surrounds the red pulp vascular
spaces.
To understand the structure and function of the red pulp
and white pulp, it is necessary to have a detailed understand-
ing of the blood circulation through the spleen. Blood vessels
are an important component of the splenic architecture and,
whereas arteries and veins are similar in all domestic animals,
the capillary beds of the red pulp differ. In cats and dogs, the
red pulp is richly supplied with an anastomosing network of
sinusoids, and the spleen is classified as a sinusoidal spleen. All
other domestic animals have a nonsinusoidal or reticular spleen
in which the penicillary arterioles empty into the red pulp
vascular space and only small numbers of venous capillaries
can be found in the red pulp. The main anatomic differences
between sinusoidal and nonsinusoidal spleens can be found
in the arterial vasculature. In dogs, arterial capillaries either
continue into the described venous sinusoids, or they termi-
nate into the red pulp vascular spaces. The continuous lining
by endothelium as observed for the circulation through the
sinusoids is called a closed system, whereas the interruption
of endothelial lining in vascular spaces is referred to as an
open system. Dogs and cats have therefore a closed and an open
system, whereas all other domestic animals have an open
system.
The spleen receives its blood from the splenic artery, which
comes from the celiac artery, and the blood drains through
the splenic vein into the portal vein. Approximately 2.5% of
the cardiac output flows through the spleen. The splenic
artery divides into multiple branches, each of which functions
as an end artery that supplies a specific segment of the spleen.
These arteries enter the spleen through the hilus and continue
within the capsule or trabeculae as trabecular arteries. Trabecu-
lar arteries have the typical wall structure of muscular arteries
and carry the splenic nerves in their adventitia. The returning
trabecular veins have an intima that is directly integrated into
the trabecular tissue. Cats have the most extensive trabecular
vascular system of all domestic animals. As the trabecular
arteries branch and enter the white pulp, they become
ensheathed, with cuffs of T lymphocytes forming the periar-
teriolar lymphoid sheaths (PALS). The arteries surrounded by
these PALS are now identified as central arterioles. Irregularly
along the length of these sheathed central arterioles, the lym-
phoid tissue widens eccentrically to the arteriole and forms
the B-cell–derived splenic follicles (Malpighian bodies).
Although the PALS are not vascularized, and oxygen is
provided through diffusion from the central artery, the splenic
follicles have their own capillary bed that provides both sus-
tenance and antigen exposure. The first branches of the central
160 CHAPTER 2  •  Hematopoietic System Spleen and Hemolymph Nodes

Periarteriolar macrophage
sheath (PAMS)

Trabecula Splenic cord

Trabecular Periarteriolar lymphoid

artery sheath (PALS)

Splenic
sinusoid
Central
arteriole Marginal
sinus

Penicillar
arterioles
Closed
circulation

Open
circulation
Splenic
follicle

Marginal zone
Red pulp with ending
capillaries

Trabecular Radial
vein arteriole

Nonsinusoidal Sinusoidal
spleen spleen
Figure 2-62  Schematic drawing of normal spleen. The left side depicts a nonsinusoidal spleen as
observed in most domestic animals; the right side shows a sinusoidal spleen characteristic of dogs.

arterioles form a vascular plexus surrounding the splenic fol-
licles. The capillaries of this plexus, the follicular capillaries, are
characterized by dense zonulae occludens. Numerous follicu-
lar capillaries emerge on the surface of the splenic follicles and
form anastomoses. These thin-walled vascular spaces separate
the splenic follicles from the periphery and are known as
marginal sinuses. Because the marginal sinuses are supplied by
the first branches of the central arterioles, the splenic follicles
are the first to be exposed to antigens transported to the
spleen.
The marginal areas surrounding the whole white pulp,
including the PALS, are called the marginal zone and are char-
acterized by a very fine reticular meshwork. The marginal
sinuses have uniform endothelial coverage on the follicular
side, but an incomplete endothelial layer on the peripheral
side. The blood percolates through the marginal sinuses via
this incomplete coverage, and enters the meshwork of the
marginal zone. Although most blood flows directly from the
marginal zone into the adjacent splenic sinusoids (fast
pathway), some blood enters the red pulp vascular spaces
(slow pathway).
Ultimately, the central arterioles terminate in the arboriz-
ing penicillar arterioles when entering the red pulp. Penicillar
arterioles can be divided into 3 segments: pulp arterioles, macro-
phage sheathed portions, and terminal arterial capillaries. Shortly
after branching, the pulp arterioles are ensheathed by macro-
phages that form the periarteriolar macrophage sheath (PAMS).
These structures have historically been called ellipsoids.
 Spleen and Hemolymph Nodes
Subsequently, penicillar arterioles lose their media and become
capillaries. The sheathed arterioles are lined by elongated
endothelial cells that are isoprismatic on cross section and
contain large numbers of vimentin filaments. Adjacent endo-
thelial cells are not joined by junctions, and they therefore
form a lattice-work through which blood and plasma cells
readily permeate. Blood passes from the capillaries into the
surrounding mantles that form up to 200 µm–long structures
sheathing capillaries. Integrated into this reticular network
composed of type III collagen reticular fibers are macrophages
that are exposed to the migrating red blood cells. Blood passes
through the reticular meshwork into the surrounding red pulp
vascular spaces. In all domestic animals, capillaries emerging
at the distal end of the splenic cords terminate in rounded
excavations (ampullae of Thoma) from which blood empties
into the red pulp vascular space. Actin filaments in the endo-
thelial cells forming these ampullae might indicate the ability
to actively regulate the blood flow. In dogs, some capillaries
enter into the splenic sinusoids as previously described. The
space between the sinusoids is occupied by loose reticular
stroma in which macrophages, lymphocytes, and plasma cells
are enmeshed, called splenic cords (cords of Bilroth). Splenic
cords are irregularly supported by encircling reticular fibers,
like the staves and hoops of a barrel. Regardless of the differ-
ences in blood flow in different animals, erythrocytes are
subject to surveillance by macrophages that are located peri-
vascularly around sinusoids, in the red pulp vascular spaces,
and splenic cords, depending on the animal species. Blood
from the red pulp vascular spaces collects in the venous capil-
laries that have an open connection to the meshwork of the
pulp reticulum. The venous capillaries and the splenic sinu-
soids drain the blood into trabecular veins and ultimately the
splenic vein. The splenic vein drains blood directly into the
portal vein, and therefore any increase in portal pressure
increases the blood volume of the spleen, leading in long-
standing conditions to congestive splenomegaly.
In domestic animals, the red pulp functions as both storage
compartment, as described previously, and the red pulp mac-
rophages together with the marginal zone macrophages have
essential functions in phagocytizing foreign material, bacteria,
and senescent or defective erythrocytes. The red pulp is com-
posed of splenic cords and vascular spaces; cylinders of pulp
spaces that are organized in a reticular meshwork, fed by a
peripheral ring of arterial capillaries, and drained by a central
venule. In dogs, it also consists of venous sinusoids. The red
pulp macrophages are derived from bone marrow monocytes
and are CD11d positive. Macrophages are integrated into the
reticular meshwork composed of type III collagen and reticu-
lar fibers. They use a number of different mechanisms to
recognize senescent erythrocytes. Although decreased meta-
bolic activity, morphologic alterations, including decreased cell
volume, and changes in cell shape predispose erythrocytes to
removal, macrophages can also detect senescent cells through
surface receptors. Such receptors can bind to Amadori
products—aminoketoses that are the result of nonenzymatic
browning, the nucleophilic addition of an amine to the car-
bonyl function of a reducing sugar, with formation of glyco-
sylamines that occurs over time on the surface of erythrocytes.
Other surface alterations in aging erythrocytes include
decreased levels of sialic acid and CD47, which result in
binding of autologous immunoglobulins and opsonins that can
be recognized by macrophages. In dogs, macrophages remove
entire erythrocytes, a process called erythrophagocytosis, but
161
also nuclear remnants and red blood cell membranes. Nuclear
remnant–containing erythrocytes are pitted of their inclusions,
such as Howell-Jolly bodies, as they squeeze through the
interendothelial slits of venous sinusoids in their passage out
of the spleen, and return to the circulation. The observation
of excessive amounts of nuclear remnants in erythrocytes
often indicates a splenic disease. In dogs, ~90% of blood passes
through the splenic sinusoids and bypasses the red pulp vas-
cular space. The remaining blood perfuses the splenic cord
areas, where sorting and processing by macrophages is carried
out to remove senescent or injured red cells. The flow rate
through the spleen is relatively large, and even with this low
sorting fraction, all of the blood passes through the splenic cord
system once per day. Because the spleen plays a central role in
removing senescent erythrocytes, it also has a central role in
iron metabolism. Iron is scavenged by macrophages when
removing senescent red blood cells from circulation and ini-
tially stored as a protein-iron complex ferritin, but ferritin can
be incorporated by phagolysosomes to form hemosiderin
granules.
The vascular system of the spleen does not include lym-
phatics. As a consequence, all antigens reach the spleen
through the blood, which determines that primary sensitiza-
tion and antibody production take place in the spleen only if
the first contact with antigen occurs by hematogenous sensitiza-
tion (Fig. 2-63). On subsequent challenge, the splenic follicles
of the spleen play an important role in the humoral anamnes-
tic response, both by local production of antibodies and by
the provision of committed B memory cells to the peripheral
lymphoid organs. The clearance of bacteria from the blood by
the spleen predominates only if systemic opsonization is defi-
cient. In other circumstances, the clearing appears to occur
mainly in hepatic Kupffer cells. Thus splenectomy increases
susceptibility to a number of blood-borne infections, including
bacterial septicemia and protozoal infections such as malaria,
anaplasmosis, and hepatotrophic mycoplasmal infection
(hemobartonellosis). All of these hazards can be prevented by
specific immunization that brings the hepatic Kupffer cells to
an alerted state following splenectomy.
The white pulp is composed of macrophages, antigen-
presenting cells, and B and T lymphocytes in the periarteriolar
Figure 2-63  Splenic babesiosis in a dog. Babesia canis can be
detected as single or paired, piriform organism within erythro-
cytes of a severely congested spleen. (Courtesy E.P. Lane.)
162 CHAPTER 2  •  Hematopoietic System
lymphoid sheaths (PALS), the splenic follicles (Malpighi bodies),
and the marginal zone. Splenic follicles are also often referred
to as lymphoid follicles. The PALS are localized around the
central arterioles as previously described. They are formed by
concentric layers of flat reticular cells and fibers forming a
reticular meshwork that is populated by a dense inner layer
of small, predominantly CD4+ T cells and a slightly less dark–
staining layer of small- to medium-sized T and B cells admixed
with macrophages. During immunostimulation, large numbers
of plasma cells may be observed in the outer layer. The splenic
lymphoid follicles are continuous with the PALS and usually
located eccentrically to the PALS at the bifurcations of the
central arterioles. Their structure is typical of a lymphoid fol-
licle. Under antigenic stimulation, these follicles develop ger-
minal centers characterized by a dark zone composed of
centroblasts and immunoblasts and a light zone of centroblasts
and centrocytes admixed with tingible body macrophages.
The germinal centers are surrounded by the mantle cell layers,
a narrow cuff of small, inactive, mature, naive B lymphocytes.
Immunophenotyping is highly useful to visualize these struc-
tures. One of the most valuable aspects of phenotypic identi-
fication of B- and T-cell areas of the spleen is the opportunity
to understand that the spleen is a highly ordered immune struc-
ture that varies in morphology as predictably as lymph nodes in
response to various stimuli. In addition, the dissection of the
phenotypic structure of the spleen clearly demonstrates the
differences between hyperplastic and neoplastic lymphoid
proliferations arising from the different portions of the splenic
follicles (see Lymphoma). Outside of the mantle cell cuff is
the marginal zone, located at the interface between white and
red pulp. A band of macrophages, the metallophilic macro-
phages, and the marginal sinus separate the marginal zone
from the PALS and lymphoid follicles. The marginal zone is
composed of a thick ring of medium-sized B cells admixed
with dendritic cells, macrophages, T cells, and reticular cells.
The marginal zone region receiving the blood exiting the
germinal center is the area most densely populated with cells
that have recently arrived from the general circulation. As a
consequence, animals with marked neutrophilia will have
many neutrophils in the area immediately surrounding the
corona of the splenic follicle. Increased neutrophils in this area
are more likely to be seen in the cat, dog, and horse, which
respond to infectious diseases such as salmonellosis by strong
neutrophilia, whereas in calves and mature cattle, bands and
metamyelocytes may be found in this area. Because many of
these animals die with neutropenia, neutrophil accumulation
in this site is less frequently observed.
Hemal nodes (hemolymph nodes) occur in ruminants, and
a similar structure occurs in rats. They are enclosed by a fibro-
muscular capsule and have a general architecture similar to
that of lymph nodes (Fig. 2-64, eFig. 2-10). Hemal nodes have
a blood vascular circulation and afferent and efferent lymphatics,
although the former are apparently much diminished in com-
parison to lymph nodes. The arteries entering through the
hilar area arborize peripherally and are followed closely by
veins that apparently communicate through a trabecular
structure similar to that of the spleen. Hemal nodes have
germinal centers, but the paracortical areas are much reduced
in comparison to those of lymph nodes. Unlike lymph nodes,
where the subcapsular sinus is poorly cellular, hemolymph
nodes contain red cells in density similar to that of peripheral
blood. Red cells are not present in the afferent lymphatics,
and very few are present in the efferent lymphatics; however,
Spleen and Hemolymph Nodes
Figure 2-64  Normal hemal node in a cow. The architecture of
hemal nodes is similar to that of lymph nodes, with a thick fibro-
muscular capsule, germinal centers with a reduced paracortical
area, and a subcapsular sinus expanded by large numbers of
erythrocytes.
the larger sinuses of the hemal nodes may be filled with blood
and more closely resemble sinus areas of the spleen. Macro-
phages occupy the trabecular areas, and phagocytosis occurs
as in the spleen. Myelopoiesis does not occur in hemal nodes
either under normal circumstances or under conditions of
hematopoietic stress.
Further reading
Cesta MF. Normal structure, function, and histology of the spleen.
Toxicol Pathol 2006;34:455-465.
Elmore SA. Enhanced histopathology of the spleen. Toxicol Pathol
2006;34:648-655.
Elmore SA. Enhanced histopathology of the immune system: a review
and update. Toxicol Pathol 2012;40:148-156.
Marx A, et al. Functional splenic pathology and differential diagnosis
in splenectomy. Pathologe 2008;29:109-114.
Wluka A, Olszewski WL. Innate and adaptive processes in the spleen.
Ann Transplant 2006;11:22-29.
Zidan M, Pabst R. Histology of the hemal nodes of the water buffalo
(Bos bubalus). Cell Tissue Res 2010;340:491-496.
Developmental diseases of the spleen
The spleen may be congenitally absent. Although this condi-
tion is common in some strains of inbred mice, it is rare in
humans and domestic animals, where it usually occurs in
conjunction with multiple other anomalies. In nude mice and
rats with thymic deficiency, there is loss of the periarteriolar
lymphoid sheaths of the spleen. The significance of splenic
aplasia in domestic animals, in particular its importance for
the immune function, is unknown. Asplenia in mice has been
associated with higher mortality and a higher risk for proto-
zoal infections. Splenectomy in adult animals increases the
risk of bacterial infections.
Accessory spleens occur quite commonly in the gastro-
splenic omentum of domestic animals (Fig. 2-65) and can
either be congenital or acquired; most commonly they repre-
sent implants of splenic parenchyma following traumatic
 162.e1

eFigure 2-10  Normal hemal node in a cow. In contrast to lymph

nodes, the subcapsular sinus is expanded by large numbers of
erythrocytes.
162.e2

Further reading
Dullmann J, et al. Lectin histochemistry of the spleen: a new lectin
visualizes the stromal architecture of white pulp and the sinuses of
red pulp. J Histochem Cytochem 2000;48:923-931.
Schmidt EE, et al. Circulatory pathways in the sinusal spleen of the
dog, studied by scanning electron microscopy of microcorrosion
casts. J Morphol 1983;178:111-123.
 Spleen and Hemolymph Nodes
Figure 2-65  Accessory spleen in a dog. The smaller spleen on the
gastrosplenic ligament most likely represents an implant of splenic
parenchyma following trauma to the spleen.
rupture of the main organ (see Rupture of the spleen). Acces-
sory splenic tissue has rarely been reported in the pancreas of
dogs and cats as an incidental finding, but care should be taken
to not misdiagnose such splenic nodules as neoplasms. Intra-
pancreatic accessory spleens appear grossly as firm, well-
demarcated, dark red nodules.
Duplication of the spleen is occasionally observed in normal
swine and as one of multiple visceral defects in nonviable
calves and lambs. Ectopic pancreatic tissue, either exocrine or
endocrine, is occasionally observed in spleens that are other-
wise developmentally normal.
Splenic hypoplasia occurs most commonly as part of severe
combined immunodeficiencies as the result of lymphoid
hypoplasia. Affected spleens are grossly small, firm, and pale
red, and histologically lack lymphoid follicles and periarterio-
lar lymphoid sheaths.
Splenic fissure is a congenital abnormality that has been
mainly described in horses and appears as a smooth elongated
indentation parallel to the splenic edge that is covered by
normal splenic capsule (Fig. 2-66).
Further reading
Knostman KA, et al. Intrahepatic splenosis in a dog. Vet Pathol
2003;40:708-710.
Ramírez GA, et al. Intrapancreatic ectopic splenic tissue in dogs and
cats. J Comp Pathol 2013;148:361-364.
Rossi F, et al. B-mode and contrast-enhanced sonographic assessment
of accessory spleen in the dog. Vet Radiol Ultrasound 2010;51:
173-177.
Degenerative diseases of the spleen
Senile atrophy affects the spleen, as it does other lymphoid
tissues, and is seen particularly in old dogs and old horses.
Atrophy also accompanies cachexia and is seen in wasting
disease caused by starvation, malignant neoplastic diseases, or
malabsorption syndromes. Chronic radiation can also cause
severe atrophy of the spleen and red pulp fibrosis. Atrophied
spleens are small, and their capsule is contracted and thick-
163
Figure 2-66  Splenic fissure in a horse. Splenic fissures occur as
smooth elongated indentations that are covered by normal splenic
capsule. (Courtesy R.L. Amorim.)
ened. Microscopically, there is lymphoid atrophy affecting
both lymphoid follicles and periarteriolar sheaths. The severity
of atrophy is an indication of the duration and severity of the
atrophic process. The sinus areas appear fibrous as a result of
condensation of the sinusoids, and lack of blood and resident
hematopoietic cells. Contraction without atrophy of lym-
phoid tissue can be observed in storage type spleens, most
commonly because of catecholamine release or activation of
the autonomic innervation. Common causes include splenic
rupture, “fight and flight” situations, heart failure, and hypo-
volemic, cardiogenic, or septic shock.
In hyperimmune states, there are characteristic changes in
the germinal centers of animals that are severely stressed.
Lympholysis occurs, and germinal centers appear epithelioid
and hypocellular. If the cell loss is acute, there will be promi-
nent tingible body macrophages, as in foals aborted because
of equid herpesvirus 1 infection. If the reaction has persisted
for more than a day, cellular debris is absent and only dendritic
cells remain. Various vascular changes occur, consisting of
hyaline changes in small arterioles that may proceed to
mineralization.
Old dogs often have yellow encrustations along the splenic
margins, or even covering most of the capsule (Fig. 2-67, eFig.
2-11). Microscopically, there is condensation and thickening
of the capsule, trabeculae, and perivascular tissue. The yellow
or brown encrustations are known as siderotic plaques or
Gamna-Gandy bodies, and represent deposits of bilirubin,
hemosiderin, and/or calcium in connective tissue, usually the
trabeculae (Fig. 2-68, eFig. 2-12). The salts become encrusted
on connective tissue and elastic fibers, which are swollen,
refractile, and stain with hematoxylin. The encrusted fibers
occasionally are misinterpreted as fungal hyphae. In associa-
tion with ceroid, these nodules likely represent the residual
effects from areas of hemorrhage. Although siderotic plaques
occur primarily in older dogs, they are often interpreted as an
incidental aging change, but their deposition on the capsular
margins suggests a dystrophic lesion secondary to previous
trauma.
Amyloidosis of the spleen occurs as part of generalized
amyloidosis and can be either primary (AL) or secondary
(AA), but the deposits in the spleen may not be detectable
 163.e1

Further reading
Movitz D. Accessory spleens and experimental splenosis. Principles of
growth. Chic Med Sch Q 1967;26:183-187.
163.e2

A
eFigure 2-12  Splenic siderotic plaques in a dog. Yellow bilirubin
pigment surrounded by extensive fibrosis.

B
eFigure 2-11  Splenic siderotic plaques in a dog. A. Yellow
encrustation along the splenic margin. B. Multiple siderotic
plaques on the capsular surface.
164 CHAPTER 2  •  Hematopoietic System Spleen and Hemolymph Nodes

A A

B B

C C
Figure 2-67  Splenic siderotic plaques in a dog. A. Yellow encrus- Figure 2-68  Splenic siderotic plaques in a dog. A. Yellow biliru-
tation along the splenic margin. B. Some cases can have extension bin pigment surrounded by extensive fibrosis. B. Extensive fibrosis
of siderotic plaques over the whole capsule. C. Depending on the is highlighted in blue with a trichrome stain. C. Hemosiderin
content of bilirubin, hemosiderin, calcium, and the extent of pigments stain blue, and bilirubin stains orange with Prussian blue
fibrosis, the color of plaques varies from white to yellow or green, staining.
as shown in is this remarkably hypoplastic spleen.

grossly or histologically without appropriate stains (Fig. 2-69).
Amyloidosis of an obvious degree is not common, and when
present it involves the germinal centers and may spare the red
pulp vascular spaces completely (eFig. 2-13). This distribution
of amyloid gives the organ the “sago-spleen” appearance (gray
or white, opaque 2-mm spherules) in which the enlarged and
waxy follicles protrude from the cut surface. The spleen is not
enlarged. The deposition of amyloid occurs first in the small
arteries of the lymphoid nodules, and minor deposits are
common.
 164.e1

B
eFigure 2-13  Splenic amyloidosis in a dog (A) and in a horse (B).
A. Cross section of spleen shows a follicular distribution of
amyloid resulting in a “sago” spleen appearance. B. Eosinophilic
hyalinized germinal center as a result of amyloid deposition.
 Spleen and Hemolymph Nodes
A A
B B
Figure 2-69  Splenic amyloidosis in a dog (A) and in a horse (B).
A. Uniformly pale orange, slightly swollen spleen, with a waxy
consistency. B. Multifocal eosinophilic hyalinized foci of amyloid
that involve the germinal centers and spare the red pulp vascular
spaces.
Most lysosomal storage diseases (see Vol. 1, Nervous system)
will ultimately result in accumulation of the undegradable
substrate in histiocytes that accumulate in the spleen and
lymph nodes. Such accumulation causes a uniformly enlarged,
firm, often pale red spleen.
Hemosiderin is a storage form of iron and the only splenic
pigment of importance (Fig. 2-70). The amount and form of
storage iron vary greatly in normal animals with age and
species. The pigment is usually present only in macrophages,
but in long-standing iron accumulation, it may be encrusted
on fibers of connective tissue. The amount of hemosiderin is
significant only when it is in excess to the point of causing
tissue injury and fibrosis, and justifies the name hemosiderosis.
Iron is absorbed in excess in any anemia where it is sufficiently
available. Increased iron is one of the splenic changes in hemo-
lytic anemias. The patterns of iron storage in the spleen and
marrow can yield information about the type of anemia. In
nonresponsive anemias, the iron is mainly aggregated into
large coarse granules within the phagocytes, which themselves
are inapparent. In responsive hemolytic anemias where there
is rapid iron turnover, there is a spectrum of iron deposition
from barely stainable, diffusely or focally distributed ferritin,
to coarse hemosiderin. In the anemias of chronic disease, there
165
Figure 2-70  Splenic hemosiderosis in a cow. A. Phagocytosed
hemosiderin appears as aggregates of large coarse granules. B.
Pigment can be confirmed as hemosiderin through blue staining
with Prussian blue.
is increased coarse splenic iron as a result of continued scav-
enging of the transferrin system by the acute phase–reacting
proteins.
Further reading
Cole PA. Association of canine splenic hemangiosarcomas and hema-
tomas with nodular lymphoid hyperplasia or siderotic nodules. J Vet
Diagn Invest 2012;24:759-762.
Murakami T, et al. Atypical AA amyloid deposits in bovine AA amyloi-
dosis. Amyloid 2012;19:15-20.
Rupture of the spleen
A normal spleen can be ruptured by the level of trauma that
cats and dogs suffer regularly in automobile collisions or
through blunt trauma, for instance, kicks. The result is more
or less severe loss of blood into the abdomen often causing
death. Pathologic rupture may occur in any species with only
minor trauma if the spleen is enlarged and the capsule thinned.
Severe diffuse splenomegaly or focal splenic nodules may
develop secondary to a variety of causes, including diffuse
passive congestion, hyperemia secondary to septicemia,
 165.e1

Further reading
Day MJ, et al. A review of pathological diagnoses made from 87 canine
splenic biopsies. J Small Anim Pract 1995;36:426-433.
166 CHAPTER 2  •  Hematopoietic System
Figure 2-71  Splenic hematoma in a dog. Localized hematoma
covered by stretched capsule with focal tear. (Courtesy S.
Kesdangsakonwut.)
Figure 2-72  Splenic scar in a dog. Healing of a focal tear resulted
in contracted scar tissue. (Courtesy E.P. Lane.)
hyperplastic or neoplastic cell proliferations, infarcts, or hema-
tomas. As a result of rupture, the spleen may be fully divided
into 2 or more parts, or have a merely focally torn capsule. In
some cases, only the red pulp will be ruptured resulting in a
localized hematoma covered by the intact capsule (Fig. 2-71).
Early hematomas appear as solitary, large, dark red, bulging
masses that are prone to rupture, causing hemoabdomen and
death resulting from hypovolemic shock. Over time, splenic
hematomas may resolve and develop into soft brown masses
that are histologically characterized by infiltrated macro-
phages that have phagocytized erythrocytes. Breakdown of
hemoglobin into bilirubin and hemosiderin is responsible for
color changes. Complete healing will usually result in con-
tracted scar tissue (Fig. 2-72). In areas of a ruptured capsule,
the spleen is normally contracted, and clotted blood may
adhere to the tear (Fig. 2-73, eFig. 2-14). Small non–life-
threatening ruptures will heal, and scarring lines of healing
can produce notches and fissures.
Spleen and Hemolymph Nodes
Figure 2-73  Splenic rupture in a dog. The ruptured spleen is
contracted and clotted blood adheres to the tear. (Courtesy R. L.
Amorim.)
Figure 2-74  Splenosis in a dog. Widespread seeding of splenic
explants onto the serosal surfaces following rupture. (Courtesy S.
Kesdangsakonwut.)
Concurrent hepatic lacerations are usually characterized by
a rise in transaminases, indicating an additional source of
abdominal hemorrhage. Following rupture of the splenic
capsule and spilling of the sinus cells into the abdominal
cavity, there may be widespread seeding of splenic explants
onto the serosal surfaces, called splenosis (Fig. 2-74). These
accessory spleens are functional, and there may be hundreds
on the omentum with a few on the peritoneum. They appear
grossly like hemal nodes and histologically like normal spleen.
Following splenectomy, any splenic implants present will
increase in size and may give some protection against infec-
tious diseases.
Further reading
Tian J, et al. Evaluation and establishment of a canine model of delayed
splenic rupture using contrast-enhanced ultrasound. Mol Med Rep
2012;6:483-487.
 166.e1

A B

C
eFigure 2-14  Splenic rupture in a dog. A. Contracted spleen with clotted blood adhering to the
tear. B. Ruptured splenic hematoma. (Courtesy R.L. Amorim.) C. Following complete rupture,
the scared splenic fragments are shown as “multiple” spleens.
166.e2

Further reading
Pollock S, et al. Traumatic subcapsular splenic cystic hematoma in the
dog/a case study. Vet Med Small Anim Clin 1978;73:600-606.
 Spleen and Hemolymph Nodes
Figure 2-75  Splenic volvulus in a dog. Volvulus of the spleen and
stomach occurs mainly in deep-chested dogs.
Volvulus of the spleen
Volvulus affecting only the spleen (commonly referred to as
splenic torsion) occurs in pigs, dogs, and humans, and rarely
in horses. Volvulus of the spleen and stomach occurs mainly in
deep-chested dogs (Fig. 2-75). When the whole spleen is twisted
around the gastrosplenic ligament, there is severe congestion
and hemorrhagic infarction because of occlusion of the vein
and ultimately blockage of the artery. When the distal portion
is twisted, only the vein tends to be occluded, causing hemor-
rhagic infarction and congestion.
Splenic volvulus in the dog occurs in large breeds and is
characterized by enlargement of the upper abdomen with
painful guarding on palpation, and the passage of dark brown
urine. Hematologically, there is a characteristic brown discol-
oration of plasma as a result of leaching of blood pigments
through the capsule as it becomes necrotic. Associated signs
consist of the postsplenectomy state with many distorted
red cells, hypersegmented neutrophils, and the presence of
Howell-Jolly bodies and red cell pits, which are unusual in the
peripheral blood of the dog with a normally functional spleen.
Grossly, the spleen is diffusely enlarged, dark blue-black, and
often folded so its visceral surfaces touch each other in a “C”-
shaped form. On surgical removal, such spleens may weigh as
much as 5 kg and contain a large proportion of the original
red cell mass. Following splenectomy, there is risk of infection
by organisms normally controlled by the spleen. In the dog,
hemotropic mycoplasmal infections and severe hemolytic
anemia are the most likely adverse sequelae.
Splenic volvulus in pigs usually occurs in sows, and
often several are affected in the same herd over a period of
a few days. Circumstantial evidence suggests that a change
from once- to twice-daily feeding will prevent further losses,
but other factors are most likely involved. Affected animals
usually die acutely. Grossly the spleen is blue-black, massively
and uniformly enlarged, and twisted 180 degrees about its
long axis.
Further reading
Grange AM, et al. Evaluation of splenectomy as a risk factor for gastric
dilatation-volvulus. J Am Vet Med Assoc 2012;24:461-466.
167
A
B
Figure 2-76  Splenomegaly in a dog (A) and a pig (B). A. Try-
panosoma evansi can cause diffuse splenic congestion, a so-called
“bloody” spleen. (Courtesy S. Kesdangsakonwut.) B. Erysipelothrix
rhusiopathiae commonly causes noncongested splenomegaly, a
so-called “meaty” spleen.
Sartor AJ, et al. Association between previous splenectomy and gastric
dilatation-volvulus in dogs: 453 cases (2004-2009). J Am Vet Med
Assoc 2013;242:1385-1391.
Weber NA. Chronic primary splenic torsion with peritoneal adhesions
in a dog: case report and literature review. J Am Anim Hosp Assoc
2000;36:390-394.
Splenomegaly and splenic nodules
Splenomegaly is a descriptive term for a diffusely enlarged
spleen and provides little information about the underlying
disease mechanism. The degree to which splenic size departs
from normal is difficult to assess, but weight is always a useful
parameter, particularly in animals that die without exposure
to barbiturate anesthetics. Splenomegaly is common, espe-
cially in animals with a storage spleen, and the interpretation
of splenic size at autopsy can be gained by reference to the
whole carcass and to the cut surface of the organ.
Enlarged spleens can be divided into congested, so-called
“bloody” spleens, or noncongested, so called “meaty” spleens
(Fig. 2-76). Congested spleens are usually dark red to blue-
black, and the red pulp bulges on cross section and oozes
 167.e1

Further reading
Neath PJ, et al. Retrospective analysis of 19 cases of isolated torsion of
the splenic pedicle in dogs. J Small Anim Pract 1997;38:387-392.
Wendt M. Stomach torsion in swine. Tierarztl Prax 1987;15:375-376.
168 CHAPTER 2  •  Hematopoietic System
blood. Noncongested spleens feel firmer, hence the descriptive
term “meaty,” and do not ooze blood on their cut surface. The
color of such enlarged noncongested spleens varies depending
on the underlying disease process, and hyperplastic follicles
may be visible grossly.
Congested splenomegaly is most commonly the result of
circulatory disturbances (see later) and the process can result
from acute hyperemia, for instance, septicemia, passive con-
gestion; or euthanasia with barbiturates, splenic volvulus,
acute hemolytic anemia; or infectious disease, for instance,
babesiosis or trypanosomiasis. Noncongested splenomegaly can
be caused by a number of different disease mechanisms,
including phagocytosis, cell proliferation, or storage material.
Each of these conditions is described in more detail under
inflammatory, neoplastic, or degenerative diseases of the
spleen. Although many acute bacterial septicemias can cause
diffuse splenic congestion, subacute to chronic infections are
often associated with noncongested splenomegaly, for instance,
salmonellosis (Fig. 2-77). The following is an arbitrary list of
diseases to be considered when congested splenomegaly is
present.
Cattle and sheep
Anthrax
Salmonellosis
Babesiosis
Hemolytic disease
Horses
Equine infectious anemia
Isoimmune hemolytic anemia
Salmonellosis
Anthrax
Barbiturates
Pigs
Volvulus
Salmonellosis
Isoimmune hemolytic anemia
Mycoplasma haemosuis
African swine fever
Figure 2-77  Congested splenomegaly in a pig. Salmonella
choleraesuis can cause severe diffuse splenic congestion. A
normal spleen is shown below for comparison. (Courtesy S.
Kesdangsakonwut.)
Spleen and Hemolymph Nodes
Dogs and cats
Barbiturates
Acquired hemolytic anemias
Leishmaniasis
Volvulus (dogs)
Trypanosomiasis
The absence of splenomegaly does not eliminate any of
these causes, but makes them unlikely. Multiple focal lesions
normally do not cause splenomegaly, although hemangiomas
of the canine spleen and metastatic melanomas of the equine
spleen are exceptions.
In contrast to diffuse splenomegaly, single or multiple
nodules are also commonly encountered in the spleen. In
similar fashion as diffuse splenomegaly, splenic nodules can
be further divided into “bloody” nodules and “firm” nodules
(Fig. 2-78). The most common types of bloody nodules are
splenic hematomas or vascular neoplasms, for instance, hem-
angioma, hemangiosarcoma (Fig. 2-79). Hematomas can occur
secondary to trauma or can be caused by vascular neoplasms.
Splenic hyperplastic nodules can also become engorged
with blood and may closely resemble hematomas grossly (Fig.
2-80, eFig. 2-15). Acute splenic infarcts may also grossly
resemble “bloody” nodules, but their marginal distribution and
shape usually allow differentiation on gross examination.
B
Figure 2-78  Splenic nodules in a dog. A. Splenic hematoma
representing a “bloody” nodule. B. Cross section of splenic nodular
hyperplasia representing a “firm” nodule.
 Spleen and Hemolymph Nodes 169

A A

B B
Figure 2-80  Splenic hyperplastic nodule in a dog. A. Hyperplas-
tic nodules can become engorged with blood and appear as
“bloody” nodules. B. A few splenic follicles surrounded by severely
congested splenic parenchyma are indicative of the pathogenesis
of this “bloody” nodule.

sarcomatous have been classified as fibrohistiocytic nodules

(see later).

Circulatory diseases of the spleen

C erysipelas of swine, and Streptococcus pneumoniae infection in
Figure 2-79  Splenic vascular neoplasms representing a “bloody”
nodule, in a dog. A. Cross section of well-circumscribed, encap-
sulated, multilocular splenic hemangioma. B. Hemangiosarcomas
can cause focal splenic hematomas. C. Neoplastic, anaplastic
endothelial cells form incomplete vascular channels.
Incompletely contracted areas of spleen may appear similar to
infarcts. Firm nodules can represent a number of different
disease processes, including hyperplastic nodules, neoplastic
nodules, abscesses, and granulomas. Hyperplastic nodules
occur most commonly in the spleen of old dogs, and lesions
that are difficult to differentiate as hyperplastic versus
Active hyperemia is common in acute systemic infections and
also occurs in some acute bacterial intoxications, such as
anthrax infections in cattle, clostridial enterotoxemia of calves,
neonatal calves and goats. Passive congestion of the spleen
may arise as a result of disturbances in the systemic and portal
circulation, and is a feature of some of the acute hemolytic
anemias. The most common cause of passive congestion is the
use of barbiturates for euthanasia or anesthesia in horses and
dogs, or the earlier described splenic volvulus in dogs or pigs
(Fig. 2-81). Central (cardiac or pulmonary) venous congestion
does not usually cause congestion of the spleen in animals,
partly because they usually do not live long enough, and partly
because the splenic capsule is not elastic as in sheep, or is
highly muscular as in the dog and horse and not easily dis-
tended. Portal obstruction, with chronic hepatic fibrosis and
hepatic congestion, causes significant splenic enlargement in
humans, but this sequence of events is rare in animals.
 169.e1

eFigure 2-15  Splenic hyperplastic nodule, in a dog. Hyperplastic

nodules may become very large and are prone to rupture.
170 CHAPTER 2  •  Hematopoietic System
Figure 2-81  Diffuse splenic congestion, in a dog. Euthanasia
with barbiturates can cause diffuse congested splenomegaly.
With acute hyperemia, the spleen becomes moderately
enlarged and oozes blood on cut section. Histologically, acute
hyperemia is first observed in the marginal zones, followed by
the splenic cords. In acute septicemia, such as anthrax, disten-
tion with blood may represent the only finding. In more
chronic cases, neutrophils and macrophages may accumulate
in marginal zones and splenic cords, and there is a marked
increase in fixed cells in the red pulp and heavy sinus coloniza-
tion with macrophages and hemosiderin. A passively congested
spleen is extremely enlarged, moderately turgid, and cyanotic
with the capsule blue-black. The normal architecture is not
discernible on the cut surface, and the pulp is red-black,
bulges, and progressively exudes blood. Microscopically, the
splenic sinuses are dilated with packed red cells, and the ger-
minal centers are widely separated, and trabeculae are thinned.
The severity of acute hyperemia may make histologic exami-
nation difficult in the horse because the tissue, being largely
clotted blood, fragments on sectioning. Splenic samples from
horses destroyed with barbiturate should be taken from a thin
marginal area where the parenchyma is supported on 2 sides
by capsule.
Spleens that are enlarged for any reason are prone to
thrombosis and infarction. Thrombosis of the splenic vein has
been observed in association with traumatic reticulitis and
portal thrombosis in cattle, arterial thrombosis in cattle with
theileriosis, and with splenic abscesses in horses. Thrombosis
of both arteries and veins is seen in hypercoagulable states,
such as immune hemolytic anemias, purpura, and hemor-
rhagic pancreatitis. Infarcts are most commonly observed in
the subcapsular areas because the red pulp in these areas is
poorly perfused and has a reduced venous return (Fig. 2-82).
Acute infarcts might be difficult to visualize because they
appear as diffusely hemorrhagic, sharply demarcated areas
that raise the splenic capsule. In more chronic stages, infarcts
appears as wedge-shaped areas, extending from the capsule
into the parenchyma, that are sharply demarcated and change
in color from dark red to gray-white as the lesion becomes
more organized (Fig. 2-83, eFig. 2-16). Ultimately, infarcts will
resolve as fibrous scars. In acute classical swine fever in pigs,
there is widespread endothelial damage and fibrinoid throm-
bosis of splenic follicular arterioles, resulting in raised dark
infarcts 0.2-2.0 cm in diameter in the splenic capsule. Severe
Spleen and Hemolymph Nodes
Figure 2-82  Splenic infarct in a pig infected with classical swine
fever virus, formalin-fixed spleen. Acute infarcts appear as dif-
fusely hemorrhagic, sharply demarcated areas that raise the
splenic capsule (bottom), whereas chronic infarcts (top) change
in color from dark red to gray-white and become more organized.
(Courtesy S. Kesdangsakonwut.)
Figure 2-83  Splenic infarct as a result of metastasizing carcinoma
in a dog. Sharply demarcated, wedge-shaped, gray-white chronic
infarct extending from the capsule into the splenic parenchyma.
endothelial damage secondary to septicemic salmonellosis has
been observed to cause splenic infarcts in pigs, and there are
a few reports of Mycoplasma haemosuis also causing splenic
infarcts. In dogs, infarcts occur most likely with various splenic
neoplasms, including myeloma, lymphoma, or myeloid leuke-
mias, as a result of thrombocytopenia, procoagulant content
of hypogranular promyelocytic leukemia, or hyperviscosity
associated with myelomas (Fig. 2-84). The spleen may suffer
infarction secondary to hyperviscosity syndrome caused by
massive hyperproteinemia most commonly associated with a
monoclonal gammopathy, but hyperviscosity can also be
caused by leukemia, polycythemia vera, essential thrombocy-
tosis, or myelodysplastic disorders. Embolism with infarction is
uncommon. The outcome has the usual dependence on
whether the emboli are septic or bland. Emboli are frequently
derived from valvular vegetative endocarditis. In dogs, Dirofi-
laria immitis may cause thromboembolism (Fig. 2-85).
Incompletely contracted splenic areas occur most com-
monly in dogs secondary to disseminated intravascular coagu-
lation, where small thrombi prevent vascular outflow of
segments of spleen. Lesions develop when spleens contract
during cardiogenic shock or following a parasympathetic
 170.e1

B
eFigure 2-16  Splenic infarcts in dogs. A. Hemangiosarcoma
causing multiple wedge-shaped infarcts along the splenic margins.
B. A metastasizing carcinoma causes vascular emboli and multifo-
cal splenic infarcts.
 Spleen and Hemolymph Nodes
A spores of most remarkable durability. It is the combination of
B natural excretions, as well as pathologic exudates, and these
Figure 2-84  Splenic thrombosis in a dog. A. Splenic infarcts in a
dog with multiple myeloma. B. Fibrin thrombus in splenic follicu-
lar arterioles, resulting in acute hemorrhagic infarcts.
Figure 2-85  Splenic infarcts in a dog. Dirofilaria immitis can
cause severe thromboembolism.
stimulus and affected areas fail to empty. Such lesions are
grossly indistinguishable from splenic infarcts and are also
located along the splenic margins.
Specific infections of lymphoid tissues
Anthrax
Anthrax is caused by Bacillus anthracis, a large, gram-positive,
spore-forming organism that is highly pathogenic for most her-
bivorous animals and humans, whereas carnivorous birds and
reptiles are resistant. Domestic animals are susceptible to B.
171
anthracis in the decreasing order of goats, sheep, cattle, horses,
pigs, and dogs. Farmed mink are highly susceptible. In rumi-
nants, the disease is usually brief and septicemic; in horses,
pigs, and dogs, it is frequently localized to the throat or intes-
tine and may be fatal before invasion of the blood occurs.
When the disease is septicemic, as it usually is in herbi-
vores, the blood and tissues of the animal swarm with vegeta-
tive organisms which, when exposed to air or oxygen, form
these 2 factors, the number of organisms and the resistance
of spores, which is of paramount importance in the epidemiology
of the disease.
Bacillus anthracis probably has limited capacity for growth
in the external environment, due in part to antagonistic soil
bacteria. Growth may occur in alkaline soils with much decay-
ing vegetable matter, and alternate periods of rain and drought
and temperatures in excess of 15.5° C may also facilitate
growth. The spores are known to remain viable in soil for at
least 15 years, and probably much longer, because they have
been noted to retain their vitality and virulence for 50 years
in the laboratory. Natural survival of vegetative organisms is
rather short, equivalent to the short duration of an infection;
vegetative organisms do not survive in a carcass, but are
rapidly killed by putrefactive bacteria. In the terminal stages
of the disease, large numbers of bacilli are excreted in all
organisms sporulate and perpetuate infections. As a general
rule, the spores are very resistant to methods of disinfection, with
the exception of chemical disinfectants that are oxidizing
agents. Spores on skin have even survived tanning processes
to become a hazard for humans.
Reliable details on how spontaneous infections are initiated
in animals are not available, but the disease in wild herbivores
often follows construction requiring excavation, such as
fencing and excavation of dug-outs for watering livestock. It
is accepted that dogs and pigs acquire the infection as a result
of eating an animal that had anthrax, and deaths in humans
have occurred after eating inadequately cooked meat from a
goat dead of anthrax. Anthrax in pigs has been traced to the
ingestion of bone meal that was not sufficiently sterilized.
Vegetative bacilli are unlikely to cause the disease because
they are rapidly destroyed in the acid medium of the stomach.
Cattle and sheep are presumed to obtain the infection by
ingestion of contaminated food and water, entry through
mucous membranes possibly being aided by local trauma.
Cutaneous infection is rare in cattle but has been reported in
India, and pulmonary anthrax resulting from the inhalation of
spore-laden dust can occur. Infection through the skin is occa-
sionally seen in sheep, and may be assisted by grass seed
infestation. Ingestion is an important mode of infection in
horses and dogs, as indicated by the common occurrence of
lesions in the throat. It is also thought that infection can be
transmitted to horses by blood-sucking insects. Intestinal
anthrax in pigs probably reflects infection by ingestion.
The pathogenesis of anthrax is an initial lymphangitis and
lymphadenitis that develops into septicemia. This sequence is
especially well illustrated in the pulmonary form of the exper-
imental disease. Spores that are inhaled are ingested by cells
lining alveoli and transported in them to the tracheobronchial
nodes, in which vegetation and true initiation of the infection
occur. Spread to the blood is via lymphatics as well as by
lymphovenous connections within lymph nodes, and numer-
ous bacilli spread in the lymph from node to node as the
172 CHAPTER 2  •  Hematopoietic System
filtering mechanism of each is successively swamped. Bacilli
that enter the blood are taken up in other parts of the mono-
nuclear phagocyte system, especially the spleen, to establish
secondary centers of infection and proliferation.
There is notably little response on the part of a susceptible
animal to the local establishment of anthrax infection. Physi-
ologic disturbances, clinical signs, and death depend on the devel-
opment of a massive septicemia. In immune animals, for instance,
guinea pigs, infection is followed by a period of 2-4 hours in
which the bacilli proliferate; the area then becomes infiltrated
by leukocytes, and the bacilli fragment with little or no phago-
cytosis of intact organisms. The lysis of bacilli, of which the
first sign is loss of capsule and staining properties followed by
fragmentation, is apparently caused by anthracidal substances
in plasma or, more likely, liberated from leukocytes. Anthrax
has always been regarded as dependent on invasiveness rather
than toxigenicity, but it appears that antitoxic immunity does,
in some manner, inhibit invasiveness.
Vegetative cells produce a small array of toxins. The organ-
isms themselves and the capsular material are virtually non-
toxic, although the capsular material may act as a spreading
factor and inhibit the activity of leukocytes. The activity of
toxins in septicemic anthrax is well illustrated by the demon-
stration that, once the degree of bacteremia passes a certain
threshold, which is ~0.3% of the usual maximum, death will
occur even though all bacilli may have been destroyed by
antibiotic therapy. The toxin consists of 3 complementary com-
ponents designated factors I, II, and III, or edema factor, protective
antigen, and lethal factor, respectively. Edema factor is an adenyl-
ate cyclase that increases cyclic AMP after activation by
calmodulin. Protective antigen is likely a receptor-binding
protein that appears to be essential for the biological effects
of edema and lethal factors. Lethal factor is a central nervous
system (CNS) depressant, but its major effects may be else-
where. The toxins have been assessed only in experimental
laboratory animals, between species and strains, of which there
are considerable differences in sensitivity. The 3 toxins are
serologically distinct and, because there is autostimulation, it
is not surprising that they do not produce lesions when
injected separately. The combined effects of the 3 toxins are
injury and inactivation of phagocytes, increased capillary per-
meability, anticomplementary activity, and impairment of
coagulation.
Immunity to anthrax appears to depend on the neutraliza-
tion of toxin. Antibodies against the bacterial cells and cap-
sules are useless, as is evident from the experience that
completely avirulent or dead bacilli are not immunogenic; in
fact, the most potent vaccines are of bacilli that proliferate
and cause acute local inflammation but do not invade the
blood. The antigen that provokes antitoxic immunity is the
protective antigen that is presently regarded as being a non-
toxic degradation product (toxoid) of the very labile toxin
found in the blood of affected animals. The vaccine of Pasteur
apparently resulted from the loss of a temperature-sensitive
plasmid that encoded for the protective antigen.
When an animal is suspected of having died of anthrax,
organisms should be detected in smears of blood or local
exudate. All bacilli in internal organs are likely to be destroyed
in 48 hours or less by putrefaction. Hence it is best to obtain
blood for diagnostic purposes from close to the coronet or the tip
of the tail, places that are likely to be involved last by putrefac-
tive processes that destroy the vegetative bacilli. Bacillus
anthracis occurs in blood in pairs or in short chains of 3-4 cells.
Spleen and Hemolymph Nodes
They are large truncate organisms that are easily observed.
They are differentiated from putrefactive bacteria by their distinct
capsule, which stains pink with old methylene blue, and by having
square ends when these are apposed. The free ends of B. anthra-
cis are often rounded as in Clostridium spp. The organism can
be cultured readily from putrefied exudates if advantage is
taken of its aerobic requirements and the heat resistance of
the anthrax spores, to separate it from nonsporulating organ-
isms and sporulating anaerobes. The organism can also be
purified in guinea pigs by applying the inoculum to scarified
skin. In nonsepticemic anthrax, such as occurs in swine and
dogs, it is best to look for the organism in the local exudates
and affected lymph nodes. For the diagnosis of anthrax, when
other methods have failed or the tissues are old and dry, the
Ascoli agar-gel precipitin test is useful but not completely
specific.
Bovine anthrax is usually septicemic, and sudden death is
usually the first indication of its presence in a herd. Even when
cattle are observed closely, they may be dead within 1 hour
of showing signs of illness, although some will show general
signs of illness for about 24 hours before death. The signs of
illness vary with the route of entry and when, as usually
happens, entry is by inhalation or ingestion with no area of
localization, the animals are depressed and listless. On exami-
nation, there is high fever, increased heart and respiratory
rates, and congested and terminally cyanotic mucosae that
show evidence of bleeding. Animals that survive for a day may
have dysentery, abortion, edematous swellings of the perineum,
throat and abdominal wall, and blood-stained milk. Although
infection may be virtually synonymous with disease and death
in goats and sheep, it is not necessarily so in cattle, in which
species some infected animals probably recover. This is suggested
by the recovery of some animals that are experimentally inoc-
ulated and by the occurrence of transient febrile reactions
attributable to no other causes, in herds in which fatal anthrax
is occurring. For most animals, however, treatment to be effec-
tive must be administered early before there is marked septi-
cemia and toxin production.
The carcass of an animal dead of this disease putrefies quickly,
becomes very rapidly distended with putrefactive gases, and
blood exudes from the natural orifices. These changes are, of
course, not diagnostic, but when they are observed in an
animal that has died suddenly in an area in which anthrax is
endemic or has at any time occurred, examination of smears of
blood should always precede autopsy. Anthrax in the fulminat-
ing disease is very largely an intravascular infection, with most
of the organisms in the blood and the rest in the spleen. Sep-
ticemia in anthrax is a terminal event, and smears of blood
may not be helpful when prepared more than a few hours
before death.
The morbid picture of the disease in cattle is characterized
by splenomegaly, multiple hemorrhages, and edematous effu-
sions in connective tissues. A very large soft spleen is the most
significant lesion, and very rarely is it absent (Fig. 2-86). Sple-
nomegaly occurs in other diseases of cattle, but rarely is it as
large in association with sudden death. In anthrax, the spleen
is soft, sometimes it ruptures spontaneously, and when it is
incised, the pulp exudes very thick black-red blood that
brightens in color on exposure to air. Smears and sections of
the spleen reveal very large numbers of bacilli if the carcass
is fresh (eFig. 2-17), but, when decomposition is advanced,
they are destroyed by putrefactive changes. In some cases,
splenomegaly is the only lesion. The histology of the spleen is
 172.e1

B
eFigure 2-17  Anthrax, in a cow. A. The red pulp is expanded by
sludged red cells admixed with numerous leukocytes and bacilli
in chains. B. Cytologic smears of sections of spleen stained with
M’Faydean stain reveal very large numbers of bacilli. (Courtesy
R.B. Moeller.)
 Spleen and Hemolymph Nodes
A The vessels are intensely congested, and hemorrhage extends
B as hard circumscribed nodules. The disease in sheep takes the
C described for swine. When septicemia occurs, the morbid
Figure 2-86  Anthrax in a cow. A. Severe splenomegaly with very
thick black-red blood draining from cross sections. (Courtesy
A.W. Layton.) B. The red pulp is expanded by sludged red cells
admixed with numerous leukocytes and bacilli in chains. C. Cyto-
logic smears of sections of spleen reveal very large numbers of
bacilli. (Courtesy R.B. Moeller.)
not revealing. The sinus areas are distended with sludged red
cells, and the lymphoid follicles are widely separated and
hypocellular, but numerous leukocytes and bacilli in chains
are present. It is typical of septicemic anthrax that the organ-
isms are always intravascular.
173
Cattle are moderately resistant to B. anthracis so that local
lesions may occur at the site of entry. Local lesions are usually
in the small intestine and take the form of ulcerative hemor-
rhagic enteritis, but acute inflammation in the abomasum and
large intestine may also occur. The most severe lesions may be
over the lymphoid tissues of the intestine, or extend for a
considerable distance from these. The mucous membrane is
intensely red, and is sprinkled with small hemorrhages. The
contents of the intestine are then deeply stained with blood.
Superficial necrosis and ulceration occur in some areas of most
intense hyperemia. The corresponding mesentery, up to the
regional nodes, is infiltrated with gelatinous fluid as a result of
acute lymphangitis, and the fluid may be stained with blood.
The regional nodes have the appearance of the spleen. They
are enlarged, red-black, and on cut surface are moist and shiny.
into the peripheral sinuses and cortex. Bacilli are numerous
and leukocytes are present, but there is no necrosis.
In some cases in which the organisms gain entry through
the oropharynx, there is hemorrhagic lymphadenitis of the
nodes of the throat and edema of the connective tissues in
these regions. The occasional case of pulmonary anthrax in
cattle is characterized by acute congestion and consolidation
of a portion of the lung with larger areas of interstitial edema,
edema of the mediastinum, and regional hemorrhagic lymph-
adenitis. The pulmonary lesion is exudative, but lacks the full
gamut of inflammatory change unless another cause of pneu-
monia is superimposed.
Sheep are more susceptible to B. anthracis than are cattle,
and local lesions do not occur except in the unusual instances
of percutaneous infection, in which the lesion may take the
form of spreading edema from the outset or initially appear
same course as that in cattle except that it is even more rapid.
Splenomegaly is not as prominent in sheep as in cattle, likely
because of the greater level of collagen in the splenic capsule
of sheep. The parenchyma is, however, dark and soft. Edema-
tous effusions do not occur in sheep.
Clinical signs of anthrax in horses may last for several days
and are characterized by colic or by large edematous swellings.
The swellings, which can be very extensive, occur on the
ventral part of the abdomen and thorax, the legs, in the peri-
neal region, and about the external genitalia. Dysentery may
accompany the acute colic. When ingestion is the route of
infection in horses, the primary lesion may be in the throat or
the intestine, and death may occur from the local reaction and
without septicemia. Intestinal lesions are similar to those
described for cattle, and pharyngeal lesions are similar to those
changes are the same as in cattle, including very prominent
enlargement and congestion of the spleen.
Pigs are relatively resistant to anthrax. They acquire the
infection from eating infected flesh, and the infection remains
localized to the throat or intestine. Because septicemia is
exceptional, splenomegaly is not a prominent part of the gross
picture. The characteristic sign is swelling of the pharyngeal
region and neck. Some pigs have diarrhea and dysentery, but
it is unusual to have intestinal localization without pharyngeal
localization. Anthrax without illness has been observed in
swine; in such cases, the disease is limited to isolated mesen-
teric or pharyngeal nodes.
The local lesion of anthrax in swine is a typical carbuncle
at the point of entry, with acute regional lymphadenitis and
174 CHAPTER 2  •  Hematopoietic System
lymphangitis. Some bacilli no doubt reach the blood, but they
do not establish septicemia. Bacteria may localize in liver,
spleen, or kidney to produce a metastatic carbuncle but,
usually, only individual nodes near the site of entry are
involved. The lymphadenitis may be diffuse or focal but in
both cases it is, in the initial stages, hemorrhagic. An intense
leukocytic infiltration occurs, all cells within the affected por-
tions of the node die, and the focus becomes encapsulated.
With necrosis, the affected tissue changes from a brick-red to
a gray friable mass that can be easily shelled-out when the
gland is incised.
In primary intestinal anthrax in pigs, the initial lesion is focal
or multifocal hemorrhagic enteritis, with a central zone of
diphtheresis that eventually ulcerates. The adjacent serosa and
mesentery are thickened with edema fluid and yellow, with
foci and streaks of hemorrhage; they are the site of focal
hemorrhagic necrosis resulting from acute necrotizing vascu-
litis and lymphangitis. These mesenteric lesions extend only
as far as the regional nodes, which show the type of lymph-
adenitis characteristic of anthrax in swine.
Dogs are reputedly quite resistant to B. anthracis, but a
number of outbreaks have been observed in kennels in which
the dogs have inadvertently been fed meat from an animal
that has died of the disease. Anthrax in dogs may pursue a
peracute course to sudden death, it may be of the pharyngeal
type in which extensive edema develops in the face, head, and
neck, or it may be of the intestinal type with signs of acute
gastroenteritis. Anthrax may occur in mink with high mortal-
ity after feeding fresh meat from infected animals.
Further reading
Biswas PK, et al. Risk factors associated with anthrax in cattle on
smallholdings. Epidemiol Infect 2012;140:1888-1895.
Epp T, et al. Case-control study investigating an anthrax outbreak in
Saskatchewan, Canada - summer 2006. Can Vet J 2010;51:
973-978.
Liu S, et al. Anthrax lethal and edema toxins in anthrax pathogenesis.
Trends Microbiol 2014;22:317-325.
Mongoh MN, et al. Risk factors associated with anthrax outbreak in
animals in North Dakota, 2005: a retrospective case-control study.
Public Health Rep 2008;123:352-359.
Sweeney DA, et al. Anthrax lethal and edema toxins produce
different patterns of cardiovascular and renal dysfunction and syn-
ergistically decrease survival in canines. J Infect Dis 2010;202:
1885-1896.
Twenhafel NA. Pathology of inhalational anthrax animal models. Vet
Pathol 2010;47:819-830.
Leishmaniasis
The genus Leishmania includes protozoal parasites within the
family Trypanosomatidae. Species of the genus are parasites of
humans, dogs, and other mammals. Sandflies of the genus Phle-
botomus in the Old World and the genus Lutzomia in the New
World are the intermediate hosts. The tick Rhipicephalus may
act as mechanical vector. Direct transmission and vertical
transmission may occur in kenneled dogs.
In the mammalian host, Leishmania is found in macro-
phages in the amastigote form. Amastigotes are round to ovoid,
2.0-5.0 µm in diameter with a basophilic, vesicular nucleus
and smaller, rod-shaped, darker-staining kinetoplast, but no
flagellum. They are best visualized by Giemsa staining.
Spleen and Hemolymph Nodes
Amastigotes proliferate by binary fission and then rupture out
of macrophages and infect new cells. When sandflies ingest
blood from infected hosts, they also ingest host cells infected
with amastigotes. In the midgut of the sandfly, amastigotes are
released from cells and transform into their flagellated, procy-
clic, promastigote form and replicate. Promastigotes are leaf-
shaped with a single flagellum arising at the anterior pole.
Following sufficient replication and a number cell surface
changes, the promastigotes transition to the infectious meta-
cyclic form that detaches from the midgut and migrates to the
mouthpart, where it is injected into the host with saliva.
Leishmaniasis is important as a disease of humans, and
wherever it occurs in humans, it may also occur in dogs.
Although the human population may be the only reservoir for
some Leishmania spp., for instance, L. donovani, for other
Leishmania spp., for instance, L. infantum or L. braziliensis,
dogs, cats, and other carnivores, including rats, serve as reser-
voir hosts. Leishmaniasis may occur in 3 clinical forms:
• Cutaneous leishmaniasis (“oriental sore”) is the most
common clinical presentation and caused by different
Leishmania spp. in different parts of the globe. In the
Eastern Hemisphere, cutaneous leishmaniasis can be
caused by L. tropica, L. major, L. aethiopica, L. infantum,
and L. donovani. In the Western Hemisphere, the disease
is caused by the L. mexicana spp. complex (L. mexicana,
L. amazonensis, L. venezuelensis) or the L. braziliensis spp.
complex (L. braziliensis, L. guyanensis, L. panamensis, L.
peruviana).
• Mucocutaneous leishmaniasis (“espundia”) represents the
spread of cutaneous infection to the naso-oropharyngeal
mucosa and is caused by the L. braziliensis spp. complex.
• Visceral leishmaniasis (“kala-azar”) is the most severe form
and can occur with a wide variety of clinical signs. It is
usually caused by the species L. donovani and L. infantum
(L. chagasi).
The species of Leishmania are not well distinguished. Spe-
ciation depends on degrees of cross-immunity, different clini-
cal manifestations, geographic location, and reservoir hosts.
However, sequence data on kinetoplast DNA on several
species permits their molecular identification. In situ hybrid-
ization is available to identify leishmanial organisms and to
speciate these in formalin-fixed tissues.
Infections of animals with Leishmania spp. are common in
endemic areas, but many dogs may have subclinical disease.
Rate of infection in dog populations is usually based on sero-
logic data combined with immune response and detection of
parasitic DNA in tissues. Both cutaneous and visceral forms of
the disease have been described in dogs, as well as cases of vis-
ceral disease in which the organisms are diffusely present in
the dermis. Leishmaniasis is a disease of the monocyte-
macrophage system, and the visceral disease mimics histoplas-
mosis. The protozoa are not cytopathogenic in the usual sense,
and destruction of host macrophages appears to be purely a
mechanical consequence of proliferation of the protozoa in
the cytoplasm.
Cutaneous leishmaniasis in dogs can occur as generalized
or focal, exfoliative dermatitis with alopecia that commonly
starts around the limbs or the face and the ears, as ulcerative
dermatitis, as multinodular dermatitis, as mucocutaneous pro-
liferative dermatitis, and as papular dermatitis. The organisms
are inoculated by the biting insect and are soon ingested by
histiocytes. Rapid proliferation of the protozoa disrupts the
phagocytes, and the released organisms are ingested by other
 174.e1

