PROCEEDINGS OF THE

NORTH AMERICAN VETERINARY CONFERENCE

VOLUME 20

JANUARY 7-11, 2006

ORLANDO, FLORIDA

SMALL ANIMAL EDITION

Reprinted in the IVIS website (http://www.ivis.org) with the permission of the NAVC.
For more information on future NAVC events, visit the NAVC website at www.tnavc.org

Small Animal Hematology

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INFECTIONS OF DOG BLOOD CELLS

John W. Harvey, DVM, PhD, Diplomate ACVP
College of Veterinary Medicine
University of Florida
Gainesville, FL
INTRODUCTION
A number of infectious agents are recognized to occur
within or upon canine blood cells. These include
intracellular protozoal (Babesia and Theileria) and
rickettsial (Ehrlichia, Anaplasma, and Neorickettsia)
parasites and epicellular hemotrophic mycoplasmas
(formerly
Haemobartonella
species).
Protozoal
organisms each have a nucleus within their cytoplasm.
Rickettsial organisms and hemotrophic mycoplasmas
are bacteria and lack nuclei. Rickettsial organisms
proliferate within cytoplasmic vacuoles forming small
colonies called morulae. Hemotrophic mycoplasmas are
attached to the outside of erythrocytes as individual
organisms.
Anemia that develops as a result of infections with
protozoa and hemotrophic mycoplasmas is generally
regenerative. In contrast, anemia that develops
secondarily to rickettsial infections is generally
nonregenerative. Thrombocytopenia is often present in
animals with protozoal or rickettsial infections, but is
generally absent in animals infected with hemotrophic
mycoplasmas.
Current serologic assays cannot definitively identify
individual species of organisms because of crossreacting antibodies to related organisms, but individual
species can be identified using PCR and sequencing of
certain parasite genes. Because genetic variability
occurs within each species, questions remain
concerning the degree of sequence identity required to
include an individual isolate in a given genus or species.
Some of the organisms discussed will likely be included
in a different genus (e.g., moved from Babesia to
Theileria) and others will likely have a new species name
assigned to them as more comprehensive molecular
biology studies are performed.
All of these infectious agents are transmitted by ticks
except N. risticii which is transmitted by infected
trematodes that parasitize snails and aquatic insects.
Dogs likely become infected with N. risticii by drinking
contaminated water. Individual dogs can be infected with
more than one infectious agent. Tetracycline antibiotics
are effective in treating dogs with hemotrophic
mycoplasmas and rickettsial infections, but not protozoal
infections.
RICKETTSIAL ORGANISMS
Species of organisms in the order Rickettsiales that
infect leukocytes and platelets appear to have fairly
strong predilections for specific blood cell types. All of
these infectious agents were previously classified in the
genus Ehrlichia, but several of these organisms have
been reclassified as Anaplasma or Neorickettsia species
based on genetic findings obtained using PCR and