Further reading
Dragon DC, et al. A review of anthrax in Canada and implications for
research on the disease in northern bison. J Appl Microbiol
1999;87:208-213.
 Spleen and Hemolymph Nodes
Figure 2-87  Leishmaniasis in a dog. Leishmania infantum can
cause severe lymphadenomegaly. (Courtesy R.C. Menezes.)
phagocytic cells to repeat the process. Lymphocytes and
plasma cells surround the lesion, and neutrophils are attracted
to the debris. When the inflammation extends to the overlying
epithelium, ulceration occurs especially over bony prominences
and along mucocutaneous junctions. Numerous parasites are
present within macrophages, and some are free in the tissue.
Many dogs will develop concurrent conjunctivitis, uveitis, and
blepharitis. Lymphadenomegaly of multiple subcutaneous
nodes is common (Fig. 2-87, eFig. 2-18). Onychogryphosis and
epistaxis develop in some affected dogs.
The clinical signs of visceral leishmaniasis in the dog are
of chronic debilitation and often recurrent oculonasal dis-
charge, with some crusting of the nose, and recurrent diarrhea.
Many dogs lose weight despite a ravenous appetite. There are
often focal scurfy skin lesions, but generally, in visceral leish-
maniasis, the presentation is of a mature dog in poor body condi-
tion with a rough haircoat. Other clinical signs may include
locomotion disturbances resulting from neuralgia, footpad
clefts, interdigital ulcers, or, rarely, paraparesis. There may be
mild enlargement of lymph nodes, and the spleen is always
symmetrically enlarged 2-3 times or more normal size and is
dark brown to black on the capsular surface (Fig. 2-88, eFig.
2-19). The oral and cervical viscera are normal and the lungs
generally have mild tan mottling but are otherwise normal, as
is the heart. The liver may contain numerous granulomas and
is symmetrically enlarged and dark brown. Renal disease is
common, and the kidneys are darker than normal, and while
of normal contour, a severe immune-complex glomerulone-
phritis can lead to chronic renal failure. The bone marrow is
uniformly reddened in midfemoral shaft in well-developed
cases, but the fat is generally of normal character. Other less
common lesions include non-erosive polyarthritis, polymyosi-
tis, osteolysis and osteoarthritis, proliferative periostitis, pan-
creatitis, meningitis, or chronic colitis.
The microscopic lesions in the spleen initially are of hemic-
lymphatic hypertrophy with macrophage proliferation and
focal granulomas. Splenic follicles are hyperplastic, often with
follicular hyalinosis. In advanced cases, there is atrophy of
lymphoid follicles, and the sinus areas may be diffusely occu-
pied by large macrophages heavily laden with intracytoplas-
mic organisms, and numerous plasma cells (Fig. 2-89). The
175
Figure 2-88  Leishmaniasis in a dog. Spleens infected with Leish-
mania infantum are symmetrically enlarged 2-3 times or more
normal size. (Courtesy R.C. Menezes.)
lesions in bone marrow may be focal, but consist of clusters
of epithelioid macrophages with phagocytosed organisms. In
the dog with a well-developed infection, the bone marrow will
have remarkable plasma cell hyperplasia that may approach
50% of cells present, and there is characteristically hypergam-
maglobulinemia. The protein is broadly polyclonal, and the
plasma cells lack atypia. Renal changes are variable and may
consist of interstitial scarring with some parasitic involvement;
however, the effects of hyperproteinemia and immune com-
plexes appear to injure the kidney indirectly. Animals with
leishmaniasis and amyloidosis have been described, and there
may be concurrent changes caused by both processes.
Diagnosis is based on the demonstration of the organism in
cytologic or in histologic preparations, the former being easily
achieved by aspiration of marrow, node, spleen, or liver. There
are now several tests based on immunoglobulin titers by
ELISA or PCR, immunohistochemistry, or in situ hybridiza-
tion to detect organisms in tissue.
Further reading
Barral-Veloso L, et al. A β-mercaptoethanol-modified enzyme linked
immunosorbent assay for diagnosis of canine visceral leishmaniasis.
J Vet Diagn Invest 2013;25:239-242.
Figueredo LA, et al. Clinical and hematologic findings in Leishmania
braziliense-infected dogs from Pernambuco, Brazil. Rev Bras Para-
sitol Vet 2012;21:418-420.
Koutinas AF, Koutinas CK. Pathologic mechanisms underlying the clini-
cal findings in canine leishmaniasis due to Leishmania infantum/
chagasi. Vet Pathol 2014;51:527-538.
Menezes RC, et al. Sensitivity and specificity of in situ hybridization for
diagnosis of cutaneous infection by Leishmania infantum in dogs.
J Clin Microbiol 2013;51:206-211.
Nascimento MS, et al. Naturally Leishmania infantum-infected dogs
display impairment of chemokine and chemokine-receptor expres-
sion during visceral leishmaniasis. Vet Immunol Immunopathol
2013;153:202-208.
Rigo RS, et al. Renal histopathological findings in dogs with
visceral leishmaniasis. Rev Inst Med Trop São Paulo 2013;55:
113-116.
 175.e1

eFigure 2-19  Leishmaniasis in a dog. Spleens infected with Leish-

mania infantum are symmetrically enlarged 2-3 times or more
normal size. (Courtesy R.C. Menezes.)
eFigure 2-18  Leishmaniasis in a dog. Leishmania infantum can
cause severe lymphadenomegaly. (Courtesy R.C. Menezes.)
176 CHAPTER 2  •  Hematopoietic System
A lestoquardi. Because T. parva and T. annularis only migrate to
B ceptibility of the cattle, and indigenous animals may have
C characterized by icterus, anemia, and occasionally hemoglo-
Figure 2-89  Leishmaniasis in a dog. (Courtesy R.C. Menezes.)
A. The splenic red pulp is expanded by large numbers of macro-
phages heavily laden with intracytoplasmic Leishmania infantum
organisms. B. Immunohistochemistry for L. infantum shows large
numbers of organisms in the cytoplasm of macrophages. C. In situ
hybridization for L. infantum shows intracytoplasmic organisms.
Theileriosis
Theileria spp. are obligate intracellular protozoal parasites of
the order Piroplasmida, family Theileriidae. The 2 most impor-
tant species in cattle and water buffalo are T. parva, which
causes East Coast fever of east and central Africa, and T.
annulata, which causes Mediterranean or tropical theileriosis
Spleen and Hemolymph Nodes
across North Africa and central Asia. Other Theileria spp.,
including T. mutans, T. velifera, T. taurotragi, T buffeli, and T.
sergenti, usually cause asymptomatic infection or can increase
severity of East Coast fever and tropical theileriosis. In small
ruminants, T. lestoquardi is the most virulent species and
occurs in Asia, the Middle East, and parts of Africa and Europe.
T. separate and T. ovis can also infect sheep and goats. Although
Theileria spp. can easily be identified with Giemsa stains in
blood smears or lymph node biopsies or at autopsy in impres-
sion smears of many different tissues, species differentiation
requires DNA-based molecular or serologic assays.
Theileria spp. are spread by biological tick vectors. Rhipi-
cephalus appendiculatus is the primary vector for T. parva, but
R. zembeziensis and R. duttoni are also carriers in parts of
Africa. Hyalomma spp. are the vectors for T. annulata and T.
the acinar cells of the tick salivary gland and mature after the
tick attaches to a host, ticks have to stay attached for a number
of days to the host prior to infecting it. Protozoal parasites are
excreted in the saliva between 3-5 days after ticks start feeding.
Under high temperatures, Theileria spp. might mature in unat-
tached ticks and can enter the host right after the tick attaches.
Transovarial transmission has not been reported. Within the
host, Theileria spp. reproduce by schizogony in lymphocytes (leu-
kocytic or tissue phase) and are subsequently found in red cells
(erythrocytic phase). A related genus is Cytauxzoon, which
reproduces by schizogony primarily in macrophages and by
fission in red cells.
Both, East Coast fever and tropical theileriosis are most
severe when susceptible animals are introduced to endemic
areas and mortalities can reach 100% and 90%, respectively.
The outcome of exposure is largely determined by the sus-
a morbidity rate of 100%, but mortality is usually <5%.
The incubation period for either disease ranges between
8-21 days.
Clinical signs are similar for East Coast fever and tropical
theileriosis and include high fever, depression, drooling,
lacrimation, diarrhea, anorexia, and weight loss, as well as
decreased milk production. Abortions are common. Petechiae
and ecchymoses occur in the conjunctiva and oral mucous
membranes. Corneal opacity is commonly observed. Severe
pulmonary edema with dyspnea and a frothy nasal discharge
is common and terminal in many animals, whereas others
become prostrate and comatose. These signs associated with
progressive and prominent enlargement of the superficial
lymph nodes give the disease a close clinical resemblance to
malignant catarrhal fever. Tropical theileriosis is furthermore
binuria because the parasites also destroy red blood cells.
Some animals with East Coast fever may develop a fatal con-
dition called “turning sickness,” which is characterized by neu-
rologic signs as a result of capillary impairment by parasites
in the CNS.
Sporozoites, of tick origin, invade and infect host lympho-
cytes and macrophages. Cell entry occurs through receptor-
mediated parasite-directed phagocytosis. Infected lymphocytes
are transformed in vitro into lymphoblastic cells. Macroschizonts
develop in the cytoplasm of the transformed cells and then
divide synchronously with the host lymphocytes to infect
their daughter cells (Fig. 2-90). After a few days, macroschiz-
onts enter the microschizont stage, in which the host cells are
destroyed and merozoites are released to invade erythrocytes.
 Spleen and Hemolymph Nodes 177

Figure 2-90  East Coast fever in a cow. Bovine lymphoblasts Figure 2-91  East Coast fever caused by Theileria parva in a cow.
containing numerous intracytoplasmic Theileria parva schizonts. Enlarged, diffusely pale lymph node with multiple petechiae.
(Courtesy Plum Island Animal Disease Center, U.S. Department (Courtesy Plum Island Animal Disease Center, U.S. Department
of Agriculture.) of Agriculture.)

Merozoite entry into red cells occurs by a similar process.

During the acute phase of the disease, more than 60% of
lymphocytes may contain schizonts, also called Koch’s bodies.
The acute disease appears to be caused by massive lympholysis
and progressive anemia. Leukopenia is progressive from the
onset of fever, and, terminally, very few leukocytes may be
seen in blood smears. The total white count is seldom >2 ×
109/L, and often only a fraction of this, with the remaining
cells primarily neutrophils and lymphocytes. There is brief
initial stimulation of myelopoiesis, but as disease progresses,
neutropenia with immaturity and toxemia develops. Ulti-
mately, there is loss of precursors and trilineage depression
with accumulation of hemosiderin in macrophages. Termi-
nally, the bone marrow is hypoplastic, and the remaining cells
consist of large blastic parasitized lymphocytes and atypical
erythroblasts. In tropical theileriosis, destruction of erythro-
cytes is intimately related to fission of the piroplasm forms in
erythrocytes. Figure 2-92  East Coast fever caused by Theileria parva in a cow.
Gross lesions in acute disease resemble those of malignant Periacinar hepatocellular necrosis and inflammation with irregular
catarrhal fever. There is enlargement of lymphoid tissues, canalicular cholestasis. (Courtesy E.P. Lane.)
including Peyer’s patches. On cut surface, the lymph nodes
are diffusely discolored with a red-brown cortex containing
focal hemorrhages, and a dark red-brown medullary area (Fig. and fibrinous exudate throughout the cortical areas of nodes.
2-91). Serous effusion and gelatinous or hemorrhagic edema Lympholysis is prominent in germinal centers, and there is a
of connective tissues are common. The spleen is enlarged in general loss of small lymphocytes, with those that remain
the acute disease, but in cases with a prolonged course, it may appearing large and blastic. There is early splenic lymphoid
be shrunken and strap-like. There is ulcerative abomasitis. The hypertrophy that is later followed by lympholysis. Germinal
so-called “infarcts” of the liver and kidney are actually prolif- centers remain prominent and are surrounded by areas of
erative foci of perivascular lymphocytes. These foci, which hemorrhage. The hypocellular follicular centers are usually
project slightly, produce a mottling of small gray-white patches occupied by fibrinous or hyaline exudate similar to that seen
visible on the surface of the liver and kidney. The lungs are in lymph nodes. There is hepatic periacinar and, to a lesser
congested and edematous with increased texture on palpation, extent, periportal lymphocytic infiltration and, in addition,
and increased weight. Small hemorrhages associated with foci there are focal infiltrations of the hepatic capsule that give rise
of hyaline degeneration occur in the muscles, and petechiae to the raised foci seen grossly. In some cases, there is periacinar
are commonly present under the tongue and in the vulva. hepatocellular necrosis and irregular canalicular cholestasis
Erosive or catarrhal enteritis overlies lymphocytic hyperplasia with foci of inspissated bile (Fig. 2-92). The kidneys are
and infiltration of the gut mucosa. remarkably congested with focal hemorrhage, and there is
Histologically, in the early stages of infection, there is diffuse interstitial infiltration with lymphocytes. The lymphocytic
lymphoid hyperplasia. In animals that have died with East infiltration is prominent around vessels, and often around the
Coast fever, there is widespread lympholysis with hemorrhage parietal layer of Bowman’s capsule. There is a variable level
 177.e1

eFigure 2-20  East Coast fever caused by Theileria parva infection

in a cow. Multifocal myocardial necrosis with intracytoplasmic
protozoa. (Courtesy E.P. Lane.)
177.e2

Further reading
Bakheit MA, et al. Serological diagnostic tools for the major tick-borne
protozoan diseases of livestock. Parassitologia 2007;49(Suppl. 1):
53-62.
Mbassa GK, et al. Theileria parva infection in calves causes massive
lymphocyte death in the thymus, spleen and lymph nodes without
initial proliferation. Vet Parasitol 2006;142:260-270.
 177.e3

Further reading
Tinkler SH, et al. Premature parturition, edema and ascites in an
alpaca with Anaplasma phagocytophilum. Can Vet J 2012;53:
1199-1202.
178 CHAPTER 2  •  Hematopoietic System
Figure 2-93  “Turning sickness” caused by Theileria parva in a
cow. Parasitized lymphocytes localize by embolism or sequestra-
tion in cerebrospinal vessels and produce hemorrhagic infarcts.
(Courtesy E.P. Lane.)
of parenchymal necrosis with the formation of hyaline casts
and brown pigmentation of the remaining epithelium. The
pulmonary changes are characteristic and consist of lympho-
cytic infiltration of the septa and interstitial tissues, resulting
in widespread severe interstitial alveolitis. The bone marrow
is hypocellular with early asynchrony of the granulocytic
system and a less severely affected erythroid system accom-
panied by proliferation of large lymphocytes similar to those
infiltrating other tissues. In cases of “turning sickness,” parasit-
ized lymphocytes localize by embolism or sequestration in
cerebrospinal vessels and produce hemorrhagic infarcts (Fig.
2-93, eFig. 2-20). Infarcts of various ages may be found in
other organs, especially kidney and spleen, but are small and
easily overlooked.
Further reading
Bishop R, et al. Theileria: intracellular protozoan parasites of wild and
domestic ruminants transmitted by ixodid ticks. Parasitol 2004;
129(Suppl.):S271-S283.
Glass EJ, et al. Living with the enemy or uninvited guests: functional
genomics approaches to investigating host resistance or tolerance
traits to a protozoan parasite, Theileria annulata, in cattle. Vet
Immunol Immunopathol 2012;148:178-189.
Shaw MK. Cell invasion by Theileria sporozoites. Trends Parasitol
2003;19:2-6.
Stockham SL, et al. Theileriosis in a Missouri beef herd caused by
Theileria buffeli: case report, herd investigation, ultrastructure, phy-
logenetic analysis, and experimental transmission. Vet Pathol
2000;37:11-21.
Tick-borne fever
Tick-borne fever of sheep and goats and pasture fever of cattle
is transmitted by the vector tick Ixodes ricinus and occurs in
Great Britain, Ireland, the Netherlands, Norway, and Finland.
The causative agent is Anaplasma phagocytophilum (for-
merly Cytoecetes phagocytophila, Rickettsia phagocytophila),
which is also the agent of human granulocytic ehrlichiosis in
Europe and North America (where the vector tick is Ixodes
Spleen and Hemolymph Nodes
scapularis or I. pacificus). The infection is transmitted stage to
stage in ticks, but not hereditarily, and other blood-sucking
arthropods may also transmit the agent. Similar diseases have
been reported in sheep and cattle in North Africa.
Tick-borne fever is a transient and mild illness that causes
some loss of condition. The disease is, however, of some sig-
nificance because it appears to predispose sheep to cerebro-
spinal inflammation in louping ill and to staphylococcal
pyemia (tick pyemia), the virus of louping ill being conveyed
by the ticks and Staphylococcus aureus being first established
as an infection in the tick bites. Tick-borne fever also appears
to predispose animals to pasteurellosis.
Clinically, adult ruminants are more susceptible to tick-
borne fever than are the young. After incubation for 2-6 days,
or a few days longer in cattle, there is sudden onset of moder-
ate fever. The fever lasts 2-3 days if the agent is of relatively
low virulence and subsides quickly. If more virulent organisms
are involved, the febrile reaction may be prolonged for nearly
2 weeks, the decline to normal temperature being gradual.
Ewes in late pregnancy may abort. In terms of laboratory
diagnosis, with the onset of fever, the organism is detectable in
circulating granulocytes and large lymphocytes. The most useful
stains are Giemsa and polychrome methylene blue. As the
fever declines, the organisms disappear from smears, and there
is progressive and quite severe neutropenia for a few days,
after which the neutrophil count returns to normal. Organ-
isms are no longer visible in smears unless there is a recrudes-
cence with fever, but they do persist in the blood in some
animals for a year or more. During fever, up to 95% of the
neutrophils may contain parasites. The parasites are restricted
to the cytoplasm and are of varied morphology, the variations
probably representing developmental stages of the parasite.
They may be round, rod-shaped, or irregular masses measur-
ing 0.75-3 µm in diameter present either singly or in clusters,
ring-shaped bodies 2-3.5 µm in size breaking up into irregular
fragments, round or oval morulae of 2.5-3.5 µm in diameter
containing many small granules, or in occasional cells, there
are small masses of ~0.5 µm in diameter.
Persistence of infection after the febrile period is associated
with a state of labile premunition, resistance to reinfection
being demonstrable at ~5 weeks after the primary infection.
Reactions to reinfection are usually much milder than those
to the initial infection, but the situation is complicated by the
immunologic differences and differences in virulence of strains
of the organism. It is probable that in endemic areas sheep
remain infected for life, reinfection occurring at each season
of tick activity.
Further reading
Adaszek L, et al. Three clinical cases of Anaplasma phagocytophilum
infection in cats in Poland. J Feline Med Surg 2013;15:333-337.
Stuen S, et al. Anaplasma phagocytophilum variants in sympatic red
deer (Cervus elaphus) and sheep in southern Norway. Ticks Tick
Borne Dis 2013;4:197-201.
Woldehiwet Z. Anaplasma phagocytophilum in ruminants in Europe.
Ann N Y Acad Sci 2006;1078:446-460.
Classical swine fever
Classical swine fever (CSF, hog cholera, European swine
fever) is caused by species Classical swine fever virus (CSFV),
an enveloped single-stranded RNA virus of positive polarity
 Spleen and Hemolymph Nodes 179

in the family Flaviviridae, genus Pestivirus. There is only one

serotype with minor antigenic variants. CSFV has a close
antigenic relationship with bovine viral diarrhea virus (BVDV)
and border disease virus (BDV) of sheep. Although CSFV has
been eradicated from the United States and Canada, it is
endemic in Asia, in the wild boar population in Europe, and
disease outbreaks have been reported in the Yucatan peninsula
of Mexico.
Pigs, including wild boars, are the only natural reservoir of
CSFV. Transmission of the virus occurs primarily through the
fecal-oral route, but infection can occur through the conjunc-
tiva, mucous membranes, skin abrasions, insemination, and
iatrogenic means. Blood, tissues, semen, secretions, and excre-
tions contain CSFV. Contact with infected pigs is the principal
source of infection with CSFV. Feeding of raw or insufficiently
cooked meat or contaminated food (access of infected wild
boars to food source) is a potent source of CSFV. Airborne
transmission is not thought to be important. Mechanical Figure 2-94  Classical swine fever in a pig. Lymph nodes with
vectors such as insects may carry CSFV; however, CSFV does diffuse edema and hemorrhage in sinusoids creating a “marbled”
not replicate in invertebrate vectors. Rodents and working appearance.
personnel play a crucial role as carriers. Transplacental infec-
tion with viral strains of low virulence often results in persis-
tently infected piglets. Persistently infected pigs do not
produce neutralizing antibodies to CSFV and have a lifelong
viremia. In a protein-rich environment, CSFV is very stable
and can survive for months in refrigerated meat and for years
in frozen meat.
Clinical signs of CSF are highly variable and strongly
determined by the virulence of the strain, age of the pigs
and to a lesser extent the breed and condition of the animals.
CSF occurs in an acute, a subacute, a chronic, or a persistent
form. Virulent and moderately virulent strains of CSF cause
the acute and subacute form of the disease, whereas strains of
low virulence induce a relatively high proportion of chronic
infections that may be inapparent or atypical. Adult pigs
tend to be less severely affected. Congenital infection by viru-
lent CSFV strains will cause abortions or small litter sizes, A
mummified, stillborn, and weak-born pigs. Congenital tremors,
malformation of the visceral organs, limb deformation, arthro-
gryposis, and cerebellar hypoplasia or aplasia occur rarely. Pigs
with acute CSF exhibit high fever, severe depression, reduced
appetite, cutaneous cyanosis, conjunctivitis, anorexia, consti-
pation followed by severe diarrhea (“cholera”), convulsions,
and ultimately die. The subacute form is characterized by
pyrexia, diarrhea, central nervous disease, and low mortality.
Chronic disease is more prolonged with intermittent periods
of depression, anorexia, and fever, alternating diarrhea and
constipation, and recovery is occasionally seen in mature
animals. Poor reproductive performance may be the only sign
of disease caused by low virulence strains of CSFV.
CSF is an acute hemorrhagic disease that is characterized by
disseminated intravascular coagulation, thrombocytopenia, and
immunosuppression. The most common lesion observed in pigs
B
dying of acute CSF is hemorrhage, especially in the kidneys
and lymph nodes. Kidneys have cortical and less commonly Figure 2-95  Classical swine fever in a pig. A. Multifocal acute
medullary petechiae or ecchymoses. Lymph nodes frequently hemorrhagic infarcts. B. Chronic splenic infarcts. (Courtesy J.P.
have diffuse edema and hemorrhage in sinusoids creating a Teifke.)
“marbled” appearance (Fig. 2-94). Frequent gross lesions also
include mucosal hemorrhage in the urinary bladder, splenic small and large intestine. Accumulations of straw-colored fluid
infarcts (Fig. 2-95), and laryngeal and epiglottal hemorrhages. in the peritoneal and thoracic cavities and in the pericardial
Characteristic, but less frequent lesions are multifocal caseous sac may be present. The lungs are congested and hemorrhagic
necrosis of tonsillar crypts (Fig. 2-96) and diffuse subserosal and have secondary bronchopneumonia. In chronic cases, the
hemorrhages and/or catarrhal mucosal inflammation in the colonic mucosa contains multifocal ulcers with white caseous
180 CHAPTER 2  •  Hematopoietic System Spleen and Hemolymph Nodes

A
Figure 2-96  Classical swine fever in a pig. Multifocal necrosis of
palatine tonsils. (Courtesy J.P. Teifke.)

necrotic centers, also known as “button ulcers” (Fig. 2-97).

Chondrodysplasia causing widening of the costochondral
junction of the ribs and at the growth plates of long bones
may be observed in growing pigs that survive more than 30
days postinfection. In pigs infected transplacentally, the most
common lesions are hypoplasia of the cerebellum, thymus
atrophy, ascites, and deformities of the head and limbs. Edema
and petechiae of the skin and the internal organs occur at the
terminal stage of the disease.
Microscopically, vasculitis and lymphoid necrosis are the
most prominent and consistent lesions (Fig. 2-98). CSFV repli-
cates in macrophages and vascular endothelial cells and high B
virulent strains cause a dramatic increase of proinflammatory Figure 2-97  Classical swine fever in a pig. A. Multiple “button”
cytokines interleukin-1 (IL-1), IL-6, and IL-8 as well as an ulcers in colon. (Courtesy J.P. Teifke.) B. Histologic appearance of
increased expression of coagulation factor, tissue factor, vascu- button ulcer characterized by mucosal ulceration and caseous
lar endothelial growth factor (VEGF), and activation of the necrotic centers in lymphoglandular complexes.
transcription factor nuclear factor kappa B (NF-κB). There is
commonly necrosis and variable hemorrhage in the subman-
dibular, gastrohepatic, renal, and mesenteric lymph nodes. The
spleen may have multifocal marginal infarcts. Endothelial
infections cause endothelial swelling in affected vessels that
may occlude the lumen. Edema and reticular cells expand the
intima. The media may be thickened by edema fluid or hyaline
through fibrinoid necrosis. Vessels may be surrounded by
hemorrhage. Glomerular capillaries and splenic and colonic
submucosal arteries are often thrombosed. In contrast, arteries
in the brain rarely contain thrombi, but are surrounded by
prominent cuffs of lymphocytes, especially in the Virchow-
Robin spaces. There may also be small multiple discrete foci
of glial proliferation in the white matter. Epithelial infections
may occur throughout the digestive tract and in the tonsils
and sometimes cause crypt necrosis and abscesses. Changes in
the small and large intestine may include catarrhal, hemor-
rhagic, or ulcerative enterocolitis.

Further reading
Ji W, et al. Studying classical swine fever virus: making the best of a Figure 2-98  Classical swine fever in a pig. Lymphoid necrosis in
bad virus. Virus Res 2015;197C:35-47. Peyer’s patches.
Lange A, et al. Pathogenesis of classical swine fever—similarities to viral
haemorrhagic fevers: a review. Berl Munch Tierarztl Wochenschr
2011;124:36-47.
 180.e1

Further reading
Gómez-Villamandos JC, et al. Morphological and immunohistochemi-
cal changes in splenic macrophages of pigs infected with classical
swine fever. J Comp Pathol 2001;125:98-109.
Gregg D. Update on classical swine fever (hog cholera). J Swine Health
Prod 2002;10:33-37.
 Spleen and Hemolymph Nodes 181

Moennig V, et al. Clinical signs and epidemiology of classical swine

fever: a review of new knowledge. Vet J 2003;165:11-20.
Petrov A, et al. Comparative analyses of host responses upon infection
with moderately virulent classical swine fever virus in domestic pigs
and wild boar. Virol J 2014;11:134.

African swine fever

African swine fever (ASF) is a viral hemorrhagic disease with
high mortality rates in domestic pigs, but clinical presentation
and pathology depend on the virulence of the viral strains/
isolates and the immunologic status of infected pigs. The
disease is caused by species African swine fever virus (ASFV),
a large, double-stranded DNA virus that is the only virus in
the family of Asfarviridae. The virus is endemic to sub-Saharan
Africa. Natural hosts include warthogs and bushpigs, in which
ASFV causes only inapparent infections. The natural cycle of
transmission of ASFV occurs between warthogs and the soft Figure 2-99  African swine fever in a pig. Renal petechiae and
tick vector Ornithodoros moubata that is infective throughout ecchymoses, and diffuse hemorrhages in lymph nodes.
the various nymph stages, and there is trans-stadial, trans-
ovarial, and sexual transmission in Ornithodoros ticks. Orni-
thodoros erraticus ticks became infected during outbreaks on
the Iberian Peninsula. Stable flies (Stomoxys calcitrans) can
harbor high levels of the virus for up to 2 days. Transmission
of disease to domestic pigs can occur by tick bites or through
ingestion of warthog tissues or secretions. In domestic pigs
with clinical disease, all body fluids and tissues contain large
amounts of virus, and transmission can be through oronasal
contact, ingestion of pig meat products, or iatrogenic means.
Transplacental infection with viral strains of low virulence
may result in persistently infected piglets. In a protein-
rich environment, ASFV is very stable and can survive for
months in refrigerated meat and for years in frozen meat.
Following inhalation or ingestion, ASFV replicates in the
tonsils as well as head and neck lymph nodes. This is followed
by marked viremia. Macrophages are the primary target cells
of infection, especially mature macrophages expressing
CD163 and CD107a. Viral glycoproteins p12, p30, and p54 Figure 2-100  African swine fever in a pig. Acute splenic hemor-
play a major role in attaching to and entering target cells. rhage and necrosis. (Courtesy J. Arzt, Agricultural Research
During later stages of disease, additional cell types, including Service–U.S. Department of Agriculture.)
megakaryocytes, tonsillar epithelial cells, hepatocytes, renal
epithelial cells, and endothelial cells, become infected. Hemad-
sorption is observed in hepatic sinusoids, lymph sinuses, organs (Fig. 2-100, eFig. 2-21); hemorrhages on serosal sur-
and splenic red pulp. ASFV induces replication of infected faces, renal cortical, medullary, and pelvic hemorrhage; hydro-
cells and secretory activation: increased levels of proinflam- pericardium; and hydrothorax. Affected lymph nodes are
matory cytokines IL-1, IL-6, and tumor necrosis factor-α. Pro- severely swollen and appear grossly like blood clots because
inflammatory activation and changes in levels of acute-phase of severe hemorrhage and necrosis. The spleen is severely, dif-
proteins are the initial causes of clinical signs and lesions, fusely enlarged, friable, and dark black (Fig. 2-101). The
including fever. meninges can be congested, edematous, or hemorrhagic. Leu-
In acute forms of ASF, affected pigs have high fever and kopenia and thrombocytopenia are both characteristic for
anorexia, lethargy, weakness, recumbency, and erythema, as acute and subacute disease. Lymphopenia is associated with
well as cyanosis of ears, tails, lower legs, and abdomen. Preg- lymphoid depletion in primary and secondary lymphoid
nant sows frequently abort. Death often occurs within 7-10 organs caused by apoptosis (Fig. 2-102, eFig. 2-22). Aborted
days. Pigs with subacute disease most commonly display fetuses can have anasarca, and livers may be mottled.
dyspnea, vomiting, and nasal and conjunctival discharges. Chronic disease is mainly characterized by low fever, loss
Fever tends to be transient, and affected pigs either die or of appetite, swollen joints, occasional diarrhea and vomiting,
recover within 3-4 weeks. ASFV induces severe vascular changes and general poor doing. Lesions include lymphoid hyperplasia,
that cause pulmonary edema, disseminated intravascular coagu- fibrous pleuritis and pericarditis, and pneumonia.
lation, and hemorrhages in different organs: melena, epistaxis,
erythema, renal petechiae, and diffuse hemorrhages in lymph
nodes (Fig. 2-99). Besides endothelial damage, hemorrhages Further reading
have also been attributed to marked thrombocytopenia. Con- Alonso C, et al. African swine fever virus-cell interactions: from virus
sistent lesions include hemorrhage and necrosis of lymphoid entry to cell survival. Virus Res 2013;173:42-57.
 181.e1

A
eFigure 2-21  African swine fever in a pig. Acute splenic hemor-
rhage and necrosis. (Courtesy J. Arzt, Agricultural Research
Service–U.S. Department of Agriculture.)

B
eFigure 2-22  African swine fever in a pig. A. Tonsil with severe
lymphoid necrosis. B. Immunohistochemistry depicting African
swine fever virus (red) in macrophages. (Courtesy J. Arzt, Agri-
cultural Research Service–U.S. Department of Agriculture.)
182 CHAPTER 2  •  Hematopoietic System Spleen and Hemolymph Nodes

A
Figure 2-101  African swine fever in a pig. Severely and diffusely
enlarged, dark black spleen. (Courtesy J. Arzt, Agricultural
Research Service–U.S. Department of Agriculture.)

Blome S, et al. Pathogenesis of African swine fever in domestic pigs

and European wild boar. Virus Res 2013;173:122-130.
Galindo I, et al. African swine fever virus infects macrophages, the
natural host cells, via clathrin- and cholesterol-dependent endocy-
tosis. Virus Res 2015;200:45-55.
Gómez-Villamandos JC, et al. Pathology of African swine fever: the
role of monocyte-macrophage. Virus Res 2013;173:140-149.
Sánchez-Vizcaíno JM, et al. An update on the epidemiology and
pathology of African swine fever. J Comp Pathol 2015;152:9-21.

B
Inflammatory diseases of the spleen
Systemic inflammation causes a regular and fairly predictable
pattern of response in the spleen. The phagocytic function of
the spleen is critical in the immune response to blood-borne
pathogens. Therefore all blood passes at least one time every
day through the spleen. Septicemia and bacteremia result in
microbial deposition in the splenic sinus areas, where the
abundance of phagocytic cells usually leads to their rapid
destruction. In septicemia and following injection of endo-
toxin or gram-negative bacteria, there is rapid accumulation
of neutrophils in the splenic marginal zone and the surround-
ing red pulp vascular spaces become congested and form a
concentric ring. There is bacterial destruction and processing
of antigen in this area followed by a predictable pattern of
migration. This consists of foreign material moving in special-
ized macrophages (metallophils) successively into the marginal C
zone and then to the small-lymphocyte corona, reaching the
germinal center in 10-12 hours. In contrast, immune com- Figure 2-102  African swine fever in a pig. (Courtesy J. Arzt,
plexes appear to move by flow and not by cellular transport. Agricultural Research Service–U.S. Department of Agriculture.)
Overwhelming infections result in acute hyperemia and A. Lymph node with severe lymphoid necrosis. B. Immunohisto-
severe lympholysis of the follicular center cells, characterized chemistry depicting African swine fever virus in macrophages.
by accumulation of nuclear debris. Severe lymphoid necrosis is C. Multichannel indirect immunofluorescence technique detects
also a feature of numerous viral diseases, for instance, rinder- African swine fever virus in macrophages in the tonsil. The virus
pest (Fig. 2-103, eFig. 2-23), canine distemper, equine herpes- (red) co-localizes to CD163-expressing macrophages (aqua)
viral infection (Fig. 2-104), canine parvoviral enteritis, feline within and subjacent to a deep epithelial crypt. Cytokeratin-
panleukopenia, and bovine viral diarrhea. The nuclear frag- expressing (green) cells are spared. Nuclei are labeled blue.
ments are rapidly removed in 1 day so that the follicular
center appears empty, with the nuclei of the dendritic cells
relatively widely spaced and surrounded by their densely pink
cytoplasm, and epithelioid macrophages. These epithelioid
 182.e1

B
eFigure 2-23  Rinderpest in a cow. A. Acute hyperemia and
severe lymphocytolysis of the follicular center cells in a splenic
follicle. B. Lymphoid necrosis with intranuclear inclusions.
 Spleen and Hemolymph Nodes
Figure 2-103  Rinderpest, in a cow. Acute hyperemia and severe
lymphocytolysis of the follicular center cells in a splenic follicle.
Figure 2-104  Equid herpesvirus, in a horse. Severe lymphoid
necrosis in the spleen.
germinal centers are seen in a variety of acute toxemic diseases
in young animals. In older animals, the changes occurring in
splenic germinal centers are often more severe and long
lasting, and consist of a depopulation of the follicular center
cells and transudation of plasma proteins into the germinal
centers to form a fairly persistent coagulum recognized as
intrafollicular hyalinosis. These fibrinous exudates may be
removed, with restitution of the follicular center cells or, if
there is extensive vascular damage, they may become mineral-
ized and result in involution of the germinal center. In very
acute diseases, such as anthrax in cattle, the reaction is almost
solely vascular. In the less severe septicemias, such as erysipelas
in swine, there is moderate reactive hyperplasia that may be
more developed than that seen in acute fulminant gram-
negative septicemias such as salmonellosis.
Marked hyperplastic changes of the sinus areas are a feature
of some diseases, such as malaria, trypanosomiasis, equine
infectious anemia, and malignant catarrhal fever, although
involution characterizes some infections. Some pyogenic bac-
teria, for instance, Francisella tularensis, Yersinia pestis, and
Brucella spp., will cause more widespread liquefactive necro-
sis. Red pulp macrophages proliferate, especially in response
183
A
B
Figure 2-105  Mycobacteriosis in a horse (A) and a beaver (B).
A. Multiple granulomas disseminated over splenic capsule.
B. Multiple granulomas throughout splenic parenchyma. (Cour-
tesy E.P. Lane.)
to intracellular bacteria, for instance, Mycobacterium spp. (Fig.
2-105); fungi (Fig. 2-106, eFig. 2-24), for instance, histoplas-
mosis; or in chronic hemolytic conditions. Blood monocytes
may also accumulate in the red pulp. Such reactions can
appear as focal or diffuse granulomatous inflammation. Some
blood parasites, for instance, Cytauxzoon felis, infect macro-
phages and can cause severe histiocytosis (Fig. 2-107).
Splenic abscesses may be miliary or large and focal, but both
types are uncommon (Fig. 2-108). Abscessation occurs most
commonly secondary to septicemia or bacteremia by pyogenic
bacteria, including Trueperella (Arcanobacterium) pyogenes,
Fusobacterium necrophorum (Fig. 2-109, eFig. 2-25), Rhodococ-
cus equi, Burkholderia pseudomallei, Corynebacterium pseudo-
tuberculosis, and Streptococcus equi. Abscesses are usually
localized in the red pulp, where they develop after failure of
the monocyte-macrophage system to kill them. They usually
bulge over the surface, stretching the splenic capsule and
contain white or yellow pus. Purulent splenitis may develop by
local extension in cattle from penetrating wounds of the retic-
ulum and, in horses, by extension from the stomach as a result
of penetrating ulcers caused by Habronema spp. Inflammation
of the abdominal serosal membranes may extend to the
 183.e1

A
eFigure 2-25  Fusobacterium necrophorum in a cow. Splenic
necrosis with sequestration.

B
eFigure 2-24  Aspergillus terreus in a dog. A. Pyogranulomatous
splenitis with intralesional fungal hyphae. B. Hyphae are difficult
to recognize on routine H&E sections and require special stains,
for instance, periodic acid–Schiff.
184 CHAPTER 2  •  Hematopoietic System
Figure 2-106  Aspergillus terreus in a dog. Pyogranulomatous
splenitis with intralesional fungal hyphae.
Figure 2-107  Cytauxzoon felis in a cat. Diffuse severe splenic
histiocytosis with intracytoplasmic organisms.
Figure 2-108  Splenic abscess in a horse. Streptococcus equi is a
rare cause of splenic abscesses.
Spleen and Hemolymph Nodes
Figure 2-109  Fusobacterium necrophorum in a cow. Multifocal
splenic necrosis with sequestration.
Figure 2-110  Feline infectious peritonitis in a cat. Spleen with
severe pyogranulomatous serositis.
splenic capsule, as feline infectious peritonitis causes severe
fibrinosuppurative splenic serositis in cats (Fig. 2-110).
Tularemia
Tularemia (deer fly fever) is caused by Francisella tularensis.
The disease was first described in Tulare county of California
and is still more common in the western United States than
elsewhere. The organism is a small, gram-negative, pleomor-
phic, non–spore forming, facultative intracellular bacillus that
is strictly aerobic and shares many cultural and epidemiologic
features with Yersinia pestis, the cause of bubonic plague.
Francisella tularensis is found worldwide except for Australia
and Antarctica, but the biovar tularensis (type A) is found only
in North America, whereas biovar holarctica (type B) occurs
throughout the Northern Hemisphere. Type A is associated
with the terrestrial tick-rabbit cycle of infection and causes
severe illness in humans, cats, and rabbits. Type B is more
commonly found in aquatic species, including beavers and
muskrats. Although both type A and B have been isolated
from cats, type A generally causes more severe disease.
The organism is abundant in nature as an infection of many
species of rodents, and it is from these, either directly or by the
 Spleen and Hemolymph Nodes
A
B
Figure 2-111  Tularemia in a beaver. Francisella tularensis causes
multifocal miliary necrosis in mesenteric lymph nodes (A) and
spleen (B).
mediation of insect vectors, that humans and domestic animals
acquire the infection. The organism is able to penetrate intact
skin and mucous membranes, but it is also infective by inges-
tion, inhalation, and inoculation by biting insects and ticks.
Affected cats usually display fever, marked depression, lymph-
adenopathy, and on palpation hepatomegaly and splenomeg-
aly. Lingual and oral ulcers, icterus, and panleukopenia are
common.
Tularemia in cats is a severe systemic disease, with various
manifestations, depending on dissemination or localization.
Grossly, tularemia in cats is recognized by the presence of
miliary white foci 2 mm or more in diameter in the liver, spleen,
and lymph nodes (Fig. 2-111). They are indistinguishable
grossly from the lesions caused by Yersinia spp. Other differ-
entials include nocardiosis, mycobacteriosis, or even crypto-
coccosis. Histologically, the lesions are characterized by focal
areas of severe necrosis (Fig. 2-112). Neutrophils and pus may
be present early and macrophages accumulate, but in slightly
older and larger lesions, there is total liquefactive necrosis with
a granularity that resembles caseation. Degenerate neutrophils
admixed with cellular debris surround areas of necrosis and,
in older lesions, fibroblasts and macrophages may demarcate
the lesion. In contrast to Yersinia pseudotuberculosis, bacteria
are difficult to visualize on regular H&E-stained sections, but
immunohistochemistry (IHC) will readily demonstrate large
185
Figure 2-112  Tularemia in a cat. Francisella tularensis causes
severe liquefactive splenic necrosis.
Figure 2-113  Tularemia in a cat. Immunohistochemistry detects
abundant Francisella tularensis organisms not recognizable on
routine histology.
clumps within necrotic centers and also within macrophages
(Fig. 2-113). The lesions in lymph nodes are often larger than
those in the liver and may be readily visible grossly as wedge-
shaped areas of cortical necrosis demarcated by a narrow zone
of intense reactive hyperemia. Lymphadenitis may be general-
ized or restricted to the nodes draining the site of infection
which, if visible, is an ulcerated papule.
Naturally acquired infections with F. tularensis have been
observed in dogs, but, as a general rule, dogs are highly resistant
to the disease. Most dogs develop short periods of listlessness
and low-grade fever; lymphadenomegaly, uveitis, and conjunc-
tivitis are rather rare. Fatalities caused by tularemia have been
observed in foals and sheep in association with heavy infesta-
tions by ticks, and it is probable that the disease is endemic
in sheep in areas where the reservoir rodents and ticks, espe-
cially Dermacentor andersoni and Amblyomma americanum,
abound. The disease in foals is characterized by a systemic and
febrile illness with, at autopsy, enlargement of liver, spleen,
and kidneys, and the presence of the typical small necrotic
foci of this disease. Affected sheep have high fever, stiffness of
gait, depression, diarrhea, and hyperpnea. Lesions may be
186 CHAPTER 2  •  Hematopoietic System
confined to the superficial lymph nodes or show the more
classic localization. Pneumonia, probably of other cause, and
anemia caused by the ticks are frequently present.
Further reading
Larson MA, et al. Francisella tularensis bacteria associated with feline
tularemia in the United States. Emerg Infect Dis 2014;20:
2068-2071.
Sjöstedt A. Tularemia: history, epidemiology, pathogen physiology, and
clinical manifestations. Ann N Y Acad Sci 2007;1105:1-29.
Wobeser G, et al. Tularemia, plague, yersiniosis and Tyzzer’s disease in
wild rodents and lagomorphs in Canada: a review. Can Vet J
2009;50:1251-1256.
Histoplasmosis
Histoplasmosis is caused by Histoplasma capsulatum, a soil-
borne, facultative intracellular, dimorphic fungus that infects
macrophages, and is characterized by diffuse involvement of
the mononuclear phagocyte system. Histoplasmosis has a
worldwide distribution. H. capsulatum var. capsulatum is fre-
quently diagnosed in the Mississippi, Ohio, and St. Lawrence
River valley areas of North America. H. capsulatum var. far-
ciminosum has been reported in Japan, and H. capsulatum var.
duboisii causes African histoplasmosis.
Like all dimorphic fungi, H. capsulatum exists in a free-
living mycelial form that produces small, smooth, globose
2-3 µm in diameter microconidia, and a large, thick-walled,
globose 5-18 µm in diameter macroconidia, and in a yeast
form. Infection originates most commonly from soil that is rich
with bird or bat feces, and occurs primarily through inhalation
of microconidia. Transmission from dog to dog has been estab-
lished, but it is not known if dogs can transmit the disease to
humans. Inhaled microconidia convert in the infected animal
to the yeast form, which reproduces through budding. Yeast
organisms are phagocytosed by the monocyte-macrophage
system and continue to undergo further intracellular replica-
tion. In most cases, organisms are killed through cytokine-
mediated macrophage killing, and infection is cleared through
T-cell immunity. However, even in immunocompetent hosts,
H. capsulatum may become dormant and is not entirely elimi-
nated from the phagocytic system. Such dormant infections can
persist for many months and years. Yeast organisms are more
resistant to host immune defenses because they are more
invasive and can impair the immune system by raising the
intracellular pH and stimulating IL-4 production. The myce-
lial form is only rarely encountered on valve leaflets from
animals with endocarditis.
Most animals are subclinically infected, and clinical disease
occurs most frequently in dogs and cats, but has been reported
in other species, including humans, swine, cattle, horses, and
birds. Cats are at least as likely as dogs to develop clinical
disease and, in fact, histoplasmosis is the second most common
systemic fungal disease in cats in North America. Persian cats
as well as Weimaraner, Pointers, Terriers, Brittany Spaniels, and
sporting or working dogs are at higher risk to develop disease.
In most cases, infection is limited to the respiratory system
and associated lymph nodes, but lymphatic and hematogenous
dissemination may occur quickly in immune-compromised
hosts or animals infected with a high dose of spores. When
histoplasmosis becomes apparent clinically, the infection is
disseminated, and the disease is then progressive and always
Spleen and Hemolymph Nodes
Figure 2-114  Histoplasma capsulatum in a dog. Diffuse severe
splenomegaly with multiple white foci.
fatal. Given that intestinal and skin lesions have been observed
without respiratory tract lesions, the intestinal tract or skin
may also represent primary routes of infection. However, in
disseminated disease, the intestine could be secondarily
infected like many other tissues.
Cats with advanced histoplasmosis show nonspecific clini-
cal signs, including weight loss, mental depression, anorexia,
dyspnea and tachypnea, and pale mucous membranes. Dogs
may have similar signs; however, most cases of disseminated
histoplasmosis will involve the intestinal tract, causing emacia-
tion, persistent large-bowel diarrhea, or even voluminous
watery diarrhea associated with protein-losing enteropathy,
tenesmus, and fresh blood in the stool. Both dogs and cats
with disseminated histoplasmosis commonly have an enlarged
spleen (Fig. 2-114, eFig. 2-26), lymph nodes, and liver, in
combination with icterus.
Animals with histoplasmosis most consistently have mild
nonresponsive, normochromic normocytic anemia. Leuko-
cytic changes vary with the reserves of the animal and the
stage of the disease. If the animal is in good body condition,
the bone marrow is competent, and there is neutrophilic
leukocytosis with some degree of monocytosis. Later with
debilitation, the leukocyte count drops to normal levels or
lower with left shift and marked toxic changes, including the
appearance of Döhle bodies. There is usually lymphopenia and
eosinopenia. The organisms are readily recognized on micro-
scopic examination, hence fine-needle aspiration of liver, spleen,
enlarged lymph nodes, marrow, or skin provides adequate mate-
rial for diagnosis in appropriate cases. Occasionally, the organ-
ism may be diagnosed in fine-needle aspirates of lung or
bronchoalveolar lavage fluids, or lavage of the turbinates in
animals with sinusitis. Cytologically, the organisms are present
in the cytoplasm of macrophages, unless the cells have been
injured, and are somewhat larger than when seen in histologic
preparations.
Grossly, the pulmonary lesions of histoplasmosis may be in
the form of gray, rounded nodules 1-2 cm in diameter, and
with a distinct tendency to become confluent, or there may
be a diffuse increase in the consistency of the lungs. Intestinal
lesions are located chiefly in the lower part of the small intes-
tine. The mucosa is the site of nodular thickenings or of cor-
rugations similar to those seen in Johne’s disease of cattle. The
 186.e1

Further reading
Gyuranecz M, et al. Tularemia of European brown hare (Lepus euro-
paeus): a pathological, histopathological, and immunohistochemi-
cal study. Vet Pathol 2010;47:958-963.
Stundick MV, et al. Animal models for Francisella tularensis and Burk-
holderia species: scientific and regulatory gaps toward approval of
antibiotics under the FDA Animal Rule. Vet Pathol 2013;50:
877-892.
186.e2

eFigure 2-26  Histoplasma capsulatum in a dog. Severe multifo-

cal granulomatous lymphadenitis.
 Spleen and Hemolymph Nodes 187

A
Figure 2-115  Histoplasma capsulatum in a horse. Severe multi-
focal granulomatous lymphadenitis.

thickenings are the result of infiltration of lymphocytes,

plasma cells, and macrophages in the lamina propria and sub-
mucosa, and they may extend also through the wall to the
subserosa, giving the gut a thickened pipestem appearance.
When the thickening is extreme, ischemic ulcerations occur.
The lymph nodes are greatly enlarged, but are discrete and
without adhesions (Fig. 2-115). There may be no indication
of normal architecture on the cut surface, with the uniformity
resembling lymphoma, except that the nodes are firm and dry.
Histologically, there are coalescing granulomas with histiocy-
tosis, and cortical replacement by the reaction. The spleen is
enlarged sometimes to several times its normal size, gray, and
firm. There is marked sinus expansion and filling by fixed cells
of stromal origin and by colonization with macrophages, many
of which contain the ingested organisms. There is lymphoid
atrophy, varying in degree with the stage of debility of the
animal. The liver is uniformly enlarged, firm, and gray. The
discoloration is diffuse and related to capsular thickening
without focal lesions. The infiltrating cells collect in miliary
foci in the portal triads and sinusoids, causing extensive dis-
placement and atrophy of the parenchyma. The adrenal glands
are often involved— the medulla, the cortex, or both—n
histoplasmosis that is allowed to run its full course. Scant
normal tissues may then remain in these organs. Approxi-
mately 1 4 of all cats with histoplasmosis will develop ocular
lesions characterized by granulomatous blepharitis, conjuncti-
vitis, chorioretinitis, panuveitis or panophthalmitis, retinal
detachment, and optic nerve neuritis. Dermatitis, osteomyeli-
tis, and arthritis, as well as myelitis, are less common.
The enlargement of organs is the result of extensive prolifera-
tion and infiltration with monocytes and epithelioid macrophages
in whose cytoplasm many of the typical yeasts in small or large
numbers are found (Fig. 2-116). The yeast bodies in sections
when stained with H&E appear as basophilic dots with a clear
halo. The halo is part of the yeast’s cell wall and can be dem-
onstrated by stains for bound glycogen, the organism then
appearing as a ring. IHC testing is available, but not necessary
when the yeasts are plentiful. Necrosis of tissues is not often
present in histoplasmosis, although central caseation without
mineralization may occur in large, dense accumulations of
macrophages. Giant cells are seldom seen, and there is only
mild fibrosis.
B
Figure 2-116  Histoplasma capsulatum in a horse. A. Spleen
with extensive infiltration of histiocytes containing abundant
intracytoplasmic yeasts. B. Immunohistochemistry to detect His-
toplasma capsulatum in histiocytic cells.
Responsibility of H. capsulatum for gross or microscopic
lesions can only be claimed when the fungus can be demon-
strated histologically in the lesion. There is seldom any diffi-
culty in demonstrating organisms in sections, although Grocott
stain, IHC, and PCR are more sensitive than H&E, and clini-
cally they are readily cultured. Culture should not be under-
taken without precautions to prevent the inhalation of
microconidia. Biopsy of enlarged lymph nodes and aspiration
biopsy of bone marrow are useful for diagnostic purposes. In
some cases, the organisms can be demonstrated in circulating
monocytes, but they are never plentiful, so examination is best
performed on buffy coat preparations.
Further reading
Aulakh HK, et al. Feline histoplasmosis: a retrospective study of 22
cases (1996-2009). J Am Anim Hosp Assoc 2012;48:182-187.
Cordeiro RA, et al. Serological evidence of Histoplasma capsulatum
infection among dogs with leishmaniasis in Brazil. Acta Trop
2011;119:203-205.
Gilor C, et al. DIC and granulomatous vasculitis in a dog with dissemi-
nated histoplasmosis. J Am Anim Hosp Assoc 2011;47:26-30.
 187.e1

Further reading
Bialek R, et al. Comparison of staining methods and a nested PCR assay
to detect Histoplasma capsulatum in tissue sections. Am J Clin
Pathol 2002;117:597-603.
Tyre E, et al. Histoplasmosis in a dog from New Brunswick. Can Vet J
2007;48:734-736.
188 CHAPTER 2  •  Hematopoietic System
Melioidosis
Melioidosis is a zoonotic infection caused by the gram-negative
bacillus Burkholderia pseudomallei. This organism is a ubiq-
uitous soil saprophyte endemic to Southeast Asia and North
Australia, and the South Pacific. In humans, infection is spread
via direct contact with broken skin, inhalation, or by ingestion,
and disease is most common in severely immunocompromised
patients. Melioidosis has been reported in many domestic and
wild animals. Susceptibility to the infection varies between
species, with sheep and goats being particularly susceptible
and bovine species being rarely infected. Dogs, horses, cats,
and pigs are also considered to have a high natural resistance
to B. pseudomallei. Cases have been reported from Vietnam,
Malaysia, and Australia.
The location of lesions is believed to be associated with the
route of infection. Inhalation of aerosols will produce abscess
formation in the bronchial region, and ingestion will affect the
mandibular lymph nodes and liver. Clinical signs are uncom-
mon, and abscesses may be detected at autopsy without prior
clinical signs. Melioidosis can occur as an acute, subacute, and
chronic form, and lung, spleen, lymph nodes, and liver are most
commonly affected. Young animals are at higher risk to develop
acute, septicemic melioidosis causing death, but localized
respiratory, gastrointestinal, or neurologic disease; arthritis
causing lameness; osteomyelitis; mastitis; and orchitis have
been reported. Clinical signs are dependent on the organ
system affected and include fever, anorexia with progressive
emaciation, discharge from nose and eyes, coughing, dyspnea,
uncoordinated gait, and diarrhea. In small ruminants, pulmo-
nary disease is most common, with sheep being more severely
affected then goats. Aortic aneurysms and mastitis occur most
commonly in goats. Although young pigs are at risk for septi-
cemic melioidosis, adult pigs typically have a more chronic
form with few clinical signs (Fig. 2-117). Sows may abort or
have stillbirths, and orchitis can occur in boars. Various forms
of melioidosis occur in horses, and signs include weakness,
emaciation, edema and lymphangitis of the limbs, mild colic,
diarrhea, coughing and nasal discharge, cutaneous eczema, or
papular lesions. Neurologic disease is rare, but septicemia fol-
lowed by death may occur. Cattle rarely contract B. pseudom-
allei, and clinical disease is usually limited to fever, dyspnea,
Figure 2-117  Melioidosis in a pig. Burkholderia pseudomallei
causes splenic abscessation. (Courtesy S. Kesdangsakonwut.)
Spleen and Hemolymph Nodes
drooling, and arthritis. Dogs may occasionally develop acute,
septicemic melioidosis associated with fever, severe diarrhea,
and fulminant pneumonia. More common are subacute or
chronic lesions characterized by dermatitis with abscessation,
lymphangitis with edema of the limbs, and lymphadenitis.
There are only a few reports of melioidosis in cats, and affected
animals displayed icterus and anemia that led to death.
B. pseudomallei causes multifocal necrotizing inflammation
in multiple organs; foci tend to coalesce to form larger
abscesses that contain thick, caseous, green to yellow or white
pus (Fig. 2-118). Mineralization, in contrast to mycobacterio-
sis, does not occur. In small ruminants, severe, suppurative
bronchopneumonia with abscessation is commonly observed.
Abscesses and ulcers also occur in the nasal mucosa. Even
though rarely reported as a clinical disease, melioidosis may
be a common cause of abscesses in various organs in older
animals. In food animals, lesions may only be detected during
slaughter and if undetected may pose a serious risk for human
infection. Mycobacterium spp. as well as Trueperella pyogenes
may cause similar lesions and definitive diagnosis of melioido-
sis requires bacterial culture.
A
B
Figure 2-118  Melioidosis in a pig. A. In the bronchial lymph
nodes, Burkholderia pseudomallei causes multiple abscesses that
contain thick, caseous, yellow or white pus. (Courtesy S. Kesdang-
sakonwut.) B. Within the center of an abscess is a small eosino-
philic, club-shaped aggregate (Splendore-Hoeppli reaction)
surrounded by degenerate neutrophils.
 Spleen and Hemolymph Nodes 189

Further reading
Najdenski H, et al. Experimental Burkholderia pseudomallei infection
of pigs. J Vet Med 2004;51:225-230.
Sprague LD, Neubauer H. Melioidosis in animals: a review on epizooti-
ology, diagnosis and clinical presentation. J Vet Med B 2004;51:
305-320.

Hyperplastic diseases of the spleen

Splenic cysts
Splenic cysts are unusual. Occasionally, Cysticercus tenuicollis
and hydatids are found. Pseudocysts may result from cystic
degeneration of hematomas. Neoplastic cysts are hemangiomas
or hemangiosarcomas. The latter are relatively common in the
dog and have a rather characteristic history of repeated bouts
of malaise and weakness, with apparent recovery over 2-3
days. Severe anemia accounts for the weakness. Pallor may be
noticed and results from rupture of the tumor with abdominal
hemorrhage. The recovery over a few days is too rapid for new
red cell production, and indicates autotransfusion of the
extravasated blood. The hematologic picture is also typical
with distorted red cells, even in the absence of abdominal
hemorrhage, because of the effects of abnormal vascular walls
and arteriovenous shunts. After hemorrhage, the red cell dis-
tortion is much worse—the formerly extravasated red cells
appear tattered and effete—and the plasma is brown as a
result of extensive hemolysis. There are usually Howell-Jolly
bodies in the blood along with hypersegmented neutrophils.
If abdominocentesis is performed, the presence of an occa-
sional rubricyte is an indication of hemorrhage from the
spleen rather than from another abdominal site.
Lymphoid hyperplasia
Lymphoid hyperplasia of the spleen may be diffuse or seg-
mental affecting only the germinal centers (Fig. 2-119). The
germinal centers in hyperimmune states increase in both size
and density, and the accentuation of the marginal zone, mantle
cell corona, and follicular center cells is increased, often result-
ing in a “target” appearance. The germinal centers increase in
size through proliferation of lymphocytes as evidenced by
high mitotic activity. The vast majority of these lymphocytes
quickly undergo apoptosis, and nuclear debris becomes promi-
nent. Debris is phagocytized by tingible body macrophages, and
immunoblasts leave follicular centers and differentiate to
plasma cells. The number of plasma cells in the spleen often
correlates with the degree of follicular hyperplasia. As the
immune response decreases, germinal centers are still enlarged,
but fewer in number and the reduction of immunoblasts
causes dendritic cells to become more visible. Such hypocel-
lular appearance can also be seen in dogs with prolonged
steroid treatment. Alternatively, there may be hyperplasia of
the periarteriolar lymphoid sheath with prominent broad
cords of lymphocytes surrounding the branches of the
medium-sized arterioles. In contrast to both of these changes,
there may be hyperplasia of marginal zone cells that promi-
nently surround all germinal centers and may fuse with
adjacent germinal centers or actually surround adjacent peri-
arteriolar lymphoid sheaths. These follicular structures are
usually not apparent on gross examination unless there is
hyperplasia or neoplasia involving some or all of these lym-
phoid compartments. Because sinus and follicular hyperplasia
A
B
Figure 2-119  Lymphoid hyperplasia of the spleen in a cow
(A) and a dog (B). A. Diffuse lymphoid hyperplasia is apparent
on cross section. (Courtesy R.L. Amorim.) B. The splenic white
pulp becomes grossly prominent as multiple white nodules.
(Courtesy S. Kesdangsakonwut.)
of the spleen tend to occur together, their concurrence results
in an overall increase in splenic weight.
Nodular hyperplasia
Nodular hyperplasia is frequently observed in the spleens of
old dogs, occasionally in old bulls, and rarely in other species
(eFig. 2-27). Most nodules are up to 2 cm in diameter and
project hemispherically above the capsule, but may be 5 cm
or more in diameter (Fig. 2-120). On cut surface, the nodules
vary from gray to pink to a variegated red and white, with
yellow necrotic areas in the larger ones. The variegated pattern
is the result of persistent islands of vascular spaces in a field
of lymphocytic proliferation. Architecturally, the nodules are
unencapsulated but compress adjacent trabeculae, and irregu-
larly meld with surrounding vascular spaces that have a much
higher density of red cells (Fig. 2-121). Lacking germinal
centers, the benign nodules consist of monomorphic medium
or large lymphocytes with moderate cytoplasmic volume and
small nucleoli. Mitoses are common. In some hyperplastic
nodules, lymphocytes can be admixed with erythroid and
myeloid cells and megakaryocytes. Hyperplastic nodules
can become engorged with blood closely resembling hemato-
mas with rare lymphofollicular structures surrounded by red
blood cells.
More solid hyperplastic nodules must be differentiated
from follicular type lymphomas. Accurate diagnosis may
require immunophenotyping and PCR for antigen receptor
 189.e1

Further reading
Choy JL, et al. Animal melioidosis in Australia. Acta Trop 2000;74:
153-158.
O’Brien CR, et al. Disseminated melioidosis in two cats. J Feline Med
Surg 2003;5:83-89.
189.e2

eFigure 2-27  Splenic nodular hyperplasia in a dog. A hyperplas-

tic nodule projects above the capsule.
190 CHAPTER 2  •  Hematopoietic System
Figure 2-120  Splenic nodular hyperplasia in a dog. A hyperplas-
tic nodule projects hemispherically above the capsule.
Figure 2-121  Splenic nodular hyperplasia in a dog. The varie-
gated pattern is the result of persistent islands of vascular spaces
in a field of lymphocytic proliferation.
rearrangements (PARR) testing (see section on Lymphoma
for more details). Architecturally, benign focal lymphoid hyper-
plasia in the spleen tends to be arranged in large irregular
islands that are loosely facetted in a relatively cohesive mass.
In contrast, lymphomas of a mantle cell or marginal zone type
can be seen to be related to the arteriolar structure of the
spleen in multiple smaller foci, some of which contain rem-
nants of germinal centers. Proliferation immediately surround-
ing fading germinal centers of small cell type is likely of mantle
cell lymphoma. If the proliferation can be seen in some foci
to surround the outside of the mantle cell cuff and is com-
posed of intermediate-sized cells, then the lesion is likely
marginal zone lymphoma. It is uncertain whether hyperplastic
nodules may progress to become follicular type lymphomas,
but the vast majority has no clinical significance. However,
severe expansion with blood may lead to formation of a
hematoma that is prone to rupture causing potential hemoab-
domen (Fig. 2-122). Focal lymphoid proliferations in the
spleen, either hyperplastic or neoplastic, may also become
clinically apparent because of effects of the expanding mass
Spleen and Hemolymph Nodes
Figure 2-122  Ruptured splenic hyperplastic nodule in a dog.
Splenic hyperplastic nodules can become engorged with blood
and are prone to rupture causing hemoabdomen.
Figure 2-123  Splenic fibrohistiocytic nodule in a dog. Grossly,
fibrohistiocytic nodules appear similar to nodular hyperplasia, but
do not undergo differentiation toward hematoma.
on adjacent vascular sinuses, resulting in vascular pooling and
thrombocytopenia.
Fibrohistiocytic nodules
The term splenic fibrohistiocytic nodule has been used by
veterinary pathologists to prognosticate those lesions in which
it is difficult to differentiate among nodular hyperplasia, follicular
type lymphoma, and splenic sarcoma. Although the use of
immunohistochemistry (IHC) and PARR testing allows for
proper identification of follicular type lymphomas, differenti-
ating splenic sarcomas from hyperplastic lesions based on
morphology alone still presents a major challenge. Grossly,
fibrohistiocytic nodules appear similar to nodular hyperplasia,
but occur as highly cellular masses that do not undergo dif-
ferentiation toward hematoma (Fig. 2-123). Histologically,
fibrohistiocytic nodules are composed of mixed populations
of lymphoid cells, histiocytes, and spindle cells, referred to as
“fibrohistiocytic” cells, admixed with hematopoietic elements
(Fig. 2-124). They essentially present a morphologic contin-
uum between nodular hyperplasia and either follicular
 Spleen and Hemolymph Nodes
Figure 2-124  Splenic fibrohistiocytic nodule in a dog. Fibrohis-
tiocytic nodules are composed of mixed populations of lymphoid
cells, histiocytes, and spindle cells referred to as “fibrohistiocytic”
cells.
lymphoma or histiocytic or splenic sarcoma (eFig. 2-28). The
proportion of lymphocytes and fibrohistiocytic cells has been
used to grade fibrohistiocytic nodules. Grade 1 nodules are
composed of 70% or more lymphocytes, and grade 2 nodules
have 40-70% lymphocytes. Both grade 1 and grade 2 fibrohis-
tiocytic nodules most likely represent nodular hyperplasia, but
follicular lymphoma should be excluded, especially for grade
1 nodules by using immunophenotyping and PARR testing.
Grade 3 nodules are composed of <40% lymphocytes and
most likely represent splenic sarcomas, but histiocytic sarco-
mas have to be excluded by using IHC for CD18 and CD204.
When combined with the mitotic index, dogs with grade 1
and 2 fibrohistiocytic nodules with >40% lymphocytes and
<10 mitoses/high-power field (HPF) had an 87% survival
probability of 12 months or more following splenectomy. In
contrast, dogs with grade 3 fibrohistiocytic nodules with >10
mitoses/HPF were associated with only 55% survival probabil-
ity at 12 months postsplenectomy.
Hematopoietic alterations
Extramedullary hematopoiesis (EMH) results when there
is cytokine induction for increased cell production and plu-
ripotent hematopoietic stem cells available. These cells nor-
mally circulate in very low numbers and, in preparation for
EMH, they return to embryonic sites of colonization, sparing
the germinal centers and periarteriolar lymphoid sheaths.
Although EMH is commonly found to some degree within
the spleen of normal dogs, extensive EMH occurs with chronic
anemia, chronic respiratory or cardiovascular disease, various
bacterial septicemias, or with certain neoplastic conditions, for
instance, hemophagocytic histiocytic sarcoma. Grossly, the
spleen appears unremarkable or mildly enlarged. Histologi-
cally, EMH is characteristically includes all 3 lineages (Fig.
2-125), but one cell line may predominate, such as the granu-
locytic system in canine pyometra or the erythroid system in
hemolytic anemia. Megakaryocytes are the most obvious
hematopoietic precursor and characteristically lie adjacent to
the smooth muscle trabeculae when stimulation is mild, but
may become diffusely distributed in the sinus areas when
stimuli are pronounced and prolonged. Splenic erythropoiesis
191
Figure 2-125  Splenic extramedullary hematopoiesis in a dog.
Histologically, extramedullary hematopoiesis includes all 3 lin-
eages, but one cell line may predominate.
is coincident with increased splenic red cell destruction in
immune hemolytic anemias.
Myeloid metaplasia in spleen and other sites of embryonic
hematopoiesis occurs in myelofibrosis (MF), myelodysplastic
syndrome (MDS), myeloproliferative neoplasia (MPN), and
combinations of MF with MDS or MPN. If the animal has
idiopathic myelofibrosis in bone marrow, myeloid metaplasia
in spleen is benign, and typically consists of all hematopoietic
lineages with synchronous maturation. Grossly, the spleen is
uniformly enlarged with turgid capsule and rounded borders.
The parenchyma is dry and light pink owing to hematopoietic
tissue, rather than dark and cyanotic as in congestion. If the
animal has MDS or MPN, there is predominance of one cell
lineage, most often granulocytes, but other cell lineages may
also be present. The predominant cell lineage typically has
dysplastic changes, and fills the splenic sinusoids. Dyspoiesis
may affect only one cell lineage, or more commonly all 3, and
may consist of multinucleated rubricytes, incipient Howell-
Jolly bodies, giant metamyelocytes and band neutrophils,
donut-shaped nuclei, and hypolobulated megakaryocyte
nuclei. Myeloid metaplasia is often accompanied by lymphoid
hyperplasia, hemosiderosis, and splenic fibrosis. With increas-
ing splenic myeloid metaplasia, there is gradual reduction in
the lymphoid compartment, and eventual lymphoid atrophy.
Small infarcts develop and are likely the result of loss of plas-
ticity, and fibrous impairment of vascular structures.
Rarely, the spleen contains a myelolipoma, which appears
grossly as a focal pale nodule on cut surface. Histologically,
myelolipomas are composed of well-differentiated adipocytes
with large fat vacuoles that are sharply demarcated from
the normal splenic parenchyma (Fig. 2-126). Adipocytes are
admixed with hematopoietic and largely erythropoietic cells
that can be sparse or dominate the neoplastic mass. Myeloli-
pomas are usually incidental findings.
Neoplastic diseases of the spleen
The vascular tumors of the spleen are described in Vol. 3,
Cardiovascular system, and leukemic diseases involving the
spleen are described with the myeloproliferative and lympho-
proliferative diseases (this chapter). The spleen is expected to
 191.e1

eFigure 2-28  Splenic fibrohistiocytic nodule in a dog. Fibrohis-

tiocytic nodules are composed of mixed populations of lymphoid
cells, histiocytes, and spindle cells referred to as “fibrohistiocytic”
cells.
192 CHAPTER 2  •  Hematopoietic System
Figure 2-126  Splenic myelolipoma in a dog. Myelolipomas are
composed of well-differentiated adipocytes with large fat vacu-
oles admixed with hematopoietic and largely erythropoietic cells,
and sharply demarcated from the normal splenic parenchyma.
be involved in systemic malignancies of the lymphoid system,
but this is not always the case or apparent. In a review of
lymphomas, there was splenic involvement in only 41 of 72
cases (57%) in dogs, 35 of 81 cases (43%) in cats, and 27 of
90 cases (30%) in cattle. Although the spleen may not be invaded
in lymphoma, it is much more likely to be involved in lymphoid
and myeloid leukemia.
The size of the spleen or the absence of splenic masses
cannot be taken in itself as evidence for the presence or
absence of neoplastic processes, especially round cell neo-
plasms. Careful microscopic examination must be carried out
to determine whether a suspect neoplastic cell population can
be detected. Even when nodular masses are detected grossly,
differentiating hematomas or blood-distended hyperplastic nodules
from hemangiosarcomas will often require examination of numer-
ous sections of tissue around the margins of the splenic mass.
Most hemangiosarcomas will appear grossly as dark red,
blood-filled cystic masses (eFig. 2-29), but small more solid,
gray-white nodules can occur (Fig. 2-127). Microscopically,
hemangiosarcomas may be characterized by well-differentiated
endothelial cells forming incomplete vascular spaces that are
supported by a collagenous and fibrous stroma and commonly
thrombosed (Fig. 2-128). Alternatively, they can appear as
sheets of haphazardly arranged, anaplastic spindle cells with
rare slit-like vascular spaces. Such lesions may require IHC for
CD31, factor 8–related antigen, or CD34 for confirmation.
Hemangiosarcomas of the spleen occur most commonly in the
dog and often result in hemoabdomen. Primary splenic hem-
angiosarcomas rapidly metastasize to the liver and often seed
the mesentery. Metastatic hemangiosarcomas from the subcu-
tis or other sites are also common. Regardless, the prognosis
is poor. Despite careful staging prior to splenectomy, there are
frequent micrometastases at the time of surgery.
When the spleen is symmetrically enlarged to a mild or
greater degree, the histologic infiltration is usually diffuse, as
occurs in leukemia, and occupies all of the vascular spaces.
There is initially, at least, mild atrophy of the germinal centers
and periarteriolar lymphoid sheaths. A diagnosis of lymphoma
in the spleen may be difficult when there is a prominent fol-
licular pattern, and IHC and PARR testing are often required.
Spleen and Hemolymph Nodes
A
B
Figure 2-127  Splenic hemangiosarcoma in a dog. A. Some hem-
angiosarcomas appear as small more solid, gray-white nodules.
B. More commonly, hemangiosarcomas appear as dark red, blood-
filled cystic masses.
In the horse and dog, lymphoma may be nodular in the spleen
and has to be differentiated from nodular hyperplasia.
Non-angiogenic, nonhematogenic splenic sarcomas occur
with some frequency in older dogs with no or vague clinical
signs (Fig. 2-129). This group of sarcomas includes a number
of morphologic variants, including osteosarcoma, fibrosar-
coma, leiomyosarcoma, liposarcoma, chondrosarcoma, myxo-
sarcoma, rhabdomyosarcoma, and undifferentiated sarcoma.
The lack of distinct morphologic characteristics among fibro-
sarcomas, leiomyosarcomas, fibroblastic osteosarcoma, or
undifferentiated sarcomas has led to the use of the common
term “splenic sarcoma” for this group (eFig. 2-30). Although
malignant fibrous histiocytoma of the canine spleen has been
proposed as a distinct entity, the term is confusing because it
is based on the microscopic feature of multinucleated giant
cells rather than histiocytic differentiation and should be
avoided. In general, splenic sarcomas appear grossly as rela-
tively well-circumscribed, gray, often multilobular masses that
are raised above the splenic capsule and commonly undergo
necrosis (Fig. 2-130). Microscopically, these neoplasms are
unencapsulated, invasive, and composed of polygonal to spin-
dyloid neoplastic cells that can become highly anaplastic with
large, bizarre nuclei, and prominent nucleoli (Fig. 2-131, eFig.
2-31). Multinucleated giant cells are common. Neoplastic
cells are often arranged in a storiform or fingerprint pattern
(Fig. 2-132, eFig. 2-32). Large areas of necrosis are commonly
observed. Neoplastic cells are positive for vimentin and vari-
ably positive for muscle specific actin, desmin, and S100,
suggesting either ambiguous morphologic findings or the
 192.e1

eFigure 2-29  Splenic hemangiosarcoma in a horse. Most com- eFigure 2-30  Splenic sarcoma in a dog. A non-angiogenic, non-
monly, hemangiosarcomas appear as dark red, blood-filled cystic hematogenic splenic sarcoma appears as a multinodular, white,
masses. firm, highly destructive mass in the splenic parenchyma.

A B
eFigure 2-31  Splenic sarcoma in a dog. A. A non-angiogenic, nonhematogenic splenic sarcoma
composed of sheets of polygonal neoplastic cells arranged in a storiform pattern that is unencap-
sulated and highly invasive. B. Although necrosis can be extensive, neoplastic cells surrounding
blood vessels tend to be more viable.
192.e2

eFigure 2-32  Splenic sarcoma in a dog. Neoplastic cells are often

arranged in a storiform or fingerprint pattern.
 Spleen and Hemolymph Nodes 193

A
Figure 2-130  Splenic sarcoma in a horse. Well-circumscribed,
firm, gray mass that is raised above the splenic capsule.

B
Figure 2-128  Splenic hemangiosarcoma in a dog. A. Well-
differentiated neoplastic endothelial cells form incomplete vascu-
lar spaces that are supported by a collagenous and fibrous stroma.
B. Thrombosis is commonly observed within hemangiosarcomas.

Figure 2-131  Splenic sarcoma in a dog. A non-angiogenic, non-

hematogenic splenic sarcoma composed of sheets of polygonal
neoplastic cells that are unencapsulated and highly invasive.

Figure 2-129  Splenic sarcoma in a dog. A non-angiogenic, non-

hematogenic splenic sarcoma appears as a multinodular, white,
firm, homogeneous mass rising above the capsular surface. (Cour- Figure 2-132  Splenic sarcoma in a dog. Neoplastic cells are often
tesy S. Kesdangsakonwut.) arranged in a storiform or fingerprint pattern.
194 CHAPTER 2  •  Hematopoietic System Spleen and Hemolymph Nodes

Figure 2-134  Sertoli cell tumor metastatic to the spleen in a

Figure 2-133  Carcinoma metastasizing to the spleen in a dog. horse. A large multinodular mass in the splenic parenchyma with
Multifocal metastases of a mammary carcinoma with “umbilica- cystic spaces and extensive areas of necrosis.
tion” caused by central necrosis. (Courtesy S. Kesdangsakonwut.)

possibility of a common histogenesis from smooth muscle

trabeculae or a distinct population of splenic myofibroblasts.
Most cases are negative for KIT, excluding a metastasizing
gastrointestinal sarcoma as a differential. In some neoplasms,
there is extensive inflammatory cell infiltration, especially
small lymphocytes and dendritic cells, making differentiation
from lymphohistiocytic hyperplastic lesions extremely diffi-
cult. IHC for CD18 and CD204 should be used to differenti-
ate splenic sarcomas from histiocytic sarcomas and CD31 and
factor 8–related antigen for differentiation from hemangiosar-
comas that both may present similar microscopic features.
Whether some splenic sarcomas arise from hyperplastic
nodules is unclear. Although one should also try to reach an
accurate diagnosis, for lesions that lack distinct features of
malignancy and appear as proliferative lesions composed of
lymphocytes, histiocytic cells, and spindle cells, classification
as a fibrohistiocytic nodule of a distinct grade may still provide
additional clinical information. Regardless, splenic sarcomas Figure 2-135  Splenic mastocytosis in a cat. The spleen is dif-
often metastasize to the liver, invade adjacent organs, and are fusely infiltrated by neoplastic mast cells that have a finely granu-
more common in small- rather than large-breed dogs. Median lated, poorly stained cytoplasm with no recognizable mast cell
survival times are only 4 months with 80-100% mortality after granules.
12 months. The mitotic index closely correlates with biological
behavior of primary splenic non-angiogenic, nonhematogenic sar-
comas, and dogs with neoplasms with a mitotic index <9 had
significantly longer survival times than dogs with neoplasms blue or Giemsa. For feline splenic MCTs, widespread dissemi-
with a higher index. nation and metastasis are common. Regardless, splenectomy
Metastatic neoplastic disease of the spleen is uncommon. It is of cats with splenic MCTs has been associated with median
not likely that migration of tumor cells to the spleen is unusual, survival times of more than a year.
but rather that the functional efficiency of the cord and sinus
macrophages usually prevents colonization. Metastatic carci-
noma involving the spleen usually arises from the pancreas, Further reading
liver, or reproductive system (Fig. 2-133). Carcinomatous Cole PA. Association of canine hemangiosarcoms and hematomas with
implants originating from other abdominal viscera can mimic nodular lymphoid hyperplasia or siderotic nodules. J Vet Diagn
peritoneal tuberculosis (Fig. 2-134). Mast cell tumors (MCT) Invest 2012;24:759-762.
in dogs and cats can metastasize to the spleen, but primary Eberle N, et al. Splenic masses in dogs. Part 1: epidemiologic, clinical
visceral MCTs affecting the spleen are not uncommon in cats. characteristics as well as histopathologic diagnosis in 249 cases
Affected spleens are enlarged and may appear mottled, (2000-2011). Tierarztl Prax Ausg K Kleintiere Heimtiere 2012;40:
nodular, or irregular. Histologically, feline neoplastic mast cells 250-260.
have a finely granulated, poorly stained cytoplasm, and typical Gamlem H, et al. Canine vascular neoplasia—a population-based clini-
mast cell granules are not usually recognizable on H&E (Fig. copathologic study of 439 tumours and tumour-like lesions in 420
2-135). Cytoplasmic granules may not stain with toluidine dogs. APMIS Suppl 2008;125:41-54.
 Spleen and Hemolymph Nodes 195

Malinckrodt MJ, Gottfried SD. Mass-to-splenic volume ratio and splenic

weight as a percentage of body weight in dogs with malignant and
benign splenic masses: 65 cases (2007-2008). J Am Vet Med Assoc
2011;239:1325-1327.
Moore AS, et al. Histologic and immunohistochemical review of splenic
fibrohistiocytic nodules in dogs. J Vet Intern Med 2012;26:1164-
1168. Erratum in J Vet Intern Med 2013;27:215.
Sharpley JL, et al. Color and power Doppler ultrasonography for char-
acterization of splenic masses in dogs. Vet Radiol Ultrasound
2012;53:586-590.
Spangler WL, Kass PH. Pathologic and prognostic characteristics of
splenomegaly in dogs due to fibrohistiocytic nodules: 98 cases. Vet
Pathol 1998;35:488-498.
Spangler WL, Kass PH. Splenic myeloid metaplasia, histiocytosis and
hypersplenism in the dog (65 cases). Vet Pathol 1999;36:
583-593. A
Watson AT, et al. Safety and correlation test results of combined
ultrasound-guided fine-needle aspiration and needle core
biopsy of the canine spleen. Vet Radiol Ultrasound 2011;52:
317-322.

Jembrana disease
Jembrana disease is a severe acute disease that occurs in Bali
cattle (Bos javanicus) in Indonesia, with a case fatality rate of
20%. Mild or subclinical disease occurs in other cattle species
and buffalo. The cause is species Jembrana disease virus
(JDV), family Retroviridae, genus Lentivirus, which is closely
related to bovine immunodeficiency virus. Virus titers are high
in blood in the acute disease, and virus is present in saliva and
milk; direct transmission may then occur to susceptible cattle
via conjunctival, intranasal, or oral routes. During the height
of viremia, virus may also be transmitted mechanically by
hematophagous arthropods. Recovered cattle may have per-
sistent low-level viremia, but are probably an infrequent
source of infection. Cattle of European breeds and sheep are
resistant to the disease, although subclinical carrier status may
persist for many months.
Clinically, following an incubation period of 5-10 days after
infection, there is transient fever, dullness, and anorexia. Mild
oculonasal discharge, pallor of the mucosae, and diarrhea with
blood-stained feces are common. Superficial lymph nodes are
visibly enlarged, whereas examination of the blood during the
febrile period usually reveals anemia, leukopenia, thrombocy-
topenia, and in some cases, which may be fatal, an elevated
blood urea level. Basophilic cytoplasmic inclusions are occa-
sionally seen in circulating mononuclear cells.
At autopsy, lesions may not be remarkable, but several are
constant and of diagnostic value. Enlargement of lymph nodes
with slight edema and blurred corticomedullary junctions are
widespread but most marked when regional to other affected
organs, for instance, lungs and liver. During the acute phase,
there is splenomegaly, and on incision the surface shows small
gray-red foci. Scattered petechiae or ecchymoses occur irregu-
larly in the serosae of the heart, gastrointestinal tract, kidneys,
and below the endocardium. The myocardium and liver
contain gray 1-3 mm foci. Pulmonary lesions are subtle, but
can be detected in the cranial lobes, which are firmer than
normal, blue-red, finely mottled, and slightly edematous.
Adjacent lobules show mild overinflation. Shallow erosions
may occur in the gastrointestinal tract and caudal tongue,
where an occasional ulcer is also possible. The kidneys are
consistently pale and have small numbers of gray foci.
B
Figure 2-136  Jembrana disease in a cow. A. Lymph node with
intense proliferation of large lymphoid cells sparing lymphoid
follicles. B. The bone marrow is infiltrated by proliferating large
lymphoid cells. (Courtesy M. O’Hara.)
The general picture may be complicated by concurrent
infections, which have included Pasteurella pneumonia and
helminths of the liver, pancreas, or alimentary tract.
Histologically, 3 phases can be recognized in the acute
disease process of ~6 weeks. During the first week, there is a
general response by the lymphoreticular system, but in the
second phase, up to the fifth week approximately, there is
intense nonfollicular proliferation of reticular and large lym-
phoid cells (Fig. 2-136, eFig. 2-33). A similar infiltrative
process occurs in the liver, kidneys, adrenal medulla, and
choroid plexus systems of the brain, although the central
nervous tissue shows little change. In the cranial lung lobes,
alveolar cells are enlarged, pleomorphic, and proliferative
alongside infiltrating mononuclear cells (Fig. 2-137, eFig.
2-34). Mid-sized pulmonary veins and arteries may be filled
with large numbers of intravascular macrophages. In the third
phase, the follicular lymphoid system is reactivated, and
plasma cells appear progressively. The sequence is consistent
with a transient immunosuppressive disease. Indeed, the
humoral immune response can be suppressed and delayed in
JDV-infected cattle. Histologic lesions regress, but traces of
change can be seen up to 60 days after infection.
Pleomorphic basophilic cytoplasmic inclusions are present in
all affected tissues. By light microscopy, clumps of minute
 195.e1

Further reading
Locke JE, Barber LG. Comparative aspects and clinical outcomes of
canine renal hemangiosarcoma. J Vet Intern Med 2006;20:
962-967.
Spangler WL, et al. Primary mesenchymal (nonangiomatous/
nonlymphomatous) neoplasms occurring in the canine spleen: ana-
tomic classification, immunohistochemistry, and mitotic activity cor-
related with patient survival. Vet Pathol 1994;31:37-47.
Yang TJ, Gawlak L. Lymphoid organ weights and organ:body weight
ratios of growing beagles. Lab Anim 1989;23:143-146.
195.e2

eFigure 2-33  Jembrana disease in a cow. The bone marrow is eFigure 2-34  Jembrana disease in a cow. Pulmonary capillaries,
infiltrated by proliferating large lymphoid cells. (Courtesy M. veins and arteries may be filled with large numbers of intravascu-
O’Hara.) lar mononuclear cells. (Courtesy M. O’Hara.)
196 CHAPTER 2  •  Hematopoietic System
Figure 2-137  Jembrana disease in a cow. Mid-sized pulmonary
veins and arteries may be filled with large numbers of intravascu-
lar mononuclear cells. (Courtesy M. O’Hara.)
coccoid bodies about 0.5 µm, and larger, pleomorphic intravac-
uolar structures occur in reticular and large lymphoid cells,
macrophages including Kupffer cells, pulmonary alveolar cells,
and occasionally in vascular endothelium.
Further reading
Desport M, Lewis J. Jembrana disease virus: host responses, viral
dynamics and disease control. Virology 2010;404:261-268.
Stewart M, et al. TaqMan real-time reverse transcription-PCR and
JDVp26 antigen capture enzyme-linked immunosorbent assay to
quantify Jembrana disease virus load during the acute phase of in
vivo infection. J Clin Microbiol 2005;43:5574-5580.
LYMPH NODES
Structure and function of normal lymph nodes
Lymph nodes, like the spleen, are variably involved in myelo-
poiesis in the fetal stage of development, and convert to
lymphopoiesis shortly after birth. The actual level of lymph
node development at birth varies widely with different species.
Calves and foals have well-developed lymph nodes at birth,
and may have germinal center formation if there has been
intrauterine infection. In contrast, nestlings at birth have
readily recognizable lymph nodes in the neck, mediastinal and
mesenteric areas; the nodes have a loose reticular structure,
very low lymphocyte density, and only limited organization
into cortex and medulla (Fig. 2-138).
The microcirculation of the mammalian lymph node is
closely related to its function, and governs the architectural
development of the node. Afferent lymphatics enter the capsule
and bathe the cortex via the peripheral sinus. Small lymphatic
ducts perforate the inner capsule of the peripheral sinus and
form spherical microanastomotic networks that correspond in
size to the germinal centers. On the medullary aspects of these
spherical networks, the lymphatics anastomose into larger
ducts that drain into the medullary sinuses and then into the
major hilar efferent lymphatics. In contrast, blood vessels both
enter (arteriole) and leave (vein) through the hilar region,
Lymph Nodes
Lymphoid follicle
in outer cortex
Paracortex
Medullary sinus
Efferent lymphatic
vessel
Vein
Artery
Paratrabecular
sinus
Subcapsular sinus
Capsule
Afferent lymphatic
vessel
Figure 2-138  Schematic drawing of lymph node. Sectioning at
various levels of lymphoid follicles will results in different histo-
logic appearances.
with small vessel arborization in the cortical area. In the outer
cortex, small arteries form meta-arterioles or precapillary arte-
rioles that perfuse the germinal centers and mantle cell corona.
The transition to capillaries occurs at this site, and these
vessels then enlarge to form the postcapillary or high-endothelial
venules (HEV) of the paracortex, across which there is heavy
traffic of lymphocytes from the blood to the lymph node
parenchyma. These vessels then unite to form progressively
larger veins that follow the medullary stroma to form the
efferent hilar vein. In pigs, the cortex and medullary areas of
lymph nodes are inverted, but the afferent lymph passes in
lymphatics to the center of the node, where the germinal
centers are normally located. Like other mammals, recirculat-
ing lymphocytes enter lymph nodes through postcapillary
HEV and distribute to T and B areas, and emigrate directly
into the peripheral venous system rather than the efferent
lymphatics as in other mammals.
The various vascular structures of lymph nodes form char-
acteristic associations with nodal tissue. The lymphatics are
identifiable as major ducts entering the peripheral sinus, but
the intranodal lymphatics are not obvious, except for the large
medullary sinuses and the efferent hilar vessels. The arteries
can be identified entering the medullary or hilar area of the
node, and they follow the fibrous trabeculae to the medullary
sinuses. In this area, the arterioles form a regular and obvious
network with the medullary cords, such that the arterioles are
centrally placed in the sinuses and connected by a network of
reticular stroma to the thin limiting membranes of the medul-
lary cords. The smaller arterioles of the paracortex are irregu-
larly visible, as are the terminal arteriolar vessels of the
germinal centers and the mantle cell cuffs. In the adjacent
paracortex, the postcapillary venules are readily identified.
These vascular structures of the blood and lymphatic systems
through their interactions form 3 endothelium-lined compart-
ments: intravascular, intralymphatic, and interstitial, which
 196.e1

Further reading
Tenaya IW, et al. Flow cytometric analysis of lymphocyte subset kinetics
in Bali cattle experimentally infected with Jembrana disease virus.
Vet Immunol Immunopathol 2012;149:167-176.
196.e2

Further reading
Belisle C, Sainte-Marie G. Blood vascular network of the rat lymph
node: tridimensional studies by light and scanning electron micros-
copy. Am J Anat 1990;189:111-126.
Spalding H, Heath T. Pathways of lymph flow through superficial ingui-
nal lymph nodes in the pig. Anat Rec 1987;217:188-195.
 Lymph Nodes
provide the functional architecture by which the lymph node
filters lymph, processes antigen, and returns lymphocytes from
blood to tissue.
The microcirculatory arrangement directs antigens enter-
ing the cortical area in lymphatics to the germinal centers and
the outer cortex. The germinal center has a specific polarity
directed at the source of antigen, which in those germinal
centers near the outer cortex can directly be seen to be ori-
ented to lymph flow from the peripheral sinus. The germinal
center is surrounded by an elliptical cuff of small B lympho-
cytes that is thicker at the superficial pole region near the
peripheral sinus and thinner on the deep pole region adjacent
to the deeper areas of paracortex. Within the germinal center
itself, the cells in the superficial area nearer to the peripheral
sinus have smaller nuclei with more abundant cytoplasm. This
produces a lighter staining area on low-power examination. In
contrast, the cells in the deep pole of the germinal center,
nearer to the paracortex, have larger nuclei with minimal
cytoplasm, resulting in this area having a darker appearance.
Within the central area of the germinal center, there are large
macrophages that contain ingested apoptotic debris, repre-
senting lymphocytes that have undergone somatic hypermu-
tation and have not been selected for further development.
Depending on the plane in which the germinal center is sec-
tioned, these large macrophages may form a complete field
with a “starry-sky” effect across the circumference of the ger-
minal center. This polarity of germinal centers is a constant
feature of lymphoid tissue in lymph nodes, tonsil, and Peyer’s
patch, and is always most readily observed in relation to
antigen in those follicles near the peripheral sinus. Recognition
of polarity is an important aspect of differentiating benign follicu-
lar hyperplasia from follicular lymphoma. Germinal centers that
lie deep within the node in cases of follicular hyperplasia do
not have an obvious orientation to the capsule, but it can be
assumed that the deep and superficial poles are oriented to
the source of antigen and the fine network of lymphatics that
drain the peripheral sinus.
Paracortical tissue may appear as a solid band encompass-
ing the germinal centers, as is typical of the young. With
maturity, paracortical areas irregularly recede and regenerate
in loosely defined, but densely aggregated, deep cortical units.
These units are also defined by cellular composition and vas-
cular supply as are the germinal centers, but, in contrast to
the latter, the deep cortical units are thymus-dependent areas
populated by T cells. There are gaps in the inner layer of the
outer sinus by which the entering lymph can pass directly to
the medullary sinuses. Presumably, this route is taken by heavy
but nonthreatening flow as occurs when frank blood enters
the lymph node during biopsy (red cells appear in the medul-
lary sinuses within minutes of the initiating surgical trauma).
The processing of antigen occurs at random in lymph node
cortex but is always associated with a background of dendritic
reticular cells that function as antigen-presenting cells of the
germinal center. A portion, if not all, of the follicular dendritic
cells arise from Langerhans cells that develop contact sensiti-
zation with antigen in epidermal areas drained by the particu-
lar lymph node. Langerhans cells in epithelial areas bind
antigen relatively inefficiently, but within the germinal centers
they undergo maturation in the process of forming tight junc-
tions with other dendritic cells, thus forming a background
network for the lymphoid follicles. Blood monocytes cultured
in the presence of macrophage colony-stimulating factor
(M-CSF) or granulocyte-macrophage CSF and interleukin-4
197
differentiate without proliferation into CD14-positive macro-
phages or CD1A-positive dendritic cells. It is believed that
this process is repeated in lymph nodes for the maturation of
Langerhans cells to dendritic cells of the germinal centers, and
monocytes for differentiation into dendritic cells of the paracortical
areas. The process of B-cell maturation then involves the
interaction of B lymphocytes that have undergone immuno-
globulin chain rearrangement in preparation for antigen-
driven selection by hypermutation. This process takes place
with the assistance of T-helper cells and on the basis of antigen
presentation in the context of major histocompatibility
complex cellular recognition. B lymphocytes bound in this
manner are selected for further division, finally to become
plasma cells or memory B cells. Those that fail to be bound
in the T-lymphocyte interaction undergo programmed cell
death and are removed by follicular macrophages.
In the development of the T and B cells there are 2 major
phases of differentiation that include antigen-independent and
antigen-dependent stages. The antigen-independent differentia-
tion occurs in the primary lymphoid organs of the bone
marrow and the thymus where there is no exposure to foreign
antigen. These early stages are the blast cells of the lymphoid
system that are capable of self-renewal. In the germinal center,
these blast cells on exposure to antigen are able to divide and
become antigen dependent and become less able to prolifer-
ate, and may become memory cells that can last for years.
Tumors of the undifferentiated B cells are of the aggressive
type, whereas those of the differentiated type are likely to be
of indolent type.
The germinal center formation may arise from 3-10 naive
B cells that proliferate to form 10-15,000 B-cells in the more
than 10 generations of cells required to form a germinal center.
The seed cells for the germinal center are derived from the
naive cells of the mantle cell cuff. These cells initially express
immunoglobulin M (IgM) or IgD that assists them in recog-
nizing antigen in collaboration with the helper T cells and
antigen expressed by the dendritic cells. These antigen-
differentiated cells proliferate to fill the dendritic cell mesh-
work in the follicle center in ~3 days after initial antigen
stimulation. These blast cells accumulate in the deep pole or
dark zone of the germinal center, where they lack surface
immunoglobulin (sIg) and switch off the gene for BCL-2
protein and become susceptible to death through apoptosis.
These blasts undergo the somatic mutation of the Ig gene that
alters the affinity for antigen of the antibody they produce.
They may also at this stage switch from IgM to IgG or IgA in
the process of the late primary or secondary immune response.
The blast cells then mature to a smaller cell as the clone
reduces from 5-10 at the centroblast stage to a few as 3 as the
degree of somatic mutation increases. These mutated cells
then mature to centrocytes and accumulate in the superficial
or light pole of the germinal center. This process of Ig muta-
tion produces a marked intraclonal diversity of antibody-
combining sites from that of only a few precursors. As well,
in the germinal center, the BCL-6 gene undergoes mutation
of the 5′ noncoding promoter region that permits these cells
to be identified as having gone through the germinal center
reaction. Those centrocytes that have reduced affinity for
antigen undergo apoptosis and are phagocytized by the ger-
minal center macrophages. Those centrocytes that have
increased affinity for antigen may undergo further differentia-
tion to plasma cells by the action of CD23 on the dendritic
cells.
198 CHAPTER 2  •  Hematopoietic System
The dendritic cells of the germinal centers are long lived and
may retain antigen on their surface membranes for weeks or
months in the process of antigen focusing, by which the speci-
ficity of a germinal center for a specific antigen is maintained.
The dendritic cells of the paracortical nodules are also long
lived and, in superficial nodes of humans and cattle, they often
contain Birbeck granules characteristic of the Langerhans cells
of the skin, consistent with an origin and previous experience
in that area. There is functional logic in this arrangement
because the follicular macrophages and dendritic cells (B area)
are efficient in the digestion and processing of large particulate
antigens, whereas those in the paracortex (T area) are more
efficient in trapping low-molecular-weight substances, such as
agents producing contact sensitization in the skin, where the
resident reticular cells likely had their initial training. In
general, the lymphocytes in the germinal centers are largely B
cells, whereas those in the paracortex are largely T cells. The
subsets of T cells are also segregated, but not as cleanly, with
the suppressor and natural killer (NK) cells confined almost
entirely to the paracortex, whereas the helper cells are largely
in the paracortex but also in the germinal centers and their
small cell corona of mantle cells.
The process by which lymphocytes exiting the marrow and
thymus are directed to specific tissues is regulated by homing
receptors on the lymphocytes, called intercellular adhesion mol-
ecules (ICAMs), which bind to tissue-specific addressins on
high-endothelial or postcapillary venules in the peripheral
lymphoid tissues. The characteristic development and binding
of hematopoietic progenitor cells is mediated by adhesive
functions of cell surfaces, called integrins and selectins, which
are members of the immunoglobulin superfamily and identi-
fied by the CD44 reagent. There are >30 types of integrins,
which are heterodimeric transmembrane proteins, and 3
major subtypes of selectins, each with an affinity for various
ligands of endothelial cells in different areas of the body. By
these means, naive B and T cells leaving the bone marrow are
guided to specific areas of maturation in the thymus, intestine,
skin, and lymph nodes, and memory cells are directed to the
endothelium of specific tissues. It is worth mentioning in
conclusion that all of the hematopoietic-derived cells resident in
lymph nodes are capable of forming malignant tumors, including
the interdigitating reticular cells. This is in contrast to earlier
concepts that the cells of lymphomas of B- and T-cell types
remain relatively confined to the initial site of malignant trans-
formation until late in tumor progression. It is now under-
stood that neoplastic lymphocytes from either bone marrow or
peripheral lymphoid tissues may circulate at low levels early in
the development of disease. Their ability to disseminate is
dependent upon the development of cell surface proteins,
which mimic those of their benign counterparts, and permit
them to bind to endothelium and extravasate to new tissue
sites.
Further reading
Baumjohann D, et al. Persistent antigen and germinal center B-cells
sustain T follicular helper cell responses and phenotype. Immunity
2013;38:596-605.
Brum JS, et al. Eosinophilic sarcoma in a pig. J Vet Diagn Invest
2012;24:807-811.
Harris NL, Ferry JA. Classification of non-Hodgkin’s lymphomas. In:
Knowles DM, editor. Neoplastic Hematopathology. 2nd ed. Lip-
pincott Williams & Wilkins ed; 2001. p. 691-753.
Lymph Nodes
Huang C, et al. Lineage-specific functions of Bcl-6 immunity and
inflammation are mediated by distinct biochemical mechanisms.
Nat Immunol 2013;14:380-388.
Developmental diseases of lymph nodes
The lesions associated with inherited diseases of the immune
system are included in developmental diseases of the thymus.
In severe combined immunodeficiency, lymph nodes lack both
germinal centers and paracortical lymphoid colonization. In
severe T-cell deficiency, germinal centers are present, but para-
cortical areas are hypoplastic and the spleen lacks periarterio-
lar lymphoid sheaths. In severe B-cell deficiency, as occurs in
agammaglobulinemic foals, germinal centers are not formed
in lymph nodes or spleen, and plasma cells are not found. The
paracortical areas have normal cellularity, and cell-mediated
immunity is intact.
Degenerative diseases of lymph nodes
Lymphoid atrophy, with or without architectural alteration,
occurs in a variety of circumstances. The lack of antigenic
stimulation will result in smaller lymph nodes, as observed in
animals that have been raised germ free. Histologically, such
nodes have fewer primary and secondary follicles. Peripheral
lymph nodes of younger animals may appear similar, because
they have not been exposed to the same constant antigenic
stimulation as those nodes associated with the alimentary or
respiratory tract. Lymphoid atrophy associated with marked
dilation of medullary sinuses, termed vascular sinus transfor-
mation, results from blockage of the efferent lymphatics, and
is exacerbated by the development of fibrosis if the venous
drainage is also obstructed. These conditions may follow surgi-
cal intervention for lymphatic cannulation or occasionally
develop in animals that have been recumbent for prolonged
periods and suffered concurrent venous infarction of muscle.
High doses of radiation cause rapid destruction of lympho-
cytes, especially B cells, and thereby result in smaller lymph
nodes. Histologically, there is severe lymphocyte apoptosis
characterized by cell shrinkage, nuclear pyknosis, and frag-
mentation with apoptotic bodies and tingible body macro-
phages. Prolonged high doses of steroids, cytotoxic cancer
drugs, hypoxia, or heat can also cause severe apoptosis (Fig.
2-139). As long as the bone marrow can supply lymphocytes,
the normal lymph node size and architecture will be restored
within a few weeks. However, prolonged radiation will cause
fibrosis. Many viral infections target lymphocytes and will cause
lymphocyte necrosis in germinal center. Histologically, there is
cell swelling with chromatin clumping, karyorrhexis and kary-
olysis, and ultimately abundant eosinophilic cellular debris.
Lymphocyte necrosis is frequently accompanied by inflamma-
tory cells, for instance, neutrophils and phagocytic macro-
phages with intracytoplasmic cellular debris. Examples of
viruses that cause severe lymphocytolysis include equine her-
pesvirus, canine distemper virus, canine parvovirus, feline pan-
leukopenia virus, rinderpest virus, and bovine viral diarrhea
virus.
Cachectic and senile atrophy lead to moderate reduction in
the overall size of body nodes. Aging changes are largely limited
to mild thickening of the capsule and medullary trabeculae,
with reduced density of germinal centers. Senile atrophy is
seldom of note in large domestic animals, and is only seen with
frequency in dogs, cats, and primates. Grossly, senile lymph
nodes are small with edematous fascia, and characteristically
 Lymph Nodes
Figure 2-139  Lymphoid atrophy in a dog. Prolonged high doses
of steroids can cause severe apoptosis of lymphocytes.
Figure 2-140  Lymphoid atrophy and anthracosis in a dog. Lymph
node with marked reduction of size and discoloration by dark
pigment.
have dark-brown pigmentation of medullary areas that may
extend in decreasing concentration to the subcapsular sinus.
Histologically, there is loss of B and T cells and lymphoid fol-
licles. Cachectic atrophy is frequently encountered in old
sheep and goats with dental attrition, but can be encountered
in any animal with chronic debilitating disease caused by
systemic cancer, malabsorption, or prolonged starvation
leading to cachexia (Fig. 2-140). Cachexia leads primarily to
a loss of T cells and decreased T-cell production with little
effect on B cells. Histologically, cachectic atrophy is character-
ized by retention of the overall architecture with marked
reduction in cell density. Germinal centers are small with a
hypocellular mantle cell corona, and paracortical areas are
sparsely populated. In old animals with cachexia, there are
numerous pigment-bearing macrophages in medullary sinuses,
and thin proteinaceous fluid may be present in medullary
cords and sinuses.
Lymphatic sinus ectasia or lymphatic cysts can affect both
the medullary and subcapsular sinuses (Fig. 2-141). Diffuse
sinus ectasia usually occurs together with lymphoid atrophy
and can occur as part of the earlier described senile atrophy
199
Figure 2-141  Lymphatic sinus ectasia in a dog. Lymphatic sinus
ectasia or lymphatic cysts can affect both the medullary and
subcapsular sinuses of lymph nodes.
Figure 2-142  Lymphoid atrophy in a dog. Severe loss of cortical
tissue and diffuse edema in a lymph node with senile atrophy.
(Fig. 2-142). Histologically, dilated or cystic sinuses are lined
by lymphendothelium and filled with pale eosinophilic lymph.
A few lymphocytes, plasma cells, and macrophages may be
admixed with the lymph.
In emphysema of lymph nodes, gas is confined to the
sinuses. It affects the mesenteric nodes of swine in association
with intestinal emphysema. Emphysema of the bronchial
nodes commonly accompanies interstitial pulmonary emphy-
sema of cattle. The lymph nodes are puffy and light. The
sinuses are distended, and their endothelium is lined by large
macrophages and even giant cells, the mobilized cells occur-
ring in small clusters of spotty distribution on the walls of the
sinuses.
Complete or focal infarction of node is rather uncommon
and occurs with obstruction of blood flow. Predisposing condi-
tions include periarteritis nodosa, but in domestic animals,
lymph node infarcts are most commonly observed with large
cell lymphoma, and may be the first sign of disease. Throm-
bosis is seldom observed.
Sinus erythrocytosis, originating from hemorrhages in
tissues drained by the node, may be visible in the cortical or
200 CHAPTER 2  •  Hematopoietic System
Figure 2-143  Sinus erythrocytosis in a pig. Resulting from hem-
orrhage in tissues drained by the node.
Figure 2-144  Lymph node hemorrhage and edema in a pig.
Salmonella choleraesuis causes severe parenchymal hemorrhage
and edema.
medullary sinuses (Fig. 2-143, eFig. 2-35). Red cells delivered
to the node through the afferent lymphatics tend to pass
through the cortex to the medullary sinuses, where they are
ingested by macrophages. The breakdown of erythrocytes
within lymph nodes results in accumulation of hemosiderin,
which is always most prominent in medullary areas. This
finding has to be separated from hemorrhage occurring within
the nodal parenchyma (Fig. 2-144). In cattle and sheep that
die with acute respiratory distress, the cervical nodes are fre-
quently red-black owing to the blood contained in them,
much of which comes from the tracheal mucosa, where
extravasations begin in the lymphoid nodules. Intrasinusoidal
erythrocytes may also be an artifact from euthanasia or tissue
dissection during autopsy, especially in the bronchial or medi-
astinal lymph nodes.
Besides hemosiderin, which is iron-containing and appears
as golden brown granular material in macrophages within the
medullary cords and lymphatic sinuses of nodes with sinus
erythrocytosis, lipofuscin can also be detected in lymph nodes.
Lipofuscin is also a golden brown, finely granular pigment that
Lymph Nodes
Figure 2-145  Anthracosis in a cow. The black pigment detected
on cross section of a pulmonary lymph node is an incidental
finding.
is derived from the breakdown products of cell membrane
lipids. Ceroid is an acid-fast and autofluorescent variant of
lipofuscin. Both pigments are difficult to distinguish from each
other with H&E stains, but can be differentiate by use of an
iron stain (Prussian blue) and a Ziehl-Neelsen acid-fast stain.
Anthracosis is a regular finding in the pulmonary nodes of
dogs that live in industrial areas, and is of lesser frequency and
degree in cats (Fig. 2-145). The carbon is retained solely in
the phagocytes, principally those of the medullary cords, and
therefore only the medulla is black. The pigment is inert and
of no consequence. Melanin maybe found within lymph
nodes. This is a normal finding in some black-skinned animals
and is not considered a lesion. Care has to be taken to dif-
ferentiate neoplastic melanocytes draining to the lymph node
from melanin-containing macrophages in dogs with oral
malignant melanoma. Immunohistochemistry for CD204,
PNL-2, and melan-A are helpful in the distinction. An uniden-
tified black pigment, in appearance like melanin, is present in
the hepatic nodes of sheep and cattle when they have, or have
had, hepatic distomiasis. The same pigment occurs in bile ducts
infested by Fasciola hepatica, and, in the case of F. magna,
wherever the fluke has wandered.
Amyloid is rarely found in lymph nodes, but appears as
amorphous, eosinophilic, and hyalinized extracellular material
that can cause atrophy of the adjacent tissue. Congo red or
thioflavine staining are helpful in the diagnosis.
Further reading
Elmore SA. Histopathology of the lymph nodes. Toxicol Pathol 2006;
34:425-454.
Lanteri G, et al. Acquired melanosis caused by acorn ingestion in the
Nero Siciliano pig. Vet Pathol 2009;46:329-333.
Perillo A, et al. Lesions associated with mineral deposition in the lymph
nodes and lungs of cattle: a case-control study of environmental
health hazard. Folia Histochem Cytobiol 2009;47:633-638.
Hyperplasia of lymph nodes
Lymphadenopathy is defined as a regional or generalized
lymph node enlargement of unknown or unspecified cause.
 200.e1

B
eFigure 2-35  Sinus erythrocytosis in a cow (A) and in a pig (B).
A. Salmonella choleraesuis causes severe parenchymal hemorrhage
and edema. (Courtesy R.L. Amorim.) B. Hemorrhage in tissues
drained by the node results in a marbled appearance.
200.e2

Further reading
Blanchard JL, et al. Generalized amyloidosis in rhesus monkeys. Vet
Pathol 1986;23:425-430.
Ozcan K, Beytut E. Pathological investigations on anthracosis in cattle.
Vet Rec 2001;149:90-92.
 Lymph Nodes
Lymphadenosis, although not commonly used, more correctly
defines generalized lymphoid hypertrophy. Local enlargement
usually reflects a pathologic process limited to the drainage
area, particularly inflammatory or neoplastic disease. General-
ized enlargement of lymph nodes is a more serious finding,
because it indicates a systemic disease or a systemic neoplastic
disease. Infectious causes of generalized lymph node enlarge-
ment include tuberculosis, brucellosis, and protozoal infec-
tions. Generalized lymphadenopathy produced by an
extraordinary degree of paracortical blastogenesis is typical of
the early phases of malignant catarrhal fever and theileriosis
in cattle. There may be mild generalized lymph node enlarge-
ment in adrenal atrophy. Lymphoid hyperplasia to some
degree is transient in most young animals and in mesenteric
lymph nodes because of stimulation with intestinal antigens.
Endocarditis or abscessation may cause generalized lymphade-
nopathy, as will lymphoma. In general, in acute septic inflam-
mation the lymph nodes are normally mobile within the
subcutaneous tissues, but may become fixed with chronic
sepsis. Similarly, lymphadenopathy resulting from lymphoma
usually does not cause fixation of the nodes in the early stages,
but there is usually perinodal colonization with loss of mobil-
ity in advanced stages.
Lymphoid hyperplasia is a benign, non-neoplastic, reactive
response to an immune stimulus. It can involve both B cells
in lymphoid follicles and T cells in the paracortex, suggesting
either a humoral or cell-mediated response, respectively.
The histologic picture in benign lymphoid hyperplasia is vari-
able, with small and large lymphocytes, with cleaved and
noncleaved nuclei, and macrophages, plasma cells, and neu-
trophils. Unless the stimulation is <10 days old, this mixture
of cells should be organized architecturally with evidence of
follicular hyperplasia. Reactive or secondary follicles are
larger than unstimulated primary follicles, and the detailed
histologic appearance has been described under lymphoid
hyperplasia of the spleen. In the early, marked, immune
response, there will be diffuse parafollicular hyperplasia
with effacement of pre-existing follicles. The paracortex has
a moth-eaten appearance owing to the mixture of cells
present, which includes many large macrophages and pale
dendritic reticular cells. In contrast, in lymphomas, there tends
to be a monomorphic or bimorphic cell population and,
in animals in which follicular lymphomas are uncommon,
finding a monomorphic cell population with diffuse architecture
suggests lymphoma. In some lymphomas, macrophages may be
present, and numerous if there is a high mitotic rate in the
tumor and a high rate of cell death. In contrast, plasma cells
and neutrophils tend to be less numerous than in lymphadeni-
tis, although the presence of plasma cells in itself is no guar-
antee of a benign reaction. There are no lymphomas with 3 or
more lymphocyte types regularly present, and this fact tends
to assist in correctly interpreting the early diffuse lymphoid
proliferations.
The most important criteria for distinguishing lymphoma from
hyperplasia are the integrity of normal architecture and the uni-
formity of cell and chromatin type. Architecturally, the periph-
eral sinus is preserved, even in reactive states where there is
perinodal colonization by benign lymphocytes. In contrast, the
outer sinus is regularly encompassed or destroyed by lym-
phoma of advanced stage. One of the most difficult distinc-
tions is between follicular hyperplasia and follicular
lymphomas, and in these cases, the decision must be based
upon both the architectural changes and the character of the
201
cells interpreted in terms of the history and clinical signs.
Benign lymphoid follicular hyperplasia has a characteristic
morphology because of the antigen-oriented polarity of the
normal germinal center that can be highlighted by immuno-
phenotyping (see Lymphoma). For some cases, additional
PCR for antigen receptor rearrangements may be required
to accurately differentiate follicular lymphomas from follicu-
lar hyperplasia. If a firm decision cannot be made, it is fully
justifiable to request a repeat biopsy in 10 days, at which time
a diffuse benign reaction will have developed a follicular
pattern.
Many infectious diseases, for instance, brucellosis and
trypanosomiasis, present no problem in distinction from lym-
phoma, and result in a regular picture of follicular hyperplasia
followed by paracortical atrophy with generalized sclerosis
and medullary cord hyperplasia. In contrast, an unusual type
of follicular hyperplasia occurs in old sheep and goats that are
in poor condition and may have been destroyed because of
debility. There is regular capsular thickening and increased
prominence of medullary trabeculae. The follicles may be
very large, angular, and facetted in an irregular fashion.
Paracortical atrophy is marked, and there is medullary
cord hyperplasia and empty medullary sinuses. The most
marked changes affect the follicular vessels, which are large,
tortuous arterioles with hyalinized media and often mineral-
ized endothelium. The follicular centers include large epithe-
lioid macrophages and often a very marked tingible body
reaction. This reaction resembles angiofollicular lymph
node hyperplasia of the hyaline vascular type as described in
humans.
A nodular reaction of the paracortical areas occurs in lymph
nodes draining malignant tumors as well as other conditions,
and appears to represent an exaggerated thymus-dependent
response. The nodules may be multiple and coexist with fol-
licular atrophy, raising concern that they represent a multifo-
cal diffuse lymphoma. The nodules represent deep cortical units.
They are centered on networks of dendritic reticular cells and
are densely cellular, but with the histologic variation in cell
type characteristic of the paracortex. These paracortical
nodules, unlike germinal centers, are irregularly defined from
the surrounding paracortex by a lighter staining border of
larger pale-staining dendritic cells and macrophages and fewer
small densely stained lymphocytes.
Plasma cell hyperplasia occurs commonly together with
B-cell hyperplasia in animals when the response to antigenic
stimulation requires antibody production. Affected animals
have often enlarged nodes that contain large numbers of
plasma cells and their precursors in the medullary cords. There
may be partial effacement of normal nodal architecture, and
a plasma cell neoplasm may be considered as a potential dif-
ferential. However, the lack of cortical and capsular infiltra-
tion, binucleated, megalokaryocytic, and atypical plasma cells
help differentiate hyperplasia from a plasma cell neoplasm.
Chronic ehrlichiosis or leptospirosis has been associated with
severe lymphoid plasmacytosis.
Macrophage hyperplasia is primarily the result of resident
macrophage proliferation in the sinuses, but can be seen as
aggregates of macrophages within any region of the lymph
node that is, cortical, paracortical, and medullary. When
aggregates of macrophages occur within the lymph node
parenchyma, other terms such as “granulomatous inflamma-
tion,” “granulomatous lymphadenitis,” “histiocyte aggregates/
infiltrates,” and “macrophage aggregates/infiltrates,” have been
202 CHAPTER 2  •  Hematopoietic System
used to describe macrophage hyperplasia. However, the
term “granulomatous” should only be used if there is evidence
of epithelioid macrophages. In addition, some pathologists
only refer to granulomatous lymphadenitis when there is
partial or complete effacement of nodal architecture. Macro-
phage hyperplasia must be differentiated from sinus histiocytosis
that occurs through drainage of large numbers of macrophages
with efferent lymphatics from a tributary site. It is often neces-
sary to closely examine the organs and tissues within the
tributary field to differentiate sinus histiocytosis from sinus
macrophage hyperplasia. Lymph nodes of the respiratory and
alimentary system are constantly stimulated by antigens and
can have both sinusoidal histiocytosis and macrophage
hyperplasia.
Some of the more difficult distinctions between hyperpla-
sia and primary malignancy of lymph nodes must be made in
hyperimmune diseases. Eosinophilic myositis causes a remark-
able alteration in lymph nodes with generalized sclerosis and
thickening of the capsule, which is irregularly colonized by
small lymphocytes and eosinophils in linear arrays, and marked
thickening of the cortical and medullary trabeculae. There is
diffuse follicular hyperplasia with paracortical atrophy, and
medullary cord hyperplasia with prominent plasma cell accu-
mulation. Germinal centers vary markedly in size and shape
and have accentuation of the large and small cell layers, and
sinuses contain a dense matrix of epithelioid macrophages and
eosinophils. Eosinophils are also prominent throughout the
collagenous trabeculae.
One of the more common indications for lymph node
aspiration in dogs and cats are those associated with chronic
dermatitis or otitis. Lymph nodes draining areas of pigmented
skin that have been chronically inflamed frequently contain
melanin-bearing macrophages in the outer cortex, and these
are frequently present in aspirates of draining nodes. Cytologi-
cally, the melanin granules in melanocytes and macrophages from
benign sources are of uniform size, shape, and pigmentary density,
and in the dog resemble tiny rice grains. In contrast, melanin
granules that are associated with pigmented melanomas are
markedly variable in size, shape, and staining density and are
more likely to be associated with a sclerotic reaction in
the node.
In cases of benign chronic pleural effusion, there may be
infiltration of the mediastinal nodes and others draining the
cavities of benign mesothelial cells. These cells have large
vesicular nuclei with irregularly prominent, single, central
nucleoli and abundant cytoplasm and may be mistaken for
metastatic tumor. The exfoliating cells distend the peripheral
and medullary sinuses and stain for cytokeratin. There is no
necrosis. In pigs with porcine proliferative enteropathy, hyper-
plastic crypts may have “metastasized” to the mesenteric
lymph nodes. Drained epithelial cells lodge in the medullary
sinuses and continue to proliferate (Fig. 2-146). Silver stains
can easily identify Lawsonia intracellularis in the apical portion
of those epithelial cells.
Further reading
Elmore SA. Enhanced histopathology of the lymph nodes. Toxicol
Pathol 2006;34:634-647.
MacNeill AL. Cytology of canine and feline cutaneous and subcutane-
ous lesions and lymph nodes. Top Companion Anim Med
2011;26:62-76.
Lymph Nodes
Figure 2-146  Lawsonia intracellularis in a pig. Drained epithe-
lial cells lodge in the medullary sinuses of a mesenteric lymph
node and continue to proliferate.
Inflammatory diseases of lymph nodes
Lymphadenitis results when an infectious agent is present in the
lymph node, as distinguished from benign lymphoid hyperplasia,
in which the node is immunologically reactive, but free of
local invasion. Lymphadenitis may result from drainage to the
node of the products of a distant inflammatory process that
may progress to involve the node directly. It may be possible
to identify the infectious agent responsible for the lymphad-
enitis or to determine the type of invading agent from exami-
nation of the changes in the node. Lymphadenitis may be acute
or chronic, suppurative, caseous, or granulomatous. Distribution
of cell types present, including large macrophages, and the
quality and character of the neutrophil nuclei are indicative
of the cause of the process, which may be apparent if the
phagocytic cells present have ingested bacteria. Needle aspi-
rates from lymph nodes may occasionally yield large numbers
of necrotic cells, and it should be kept in mind that focal
lymphadenopathy may be the result of metastatic tumor.
Grossly in acute lymphadenitis, the lymph nodes are
enlarged, soft, and hyperemic to a variable degree. The capsule
is taut and thinned given the influx of cells, and on the cut
surface, the parenchyma bulges and may exude blood or pus
or lymph. Thus, if imprint preparations are made, it is neces-
sary to repeatedly remove the exuding lymph from the cut
surface to obtain enough adhesion for cellular exfoliation.
There may be focal discoloration resulting from previous pig-
mentation or infarction.
Histologically, acute lymphadenitis is characterized by
marked hyperemia with unusual prominence of small vessels
in cortical areas. Neutrophils are often present, both through
drainage from the subcapsular sinus and by migration from
the postcapillary venules. Some useful distinction can be made
between granulocytes arriving from these 2 sources. In general,
neutrophils in the peripheral and medullary sinuses indicate
inflammation in the tissue area drained and are not necessarily
indicative of local sepsis. On the other hand, foci of neutrophils
in cortical areas are of hematogenous origin and almost always
are reactions to bacterial colonization, as is likely to occur in
the mesenteric node in bacterial enteritis. When the inflam-
mation is caused by pyogenic organisms, abscessation is likely.
Depending on the disease process, eosinophils may constitute
 202.e1

Further reading
Langenbach A, et al. Sensitivity and specificity of methods of assessing
the regional lymph nodes for evidence of metastasis in dogs
and cats with solid tumors. J Am Vet Med Assoc 2001;218:
1424-1428.
 Lymph Nodes
25% or more of the cells present. The chromatin pattern of
lymphocytes in acute lymphadenitis differs from lymphoma
in that large chromocenters persist in small lymphocytes, and
larger lymphocytes, which are presumed to be the prolifera-
tive population, maintain larger chromocenters in benign con-
ditions, but tend to have a more uniform chromatin pattern
characteristic of cells in cycle in lymphomas. Suppurative
lymphadenitis is commonly encountered with bacterial septi-
cemias, for instance, Streptococcus equi, Brucella spp., or True-
perella pyogenes. Although severe pyogenic bacterial infections,
for instance, Francisella tularensis or Yersinia pestis, cause
focally extensive to severe liquefactive necrosis, focal areas of
necrosis are common with many infections, including parasitic
infections, for instance, toxoplasmosis (Fig. 2-147) or bacterial
infections, for instance, salmonellosis or Tyzzer’s disease.
In chronic lymphadenitis, hyperemia and edema are irreg-
ularly present, and the infected nodes are large and firm, and
may be fixed to local tissues if there has been cellulitis. The
capsule is thickened as are the internal trabeculae, and with
prolonged inflammation the node becomes dry and hard.
Lesions can occur as well-encapsulated abscesses (Fig. 2-148,
eFig. 2-36), granulomatous inflammation that can be focal or
diffuse (Fig. 2-149), or a mixed inflammatory reaction com-
Figure 2-147  Toxoplasma gondii in a cat. Necrotizing lymphad-
enitis with intralesional protozoal organisms.
Figure 2-148  Lymph node abscess in a dog. Large amounts of
yellow green pus surrounded by a thick fibrous capsule.
203
posed of follicular hyperplasia, microabscesses, sinus histiocy-
tosis, and fibrosis. Such chronic mixed lymphadenitis is
characteristically present in the supramammary lymph nodes
of cows with brucellosis of long standing, and to a lesser extent
in animals with chronic or recurrent bacterial mastitis. In the
latter cases, marked proliferation of the collagenous septa of
the medulla may completely displace the medullary cords.
Examples of chronic suppurative lymphadenitis leading to
abscessation include streptococcal infections in different
animal species, localized Trueperella pyogenes infections, or
foreign bodies in small animals causing severe peritonitis (Fig.
2-150). A specific example of a caseous lymphadenitis is caused
by Corynebacterium pseudotuberculosis. The classic example of
focal granulomatous lymphadenitis is caused by mycobacterial
infections. Although Mycobacterium bovis causes caseating
granulomas in lymph nodes of the digestive or respiratory tract
in ruminants or humans (Fig. 2-151, eFig. 2-37), Mycobacte-
rium avium complex can cause similar lesions in a wide variety
of species. Most commonly, granulomas appear caseous and
single or multifocal throughout the affected node. Histologi-
cally, they are characterized by caseous necrotic centers that
often become mineralized and are surrounded by epithelioid
macrophages and multinucleated giant cells of Langhans type
(Fig. 2-152). These granulomas are commonly surrounded by
A
B
Figure 2-149  Granulomatous lymphadenitis in a cow (A) and a
horse (B). A. Mycobacterium bovis can cause severe granulomatous
inflammation or caseous necrosis. B. Rhodococcus equi causing
multifocal granulomas.
 203.e1

A
eFigure 2-37  Tuberculosis in a cow. Multifocal caseous necrosis
of lymph node caused by Mycobacterium bovis. (Courtesy R.L.
Amorim.)

B
eFigure 2-36  Lymph node abscess in a cat (A) and a deer (B).
A. The lymph node parenchyma has been replaced by yellow pus
surrounded by a thick fibrous capsule. B. Trueperella pyogenes most
commonly causes abscesses in a variety of species.
204 CHAPTER 2  •  Hematopoietic System
Figure 2-150  Suppurative lymphadenitis in a dog. Severe sup-
purative peritonitis caused by a penetrating foreign body.
A sheep wherever they are raised, but horses, camels, deer,
B of spread in sheep and goats that are routinely corralled on
Figure 2-151  Tuberculosis in a cow. A. Diffuse caseous necrosis
of pulmonary lymph node caused by Mycobacterium bovis. B. M.
bovis causing miliary granulomatous inflammation of retropharyn-
geal lymph node.
Lymph Nodes
rims of lymphocytes, plasma cells, and fibroblasts. In contrast
to tuberculosis, Johne’s disease, caused by Mycobacterium
avium subsp. paratuberculosis, is characterized by noncaseous
granulomas in mesenteric lymph nodes of ruminants (Fig.
2-153, eFig. 2-38). Rhodococcus equi can cause severe granu-
lomatous lymphadenitis in horses that may ultimately result
in diffuse lymph node necrosis (Fig. 2-154, eFig. 2-39). Feline
infectious peritonitis can cause severe, focal, pyogranuloma-
tous lymphadenitis, and lesions may coalesce and efface the
whole node (Fig. 2-155). Many fungal infections, for instance,
blastomycosis (Fig. 2-156), cryptococcosis (Fig. 2-157), and
histoplasmosis, can cause severe diffuse granulomatous lymph-
adenitis. The architecture of an affected node is commonly
completely obliterated and replaced by pyogranulomatous to
granulomatous inflammation with accumulation of epitheli-
oid macrophages admixed with variable numbers of multi-
nucleated giant cells, neutrophils, lymphocytes, and plasma
cells.
Lymphadenitis may be expressed by changes that are
largely degenerative and affect the architecture of the tissue.
Thus, in some acute viral infections, there is rapid lysis of
lymphocytes, as in canine parvoviral infection and in rinder-
pest. In dogs, canine distemper virus causes marked necrosis
of lymphocytes with simultaneous formation of syncytial cells
(Fig. 2-158). In salmon poisoning of dogs, the nodes may be
much enlarged with follicular hyperplasia and accumulation
of macrophages. In some chronic infections, particularly with
the viruses causing immunodeficiency, the nodes are atrophic
with wrinkled capsule and depletion of lymphocytes, and with
sinus histiocytosis if the production of monocytes by the bone
marrow remains competent.
Caseous lymphadenitis
Caseous lymphadenitis (CLA) is a suppurative infection of the
lymph nodes, primarily of sheep and goats, caused by Coryne-
bacterium pseudotuberculosis (ovis). The disease occurs in
mules, and rarely cattle and humans may be affected. Cattle
can develop a pathologic syndrome that resembles that in
sheep, but they do so quite rarely, and the infection generally
remains localized to 1-2 regional nodes draining an infected
surface wound or a segment of intestine. C. pseudotuberculosis
is also the cause of ulcerative lymphangitis in cattle and horses,
and of pectoral abscesses in horses.
There are 2 serotypes, with type I in ovine, caprine and
occasionally bovine isolates, and type II in buffalo and most
infections in cattle. An exotoxin that consists of a phospholi-
pase D is an important aspect of virulence, with effects includ-
ing intravascular hemolysis, necrosis, pulmonary edema, and
shock.
C. pseudotuberculosis survives briefly in the environment
and is able to spread indirectly, which is an important means
the same bed ground to avoid predation. Infection also occurs
in shearing wounds in sheep and butting abrasions in males.
In horses, the disease occurs as ulcerative lymphangitis on
the fetlocks, which is consistent with the concept that skin
abrasions are important in spread of the organism. The sea-
sonal incidence of abscesses in the pectoral and other regions
of horses suggests that the organism may also be borne by
arthropods.
The disease in goats can be more severe than in sheep, the
most frequent lesions being in the lymph nodes of the head
 204.e1

eFigure 2-38  Johne’s disease in a goat. Mycobacterium avium eFigure 2-39  Rhodococcus equi in a horse. Multifocal granulo-
subsp. paratuberculosis causes noncaseous granulomas in mesen- matous inflammation in mesenteric lymph node.
teric lymph nodes.
 Lymph Nodes 205

A A

B B
Figure 2-153  Johne’s disease in a goat. A. Mycobacterium avium
subsp. paratuberculosis causes noncaseous granulomas in mesen-
teric lymph nodes. B. Multifocal granulomatous inflammation
with multinucleated giant cells of Langhans type.

a function of age. Caseous lymphadenitis is widespread in goats,

C enteric or mediastinal nodes grossly.
Figure 2-152  Tuberculosis in a cow. A. Caseating granulomatous
lymphadenitis with dystrophic mineralization of the necrotic
center. B. Large numbers of multinucleated giant cells of Lang-
hans type surround necrotic centers. C. Acid-fast bacilli can be
detected in the cytoplasm of epithelioid macrophages.
and neck. The lesions in goats closely resemble those of pseu-
doglanders or melioidosis (Burkholderia [Pseudomonas] pseu-
domallei), and it is important to distinguish these diseases.
Female goats and intact males have more extensive lesions
than castrated males. In sheep and goats, there is slow spread
of the disease, with the prevalence of abscesses increasing as
and the disease differs from sheep in the distribution of the
abscesses. In goats, the mandibular lymph nodes, followed by
the parotid nodes, are most often involved suggesting that the
organism is acquired through the buccal mucosa, as well as
through skin wounds. Superficial abscesses of the jaw and neck
region are common, and infection is probably acquired from
feed stalls or collars carrying the bacterium. In only a small
proportion of goats does infection spread to involve the mes-
Caseous lymphadenitis (CLA) in sheep almost always
follows a wound infection, usually a shearing wound. The
organism can penetrate the intact skin of freshly shorn sheep,
however, and may be transmitted by dipping fluids. Docking
and castration wounds and the umbilicus are of minor impor-
tance. Occasionally, the infection may be acquired by inges-
tion, as indicated by confinement of the lesions to the nodes
draining the buccal cavity (eFig. 2-40). Less commonly, the
organism is inhaled, producing lung abscesses. Parasitic wounds
of the lower alimentary tract are not a portal of infection.
Although the phospholipase toxin is an important aspect
of pathogenicity, the natural infection in sheep, even when
generalized, gives scant clinical evidence of intoxication,
although the exotoxin facilitates the spread of infection from
 205.e1

eFigure 2-40  Corynebacterium pseudotuberculosis in a sheep.

Severely enlarged lymph node because of abscessation.
206 CHAPTER 2  •  Hematopoietic System Lymph Nodes

A A

B B
Figure 2-154  Rhodococcus equi in a horse. A. Multifocal granu- Figure 2-155  Feline infectious peritonitis in a cat. A. Multiple
lomatous inflammation in mesenteric lymph node. B. Diffuse pyogranulomas distributed throughout the mesenteric lymph
caseous necrosis of lymph node parenchyma. node and along the peritoneal surface. B. Severe pyogranuloma-
tous lymphadenitis.

the primary site. Vaccination with the exotoxin provides a

degree of protective immunity. The leukotoxic surface lipid
allows persistence within inactivated macrophages in which
the organism is effectively a facultative intracellular parasite.
The acquisition of host resistance may also be in part the
result of the selection of a population of macrophages resistant
to and capable of destroying the toxic surface lipids of the
bacteria.
The sequence of events in progressive CLA is infection of a
superficial wound, spread of infection to the local lymph
nodes, which suppurate, and then lymphogenous and hema-
togenous extension to produce abscesses in internal organs.
The progression is slow and may reach the bloodstream only
in older animals, whereas in young animals the disease tends
to be confined to the superficial lymph nodes, most commonly
the precapsular and precrural.
Spontaneous recovery from the primary infection of cuta-
neous wounds, even when they suppurate, is more common Figure 2-156  Blastomyces dermatitidis in a dog. Severe pyo-
than is progression of the infection to the lymph node, as granulomatous lymphadenitis with large numbers of intralesional
judged by results in the experimental disease in which only yeast organisms that exhibit broad-based budding.
~20% of sheep develop abscesses in the nodes. Even when
abscesses develop in the nodes, the primary cutaneous lesion
heals, but secondary cutaneous lesions may develop as fistulae
when the nodal abscess ruptures to the exterior. This occurs
 Lymph Nodes
Figure 2-157  Cryptococcus neoformans in a cat. Granulomatous
lymphadenitis with typical “soap-bubble” appearance caused by
the thick gelatinous capsule of the yeast organisms.
Figure 2-158  Canine distemper virus infection in a dog. Lymph
node with severe lymphoid depletion and formation of multi-
nucleated syncytial cells.
more often in goats than in sheep. As a general rule, once the
infection gains the nodes, it is persistent, although a few infec-
tions may be cleared when the node ruptures and discharges
on to the skin.
The initial lesion in lymphoid tissues is diffuse lymphadeni-
tis that is probably the result of the soluble exotoxin (Fig.
2-159). When the organism reaches the nodes, multiple
microscopic abscesses form in the cortex. Eosinophils are
prominent in the reaction and cause the pus to be green.
These foci rapidly coalesce, and the central areas caseate to
form a structureless mass that contains fragments of nuclear
material and discrete clumps of bacteria. The abscesses are
rapidly encapsulated, and when this occurs, the acute reaction
in the surrounding tissues subsides, but the abscesses continue
to enlarge. With enlargement, there is progressive necrosis and
re-formation of the capsule, which gives the lesion a very
characteristic structure of concentric lamellations (Fig. 2-160).
These are particularly prominent when calcareous granules
are deposited in successive layers at the margin of the expand-
ing lesion. In old lesions, the contents lose their green color,
207
A
B
Figure 2-159  Corynebacterium pseudotuberculosis in a sheep.
A. Diffuse severe, suppurative lymphadenitis resulting in absces-
sation. B. Microscopic abscesses in the cortex of a lymph node.
become inspissated, and resemble putty. The lamellation is
specific to the organism, not the organ involved. The nodal
lesions often attain a diameter of 4-5 cm, and exceptionally,
they may reach 15 cm. The larger lesions in superficial nodes
cause pressure atrophy and depilation of the overlying skin;
they frequently rupture to discharge chronically through a
narrow fistula.
Caseous lymphadenitis is rarely fatal, and indeed, it seldom
even causes debility. Its economic importance results from
regulations concerning the trading of carcasses that show evi-
dence of the disease. When fatalities do occur, they are caused
principally by large pleuropulmonary abscesses. Rarely, C.
pseudotuberculosis causes small outbreaks of polyarthritis in
lambs, but they recover spontaneously.
The mature lesion of CLA is an encapsulated abscess with
pus of a distinct green hue and of caseous or caseopurulent con-
sistency. The initial cutaneous lesion may not be evident,
having resolved, but it is noted that occasional subcutaneous
abscesses, principally of the face and belly, do occur without
relation to known aggregations of lymphoid tissue. Mastitis, as
occasionally observed in sheep and often observed in goats,
probably represents an extension from a wound of the overly-
ing skin; when the mastitis is early and acute, it is diffuse and
suppurative, and when chronic, it is localized to encapsulated
208 CHAPTER 2  •  Hematopoietic System
A
B
Figure 2-160  Corynebacterium pseudotuberculosis in a sheep.
A. Lymph node with multifocal encapsulated abscesses with
characteristic concentric lamellations. (Courtesy S. Kesdangsakon-
wut.) B. Concentric lamellations are also recognizable on micro-
scopic examination. (Courtesy S.D. Fitzgerald.)
abscesses. The disease is easily diagnosed by fine-needle aspira-
tion of the focal lesions, which allows both cytopathology and
bacterial culture.
Spread from the lymph nodes produces lesions in the lungs,
and these are rather common with advancing age. They also
occur in young lambs, in which the progression is more rapid
than in adults. The pulmonary lesions may consist of extensive
bronchopneumonia, when abscesses rupture into bronchi in
which there are soft caseopurulent foci, or there may be dis-
crete nodules of various sizes and numbers. In cases of bron-
chopneumonia, and overlying those nodules that are
immediately subserosal, there is pleuritis, often with adhe-
sions. When the adhesions are few and localized adjacent to
the nodules, the remaining pleural cavity may be normal.
When the adhesions are more diffuse, there is a large amount
of serous fluid in the cavities and a thin layer of fibrin on the
pleura. The nodular lesions in the lung are similar to those in
lymph nodes, and have a narrow zone of bronchopneumonia
outside the capsule. With time, the pulmonary nodules
become sharply circumscribed, encapsulated, subpleural
abscesses. The pulmonary lesions are associated with charac-
teristic lesions in bronchial lymph nodes, which may be much
Lymph Nodes
Figure 2-161  Strangles in a horse. Streptococcus equi var. equi
causing suppurative lymphadenitis of the retropharyngeal lymph
node.
enlarged. Dissemination of the infection from the lungs to
other viscera is uncommon. Lesions are occasionally observed
in the renal cortex as discrete abscesses or as descending
pyelonephritis. Other viscera, chiefly the liver and spleen, may
contain solitary abscesses of the typical form. The organism is
sensitive to a number of antimicrobials, but response to
therapy is poor, both because of the intracellular location of
the organism and the characteristic formation of abscesses into
which drug penetration is ineffective.
Further reading
Baird GJ, Fontaine MC. Corynebacterium pseudotuberculosis and its
role in ovine caseous lymphadenitis. J Comp Pathol 2007;137:
179-210.
Dorella FA, et al. Corynebacterium pseudotuberculosis: microbiology,
biochemical properties, pathogenesis and molecular studies of viru-
lence. Vet Res 2006;37:201-218.
Hoelzle LE, et al. Differences in the antigen structures of Corynebacte-
rium pseudotuberculosis and the induced humoral immune
response in sheep and goats. Vet Microbiol 2013;164:359-365.
Streptococcal adenitis in swine
Jowl abscess is a cervical adenitis caused by Streptococcus
porcinus, and like strangles of horses (Fig. 2-161, eFig. 2-41),
the deep infection follows colonization of the oral cavity and
likely the tonsils. The disease has become less common with
the introduction of better hygiene, age-matched groups, and
feeding equipment designed to avoid sharp projections.
The organism has 8 serotypes, based on carbohydrate antigens,
of which type 4 is most frequently isolated from cervical
lymphadenitis of swine. Baby pigs have passive immunity, and
transmission occurs through direct contact with infected
animals that can shed organisms for months, and also from
contaminated environment. Invasion of the nasopharynx is
followed by mild fever and leukocytosis that resolve, and by
lymph node enlargement in 2 weeks, which less often pro-
gresses to open drainage. The mandibular nodes are most often
involved, followed by the retropharyngeal and parotid nodes.
 208.e1

Further reading
Windsor PA. Control of caseous lymphadenitis. Vet Clin North Am Food
Anim Pract 2011;27:193-202.
208.e2

eFigure 2-41  Strangles in a horse. Streptococcus equi var. equi

causing suppurative lymphadenitis of the retropharyngeal lymph
node.
 Lymph Nodes
Figure 2-162  Jowl abscess in a pig. Streptococcus porcinus causing
cervical lymph node abscesses. (Courtesy S. Kesdangsakonwut.)
The disease is not often of clinical importance, and affected
animals grow well, but losses result from condemnation at
slaughter. Bacterins are less effective than live oral culture in
prevention.
The abscesses are usually multiple and measure 1-10 cm
in diameter. The pus is typically without odor, green, and
creamy in consistency (Fig. 2-162). Streptococcus porcinus, a
β-hemolytic Streptococcus that belongs to Lancefield’s group
E, can be isolated regularly from the pus.
Rhodococcus equi is often present in the submaxillary
lymph nodes of swine and has been proposed as the cause of
lesions typical of tuberculosis in these nodes. Evidence sug-
gests, however, that R. equi is not responsible for the tubercle-
like lesions, which are thought to be caused by Mycobacterium
spp. of one or other variety. Rhodococcus equi has also been
isolated from lymphadenitis in cattle.
Streptococcal adenitis in dogs
Streptococcal adenitis of dogs is similar to the disease in swine.
It occurs in minor endemics in kennels and is characterized
by pharyngitis, fever, conjunctival discharge, and enlargement
of the submaxillary nodes. The illness is transient in most
cases, but in ~10% of dogs, the course is prolonged for 2 weeks
or more, and in these there is a tendency for the nodes to
suppurate. The abscesses may be encapsulated and sterilized,
or they may fistulate on to the skin and heal. Dysphagia and
occasionally asphyxia occur in puppies. Typically, the Strepto-
coccus belongs to Lancefield’s group G and, coincident with
the pharyngeal infection, bitches may have inflammation of
the genital tract caused by the same organism. Cervical adeni-
tis caused by the group G organism may occur in kittens.
Further reading
Martin-Vaquero P, et al. Presumptive meningoencephalitis secondary to
extension of otitis media/interna caused by Streptococcus equi
subspecies zooepidemicus in a cat. J Feline Med Surg 2011;13:
606-609.
Neis C, et al. Investigation of a possible chain of Streptococcus suis
infection in a pig breeding community using PCR-based genotyp-
ing. Berl Munch Tierartzl Wochenschr 2007;120:202-206.
209
Figure 2-163  Yersinia pestis in a cat. Lymph node with focally
extensive liquefactive necrosis with intralesional bacterial colo-
nies and fragmented leukocytes surrounded by macrophages.
Pseudotuberculosis
Yersinia pseudotuberculosis occurs worldwide in wild rodents
and birds and is widespread in nature, recoveries having been
made from soil, milk, and feed. The organism regularly pro-
duces disease, often in epidemic proportions, only in rodents
and birds. Sporadic infections with this organism and occa-
sional outbreaks of disease occur in domestic animals. Cats,
because of their contact with rodents and birds, are the
domestic species most apt to be secondarily involved by out-
breaks of the disease in its natural hosts. Losses of serious
proportion, however, occur in sheep that are exposed to large
numbers of organisms during outbreaks of the disease in
rodents during cold weather. The ovine disease is known as
“pyemic hepatitis.”
The general pattern of this disease is the same in all
species, the variation that occurs with respect to severity and
duration being related to species susceptibility, and numbers
and virulence of the organisms. Y. pseudotuberculosis is a fac-
ultative intracellular parasite, which explains the latent carrier
state and the need for strong cell-mediated immunity for
protection from infection. The route of transmission is by
ingestion and, in susceptible animals, organisms enter the body
through the intestine. Small necrotic foci develop in the
Peyer’s patches of the ileum and colon, and extend as lym-
phangitis to the regional nodes. The organism becomes septi-
cemic and may kill susceptible rodents at this stage; more
typically, and in all domestic species, caseonecrotic foci form in
the mesenteric nodes, spleen, and liver, often in association with
fibrinohemorrhagic inflammation in the small intestine. The
hepatic foci, which are the most obvious, are 1-10 mm in
diameter, white, and have no or scant tendency to encapsula-
tion or softening. They are interspersed with irregular areas of
parenchymal collapse that probably result from vasculitis and
thrombosis. Microscopically, there is necrosis, with bacterial
colonies and fragmented leukocytes surrounded by macro-
phages (Fig. 2-163). Giant cells are absent, even from later-
contracting granulomas. The mesenteric nodes and spleen
contain similar foci, and are enlarged by lymphoid and histio-
cytic hyperplasia. The mesenteric nodes in the cat may be
2-4 cm in diameter and can be grossly confused with intestinal
toxoplasmosis or lymphomatosis.
 209.e1

Further reading
Chalker VJ, et al. Genetic diversity of Streptococcus equi subsp. zooepi-
demicus and doxycycline resistance in kenneled dogs. J Clin Micro-
biol 2012;50:2134-2136.
Jaeger G, et al. Haemorrhagic pneumonia in sled dogs caused by
Streptococcus equi subsp. zooepidemicus—one fatality and two
full recoveries: a case report. Acta Vet Scand 2013;55:67.
Pesavento PA, Murphy BG. Common and emerging infectious diseases
in the animal shelter. Vet Pathol 2014;51:478-491.
210 CHAPTER 2  •  Hematopoietic System
Figure 2-164  Yersinia pestis in a sheep. Abscessation of mesen-
teric lymph node.
Yersiniosis has emerged as a significant cause of disease in
farmed ruminants, including deer. Affected deer may be found
moribund or dead, but animals under observation are systemi-
cally ill with profuse diarrhea. The incidence of disease in
younger age groups rivals that of salmonellosis. In addition to
the mesenteric lymphadenitis (Fig. 2-164) and hepatitis, enteritis
is consistently present and characteristic in its histologic
expression. Numerous bacterial colonies are present in the
lamina propria, associated with multiple suppurative foci or
diffuse suppurative enteritis.
In view of the extraordinarily wide host range of Y. pseu-
dotuberculosis in natural infections, it is surprising that the
infection is not recorded more often. This may be in part
because of a preference of the organism for growth at room
temperature rather than at 37° C. Although susceptible labo-
ratory species, such as guinea pigs and mice, are readily
infected experimentally, there are few pathogenetic studies in
domestic species. The frequency of natural infection in cats
contrasts with the difficulty in establishing experimental
infection in this species, which suggests the need for some
predisposing alteration in the alimentary mucosa. Natural
infection in cats is not treatable with current antimicrobials
and is usually fatal.
The related organism Yersinia enterocolitica causes gastroen-
teritis and mesenteric adenitis in a wide variety of species,
including wild and domestic animals, and is increasingly iso-
lated from cattle. The organism has been isolated from deer,
rabbits, dogs, pigs, horses, mink, various avian species, as well
as sheep and goats. Y. enterocolitica shares an antigen with
Brucella abortus that may result in false-positive agglutination
reactions in serologic testing for brucellosis.
Further reading
Byun JW, et al. Hepatic yersiniosis caused by Yersinia enterocolitica
4:03 in an adult dog. J Vet Diagn Invest 2011;23:376-378.
Warth JF, et al. Yersinia pseudotuberculosis O III causes diarrhea in
Brazilian cattle. Adv Exp Med Biol 2012;954:107-110.
Wobeser G, et al. Tularemia, plague, yersiniosis and Tyzzer’s disease in
wild rodents and lagomorphs in Canada: a review. Can Vet J
2009;50:1251-1256.
Lymph Nodes
Figure 2-165  Postweaning multisystemic wasting syndrome in a
pig. Porcine circovirus 2 causes palpable lymphadenopathy affect-
ing the inguinal lymph nodes. (Courtesy S. Kesdangsakonwut.)
Porcine circovirus-associated diseases
Porcine circovirus 2 (PCV-2)-associated diseases (PCVAD) in
pigs encompass a broad spectrum of diseases, including post-
weaning multisystemic wasting syndrome (PMWS), porcine
dermatitis and nephropathy syndrome (PDNS), congenital
tremors in newborn piglets, and a number of other respiratory,
reproductive, enteric, nervous, and cardiovascular disorders.
Species Porcine circovirus 2 is a member of the family Circo-
viridae and one of the smallest (17-nm diameter) animal
viruses with a non-enveloped capsid and a single-stranded,
circular genome. There are currently 2 types of porcine circo-
virus, PCV-1 and PCV-2. PCV-1 was detected as a contami-
nant of the PK-15 porcine kidney cell line and may also be
the potential cause of congenital tremors in newborn piglets.
PCV-2 was initially identified in the mid to late 1990s in
weaned pigs from North America and Europe with PMWS.
Two subtypes of PCV-2, a and b, have been characterized, and
some reports suggest that PCV-2b may be more virulent or
may escape existing herd immunity stimulated by the previ-
ously circulating subtype PCV-2a. This section will only focus
on PMWS with its characteristic lesions in lymphoid tissues.
PMWS is a wasting syndrome that often occurs after
weaning, when pigs are 4-14 weeks of age. The disease also is
seen in older pigs, particularly finishing stage pigs that weigh
45-70 kg. Morbidity usually ranges from 5-20% in nursery or
finishing pigs, and mortality in pigs that show signs of PMWS
often exceeds 50%. Affected pigs have generalized lymphade-
nopathy, decreased weight gain or wasting in combination
with variably occurring, less specific clinical signs, such as
dyspnea, coughing, fever, diarrhea, pallor, icterus, central
nervous signs, and sudden death.
Characteristic gross lesions include pallor, icterus, diarrhea,
and palpable lymphadenopathy (Fig. 2-165). Inguinal, popli-
teal, internal iliac, mesenteric, bronchiolar, and mediastinal
lymph nodes may be substantially enlarged (Fig. 2-166).
Enlarged lymph nodes are pale and homogeneous on cut
surface (Fig. 2-167). Gross lesions in the lungs may include
failure to collapse, firmness, diffuse pulmonary edema, mot-
tling, and consolidation. The liver may appear icteric and/or
atrophic and have prominent interlobular connective tissue.
The most characteristic microscopic lesion in pigs with PMWS
 210.e1

Further reading
Backhans A, et al. Occurrence of pathogenic Yersinia enterocolitica and
Yersinia pseudotuberculosis in small wild rodents. Epidemiol Infect
2011;139:1230-1238.
Bush JM, et al. Disease transmission from companion parrots to dogs
and cats: what is the real risk. Vet Clin North Am Small Anim Pract
2011;41:1261-1272.
 Lymph Nodes
Figure 2-166  Postweaning multisystemic wasting syndrome in a
pig. Severe lymphadenopathy affecting sublingual and retropha-
ryngeal lymph nodes.
Figure 2-167  Postweaning multisystemic wasting syndrome in a
pig. Enlarged lymph nodes are pale and homogeneous on cut
surface. (Courtesy S. Kesdangsakonwut.)
is granulomatous inflammation with or without unique globular
intracytoplasmic viral inclusion bodies in macrophages (Fig.
2-168). However, severe lymphoid depletion may be the only
lesion in lymphoid organs affecting both lymphoid follicles
and parafollicular zones. Granulomatous inflammation in lym-
phoid tissues is characterized by epithelioid macrophages and
multinucleated giant cells that may contain sharply demar-
cated, spherical, basophilic, often botryoid cytoplasmic inclu-
sion bodies. Less consistent lesions include interstitial
pneumonia, interstitial nephritis, myocarditis, hepatitis with
hepatic atrophy and/or icterus, and perivasculitis in a number
of tissues. Liver lesions have been identified as a frequent
finding and are the most likely cause of icterus and wasting.
Although previous reports indicated that PCV-2 capsid pro-
teins localized predominantly in the nuclei of infected cells,
abundant amounts of PCV-2 capsid proteins were observed
in the cytoplasm of many cells. Vasculitis has been recently
described as a hallmark lesion of the severe form of systemic
PCVAD, and experimental infections with PCV-2b directly
caused acute vasculitis, whereas chronic vasculitis may in part
211
A
B
Figure 2-168  Postweaning multisystemic wasting syndrome in a
pig. A. Granulomatous lymphadenitis with multinucleated giant
cells and characteristic botryoid inclusions. B. Amphophilic intra-
cytoplasmic inclusion bodies can vary from botryoid clusters to
fine cytoplasmic dusting.
be mediated by the immune system. The diagnosis of PMWS
requires detection of (1) clinical signs: wasting or ill thrift with
or without the other earlier described clinical signs; (2) histo-
logic lesions: depletion of lymphoid organs and/or granuloma-
tous inflammation in any organ; and (3) detection of PCV2
infection within characteristic lesions (Fig. 2-169).
PMWS has been reproduced with combined PCV-2 and
porcine parvovirus inoculation, combined PCV-2 and PRRSV
infection, prenatal PCV-2 infection and postnatal porcine par-
vovirus infection, and dual infections with torque-teno virus
(TTV) and PCV-2. PMWS been reproduced in gnotobiotic
pigs with PCV-2 alone, following administration of keyhole
limpet hemocyanin in incomplete Freund’s adjuvant, and has
been reproduced with PCV-2 alone in cd/cd pigs. Intramus-
cular injection of pigs with a vaccine against Mycoplasma
hyopneumoniae or a nonspecific immunomodulating drug
(Baypamun) caused clinical signs, moderate to severe gross
and histologic lesions of PMWS. Vaccination with selective
bacterins increased the severity of lesions in conventional pigs
infected with PCV-2. It has been shown that monocyte
chemoattractant protein-1 (MCP-1) expression, but not
interleukin-8, may play a role in the pathogenesis of granulo-
matous inflammation in pigs with PMWS. There are also
212 CHAPTER 2  •  Hematopoietic System
A
B Vol. 2, Respiratory system) are, in some countries, very
Figure 2-169  Postweaning multisystemic wasting syndrome in a
pig. A. Granulomatous inflammation with multinucleated giant
cells in Peyer’s patches. B. In situ hybridization for porcine circo-
virus 2 shows large amounts of viral DNA in Peyer’s patches.
breed-dependent differences to PCV-associated disease and
lesions.
Further reading
Darwich L, Mateu E. Immunology of porcine circovirus type 2 (PCV2).
Virus Res 2012;164:61-67.
Opriessnig T, Langohr I. Current state of knowledge on porcine circo-
virus type 2-associated lesions. Vet Pathol 2013;50:23-38.
Parasitic infestations of lymph nodes
The lymph nodes are not a final habitat for any nematode, but
some species traverse them in migration, and some lodge there
accidentally. The larvae of lungworms travel by lymphatics
from the intestine to the lungs, but cause little injury. The
first-stage larvae of those lungworms that reside in the alveolar
spaces, Muellerius spp. and Protostrongylus spp. particularly,
often find their way to the bronchial nodes and cause granu-
lomatous lymphadenitis. The nodes become enlarged, hard,
and irregular, and have many firm granulomas and encapsu-
lated caseocalcareous nodules. The larvae of Strongylus spp. in
horses occasionally produce hemorrhagic lymphadenitis of the
abdominal lymph nodes that, when secondarily infected, may
Lymph Nodes
Figure 2-170  Metastatic squamous cell carcinoma in a cat. Clus-
ters of neoplastic epithelial cells in subcapsular sinus of retropha-
ryngeal lymph node.
develop suppurative lymphadenitis. Larval trematodes, prob-
ably Fasciola hepatica, are often found in the mesenteric nodes
of ruminants, as are larvae of Oesophagostomum columbianum
in sheep. Nodules develop in the cortex or medulla, as well
as in capsular tissues. They are 2-5 mm in size and contain
yellow-green pus, which in older lesions becomes caseous and
mineralized. The pus is largely the debris of eosinophils, and
it is surrounded by giant and epithelioid macrophages and a
well-defined capsule. A very similar lesion is produced by the
larvae of Linguatula serrata. The larvae of this parasite (see
common in the mesenteric nodes of sheep and cattle. Viable
larvae can be found in normal nodes, but usually the nodes
show focal or diffuse hyperplasia and edema, with the larvae
lying in cavities that contain a milky fluid resembling chyle.
Apparently, the larvae die in the nodes and become encysted
in abscesses, which may mineralize. In active cutaneous infes-
tations by Demodex spp. in dogs, the parasite is often found in
the peripheral lymph nodes.
Neoplastic metastatic diseases of lymph nodes
The general reaction of lymph nodes to nonlymphoid cancer
is atrophy of greater or lesser degree because of the systemic
effects of cancer, apparently mediated by tumor necrosis
factor. If the tumor causes inflammatory drainage to local
nodes, these will have changes of reactive hyperplasia and
lymphadenitis, as occurs in mesenteric nodes draining an
area of ulceration owing to carcinoma. It is significant that
the specific reaction of local nodes to a local neoplasm is
usually little more substantive than to distant or systemic
malignancies.
In general, the response of local nodes to a local neoplasm is
sinus histiocytosis. If metastases are present, they will originally
consist of single cells and small clusters in the peripheral and
medullary sinuses (Fig. 2-170). The tumor cells appear to pass
through the cortex to the medulla through the cortical gaps,
and largely avoid processing through the cortical system of
antigen surveillance. The sinus macrophages do not appear to
be specifically attracted to the neoplastic cells, even when they
invade the cortical parenchyma. There are minor variations in
the reaction of affected lymph nodes to specific neoplastic cell
 212.e1

Further reading
Madec F, et al. Post-weaning multisystemic wasting syndrome and
other PCV2-related problems in pigs: a 12-year experience. Trans-
bound Emerg Dis 2008;55:273-283.
Segalés J, et al. Porcine circovirus diseases. Anim Health Res Rev
2005;6:119-142.
 Lymph Nodes
types. Highly secretory adenocarcinomas may induce a stron-
ger reaction to their secretory products than to the neoplastic
cells themselves. Metastatic mast cells cause the same reaction
in nodes as in primary sites, but tend not to incite a strong
cellular and stromal reaction even when highly granulated
(Fig. 2-171). As noted earlier, the medullary cords of lymph
nodes form a tissue-vascular boundary similar to marrow, and
extramedullary hematopoiesis will colonize these sites. In leu-
kemias, the neoplastic cells colonize the medullary cords and
benign cells are displaced, in this case to the adjacent medul-
lary sinuses. In the acute myeloid leukemias, the primitive
neoplastic cells have round nuclei and resemble the surround-
ing residual lymphocytes, making recognition of the meta-
static disease difficult with H&E stains. Diagnosis is aided by
the identification of megakaryocytes in either the cords or
sinuses.
Interestingly, detection of regional lymph node metastasis
has not been found to correlate with remission length or
survival times of dogs with various malignancies, for instance,
oral malignant melanomas (Fig. 2-172, eFig. 2-42). This is
Figure 2-171  Metastatic mast cell tumor in a dog. Diffusely
enlarged hepatic lymph node caused by infiltration with neoplas-
tic mast cells.
Figure 2-172  Metastatic malignant oral melanoma in a dog. The
lymph node parenchyma has been diffusely replaced by the
expansile growth of the pigmented melanocytic neoplasm.
213
most likely because of the failure to histologically detect nodal
metastases that were present in some nodes, as well as excision
of nodes based on anatomic location rather than evidence of
drainage. In human medicine, sentinel node detection is com-
monly applied to detect those lymph nodes that actually drain
the area where the primary tumor is localized. Lymphatic
mapping strategies have been devised using a combination of
radio-labeled and colored colloids to define drainage areas
from a particular anatomic site. Serial sectioning of draining
lymph nodes and immunohistochemical labeling for neoplas-
tic cells, for instance, cytokeratin for carcinomas, as well as
RT-PCR for detecting mRNA specific for neoplastic cells, for
instance, mammaglobin, have been used in human medicine
to dramatically improve detection of even single neoplastic
cells in draining lymph nodes.
Further reading
Nyman HT, et al. A review of the sonographic assessment of tumor
metastases in liver and superficial lymph nodes. Vet Radiol Ultra-
sound 2004;45:438-448.
Worley DR. Incorporation of sentinel lymph node mapping in dogs with
mast cell tumours: 20 consecutive procedures. Vet Comp Oncol
2014;12:215-226.
Lymphoid neoplasms
Lymphomas are neoplasms of the hematopoietic system and
can arise in lymph nodes or extranodal locations. In human
medicine, they are divided into Hodgkin- and non-Hodgkin
lymphomas. Although a Hodgkin-like lymphoma has been
reported in cats, the vast majority of lymphomas in domestic
animals closely resemble non-Hodgkin lymphomas in humans
and are simply referred to as lymphoma or malignant lym-
phoma. Although the term “lymphosarcoma” has also been
used in veterinary medicine, it should be avoided especially
for comparative reasons. Historically, lymphoid leukemias and
lymphomas have been viewed as separate entities. The World
Health Organization (WHO) classification postulates that pre-
cursor neoplasms occurring as solid tumors, and those lymphomas
with marrow and blood involvement, are biologically the same
disease with different clinical presentations. An artificial division
using different names has therefore been avoided by using
lymphoma/leukemia for entities that can occur in either form, for
instance, acute lymphoblastic leukemia/lymphoblastic lym-
phoma. Essentially, the presence of neoplastic cells in the bone
marrow and peripheral blood is principally a prognostic factor
that reflects a stage of disease rather than a difference in clas-
sification. However, the biological basis for the different clini-
cal presentations is not fully understood. Because most
precursor lymphoid neoplasms develop in the bone marrow
and are seen as leukemia, the term acute lymphoid leukemia
is still used for the leukemic phase of precursor neoplasms of
T and B cells. For a detailed review of those lymphomas
appearing primarily as leukemias, see Acute lymphocytic leu-
kemia and Chronic lymphocytic leukemia previously.
More important, the WHO classification recently adapted for
animals characterizes different types of lymphomas as specific
disease entities rather than considering lymphoma as a single
biological entity of various morphologic presentations. Although
the previous lymphoma classifications used morphologic fea-
tures such as cell size to grade lymphomas and to predict their
behavior, the WHO classification establishes a new paradigm
 213.e1

eFigure 2-42  Metastatic malignant melanoma in a horse. Axillary

lymph node with multinodular, pigmented, neoplastic masses
replacing the parenchyma.
213.e2

Further reading
Kurosumi M, Takei H. Significance and problems of histopathological
examination and utility of real-time reverse transcriptase-polymerase
chain reaction method for the detection of sentinel lymph node
metastasis in breast cancer. Breast Cancer 2007;14:342-349.
Stroup SP, et al. Preoperative sentinel lymph node mapping of the
prostate using PET/CT fusion imaging and Ga-68-labeled tilmano-
cept in an animal model. Clin Exp Metastasis 2012;29:673-680.
214 CHAPTER 2  •  Hematopoietic System
that identifies lymphomas according to their unique phenotype
and genotype. However, each lymphoma type may appear with
a variety of morphologic features and a range of clinical
behaviors.
Clinical features of lymphomas
Lymphomas are commonly classified according to their ana-
tomic location. The main types of lymphoma include multi-
centric, thymic/mediastinal, gastrointestinal, cutaneous,
extranodal, and central nervous system (CNS). With the adap-
tation of the new WHO classification that integrates clinical
findings with cell morphology and molecular features, such
gross anatomic classification becomes less important. Common
gross presentations and organ systems involved are described
in more detail for each individual species later in this chapter.
Clinically, most commonly a diagnosis of lymphoma is
based on enlargement of lymph nodes or other organs, for
instance, hepatosplenomegaly, or ultrasound findings, for
instance, thickening of the intestinal wall with intestinal lym-
phoma. Clinical signs are based on the primary organ system
affected and range from anorexia, lethargy, and weight loss;
to dyspnea, coughing, and caval syndrome with thymic/
mediastinal lymphoma; vomiting and diarrhea with gastroin-
testinal lymphoma; cutaneous nodules and plaques with cuta-
neous lymphoma; and paralysis, seizures, or paresis with
lymphomas of the CNS. Polyuria and polydipsia are com-
monly observed because of paraneoplastic hypercalcemia of
malignancy. Clinically, staging has been used to prognosticate
multicentric lymphoma:
• Stage I: Involvement restricted to a single lymph node or
organ (not bone marrow)
• Stage II: Regional lymph node involvement (restricted to
one side of diaphragm)
• Stage III: Generalized lymph node involvement
• Stage IV: Involvement of the liver and/or spleen and gen-
eralized lymph node involvement
• Stage V: Involvement of blood and bone marrow and/or
other extranodal sites
As noted earlier, the more recent classification of lympho-
mas according to the WHO provides more accurate prognos-
tic information and therapeutic guidance because it recognizes
lymphomas as a heterogeneous group of distinct diseases that
are unrelated to one another, and not grades of a single disease
entity. The WHO classification predicts the biological behav-
ior of different types of lymphoma by pathologic features (cell
size, proliferation) or clinical features, and thereby fosters
establishment of entity-specific therapeutic approaches.
Laboratory findings are highly variable. Nonregenerative
anemia of various severities is commonly observed. Both lym-
phopenia and lymphocytosis may occur with different types
of lymphomas, and hypercalcemia of malignancy owing to
secretion of parathyroid hormone-related peptide (PTHrP) or
bone lysis has been commonly observed with some lymphoma
entities. More detailed descriptions are provided with each
lymphoma entity.
Diagnosis of lymphomas
The accurate diagnosis of malignant lymphoma requires
proper selection and handling of tissues, and ancillary tests are
essential. Ideally, a combination of cytology and histology is
desired. Touch preparations are important for cytologic assess-
ment; submission of surgical biopsies fixed in 10% neutral
buffered formalin is a good all-purpose approach to allow
Lymph Nodes
histologic evaluation of tissue architecture, immunophenotyp-
ing, and clonality assessment. Clinical pathologists often prefer
B5 fixative for bone marrow sections because the nuclear
morphology will be better preserved; however, standardized
immunohistochemistry (IHC) protocols for B5 fixed, demin-
eralized tissues are lacking. A high quality, 2-4 µm thick,
H&E-stained slide is essential for lymphoma evaluation
because thicker sections make it difficult to assess cytologic
detail. Some pathologists prefer Giemsa stains for more
nuclear detail. The integration of flow cytometry will become
more commonplace in routine lymphoma diagnostics as will
cytogenetic studies.
Lymphoid tissue is very fragile, and artifacts are commonly
induced through tissue compression, delayed fixation, or
drying of tissues. Histologically, the center of a formalin-fixed
lymph node is commonly underfixed or primarily ethanol
fixed, and immunohistochemical stains appear light, or there
is a lack of labeling. Nuclei may be slightly larger with less
dense chromatin. These features are in complete contrast to
the lymph node rim, where overfixation or drying out of the
tissue may result in smaller, more basophilic nuclei, and
intense immunohistochemical labeling. Suboptimal fixation
can be compounded by extensive areas of necrosis caused by
infarction or focal cellular lysis. Identifying areas with good-
quality cell morphology that are representative for the disease
process is essential, because determining tissue architecture, cell
size, and mitotic index are all parts of reaching an accurate
diagnosis. After differentiating a diffuse from a follicular
growth pattern, pathologists should determine cell size. The size
of neoplastic lymphoid cells is based on the size of their nuclei
compared to erythrocytes. Small lymphoid cells have nuclei
1-1.25 times the size of erythrocytes, intermediate lymphoid
cells have nuclei ~1.5 times the size of erythrocytes, and large
cells have nuclei at least 2 times larger than erythrocytes. The
mitotic index is determined as the average number of mitotic
figures in 10 random high-power (40×) fields (HPF) in the
areas of highest mitotic activity. A low mitotic index is defined
as 0-5 mitoses/HPF, a medium mitotic index as 6-10 mitoses/
HPF, and a high mitotic index as >10 mitoses/HPF.
Immunophenotyping is an essential part of an accurate
diagnosis of lymphomas. In domestic animals, CD3 is used to
detect T cells and CD79a for B cells. In dogs and cats, CD20
and Pax-5 are commonly used instead of CD79a because
CD79a commonly causes artifactual nuclear binding. Immu-
nophenotyping is required to reach a conclusive diagnosis
because a number of lymphoma entities are morphologically
homogeneous, but are phenotypically heterogeneous, for
instance, small lymphocytic lymphoma, lymphoblastic lym-
phoma, marginal zone lymphoma versus T-zone lymphoma,
or diffuse large B-cell lymphoma versus peripheral T-cell lym-
phoma. Additional antibodies used for lymphoma diagnosis
include CD45, CD45RO, CD18, CD204, CD34, CD30,
CD90, BCL-2, Ki-67, granzyme B, perforin, CD10, BCL-6,
GCET1, FOXP1, CD204, MUM-1, immunoglobulin and
light-chain antibodies, and, in frozen sections, CD4 and CD8.
Details on their application are listed later under the indi-
vidual lymphoma entities. Although the list appears long,
compared to human medicine, veterinary pathology stills lacks
a number of essential, species-specific antibodies to more
accurately diagnose lymphomas. It is also important to recog-
nize that antibodies targeting epitopes in a particular species
may not react with another species at all and either do not
label target cells or, often even more dangerous, label epitopes
 Lymph Nodes
expressed on other cell types. A good example is Bla.36, which
labels B cells in a variety of species, but will also detect den-
dritic cells in most domestic animals, for instance, histiocytic
sarcomas in dogs. Regardless, immunohistochemical labeling
can only be interpreted in association with the cell morphol-
ogy and has to be correlated to the morphologic diagnosis.
Having a reactive cell background or diffuse infiltration with
T cells or dendritic cells is common, and the pathologists need
to carefully determine immunohistochemical labeling of the
neoplastic cell population. This also includes assessment of the
proper type of labeling, for instance, membranous versus intra-
cytoplasmic versus nuclear. As an example, nuclear labeling
for CD79a is a common artifact and has to be disregarded
when evaluating tissues microscopically.
Numerous lymphoid neoplasms are difficult to differenti-
ate from inflammatory conditions. In particular, enteropathy-
associated T-cell lymphomas of small cell type, or cutaneous
lymphocytosis in cats, and lymphohistiocytic proliferations in
the skin or lymphofollicular proliferations in lymph nodes or
spleen of dogs pose a morphologic challenge to the patholo-
gist. Detection of clonality using the PCR for antigen receptor
rearrangements (PARR) assay of the T-cell receptor γ and/or
immunoglobulin heavy chains has become the gold standard
for differentiating neoplastic (monoclonal) from reactive
(polyclonal) lymphocytes in dogs and cats (Fig. 2-173).
However, PARR testing should never be used independently of
the histologic features, immunophenotype, and clinical findings.
PARR should only be applied after preliminary differential
diagnoses have been established. Testing should be done using
capillary gel electrophoresis to achieve sufficient resolution for
properly differentiating monoclonal from polyclonal popula-
tions. False-negative results can occur because of the limits of
the qualitative sensitivity (the ability of the assay to detect
clonal rearrangement) or quantitative sensitivity (the ability of
the assay to detect a clonal rearrangement in a background of
non-neoplastic lymphocytes). Qualitative sensitivity is espe-
cially affected by the use of species-specific primers and early
primer sets that had been developed based on limited sequence
data. Even the most recently published multiplex PCRs do
not detect all potential gene rearrangements, and aiming for
100% sensitivity may be simply cost prohibitive with the
current methodology. The qualitative sensitivity is most com-
monly reduced by either having a large population of inflam-
matory lymphocytes infiltrating the tissue to be tested or by
having numerous other tissue samples included with the test
material, for instance, numerous endoscopic biopsy samples
of small intestine in a single tissue block with only a few
samples being suspect for lymphoma. In general, PARR testing
has a higher sensitivity in nonlymphoid tissues compared to
lymphoid tissues because the rearranged sequences from
normal lymphocytes compete with amplification of the neo-
plastic DNA. Although PARR assays have been shown to be
highly specific, false-positive results can also occur. Very small
number of inflammatory lymphocytes within a sample or
certain inflammatory conditions, for instance, infection with
Ehrlichia canis, may result in a clonal PCR result. The most
common false-positive results are cause by a procedural error
that causes pseudoclones that occur in up to 10% of per-
formed tests. To avoid pseudoclones, all PARR testing should
be run in duplicate, along with both native and denatured
forms. Furthermore, it is essential not to mistake clonality results
for immunophenotype. The phenotype of a lymphoma should
always be based on expression of CD molecules as determined
215
by IHC or flow cytometry not PARR testing, as neoplastic
lymphocytes may show cross-lineage clonal rearrangement.
The incidence of cross lineage rearrangement in different lym-
phomas has been reported between 7-10%. Even exclusive
amplification of the cross-lineage receptor can be observed
either because of failure of the PARR assay primers to recog-
nize the specific rearrangement within receptor matching the
lymphoma phenotype, or because of chromosomal alterations
within the neoplastic lymphocytes that distorted the sequence
of the primer binding site and resulted in binding failure.
Unfortunately, it is not uncommon practice to aspirate
enlarged lymph nodes and to diagnose a B- or T-cell lym-
phoma based on single-run PARR testing. Failure to properly
diagnose the aspirate based on cell morphology and to deter-
mine the immunophenotype by antibody reaction as well as
not performing PARR testing in duplicate and for both recep-
tors will result in a risk of at least 20% false-positive results.
Alternatively, if the histologic features and immunopheno-
typical results are consistent with a diagnosis of lymphoma
despite a polyclonal PCR result, a diagnosis of a suspected
lymphoma should still be made and additional biopsy sample
from a later date should be tested with PARR.
Classification of lymphomas
Throughout the last century, veterinary pathologists have used
classifications that were developed for non-Hodgkin lympho-
mas in humans to classify malignant lymphomas in domestic
animals, especially dogs. Whereas early classification systems
were based entirely on the morphologic characteristics of
malignant lymphocytes, the ability to further differentiate
cells immunohistochemically led to a revision of the historical
classification systems.
The Rappaport classification from 1966 is one of the earli-
est classification systems applied to canine malignant lympho-
mas and according to this classification, a large number of
canine malignant lymphomas were classified as histiocytic.
With increasing knowledge of the immunologic aspects of
malignant lymphomas, a better understanding of maturation
and differentiation of lymphoid cells, and the advance of
chemotherapy, new classification systems were developed.
Almost simultaneously, the Lukes-Collins classification in
North America and the Kiel classification in Europe were
published. Both systems were based on immunologic concepts
and only differed significantly in the identification of 2 of 13
entities. This difference was mainly because of the different
position of centrocytes within the line of maturation in each
classification. Despite these differences, a translation from one
to the other classification was still possible. In particular, the
Kiel classification was easily adapted for canine malignant
lymphomas.
To avoid further confusion and in attempt to unify the
European and North American classifications, the National
Cancer Institute initiated a multi-institutional study. As a
result, the Working Formulation was published in 1982. In
contrast to the previously described classifications, the working
formulation represents a form of lymphoma “Esperanto,” a
translational system for the existing human non-Hodgkin lym-
phoma classifications. The Working Formulation was strictly
oriented on the clinical outcome and not based on a morpho-
logic principle. The various lymphoma entities in this classifi-
cation were based on groups of human patients with similar
survival curves, and the morphologic features for each group
were described following a classification according to survival.
 [bp] A1 A2 A3 A4 A5 A6 [bp]

300 300

250 250

200 200

150 150

Peak size

Peak size
100 100

75 75
50 50

25 25
15 15

1.400
1.300
15 bp
1.200 105 bp
1.100
Relative fluorescence

1.000
units [RFU x 180]

0.900
0.800 3,000 bp
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
[bp]
15
50
100
150
200
250
300

400
500

3,000

Size
1.500
1.400
15 bp
1.300
1.200
1.100
Relative fluorescence

3,000 bp
units [RFU x 180]

1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300 109 bp
0.200
0.100
0.000
[bp]
15
50
100
150
200
250
300

400
500

3,000

Size
Figure 2-173  PCR for antigen receptor rearrangement for T-cell receptor γ in a cat. A. Duplicate
runs with capillary gel electrophoresis result in single strong bands for monoclonal (neoplastic)
cell populations (lanes A1 and A2) or smear for polyclonal (inflammatory) cell populations (lanes
A3 and A4). Negative control (lane A5), positive control (lane A6). B. In the electrophoretogram,
monoclonal (neoplastic) cell populations are characterized by a single dominant narrow peak
(105 bp). C. Polyclonal (inflammatory) cell populations produce a curve or broader peak that is
at least 2 3 shorter (109 bp).
 Lymph Nodes 217

Numerous studies addressed this weakness of the Working

Formulation and strongly opposed use of the Working Formu-
Table • 2-5 
lation as a primary morphologic classification. Unfortunately,
the simplified morphologic categories of the Working Formu-
The classification of lymphoid neoplasms
lation led to adaptation of this classification to canine malig- adopted by the WHO, as applied for use
nant lymphomas and use in daily veterinary oncologic practice in animals
until this day. Such adaptation for canine malignant lympho-
• B-cell neoplasms
mas is even more questionable, given that all survival data
• Precursor B-cell neoplasms
were based on human trials using uniform therapeutic
• B-lymphoblastic leukemia/B-lymphoblastic lymphoma
approaches, and similar studies have rarely been performed in
• Mature (peripheral) B-cell neoplasms
veterinary medicine.
• B-cell chronic lymphocytic leukemia/small lymphocytic
The original Kiel classification had failed to classify ~12%
B-cell lymphoma
of human non-Hodgkin lymphomas, but with the ability
• Diffuse large B-cell lymphoma
to immunohistochemically characterize lymphoid cells, an ■ Centroblastic
“updated” version of the Kiel classification was introduced. ■ Immunoblastic
This “updated” Kiel classification was shown to be applicable ■ Anaplastic
to canine malignant lymphomas, and multiple studies docu- ■ T-cell rich
mented its prognostic significance. This “updated” Kiel classi- ■ Lymphomatoid granulomatosis
fication represented until recently the most valuable prognostic
• Follicular B-cell lymphoma
classification of canine malignant lymphomas. ■ Grades I, II, and III
In the last decade of the past century, the rapidly develop-
• Marginal zone B-cell lymphoma
ing oncology in both human and veterinary medicine high- ■ Nodal marginal zone lymphoma
lighted the pressing need for consistency of diagnostic and ■ Splenic marginal zone lymphoma
prognostic classifications and terminology across various insti- ■ MALT
tutions and also in publications. The WHO system of classifi-
• Mantle cell lymphoma
cation of hematopoietic neoplasms was largely based on
• Burkitt-like lymphoma
proposals made by the International Lymphoma Study Group
• Extramedullary plasmacytoma
(ILSG) who proposed a Revised European-American Classi-
• Multiple myeloma
fication of Lymphoid Neoplasms (REAL). The objectives of
• T-cell and NK-cell neoplasms
the ILSG were to devise a system that did not have obvious
• Precursor T-cell neoplasms
ties to any country or region and to greatly expand criteria for
• T-lymphoblastic leukemia/T-lymphoblastic lymphoma
disease recognition. According to the ILSG proposal, lym-
• Mature (peripheral) T-cell neoplasms
phoid neoplasms were identified for the first time as disease
• Nodal T-cell lymphoma
entities and not simply based on cell types reflecting normal ■ T-zone lymphoma
counterparts of neoplastic cells. This represents a completely ■ Peripheral T-cell lymphoma, unspecified
new paradigm in lymphoma classification because diagnostic ■ Anaplastic large T-cell lymphoma
criteria consist now of all relevant information, including cel- ■ Angioimmunoblastic T-cell lymphoma
lular morphology and cell lineage, lesion topography, and also
• Enteropathy-associated T-cell lymphoma (EATL)
general biology of each neoplasm that defined it as a specific ■ Large cell type: EATL type 1
disease entity. The lack of grading in this system is made up ■ Small cell type: EATL type 2
for by each subtype of lymphoma now representing a well-
• Extranodal T-cell lymphoma
characterized disease. The efficacy of the ILSG and subse- ■ Hepatosplenic T-cell lymphoma
quent WHO system of classification was demonstrated by a ■ Hepatocytotropic T-cell lymphoma
group of MD pathologists of the Non-Hodgkin’s Lymphoma ■ Peripheral T-cell lymphoma, unspecified
Classification Project who achieved an 85% consensus in iden-
• Cutaneous T-cell lymphoma
tifying the major types of lymphoma by using the criteria of ■ Cutaneous epitheliotropic T-cell lymphoma
the ILSG classification system. These results immediately ren-
• Mycosis fungoides
dered other systems of classification obsolete and pointed to
• Pagetoid reticulosis
the need for a similar study to demonstrate the applicability
• Sézary syndrome
of the WHO system to animal neoplasms. Such a study was ■ Cutaneous non-epitheliotropic T-cell lymphoma
performed for canine malignant lymphomas, and the WHO
• Peripheral T-cell lymphoma, unspecified
classification was adapted to canine lymphomas with similar
• Subcutaneous “panniculitis-like” T-cell lymphoma
intraobserver and interobserver consistencies as reported by
• Anaplastic large T-cell lymphoma
the ILSG (Fig. 2-174). In recent years, the WHO classification
• T-cell large granular lymphocytic leukemia
has been applied to lymphomas in other animal species either
• Acute T-cell large granular lymphocytic leukemia
in similar large reviews, for instance, equine lymphomas, or
• Chronic T-cell large granular lymphocytic leukemia
for specific commonly occurring entities, for instance, gastro-
intestinal lymphomas in cats. MALT, mucosa-associated lymphoid tissue; NK, natural killer;
The current WHO classification of canine lymphomas WHO, World Health Organization.
(Table 2-5) is based on the principle that lymphoma subtypes
cannot be classified simply based on their cell morphology,
but accurate identification of each entity requires inclusion of
additional data, in particular the cell phenotype, but also
218 CHAPTER 2  •  Hematopoietic System Lymph Nodes

Follicular Color of fill

Benign follicular hyperplasia
Retention of mantle: cuff of
small dark-staining cells.
Cells have antigen-related
polarity: deep dark zone and
light superficial zone, best
seen in follicles near capsule.
Follicular tingible body
macrophages numerous.
Follicular lymphoma (B-cell)
No mantle cell cuff, with
vessels compressed between
follicles.
Uniform proportion of large to
intermediate cells
(centroblasts to centrocytes)
in all follicles.
Follicular tingible body
macrophages absent.
Grade based on centroblasts
per 400: FL1  0-5;
FL2  6-15; FL3  15.
Marginal zone lymphoma (B-cell)
Primary in spleen or node.
Splenic are locally extensive
arising on end arterioles and
coalescing. Node/spleen follicles
arise on fading GCs. Round
intermediate-sized nuclei with
peripheralized chromatin, very
large prominent single central
nucleoli, abundant lightly stained
cytoplasm, distinct cell boundaries.
Mitoses absent in early cases.
(hyperplasia  mixed with mantle
cells)
Mantle cell lymphoma (B-cell)
Primarily spleen, rare in node.
intermediate-sized nuclei,
smaller than MZL: round,
dense chromatin, small or
inapparent nucleoli, little
cytoplasm, no mitoses in early
cases. Larger nuclei with
nucleoli in blastoid cases.
Resemble large fading GCs.
Surrounded by variable
numbers of MZL cells and
plasma cells.

Nodal T-zone lymphoma

Nuclear size (vs. RBC) Mitotic rate per 400 field Early cases: Small nuclei with no mitoses, dense chromatin without
Color of border Color of font nuclear detail.
Large (2  RBC) Advanced cases: Intermediate-sized nuclei, dense chromatin, faint
Low (0-5/HPF) nucleoli, abundant light cytoplasm, few mitoses, sinus ectasia.
Intermediate (1.5  RBC) Medium (6-10/HPF) Filling of paracortex and cords, does not efface medullary architecture
or subcapsular sinus, prominent postcapillary venules, B follicles are
High (10/HPF) compressed against trabeculae.
Small (1-1.25  RBC)
(Note: Hyperplasia also has DCs, Mq).

Diffuse large B-cell lymphoma (B cell)
Uniform, round or cleft nuclei.
Centroblastic Uniformly shaped nuclei,
multiple nucleoli impinging on nuclear
membrane, scant cytoplasm.
Immunoblastic 90% of cells have
uniform round or oval nuclei with single
central nucleolus.
T-cell–rich large B-cell lymphoma.
Few large B cells amid many small
non-neoplastic T cells. Abundant stroma
with focal ischemic necrosis.
Lymphoblastic lymphoma
intermediate-sized nuclei,
round to oval, moderate
anisokaryosis. Chromatin
dense and dispersed,
multiple obscured small
nucleoli, scant basophilic
cytoplasm. Consistently
high mitotic rate with
chromosomes not sharply
defined.
B cell or T cell (most
T cell).
Anaplastic large cell Peripheral T-cell lymphoma
lymphoma anisokaryosis and poikilokaryosis
Uncommon.
Always some
Lymphoplasmacytic
multinucleated cells, very
lymphoma rare. Nodal, small
irregularly shaped nuclei
nuclei, deeply red cytoplasm,
(horseshoe, elongated);
few mitoses.
often vacuolated cytoplasm.
Mostly T cell, can be B cell.
R/O histiocytic sarcoma. CLL & small lymphocytic
lymphoma rare. Small nuclei,
few mitotic figures.

Diffuse Color of fill

Figure 2-174  Schematic flowchart for the diagnosis of the most common canine malignant lym-
phoma entities. (Based on concept created by J.L. Caswell.) Follicular versus diffuse lymphomas
are divided based on their yellow and blue background color, nuclear sizes are divided based on
the purple, green, and red colors outlining the text boxes, and the mitotic rate is divided based
the red, green, and blue font color. CLL, chronic lymphocytic leukemia; DC, dendritic cell; FL,
follicular lymphoma; GC, germinal center; HPF, high-power field; MΦ, macrophages; MZL, mar-
ginal zone lymphoma; RBC, red blood cell; R/O, rule out.

location of the lesion, its clinical course, and tissue architec-
ture. This understanding was largely driven by the advent of
IHC, which demonstrated that cells with similar morphology
might have a differing phenotype and markedly different
biology in both normal and neoplastic presentations.
The WHO classification of human non-Hodgkin lympho-
mas has been tested clinically and for each lymphoma entity
its frequency of occurrence, median age of affected patients,
ratio of males to females, stage distribution, survival, and
therapeutic response are well known. Basically, the WHO
classification identifies 3 major categories of lymphoma based
on their biological behavior: the indolent, the aggressive, and the
highly aggressive entities. This classification does not necessarily
reflect survival of human patients because current therapeutic
approaches result in a better outcome/curability for
the biologically more aggressive diseases. It is of the utmost
importance that veterinarians do not simply extrapolate survival
and therapeutic response data from human medicine to the spe-
cific lymphoma entities that are now recognized in domestic
animals according to WHO criteria. In human medicine, an
International Prognostic Index (IPI) has been established that
is used concurrently with the WHO classification to accu-
rately prognosticate the various lymphoma entities. This index
includes stage, patient age, number of extranodal sites, perfor-
mance status, and serum lactate dehydrogenase (LDH) levels.
Such index has not been established for domestic animals.
The WHO classification is not a rigid classification, and a
number of disease entities, for instance, cutaneous anaplastic
lymphomas and Burkitt-like lymphomas, still pose difficulties
regarding their prognosis or reproducibility of their diagnosis,
 Lymph Nodes
respectively. Advances in genetics and IHC have helped to
further categorize diffuse large B-cell lymphomas into cases
that respond well or poorly to classic CHOP (cyclophospha-
mide, hydroxydaunorubicin [also known as doxorubicin or
adriamycin], Oncovin [vincristine], and prednisone) therapy.
In a similar manner, the original study of canine lymphomas
focused mainly on nodal lymphomas, because they are the
most common entities in dogs, and ignored other lymphoma
types. Individual studies later reviewed other entities in
domestic animals, for instance, gastrointestinal lymphomas in
dogs and cats, and showed efficacy of the WHO classification
for these entities as well. As a pathologist or oncologist diag-
nosing or treating lymphomas, the most current publications
should be reviewed pertaining to the current understanding
of classification and associated behavior and therapeutic
response of any particular lymphoma entity in any animal
species.
The following text characterizes each lymphoma entity
according to the WHO criteria as far as they have been estab-
lished for animal species (see Table 2-5).
Hodgkin-like lymphoma.  Hodgkin-like lymphoma (HLL)
that meets acceptable criteria as described for Hodgkin lym-
phoma in humans does not occur in animals. Hodgkin-like
lymphoma has been suggested to occur in cats and less com-
monly in dogs. The diagnosis of Hodgkin lymphoma (HL)
rests on the demonstration of one of the types of Reed-
Sternberg cell in a background appropriate for the specific sub-
types of Hodgkin disease, lymphocyte-predominant (LPHL) or
classic, with the subtypes lymphocyte-rich, mixed-cellularity,
nodular-sclerosis, and lymphocyte-depleted HL. These criteria
have not been met in suspected cases of HLL in domestic animals.
The Reed-Sternberg cell has been shown to be of B-cell type
by analysis of single cells derived from human cases, and is
Pax-5 positive. Most cases reported as Hodgkin lymphoma in
dogs and cats most likely represent T-cell–rich B-cell lym-
phoma, a form of diffuse large B-cell lymphoma. Differentiat-
ing T-cell–rich B-cell lymphoma from HLL is extremely
difficult even when using IHC. Although Reed-Sternberg cells
tend to be CD20 and CD79a negative and Pax-5, CD30, and
CD15 positive in classic forms of Hodgkin disease, in LPHL,
CD30 negative, CD20-, and CD79a-positive Reed-Sternberg
cells have been reported. Uniformly strong positive labeling
of the large cell population for CD20 confirms a diagnosis of
T-cell–rich B-cell lymphoma.
HLL has been best described in cats with unilateral man-
dibular or cervical lymphadenopathy. In this study, 9 of 20
cats were classified as having LPHL-type disease and the
remaining 11 cats as classic forms of Hodgkin disease. All cases
had labeling of small- to intermediate-sized lymphocytes for
CD79a, and the different types of Reed-Sternberg cells were
negative for CD79a, CD3, Bla.36, and Mac387. Unfortu-
nately, IHC was not performed for Pax-5, CD20, CD30, and
CD15. Although lesions in these cats were classified according
to the different subtypes of Hodgkin lymphoma in humans,
there were a number of differences. The reported immuno-
histochemical labeling is in contrast to human LPHL where
detection of CD20- or CD79a-positive large cells in a nodule
of CD20- or CD79a-positive small cells is a characteristic
histologic feature. Furthermore, diagnostic Reed-Sternberg
cells were rare in cases of feline mixed cellularity and nodular
sclerosis HLL compared to human Hodgkin disease. In addi-
tion, lymphohistiocytic cells were found in conjunction
with classic Reed-Sternberg cells in mixed-cellularity and
219
nodular-sclerosis feline cases, and lacunar cells were noted in
small numbers in feline LPHD. Whether such cases truly
represent a unique type of HLL in cats or a type of T-cell–rich
B-cell lymphoma requires further studies. Regardless, cats
with solitary lymph node enlargement and a diagnosis of
suspected HLL or T-cell–rich B-cell lymphoma should be
treated differently than cats with diffuse large B-cell lym-
phoma. This entity has been suggested to represents a less
aggressive neoplasm that may not require chemotherapy.
Additional studies are needed to more accurately establish the
behavior and therapeutic response of these neoplasms.
Precursor lymphoblastic leukemia/lymphoma.  Lympho-
blastic lymphomas (LBL) are highly aggressive lymphomas of
B, T, or rarely natural killer (NK) cell lineage that can occur
in the lymph nodes, spleen, and most commonly thymus/
mediastinum in all domestic animals. In dogs, they are most
frequently of T-cell origin (Fig. 2-175). Neoplastic lymphoid
cells form diffuse, dense infiltrates of monomorphic,
intermediate-sized cells that are characterized by oval or folded
convoluted nuclei with a thin nucleolemma, dispersed chromatin,
and indistinct nucleoli. The absence of any parachromatin clear-
ing makes these tumors appear darker when viewed histologi-
cally at low magnification. The volume of the deeply stained
cytoplasm is minimal. They have a high mitotic rate of usually
>10 mitoses/HPF, but the mitotic figures are not as distinct as
in other types of lymphoma. When neoplastic lymphoid cells
infiltrate outside lymphoid tissues, they often appear in a
single-file pattern. Immunophenotyping may be difficult,
because CD20 and CD3 may be negative in B- or T-cell lym-
phoblastic lymphomas, respectively, and a small subset will be
bi-phenotypic, expressing both CD20 and CD3.
With lymphomas of LBL type, it is very important to make
the identification of cell type near the edge of the tissue,
where fixation is rapid for the chromatin to display the dis-
persed quality. One of the values of the Tru-cut biopsy is its
small size, permitting rapid fixation of the whole biopsy. Lym-
phoblastic lymphoma and lymphoblastic leukemia most likely
represent different ends of a spectrum of a single disease
entity. Although the T-cell lymphoblastic lymphoma occurs
commonly in the thymus, the vast majority of B-cell lympho-
blastic neoplasms occur as acute leukemia originating in bone
marrow (see section on Hematopoietic neoplasia). Clinically,
animals with T-LBL frequently have hypercalcemia (Fig.
2-176). Often animals in good body condition suddenly lose
appetite and appear depressed and may show signs of labored
breathing on physical examination.
Mature (peripheral) B-cell neoplasms
B-cell chronic lymphocytic leukemia/small lymphocytic
lymphoma.  B-cell chronic lymphocytic leukemia/small lym-
phocytic lymphoma (B-CLL/SLL) is characterized by prolif-
eration of mature-looking, small lymphoid cells, and only rare
cases occur as lymphomas, whereas the majority occur as
leukemias. A detailed description is therefore provided under
leukemia. Nodal forms of B-SLL efface the lymph node archi-
tecture and are originally localized in the interfollicular zones.
The dark-staining, small lymphoid cells surround lighter-
staining foci that represent proliferation centers. Neoplastic
cells commonly infiltrate the perinodal tissue. Nuclei of neo-
plastic lymphoid cells are small, round, have condensed chro-
matin, and inconspicuous nuclei. The cytoplasm is scant and
the mitotic index is low. Slightly larger, prolymphocytic cells
are dispersed throughout the sheets of neoplastic cells. Immu-
nophenotyping with CD79a will identify B-cell lineage, but
220 CHAPTER 2  •  Hematopoietic System
A
B tinct biological behavior.
C tingible body macrophages. Neoplastic cells are large, and the
Figure 2-175  Precursor T-cell lymphoblastic lymphoma in a dog.
A. Diffuse, dense infiltrates of monomorphic, intermediate-sized
cells that are characterized by oval or folded convoluted nuclei
with a thin nucleolemma, dispersed chromatin, and indistinct
nucleoli. B. Neoplastic T cells are diffusely positive for CD3 by
immunohistochemistry (IHC). C. Rare CD20-positive B cells can
be detected by IHC.
labeling may be weak. Cells may not label with CD20. B-SLL
have a low proliferation activity and the Ki67 index tends to
be low except in the proliferation centers.
Diffuse large B-cell lymphoma.  Diffuse large B-cell lympho-
mas (DLBCLs) are aggressive, rapidly growing neoplasms that
Lymph Nodes
Figure 2-176  Hypercalcemia of malignancy in a dog. Metastatic
mineralization of the kidney in a dog with precursor T-cell lym-
phoblastic lymphoma. (Courtesy K.G. Thompson.)
represent the most frequently encountered lymphoma in most
domestic animals. There are numerous subtypes of DLBCL.
Centroblastic and immunoblastic cell types represent mere
morphologic variants that commonly occur as heterogeneous
populations within the same neoplasm, but distinguishing
these subtypes has not been associated with differences in
clinical behavior or therapeutic response. In contrast, other
subtypes such as T-cell–rich B-cell lymphoma (TCRBCL),
anaplastic large cell lymphomas, or lymphomatoid granuloma-
tosis may pose a major diagnostic challenge, and have a dis-
DLBCL can be found in any tissue, but are most often
found in lymph nodes. They are the most common lymphoma
in dogs (Fig. 2-177). DLBCL can also occur as a mediastinal
mass or primary gastric or ocular lymphoma. Most cases arise
in the dark zone of the germinal center from centroblasts.
They expand diffusely and quickly destroy the lymph node
architecture. Although lymph nodes with DLBCL are most
commonly characterized by generalized enlargement, in the
spleen, DLBCL expands within white pulp areas and com-
presses the red pulp, which leads to grossly visible nodules
that over time may coalesce. Neoplastic cells commonly
invade vessels, and necrosis and infarction are common occur-
rences in larger neoplastic masses.
Histologically, neoplastic cells form large sheets that often
resemble a “starry-sky,” which is the result of interspersed
shape of centroblastic nuclei is highly variable, round, indented,
and irregularly folded. In contrast, the large immunoblasts are
primarily rounded. Regardless, both cell types have euchro-
matic nuclei with vesicular or coarsely granular chromatin and
easily recognizable large, amphophilic nucleoli that appear as
multiple, nucleolemma-associated nucleoli in centroblasts, but
are solitary and centrally located in immunoblasts. Mitoses are
common and atypical forms may be observed. Neoplastic cells
readily label with CD79a or CD20, and the Ki67 labeling
index is very high.
Because DLBCL is the most common form of nodal lym-
phoma in dogs and clinically is commonly seen as generalized
lymphadenopathy, the diagnosis is often based on fine-needle
aspirates detecting large lymphoid cells combined with PARR
 Lymph Nodes 221

A B

C D
Figure 2-177  Diffuse large B-cell lymphoma in a dog. A. The neoplasm expands diffusely and
quickly, destroying the tonsillar architecture. B. Sheets of large centroblastic or immunoblastic B
cells that are interspersed with tingible body macrophages resembling a “starry-sky.” C. Neoplastic
B cells are diffusely positive for CD20 by immunohistochemistry (IHC). D. Rare CD3-positive
non-neoplastic T cells can be detected by IHC.

testing and no immunophenotyping. This practice is very
unfortunate because it carries a risk of misdiagnosis. Peripheral
T-cell lymphomas can appear histologically similar to DLBCL
and account for up to 30% of nodal lymphomas. Fine-needle
aspirates also do not allow for evaluation of the tissue archi-
tecture, and differentiating high-grade follicular lymphomas
or even hyperplastic lesions from DLBCL may present a chal-
lenge. Considering that the sensitivity of PARR testing for IgH
receptor has only a sensitivity of ~65% and that cross-lineage
rearrangement occurs in ~10% of cases, making a diagnosis of
DLBCL based on cytology and PARR testing alone without
immunophenotyping is not acceptable for diagnostic purposes.
DLBCL also pose a therapeutic challenge. Chemotherapy
most commonly consists of CHOP treatment and large number
of cases respond to therapy. However, a subset of DLBCL will
fail therapy during initiation. There are currently no morpho-
logic features that correlate the neoplastic phenotype to thera-
peutic response. In human medicine, DLBCL have been
subclassified into activated B-cell–like (ABC DLBCL) and ger-
minal center B-cell–like (GCB DLBCL) based on gene expres-
sion profiling. Currently, an immunohistochemical algorithm
published by Choi and colleagues (see Further reading, this
section) is used to identify these 2 subtypes by using
antibodies against CD10, GCET1, BCL-6, MUM-1, and
FOXP1. Differentiating these 2 entities is clinically important
because ABC DLBCLs have significantly reduced survival
time despite CHOP therapy, but survival improves when
treated with bortezomib. A similar algorithm has recently
been established for dogs, and preliminary data indicate that
labeling, especially with GCET1 and CD10, predicts CHOP
response in canine DLBCL.
Anaplastic DLBCLs are uncommon lymphoid neoplasms
in dogs that are characterized by neoplastic B cells that have
large, often bizarre nuclei, and abundant amounts of cyto-
plasm. These lymphomas are indistinguishable from the more
common anaplastic large T-cell lymphomas, and also need to
be differentiated from histiocytic sarcomas by IHC. In human
medicine, the diagnosis of anaplastic T-cell lymphomas is
based on positive labeling with anaplastic lymphoma kinase
(ALK), which is usually negative in anaplastic B-cell lympho-
mas, thereby justifying their classification as DLBCL.
TCRBCL is a variant of DLBCL that represents the
most common lymphoma in horses and the most common
nodal lymphoma in cats, but has been reported in dogs (Fig.
2-178). In contrast to DLBCL, TCRBL are primarily com-
posed of sheets of small, reactive T cells admixed with
222 CHAPTER 2  •  Hematopoietic System
A not been performed in dogs or cats. Histologically, these neo-
B and benign follicular hyperplasia is required (Fig. 2-179). Lym-
C inconspicuous. Marginal zone lymphocytes are intermediate-
Figure 2-178  T-cell–rich B-cell lymphoma in a horse. A. Sheets
of small, reactive T cells admixed with histiocytic cells and neo-
plastic, large B cells that represent <10% of the cell population.
B. The reactive T cells are positive for CD3 by immunohisto-
chemistry (IHC). C. Small numbers of large neoplastic B cells are
strongly positive for CD79a by IHC.
histiocytic cells and eosinophils and neoplastic, large B cells
that are usually <10% of the cell population. Because of the
large number of histiocytic cells, the name T-cell/histiocyte–rich
B-cell lymphoma has also been used. The large B cells resemble
centroblasts and immunohistochemically label with CD20 or
Lymph Nodes
CD79a. Although CD79a may work poorly in horses, labeling
with Bla.36 should be avoided because it also labels histiocytic
cells. Clinically, TCRBCLs in horses occur most commonly as
abdominal or cutaneous masses that may wax and wane for
years before progressing to more aggressive DLBCLs that
disseminate.
There is an uncommonly encountered lymphoma in dogs
and rarely in cats that is of mixed cell type and most com-
monly occurs as large neoplastic nodules in the lungs. This
entity is called lymphomatoid granulomatosis (LYG), and it
can also occur in the skin, kidney, or other nonlymphoid loca-
tions. Although earlier studies classified this entity as a T-cell
lymphoma, the morphologic features and recent immunohis-
tochemical studies are more consistent with a special form of
a TCRBCL. However, assessment of clonal rearrangements has
plasms are characterized by a mixed infiltrate of lymphoid
cells composed of small T cells and atypical large B cells
admixed with histiocytic cells. Formation of granulomas is not
uncommon. The large atypical B cells resemble centroblasts
and immunoblasts and are positive for CD20 and CD79a and
often Ki67. CD20-negative Reed-Sternberg-like (multiple or
multilobed nuclei) cells that are positive for CD15 and CD30
have also been reported. There is prominent angiocentricity and
angioinvasion leading to vascular destruction and thrombosis,
followed by severe coagulative necrosis. In humans, LYG has
been variably associated with Epstein-Barr virus infections,
but no viral etiology has been established in animals.
Follicular-derived B-cell lymphomas.  To accurately diag-
nose follicular-derived lymphomas, including marginal zone
lymphomas, mantle cell lymphomas, and follicular lympho-
mas, some understanding of the structure of lymphoid follicles
phoid follicles have a germinal center with a dark and a light
zone that is surrounded by a mantle cuff, followed by a mar-
ginal zone. In lymph nodes, the marginal zone only develops
in long-standing benign hyperplasia. The darker zone of the
germinal center is primarily composed of centroblasts, whereas
the lighter zone is composed of a mixture of cells, including
many centrocytes. Centroblasts have been described under
DLBCL. Centrocytes are intermediate-sized cells with abun-
dant pale-staining cytoplasm and folded, angulated, or
indented nuclei. Nuclei have dense, stippled chromatin that
is lighter-staining than small lymphocytes. Mantle cuff lympho-
cytes are intermediate-sized cells that are slightly larger than
small lymphocytes. The amount of cytoplasm is scant. The
nuclei have variable degrees of indentation and angulation and
can even be round. Chromatin is very dense, and nucleoli are
sized cells with abundant, lightly stained cytoplasm and dis-
tinct borders. The nuclei are mildly vesiculated, with their
chromatin peripheralized against the nuclear membrane and
with a large single central nucleolus. This morphology resem-
bles to some degree immunoblasts that are large cells and need
to be differentiated from marginal zone lymphocyte to avoid
misdiagnosing a DLBCL. The marginal zone stains more
lightly than the mantle cuffs.
Follicular hyperplasia is characterized by follicular polarity
reflected by an eccentric cuff of small mantle cells that are
thicker over the peripherally oriented lighter zone of the
germinal center (Fig. 2-180). Tingible macrophages are usually
visible in germinal centers of hyperplastic follicles. A darkly
staining, well-defined mantle cell cuff is characteristic for
 Lymph Nodes 223

Marginal zone
Marginal zone
Marginal zone lymphoma

Intermediate cell:
Mantle zone
Marginal zone lymphocyte

Mantle zone
Mantle cell lymphoma

Intermediate cell:
Mantle cell lymphocyte
Germinal center - light zone

Germinal center - light zone

Follicular B cell lymphoma

Intermediate cell: Large cell:

Centrocyte Centroblast

Germinal center - dark zone

Germinal center - dark zone Diffuse large B cell lymphoma

Large cell: Large cell:

Immunoblast Centroblast

Figure 2-179  Schematic drawing of origin of B-cell lymphomas based on normal follicle architec-
ture. (Based on concept created by P.F. Moore.)

A B
Figure 2-180  Follicular hyperplasia in a dog. A. Follicular polarity reflected by an eccentric cuff
of small mantle cells that are thicker over the peripherally oriented lighter zone of the germinal
center. B. Immunohistochemistry for BCL-2 highlights polarity by labeling mantle cells, but not
follicular center cells.

follicular hyperplasia. Polarity and the eccentric mantle cell
cuff are even more easily recognizable when labeling tissues
with CD20. The follicles are discrete and separated by at least
some interfollicular lymphoid tissue. Involution and “fading”
of follicles are commonly observed with follicular-derived
lymphomas. The involution of germinal centers results in for-
mation of aggregates of mantle cells that have collapsed into
the dendritic bed of fading germinal centers. This feature has
been described as fading follicular hyperplasia (FFH). Regard-
less, clonality testing may be required to differentiate neoplas-
tic from hyperplastic follicular proliferations.
Follicular lymphoma (FL) is an uncommon follicle-derived
lymphoma that has been reported in lymph nodes, spleen,
and extranodal tissues of dogs (Fig. 2-181). As with all
224 CHAPTER 2  •  Hematopoietic System Lymph Nodes

A B

C D
Figure 2-181  Follicular lymphoma in a dog. A. Follicles are usually large, uniform in size, lack a
mantle cell cuff, and show no polarity. B. Absence of tingible body macrophages and uniform
proportions of centrocytes and centroblast through follicle, with an absence of a dark zone.
C. Neoplastic B cells in individual and confluent follicles are diffusely and uniformly positive for
CD20 by immunohistochemistry. D. Small rims of immunohistochemically CD3-positive T cells
surround follicles.

follicular-derived lymphomas, FL must be differentiated from
follicular hyperplasia. Low-magnification examination is
essential when differentiating follicular-derived lymphoma
from follicular hyperplasia. A pattern of densely packed fol-
licles throughout the whole parenchyma of a lymph node is
characteristic for follicular lymphoma. The follicles are usually
large, uniform in size, lack a mantle cell cuff (“bare”) and show
no polarity. Paracortical high-endothelial venules are com-
pressed between opposing follicles and cannot be found
within them. Other important features include an absence of
tingible body macrophages and uniform proportions of cen-
trocytes and centroblasts through all follicles, with an absence
of a dark zone. The cellular features of centrocytes and cen-
troblasts have been described previously. Both centroblasts
and centrocytes are positive for CD79a and CD20 and nega-
tive with CD3. The Ki67 index is low. Immunohistochemical
labeling with BCL-2 is used in human FL because normal
follicular center cells are negative for BCL-2. Although similar
BCL-2 labeling has been confirmed in hyperplastic lesions in
dogs, data on canine FL are missing. Vascular invasion, extra-
nodal invasion, and loss of tissue architecture are more obvious
features of FL. In humans, FLs are graded based on the
percentage of centrocytes and centroblasts, with larger
numbers of centroblasts indicating more aggressive biological
behavior. There are no data available for domestic animals and
grading of FL is not performed routinely. Whether high-grade
FLs require more aggressive CHOP-based therapy is unknown.
Marginal zone lymphoma (MZL) is a follicular-derived
B-cell lymphoma that has been described in dogs in the lymph
nodes, spleen, and bronchus-associated lymphoid tissue and
mucosa-associated lymphoid tissue of the respiratory and
intestinal tracts (Fig. 2-182), respectively. In the spleen, MZL
often develops as a nodular mass. It is one of the 6 most
common types of nodal lymphomas in dogs, accounting for
~15% of canine lymphomas. Dogs with MZL usually are in
apparent good health and with an enlarged node or single
nodule in spleen, intestine, or lungs at the early stage of the
disease, progressing to multiple nodes, spleen, and tonsils
being involved in late-stage disease. MZL are considered indo-
lent, slowly progressive lymphomas that should not be treated
with regular CHOP therapy.
MZL originate from the marginal zone of the splenic fol-
licles or from the marginal zone that appears in lymph nodes
in chronic hyperplasia (Fig. 2-183). MZL must be differentiated
 Lymph Nodes 225

A B
Figure 2-182  Mucosa-associated lymphoid tissue (MALT)oma in a cat. A. Intermediate-sized
lymphoid cells forming clusters within crypt epithelium of the small intestine. B. Neoplastic B
cells are positive for CD20 by immunohistochemistry.

A B

C D
Figure 2-183  Marginal zone lymphoma in a dog. A. Homogeneous cuffs of neoplastic marginal
zone lymphocytes surround faded and hemorrhagic germinal centers. B. Cuff of intermediate-sized
marginal zone lymphocytes with mildly vesiculated nuclei with peripheralized chromatin and
a large single central nucleolus. C. Neoplastic marginal zone lymphocytes surround regressing
germinal center. D. Rim of marginal zone lymphocytes is strongly positive for CD20 by
immunohistochemistry.
226 CHAPTER 2  •  Hematopoietic System
from marginal zone hyperplasia (MZH). Both entities are char-
acterized by proliferation of marginal zone lymphocytes in
lymphoid follicles with their germinal centers in regression.
Some degree of MZH may also be observed with prominent
follicular hyperplasia. Usually MZH appears often as discon-
tinuous cuffs of marginal zone lymphocytes admixed with
smaller mantle cells and centroblasts. Cellular features of mar-
ginal zone lymphocytes have been described previously. In
MZL, the proliferating neoplastic cells form more homogeneous
cuffs of marginal zone lymphocytes surrounding a fading germinal
center (eFig. 2-43). During tumor progression, perifollicular
proliferating areas coalesce and commonly cause paracortical
atrophy and marked sinus dilation in the spleen. Cavities are
filled with erythrocytes and neoplastic cells. MZL have been
classified as “early,” “mid,” or “late” MZL according to their
stage of development, which is reflected by the degree of
coalescence of marginal zones, the amount of tissue affected,
and the number of mitoses, which are absent in early cases.
However, the overall mitotic index remains low even in late
cases. Neoplastic lymphoid cells are strongly positive for
CD20 or CD79a and negative for CD3. MZL should be easily
differentiated from DLBCL by its follicular architecture,
except in late cases, and size and amount of cytoplasm of
neoplastic cells. Cellular morphology and the orientation around
fading germinal centers are the main features to differentiate MZL
from FL.
Mantle cell lymphoma (MCL) is a rare follicular-derived
lymphoma that has been described in the spleen of dogs. MCL
present as solitary masses and are far less common than other
follicular-derived B-cell lymphomas (Fig. 2-184). The appear-
ance of MCL in the spleen is determined by the splenic end
arterioles that dictate the regular periodicity of neoplastic
nodules. The neoplasm arises from the inner mantle cuff and
differentiation from splenic follicular hyperplasia is difficult
because both lesions consist predominantly of mantle cells. As
with other follicle-derived B-cell lymphomas, in contrast to
hyperplastic lesions, neoplastic cells surround fading germinal
centers that are commonly hyalinized. The cell morphology
of mantle cells has been described previously. Mitoses are rare.
The MCL cells are B cells and label positive with CD79a and
CD20 and are negative with CD3.
Clinically, MCL has been considered to be an indolent
lymphoma and is grouped with MZL, but small case numbers
are probably the main reason that published evidence is
missing. Classification of MCL as indolent lymphomas is also
based on data from human medicine, where MCLs occur as
an incurable disease with a median survival time of 3-5 years.
Older age, a high Ki67 index, a high mitotic rate (1-3/HPF),
blastoid cell morphology, and mutations in p53 have been
associated with poor prognosis in humans. None of these
factors has been studied in dogs.
Burkitt-like lymphoma (BKL).  Although Burkitt lympho-
mas represent a well characterized entity in humans, Burkitt-
like lymphoma (BKL) is a highly controversial entity that has
been reported in dogs. Burkitt lymphomas in humans are highly
aggressive lymphomas that exist as an endemic form in Africa,
and are strongly associated with Epstein-Barr virus (EBV). In
the sporadic form, the association with EBV is highly variable,
and in acquired immunodeficiency syndrome (AIDS)-associ-
ated Burkitt lymphomas, there is only an association with EBV
in 30% of cases. In addition, the vast majority of Burkitt lym-
phomas have c-Myc translocations. Although seroconversion
to EBV has been reported in dogs and cats, viral DNA was
Lymph Nodes
not detected in blood of seropositive animals. Neither have
mutations in c-Myc been detected in BKL in dogs.
Histologically, canine BKL are diffuse neoplasms that give,
on low-power examination, the impression of a “starry sky”
because of the large number of tingible body macrophages. Neo-
plastic lymphoid cells are of intermediate size with round
nuclei that have a vesicular appearance. Aggregated chromatin
is clumped on the small, but prominent nucleoli and the
nucleolemma with irregular parachromatin clearing. Although
3-5 nucleoli have been reported in human Burkitt lymphoma,
there are usually fewer nucleoli in canine BKL. There is mild
anisokaryosis. The cytoplasm is of moderate volume and stain-
ing density. The mitotic rate is 10 or greater/HPF. During a
blinded review of canine nodal lymphomas, participating
pathologists failed to consistently differentiate BKL from
DLBCL, leading to exclusion of the BKL as an entity from the
review process.
Extramedullary plasmacytoma.  Extramedullary plasma-
cytomas (EMP) occur most commonly in the skin (digits and
ears) of dogs (Fig. 2-185), but are also common in the oral
and colorectal mucosa. Rare cases have been reported in
cats. Overall, EMPs are benign neoplasms with a low Ki67
index, and complete surgical removal of cutaneous masses is
curative. Plasmacytomas occurring in the facial skin have a
higher risk to invade into the underlying parenchyma, includ-
ing bone, but aggressive surgical removal is curative and
metastasis rarely occurs. Regardless, malignant variants occur
and may metastasize widely, but the incidence is <10%.
Colorectal plasmacytomas often pose a greater surgical chal-
lenge, and local recurrence has been reported. Some cases
occur as multiple plasmacytomas throughout the digestive
tract mucosa. Clinically, canine colorectal plasma cell tumors
have been reported to cause hematochezia, tenesmus, and
rectal prolapse. Macroscopically, cutaneous plasmacytomas
are solitary, broad-based, spherical or dome-shaped, hairless
neoplasms up to 3 cm in diameter. Some cutaneous EMPs may
be pedunculated, and ulceration is common. Intestinal EMPs
form solitary, red, raised and smooth, rapidly growing nodules
up to 5 cm in size. EMPs occur in middle-aged to older dogs,
and there is no sex or breed predilection. Urinary Bence-Jones
protein has not been detected in dogs with cutaneous or
intestinal EMPs, and hypergammaglobulinemia, which is com-
monly observed with multiple myeloma, is not a typical
feature of canine EMPs.
Most EMPs are circumscribed but not encapsulated, and
primarily located in the superficial dermis or the intestinal
submucosa, but may extend into the mucosa. Cutaneous neo-
plasms typically create a Grenz zone, and do not involve the
overlying epithelium. EMPs are densely cellular tumors com-
posed of densely packed, round to oval neoplastic cells that
are commonly arranged in cords and single files. A fine reticu-
lar stromal network of capillaries separates the cellular neo-
plastic cells. Neoplastic cells have indistinct cell borders and
round nuclei that are commonly eccentrically placed, and
have coarsely clumped chromatin arranged in a “clockface”
pattern and small solitary nucleoli. Megalokaryosis as well as
binucleate and rare multinucleate cells are a characteristic
feature (eFig. 2-44). Anisocytosis and anisokaryosis are moder-
ate and the mitotic index is usually low (0-1 mitoses/HPF).
There are variable amounts of often deeply amphophilic cyto-
plasm with perinuclear pallor. Some cells contain large eosino-
philic, intracytoplasmic aggregates of immunoglobulin (Russell
bodies). Cells toward the periphery of the neoplasm often
 226.e1

A A

B B
eFigure 2-43  Marginal zone lymphoma in a dog. A. Homoge- eFigure 2-44  Cutaneous plasmacytoma in a dog. A. Rows of
neous cuffs of neoplastic marginal zone lymphocytes surround neoplastic plasma cells surround aggregates of extracellular
faded and hemorrhagic germinal centers. B. Cuff of intermediate- amyloid. B. Megalokaryosis is a common feature of cutaneous
sized marginal zone lymphocytes with mildly vesiculated nuclei plasmacytomas.
with peripheralized chromatin and a large single central
nucleolus.
 Lymph Nodes 227

A B

C D

E
Figure 2-184  Mantle cell lymphoma in a dog. A. Homogeneous cuffs of neoplastic mantle cuff
lymphocytes surround fading germinal centers. B. Splenic end arterioles dictate the regular peri-
odicity of neoplastic nodules. C. Intermediate-sized mantle cuff lymphocytes, which are slightly
larger than small lymphocytes and have chromatin-dense nuclei and inconspicuous nucleoli, form
cuffs around regressed germinal centers. D. Mantle cuff lymphocytes are strongly positive for
CD20 by immunohistochemistry (IHC). E. No CD3-positive T-cells are detected within neoplastic
nodules by IHC.

closely resemble non-neoplastic plasma cells. Different mor-
phologic types have been reported for cutaneous EMPs in
both dogs and cats, including lymphoid, histiocytic, and pleo-
morphic subtypes, this classification has not been applied to
intestinal EMPs. Submucosal, perivascular, and intracellular
amyloid may be demonstrated with thioflavine T or Congo
red stains, and it is most commonly of λlight-chain origin.
Macrophages and foreign-body giant cells usually surround
amyloid deposits. A few cases with metaplastic cartilage and
bone within the amyloid have been reported. Ultrastructural
228 CHAPTER 2  •  Hematopoietic System Lymph Nodes

A B

C D
Figure 2-185  Cutaneous plasmacytoma in a dog. A. Neoplastic plasma cells have indistinct cell
borders and round nuclei that are commonly eccentrically placed; megalokaryosis as well as
binucleate cells are characteristic features. B. Extracellular and intracellular amyloid of λ light-
chain origin can be found in extramedullary plasmacytomas. C. Approximately 80% of cutaneous
plasmacytomas are positive for CD79a by immunohistochemistry (IHC). D. Nuclear labeling
for multiple myeloma oncogene-1 (MUM-1) can be detected by IHC in most cutaneous
plasmacytomas.

studies demonstrated large profiles of rough endoplasmic

reticulum.
Confirmation of the diagnosis of EMP is generally possible
using IHC to detect Mum-1 and immunoglobulins as well as
immunoglobulin light chains, but a combination with CD79a,
CD20, and Pax-5 for B-cells and CD3 for T cells is often
helpful in determining the cell of origin. Approximately 80%
of cutaneous plasmacytomas are positive for CD79a and only
60% for CD20.
Multiple myeloma is a malignant neoplasm of plasma cells
that typically occurs at multiple intramedullary sites (Fig.
2-186) and involves production of a clonal immunoglobulin.
The tumor is described in detail previously under Other
hematopoietic conditions.
T-cell and NK-cell neoplasms
Mature (peripheral) T-cell neoplasms.  Mature T-cell neo-
plasms are lymphomas of mature T cells in contrast to T-cell
lymphoblastic lymphomas. This is an umbrella diagnosis for Figure 2-186  Multiple myeloma in a dog. Neoplastic mass located
numerous T-cell lymphomas, and the WHO classification has in the medullary cavity of femur.
been expanded over the last decade to more accurately
 Lymph Nodes 229

characterize some of the heterogeneous subtypes. Classifying

subtype groups by the primary location of the neoplasm
has resulted in the identification of nodal, extranodal,
enteropathy-associated, and cutaneous T-cell lymphomas. Large
granular lymphocytic T-cell lymphomas have been characterized
as a specific subtype. The remaining group of mature T-cell
lymphomas is simply identified as unspecified peripheral T-cell
lymphomas.
Unspecified, peripheral T-cell lymphomas (PTCL) have
been reported in all species because this heterogeneous group
represents the “basket”-diagnosis for all nonspecific mature
T-cell lymphomas. PTCL can occur in lymph nodes, spleen,
and extranodal tissues, such as the skin or subcutis. In dogs,
up to 20% of nodal lymphomas have been classified as PTCL
(Fig. 2-187). PTCLs are highly aggressive lymphomas that
respond poorly to chemotherapy and together with T-LBLs A
are thereby largely responsible for the reputation of a poor
prognosis of T-cell lymphomas. As individual entities within
the group of PTCL will get recognized over time, some of
these entities may actually carry a different diagnosis, as has
been documented for T-zone lymphomas.
In dogs, about 2 3 of PTCL occur as a single-node enlarge-
ment and the rest with multiple-node involvement. In lymph
nodes, the neoplastic cell infiltrates are paracortical or diffuse.
The cellular composition of PTCL varies from cases to case
and ranges from small to intermediate to large cells, with large
cell PTCL being the most common. Some neoplasms have a
monomorphic cellular infiltrate, whereas others may contain
various inflammatory components, including eosinophils, his-
tiocytes, and plasma cells. Nuclei often have prominent inden-
tations, convolutions, or multilobulation, and chromatin is
granular or coarse. The cytoplasm is moderate to abundant in
volume and can be clear, eosinophilic, amphophilic, or deeply B
stained, with cell boundaries generally being distinct.
Nodal T-cell lymphoma.  Nodal T-cell lymphoma includes
T-zone lymphomas, anaplastic large T-cell lymphomas, and
angioimmunoblastic T-cell lymphomas, and the remaining
variants are currently classified as unspecified peripheral T-cell
lymphomas, which have been discussed previously.
T-zone lymphomas (TZL) are indolent, nodal lymphomas
that have been well characterized in dogs and rarely been
described in horses. Affected dogs almost invariably retain
normal appetite and activity and have one or sometimes more
nodes enlarged at the time of diagnosis. Generalized lymph-
adenopathy may occur. Dogs frequently develop a low-grade
lymphocytosis of 5-15 × 109/L that does not seem to influence
the progression of the lymphoma, which takes an indolent
course. Rarely epitheliotropic proliferations of neoplastic T
cells may occur in different organs during the late course of C
disease. The diagnosis of TZL is primarily based on their char-
acteristic architecture of paracortical proliferation of neoplastic T Figure 2-187  Peripheral T-cell lymphoma in a dog. A. Mixture
cells that causes compression and fading of germinal centers, of large and intermediate-sized neoplastic T cells that have mod-
which are peripheralized against the capsule (Fig. 2-188). The erate amounts of cytoplasm and often indented nuclei with stip-
capsule is thin and peripheral, and cortical sinuses are irregu- pled chromatin and indistinct nucleoli. B. Neoplastic T cells are
larly dilated with low cellularity. There is no involvement of positive for CD3 by immunohistochemistry (IHC). C. Small rem-
perinodal tissues. The proliferating paracortex may on low nants of lymphoid follicular B cells are positive for CD20 by IHC.
magnification be misinterpreted as a follicle-derived B-cell
proliferation, but the proliferating neoplastic cells do not sur-
round fading germinal centers as in marginal zone prolifera- mitotic rate is very low, and no mitoses are seen in single HPFs
tions. High-endothelial venules are prominent. Neoplastic in most cases. In contrast, T-zone hyperplasia is characterized
cells are uniform, small T cells with moderate amounts of pale, by a more heterogeneous population of small lymphocytes,
often water-clear cytoplasm, and densely stained nuclei that macrophages, and dendritic cells resulting in a moth-eaten
have shallow, sharp indentations and inapparent nucleoli. The appearance at low magnification. IHC helps to identify the
230 CHAPTER 2  •  Hematopoietic System Lymph Nodes

A B

C D
Figure 2-188  T-zone lymphoma in a dog. A. Paracortical proliferation of neoplastic T cells causes
compression and fading of germinal centers, which are peripheralized against the capsule.
B. Uniform, small neoplastic T cells with moderate amounts of water-clear cytoplasm and densely
stained nuclei that have shallow, sharp indentations and inapparent nucleoli. C. T cells that
are CD3 positive by immunohistochemistry (IHC) compress and peripheralize germinal centers.
D. Subcapsular remnants of germinal centers are positive for CD20 by IHC.

fading and peripheralized germinal centers that label with
CD79a and CD20. The neoplastic paracortical areas are
eccentric to fading germinal centers and are composed of
solidly CD3-positive neoplastic cells.
Anaplastic large T-cell lymphoma (ALTCL) is a rare
nodal lymphoma that has been reported in dogs and cats, and
carries a poor prognosis. Neoplastic cells have abundant cyto-
plasm and large, irregular, often bizarre, nuclei that are respon-
sible for the name of this entity. Affected nodes are partially
or completely involved, and predominantly sinusoidal growth
is a common feature. Fibrosis of the capsule and parenchyma
is common. Secondary infiltrates of small lymphocytes, plasma
cells, and eosinophils may occur. Histologically, ALTCLs are
indistinguishable from the anaplastic variant of DLBCLs.
However, as discussed previously, in humans, only ALTCLs are
consistently positive for anaplastic lymphoma kinase (ALK).
Neoplastic cells are commonly negative for CD3 and clonal
rearrangements in T-cell-receptor γ may help in the diagnosis.
Neoplastic cells consistently express CD30 and contain cyto-
toxic granule–associated proteins, such as perforin and gran-
zyme B, suggesting a cytotoxic T-cell phenotype. Unfortunately,
expression of ALK and CD30 has not been studied in animals.
ALTCL also have to be differentiated from histiocytic
sarcomas.
Angioimmunoblastic T-cell lymphoma is another nodal
lymphoma rarely reported in dogs. It is characterized by poly-
morph lymphoid infiltrates of affected nodes and associated
systemic disease. Although the node parenchyma is commonly
destroyed by the neoplastic cell proliferation, subcapsular
sinuses often remain open. Prominent high-endothelial capil-
laries spread through the node. Few fading germinal centers
may be observed. The neoplastic cells are of intermediate size
and have round nuclei and often clear cytoplasm. A charac-
teristic feature of this entity is clusters of immunoblastic cells
around vessels that also migrate into the vessels. Although the
neoplastic cells are consistently positive with CD3, infiltration
of B cells and eosinophils is common. Dendritic cells may
form perivascular, spindyloid proliferations. There are limited
data regarding the prognosis of these cases, and an aggressive
behavior has been suggested.
Enteropathy-associated T-cell lymphoma (EATL). 
Enteropathy-associated T-cell lymphomas (EATL) are intesti-
nal tumors of intraepithelial lymphocytes. They are further
subdivided into EATL type 1 (large cell neoplasms) that are
 Lymph Nodes 231

Figure 2-189  Enteropathy-associated T-cell lymphoma type 1 in Figure 2-190  Large granular lymphocyte lymphoma in a cat.
a dog. Neoplastic T cells infiltrating the jejunal mucosa have large Neoplastic T cells in many enteropathy-associated T-cell lym-
round or irregularly folded nuclei with parachromatin clearing phoma type 1 in cats have intracytoplasmic eosinophilic
and prominent central nucleoli of centroblastic nuclear type. granules.

often associated with necrosis and an inflammatory back-

ground and EATL type 2 (small to intermediate cell neoplasms)
that have no inflammatory background and rare necrosis. The
intraepithelial neoplastic cells are αβ T-cells and are consis-
tently CD3 positive and CD20 and CD79a negative. As
expected, EATL type 1 has a significantly higher Ki67 labeling
index than EATL type 2. Regardless of the type, animals with
EATL most commonly appear with weight loss, anorexia,
vomiting, diarrhea, lethargy, polydipsia, and polyuria. The
prognosis of EATL type 1 is much worse than for EATL type
2, which usually occurs as an indolent disease. Animals affected
with EATL type 1 respond poorly to therapy and survival is
limited to a few weeks or months.
EATL type 1 occurs most commonly in older dogs and is
often associated with transmural infiltration (Fig. 2-189). Sim-
ilarly, transmural EATLs in cats have been reported to be most A
commonly type 1 too, and in both species neoplasms occur
most frequently in the jejunum. The neoplastic lymphoid cells
have large round or irregularly folded nuclei with apparent
parachromatin clearing and prominent central nucleoli of cen-
troblastic nuclear type. Strong eosinophilic infiltrates may be
observed in some cases. Neoplastic cells in EATL type 1 often
have intracytoplasmic eosinophilic granules that express the
cytotoxic granule protein, granzyme B, consistent with a diag-
nosis of large granular lymphocyte lymphoma (LGL) (Fig.
2-190). The neoplastic cells of LGL often have abundant
cytoplasm, and IHC for perforin can also be used to aid in the
diagnosis. LGL may be a subentity of EATL type 1, but it is
commonly associated with leukemia.
EATL type 2 has primarily been reported in older cats and
occurs most commonly as intestinal mucosal lymphoma in the
jejunum (Fig. 2-191). EATL type 2 also occurs commonly in B
horses, where it affects the rectum. In both species, EATL type
2 can be diagnosed with full thickness or endoscopic biopsies, Figure 2-191  Enteropathy-associated T-cell lymphoma type 2 in
especially when combining morphologic evaluation with IHC a cat. A. Epitheliotropism is an important feature for diagnosing
and clonality testing. Morphology alone is often unable to this small-cell lymphoma and intraepithelial nests and/or plaques
differentiate the small, dense neoplastic T cells from similar of lymphocytes are highly suggestive of enteropathy-associated
appearing inflammatory T cells. In cats with EATL type 2, the T-cell lymphoma type 2. B. Immunohistochemistry for CD3
pattern of mucosal infiltration of neoplastic lymphocytes greatly enhances the ability to evaluate epitheliotropism of neo-
ranges from discrete, patchy areas to total effacement of the plastic T cells.
232 CHAPTER 2  •  Hematopoietic System
lamina propria. Early cases often show only infiltration of the
lamina propria of individual villi by large number of lympho-
cytes, and adjacent villi may be uninvolved. In other cases,
lymphocytic infiltration may occur as a band that spans the
crypt-villus junction. In advanced cases, the infiltrating neo-
plastic lymphocytes obliterate both the villus and crypt lamina
propria and form a band beneath the crypt epithelium, but
above the muscularis mucosae (eFig. 2-45). Such cases are
commonly associated with villous blunting and fusion as well
as crypt effacement.
Epitheliotropism is an important feature for diagnosing
EATL type 2, and the presence of intraepithelial nests (clusters
of more than 5 intraepithelial lymphocytes) or plaques (5
adjacent epithelial cells overrun by lymphocytes) of lympho-
cytes is highly suggestive of lymphoma. IHC for CD3 greatly
enhances the ability of the pathologist to evaluate epitheliot-
ropism. Although most epitheliotropic lymphocytes are
observed in villi, infiltration of crypt epithelium is an even stron-
ger indication of lymphoma. However, intraepithelial lympho-
cytes are a common immunologic response in the intestine,
and individual intraepithelial lymphocytes are commonly
found in inflammatory conditions, for instance, cats with
inflammatory bowel disease (IBD). The prognosis of cats with
EATL type 2 is still somewhat controversial, and large-scale
prospective studies using a standardized therapeutic approach
are lacking. Data from many studies are also hampered by the
lack of an accurate diagnosis of EATL type 2 versus IBD
because of the lack of TCRG clonality testing. Regardless,
EATL type 2 is considered a slowly progressing, smoldering
disease that may not require aggressive therapy. Overexpression
of Bcl-2 has recently been documented in EATL in cats, which
may prove useful as a therapeutic target.
Extranodal T-cell lymphoma.  Although the majority of
extranodal T-cell lymphomas in most animal species are cur-
rently classified as unspecified peripheral T-cell lymphomas,
hepatosplenic T-cell lymphomas rarely occur in dogs and horses.
Furthermore, hepatocytotropic T-cell lymphoma has recently
been described as an even more rare disease in dogs.
Hepatosplenic T-cell lymphoma (HS-TCL) causes a rapidly
progressive disease in dogs that clinically have lethargy,
anorexia, weight loss, diarrhea, and vomiting. Hepatomegaly
and splenomegaly, regenerative anemia, thrombocytopenia,
and hypoproteinemia are commonly observed. Dogs have
mild icterus and mild toxic changes in neutrophils. Horses
have only been diagnosed after autopsy, and their clinical signs
are not known. Neoplastic T cells are found primarily within
hepatic and splenic sinusoids, ranging from small clusters to
large aggregates that expand sinusoids and in the liver com-
press hepatic cords. Periportal and centrilobular neoplastic cell
infiltrates are also common in the liver. Erythrophagocytic
macrophages, extramedullary hematopoiesis, and fibrin
thrombi with associated ischemic necrosis are all features
commonly observed. Neoplastic lymphocytes have mild to
moderate amounts of eosinophilic to clear cytoplasm, inter-
mediate to large size, round to oval nuclei with indistinct
nucleoli, and variably distinct cell borders. Anisocytosis and
anisokaryosis are mild, and the mitotic index is low with 0-2
mitoses/HPF. Bone marrow and lungs are also consistently
affected. Immunohistochemically, neoplastic T cells are posi-
tive for CD3, T-cell-receptor γδ (TCRγδ), CD11d, and gran-
zyme B and negative for TCRαβ.
Only 2 cases of hepatocytotropic T-cell lymphoma (HC-
TCL) have been described in dogs. The liver was affected in
Lymph Nodes
both cases, whereas involvement of spleen and lung was
minimal. In contrast to HS-TCL, neoplastic lymphocytes in
HC-TCL are not confined to hepatic sinusoids, but invade
hepatic cords individually or in clusters. Neoplastic T cells are
morphologically similar to those described with HS-TCL, but
are CD11d negative. Bile duct epithelium is also infiltrated by
lymphocytes. However, no degenerative changes are apparent
in hepatocytes. Erythrophagocytosis is not an important
feature, and extramedullary hematopoiesis and fibrin thrombi
are absent. Further studies are needed to confirm HC-TCL as
a specific entity.
Cutaneous T-cell lymphoma.  Cutaneous T-cell lympho-
mas (CTCL) are a heterogeneous group of lymphomas
that include epitheliotropic and non-epitheliotropic lymphomas.
Epitheliotropic CTCLs appear as mycosis fungoides (MF),
Pagetoid reticulosis (PR), or Sézary syndrome (SS). Non-
epitheliotropic CTCLs appear as a subcutaneous “panniculitis-
like” T-cell lymphoma or anaplastic large T-cell lymphoma; all
other non-epitheliotropic CTCLs are currently classified as
unspecified peripheral T-cell lymphomas, which have been
described earlier.
Mycosis fungoides (MF) is a disease of older cats, dogs, and
horses. MF may exist originally as patches or plaques in the
skin that commonly progress rapidly to larger neoplastic
masses that invade the deeper dermis and potentially cause
local and distant metastases. In dogs and cats, MF occurs most
commonly around mucocutaneous junctions and feet or other
skin sites. Many affected dogs have a history of chronic der-
matitis, and erythema is the most common clinical sign. Pru-
ritus is sometimes associated with erythema and scaling;
ulcerations, hypopigmentation and alopecia can occur. In
horses, the skin may not be affected, but the mucous mem-
branes are the primary sites. In advanced cases, MF extends to
lymph nodes causing them to become enlarged and firm.
Histologically, in contrast to humans, MF in animals is char-
acterized by a strong tropism of neoplastic T cells for the epider-
mis and adnexal structures, including hair follicles, apocrine
sweat, and sebaceous glands (Fig. 2-192). T cells invading the
epidermis commonly form cavities filled with lymphocytes
called Pautrier’s microabscesses. Early lesions typically appear
as small numbers of T cells infiltrating along the basal level of
the epidermis. As disease progresses, the neoplastic cells
invade the deeper dermis and panniculus, causing larger
nodular masses. Neoplastic T cells are intermediate-sized cells
with a round, hyperchromatic and convoluted nuclei that
occasionally have sharp shallow indentations and large nucle-
oli that are difficult to visualize. The amount of cytoplasm is
minimal and generally water clear. Mitoses are rare.
Pagetoid reticulosis (PR) is considered a more epitheliotro-
pic variant of MF in which the neoplastic T cells form large
plaques that are almost entirely confined to the epidermis (Fig.
2-193). Morphologically, neoplastic cells are similar to those
described with MF. Sézary syndrome is the generalized stage of
MF with secondary lymphadenopathy and leukemia. The immu-
nophenotype of canine epitheliotropic CTCL is markedly dif-
ferent from its human counterpart. In canine epitheliotropic
CTCL, T cells are consistently positive for CD3. In MF, neo-
plastic T cells express CD8, and a smaller percentage of cases
are CD4 and CD8 negative, but CD4 expression has not been
reported. TCRαβ and TCRγδ are expressed in approximately
equal numbers of cases. This is in contrast to human MF,
which is primarily a disease of CD4- and TCRαβ-positive T
cells. Interestingly, T cells in canine PR have a slightly different
 232.e1

B
eFigure 2-45  Enteropathy-associated T-cell lymphoma type 2 in
a cat. A. Epitheliotropism characterized by intraepithelial nests
and/or plaques of lymphocytes is highly suggestive of enteropathy-
associated T-cell lymphoma type 2. B. Immunohistochemistry for
CD3 highlights the marked epitheliotropism of neoplastic T cells.
 Lymph Nodes 233

A B

C D
Figure 2-192  Mycosis fungoides in a dog. A. Epitheliotropic cutaneous T-cell lymphoma that
often infiltrates along the basal level of the epidermis. B. Strong tropism of neoplastic T cells for
the epidermis and adnexal structures, including hair follicles. C. Neoplastic T cells in the dermis
and epidermis are CD3 positive by immunohistochemistry. D. Epitheliotropism of immunohisto-
chemically CD3-positive T cells affecting adnexal structures.

A B
Figure 2-193  Pagetoid disease in a dog. A. A more epitheliotropic variant of mycosis fungoides
in which the neoplastic T cells form large plaques that are almost entirely confined to the epider-
mis. B. Intraepidermal T cells are strongly positive for CD3 by immunohistochemistry.
234 CHAPTER 2  •  Hematopoietic System
immunophenotype characterized also by expression of CD8
in most cases or being negative for CD4 and CD8, but in
contrast to canine MF, all cases of PR are TCRγδ positive.
Subcutaneous “panniculitis-like” T-cell lymphoma is rarely
seen in animals and has only been recognized in the dog (Fig.
2-194). Most cases take an indolent course, and removal seems
A ing from the epithelial basement membrane and irregularly invad-
C
Figure 2-194  Subcutaneous “panniculitis-like” T-cell lymphoma
in a dog. A. Neoplastic T cells infiltrate the panniculus in a lace-
like pattern often rimming large vacuolated adipocytes. B. Karyor-
rhexis and heavy infiltration with histiocytic cells are prominent
features. C. Neoplastic T cells rimming adipocytes are strongly
positive for CD3 by immunohistochemistry.
Lymph Nodes
to be curative. Lesions usually occur as multiple nodules on
the trunk or limbs that are localized in the panniculus and
almost completely spare the dermis. Histologically, neoplastic
T cells infiltrate the panniculus in a lace-like pattern, often
rimming large vacuolated adipocytes. Neoplastic T cells are
usually small- to intermediate-sized cells, but large cells may
dominate. Karyorrhexis is a prominent feature, and some cases
have extensive necrosis. This often leads to heavy infiltration
with histiocytic cells, and there is prominent phagocytic activ-
ity. The histiocytic infiltrates may mask the lesion as a histio-
cytic panniculitis, and IHC and clonality testing are helpful in
reaching an accurate diagnosis. Neoplastic cells are CD3 and
CD8 positive. A CD4-negative cytotoxic phenotype has been
described in humans, but has not been investigated in dogs.
Cutaneous anaplastic large T-cell lymphoma has been
described in middle-aged to older dogs as a highly aggressive
form of non-epitheliotropic CTCL with a poor prognosis (Fig.
2-195). Originally, lesions occur as single or multiple nodules
in the skin that progress remarkably rapidly and cause general-
ized lymphadenopathy. A disseminated form has also been
observed, and the nodal form has been described previously.
The neoplasm occurs as a solid mass of neoplastic T cells extend-
ing the subcutaneous fat. The neoplastic cells grow in solid
B
Figure 2-195  Cutaneous anaplastic large T-cell lymphoma in a
dog. A. Neoplastic T cells have large, often bizarre nuclei, and
abundant amounts of cytoplasm. B. Neoplastic T cells are strongly
positive for CD3 by immunohistochemistry.
 Lymph Nodes
sheets displacing hair follicles and collagen fibers. In deeper
areas, neoplastic T cells surround vessels and infiltrate between
fat cells, forming solid areas of tumor. The morphologic and
immunohistochemical features of the cutaneous form are
identical to the earlier described nodal form. Histiocytic sarco-
mas are the primary differential, and IHC is required for an
accurate diagnosis.
T-cell large granular lymphocytic leukemia.  Large granu-
lar lymphocytic lymphomas (LGL) have already been
described as a special form of EATL type 1, but they can also
occur as a leukemia in dogs. For a detailed description of the
leukemic disease, please see the paragraph on LGL leukemias.
For intestinal LGLs, please see description of EATL type 1.
Viral etiology and lymphomagenesis.  The etiology of most
lymphomas in domestic animal species is unknown, and there
are limited genetic studies. However, feline and bovine retro-
viruses have been shown to cause different types of lympho-
mas in cats and cattle, respectively.
Feline lymphoma may be caused by the horizontally trans-
mitted species Feline leukemia virus (FeLV), genus Gamma-
retrovirus in the oncovirus subfamily of family Retroviridae.
The virus has several subgroups of which FeLV-A, FeLV-B,
and FeLV-C are the most important. Only FeLV-A is conta-
gious and spreads horizontally among cats. The other sub-
groups result from recombination or mutation of FeLV-A in
infected cats and can only replicate under natural conditions
with the help of FeLV-A. The pathogenicity is higher for
multiple subgroup infections, and the envelop proteins of the
different subgroups determine lesion development. FeLV-B
is primarily responsible for development of thymic lympho-
mas in cats.
Neoplastic transformation is caused by insertional mutagen-
esis. Although some retroviruses insert a viral oncogene into
the host genome, causing rapid neoplastic transformation,
FeLV is a slowly transforming oncovirus. When FeLV becomes
integrated near a cellular proto-oncogene, transcription of this
gene can be upregulated by the promoter and enhancer func-
tion of viral long-term repeats (LTR). In cats, insertion occurs
most commonly near c-myc, which leads to uncontrolled
clonal proliferation of the affected cell without inducing an
immune response. Because insertion is random at one of hun-
dreds of proviral common integration sites, not all cats will
develop lymphoma, and neoplastic transformation may only
occur after long-standing infection. In addition to c-myc, other
common insertion loci associated with oncogenesis include
flvi-1, flvi-2 (contains bmi-1), fit-1, pim-1, and flit-1. Although
most insertion sites have been associated with formation of
thymic T-cell lymphoblastic lymphomas, insertion at flvi-1
results in B-cell lymphomas. Although c-myc is an oncogene,
bmi-1, and pim-1 are c-myc collaborators, and fit-1 has been
closely linked to myb. Flit-1 is associated with overexpression
of activin-A receptor type II–like 1 (ACVRL1). Flvi-2 contains
a gene that encodes feline homologs to bmi-1 and pmi-1. A
feline oncovirus cell membrane antigen (FOCMA) has been
detected on the surface of many transformed neoplastic cells,
and many FeLV infected cats will develop antibodies against
FOCMA. The role of FOCMA in protective immunity and its
function are still largely unknown.
Species Bovine leukemia virus (BLV) is an oncogenic del-
taretrovirus. Similar to cats, BLV is a slowly transforming
retrovirus, but does not causes bovine lymphomas through
insertional mutagenesis. It lacks a known oncogene and does
not integrate into preferred sites in the bovine genome. In
235
contrast to simple retroviruses, deltaretroviruses carry addi-
tional genes that encode for several regulatory and accessory
proteins. One of these proteins, Tax, plays a major role in BLV
lymphomagenesis. Tax is an activator of viral gene transcrip-
tion from the LTR and modulates the expression of several
cellular genes. It also induces immortalization of infected cells.
This may be the first step in the BLV-mediated neoplastic
transformation. In contrast to cats, in which FeLV virus pri-
marily causes thymic T-cell lymphomas, BLV infects B lym-
phocytes that express surface immunoglobulin M and causes
B-cell lymphomas. IgG heavy chains are frequently found on
neoplastic mature B cells. Tax immortalizes CD5-positive
IgM-positive B cells and causes their polyclonal expansion.
Missense mutations at codons 206, 207, 241, or 242 of p53
have been identified in more than 50% of BLV-induced lym-
phomas, and suppression of p53 most likely represents the
crucial step in neoplastic transformation in many cases.
However, other genes are most likely involved. Bovine leuko-
cyte antigen (BoLA) significantly contributes to the progres-
sion of BLV infection and the host’s immune response.
Polymorphisms of BoLA influence resistance and susceptibil-
ity to a wide variety of infectious diseases, including BLV, and
the monoclonal expansion of BLV-infected B cells is associated
with specific alleles of BoLA. Furthermore, a polymorphism
in the promoter region of the tumor necrosis factor-α gene
has been associated with the development of bovine
lymphoma.
Species differences.  The general pathology and classifica-
tion of lymphomas are discussed in the preceding sections.
The following sections deal with the clinical and topographic
aspects of lymphoma in the various domestic animal species.
Bovine lymphoma.  In cattle, bovine leukemia virus (BLV)-
associated lymphomas are predominantly diffuse large B-cell
lymphomas. Sporadic cases of precursor T-cell lymphoblastic
lymphomas occur multicentrically or in the thymus, and epi-
theliotropic T-cell lymphomas occur in the skin.
Lymphoma in adult cattle is mainly the enzootic bovine
leukosis form caused by an infection with BLV. The viral onco-
genesis has been described earlier. Because almost any organ
system may be involved with leukemia-associated lymphoma,
it is usually referred to as multicentric. BLV is transmitted
horizontally by direct contact. Small numbers of BLV-infected
lymphocytes can be transmitted through natural breeding,
blood-sucking arthropods, or iatrogenic by contaminated
needles, ear-tagging, and dehorning equipment, and the use of
whole-blood vaccines. Disease is much less common in beef
compared to dairy cattle, where husbandry practices favor
transmission, but shorter life-span may also contribute to a
lower incidence. BLV does not persist outside the animal, so
contact or environmental contamination is not a source of
infection. Infection, once established, is lifelong and character-
ized by the development of circulating antibodies, which are
also present throughout life. Antibody levels tend to increase
with the number of viral antigens recognized, as the animal
passes from an asymptomatic carrier stage to lymphocytosis
and to the tumorous state. Almost all infected cows will
develop detectable serum antiviral antibodies ~5 weeks after
infection. Although lymphocytosis develops in ~30% of
infected cows, lymphoma occurs in <5%. Cows develop neo-
plastic masses usually between 4 to 8 years of age. The low
incidence of neoplastic disease, and even lymphocytosis, has
hindered eradication of the disease. With the development of
an agarose gel immunodiffusion test that can detect antibodies
236 CHAPTER 2  •  Hematopoietic System Lymph Nodes

Figure 2-196  Enzootic bovine leukosis in a cow. Malignant lym- Figure 2-197  Enzootic bovine leukosis in a cow. Diffuse thicken-
phoma in the wall of the uterus. ing of the abomasal submucosa by an infiltrating malignant
lymphoma.

against BLV antigen and culling of infected animals, herds can

be maintained free of BLV.
BLV-associated lymphoma most commonly appears as
multiple enlarged lymph nodes that may appear anywhere in
the body or can be identified by rectal palpation. Enlarged
lymph nodes in the retrobulbar area may cause unilateral or
bilateral proptosis, and lymphadenopathy in the pharyngeal
area can result in dysphagia and stertorous respiration. Other
commonly affected organs include the abomasum, heart, spinal
cord, kidney, and uterus (Fig. 2-196, eFig. 2-46). Abomasal
lymphoma is characterized by diffuse thickening of the
abomasal submucosa with eventual ulceration leading to hem-
orrhagic anemia (Fig. 2-197, eFig. 2-47). Cattle with abomasal
lymphoma may also have vagal indigestion or diarrhea. There
is irregular involvement of the mesenteric nodes associated
with abomasal lymphoma, and these may, at times, be large
enough to cause obstruction. Lymphoma in the myocardium
may cause congestive heart failure (Fig. 2-198, eFig. 2-48).
Hindlimb lameness, which progresses quickly to weakness and Figure 2-198  Enzootic bovine leukosis in a cow. Malignant lym-
paraplegia, reflects the spread of neoplastic cells from sublum- phoma protruding as multinodular masses from the myocardium.
bar nodes to the spinal canal, where there is a relatively diffuse
infiltration into the perineural fat, leading to compression of
the lumbar spinal cord or spinal nerves. Grossly, the perineural
infiltration has only a slight fleshy pinkness to distinguish it
from normal fat, although the distinction is readily made by
an imprint of the affected tissues (Fig. 2-199, eFig. 2-49).
Occasionally, the kidneys are the primary site of gross mani-
festation of lymphoma. Liver and spleen are less commonly
affected, but some cows die rapidly because of splenomegaly
with rupture and abdominal hemorrhage.
The sporadic forms of bovine lymphoma are not associated
with BLV infection. The juvenile multicentric form most com-
monly occurs in calves between 3-6 months of age, but may
be present at birth. It is characterized by symmetrical lymph-
adenopathy and leukemia with leukocyte counts varying from
10-100 × 109/L. Affected animals often have nonresponsive,
normochromic normocytic anemia and thrombocytopenia.
Terminally, there is virtually complete myelophthisis and
marrow infarction with neutropenia and thrombocytopenia,
but without hemorrhage. At autopsy, virtually all nodes are Figure 2-199  Enzootic bovine leukosis in a cow. Malignant lym-
enlarged up to 10 times normal size, and the kidneys are phoma infiltrating the perineural fat, leading to compression of
usually diffusely involved but may not be markedly enlarged. spinal nerves and eventually the lumbar spinal cord.
 236.e1

A A

B B
eFigure 2-46  Enzootic bovine leukosis in a cow. A. Malignant
lymphoma in the wall of the uterus. B. Multifocal infiltrates of
malignant lymphoma throughout the liver. (Courtesy K.G.
Thompson.)

C
eFigure 2-47  Enzootic bovine leukosis in a cow. A. Diffuse thick-
ening of the abomasal wall by malignant lymphoma. B. The
kidneys have multifocal infiltrates of neoplastic lymphocytes
extending through the whole thickness of the cortex. C. Cross
section of spleen with multiple white nodules consistent with
malignant lymphoma.
236.e2

eFigure 2-48  Enzootic bovine leukosis in a cow. Malignant lym- eFigure 2-49  Enzootic bovine leukosis in a cow. Malignant ret-
phoma in the myocardium extending into papillary muscles. robulbar lymphoma. (Courtesy D.G. Sledge.)
 Lymph Nodes
Figure 2-200  Cutaneous lymphoma in a cow. Plaque-like, round,
raised lesions in the skin. (Courtesy K.G. Thompson.)
The liver and spleen may be massively involved, or relatively
spared. Calves may be born with lymphoma with the liver so
enlarged at parturition as to cause dystocia.
Thymic lymphoma occurs in cattle <2 years of age. Clini-
cally, it commonly causes presternal swelling, jugular disten-
tion, and local edema, as well as compression of the esophagus
and respiratory distress owing to displacements of the lungs
by a very large mediastinal mass. Often large areas of the
tumor may be infarcted.
The skin form is the least common type of bovine lym-
phoma, and occurs most often in 2- to 3-year-old cattle (Fig.
2-200). It is characterized by plaque-like, round, raised lesions
that appear on the head, sides, and perineum (eFig. 2-50). The
lesions become depilated, and are frequently ulcerated. The
lesions wax and wane over a period of months with some
regressing entirely and new lesions appearing. Ultimately,
there is deep organ involvement, and the visceral lesions are
then grossly indistinguishable from those of the enzootic type
of bovine lymphoma. This form of mycosis fungoides often
occurs in the advanced state with leukemia (Sézary
syndrome).
Ovine and caprine lymphoma.  Ovine lymphomas most
commonly occur as multicentric disease without leukemia and
bone marrow involvement (Fig. 2-201). A retrovirus indigenous
to sheep shares major proteins with BLV; however, most spon-
taneous lymphomas of sheep, and virtually all of those pro-
duced experimentally, are caused by BLV. Experimentally
infection with BLV causes lymphoma in 30-60% of sheep 2-3
years after infection. In sheep, as in cattle, infection with BLV
once established is lifelong, and the virus persists in the face
of a strong and persistent antibody response that can be
detected with the bovine agarose gel immunodiffusion test.
BLV produces a moderate persistent lymphocytosis in 50% of
sheep at 10-13 months postinfection, and causes neoplastic
masses in ~40% of sheep within 6 years postinfection.
In goats, sporadic forms of lymphoma occur more fre-
quently, and multicentric disease is much less common com-
pared to sheep. Disease presentation can be highly variable,
and lymphoma should be considered as a differential in any
goat >2 years of age with a fast progressing debilitating disease.
The gross and microscopic presentation of BLV-associated
lymphoma in sheep and goats is very similar to that described
237
Figure 2-201  Renal lymphoma in a sheep. The cortex is diffusely
infiltrated by neoplastic lymphocytes.
in cattle. Generalized lymphadenopathy is most commonly
encountered in sheep, and exophthalmos secondary to retro-
bulbar lymphadenopathy has especially been reported in
goats. Lymphoma can also be commonly found in the aboma-
sum, heart, and uterus. Small neoplastic masses are also not
uncommon in the spleen, whereas kidney and liver are rarely
involved. Thymic and cutaneous lymphoma also occur in
sheep and goats, and in goats, thymic lymphoma has to be
differentiated from thymoma because goats have a high preva-
lence of thymoma B1 as previously described. Furthermore,
lymphomas of the central nervous system (CNS) and synovial
lymphomas have been described in goats.
Equine lymphoma.  Lymphoma in the horse occurs primarily
as a subentity of diffuse large B-cell lymphoma, the T-cell–rich
large B-cell lymphoma (TCRLBCL). Peripheral T-cell lym-
phoma and other diffuse large B-cell lymphomas are also
commonly encountered. Lymphomas of the large intestine are
most commonly enteropathy-associated T-cell lymphomas of
small cell type (EATL type 2) and cutaneous lymphomas are
epitheliotropic T-cell lymphomas. Individual cases of other
entities have been reported. The cases reviewed covered 24
breeds, with Thoroughbreds the most prevalent, and the ages
ranged from 2 months to 31 years.
Equine lymphomas do not label well with CD79a, and B
cells in the horse are more efficiently marked with CD20.
Numerous studies used Bla.36 to label horse B cells, but the
antibody has been shown to also label macrophages and den-
dritic cells. The large number of T cells in TCRLBCL and
difficulties in detecting neoplastic B cells in the horse may
have led to misdiagnosis of some cases of multicentric lym-
phoma. Similarly, the large number of Bla.36-positive non-
lymphoid cells in sections of large intestine with EATL type
2 and the small size of neoplastic T cells and their close resem-
blance to inflammatory T cells may have caused an over-
reporting of intestinal B cell lymphomas in the older
literature.
Lymphoma is the most common malignant neoplasm in the
horse. Regardless of the anatomic location, lymphomas in
horses cause a general wasting disease with anorexia and
depression and ventral edema with or without reported hypo-
proteinemia. Pyrexia and anemia are common, and diarrhea
and colic can occur with any form of lymphoma. Multicentric
 237.e1

eFigure 2-50  Cutaneous lymphoma in a cow. Plaque-like, round,

raised lesions in the skin. (Courtesy K.G. Thompson.)
238 CHAPTER 2  •  Hematopoietic System
A
B
Figure 2-202  Abdominal lymphoma in a horse. A. Large, multi-
nodular intestinal masses. B. Neoplastic masses are white, firm,
and homogeneous on cross section.
lymphoma is the most common anatomic form, and the
vast majority of cases are TCRLBCL. In contrast to dogs,
multicentric lymphoma in the horse more commonly occurs
as masses in the abdominal (Fig. 2-202, eFig. 2-51) and tho-
racic cavity, as well as multiple subcutaneous, non-ulcerative
nodules on the trunk or limbs, rather than generalized lymph-
adenopathy. Skin nodules may be very numerous, and the skin
is mobile over these lesions that may wax and wane and
eventually become systemic. Other organs affected include
the pancreas, thyroid gland, spinal cord, and mediastinal
nodes, as well as the liver, spleen, and the gastrointestinal tract.
Although gastrointestinal TCRLBCL occurs as large intestinal
masses, EATL type 2 causes uniform thickening of the intes-
tinal mucosa and affects primarily the large intestine (Fig.
2-203). Rectal biopsies combined with immunohistochemis-
try (IHC) have been shown to be helpful in differentiating
EATL type 2 from lymphoplasmacytic proctitis antemortem.
Subcutaneous nodules are most commonly TCRLBCL, but
epitheliotropic T-cell lymphomas also occur in horses, and
some cases progress to Sézary syndrome.
There is some breed disposition regarding the type of lym-
phoma. Quarter Horses and Thoroughbreds most commonly
develop multicentric lymphomas, followed by cutaneous lym-
phomas; gastrointestinal lymphomas only represent up to 15%
of lymphomas. In contrast, Standardbreds have a higher
Lymph Nodes
Figure 2-203  Intestinal lymphoma in a horse. Multiple white
raised nodules protrude over a uniformly thickened intestinal
mucosa.
Figure 2-204  Nodal lymphoma in a dog. Generalized nodal lym-
phoma is the most common form of malignant lymphoma in dogs.
incidence of gastrointestinal lymphomas and rarely develop
cutaneous lymphoma. EATL type 2 is also much more com-
monly observed in Standardbreds compared to TCRLBCL in
Quarter Horses or Thoroughbreds.
Canine lymphoma.  Lymphomas are the most common
hematopoietic neoplasm in dogs with a reported incidence of
~0.025%. Lymphoma occurs primarily in middle-aged to
older dogs, and large-breed dogs are much more likely to
develop lymphomas. There is no sex predisposition. Breeds
most commonly affected include Golden Retrievers, Labrador
Retrievers, Boxers, Terriers, Cocker Spaniels, Beagles, and
Shih-Tzus.
Generalized nodal lymphoma is the by far the most common
clinical presentation, accounting for ~84% of all canine lym-
phomas (Fig. 2-204, eFig. 2-52). In dogs, the 3 most common
nodal lymphomas are the diffuse large B-cell lymphoma (~50-
60%), the peripheral T-cell lymphoma (~15-20%), and the
T-zone lymphoma (~10%). Other entities, such as lympho-
blastic T- or B-cell lymphomas, follicular B-cell lymphomas,
or marginal zone lymphomas are less common. Most nodal
 238.e1

B
eFigure 2-51  Malignant lymphoma in a horse. A. Mesenteric
lymph node and mesentery are diffusely infiltrated by a homoge-
neous neoplastic mass. B. The malignant lymphoma in the spleen
is white, firm, and has central areas of ischemic necrosis.
238.e2

eFigure 2-52  Nodal lymphoma in a dog. Cross section of lymph

node with diffuse, white homogeneous neoplastic tissue replacing
normal parenchyma.
 Lymph Nodes
lymphomas are clinically silent during the early stages of
disease.
Intestinal lymphomas are not as common in dogs as in cats,
and account for ~6% of all lymphomas. They are most com-
monly enteropathy-associated T-cell lymphomas of the large
cell type (type 1). Clinical signs include weight loss and leth-
argy, vomiting, and diarrhea, and blood may be present in
vomitus or stool. Boxers and Shar Peis are over-represented.
Cutaneous lymphomas account for ~6% of canine lympho-
mas, and are most commonly epitheliotropic T-cell lympho-
mas (mycosis fungoides). The disease is usually seen in dogs
>10 years of age, but the clinical presentation can vary from
single to diffuse disease and from flat plaques to cutaneous
nodules (Fig. 2-205, eFig. 2-53). Half of the affected dogs will
have pruritus.
Mediastinal and extranodal lymphomas are the least
common canine lymphomas, accounting together for ~4% of
canine lymphomas. Mediastinal lymphomas are most com-
monly lymphoblastic T-cell lymphomas arising from the
thymus, and clinically cause respiratory signs and effusions as
well as precaval syndrome. Polyuria and polydipsia occur in
~40% of dogs because of paraneoplastic hypercalcemia of
malignancy. In the spleen, marginal zone lymphomas, and
rarely mantle cell lymphomas, can be encountered,
A
B
Figure 2-205  Cutaneous lymphoma in a dog. (Courtesy M.
Umstead.) A. Erythematous, exfoliative dermatitis of the foot-
pads. B. Similar lesions in the nasal planum with mucocutaneous
depigmentation.
239
representing specific types of follicular lymphomas (Fig.
2-206). Recently, hepatosplenic and hepatocytotropic types
have been characterized. Other primary locations include the
eye (Fig. 2-207, eFig. 2-54), the spinal cord (Fig. 2-208), or
the kidney (Fig. 2-209, eFig. 2-55), and clinical signs depend
on the organ system affected.
Feline lymphoma.  Lymphomas are one of the most common
malignant neoplasms in cats. Historically, ~30% of feline neo-
plasms were lymphomas, and an incidence of 0.2% was
reported. Up to 80% of lymphomas were reported to be asso-
ciated with species Feline leukemia virus (FeLV). However,
there has been a dramatic decrease of the prevalence of
FeLV in cats after cats became routinely vaccinated against
FeLV, and testing and elimination programs have been estab-
lished. The majority of FeLV-associated lymphomas were lym-
phoblastic T-cell lymphomas occurring in the thymus/
mediastinum, especially in cats <3 years of age. FeLV does not
represent a single genomic species, but a family of closely
related viruses, and the genetic variation among these viruses
causes a highly variable disease outcome, including thymic
Figure 2-206  Splenic lymphoma in a dog. Multiple nodules in
the spleen.
Figure 2-207  Ocular lymphoma in a dog. Malignant lymphoma
arising from and largely obliterating the anterior uveal tract and
filling the anterior and posterior chambers. (Courtesy D.G.
Sledge.)
 239.e1

A
eFigure 2-54  Ocular lymphoma in a dog. Malignant lymphoma
arising from and largely obliterating the anterior uveal tract and
filling the anterior and posterior chambers. (Courtesy D.G.
Sledge.)

C
eFigure 2-53  Cutaneous lymphoma in a dog. A. Multinodular
white, homogeneous masses along the lip fold margin. (Courtesy
M. Umstead.) B. Erythematous, exfoliative dermatitis suggestive
of mycosis fungoides. (Courtesy K. Loft.) C. Similar lesions on
the nasal planum and periocular area with mucocutaneous depig-
mentation. (Courtesy M. Umstead.)
239.e2

C
eFigure 2-55  Malignant lymphoma in a dog. A. Multiple white
nodules randomly distributed throughout the myocardium.
(Courtesy S. Kesdangsakonwut). B. Malignant lymphoma in the
urinary bladder occurring as numerous ulcerated intraluminal
nodules. C. Disseminated malignant lymphoma in liver, spleen,
lymph nodes, and kidney.
240 CHAPTER 2  •  Hematopoietic System
Figure 2-208  Spinal cord lymphoma in a dog. Malignant lym-
phoma encompassing the nerve roots of the caudal spinal cord.
Figure 2-209  Renal lymphoma in a dog. A single sharply demar-
cated, white, firm homogeneous mass protrudes from the renal
cortex.
lymphoma of T-cell origin, non–T-cell multicentric lymphoma,
myeloproliferative disorder, or anemia. In one study, a particu-
lar isolate of FeLV caused exclusively multicentric B-cell lym-
phoma affecting liver, kidney, and lungs, but not thymus.
About 15% of cats infected with FeLV are also infected
with feline immunodeficiency virus (FIV); however, the latter
agent is not nearly as infectious as FeLV or as likely to cause
disease. FIV plays an indirect role in lymphomagenesis and
increases the risk of cats to develop lymphoma. FIV-associated
lymphomas are typically of B-cell phenotype and occur most
commonly in the intestine, but the overall incidence of FIV-
associated lymphoma is low.
Up to 90% of cats with thymic/mediastinal lymphomas were
found to be FeLV antigen positive (Fig. 2-210). Most cats with
thymic lymphoma develop pleural effusions, and clinical signs
include dyspnea and regurgitation because of pressure of the
neoplasm on lung and esophagus, respectively. Horner syn-
drome is caused by pressure on sympathetic nerves.
Since the 1990s, the incidence of thymic lymphoma has
decreased, and the most common type of lymphoma in cats
is now intestinal lymphoma (Fig. 2-211, eFig. 2-56). Although
Lymph Nodes
Figure 2-210  Thymic lymphoma in a cat. Thymic lymphomas
expand the cranial mediastinum causing caudodorsal displace-
ment of the lungs.
Figure 2-211  Intestinal lymphoma in a cat. Diffuse circumferen-
tial thickening of the intestinal wall causing luminal obstruction.
(Courtesy J.S. Munday.)
the reported higher incidence of intestinal lymphoma may
partially result from the shift of the proportion of lymphomas
in cats because of a decrease of FeLV, it more likely reflects
our ability to better diagnose intestinal lymphomas in cats.
The majority of intestinal lymphomas in cats are enteropathy-
associated T-cell (EATL) lymphomas of small cell type (type
2) that histologically resemble inflammatory bowel disease
and often require IHC and clonality testing for an accurate
diagnosis. Historically, most intestinal lymphomas have been
reported as B-cell lymphomas. With the establishment of diag-
nostic algorithms to accurately diagnose EATL type 2 based
on molecular techniques even in endoscopic biopsies, it is now
certain that EATL type 2 is by far the most common form of
intestinal lymphoma in cats. Another intestinal lymphoma in
cats is EATL of large cell subtype (type 1), which, like EATL
type 2, occurs most frequently in the jejunum. EATL type 1
is often characterized by intracytoplasmic eosinophilic gran-
ules that express the cytotoxic granule protein, granzyme B,
consistent with a diagnosis of large granular lymphocyte
(LGL) lymphoma. In contrast, gastric lymphomas are most
commonly diffuse large B-cell lymphomas (Fig. 2-212,
 240.e1

B
eFigure 2-56  Intestinal lymphoma in a cat. A. Multifocal circum-
ferential thickening of the intestinal wall and enlargement of the
mesenteric lymph node. B. Thickening of the intestinal mucosa
with linear ulceration over Peyer’s patches.
 Lymph Nodes
Figure 2-212  Gastric lymphoma in a cat. Submucosal thickening
by a diffuse, white, homogeneous infiltrate.
Figure 2-213  Renal lymphoma in a cat. Numerous, sharply
demarcated, white, firm homogeneous masses distributed
throughout the renal cortex.
eFig. 2-57), and this type of lymphoma can also occur at the
ileo-ceco-colic junction or synchronous at both locations.
Multicentric lymphoma is another common type of lym-
phoma in cats, and historically, 70% of cases were positive for
FeLV. The vast majority of cases affect internal organs such as
kidney (Fig. 2-213, eFig. 2-58) or liver, and generalized lymph-
adenopathy is uncommon. Clinical signs vary depending on
the organ system affected. A specific type of nodal lymphoma,
Hodgkin-like lymphoma, is seen most commonly as unilateral
mandibular or cervical lymphadenopathy, but other lymph
nodes may also be the primary sites.
Lymphoma is the most common feline upper respiratory
tract neoplasm. Nasal and nasopharyngeal lymphomas can
occur individually at each location or synchronous at both
sites (Fig. 2-214). The majority of cases are diffuse large B-cell
lymphomas, but epitheliotropic T-cell lymphomas and small
lymphocytic B-cell lymphomas have also been observed.
Interestingly, epitheliotropism has also been described for
some B-cell lymphomas, and FeLV p27 or gp70 antigens have
been detected in ~50% of cases regardless of phenotype.
241
A
B
Figure 2-214  Nasal and nasopharyngeal lymphoma in a cat.
A. Expansile, white firm homogeneous mass that protrudes from
the frontal sinus causing destruction of bone. (Courtesy S. Kes-
dangsakonwut.) B. Plaque-like tonsillar lymphoma. (Courtesy
K.G. Thompson.)
Other extranodal lymphomas in cats include primarily lym-
phomas of the CNS, peripheral nerves (neurolymphomatosis),
cutaneous lymphomas (eFig. 2-59), and ocular lymphomas
(see eFig. 2-58), and account for ~20% of all feline lympho-
mas. None of these types of lymphoma has been associated
with FeLV or FIV infections. The clinical signs vary based on
the organ system affected, for instance, paraplegia with
neurolymphomatosis.
Porcine lymphoma.  Lymphoma is the most common neo-
plasm in pigs. There are no published data that apply the
WHO classification to a large series of porcine lymphomas.
All ages and both sexes are affected, but there is a higher
incidence in female pigs. The multicentric form is most com-
monly characterized by generalized lymphadenopathy affect-
ing visceral lymph nodes rather than peripheral lymph nodes.
Multicentric lymphoma may also primarily affect internal
organs, such as kidney (eFig. 2-60), spleen, and liver (Fig.
2-215). Leukemia occurs commonly at the terminal stage. The
thymic form most commonly occurs as lymphoblastic T-cell
lymphoma and is the most common form of lymphoma in
piglets as young as 3 weeks of age. An autosomal recessive
trait has been reported to have caused B-cell lymphomas in
25% of animals from herds of Large White pigs bearing this
 241.e1

A
eFigure 2-57  Gastric lymphoma in a cat. Cross section of severely
thickened gastric wall by a diffuse white homogeneous
infiltrate.

B
eFigure 2-58  Malignant lymphoma in a cat. A. Ocular lym-
phoma. (Courtesy D.G. Sledge.) B. Renal lymphoma character-
ized by diffuse infiltration of the cortex by neoplastic
lymphocytes.
241.e2

eFigure 2-59  Lymphocytosis in a cat. Solitary area of alopecia eFigure 2-60  Malignant lymphoma in a pig. White, raised nodules
with scaling, erythema, and ulceration. (Courtesy M. Umstead.) randomly distributed throughout the kidney.
242 CHAPTER 2  •  Hematopoietic System
A Durham AC, et al. Two hundred three cases of equine lymphoma clas-
B Mazoub M, et al. Histopathologic and immunophenotypic character-
C Ogihara K, et al. Lymphoid neoplasms in swine. J Vet Med Sci
Figure 2-215  Malignant lymphoma in a pig. A. White, raised
nodules randomly distributed throughout the liver. B. Similar
lesions throughout the renal cortex. C. A large multinodular mass
expands the gastric wall.
defect. Lymphomas were detectable at 2-3 months of age, and
the average survival time was 4-6 months. The disease was
characterized by generalized lymph node enlargement, sple-
nomegaly, hepatomegaly, and involvement of the mesenteric
lymph nodes, stomach, and intestines. There was late-stage
involvement of the bone marrow, and many pigs died as a
result of leukemia, and had widespread hemorrhages because
Lymph Nodes
of thrombocytopenia. Pigs with the inherited form of lym-
phoma have a heavy-chain gammopathy with appearance of
this fragment in the urine.
Further reading
Aida Y, et al. Mechanisms of pathogenesis induced by bovine leukemia
virus as a model for human T-cell leukemia virus. Front Microbiol
2013;4:328.
Carrasco V, et al. Distinguishing intestinal lymphoma from inflamma-
tory bowel disease in canine duodenal endoscopic biopsy samples.
Vet Pathol 2014 Dec 8. [Epub ahead of print].
Choi WW, et al. A new immunostain algorithm classifies diffuse large
B-cell lymphoma into molecular subtypes with high accuracy. Clin
Cancer Res 2009;15:5494-5502.
sified according to the World Health Organization (WHO) classifica-
tion criteria. Vet Pathol 2013;50:86-93.
Fournel-Fleury C, et al. Canine T-cell lymphomas: a morphological,
immunological, and clinical study of 46 new cases. Vet Pathol
2002;39:92-109.
Fujino Y, et al. Molecular pathogenesis of feline leukemia virus-induced
malignancies: insertional mutagenesis. Vet Immunol Immunopathol
2008;123:138-143.
Keller SM, et al. Hepatosplenic and hepatocytotrophic T-cell lymphoma:
two distinct types of T-cell lymphoma in dogs. Vet Pathol 2012;
50:281-290.
Kiupel M, et al. Diagnostic algorithm to differentiate lymphoma from
inflammation in feline small intestinal biopsy samples. Vet Pathol
2011;48:212-222.
Little L, et al. Nasal and nasopharyngeal lymphoma in cats: 50 cases
(1989-2005). Vet Pathol 2007;44:885-892.
ization of extramedullary plasmacytomas in nine cats. Vet Pathol
2003;40:249-253.
McDonough SP, et al. Clinicopathological and immunophenotypical
features of canine intravascular lymphoma (malignant angioendo-
theliomatosis). J Comp Pathol 2002;126:277-288.
Meyer J, et al. Clinical, laboratory, and histomorphologic features of
equine lymphoma. Vet Pathol 2006;43:914-924.
Moore PF, et al. Canine epitheliotropic cutaneous T-cell lymphoma: an
investigation of T-cell receptor immunophenotype, lesion topogra-
phy and molecular clonality. Vet Dermatol 2009;20:569-576.
Moore PF, et al. Feline gastrointestinal lymphoma: mucosal architec-
ture, and molecular clonality. Vet Pathol 2012;49:658-668.
Norsworthy GD, et al. Diagnosis of chronic small bowel disease in cats:
100 cases (2008-2012). J Am Vet Med Assoc 2013;243:
1455-1461.
2012;74:149-154.
Pohlman LM, et al. Immunologic and histologic classification of 50
cases of feline gastrointestinal lymphoma. Vet Pathol 2009;46:
259-268.
Rola-Łuszczak M, et al. Development of an improved real time PCR for
the detection of bovine leukaemia provirus nucleic acid and its use
in the clarification of inconclusive serological test results. J Virol
Methods 2013;189:258-264.
Santagostino SF, et al. Feline upper respiratory tract lymphoma: site,
cyto-histology, phenotype, FeLV expression, and prognosis. Vet
Pathol 2014;52:250-259.
Valli VE, et al. Classification of canine malignant lymphomas according
to the World Health Organization criteria. Vet Pathol 2012;48:
189-211.

Further reading
Buczinski S. Echocardiographic findings and clinical signs in dairy cows
with primary cardiac lymphoma: 7 cases (2007-2010). J Am Vet
Med Assoc 2012;241:1083-1087.
Djilali S, Parodi AL. The BLV-induced leukemia-lymphosarcoma complex
in sheep. Vet Immunol Immunopathol 1989;22:233-244.
Fend F. Classical Hodgkin’s lymphoma. In: Jaffe ES, et al., editors.
Hematopathology. Philadelphia: Saunders/Elsevier; 2011. p.
454-472.
Flatland B, et al. Large anaplastic spinal B-cell lymphoma in a cat. Vet
Clin Pathol 2008;37:389-396.
Fondevila D, et al. Primary central nervous system T-cell lymphoma in
a cat. Vet Pathol 1998;35:550-553.
Fry MM, et al. Hepatosplenic lymphoma in a dog. Vet Pathol
2003;40:556-562.
Gilbert S, et al. Clinical, morphological and immunohistochemical char-
acterization of cutaneous lymphocytosis in 23 cats. Vet Dermatol
2004;15:3-12.
Greenlee PG, et al. Lymphomas in dogs. A morphologic, immunologic
and clinical study. Cancer 1990;66:480-491.
Grindem CB, et al. Immunophenotypic comparison of blood and lymph
node from dogs with lymphoma. Vet Clin Pathol 1998;27:16-20.
Hadlow WJ. High prevalence of thymoma in the dairy goat report of
seventeen cases. Vet Pathol 1978;15:153-169.
Harris N, et al. The World Health Organization classification of neoplas-
tic diseases of the haematopoietic and lymphoid tissues: report of
the clinical advisory committee meeting—Airlie House, Virginia,
November 1997. J Clin Oncol 1999;17:3835-3849.
Hayashi M, et al. Histological classification of malignant lymphomas in
slaughtered swine. J Comp Pathol 1988;98:11-21.
Hejazi R, Danyluk AJ. Two cases of multicentric lymphosarcoma in
swine: gross and histopathological findings. Can Vet J
2005;46:179-180.
Henson KL, et al. Immunohistochemical characterization of estrogen
and progesterone receptors in lymphoma of horses. Vet Clin Pathol
2000;29:40-46.
Higgins MA, et al. B-cell lymphoma in the peripheral nerves of a cat.
Vet Pathol 2008;45:54-57.
Holmberg CA, et al. Canine malignant lymphomas: comparison of
morphologic and immunologic parameters. J Natl Cancer Inst
1976;59:125-135.
Kelley LC, Mahaffey EA. Equine malignant lymphomas: morphologic
and immunohistochemical classification. Vet Pathol 1998;5:
241-252.
Kiupel M, et al. Prognostic factors for the treatment of canine malig-
nant lymphoma. Vet Pathol 1999;36:292-300.
Lennert K, et al. Concordance of the Kiel and Lukes-Collins classifica-
tion of non-Hodgkin’s lymphomas. Histopathol 1983;7:549-559.
Lennert K, Feller AC. Histopathologie der non-Hodgkin-lymphome.
2nd ed. Berlin: Springer-Verlag; 1990.
Lennert K, Mohri N. Malignant Lymphomas Other Than Hodgkin’s
Disease: Histopathology and Diagnosis of Non-Hodgkin’s Lympho-
mas. New York: Springer-Verlag; 1978. p. 111.
Lieberman PH, et al. Evaluation of malignant lymphomas using three
different classifications and the Working Formulation. 482 cases
with median follow-up of 11.9 years. Am J Med
1986;81:365-379.
Lukes RJ, Collins RD. Immunologic characterization of human malig-
nant lymphomas. Cancer 1974;34:1488-1503.
McDonough SP, Moore PF. Clinical, hematologic, and immunologic
characterization of canine large granular lymphocytosis. Vet Pathol
2000;37:637-646.
242.e1
National Cancer Institute. National Cancer Institute sponsored study
of classifications of non-Hodgkin’s lymphomas: summary and
description of a working formulation for clinical usage. The Non-
Hodgkin’s Lymphoma Pathologic Classification Project. Cancer
1982;49:2112-2135.
Neta M, et al. Perforin expression in feline epitheliotropic cutaneous
lymphoma. J Vet Diagn Invest 2008;20:831-835.
Park HM, et al. Pulmonary lymphomatoid granulomatosis in a dog:
evidence of immunophenotypic diversity and relationship to human
pulmonary lymphomatoid granulomatosis and pulmonary Hodg-
kin’s disease. Vet Pathol 2007;44:921-923.
Parodi A, et al. Histological classification of canine malignant lympho-
mas. J Vet Med A 1988;35:178-192.
Patnaik AK. Histologic and immunohistologicial studies of granular cell
tumors in seven dogs, three cats, one horse and one bird. Vet Pathol
1993;30:176-185.
Rafferty AL, et al. Acute lymphoblastic leukaemia in a 5-month-old
boar. N Z Vet J 2007;55:244-247.
Rappaport H. Tumours of the hematopoietic system. In: Rappaport H,
editor. Atlas of Tumour Pathology. Section 3, Fascicle 8. Washington
DC: Armed Forces Institute of Pathology; 1966.
Rebhun WC, Del Piero F. Ocular lesions in horses with lymphosarcoma:
21 cases (1977-1997). J Am Vet Med Assoc 1998;212:852-854.
Roccabianca P, et al. Hepatosplenic T-cell lymphoma in a mare. Vet
Pathol 2002;39:508-511.
Smith DA, Barker IK. Four cases of Hodgkin’s disease in striped skunks
(Mephitis mephitis). Vet Pathol 1983;20:223-229.
Stefanello D, et al. Splenic marginal zone lymphoma in 5 dogs (2001-
2008). J Vet Intern Med 2011;25:90-93.
Stein H, et al. Morphology and immunohistology of malignant lympho-
mas. In: Yohn DS, Blakeslee JR, editors. Advances in Comparative
Leukemia Research. New York: Elsevier North Holland; 1981. p.
479-485.
Teske E, et al. Prognostic factors in canine non-Hodgkin’s lymphoma:
a prospective study in 138 dogs. Proefschrift Universiteit Utrecht
1993;83-100.
Teske E, et al. Histological classification and immunophenotyping of
canine non-Hodgkin’s lymphoma. An unexpected high frequency
of T-cell lymphomas with B-cell morphology. Proefschrift Universiteit
Utrecht 1993;19-36.
Valli VE, et al. Canine indolent nodular lymphoma. Vet Pathol
2006;43:241-256.
Vernau W, et al. Classification of 1,198 cases of bovine lymphoma
using the National Cancer Institute Working Formulation for human
non-Hodgkin’s lymphomas. Vet Pathol 1992;29:183-195.
Vernau W, et al. The immunophenotypic characterization of bovine
lymphomas. Vet Pathol 1997;34:222-225.
Walton RM, Hendrick MJ. Feline Hodgkin’s-like lymphoma: 20 Cases
(1992-1999). Vet Pathol 2001;38:504-511.
 Histiocytic Proliferative Diseases
Valli VE, et al. Canine lymphomas: association of classification type,
disease stage, tumor subtype, mitotic rate, and treatment with
survival. Vet Pathol 2013;50:738-748.
HISTIOCYTIC PROLIFERATIVE DISEASES
Proliferative histiocytic diseases in domestic animals occur
largely in dogs and to a lesser extent in cats. They are likely
present in other species, but have not been adequately
described. It is important to recognize that usage of the term
“histiocyte” has changed over time and that the term “histiocytic
diseases” now encompass all proliferative disease processes of
dendritic cells or macrophages. Gross or histopathology is often
insufficient to properly diagnose these disease processes, and
a basic understanding of the cell biology of histiocytes is
required to use immunohistochemistry (IHC) as a means to
reach an accurate diagnosis.
Histiocytes are derived from hematopoietic CD34+ stem cells
and differentiate into macrophage, nonlymphoid dendritic cell,
and lymphoid dendritic cell lineages. Nonlymphoid dendritic
cells include interstitial and epithelial (Langerhans) dendritic
cells, whereas lymphoid dendritic cells of the T-cell domains
in lymphoid organs, such as spleen or lymph nodes, are called
interdigitating dendritic cells. Langerhans cells initially reside in
the epithelia of the skin, digestive, respiratory, and reproduc-
tive tract as intraepithelial dendritic antigen-presenting cells.
Interstitial dendritic cells can be found in most organs around
vessels, with the exception of the brain. The dendritic cells in
lymph nodes comprise both resident interdigitating dendritic
cells as well as migratory epithelial and interstitial dendritic
cells that have been drained via the lymphatics from their
tributary area. All dendritic cells are part of the adaptive
immune response and act as potent antigen-presenting cells.
By presenting antigens to naive T-cells in the paracortex of
lymph nodes, dendritic cells initiate an immune response.
Most histiocytic diseases in domestic animals originate from
interstitial dendritic cells or Langerhans cells. Only hemophago-
cytic histiocytic sarcomas in dogs have been demonstrated to
originate from macrophages, in particular red pulp macro-
phages from the spleen. The characterization of histiocytic
cells in formalin-fixed tissues is limited by the availability of
species-specific antibodies that will detect the appropriate
epitopes. A number of such antibodies have been developed
for canine and feline tissues and can be used in routinely
formalin-fixed tissues, whereas others are currently only avail-
able for use in frozen sections. Antibodies that can be used to
characterize histiocytic proliferative diseases and function in
formalin-fixed tissues include CD18, CD11d, CD204, CD90,
major histocompatibility complex II (MHC II), and E-cadherin.
Additional antibodies of importance that only work in frozen
sections include CD1a, CD11c (dog only), and CD4. All
dendritic cells express CD1a that, together with MHC I and
MHC II molecules, presents antigens to T cells. Another mol-
ecule that is useful in the diagnosis of histiocytic proliferative
diseases is CD18. CD18 is part of the heterodimeric β-2
integrin adhesion molecule that is composed of 1 of 4 differ-
ent CD11 (a-d) subunits and the CD18 subunit. Therefore all
histiocytic cells express CD18. CD11c is found in all epithe-
lial and interstitial dendritic cells; CD11d can be detected in
macrophages. Macrophage scavenger receptor 1 (CD204) is
expressed on macrophages and interstitial dendritic cells,
where it mediates endocytosis, but not in epithelial or
243
interdigitating dendritic cells or lymphocytes. Furthermore,
epithelial dendritic cells and interstitial dendritic cells can be
differentiated by the expression of CD90 (Thy-1) in intersti-
tial dendritic cells and E-cadherin in epithelial dendritic cells.
Activated dendritic cells express CD4.
Numerous well-defined histiocytic proliferative diseases
have been characterized in dogs and cats. Canine cutaneous
histiocytomas and canine Langerhans cell histiocytosis (LCH)
are both diseases of Langerhans cells with different biological
behavior. A pulmonary form of LCH has rarely been reported
in cats; reports of feline cutaneous histiocytomas are only
anecdotal. Diseases of interstitial dendritic cells include the
cutaneous or systemic forms of reactive histiocytosis, an
inflammatory disorder, and the various entities of histiocytic
sarcoma complex in dogs, as well as histiocytic sarcomas and
feline progressive histiocytosis in cats. Canine hemophago-
cytic histiocytic sarcoma is the only macrophage-derived entity,
and a feline counterpart has recently been suggested. A con-
genital histiocytosis of non–Langerhans cell origin has rarely
been reported in pigs, and the condition most closely resem-
bles juvenile xanthogranuloma in humans, a generally skin-
limited non-Langerhans histiocytic disorder.
A combination of gross presentation, histopathology, and
IHC is sufficient to accurately differentiate most canine his-
tiocytic diseases. In rare cases, additional frozen sections will
be required to confirm the diagnosis. A summary of the immu-
nohistochemical differences of the canine histiocytic diseases
can be found in Figure 2-216.
Canine cutaneous histiocytoma
Cutaneous histiocytoma is a benign common skin disease of
young dogs that usually appears as a single, focal, bulging mass
frequently on the head, ears, and limbs. Although most
common in dogs <3 years of age, histiocytomas can occur in
dogs of all ages. All breeds are affected, but there is a predis-
position in brachycephalic breeds, such as Boxers; Dachs-
hunds, Doberman Pinschers, and Cocker Spaniels are also
more commonly affected. The proliferation is of epidermal
Langerhans cells and usually ulcerates and regresses
spontaneously.
Grossly, canine cutaneous histiocytomas appear as round
1-2 cm dome-shaped skin protrusions that are usually hairless
and may be irritating to the dog, because they are frequently
abraded and ulcerated (Fig. 2-217, eFig. 2-61). Much less
frequently, the lesion may be a flat thickened plaque. If lesions
are left unattended, they usually involute spontaneously, with
complete healing in young dogs, but in older dogs, they are
more persistent. Surgical excision tends to be curative and
recurrence or development of additional masses is rare. Mul-
tiple histiocytomas are also uncommon, but Shar Peis are
more prone to develop multiple lesions. If dogs develop
numerous histiocytomas, such lesions should be better con-
sidered as a form of LCH (see later). Rare spread to lymph
nodes followed by regression has been reported in individual
cases, but may represent a Langerhans cell histiocytosis. Spon-
taneous regression is a major characteristic of canine cutaneous
histiocytomas, but the mechanisms are poorly understood.
Lysis of the histiocytic cells mediated by infiltrating lympho-
cytes, especially CD8+ lymphocytes, has been postulated as
the primary mechanism of regression. Some authors speculate
that tumor cells could facilitate their own destruction by
functioning as antigen-presenting cells, but infiltrating dermal
dendritic cells may also play a major role.
 243.e1

eFigure 2-61  Cutaneous histiocytoma in a dog. A single, pink,

raised, sharply demarcated nodule in the skin. (Courtesy R. Torres
Neto.)
244 CHAPTER 2  •  Hematopoietic System Histiocytic Proliferative Diseases

Histiocytic sarcoma

Cutaneous Hemophagocytic
histiocytoma histiocytic sarcoma

Reactive histiocytosis

Langerhans
histiocytosis

Langerhans DC Interstitial DC Macrophage

CD18 CD204 CD18 CD204 CD18 CD204
CD11c CD11d CD11c CD11d CD11c CD11d
E-cad Thy-1 E-cad E-cad Thy-1
Thy-1, CD4 (CHSH)

Figure 2-216  Classification of canine histiocytic diseases based on cell morphology and immu-
nohistochemical phenotype.

multinucleated cells can be occasionally seen. However,

Figure 2-217  Cutaneous histiocytoma in a dog. A single, pink,
raised, sharply demarcated nodule in the upper eyelid. (Courtesy
M. Coster.)
The histology of the canine cutaneous histiocytomas is very
characteristic and changes with the degree of regression. All
lesions are located in the superficial dermis and line up below
the basement membrane, giving the lesion its typical dome-
shaped appearance (Fig. 2-218). Histiocytic cells are densely
packed in the deep dermis and are more loosely arranged and
in rows near the epidermis (eFig. 2-62). The cells extend from
the dermoepidermal junction to the deep dermis and pannicu-
lus. The neoplasm surrounds dermal vessels, and displaces but
does not destroy adnexal structures. The proliferating cells
may penetrate the surface epithelium, and epitheliotropism is
observed in ~30% of cases. Especially in older dogs, such
tropism may raise concerns of a cutaneous epitheliotropic
T-cell lymphoma as the primary differential. Histiocytic cells
have a highly variable morphology. Nuclei tend to be vesicular
and are ~1.5-2.0 times the diameter of red cells. Round to
oval, reniform, or convoluted shapes are common, and even
marked anisokaryosis or megalokaryosis are not features of
cutaneous histiocytomas. The cytoplasm is relatively abun-
dant, lightly eosinophilic, and, unlike lymphocytes, is of the
same density from the nucleus to the cell margin. The cell
boundaries are generally distinct near the skin surface. The
mitotic index can be highly variable, but is commonly very
high. Although Birbeck granules are a characteristic ultrastruc-
tural feature of Langerhans cells in humans, cats, and many
other species, they have not been detected in canine cutaneous
histiocytomas. It has been speculated that cutaneous histiocy-
tomas in dogs arise from CD14+ dermal precursors of Lang-
erhans cells that invade the epidermis and lack Birbeck
granules.
As lesions regress, deep infiltration of small lymphocytes that
progresses throughout the nodule can be observed. The lympho-
cytes are predominantly CD3+, CD8+ cytotoxic T cells. Infil-
tration of T-cells is mirrored by necrosis of histiocytes. There
may also be mild neutrophilia if the lesion is allowed to prog-
ress to an ulcerated skin lesion. In late stages of regression, 
the massive lymphoid infiltrate may be mistaken as a non-
epitheliotropic lymphoma because only a few recognizable
histiocytes remain. Even analysis of clonality of the T-cell
receptor γ will not help in differentiating these 2 entities
because clonal expansion of the cytotoxic T cell has been
documented in 50% of canine cutaneous histiocytomas.
Because the resolution of the infiltrate is the result of the
action of the adaptive immune system, any treatment with
steroids is contraindicated. In both cytologic and histologic
preparations, the proliferating cells of cutaneous histiocytoma
are strongly positive for CD18. The deep infiltrating lympho-
cytes are strongly CD3 positive. Langerhans cells in canine
cutaneous histiocytomas express CD1a, CD11c, CD18, MHC
II, and E-cadherin, but are negative for CD90 and CD204. To
differentiate epitheliotropic T-cell lymphoma from cutaneous
histiocytoma, a panel of CD18, CD3, CD20, and E-cadherin
is most commonly used. It is important to recognize that
Langerhans cells express E-cadherin along the cell membrane,
and to use antibodies that detect the relevant E-cadherin.
 244.e1

B
eFigure 2-62  Cutaneous histiocytoma in a dog. A. Histiocytic
cells have highly variable morphology, and nuclei tend to be round
to oval, but reniform or convoluted shapes are also common.
B. Epitheliotropism occurs in 30% of cases.
 Histiocytic Proliferative Diseases 245

Dermal
vessels

Histiocytic
infiltrate
Subcutis
A B

C D
Figure 2-218  Cutaneous histiocytoma in a dog. A. Cutaneous histiocytomas are located in the
superficial dermis and extend to the basement membrane, resulting in a typical dome-shaped
appearance. B. Schematic drawing of the dermal location of cutaneous histiocytomas that surround
dermal vessels and cause epitheliotropism in 30% of cases. (Adapted from a graphic by P.F. Moore.)
C. Histiocytic cells are more loosely arranged and in rows near the epidermis. D. Immunohisto-
chemistry for E-cadherin labels both epidermal epithelial cells and neoplastic Langerhans cells.

Commonly, expression of E-cadherin is limited to histiocytes
in the superficial portion of the lesion just below the epider-
mis. It may be impossible to detect E-cadherin in severely
ulcerated and regressing tumors, and a panel of antibodies will
be required to accurately diagnose such lesions.
Canine cutaneous Langerhans cell histiocytosis
Although most histiocytomas occur as solitary lesions, some
dogs may develop multiple tumors over time. In dogs with
numerous cutaneous neoplasms that histologically and immu-
nohistochemically resemble cutaneous histiocytomas, a canine
cutaneous LCH should be considered as the primary differen-
tial (Fig. 2-219). LCH always starts as a cutaneous disease
process, but it can also extend to draining lymph nodes and
rarely involve other internal organs. The cutaneous and sys-
temic forms of LCH closely resemble their human counter-
parts. Affected dogs may have hundreds of cutaneous masses,
and secondary self-inflicted trauma commonly leads to severe
inflammation and ulceration. Shar Peis are over-represented,
but other breeds can be affected. The prognosis of LCH is less
favorable than for solitary cutaneous histiocytomas. Delayed
regression of the cutaneous lesions for up to 10 months
commonly leads to extensive ulceration, and euthanasia of
50% of affected dogs has been reported because of secondary
complications. Dogs with systemic spread of LCH have a poor
prognosis, and the reported mortality is 100%, including
euthanasia. Systemic LCH tends to spread to the lymph nodes
first, followed by the lungs, and variably other organs. A peri-
bronchial pattern is most commonly observed in the lungs
(Fig. 2-220).
Histologically, the individual cutaneous masses resemble
cutaneous histiocytomas. Although cutaneous lesions of LCH
tend to be larger and may extend into the subcutis and under-
lying muscle, the occurrence of multiple nodules and delayed
regression are the primary indicators of a LCH versus a cuta-
neous histiocytoma. Some cases have a higher degree of aniso-
karyosis, and multinucleated cells may be observed, but the
cellular morphology most closely resembles those of Langer-
hans cells in cutaneous histiocytomas rather than the highly
anaplastic features of neoplastic interstitial dendritic cells in
histiocytic sarcomas. Lymphatic invasion indicates lymphatic
spread and carries a worse prognosis.
Immunohistochemically, proliferating histiocytic cells in
LCH have features similar to Langerhans cells in cutaneous
246 CHAPTER 2  •  Hematopoietic System
A
B Feline pulmonary Langerhans cell histiocytosis
Figure 2-219  Langerhans histiocytosis in a dog. (Courtesy S.
Kesdangsakonwut.) A. Large numbers of cutaneous neoplasms
each individually resembling canine cutaneous histiocytoma.
B. Cross section of these nodules shows the sharp demarcation
and localization in the superficial dermis.
Figure 2-220  Langerhans cell histiocytosis in a dog. A peribron-
chial pattern is most commonly observed in the lungs.
Histiocytic Proliferative Diseases
Figure 2-221  Pulmonary Langerhans cell histiocytosis in a cat.
Nodular proliferations throughout the pulmonary parenchyma
that coalesce and extend to the pleural surface. (Courtesy F.
Taylor.)
histiocytomas. The cells are positive for CD18 and E-cadherin,
but negative for CD90 and CD204. As described earlier, his-
tiocytic cells in the deeper portion of the cutaneous masses
and those migrating to lymph nodes and internal organs may
lose expression of E-cadherin, and a combination of cell mor-
phology, positive labeling for CD18, but negative labeling for
lymphoid markers and CD204 and CD90 will help differenti-
ate LCH from reactive histiocytosis or histiocytic sarcoma.
In cats, only a pulmonary form of a LCH has been characterized;
cutaneous manifestations of Langerhans cell proliferations
have not been reported. Pulmonary LCH occurs primarily in
cats >10 years of age, and causes progressive respiratory disease
that is ultimately fatal. Clinical signs vary from acute respira-
tory distress, including tachypnea, labored breathing, and open
mouth-breathing, to prolonged chronic pulmonary disease.
Similar to the pulmonary lesions in canine LCH, histiocytic
proliferations affect first the peribronchial parenchyma,
causing nodular proliferations that vary from 2-5 mm in diam-
eter. As the disease progresses, nodules coalesce and lesions
extend to the pleural surface. In advanced cases, the whole
lungs tend to be affected, and all lobes appear diffusely firm
(Fig. 2-221). The tracheobronchial lymph nodes are com-
monly diffusely enlarged and white, firm, and homogeneous
on cut section. In some cats, nodular masses have also been
described in the pancreas and kidneys and their draining
lymph nodes.
Histologically, the nodular masses are centered around ter-
minal bronchioles. Proliferating histiocytes are similar to those
described in canine LCH; however, there is a higher degree of
cellular and nuclear pleomorphism. Aggregates of histiocytes
are usually cohesive, and proliferating cells extend from the
peribronchial parenchyma into the surrounding alveolar septa
and alveoli, eventually effacing the pulmonary parenchyma
(Fig. 2-222, eFig. 2-63). Ultrastructurally, Birbeck granules,
which represent the internalized cell surface receptor langerin
(CD207) cross-linked by an antibody, have been reported in
the cytoplasm of proliferating histiocytic cells, confirming a
Langerhans cell origin. This is in contrast to canine Langerhans
 246.e1

B
eFigure 2-63  Pulmonary Langerhans cell histiocytosis in a cat.
A. Histiocytic aggregates are cohesive, and proliferating cells oblit-
erate the lumen of a respiratory bronchiole and extend into the
surrounding alveoli and alveolar septa. B. Immunohistochemistry
for CD18 detects the infiltrating Langerhans cells.
 Histiocytic Proliferative Diseases
A supported by the clinical behavior of these lesions, which may
B Nodules range in size up to 4 cm in diameter, and solitary
Figure 2-222  Pulmonary Langerhans cell histiocytosis in a cat.
A. Histiocytic aggregates are cohesive, and proliferating cells oblit-
erate the lumen of a respiratory bronchiole and extend into the
surrounding alveoli and alveolar septa. B. Immunohistochemistry
for CD204 fails to detect the infiltrating Langerhans cells, but
labels intra-alveolar macrophages.
cells, which do not have Birbeck granules. Immunohistochem-
ically, histiocytic cells of feline LCH are positive for CD18
and E-cadherin and negative for CD204 in formalin-fixed
tissues. Frozen tissues of feline LCH have not been studied,
and the number of cat-specific antibodies that can be used to
characterize histiocytic cells is limited.
Canine reactive histiocytosis
Reactive histiocytic diseases have only been reported in dogs.
Two forms have been reported based on their clinical presen-
tations: cutaneous reactive histiocytosis is limited to the skin and
draining lymph nodes; systemic reactive histiocytosis affects the
skin and internal organs. It is important to include the term
“reactive” when discussing these disease entities rather than
abbreviating these diseases as cutaneous and systemic histio-
cytosis. This will help preventing a mix-up with the obsolete
term “malignant histiocytosis.” Malignant histiocytosis has for-
merly been used to characterize disseminated histiocytic sar-
comas (see later) and should be avoided, especially since
systemic reactive histiocytosis and disseminated histiocytic
sarcomas both occur commonly in Bernese Mountain dogs,
but represent entirely different diseases and progression from
247
systemic reactive histiocytosis to histiocytic sarcoma has not
been observed.
Most likely, the cutaneous and systemic forms of reactive
histiocytosis emerge from the same cutaneous lesion and rep-
resent an inflammatory disease caused by immune dysregulation
rather than a neoplastic process. In contrast to cutaneous his-
tiocytomas or Langerhans cell proliferative diseases, the pro-
liferating cells in reactive histiocytosis are activated dermal
interstitial dendritic cells and CD8+ T cells. Regression of the
lesion caused by CD8+ T cells, as observed in cutaneous his-
tiocytomas, does not occur in reactive histiocytosis. Although
the lesions appear to be antigen driven, the antigen itself is
unknown, and the T cell may drive the activation of the den-
dritic cells via tumor necrosis factor -α and granulocyte-
macrophage colony-stimulating factor. This hypothesis is
involve spontaneous remission of early cutaneous lesions and
a positive response to immunomodulatory drugs.
Reactive histiocytosis is a lesion of activated dermal interstitial
dendritic cells that are immunohistochemically positive with
CD1a, CD11c, CD18, and MHC II, as well as CD90 and
CD204. They are negative for E-cadherin. Furthermore, the
proliferating cells express CD4, a marker of dendritic cell
activation that is not expressed in histiocytic sarcomas or
Langerhans cell proliferations. Unfortunately, this antibody is
currently only available for frozen sections.
Cutaneous reactive histiocytosis
Cutaneous histiocytosis is a common disease of dogs that is
limited to the skin and subcutis, but may involve draining
lymph nodes (eFig. 2-64). Dogs clinically have multiple
nodules that are grossly similar to cutaneous histiocytomas.
lesions are rare. Numerous breeds are affected, but no predis-
positions have been reported. The disease affects a broad age
range, and affected areas tend to be roughly similar to those
of cutaneous histiocytoma, with an anterior predilection for
skin of the head and neck as well as for the caudal areas of
flanks, perineum, and the scrotum (Fig. 2-223). Occasionally,
mucus membranes of the nasal and conjunctival areas may be
involved. The blood and bone marrow tend to be unaffected
unless there is ulceration sufficient to cause systemic neutro-
philia. Cutaneous lesions may regress at one site and spontane-
ously appear at another site simultaneously.
Histologically, the cutaneous lesions of reactive histiocyto-
sis, both the cutaneous form and the systemic form, have a
distinct morphologic pattern that allows differentiation from
proliferative Langerhans cell diseases. The skin lesions tend to
be deep and angioinvasive, frequently presenting as a lympho-
histiocytic vasculitis. There is often sparing of the superficial
dermis, with the lesions tending to be “bottom heavy” in con-
trast to the characteristic superficial, “top-heavy” topography
of cutaneous histiocytomas (Fig. 2-224). Commonly, lesions
extend into the subcutis, where they may coalesce. Likely
because of the tendency to surround and invade arterioles,
there are small irregular foci of ischemic necrosis that may
contribute to superficial ulceration. The cellular infiltrates are
dominated by lymphocytes and dermal dendritic cells, but
commonly admixed with neutrophils and eosinophils. Prolif-
erating histiocytic cells have abundant cytoplasm of even
density throughout, and their cell boundaries are indistinct.
Their nuclei tend to be round to oval, but can be twisted,
indented, or folded, and there is a fine chromatin distribution
 247.e1

A B

C D
eFigure 2-64  Reactive histiocytosis in a dog. A. Cutaneous reactive histiocytosis characterized by
large numbers of cutaneous nodules widely disseminated throughout the skin. (Courtesy K. Loft.)
B. Systemic reactive histiocytosis with sharply demarcated nodule on the epiglottis. C. Enlarge-
ment of lymph nodes can be observed in either cutaneous or systemic reactive histiocytosis.
D. Large numbers of nodules in the oral cavity of a dog with systemic reactive histiocytosis.
248 CHAPTER 2  •  Hematopoietic System Histiocytic Proliferative Diseases

A B
Figure 2-223  Cutaneous reactive histiocytosis in a dog. (Courtesy K. Loft.) A. Large numbers of
cutaneous nodules widely disseminated throughout the skin. B. Similar sharply demarcated
nodules in the skin of the lower extremities.

A B

Dermal
vessels

Histiocytic
Subcutis infiltrate

C D
Figure 2-224  Cutaneous reactive histiocytosis in a dog. A. Deep dermal and angioinvasive cellular
infiltrates that are dominated by lymphocytes and dermal dendritic cells extend into the subcutis
where they may coalesce. B. Immunohistochemistry for Thy-1 labels activated dermal interstitial
dendritic cells. C. Schematic drawing depicting the “bottom-heavy” topography of reactive histio-
cytosis in contrast to of the superficial, “top-heavy” topography of cutaneous histiocytomas.
(Adapted from a graphic by P.F. Moore.) D. Proliferating histiocytic cells have abundant cytoplasm,
indistinct cell boundaries, and monomorphic round to oval nuclei that can be twisted, indented,
or folded, but features of anaplasia characteristic for histiocytic sarcoma are absent.
 Histiocytic Proliferative Diseases
and small but prominent central nucleoli. Multinucleated or
highly anaplastic cells as observed in histiocytic sarcomas are
not a feature of reactive histiocytosis. Mitotic figures are
absent or rare, and bizarre forms are lacking. Proliferating
histiocytic cells may be found in draining lymph nodes, where
they selectively invade the sinuses and paracortex.
Cutaneous reactive histiocytosis must be differentiated
from Langerhans cell proliferation (cutaneous histiocytoma
and LCH) and from more aggressive types of histiocytic pro-
liferations (histiocytic sarcomas). The clinical presentation
(multiple vs. solitary lesions), the distinct morphologic pattern
(vasocentric “bottom-heavy” vs. commonly epitheliotropic
“top-heavy”), the cellular morphology (well-differentiated his-
tiocytic cells vs. marked anaplasia), and the immunohisto-
chemical features (positive for CD4 [frozen only] CD90, and
CD204 (eFig. 2-65), but negative for E-cadherin) allow for an
accurate differentiation among these 3 entities (Fig. 2-225).
Another major differential diagnosis for cutaneous reactive
histiocytosis is an inflamed cutaneous non-epitheliotropic
T-cell lymphoma. The large component of CD3+ T-cells in
cutaneous reactive histiocytosis may lead to confusion between
A and renal disease have all been reported. As with the cutane-
B
Figure 2-225  Cutaneous reactive histiocytosis in a dog.
A. Lesions are frequently vasocentric and composed of dense
infiltrates of interstitial dendritic cells admixed with focal small
lymphocytic aggregates. B. Immunohistochemistry demonstrates
expression of CD4 in interstitial dendritic cells, a marker of
dendritic cell activation that is not expressed in histiocytic sarco-
mas or Langerhans cell proliferations.
249
both entities. Clusters of atypical T cell will aid a diagnosis of
lymphoma, but in many cases, a definite diagnosis has to rely
on detection of a clonal T-cell expansion in lymphoma cases
by T-cell receptor γ gene rearrangement.
Systemic reactive histiocytosis
Systemic reactive histiocytosis is a much less common disease
of dogs than cutaneous reactive histiocytosis, and essentially
represents a progression of the cutaneous disease to involve inter-
nal organs. Like its cutaneous counterpart, systemic reactive
histiocytosis is also characterized by cutaneous nodules with
the earlier-described histologic features of a “bottom-heavy”
histiocytic proliferation. Although no breed association has
been established for the cutaneous form, the systemic form
has a familial association in Bernese Mountain dogs. It is
important not to misdiagnose systemic reactive histiocytosis
as a disease process in the histiocytic sarcoma complex, and
there is no evidence of progression of this inflammatory condi-
tion into a malignant neoplasm. Systemic reactive histiocytosis
also has a familial component in Irish Wolfhounds and has
been reported to occur in other large breeds, for instance,
Rottweilers and Labrador Retrievers. Young to middle-aged
dogs are most commonly affected. Skin nodules occur most
frequently around the nose (planum, apex), scrotum, and
eyelids, similar to what has been described for the cutaneous
form. This distribution makes distinction between the cutane-
ous and systemic form very difficult during the early stages of
a systemic reactive histiocytosis. As with the cutaneous form,
ulceration of skin masses is very common and occurs most
likely secondary to vascular lesions that cause infarction, as
described earlier. Besides the skin, nodular masses are often
observed in the ocular and nasal mucous membranes, and
lymph nodes are typically enlarged (Fig. 2-226). Involvement
of internal organs is quite variable, and lesions in the lungs,
liver, spleen, bone marrow, kidney, and testis have been
reported. The wide range of internal organs that can be
affected drives the variation of clinical signs and the degree of
their severity. Weight loss, anorexia, respiratory distress, ocular,
ous form, remission and relapse are also features of systemic
reactive histiocytosis.
Figure 2-226  Systemic reactive histiocytosis in a dog. The
lymph node is infiltrated by large numbers of activated dermal
interstitial dendritic cells that are positive for CD90 by
immunohistochemistry.
 249.e1

B
eFigure 2-65  Cutaneous reactive histiocytosis in a dog.
A. Immunohistochemistry for CD204 labels activated dermal
interstitial dendritic cells. B. These cells are negative for E-
cadherin by immunohistochemistry, thus excluding a cutaneous
histiocytoma.
250 CHAPTER 2  •  Hematopoietic System Histiocytic Proliferative Diseases

Histologically, systemic reactive histiocytosis is also char-

acterized by proliferation of activated dermal interstitial den-
dritic cells that form deep-seated cutaneous mass identical to
those described with the cutaneous form. Lesions in internal
organs are also vasocentric, and the mixture of lymphocytes
and histiocytic cells tends to expand from the perivascular
location into the surrounding parenchyma. The immunohis-
tochemical features of the proliferating histiocytic cells are
identical to those described with cutaneous reactive histiocy-
tosis, but only dermal dendritic cells express CD90. Although
histiocytic sarcoma may be considered as a potential differen-
tial based on lesion distribution, histiocytic sarcomas tend to
form significantly larger nodules, whereas systemic reactive
histiocytosis may only manifest histologically in internal
organs. Regardless, the degree of cellular pleomorphism and
anaplasia observed with histiocytic sarcomas is always absent A
in reactive histiocytosis.

Histiocytic sarcoma
Histiocytic sarcomas are principally neoplasms of interstitial den-
dritic cells. However, one distinct entity, hemophagocytic his-
tiocytic sarcoma, is an aggressive malignant neoplasm of
macrophage origin in dogs. This neoplastic entity causes a
distinct disease syndrome that differs in many aspects from
other histiocytic sarcomas of dendritic cell origin and will
therefore be discussed as a separate entity.
Histiocytic sarcoma is a relatively frequently occurring
aggressive malignant neoplasm primarily of dogs and much
less commonly cats. The disease presentation is essentially the
same in both species. Lesions may be localized arising at a
single site or organ or disseminated after metastasis to distant
sites (eFig. 2-66). It is uncertain whether multiple simultane-
ously occurring neoplasms in different organs represent a mul- B
ticentric disease process or distant metastases of a primary Figure 2-227  Histiocytic sarcoma in a dog. A. Small white
mass. The term “malignant histiocytosis” has historically been nodules, which in some areas almost become confluent, randomly
applied to disseminated histiocytic sarcomas, but should be distributed throughout the spleen. (Courtesy K.G. Thompson.)
avoided to prevent confusion of histiocytic sarcomas with the B. Discrete neoplastic masses that protrude above the splenic
earlier described inflammatory disease processes of reactive capsule (Courtesy R.L. Amorim.)
histiocytosis.
Histiocytic sarcomas may develop at various locations, but
primary masses are commonly observed in the spleen (Fig.
2-227), lymph nodes (Fig. 2-228), lung, bone marrow, skin
(Fig. 2-229), the periarticular tissue of the extremities (eFig.
2-67), and even the central nervous system (CNS) (Fig.
2-230). Periarticular histiocytic sarcomas occur most com-
monly around the stifle and elbow joints, but carpus and
coxofemoral joints can also be affected. Masses in the brain
most commonly occur as focal, solitary, subdural masses, and
occasionally as diffuse meningeal infiltrates (Fig. 2-231, eFig.
2-68). Histiocytic sarcomas typically appear as white/cream
to tan, homogeneous masses with a smooth cut surface, but
diffuse organ enlargement by infiltrating neoplastic histiocytic
cells can also occur. Nodules can be solitary or multiple at the
primary site and tend to be highly destructive. Metastatic sites
can include any organ system, but liver (eFig. 2-69), kidneys
(eFig. 2-70), lungs (Fig. 2-232, eFig. 2-71), and draining lymph
nodes are most commonly affected.
Numerous dog breeds have a familial predisposition,
including Bernese Mountain dogs, Golden Retrievers, Labra-
dor Retrievers, Flat-coated Retrievers, and Rottweilers. Periar-
ticular histiocytic sarcomas have been most commonly Figure 2-228  Histiocytic sarcoma in a dog. Cross section of a
reported in Rottweilers and Flat-coated Retrievers. A poly- white, firm, and homogeneous splenic nodule. (Courtesy S.
genic mode of inheritance has been postulated for Bernese Kesdangsakonwut.)
 250.e1

eFigure 2-67  Periarticular histiocytic sarcoma in a dog. Periar-

ticular histiocytic sarcomas appear as multinodular masses around
joint capsules.

B
eFigure 2-66  Histiocytic sarcoma in a dog. A. Small white
nodules randomly distributed throughout the spleen. B. Discrete
neoplastic masses that protrude above the splenic capsule, but
their color resembles the splenic parenchyma.

A B
eFigure 2-68  Histiocytic sarcoma in a dog. A. Submeningeal infiltrates of distinct large, highly
anaplastic histiocytic cells with abundant eosinophilic cytoplasm. B. Neoplastic cells expand
Virchow-Robin spaces and invade the cerebral cortex.
250.e2

A B
eFigure 2-69  Histiocytic sarcoma in a dog. A. Multifocal, sharply demarcated neoplastic nodules
randomly distributed throughout the liver. B. A discrete neoplastic nodule with less well-defined
borders in the liver. (Courtesy S. Kesdangsakonwut.)

eFigure 2-70  Histiocytic sarcoma in a dog. Well-circumscribed eFigure 2-71  Histiocytic sarcoma in a dog. Numerous pulmonary
neoplastic masses in the renal cortex. vessels are obstructed by large numbers of neoplastic histiocytic
cells that are highly pleomorphic.
 Histiocytic Proliferative Diseases 251

A
Figure 2-229  Histiocytic sarcoma in a dog. Severely ulcerated
discrete and coalescing cutaneous nodules. (Courtesy S.
Kesdangsakonwut.)

Figure 2-230  Histiocytic sarcoma in a dog. Well-circumscribed

mass in the cerebral cortex with central necrosis.

C
Figure 2-232  Histiocytic sarcoma in a dog. A. Numerous peri-
bronchial vessels are obstructed by large numbers of neoplastic
histiocytic cells that are highly pleomorphic. B. Multinucleated
giant cells and neoplastic cells with megalokaryosis infiltrating the
hepatic sinusoids. C. Neoplastic histiocytic cells are positive for
CD18 by immunohistochemistry.

Figure 2-231  Histiocytic sarcoma in a dog. Submeningeal infil-

trates of distinct large, highly anaplastic histiocytic cells with
abundant eosinophilic cytoplasm.
252 CHAPTER 2  •  Hematopoietic System
Figure 2-233  Histiocytic sarcoma in a dog. Lymph node with
diffuse infiltrate of highly anaplastic, large, pleomorphic histio-
cytic cells with abundant eosinophilic, often vacuolated cyto-
plasm. Multinucleated cells, megalokaryosis, and bizarre nuclei are
common.
Mountain dogs, and recent research suggests mutations in
tumor suppressor genes PTEN, CDKN2A/B, and RB1 in his-
tiocytic sarcoma genesis in Bernese Mountain dogs and Flat-
coated Retrievers.
Clinical signs reflect the wide range of organ systems
affected and range from anorexia, weight loss, and lethargy, to
respiratory distress, including coughing and dyspnea; neuro-
logic signs, including seizures and paralysis; and lameness with
periarticular neoplasms. Few cases of histiocytic sarcomas
developed hypercalcemia of malignancy. Neoplastic cells are
rarely encountered in peripheral blood, and only a few cases
of dendritic cell leukemia have been reported. Approximately
30,000 to 60,000 atypical histiocytic cells were observed per
microliter.
Histologically, histiocytic sarcomas are composed of sheets
of distinct large, pleomorphic, often highly anaplastic cells
with abundant amounts of often vacuolated or atypical cyto-
plasm (Fig. 2-233). Multinucleated giant cells are common as
are megalokaryosis and bizarre nuclei. The mitotic index is
usually high, and atypical mitotic figures are easily recogniz-
able. Some histiocytic sarcomas, especially those around peri-
articular and articular tissues and in the subcutis can also have
sheets of spindle cells with no distinct cell margins and an
absence of marked cellular pleomorphism (Fig. 2-234). Such
neoplasms are best differentiated from soft tissue spindle cell
sarcomas by their immunohistochemical staining pattern.
Periarticular histiocytic sarcomas commonly have a micronod-
ular growth pattern and are located beneath the intact syno-
vial lining. Secondary inflammatory infiltrates composed of
neutrophils and lymphocytes as well as plasma cells are
common in histiocytic sarcomas around joints and in the skin
and may obscure the primary neoplastic cell population. Large
numbers of mixed inflammatory cells are also commonly
observed in histiocytic sarcomas in the CNS, and lesions must
be differentiated from granulomatous meningoencephalitis.
Histiocytic sarcomas originate primarily in the leptomeninges,
and atypical histiocytic cells can be observed.
Immunohistochemically, histiocytic sarcomas are uni-
formly strongly positive for CD18, CD204, and MHC II, and
Histiocytic Proliferative Diseases
A
B
Figure 2-234  Periarticular histiocytic sarcoma in a dog. A. Some
histiocytic sarcomas occur in the periarticular and articular tissues
around appendicular joints. B. Periarticular histiocytic sarcomas
are often composed of sheets of spindle cells with no distinct cell
margins and an absence of marked cellular pleomorphism.
variably positive for Cd163 in formalin-fixed tissues, and in
frozen sections they are positive for CD1 and CD11c. They
are negative for CD4 and E-cadherin. Only cutaneous histio-
cytic sarcomas express CD90. Periarticular histiocytic sarco-
mas also express the typical interstitial dendritic cell
phenotype, and are CD1a and CD11c positive rather than
CD1a negative and CD11b positive, which would be consis-
tent with synovial type A cells.
Histiocytic sarcoma may occur as solitary or multiple pro-
liferations that are all characterized by very rapid progression
and metastatic spread. Only histiocytic sarcomas in the brain
have not been shown to cause extracranial metastasis, whereas
metastases to the brain are not uncommon. Early surgical
excision of cutaneous or periarticular histiocytic sarcomas
might be potentially curative, but widespread metastasis
occurs early in the course of disease and average survival times
are 5-8 months. Therefore the overall prognosis is grave and
response to chemotherapy usually poor. Recent studies suggest
that histiocytic sarcomas may respond to some tyrosine kinase
inhibitors, for instance, dasatinib. Rupture of the cranial cruci-
ate ligament or traumatic joint injury has been reported as a
sequel to periarticular histiocytic sarcoma.
 Histiocytic Proliferative Diseases 253

Histiocytic sarcoma must be differentiated from reactive

histiocytosis, which is usually accomplished on the basis of the
extent of the lesion and the high degree of anaplasia of the
neoplastic cell population in histiocytic sarcomas. Hemo-
phagocytic histiocytic sarcomas are characterized by erythro-
phagia and express CD11d in formalin-fixed tissues, but not
CD11c in frozen sections. Depending on the primary site of
the histiocytic sarcoma, other differentials include nonlym-
phogenic, non-angiogenic sarcomas in the spleen and synovial
sarcomas or other soft tissue spindle cell sarcomas in periar-
ticular tissue or skin and subcutis, respectively. Historically,
most intra-articular and periarticular sarcomas have been diag-
nosed as synovial sarcomas, but immunohistochemical staining
with CD18 confirmed that the vast majority of these neoplasms
are periarticular histiocytic sarcomas. Immunohistochemistry
(IHC) with CD18 and CD204 is usually sufficient to accu-
rately diagnose histiocytic sarcomas. However, especially syno-
vial sarcomas or soft tissue spindle cell sarcomas can be heavily Figure 2-235  Feline progressive histiocytosis. Alopecic nodules
infiltrated by CD18-positive histiocytic cells and may closely with focal ulcerations on the face of a cat.
resemble histiocytic sarcomas on low magnification. It is
important to closely examine these neoplasms on higher mag-
nification because a population of CD18-negative spindle cells
will be recognizable in nonhistiocytic sarcomas. In our experi-
ence, CD204 often provides a more distinct differentiation
between histiocytic and nonhistiocytic cells. Another soft
tissue spindle cell sarcoma that is characterized by large
numbers of multinucleated giant cells is actually not a neo-
plasm of histiocytic cell lineage, but has been mislabeled as
“malignant fibrous histiocytoma.” The neoplastic cells of this
particular entity most likely represent myofibroblasts that are
negative for CD18 and CD204. Because lymphomas may
express CD18, especially subcutaneous histiocytic sarcomas
should be differentiated from anaplastic large cell lymphomas
by staining for T- and B-cell markers CD3, CD79a, CD20, and
Pax-5.

Feline progressive histiocytosis

Feline progressive histiocytosis (FPH) is a disease of mature
cats 7-17 years of age, with most animals older than 10 years
at presentation. There is no apparent breed predilection, but
female cats tend to be affected more than male. Lesions
develop most commonly on the head, including the face (eFig.
2-72), nose, eyelids, lips, and ears, as well as around the legs
and feet, and with lesser frequency in other areas of the skin
(Fig. 2-235). The disease is initially indolent and may persist
in the skin for prolonged periods before there is involvement
of internal organs, including lymph nodes, lungs, liver, kidney,
and spleen. First signs of FPH most commonly consist of soli-
tary or multiple nodules, plaques, or papules that are neither
painful nor pruritic. Nodules may be up to 1.5 cm in diameter.
During these early disease stages, cats are not irritated by the
lesions and appear to be in good health. As the disease pro-
gresses, the cutaneous lesions tend to coalesce and eventually
to ulcerate. Although some lesions wax and wane, complete
regression as observed with canine cutaneous histiocytomas is not
a feature of FPH. As the disease becomes more invasive, no
treatment appears to be effective, and the animals are eutha-
nized when they develop systemic illness.
In essence, FPH resembles a low-grade histiocytic sarcoma
originating from cutaneous interstitial dendritic cells. Histologi-
cally, the early cutaneous lesions bear some resemblance to
canine cutaneous histiocytomas and are characterized by
nodular to diffuse histiocytic infiltrates in the superficial
Figure 2-236  Feline progressive histiocytosis. Nodular histio-
cytic infiltrate in the superficial portion of the dermis that is
in contact with the basement membrane and causes focal
epitheliotropism.
portion of the dermis (Fig. 2-236). Most infiltrates are in
contact with the basement membrane that is irregularly
breached (epitheliotropism) in ~40% of cases. Some masses
extend into the subcutis (eFig. 2-73). During the initial phase
of FPH, infiltrating histiocytes are well differentiated and
closely resemble inflammatory cells. Special stains for intracel-
lular bacteria, including mycobacteria, and macerated tissue
culture are recommended to exclude an infectious disease
process. As the disease progresses, the degree of anisokaryosis
increases, and the lesion more closely resembles canine histio-
cytic sarcomas (Fig. 2-237). At this stage, neoplastic cells have
irregular, euchromatic nuclei with finely dispersed chromatin
and typically a single moderate-sized central nucleolus. The
cytoplasm is abundant and deeply and uniformly stained, with
cell boundaries generally distinct. More multinucleated cells
are present as is megalokaryosis and a few bizarre nuclei. The
mitotic index varies, and atypical mitotic figures may be
present. The extent of inflammatory cell infiltrates composed
of small lymphocytes and neutrophils varies between indi-
vidual cases. Immunohistochemically, histiocytic cells of FPH
 253.e1

eFigure 2-72  Feline progressive histiocytosis. Alopecic nodules eFigure 2-73  Feline progressive histiocytosis. Deep histiocytic
with focal ulcerations on the face of a cat. infiltrates into the underlying muscle occur during disease
progression.
254 CHAPTER 2  •  Hematopoietic System
Figure 2-237  Feline progressive histiocytosis. As lesions progress,
cellular pleomorphism, nuclear anisokaryosis, and mitotic activity
increase.
have a characteristic interstitial dendritic cell phenotype, and are
positive for CD1a, CD18, and MHC II. Interestingly, some of
the cases express E-cadherin at an earlier disease stage, which
would indicate a Langerhans cell differentiation. About half
the cases in one study expressed CD5. Antibodies specific for
feline CD molecules are limited, and only an antibody specific
for feline CD18 is available for formalin-fixed tissue. The
canine-specific CD18 will not cross react with feline dendritic
cells. The infiltrating lymphocytes are CD3 and CD8 positive.
The location of the lesions, the progression of the disease
process and the cell morphology at the different stage of FPH
in combination with positive IHC for CD18 are used to reach
an accurate diagnosis. Although inflammatory lesions must be
excluded at an early stage of the disease, an atypical large cell
lymphoma should be excluded at late stage by using IHC for
CD3, CD79a, CD20, and Pax-5.
Hemophagocytic histiocytic sarcoma
Hemophagocytic histiocytic sarcoma is the only malignancy of
macrophage origin and occurs primarily in dogs; a few cases have
been reported in cats. There is no known breed or age predis-
position, but commonly affected breeds are similar to those
affected by histiocytic sarcoma of dendritic cell origin. It is
therefore important to recognize that hemophagocytic histio-
cytic sarcoma may occur simultaneously to histiocytic sarcoma,
especially in breeds with a predisposition for the latter disease.
Hemophagocytic histiocytic sarcoma has a unique clinical
picture that differs from that caused by other histiocytic sar-
comas and most closely resembles an immune-mediated hemo-
lytic anemia, or, more specifically, Evans syndrome because
there is concurrent thrombocytopenia. Affected dogs have
splenomegaly and hepatomegaly (Fig. 2-238, eFig. 2-74) and
rapidly progressive, regenerative hemolytic anemia and throm-
bocytopenia, but are Coombs test negative (no erythrocyte-
bound IgG). The pathogenesis of the disease is the result of
the massive level of erythrophagocytosis by the neoplastic histio-
cytes that are primarily located in the spleen, liver, and bone
marrow. Many dogs are icteric at initial presentation and have
marked hyperbilirubinemia. Hypoalbuminemia and hypocho-
lesterolemia are also commonly observed. Because dogs with
hemophagocytic histiocytic sarcomas lack grossly visible
Histiocytic Proliferative Diseases
Figure 2-238  Hemophagocytic histiocytic sarcoma in a dog.
Diffuse splenomegaly with ill-demarcated neoplastic nodules pro-
truding over the splenic capsule.
masses, a neoplasm is often considered less likely as the
primary cause than an immune-mediated disease, and the
ultimate diagnosis is often made through autopsy and histo-
logic examination of the spleen and liver. Adding the poor
response of hemophagocytic histiocytic sarcomas to standard
chemotherapy to the difficulties in reaching an accurate diag-
nosis may help to explain reported median survival times of
<30 days.
Hemophagocytic histiocytic sarcomas are caused by prolif-
eration of neoplastic macrophages that are avidly erythrophago-
cytic (Fig. 2-239). Primary sites are the spleen and the bone
marrow with secondary spread to lung and liver and rarely
kidneys (eFig. 2-75). Grossly, the spleen is markedly diffusely
enlarged with few to many, discolored green-tan to white
splenic infarcts that are visible through the splenic capsule. In
cats, the involvement of the liver may be of solid foci of neo-
plastic cells.
Microscopically, in the spleen, neoplastic cells differentiate
toward red pulp macrophages and cause locally extensive to
diffuse expansion of the red pulp sinuses and frequently oblit-
erate the adjacent white pulp. Focal areas of ischemic necrosis
secondary to vascular thrombosis are common in the splenic
sinuses. Most cases are accompanied paradoxically by inter-
spersed foci of extramedullary hematopoiesis. The neoplastic
histiocytes migrate to the lung and liver, where they insidi-
ously invade hepatic sinusoids. In the lungs, hemophagocytic
neoplastic cells are found primarily in the vasculature. The
bone marrow is also infiltrated by neoplastic hemophagocytic
histiocytes. Most neoplastic cells are severely hemophagocytic
or contain hemosiderin, but it is common to find areas in the
spleen where cells exhibit less erythrophagocytosis. Some neo-
plastic cells may also phagocytose red cell precursors and
granulocytes. Although some atypia of the neoplastic cells is
common, especially in the spleen, in contrast to the highly
pleomorphic cells in histiocytic sarcomas of interstitial den-
dritic cell origin, neoplastic cells of macrophage origin in hemo-
phagocytic histiocytic sarcomas are surprisingly well differentiated.
Especially within the bone marrow, but also liver and lung,
neoplastic cells closely resemble their normal counterpart
except for erythrophagocytosis. In the spleen, occasional
anisocytosis, anisokaryosis, and hyperchromatic nuclei are
 254.e1

eFigure 2-74  Hemophagocytic histiocytic sarcoma in a dog. eFigure 2-75  Hemophagocytic histiocytic sarcoma in a dog. In
Diffuse splenomegaly with ill-demarcated neoplastic nodules pro- the spleen, there is proliferation of neoplastic macrophages that
truding over the splenic capsule. are avidly erythrophagocytic.
 Disorders of Hemostasis
A Valli VE. Histiocytic Neoplasms. Veterinary Comparative Hematopathol-
B designed to ensure that blood clotting is tightly regulated,
Figure 2-239  Hemophagocytic histiocytic sarcoma in a dog.
A. In the spleen, there is proliferation of neoplastic macrophages
that are avidly erythrophagocytic. B. Neoplastic splenic macro-
phages are CD11d positive by immunohistochemistry.
observed, as well as a few multinucleated giant cells. The
abundant cytoplasm is eosinophilic and often vacuolated.
Nuclei are most frequently ovoid or cleaved, and bizarre forms
are rare. The mitotic index is low. Because neoplastic cells
appear well differentiated, the early pattern of invasion into
the liver may be misdiagnosed on fine-needle biopsies as
Kupffer cell hyperplasia or an inflammatory process. IHC has
been helpful for characterizing the distribution of neoplastic
cells in the spleen and liver.
Immunohistochemically, the neoplastic hemophagocytic
histiocytes most closely resemble macrophages of the splenic
red pulp and bone marrow, are positive for CD11d and CD18,
and have variable expression of CD1a, but are negative for
CD11c, which is positive in interstitial dendritic cells. Although
antibodies for canine CD11d are available for formalin-fixed
tissues, there is no feline counterpart and definite confirmation
of hemophagocytic histiocytic sarcomas in cats is still missing.
Further reading
Affolter VK, Moore PF. Localized and disseminated histiocytic sarcoma
of dendritic cell origin in dogs. Vet Pathol 2002;39:74-83.
Affolter VK, Moore PF. Feline progressive histiocytosis. Vet Pathol
2006;43:646-655.
255
Busch MD, et al. Feline pulmonary Langerhans cell histiocytosis with
multiorgan involvement. Vet Pathol 2008;45:816-824.
Friedrichs KR, Young KM. Histiocytic sarcoma of macrophage origin in
a cat: case report with a literature review of feline histiocytic malig-
nancies and comparison with canine hemophagocytic histiocytic
sarcoma. Vet Clin Pathol 2008;37:121-128.
Fulmer AK, Mauldin GE. Canine histiocytic neoplasia: an overview. Can
Vet J 2007;48:1041-1043, 1046-1050.
Moore PF. A review of histiocytic diseases of dogs and cats. Vet Pathol
2014;51:167-184.
Moore PF, et al. Canine hemophagocytic histiocytic sarcoma: a prolif-
erative disorder of CD11d+ macrophages. Vet Pathol 2006;43:
632-645.
Moore PF, Rosin A. Malignant histiocytosis of Bernese Mountain dogs.
Vet Pathol 2006;23:1-10.
ogy. Ames, Iowa: Blackwell; 2007. p. 505-524.
DISORDERS OF HEMOSTASIS
R. Darren Wood
Hemostasis is the process by which interactions between a
series of plasma proteins and cells, including platelets, endo-
thelial, and white blood cells, combine to provide a balance
between anticoagulant and procoagulant functions. It is essen-
tially a host defense mechanism that protects the integrity of
the vascular system after injury. In recent years, the impor-
tance of activated cell membranes to the hemostatic process
has become well understood, and the entire process is now
frequently referred to as the cell-based model of hemostasis.
During health, anticoagulant mechanisms, which are
predominate. When injury to, or activation of, endothelium
occurs, procoagulant proteins are released from these cells,
and circulating platelets and plasma proteins become exposed
to subendothelial tissues, including collagen and fibroblasts.
This promotes a tipping of the balance toward procoagulation,
where platelet-mediated hemostasis initially stems the loss of
blood at the site of the vascular injury via formation of a
platelet plug. Once sufficient platelet activation occurs, initial
thrombin production activates additional platelets, promotes
the conversion of fibrinogen to fibrin, provides positive feedback
amplification to produce more fibrin, and stimulates antico-
agulants and the fibrinolytic system to regulate overall clot
formation. Thrombin has additional roles in cell proliferation,
inflammation, and repair that are mediated through its ability
to engage protease-activated receptors (PARs) on target cells
(Fig. 2-240).
During disease, there are many possible derangements that
can occur, depending on the nature of the abnormality, and
whether it remains localized or becomes disseminated sys-
temically. As a result, disorders of the hemostatic system can
range from poorly detectable but significant pathology, to
overt and potentially catastrophic hemorrhage and/or throm-
bosis. A brief review of important physiology, pathophysiol-
ogy, and disorders follows.
Platelet plug formation
When endothelial cells are injured, or exposed to inflamma-
tory mediators, the cell membrane changes from expressing
an anticoagulant phenotype to one more procoagulant in
nature. The expression of platelet surface receptors that interact
 255.e1

Further reading
Hélie P, et al. Congenital cutaneous histiocytosis in a piglet. Vet Pathol
2013;51:812-815.
Ide K, et al. Disseminated histiocytic sarcoma with excessive hemo-
phagocytosis in a cat. J Vet Med Sci 2009;71:817-820.
Kato Y, et al. The class A macrophage scavenger receptor CD204 is a
useful immunohistochemical marker of canine histiocytic sarcoma.
J Comp Pathol 2013;148:188-196.
Moore PF, et al. Canine cutaneous histiocytoma is an epidermotropic
Langerhans cell histiocytosis that expresses CD1 and specific beta
2-integrin molecules. Am J Pathol 1996;148:1699-1708.
Pinard J, et al. Histiocytic sarcoma in the tarsus of a cat. Vet Pathol
2006;43:1014-1017.
Weiss DJ. Flow cytometric evaluation of hemophagocytic disorders in
canine bone marrow. Vet Clin Pathol 2002;31:36.
256 CHAPTER 2  •  Hematopoietic System
Platelet aggregation
Procoagulant
Inflammation
Kallikrein/bradykinin
THROMBIN
PAF synthesis
Neutrophil activation
Anticoagulation
Activation of
TM–protein
TM – protein C–protein
C – proteinSS
Figure 2-240  Multiple functions of thrombin that include modulation of coagulation, anticoagula-
tion, inflammation, and tissue repair. PAF, platelet activating factor; TM, thrombomodulin.
with von Willebrand factor (vWF) released from endothelial
cells, initiates platelet adhesion, aggregation, and granule
content release at the site of vascular injury. Simultaneously,
tissue factor and procoagulant platelet phospholipids become
available on cell surfaces to initiate thrombin, and then fibrin,
formation.
Adhesion
Platelets are derived from megakaryocytes and circulate as
small discoid cells in an unactivated state. When activated,
they undergo a shape change, becoming more spherical and
with extensions, which is mediated by actin and myosin fila-
ments in the cytoplasm. This is accompanied by alterations in
the cell membrane conformation that activates platelet mem-
brane glycoprotein (GP), or integrin, receptors. These receptors
mediate platelet adhesion to exposed tissue that then pro-
motes further recruitment and adhesion of additional
platelets.
The 2 main proteins required for adhesion of platelets to
each other and to damaged endothelial cells and exposed
fibroblasts are fibrinogen and vWF. Glycoprotein Ib-IX-V is a
constitutively active receptor that causes rapid platelet adhe-
sion to exposed vWF. This receptor is important in blood
vessels with high shear forces because it facilitates stabiliza-
tion of platelet aggregates. The most abundant platelet integrin,
αIIbβIII (or GPIIb/IIIa), undergoes a conformational change
during platelet activation to bind fibrinogen, and to a lesser
extent vWF. This receptor is also expressed in the open cana-
licular system, a feature of most mammalian platelets, which
expands the platelet surface area once shape change occurs.
When fibrinogen binds to αIIbβIII, it connects adjacent plate-
lets, which is critical for the development of platelet
aggregates.
Platelets possess specific membrane thrombin receptors.
These receptors are members of the G-protein–coupled
receptor family that are characterized by a single polypeptide
with a 7-transmembrane domain. Thrombin receptors are
referred to as PARs, because the receptors only acquire trans-
membrane signaling functions after thrombin has cleaved
a peptide from the extracellular domain. Although there
Disorders of Hemostasis
Fibrin formation
Tissue repair
Release of
growth factors
Activation of
fibrinolysis
are others, thrombin is considered the most potent platelet
agonist.
Aggregation
Further recruitment of circulating platelets and their incorpo-
ration into developing platelet aggregates around the site of
already adherent platelets depends on the local accumulation
of platelet agonists and the generation of procoagulant activity
provided by integrin and phospholipid exposure on the surface
of activated platelets. Local agonists include adenosine diphos-
phate (ADP), serotonin, and epinephrine, which are pre-
formed in platelet-dense granules, and newly synthesized
agonists, such as platelet-activating factor (PAF) and thrombox-
ane A2 (TxA2). These agonists interact with their own
G-coupled protein receptors to initiate inside-out signaling.
The activator-receptor interactions increase the avidity of the
glycoprotein-integrin receptors for their ligands and addition-
ally initiate intracellular downstream signaling via several
pathways involving the activation of phospholipases, protein
kinase K (PKC), and adenylate cyclase.
Platelets are maintained in an unactivated state by
regulation of cytoplasmic levels of calcium and cyclic AMP
(cAMP). Upon activation, phospholipase C–mediated hydro-
lysis of the membrane phospholipid phosphatidylinositol-4,5-
bisphosphate (PIP2), results in the release of 2 secondary
messengers, inositol-1,4,5-triphosphate (IP3) and diacylglyc-
erol (DAG); phospholipase A2 hydrolyses phosphatidylcho-
line to PAF and the TxA2 precursor, arachidonic acid.
Intracellular IP3 directly induces calcium release from storage
pools in dense granules; DAG indirectly induces the same
response through activation of PKC. An increase in free cyto-
plasmic calcium, with concurrent reduction in intracellular
cAMP, is a universal response among mammalian platelets and
explains why drugs that function as calcium channel blockers
impact platelet aggregation. Nonsteroidal anti-inflammatory
drugs such as aspirin, which inhibit reactivity of cyclooxygen-
ase enzyme (COX-1), do not interfere with the aggregation
response in platelets from those species, for instance, cow, rat,
and some dogs, that are relatively insensitive to TxA2 as an
agonist.
 Disorders of Hemostasis 257

Vascular Tissue
damage repair Vessel wall
Endothelium
Vascular lumen
TF
FVII PL
FVIIa FIX FIXa FIX
FVIIIa FVIII PDGF
TF-FVIIa Ca2+ EGF

PL
FX FXa FX
Secretion
FVa FV
Ca2+

Prothrombin Thrombin Platelet activation

Fibrinogen Fibrin
(soluble) Adhesion
FXIII FXIIIa
Fibrin Aggregation
(insoluble)

Thrombus formation

Figure 2-241  The tissue factor (extrinsic) pathway that leads to thrombin generation and platelet
activation following vascular damage. The oval structures represent activated phospholipid-
expressing cells. EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PL, phos-
pholipid; TF, tissue factor.

Platelets are additionally essential for endothelial repair
and eventual clot retraction. During aggregation, mediators
such as platelet-derived growth factor (PDGF) and epidermal
growth factor (EGF) are released from α-granules (Fig. 2-241).
Platelet cytoskeleton proteins actin and myosin, in the presence
of increased cytoplasmic calcium, interact in a manner similar
to that in myocytes. The activation of these contractile pro-
teins results in the shrinkage of the platelet–fibrin meshwork,
which results in contraction of the clot. In conjunction with
fibrinolysis, the fibrin clot is eventually removed when repair
is complete.
Further reading
Boudreaux MK. Platelet structure. In: Weiss DJ, Wardrop KJ, editors.
Schalm’s Veterinary Hematology. 6th ed. Ames, Iowa: Wiley-
Blackwell; 2010. p. 561-568.
Boudreaux MK, Catalfamo JL. Platelet biochemistry, signal transduc-
tion, and function. In: Weiss DJ, Wardrop KJ, editors. Schalm’s
Veterinary Hematology. 6th ed. Ames, Iowa: Wiley-Blackwell; 2010.
p. 569-575.
Goggs R, Poole AW. Platelet signaling—a primer. J Vet Emerg Crit Care
2012;221:5-29.
Platelet disorders
Bleeding associated with decreased numbers of functional
platelets is characterized by petechial and ecchymotic hemor-
rhages, which form small pinpoint to coalescing endothelial
lesions in mucous membranes and capillary beds. Epistaxis,
melena, hematuria, and cutaneous bruising can also be
observed. This type of bleeding is not observed in coagulation
disorders involving predominantly thrombin and fibrin gen-
eration because platelet adhesion and aggregation responses
are adequate to seal small lesions in vessels low blood pressure.
For larger wounds, and in vessels with higher blood pressure,
hemostasis requires fully functional platelet aggregation and
fibrin formation so that a stable thrombus can form. Platelet
function disorders associated with prolonged bleeding times,
and normal platelet counts and coagulation profiles, may be
the result of defects in platelet adhesion, aggregation, or
granule release.
Evaluation of platelet function
Platelet concentration is assessed with an automated platelet
count and blood smear examination, typically performed as
part of a complete blood count (CBC). However, enumeration
alone is not sufficient to explain all platelet-related bleeding
disorders, because functional defects can occur despite adequate
numbers.
Platelet function tests are best reserved for patients with
normal platelet concentration, but clinical signs supportive of
a platelet disorder. The buccal mucosal bleeding time (BMBT)
is the time for bleeding to stop after a standardized cut is
made with a commercial spring-loaded device in the mucosal
surface of the upper lip. The BMBT is prolonged when there
is decreased concentration and/or dysfunction of platelets, or
if there is reduced activity of adhesive proteins, such as vWF.
This is a simple and inexpensive test, but difficult to standard-
ize because of inter-operator and animal variability.
The gold standard for assessment of platelet function is the
platelet aggregation test. The ability of platelets to aggregate in
response to in vitro stimulation with various agonists can be
determined in citrated whole blood (impedance) or
258 CHAPTER 2  •  Hematopoietic System
platelet-rich plasma (light transmission), but because this
requires specialized instrumentation and timely evaluation, its
usefulness tends to be limited to research laboratories.
The Platelet Function Analyzer (PFA) 100 and 200 models
provide a benchtop point-of-care alternative to the BMBT, but
tend to be only available in larger referral centers. Similarly,
platelet function can be assessed using viscoelastic technology,
such as thromboelastography (TEG), although the contribution
of platelets to the overall tracing can be difficult to easily
discern and may be compensated for by other components of
hemostasis in this in vitro setting.
Thrombocytopenia
In the absence of any other underlying coagulopathy or plate-
let dysfunction, petechial hemorrhages are usually not
observed in animals unless the platelet count falls below
25-30 × 109/L. In healthy animals, ~30-40% of the total plate-
let pool is sequestered in the spleen, so apparent thrombocy-
topenia may be observed when conditions causing splenomegaly
occur, by resulting in an increase in the splenic platelet pool.
However, because overall platelet mass is not decreased, clini-
cal signs should not occur in this situation.
Inherited thrombocytopenia has been reported in Cavalier
King Charles Spaniels. In this breed, the autosomal recessive
trait in β1-tubulin is asymptomatic, and is characterized by
fewer, but larger, platelets (macrothrombocytopenia). No bleed-
ing occurs because the cells, although decreased in number,
have increased volume. Overall platelet mass is maintained.
Other breeds, including Norfolk Terriers, may have a similar
defect. May-Hegglin anomaly is another disorder that may
result in macrothrombocytopenia. A single Pug dog was
reported to have neutrophil inclusions and large platelets
typical of those observed in people. The lesion is the result of
a mutation in the MYH-9 gene encoding nonmuscle myosin
heavy-chain IIA.
Acquired thrombocytopenia is one of the most commonly
recognized hematologic disorders. Thrombocytopenia associ-
ated with decreased platelet production in the bone marrow
may be the result of megakaryocyte hypoplasia for various
reasons. However, thrombocytopenia can also occur because
of decreased platelet survival in peripheral blood that can be
immune-mediated, infectious agent–mediated, or drug-
mediated. If mean platelet volume (MPV) is increased in a
thrombocytopenic animal, and is not associated with a breed-
associated defect, then active thrombopoiesis is suggested, and
a bone marrow disorder is less likely.
Immune-mediated thrombocytopenia (ITP) has been reported
in many breeds of dogs and in both young and old animals,
but may be over-represented in middle-aged females. Primary
idiopathic, or autoimmune thrombocytopenia, is caused by the
development of autoantibodies without an apparent underly-
ing disease, and may be the result of a loss of immune system
tolerance for platelet antigens. Dogs develop severe thrombo-
cytopenia and variable anemia, because of hemorrhage. There
is currently no specific diagnostic test for ITP, and it continues
to be one of exclusion of other causes.
Neonatal alloimmune thrombocytopenia has been reported
in pigs and horses. In pigs, the disorder usually occurs after 2
or more pregnancies. Piglets are frequently born healthy but
develop thrombocytopenia by 1 week of age. Diagnosis of the
disorder is one of exclusion, sepsis being the usual differential,
because infections are the most common cause of thrombo-
cytopenia in this species.
Disorders of Hemostasis
Secondary immune-mediated thrombocytopenia can be
caused by antiplatelet antibodies or may be the result of anti-
body binding to non–self-antigens that become absorbed onto
platelet membranes. These antibodies are produced in response
to infectious agents, drug exposure, or neoplasia. For example,
many rickettsial agents are associated with thrombocytopenia,
and development of petechiae and ecchymoses can occur in
dogs with these infections. Thrombocytopenia associated with
systemic lupus erythematosus (SLE) has been reported in
dogs, cats, and horses. Thrombocytopenia may accompany
immune-mediated hemolytic anemia in dogs for a few reasons,
but it is suspected some dogs have concurrent immune-
mediated platelet destruction.
Drug-induced thrombocytopenia occurs when administration
of medications either suppresses platelet production in the
bone marrow, or increases the rate of platelet destruction as
a consequence of drug-dependent antibodies that bind to
complementary antigenic sites on platelet membranes. Mac-
rophages in the liver and spleen recognize, engage, and remove
these altered platelets, resulting in thrombocytopenia.
Further reading
Botsch V, et al. Retrospective study of 871 dogs with thrombocytope-
nia. Vet Rec 2009;164:647-651.
Christopherson PW, et al. Evaluation and clinical application of platelet
function testing in small animal practice. Vet Clin North Am Small
Anim Pract 2012;42:173-188.
Gelain ME, et al. A novel point mutation in the (1-tubulin gene in
asymptomatic macrothrombocytopenic Norfolk and Cairn Terriers.
Vet Clin Pathol 2014;43:317-321.
Scott MA, Jutkowitz LA. Immune-mediated thrombocytopenia. In:
Weiss DJ, Wardrop KJ, editors. Schalm’s Veterinary Hematology. 6th
ed. Ames: Wiley-Blackwell; 2010. p. 586-595.
von Willebrand disease
von Willebrand disease (vWD) occurs because of a deficiency or
functional variation in von Willebrand factor (vWF). Considered
an extrinsic platelet disorder, bleeding in vWD is the result of
failure of platelets to adhere and form aggregates at sites of
vascular injury. Although rare in cats, horses, and cattle, vWD
is the most common inherited bleeding disorder in dogs. It occurs
in many breeds, with highest prevalence in Doberman Pin-
schers. In dogs, as in people, it is a heterogeneous group of
disorders that are classified into 3 types on the basis of severity
of bleeding, mode of inheritance, and specific abnormalities of
the vWF protein. The vWF protein is primarily synthesized in
endothelial cells as 2 subunits that subsequently undergo post-
translational assembly into dimers and multimers ranging up
to 100 vWF subunits that are stored in and subsequently
released from Weibel-Palade bodies in endothelial cells. The
larger-molecular-weight multimers are most effective in pro-
moting platelet adhesion.
The release of vWF following endothelial cell stimulation
occurs with a variety of compounds, such as thrombin, hista-
mine, estrogen, thyroxine, and the vasopressin analogue
1-desamino-8-D-arginine vasopressin (DDAVP). Strenuous
exercise also induces an increase in plasma vWF activity. In
some species, such as humans and cats, platelets act as an
additional pool of circulating vWF because they contain
megakaryocyte-derived vWF in their α-granules. However,
canine platelets contain only very small amounts of vWF.
 258.e1

Further reading
Herring J, McMichael M. Diagnostic approach to small animal bleeding
disorders. Top Compan Anim Med 2012;27:73-80.
Pedersen HD, et al. Idiopathic asymptomatic thrombocytopenia in
Cavalier King Charles Spaniels is an autosomal recessive trait. J Vet
Intern Med 2002;16:169-173.
 Disorders of Hemostasis
Plasma vWF circulates in a noncovalent complex with clotting
factor VIII (FVIII), which stabilizes and prolongs the circulat-
ing half-life of FVIII.
Type 1 vWD, which is characterized by decreased activity
of vWF antigen and a proportional decrease in all multimeric
forms, is transmitted in an incompletely dominant pattern. In
contrast, the more clinically severe forms of vWD, types 2 and
3, are recessive traits. In type 3, plasma vWF is virtually absent,
whereas in type 2, high-molecular-weight multimers are dis-
proportionately decreased, and plasma vWF antigen activity
is reduced. In all forms of the disease, bleeding may be exac-
erbated by concurrent thrombocytopenia or administration of
drugs, such as nonsteroidal anti-inflammatory drugs, which
impair platelet function.
Acquired vWD has been reported as a result of increased
degradation of multimers by the enzyme ADAMTS13 (a dis-
integin and metalloproteinase with thrombospondin repeats),
primarily in high-shear conditions, such as those induced with
cardiac valve disease. In ponies, administration of hydroxyeth-
ylene starch solutions as blood volume expanders can also
induce a transient decrease in circulating vWF levels. Tetra-
starch administration in dogs with systemic inflammation
induced a similar effect.
Prolonged BMBT accompanied by a normal platelet count,
and coagulation profile is suggestive of vWD. A prolonged
closure time with the PFA analyzer is also a supportive, but
not specific, result. Diagnosis can only be confirmed by specific
assay of plasma vWF antigen activity by ELISA. A collagen-
binding assay is also available at specialty laboratories to assist
in confirmation of the functional defect that occurs in type 2
vWD. Because vWF is antigenically distinct among mamma-
lian species, plasma samples must be analyzed with appropri-
ate reagents. Molecular testing for vWF based on known
mutations is available for some breeds of dogs.
Further reading
Brooks MB, Catalfamo JL. von Willebrand disease. In: Weiss DJ,
Wardrop KJ, editors. Schalm’s Veterinary Hematology. 6th ed.
Ames, Iowa: Wiley-Blackwell; 2010. p. 612-618.
Burgess HJ, et al. Evaluation of laboratory methods to improve char-
acterization of dogs with von Willebrand disease. Can J Vet Res
2009;73:252-259.
Lozier JN, Nichols TC. Animal models of hemophilia and related bleed-
ing disorders. Semin Hematol 2013;50:175-184.
Intrinsic disorders of platelet function
Inherited intrinsic platelet function disorders are relatively
rare in domestic animals, but have been identified in several
breeds of dogs, horses, and in cattle. Four types of platelet
dysfunction are recognized: disorders of platelet membrane gly-
coproteins, storage pool deficiencies, impairment of intracellular
signaling, and defective platelet procoagulant activity. All appear
to be inherited as autosomal traits. Because each results in
platelet dysfunction, clinical signs are similar among the
defects and cause mucosal bleeding and can be more diffuse
than the petechial hemorrhages observed with thrombocyto-
penia alone. Gingival hemorrhage can occur during shedding
of deciduous teeth in puppies. Epistaxis, cutaneous ecchymo-
ses, hematuria, and prolonged or excessive bleeding at sites of
surgery or trauma are common in adult dogs. In horses and
259
cattle, subcutaneous hematomas and prolonged hemorrhage
from puncture wounds, such as vaccination, ear tagging, and
insect bites, are typically encountered.
Glanzmann thrombasthenia has been documented predom-
inantly in the Otterhound and Great Pyrenees dog breeds,
with a few isolated cases reported in horses. The disorder is
characterized by a decrease in the number of platelet αIIbβ3
(GPIIb/IIIa) membrane receptors for fibrinogen. Therefore
platelets from homozygous-deficient animals exhibit normal
shape-change response following stimulation with agonists
such as ADP, collagen, thrombin, or PAF, but fail to form
stable aggregates. Clot retraction is also impaired or absent.
Because platelet morphology and concentration is normal, a
definitive diagnosis requires the analysis of platelet mem-
branes by flow cytometry, using antibodies specific for α- and
β-subunits of the receptor. All the mutations described thus
far are in the gene encoding GPIIb.
The bleeding disorder component of Chediak-Higashi syn-
drome (CHS) is the result of a dense-granule storage pool
deficiency. The disease has been identified in Hereford,
Brangus, and Japanese Black cattle, in Aleutian mink, Persian
cats, blue and silver foxes, killer whales, and mice. Dense
granules are either absent or indistinct in platelets from
affected animals. The in vitro aggregation response to ADP is
reduced as a result of a decrease in the secretion of ATP, ADP,
and serotonin, which may or may not be accompanied by a
decrease in intracellular calcium mobilization.
A defect in the P2Y12 receptor for ADP on the platelet
surface membrane has been recently described in a Greater
Swiss Mountain dog. There had been no prior bleeding epi-
sodes, but excessive hemorrhage was noted after ovariohyster-
ectomy, which prompted further investigation. The same
defect was present in both related and unrelated dogs of the
same breed.
Defective intracellular signaling transduction pathways have
been identified as the cause of platelet dysfunction in Basset
hounds, Landseer–European Continental Type (ECT), and
Eskimo Spitz dogs, as well as in both purebred and crossbred
Simmental cattle. These result from various mutations in the
gene encoding calcium diacylglycerol guanine nucleotide
exchange factor I (CalDAG-GEFI). This factor is required for
activation of Rap1b, which then produces a conformation
change in GPIIb-IIIa, which permits fibrinogen binding. All
have impaired aggregation responses to most agonists, except
for thrombin. In Basset Hounds, heterozygote prevalence
approaches 25-30%. Only Landseer-ECT dogs are affected,
and the prevalence in this breed is ~10%. In the Spitz dog,
the mutation is distinct from that in Basset Hounds and Land-
seers, and much less prevalent. Affected Simmental cattle may
have mild to severe bleeding, and also have a distinct CalDAG-
GEFI mutation from the dog breeds.
A defect in platelet procoagulant activity has been identified
in a colony of German Shepherd dogs. Similar to the disorder
in people called Scott syndrome, platelet morphology is
normal, and platelet function testing determined by BMBT,
clot retraction, PFA, thromboelastography, and aggregation is
unaffected. The defect is in the exposure of phosphatidylser-
ine on the platelet surface during activation. Because of this,
the platelets cannot provide an appropriate phospholipid
context for the formation of the tenase and prothrombinase
complexes, which reduces the rate of thrombin generation and
fibrin formation. The syndrome therefore causes bleeding more
typically observed with thrombin and fibrin formation
 259.e1

Further reading
Gauthier V, et al. Effect of synthetic colloid administration on coagula-
tion in healthy dogs and dogs with systemic inflammation. J Vet
Intern Med 2015;29:276-285.
260 CHAPTER 2  •  Hematopoietic System
impairment, such as epistaxis, hyphema, hematoma, and pro-
longed bleeding with cutaneous bruising after surgery.
Further reading
Boudreaux MK. Inherited intrinsic platelet disorders. In: Weiss DJ,
Wardrop KJ, editors. Schalm’s Veterinary Hematology. 6th ed.
Ames, Iowa: Wiley-Blackwell; 2010. p. 619-625.
Gentry PA, et al. An inherited platelet function defect in a Simmental
crossbred herd. Can J Vet Res 1997;61:128-133.
Acquired platelet disorders
Besides purposeful inhibition of platelet function with anti-
platelet drugs, acquired platelet dysfunction can occur secondary
to other underlying disorders. In many instances, transient
defects in platelet responses likely occur, but are rarely docu-
mented. This is in part because of the relatively short half-life
of a circulating platelet, so that new, fully functional platelets
are replenishing the circulating pool, and to the fact that
although one intracellular signaling pathway may be altered,
another may compensate. Furthermore, without other abnor-
malities such as vWD or thrombocytopenia, partial or tran-
sient suppression of platelet reactivity may not be enough to
induce clinical signs.
Uremia resulting from renal failure has been identified as
a risk factor for platelet dysfunction. Although abnormal
BMBT, platelet adhesion, and aggregation have been reported
in uremic dogs, the specific mechanisms involved have not yet
been identified.
Certain infectious agents have been implicated as causing
platelet dysfunction. In calves infected with noncytopathic
type 2 bovine viral diarrhea virus, bleeding was thought to
occur because of combined thrombocytopenia and impaired
aggregation. Dogs infected with Ehrlichia canis or Ehrlichia
platys develop thrombocytopenia and exhibit a reduction in
both platelet adhesiveness and aggregation. The mechanism is
not entirely clear, but may be the result of antiplatelet
antibodies.
Other potential causes of platelet dysfunction include
receptor-binding interference from increased fibrinolytic products,
such as occur with disseminated intravascular coagulation and
liver disease; defective adhesion and aggregation associated
with leukemias; and coating of platelets with protein in condi-
tions causing monoclonal gammopathy.
Many drugs are known to inhibit platelet function, either
as an intentional therapeutic benefit, or as an undesired side
effect. Nonsteroidal anti-inflammatory drugs, such as aspirin
and meloxicam, that inhibit the endogenous platelet cyclo-
oxygenase enzymes, inhibit platelet aggregation in most
species. There is species variation in this response, and bovine
platelets tend to be unresponsive to the inhibitory effects of
aspirin. Drugs such as clopidogrel inhibit ADP-induced plate-
let activity by irreversibly binding to P2Y12 receptors. Penicil-
lin antibiotics can bind to platelet membranes and inhibits
glycoprotein receptor-mediated adhesion and aggregation.
There are many drugs that can impact platelet function,
including barbiturates, calcium-channel blockers, hydroxy-
ethyl starches, to name a few.
Although not given as much consideration, platelet hyper-
reactivity can contribute to abnormal hemostasis through
enhanced platelet function, and may result in thrombosis.
Disorders of Hemostasis
Disorders causing hyper-reactivity of platelets include sys-
temic inflammatory disease, neoplasia, protein-losing nephrop-
athy, and diabetes mellitus.
Further reading
Fry MM. Acquired platelet dysfunction. In: Weiss DJ, Wardrop KJ,
editors. Schalm’s Veterinary Hematology. 6th ed. Ames, Iowa:
Wiley-Blackwell; 2010. p. 626-631.
Walz PH, et al. Effect of experimentally induced type II bovine viral
diarrhea virus infection on platelet function in calves. Am J Vet Res
1999;60:1396-1401.
Thrombin formation
Initiation
Exposure of blood to tissue factor (TF), which becomes
expressed on the membrane of activated endothelial cells or
monocytes at sites of vascular injury or during inflammation,
respectively, initiates coagulation in vivo. This mechanism of
thrombin generation is termed the tissue factor or extrinsic
pathway.
In the resting state, TF is a transmembrane glycoprotein
that is sequestered or inactive in endothelial cells and mono-
cytes (and possibly other leukocytes), perhaps because of lack
of appropriate procoagulant phospholipids on the membrane
surface. Upon activation, the extracellular domain of TF is
expressed on the cell surface in a configuration that facilitates
binding of TF to inactive factor VII (FVII), or the active form
of the enzyme, FVIIa. Although FVII is the predominant form
of the protein in blood, ~1% circulates as FVIIa. Binding of
FVII and FVIIa with TF initiates coagulation by activating
additional TF-FVIIa molecules and by converting both factor
IX (FIX) and factor X (FX) to their activated forms, FIXa and
FXa, respectively (see Fig. 2-241).
Factors IXa and Xa can either remain cell associated or
they can diffuse into blood and bind to the surface of nearby
activated platelets that have aggregated at the site of vascular
damage. If they diffuse further away from the developing clot,
an inhibitor inactivates them, to prevent widespread clot for-
mation. Prothrombin (factor II) is rapidly converted to throm-
bin by the prothrombinase complex that consists of FXa,
phospholipid, calcium, and cofactor factor V (FV). During
platelet activation, negatively charged phospholipids, such as
phosphatidylserine, become exposed on the platelet surface.
These phospholipids serve as binding sites for complex forma-
tion of coagulation factors, protein cofactors, and calcium,
which are essential for the rapid propagation of fibrin and
thrombus formation.
The small amount of thrombin generated by the prothrom-
binase complex initiates a positive feedback reaction that
accelerates the rate of prothrombin conversion and causes an
increase in the amount of thrombin generated that subse-
quently increases the rate of fibrin formation (see Fig. 2-241).
Thrombin also directly increases the activity of the prothrom-
binase complex by activating FX to FXa and via proteolytic
cleavage of FV to a more reactive form (FVa). Thrombin
further facilitates thrombus formation at a site of vascular
damage by promoting localized platelet activation, adhesion,
and secretory reactions that culminate in platelet aggregation
(see Fig. 2-241).
 260.e1

Further reading
Boudreaux MK. Characteristics, diagnosis, and treatment of inherited
platelet disorders in mammals. J Am Vet Med Assoc 2008;
233:1251-1259.
Boudreaux MK, Lipscomb DL. Clinical, biochemical, and molecular
aspects of Glanzmann’s thrombasthenia in humans and dogs. Vet
Pathol 2001;38:249-260.
260.e2

Further reading
Kavanagh C, et al. Coagulation in hepatobiliary disease. J Vet Emerg
Crit Care (San Antonio) 2011;21:589-604.
Mullins KB, et al. Effects of carprofen, meloxicam and deracoxib on
platelet function in dogs. Vet Anaesth Analg 2012;39:206-217.
 Disorders of Hemostasis
Amplification of thrombin generation
The small amount of thrombin formation in the initiation
phase cannot maintain further thrombin production enough
to ensure that sufficient fibrin is made to prevent hemorrhage.
This is because of the anticoagulant protein tissue factor
pathway inhibitor (TFPI) that circulates in blood bound to
lipoproteins and platelets, and is present on endothelial cell
membranes with heparan sulfate. TFPI binds to FXa, and then
forms the FXa-TFPI-FVIIa complex, that effectively inhibits
TF pathway thrombin generation. However, the other path-
ways that result in activation of FX, and hence thrombin
production, drive its further production instead (see Fig.
2-241).
Thrombin feedback not only activates FV, but it also acti-
vates factor IX, which converts FVIII to its active form, FVIIIa
(see Fig. 2-241). Factor VIII usually circulates bound to von
Willebrand factor (vWF), but after activation by thrombin,
FVIIIa dissociates and forms a complex with FIXa, calcium,
and phospholipid exposed on the surface of thrombin-
activated platelets. This is referred to as the tenase complex,
which cleaves FX at the same reactive site as TF-VIIa and
hence also produces FXa. This FXa generation results in for-
mation of further prothrombinase complexes, providing a
rapid burst of thrombin formation.
A tenase complex leading to thrombin formation can also
be generated by the contact activation or intrinsic pathway.
The contact activation pathway includes factor XII (FXII) and
factor XI (FXI), prekallikrein (PK), and the protein cofactor
high-molecular-weight kininogen (HMWK) (Fig. 2-242). This
pathway is initiated when FXII interacts with a negatively
charged surface (such as damaged endothelium, activated
neutrophils, polyphosphates) and becomes activated (XIIa),
HMWK PolyP
FXIIa tPa
FXII
FXIa FXI
FIX FIXa
Prothrombin Thrombin
tPa
TAFI
Plasminogen
(fibrin bound)
Plasmin
2-Antiplasmin
Fibrin
Figure 2-242  Interactions between the contact activation (intrinsic) pathway and the fibrinolytic
system. Solid lines represent activation reactions; dashed lines represent inhibitory reactions.
HMWK, high-molecular-weight kininogen; PAI-1, plasminogen activator inhibitor-1; PK, prekal-
likrein; PolyP, polyphosphates; TAFI, thrombin-activatable fibrinolysis inhibitor; tPA, tissue-type
plasminogen activator.
261
resulting in ability to cleave FXI to its proteolytically active
form, FXIa, and to convert PK to kallikrein. This reaction is
accelerated by HMWK. FXIIa can convert HMWK to a modi-
fied form, HMWKa, that exhibits high surface-binding affinity
and brings large amounts of both FXI and PK to the charged
surface in proximity to already adherent FXIIa. FXIa, in the
presence of calcium, is a potent activator of circulating FIX
and thus promotes downstream thrombin formation. Direct
activation of FXI by thrombin is another amplification loop,
which, in turn, results in the generation of additional FIXa
(see Fig. 2-242).
The complete role of the contact activation pathway is not
fully understood because it does not appear to be important
for most circumstances requiring in vivo thrombin generation.
Some species, including cetacean and nonmammalian verte-
brates do not have detectable FXII activity; cats with severe
FXII deficiency do not exhibit a bleeding phenotype. However,
kallikrein not only functions as an activator of the fibrinolytic
system, but it also stimulates both chemotaxis and degranula-
tion of neutrophils attracted to sites of vascular injury (see Fig.
2-242). Hence the contact activation pathway links inflamma-
tion, thrombin formation, and fibrinolysis.
There is some evidence to suggest that this pathway may
be involved in pathologic thrombus development, because FXII-
deficient mice were protected from thrombus formation in an
atherosclerosis model. This has also been observed in primates
where FXII activity was inhibited. Other studies have dem-
onstrated that bacteria may directly engage the contact
pathway via polyphosphate binding. These phosphates are
similar to those contained within platelet-dense granules.
Polyphosphates and histamine have both been demonstrated
to directly active FXII (see Fig. 2-242).
Vessel wall
Endothelium
Vascular lumen
HMWK PolyP
FXIIa
FXII
PK
scuPA
(prourokinase)
Kallikrein
uPA
(urokinase)
Inflammatory
PAI-I responses
Plasminogen
(soluble)
Fibrin degradation
products/D-dimer
Thrombus dissolution
262 CHAPTER 2  •  Hematopoietic System Disorders of Hemostasis

Fibrin formation Laboratory evaluation of thrombin

One of the final steps in clot formation is the conversion of
fibrinogen to an insoluble fibrin strand meshwork in a process
mediated by thrombin and activated factor XIII (FXIIIa) (see
Fig. 2.241). Fibrinogen is a large molecule with 2 pairs of
3-protein chains (Aα, Bβ, γ γ) forming a complex structure
linked via disulfide bonds. Thrombin first cleaves 2 small
peptide chains, fibrinopeptides A and B, from the amino-
terminal end of the Aα and Bβ chains, respectively, which
alters the surface charge of the protein. This produces soluble
fibrin monomers that polymerize by arranging end-to-end and
side-to-side to form a loose mesh connected via covalent
bonds. This net is then converted to a more insoluble fibrin
scaffolding that facilitates stable thrombus formation and
underlying repair. Factor XIIIa, a transpeptidase enzyme acti-
vated by thrombin cleavage of its precursor zymogen, FXIII,
catalyzes the formation of the stable internal peptide bonds
between adjacent γγ and α fibrin chains, in a calcium-dependent
reaction.
Thrombin is additionally important for activation of
thrombin-activatable fibrinolysis inhibitor (TAFI). This inhibitor
prevents premature degradation of the fibrin clot and partici-
pates in the activation of the protein C anticoagulant pathway
(TM-protein C-protein S) that minimizes thrombin genera-
tion outside the localized clot (Fig. 2-243).
Further reading
Licari LG, Kovacic JP. Thrombin physiology and pathophysiology. J Vet
Emerg Crit Care (San Antonio) 2009;19:11-22.
Renné T, et al. In vivo roles of factor XII. Blood 2012;120:4296-4303.
and fibrin formation
Preanalytical considerations, including careful blood sample
collection and sample handling techniques, are essential for
reliable in vitro evaluation of hemostasis. Although blood anti-
coagulated with ethylenediaminetetraacetic acid is required
for accurate platelet counts, citrated plasma is the preferred
sample type for general testing of the coagulation system and
measuring individual components of the procoagulant, fibri-
nolytic, and inhibitory pathways. The volume of blood to
citrate in the tube should be at a 9 : 1 ratio, because over- or
under-citration of the sample may produce erroneous results.
Patient samples should be interpreted in the context of appro-
priately developed species and laboratory-specific reference
values.
Routine hemostatic testing should include determination of
automated platelet concentration and mean platelet volume,
the one-stage prothrombin time (OSPT or PT), the activated
partial thromboplastin time (APTT), and fibrinogen concentra-
tion. The PT assay measures the integrity of the tissue factor
or extrinsic pathway, and the common pathway. The proce-
dure simply involves the addition of a thromboplastin (TF)-
phospholipid reagent to the patient’s plasma, followed by
determination of the time in seconds for a fibrin clot to form
once the sample has been recalcified. For the APTT assay, a
FXII activating agent, such as celite or kaolin, mixed with
phospholipid devoid of TF (partial thromboplastin) is added
to the patient’s plasma prior to recalcification. This assay
therefore determines the integrity of the contact activation or
intrinsic pathway, which includes the formation of the tenase
and prothrombinase complexes. Like the PT assay, it will also
be prolonged if there is a deficiency in the common pathway.

Vessel wall
Endothelium
Vascular lumen
TF FVIIa Thrombin Thrombomodulin

TFPI
Delay of
TM-T TAFI activation
fibrinolysis

FX FXa
APC Protein C
PS
AT
PS
FVIIIai FVIIIa FIXa
FVa FVai
FXa FX

Prothrombin Thrombin
Antithrombin/
heparans

Thrombin-antithrombin

Fibrinogen Fibrin

Figure 2-243  The major anticoagulant pathways. Solid lines represent activation reactions, and
dashed lines represent inhibitory reactions. APC, activated protein C; AT, antithrombin; FV, factor
V; FVai, inhibitory form of FV; FVIIIai, inhibitory form of FVIII; PS, protein S; TAFI, thrombin-
activatable fibrinolysis inhibitor; TFPI, tissue factor pathway inhibitor; TM-T, thrombomodulin-
thrombin complex.
 262.e1

Further reading
Hoffman M, Monroe DM. A cell-based model of hemostasis. Thromb
Haemost 2001;85:958-965.
 Disorders of Hemostasis
Clotting times for either assay become prolonged once activity
of one or more factors that the test assesses decreases to below
~30% activity of normal pooled plasma. Prolonged clotting
times will also occur if inhibitors are present in the patient’s
plasma.
The activated clotting time (ACT) is similar to the APTT in
that it also assesses integrity of the contact activation or intrin-
sic pathway, and common pathway, but is less sensitive for
detection of deficient activity. For this assay, whole blood is
added directly to a tube containing a contact activator, the
sample is incubated at 37o C, and the time in seconds for initial
clot formation is noted. There is no need to add a source of
phospholipid because this is provided by the patient’s own
platelets in the sample. Therefore severe thrombocytopenia
may produce a longer ACT. The result for this assay becomes
prolonged once activity of one or more factors that the test
assesses decreases to below ~5% activity of normal pooled
plasma.
Fibrinogen concentration can be quantitated by several
methods, including physical and immunologic procedures. A
modified thrombin time assay, which quantifies the amount
of fibrinogen in a diluted test sample on the basis of the rate
of formation of fibrin, is currently most commonly used in
veterinary laboratories.
The individual activity of FXII, FXI, FIX, and FVIII are
determined based on a modification of the APTT assay,
whereas the activity of FVII, FX, and FV are determined
with a modification of the PT assay. The ability of the
patient’s plasma sample to correct the clotting time of a spe-
cific factor-deficient plasma relative to a reference standard is
used to quantitate the activity of a specific factor. Such assays
developed for domestic animals use human factor–deficient
plasma, as species-specific reagents are not readily available.
Some clotting factors can also be assessed using colorimetric
methods, but these are developed for use with human plasma
samples and have not been thoroughly evaluated for use in
animals.
Global assays of hemostasis have recently been validated to
a greater extent in veterinary medicine. Although the technol-
ogy has been available for some time, particularly for visco-
elastic assessment of thrombus formation, widespread use for
diagnosis of hemostatic abnormalities in animals did not occur
until the last decade. The advantages of methods such as
thromboelastography include use of whole blood, rapid analysis,
and assessment of the entire clotting process from clot initia-
tion to fibrinolysis with a single sample. Disadvantages include
requirement for immediate analysis, and poor standardization
between laboratories. It is also unclear exactly how well results
from these methods correlate with those from the traditional
tests of hemostasis. Plasma-based thrombin generation assays
also provide global hemostatic data, but have minimally been
used for animal samples to date.
Further reading
Brooks MB, Catalfamo JL. Current diagnostic trends in coagulation
disorders among dogs and cats. Vet Clin North Am Small Anim
Pract 2013;43:1349-1372.
Kol A, Borjesson DL. Application of thrombelastography/
thromboelastometry to veterinary medicine. Vet Clin Pathol
2010;39:405-416.
263
Disorders of thrombin formation
Inherited disorders
Factor VIII deficiency (hemophilia A) is one the most common
inherited coagulopathies in animals, and has been reported in
many breeds of dogs, in cats, and occasionally in horses and
cattle. It is an X-chromosomal recessively inherited disease in
which heterozygous females, the carriers of the disease, are
clinically asymptomatic. In homozygous-deficient males, the
clinical severity of the disorder varies with the extent of reduc-
tion of FVIII activity. Unlike severely affected animals (<2% of
FVIII activity), mildly affected animals (>5% of FVIII activity)
seldom exhibit spontaneous bleeding. Stress, exercise, vaccina-
tion, surgery, and trauma may induce bleeding episodes. In
puppies, the first clinical signs may be hematomas and gingival
bleeding as permanent teeth erupt. As the dog ages, intermit-
tent shifting leg lameness resulting from hemarthrosis, particu-
larly in the stifle joints, and subcutaneous and intramuscular
hematomas are common. Excessive bleeding resulting in large
scrotal hematomas following castration also occurs. Mildly
affected male dogs can reach sexual maturity, and if they are
bred with a heterozygous female, homozygous-deficient
bitches can result. These animals may exhibit protracted
estrus or postpartum hemorrhage.
Hemophilia A is suggested by signalment, clinical signs,
prolonged ACT or APTT with normal PT, and confirmed by
decreased plasma FVIII activity.
Factor IX deficiency (hemophilia B) is less common than
FVIII deficiency and has been reported in dogs and cats. It has
been described in several breeds of dogs, and is the second
most common inherited disorder in British Shorthair cats.
Like hemophilia A, it is inherited as an X-chromosomal reces-
sive trait and has similar clinical signs, with both the circulat-
ing plasma activity of FIX and the size of the animal influencing
the severity of bleeding. The reduced plasma FIX activity
results in prolonged ACT and APTT, but normal PT. Confir-
mation of reduced FIX activity is required to make a definitive
diagnosis.
Prolonged APTT and normal PT is also expected for
animals with inherited defects involving the other proteins of the
contact activation or intrinsic pathway, including, FXI, FXII,
and PK. The defect in each case is inherited in an autosomal
recessive manner. Of these, only FXI deficiency may cause
affected animals to exhibit spontaneous bleeding, most com-
monly from the urogenital tract. It has been identified in
several dog breeds, including English Springer Spaniels, Kerry
Blue Terriers, and Great Pyrenees dogs, and in Holstein and
Japanese Black cattle. Homozygous cattle have <5% of normal
plasma FXI activity, but the response to trauma or surgery
ranges from almost non-existent to severe hemorrhage devel-
oping 12-48 hours later.
Prekallikrein deficiency is minimally documented, partly
because it is usually clinically asymptomatic. It was only found
fortuitously in a German Short-haired Pointer bitch that sub-
sequently underwent uneventful ovariohysterectomy. Even in
large animals with PK deficiency, such as Miniature and
Belgian horses, bleeding is rare, although it has been reported
in a horse following castration.
Factor XII deficiency is inherited as an autosomal recessive
trait, and has been observed in dogs and cats. Bleeding does
not occur in affected animals, and the defect is often discov-
ered by chance in cats that have an APPT performed for
another reason. The absence of hemorrhage is consistent with
 263.e1

Further reading
Herring J, McMichael M. Diagnostic approach to small animal bleeding
disorders. Top Compan Anim Med 2012;27:73-80.
Lubas G, et al. Laboratory testing of coagulation disorders. In: Weiss
DJ, Wardrop KJ, editors. Schalm’s Veterinary Hematology. 6th ed.
Ames, Iowa: Wiley-Blackwell; 2010. p. 1082-1100.
264 CHAPTER 2  •  Hematopoietic System
observations that FXII activity is absent in nonmammalian
species such as birds, reptiles, and amphibians.
Factor VII deficiency has only been documented in dogs and
people. It has been reported in Beagles, Alaskan Malamutes,
Boxers, Bulldogs, Miniature Schnauzers, and mixed-breed
dogs, and is inherited as an autosomal trait. Adult dogs with
the trait usually have plasma FVII activity of at least 5% of
normal pooled plasma, which appears sufficient to initiate
thrombin generation and induce fibrin formation unless there
are other concurrent abnormalities. For example, FVII defi-
ciency was diagnosed in a mixed-breed dog that exhibited
abnormal bleeding following routine castration and failed to
respond to vitamin K therapy. FVII deficiency is generally mild
and characterized by bruising. Prolonged vaginal bleeding after
whelping can occur in affected bitches. The diagnosis is sus-
pected based on signalment and history, with prolonged PT
and normal APTT. Decreased FVII activity can be documented
with a specific factor assay.
Severe factor X deficiency is inherited as an autosomal
recessive trait, and tends to be lethal in affected dogs because
of its importance for thrombin generation. Dogs with <10%
of normal plasma FX activity are usually stillborn or die
from bleeding as neonates. The defect has been reported in
Jack Russell Terriers and in mixed-breed dogs. Because FX is
part of the common pathway, both the APTT and PT are
prolonged.
Vitamin-K-dependent γ-glutamyl carboxylase deficiency has
been identified in Devon Rex cats, Rambouillet sheep, and in
a single Labrador Retriever dog. Lack of adequate activity of
this enzyme results in synthesis of quantitatively normal, but
nonfunctional, vitamin K–dependent coagulation proteins
(FII, FVII, FIX, FX). In homozygous-deficient sheep, low
levels of activity of all 4 affected proteins are typically found,
whereas heterozygous carrier animals may have normal factor
activities. Lamb mortality can be high, and is related to umbil-
ical and subcutaneous tissue hemorrhages. In affected Devon
Rex kittens, bleeding is severe or fatal and typically manifests
as hematomas and hemarthroses, as well as hemothorax and
hemoabdomen. Administration of vitamin K1 results in rapid
recovery of circulating vitamin K–dependent factor activity to
near-normal levels. In the affected dog, circulating activity
only increased to 50-60% of normal canine pooled plasma
even after 3 months of vitamin K1 therapy.
Further reading
Barr JW, McMichael M. Inherited disorders of hemostasis in dogs and
cats. Top Compan Anim Med 2012;27:53-58.
Bender DE, et al. Molecular characterization of cat factor XII gene and
identification of a mutation causing factor XII deficiency in a
domestic shorthair cat colony. Vet Pathol 2014;52:312-320.
Giger U. Hereditary blood diseases. In: Feldman B, et al., editors.
Schalm’s Veterinary Hematology. 5th ed. Philadelphia: Lippincott
Williams & Wilkins; 2000. p. 955-959.
Lozier JN, Nichols TC. Animal models of hemophilia and related bleed-
ing disorders. Semin Hematol 2013;50:175-184.
Acquired disorders
Vitamin K antagonism.  The primary function of vitamin K
in animals is as a cofactor for γ-glutamyl carboxylase, the enzyme
required for post-translational carboxylation of glutamate
(Gla) residues into γ-carboxyglutamate. Vitamin K hydroqui-
Disorders of Hemostasis
none is the active coenzyme, and its oxidation into vitamin K
2,3 epoxide produces the carboxylation reaction. Vitamin K
is a fat-soluble vitamin, and is widely available in green leafy
plants and vegetables oils and, like γ-glutamyl carboxylase, is
widely distributed throughout the body. Impaired carboxyl-
ation of vitamin K–dependent coagulation proteins is occa-
sionally the result of dietary deficiency or impaired
gastrointestinal absorption of vitamin K, but is more often
caused by ingestion of certain anticoagulant rodenticides.
These vitamin K antagonists inhibit the recycling of vitamin
K 2,3 epoxide back to the hydroquinone form through inhibi-
tion of vitamin K epoxide reductase. The gradual reduction in
vitamin K hydroquinone leads to a decrease in the active
forms of vitamin K. Because the function of Gla residues in
FVII, FIX, FX, and prothrombin is to facilitate binding to
negatively charged phospholipids on the surface of activated
endothelium or platelets, the rate of thrombin formation is
reduced. The nonfunctional forms of these proteins are col-
lectively referred to as PIVKAs (proteins induced by vitamin K
antagonism). There is a plasma-based assay for detecting these
inactive forms.
Rodenticide intoxication is the most common cause of vitamin
K antagonism in dogs, but it has also been reported in cats,
horses, and pigs. The first generation rodenticides (such as
warfarin) had relatively low toxicity and biological half-lives
of <24 hours, which meant that vitamin K therapy often
resulted in rapid and effective resolution of signs. The devel-
opment of rodent resistance to these compounds led to the
development of subsequent-generation agents, such as broma-
diolone, brodifacoum, and diphacinone, which are more
potent, have longer half-lives, and which therefore require
more aggressive longer-term vitamin K therapy in affected
animals.
Clinical signs are dependent on the quantity and type of
agent ingested, and can range from mild hemorrhage to severe
spontaneous bleeding in the form of epistaxis, melena, and
hematuria. It may be several days after ingestion before clini-
cal signs occur and appear acute in onset. If bleeding is severe,
anemia, weakness, and pallor develop; dyspnea can be associ-
ated with hypovolemia from hemothorax. Hemarthrosis has
been reported to occur in some cases.
Cattle, pigs, and rabbits are susceptible to vitamin K antag-
onism if hay or silage containing moldy sweet clover is ingested.
Sweet clover contains coumarin, which is converted to dicu-
marol. Young calves are more susceptible to dicumarol than
are the dams, whereas sheep and horses appear resistant to
this form of vitamin K antagonism.
Because FVII has the shortest circulating half-life (~4-6
hours), it is the first factor to have decreased biological activ-
ity. Therefore, in the initial stages of vitamin K antagonism,
the PT may be prolonged, whereas the APTT is within refer-
ence intervals. However, the APTT soon becomes prolonged
as well because FIX and FX have plasma half-lives of only
14-18 hours. Toxicologic evaluation of stomach contents can
determine the specific agent ingested, which can be useful
information for deciding on the duration of therapy.
Liver disease.  Although many coagulation proteins are
made in hepatocytes, hemorrhage related to liver disease is
infrequent in domestic animals. This may be in part because
severe loss of hepatic parenchyma (<30% remaining) must
have occurred before impairment of protein synthesis is suf-
ficiently affected to prolong the PT and APTT. Additionally,
synthesis of inhibitory proteins, such as antithrombin and
 264.e1

Further reading
Baker DC, et al. Hereditary deficiency of vitamin-K-dependent coagula-
tion factors in Rambouillet sheep. Blood Coag Fibrinol 1999;10:
75-80.
Gentry PA, Ross ML. Coagulation factor XI deficiency in Holstein cattle:
expression and distribution of factor XI activity. Can J Vet Res
1995;58:242-247.
Macpherson R, et al. Factor VII deficiency in a mixed breed dog. Can
Vet J 1999;40:503-505.
Mason DJ, et al. Vitamin-K dependent coagulopathy in a Black Labra-
dor Retriever. J Vet Intern Med 2002;16:485-488.
Soute BAM, et al. Congenital deficiency of all vitamin K-dependent
blood coagulation factors due to a defective vitamin K dependent
carboxylase in Devon Rex cats. Thromb Haemost 1992;68:
521-525.
 Disorders of Hemostasis
protein C, and fibrinolytic proteins, such as antiplasmin, will
also be reduced, potentially resulting in a rebalanced system.
Also, in some inflammatory diseases, the liver can increase the
rate of synthesis of specific clotting factors (acute phase
response), including fibrinogen, FVIII, and vWF, which together
can promote the efficiency of clot formation. Despite this, it
is commonly advised to screen for hemostatic competency
with PT and PTT prior to liver biopsy, because complications
of underlying disease may easily disturb this rebalanced
system. However, there is often no clear correlation between the
results of the screening tests and risk of bleeding postbiopsy.
In a study of 45 cats with liver disease, 98% exhibited at
least one coagulation test abnormality. The more severe the
liver disease, the more likely the cat was to develop clinical
bleeding. Interestingly, one of the more frequent abnormalities
noted was decreased FXIII activity, which was present with
both inflammatory and neoplastic processes. Because of a con-
stellation of other findings, it was concluded that, overall,
coagulation abnormalities in this cohort of cats with liver
disease were the result of consumption rather than decreased
synthesis.
Coagulation was assessed with thromboelastography
(TEG) in 10 dogs with extrahepatic bile duct obstruction. It
was concluded that many of these dogs were actually in a
hypercoagulable state rather than being predisposed to hem-
orrhage. Decreased production of inhibitor proteins may be
to blame. These findings reinforce the idea that hemostatic altera-
tions in hepatobiliary disease are not necessarily predictable, and
are likely multifactorial.
Cholestasis resulting in poor vitamin K absorption, and
therefore formation of inactive FII, FVII, FIX, and FX, may
occur in animals with chronic partial or complete biliary
obstruction. Vitamin K therapy given parenterally can restore
production of activatable proteins.
Further reading
DeClementi C, Sobczak BR. Common rodenticide toxicoses in small
animals. Vet Clin North Am Small Anim Pract 2012;42:349-360.
Dircks B, et al. Haemostatic abnormalities in cats with naturally occur-
ring liver diseases. Vet J 2012;193:103-108.
Mayhew PD, et al. Evaluation of coagulation in dogs with partial or
complete extrahepatic biliary tract obstruction by means of throm-
boelastography. J Am Vet Med Assoc 2013;242:778-785.
Disorders of fibrin formation
Hereditary fibrinogen defects are rare, and have only been rec-
ognized in goats and dogs. Afibrinogenemia has been reported
in a herd of Saanen goats, in which it was associated with
severe umbilical bleeding, hemarthroses, and subcutaneous
and oral bleeding. Inherited hypofibrinogenemia has been
diagnosed in Bernese Mountain, Lhasa Apso, Vizsla, and Collie
dogs. Similar to dysfibrinogenemia in an inbred Borzoi dog
family and in a Border Leicester lamb, these fibrinogen abnor-
malities cause only mild bleeding that can be exacerbated by
stress, trauma, or surgery. Decreased fibrinogen concentration
is more commonly because of decreased synthesis from severe
liver disease, or increased consumption with disseminated
intravascular coagulation.
Hyperfibrinogenemia as a positive acute-phase response is
common in many domestic animals; however, thromboem-
bolic complications are infrequently reported in veterinary
265
medicine. Thromboembolism (TE) does occur in animals,
usually as a secondary consequence of cardiac disease, immune-
mediated hemolytic anemia, neoplasia, hyperadrenocorticism,
protein-losing nephropathy and enteropathy, and sepsis.
Canine immune-mediated hemolytic anemia is a known risk
factor for development of pulmonary TE. Increased expres-
sion of tissue factor, phospholipid-expressing microparticles,
and platelet hyperactivity have been proposed as underling
predisposing mechanisms. Immunosuppressive doses of gluco-
corticoids used for treatment of this disease have also been
suggested to promote hypercoagulability, but the association
and mechanism has not been definitively proven to date.
Elevated fibrinogen concentration, and hypercoagulable
TEG tracings have been observed in dogs with hyperadreno-
corticism, but the overall risk of TE remains unclear. Decreased
antithrombin has been proposed in the development of a
hypercoagulable state in dogs with this disease, but this has
not been borne out in some studies.
Further reading
Kidd L, Mackman N. Prothrombotic mechanisms and anticoagulant
therapy in dogs with immune-mediated hemolytic anemia. J Vet
Emerg Crit Care (San Antonio) 2013;23:3-13.
Mendez-Angulo JL, et al. Thromboelastography in healthy, sick non-
septic and septic neonatal foals. Aust Vet J 2011;89:500-505.
Park FM, et al. Hypercoagulability and ACTH-dependent hyperadreno-
corticism in dogs. J Vet Intern Med 2013;27:1136-1142.
Regulation of coagulation:
endogenous anticoagulants
To prevent widespread and over-exuberant clot formation
when procoagulant mechanisms are invoked, inhibition of acti-
vated enzymes, such as FXa and thrombin, is essential because
they can diffuse away from a developing thrombus into the
general circulation. There are 3 important mechanisms that
perform this function, including antithrombin/heparin, the
protein C pathway, and tissue factor pathway inhibitor (TFPI).
Antithrombin (AT) is the major inhibitor of both thrombin
and thrombin generation. It is a member of the serine protease
inhibitor family of proteins that also includes α1-antitrypsin,
α2-antiplasmin, and α2-macroglobulin. Plasma AT is an α2-
globulin produced by hepatocytes and endothelial cells. It
predominantly inhibits thrombin, FIXa, and FXa, with addi-
tional weak inhibition of other activated factors, including the
TF-FVIIa complex. The inhibitory action of AT arises from
the interaction of its reactive arginine residue with a serine
residue at the reactive site on thrombin, in a 1 : 1 ratio. The
thrombin-AT (TAT) complex prevents thrombin’s ability to cleave
fibrinogen to fibrin (see Fig. 2-243). The inhibitory effect of AT
is enhanced by heparan sulfate complexes on the surface of
activated endothelial cells. Intravenously administered heparin,
or heparin released from mast cells, can also increase the rate
of formation of TAT complexes. Any TAT complexes are
removed from circulation via the mononuclear phagocyte
system or by becoming bound to the endothelial cells, which
results in endocytosis and lysosomal degradation.
The protein C pathway regulates coagulation by inactivating
FVa and FVIIIa. Protein C is synthesized in the liver as a
vitamin K–dependent zymogen. It is converted to an activated
form, APC, on the surface of endothelial cells by thrombin
that has become bound to a membrane protein called
 265.e1

Further reading
Kavanagh C, et al. Coagulation in hepatobiliary disease. J Vet Emerg
Crit Care (San Antonio) 2011;21:589-604.
Waddell LS, et al. Anticoagulant rodenticide screening in dogs: 123
cases (1996-2003). J Am Vet Med Assoc 2013;242:516-521.
265.e2

Further reading
Goodwin LV, et al. Hypercoagulability in dogs with protein-losing enter-
opathy. J Vet Intern Med 2011;25:273-277.
Rose L, et al. Effect of canine hyperadrenocorticism on coagulation
parameters. J Vet Intern Med 2013;27:207-211.
266 CHAPTER 2  •  Hematopoietic System
thrombomodulin (TM) in a thrombin-TM complex (see Fig.
2-243). Thrombomodulin is present on endothelial cells in
arteries, capillaries, veins, and lymphatic vessels. It is present
in high amounts on capillaries where blood flow is slower and
thrombin concentrations are highest, such as in the lungs. APC
cleaves phospholipid membrane-bound cofactors to inactivate
FVa and FVIIIa that then result in suppression of both pro-
thrombinase and tenase activity. Cleavage of these factors
within the phospholipid-enzyme complexes is facilitated by a
vitamin K–dependent cofactor, protein S, that forms a
membrane-bound complex with APC. Only the free circulat-
ing form of protein S binds with APC. This form represents
only ~30% of the total plasma concentration, as most circu-
lates bound to complement regulator C4-binding protein.
Also part of the protein C pathway is endothelial protein C
receptor (EPCR), which amplifies the activation of protein C
by TAT complexes, and also stimulates downstream anti-
inflammatory and cytoprotective effects.
Although antithrombin and the protein C pathway are
more important for regulating elements of the contact or
intrinsic pathway, tissue factor pathway inhibitor (TFPI) per-
forms a similar function for the tissue factor or extrinsic
pathway. TFPI is produced in endothelial cells and inhibits
both TF-FVIIa and TF-FVIIa-FXa complexes. Its activity is
enhanced by heparin-like glycosaminoglycans as well as
protein S. TFPI functions by rapidly inhibiting any free FXa
generated by the TF-FVIIa complex on subendothelial fibro-
blasts during the initiation phase of coagulation.
Inhibitor disorders
Inherited antithrombin deficiency has been described in
people, but it has not been identified in domestic animals.
Acquired deficiencies in AT activity occur in animals with
protein-losing nephropathy, sepsis, and disseminated intravas-
cular coagulation (DIC). Decreased AT activity can be accom-
panied by an increase in TAT complexes. In dogs, the
hypercoagulable state associated with these disorders is at
least partly attributed to decreased plasma AT activity.
Reduced plasma AT was found in a foal with thrombosis of
the brachial artery, and occurs in horses with colic. There are
laboratory assays available for quantification of AT in plasma.
Human-based colorimetric and coagulation laboratory
assays have been validated and are available to quantify the
protein C component of the protein C pathway in dogs. Protein
C activity may be reduced in cases of vitamin K deficiency or
antagonists. DIC-induced consumption of protein C has been
reported in dogs and primates. Protein C concentrations have
been used to facilitate diagnosis of congenital and acquired
liver disease in dogs.
Tissue pathway factor inhibitor activity is not routinely
measured in animals.
Further reading
Bentley AM, et al. Alterations in the hemostatic profiles of dogs with
naturally occurring septic peritonitis. J Vet Emerg Crit Care
2013;23:14-22.
Kubier A, O’Brien M. Endogenous anticoagulants. Top Companion
Anim Med 2012;27:81-87.
Smith S. Overview of hemostasis. In: Weiss DJ, Wardrop KJ, editors.
Schalm’s Veterinary Hematology. 6th ed. Ames, Iowa: Wiley-
Blackwell; 2010. p. 635-653.
Disorders of Hemostasis
Fibrinolysis
The fibrinolytic system results in clot dissolution via proteolytic
degradation of fibrin, once underlying tissue repair has occurred.
The central enzyme mediating fibrinolysis is plasmin, which is
formed from the precursor, plasminogen, by 1 of 2 endogenous
activators: tissue-type plasminogen activator (tPA) and urokinase
(uPA) (see Fig. 2-242). Tissue-type plasminogen activator pre-
dominantly activates plasminogen within the vascular system,
whereas uPA is responsible for extravascular plasminogen acti-
vation. Plasminogen is synthesized by hepatocytes and is a
trypsin-like enzyme with broad substrate specificity. In addi-
tion to degrading fibrin strands, it can degrade fibrinogen, FV
and FVIII, extracellular matrix components, and can activate
other proteases such as matrix-metalloproteinases. Plasmin
formation from plasminogen and its activity are highly regu-
lated. For example, tPA and uPA have very short circulating
half-lives relative to plasminogen, partly because of their rapid
clearance via endothelial cell surfaces. Also, the balance between
inhibitors and activators of plasminogen typically favors inhibi-
tion of plasmin formation in healthy animals. The major inhibi-
tor of plasminogen activation is plasminogen activator
inhibitor-1 (PAI-1), which can block the action of both tPA
and uPA (see Fig. 2-242). PAI-1 is typically in excess over tPA,
so most plasma tPA circulates as an inactive tPA–PAI-1
complex. The balance can be altered by the increased release
of tPA from endothelial cells in response to exercise, vascular
occlusion, or in response to locally generated thrombin at a
site of vascular damage. In plasma, the primary inhibitor of
plasmin activity is α2-antiplasmin, but α2-macroglobulin and
α1-antitrypsin are also capable of acting as inhibitors.
Endothelium-mediated fibrinolysis
Initially during clot formation, activation of plasminogen is
suppressed so that fibrin strands develop. Although thrombin
potently induces synthesis and secretion of endothelial tPA,
this profibrinolytic action is opposed by localized activity of
tissue-activatable fibrinolysis inhibitor (TAFI) (see Figs. 2-242,
2-243). High concentrations of thrombin generated both by
the positive feedback activation of FXI to FXIa and activation
of the tenase complex, combined with binding of thrombin
to thrombomodulin, enhances the activation of TAFI, which
inhibits tPA activation of plasminogen. Most PAI-1 is present
in platelets, which is released from α-granules during platelet
activation.
Once sufficient fibrin has been formed, plasmin formation and
fibrinolysis is promoted. Most fibrinolytic proteins in thrombi
are at concentrations representing 10-30% of their concentra-
tion in plasma, consistent with trapping of plasma proteins in
the developing thrombus. Fibrin itself potentiates fibrinolysis
by bringing together plasminogen and tPA on its surface,
which facilitates enzyme-substrate interactions and protects
tPA from inhibition by TAFI and PAI-1. Plasmin interacts with
fibrin strands at both its active site and at specific lysine-
binding sites, which decreases the ability of α2-antiplasmin to
interact with and inhibit plasmin generated within the fibrin
meshwork (see Fig. 2-242).
Plasma-mediated fibrinolysis
Unlike tPA, uPA readily activates plasminogen in plasma, even
in the absence of fibrin. It is unclear how uPA participates in
hemostasis, but it is evident from transgenic and knockout
mouse models that the tPA and uPA activators can compen-
sate for each other. Fibrin is rapidly deposited after injury, not
 266.e1

Further reading
Fry MM, et al. Protein C activity in dogs: adaptation of a commercial
human colorimetric assay and evaluation of effects of storage time
and temperature. Vet Med Int 2011;2011:751849.
Toulza O, et al. Evaluation of plasma protein C activity for detection of
hepatobiliary disease and portosystemic shunting in dogs. J Am Vet
Med Assoc 2006;229:1761-1771.
 Disorders of Hemostasis
only to prevent blood loss but also to provide a scaffold for
cells required during the subsequent healing process. Plasmin
and uPA are important for the slow dissolution of fibrin that
occurs once underlying tissue repair has occurred. Also in
contrast to tPA, uPA has no specific affinity for fibrin and
thus can activate circulating and fibrin-bound plasminogen
indiscriminately.
Initially, any plasmin generated outside a fibrin clot is
quickly neutralized by α2-antiplasmin. However, if the forma-
tion of plasmin exceeds inhibitor availability, it will begin to
degrade other plasma proteins, such as fibrinogen, FV, and
FVIII. Some bacteria produce plasminogen activators that can
act exogenously, thereby inducing inappropriate plasmin for-
mation. An example is streptokinase, which is produced by
strains of streptococci that use plasmin to their advantage to
increase invasiveness. In many mammals, streptokinase acts by
forming a 1 : 1 complex with either plasminogen or plasmin.
In this form, the general proteolytic action of streptokinase is
decreased, but its plasminogen-activating activity is increased
several fold.
Primary disorders of the fibrinolytic system have not been
characterized in domestic animals. However, alterations of
fibrinolysis associated with severe inflammation are frequently
recognized with disseminated intravascular coagulation.
Laboratory assessment of fibrinolysis in animals is limited to
a few assays, and include determination of fibrin(ogen) degra-
dation products (FDPs) and D-dimers. FDPs derive from both
fibrinogen and fibrin strands; D-dimers form only when cross-
linked fibrin is degraded. Fibrinolysis can also be assessed using
global assays of hemostasis such as thromboelastography.
Further reading
Schaller J, Gerber SS. The plasmin-antiplasmin system: structural and
functional aspects. Cell Mol Life Sci 2011;68:785-801.
Smith S. Overview of hemostasis. In: Weiss DJ, Wardrop KJ, editors.
Schalm’s Veterinary Hematology. 6th ed. Ames, Iowa: Wiley-
Blackwell; 2010. p. 635-653.
Stokol T, et al. Evaluation of latex agglutination kits for detection of
fibrin(ogen) degradation products and D-dimer in healthy horses
and horses with severe colic. Vet Clin Pathol 2005;34:375-382.
Systemic inflammation and neoplasia:
dysregulation of hemostasis
Inflammatory mediators, especially if disseminated widely, can
alter hemostatic equilibrium and cause either hemorrhagic or
thrombotic complications. The role of the contact activation or
intrinsic pathway as a link between hemostasis and inflamma-
tion has long been recognized. In addition to initiating throm-
bin formation through the FXI-tenase pathway (see Fig.
2-242), activation of FXII results in conversion of PK to kal-
likrein, which converts high-molecular-weight kininogen to the
inflammatory mediator bradykinin. Recent reports suggest
that both factor XII and kallikrein can promote activation of
FIX and prothrombin via FXIa, in the presence of polyphos-
phates, such as those found in bacteria or in platelet
granules.
An increase in TF expression on the cell membrane of
monocytes and endothelial cells occurs in response to both
gram-negative and gram-positive bacteria. This can result in
activation of FVII, FX, and thrombin, setting off a series of
intracellular signaling cascades in leukocytes. Inflammatory
267
responses induced include leukocyte recruitment, release of
additional cytokines, and production of reactive oxygen
species. Systemic thrombin generation can result in widespread
cleavage of fibrinogen and deposition of fibrin strands in the
microvasculature of various end organs. The formation of fibrin
strands can benefit the host by encapsulating bacteria; however,
if large numbers of bacteria are protected from phagocytosis
once they are sequestered, additional bacterial proliferation
can occur with further damaging effects. Intravascular fibrin
strands can also have other deleterious effects, including alter-
ation of blood flow and erythrocyte fragmentation.
Platelets are also recognized as inflammatory cells, through
release of certain granule contents, for instance, histamine and
serotonin, or through the formation of cell membrane–derived
neutrophil and monocyte agonists, for instance, PAF and leu-
kotrienes. After activation, mononuclear phagocytes can produce
and release a wide range of inflammatory mediators, including
interleukin-6, which can stimulate increased hepatocyte
fibrinogen synthesis; PAF and prostaglandins, which can induce
further platelet activation; and tumor necrosis factor, which
can initiate fibrinolysis through the release of plasminogen
activators. Thus, in the initial phase of inflammation, a balance
between procoagulant activity, fibrin formation, and the lysis of
formed fibrin can be maintained. However, within several hours
of the initiation of an inflammatory response, the fibrinolytic
system is suppressed as a result of the increased synthesis and
release of PAI-1. The fact that thrombosis is not a consistent
clinical observation during inflammation can be explained by
the thrombin- and/or cytokine-induced activation of the anti-
coagulant protein C pathway (see Fig. 2-243). The suppres-
sion of both thrombin formation and plasmin generation helps
maintain hemostatic balance.
Neoplasia can also precipitate dysequilibrium of procoagulant,
anticoagulant, and fibrinolytic proteins, similar to that which
occurs during systemic inflammation. The association between
hemostatic abnormalities and neoplasia is less well understood
in domestic animals, although bleeding can be a clinical sign
in animals with tumors such as hemangiosarcoma, multiple
myeloma, and mast cell tumors. Hemostatic laboratory abnor-
malities are frequently present in animals with neoplasia. For
example, in a study of 71 dogs with various tumors, the major-
ity exhibited evidence of hypercoagulability on TEG, along
with prolonged APTT and increased fibrinogen. Dogs with
metastatic disease tended to have increases in fibrinolysis indi-
cators. Chemotherapeutic drugs and radiation can also impair
the hemostatic process. For example, L-asparaginase, which
inhibits protein synthesis, and actinomycin D, which directly
antagonizes vitamin K, can produce transient reductions in
activity of multiple clotting factors. Tumor-induced inhibitory
antibody production can also contribute to hemorrhage. These
inhibitors, or circulating anticoagulants, may either be immu-
noglobulins directed against functional epitopes of clotting
factors or antiphospholipid antibodies that interfere with the
normal formation of the tenase and prothrombinase com-
plexes (see Fig. 2-241).
Disseminated intravascular coagulation (DIC) is an
acquired syndrome that occurs when excessive systemic acti-
vation of procoagulant mechanisms result in widespread fibrin
formation, which may or may not eventually be accompanied
by inhibitor consumption and altered fibrinolysis (see also Vol.
3, Cardiovascular system). Thrombosis and/or hemorrhage
may occur at multiple sites, which often cause end-organ
dysfunction or complete failure. DIC is a potential
268 CHAPTER 2  •  Hematopoietic System
complication of any underlying disorder that results in an
increase in expression of tissue factor, or activation of other
proteases that can activate clotting on a systemic basis. In
horses, DIC has been reported most commonly as a sequel to
intestinal disease and colic. In dogs, almost half of those with
immune-mediated hemolytic anemia exhibit evidence of a
hypercoagulable state at the time of clinical presentation.
Animals with sepsis and systemic inflammatory response syn-
drome are at increased risk for DIC and frequently have many
abnormal hemostasis assay results.
In the initial stages of DIC, increased thrombin generation
is compensated for by the anticoagulant systems, such as TFPI,
APC, and antithrombin (see Fig. 2-243). Laboratory results at
this stage can vary, but a hypercoagulable tracing may be
observed with thromboelastography. Further supportive labo-
ratory diagnosis at this stage would require evaluation of
markers such as thrombin-antithrombin (TAT) complexes
(see Fig. 2-243) and peptide fragments such as prothrombin
fragment 1 + 2, which are released during activation of pro-
coagulant factors. These latter tests are, however, not widely
available for animals.
If increased thrombin generation persists, DIC may progress
to an overt hypocoagulable phase. When inhibitor consumption
outweighs production because of increased attempts to con-
strain further clotting, thrombin generation and fibrin forma-
tion proceed in an unrestricted manner. This results in
thrombocytopenia and consumption of all procoagulant pro-
teins, hence the use of the term consumptive, or consumption,
coagulopathy to characterize this stage of DIC. Ultimately, this
results in multisite spontaneous bleeding, and anemia can develop
because of blood loss. Laboratory abnormalities are more pre-
dictable at this stage, and include decreased platelet count,
prolonged PT and APTT, decreased fibrinogen concentration,
decreased antithrombin and protein C activity, and increased
Disorders of Hemostasis
fibrin degradation products (FDPs) and D-dimers. A hypoco-
agulable tracing would be apparent on thromboelastography
with prolonged R time and decreased maximum amplitude
(MA).
The fibrinolytic system is also activated in the initial phase of
DIC, resulting in increased amounts of intravascular plasmin
being generated. Excessive fibrinolysis may exacerbate bleed-
ing in later stages. During plasmin proteolysis of fibrinogen
and fibrin, peptide fragments, known as FDPs and D-dimers,
are released (see Fig. 2-243). Excessive generation of plasmin
and production of FDPs both contribute to end-organ failure,
which is often the ultimate cause of death in animals with
advanced DIC. For example, plasmin can induce vascular and
cellular damage through activation of matrix metalloprotein-
ases that degrade a wide range of cytoskeletal filaments. FDPs
can enhance local inflammatory reactions by triggering the
release of interleukin-1 (IL-1) and IL-6 from monocytes and
macrophages, and can also interfere with platelet function.
Further reading
Andreasen EB, et al. Haemostatic alterations in a group of canine
cancer patients are associated with cancer type and disease pro-
gression. Acta Vet Scand 2012;54:3.
Cesarini C, et al. Association of admission plasma D-dimer concentra-
tion with diagnosis and outcome in horses with colic. J Vet Intern
Med 2010;24:1490-1497.
Piek CJ, et al. High intravascular tissue factor expression in dogs with
idiopathic immune-mediated haemolytic anaemia. Vet Immunol
Immunopathol 2011;144:346-354.
For more information, please visit the companion site:
PathologyofDomesticAnimals.com.
 268.e1

Further reading
Puy C, et al. Factor XII promotes blood coagulation independent of
factor XI in the presence of long-chain polyphosphates. J Thromb
Haemost 2013;11:1341-1352.
Ralph AG, Brainard BM. Update on disseminated intravascular coagula-
tion: when to consider it, when to expect it, when to treat it. Top
Compan Anim Med 2012;27:65-72.