sequencing of the 16S rRNA gene. Rickettsial organisms

that infect mononuclear phagocytes of dogs include
E. canis, E. chaffeensis, and N. risticii (formerly
E. risticii). Rickettsial species infecting granulocytes
include E. ewingii and A. phagocytophilum (formerly
E. equi, E. phagocytophila, and human granulocytic
Ehrlichia). A. platys (formerly E. platys) is a rickettsial
parasite that specifically infects platelets in dogs. A new
Ehrlichia genotype that is closely related to E. ruminatum
has been identified in blood from South African dogs by
PCR, but morulae were not identified in blood cells.
Clinical Findings
E. canis appears to be the most clinically important
rickettsial agent infecting dog blood cells. Consequently,
clinical signs attributable to E. canis infections will be
emphasized. Clinical signs often go unnoticed during
early infection, but fever, malaise, anorexia, weight loss
and hemorrhage may occur. Thrombocytopenia is
usually present, but anemia and leukopenia occur less
consistently.
The chronic phase of disease may be mild or severe.
In mild cases, dogs remain infected but little evidence of
illness is recognized. There is a gradual increase in cell
counts in cytopenic animals. Many dogs infected with
this agent in the United States have unapparent
infections or exhibit only mild clinical signs.
In severe cases, marked thrombocytopenia and
leukopenia develop 40 to 80 days after infection.
Animals are often febrile with outward signs of illness
that may include lethargy, anorexia, weight loss,
bleeding disorders, and pale mucous membranes.
Peripheral
lymphadenopathy,
hepatomegaly,
splenomegaly, uveitis, retinal lesions, and CNS signs
may be recognized in some cases. Bone marrow is
hypoplastic (or rarely aplastic). Affected dogs may die of
secondary bacterial infections or massive internal
hemorrhages.
The severity of disease varies with the pathogenicity
of specific strains of the organism, individual differences
in host defensive mechanisms (e.g., pups generally have
more severe disease than adults), the presence of
concomitant diseases, and the breed of dog affected
(e.g., more severe disease in German Shepherds than in
Beagles).
Dogs are susceptible to infection with E. chaffeensis,
but clinical signs have been mild or unapparent in
experimental infections. More severe clinical signs
including epistaxis, lymphadenopathy, and vomiting
have been reported in naturally infected dogs.
A subspecies of N. risticii has been reported to produce
clinical findings similar to other rickettsial infections
discussed. Clinical signs associated with E. ewingii
infections
include
fever,
malaise,
neurologic
abnormalities, and swollen joints secondary to
polyarthritis. Similar clinical signs have been associated
with A. phagocytophilum infections, but polyarthritis is
less common. Clinical signs associated with A. platys
infections in the US are generally mild, but clinical signs
similar to other rickettsial infections have been reported
in dogs in Greece and Israel.

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Laboratory Findings
hrombocytopenia
and
mild
to
moderate
nonregenerative anemia are present in most cases of E.
canis infections. Leukopenia and neutropenia are
present in less than half of the cases and some animals
may even exhibit leukocytosis and/or monocytosis.
Lymphopenia is common. Hyperglobulinemia is often
present, and it is usually polyclonal, but monoclonalappearing hyperglobulinemia has been reported.
Proteinuria and mild increases in serum ALT and
alkaline phosphatase are common in ill animals. The
bone marrow is normal to hypercellular in acute cases
and hypocellular in severe chronic cases. Similar
laboratory findings may be observed in dogs with other
rickettsial infections except that hypoplastic bone
marrow has not been reported. Cyclic thrombocytopenia
may be observed in acute A. platys infections, but mild
persistent thrombocytopenia is more likely in chronic
infections. Joint fluid cytology reveals increased protein
and neutrophils in dogs with arthritis secondary to
E. ewingii infections.
Diagnosis
A diagnosis of a rickettsial infection may be made by
visualization of morulae in stained blood films. Those
that invade granulocytes are seen more frequently
observed than those that develop in mononuclear cells
or platelets. The diagnosis of a rickettsial infection is
generally suspected based on history, clinical findings,
and routine laboratory findings and confirmed using
serology. However, one must remember that a positive
serologic test only indicates exposure.
Indirect
fluorescent antibody (IFA) tests have been developed for
all species discussed except
E. ewingii. The IFA
test for E. canis has been used for many years to assist
in the diagnosis of E. canis infections, but E. chaffeensis
and E. ewingii antibodies also cross react in this assay.
ELISA assays have also been developed to detect
antibodies against several rickettsial organisms. One
ELISA assay that is available for in clinic use in dogs as
a component of the Snap 3Dx (IDEXX Laboratories,
Inc., Maine, USA) test. The Snap 3Dx test will be
positive in dogs previously exposed to E. canis and E.
chaffeensis, but it should not detect antibodies to the
other rickettsial agents discussed. Current serologic
assays for A. phagocy-tophila and A. platys will likely
cross react with one another. All of these rickettsial
agents can be differentiated from one another using
PCR and 16S rRNA sequencing. A positive PCR test
indicates current infection, not simply exposure.
Unfortunately, PCR assays are relatively expensive and
take more time to perform than serologic tests.
Therapy
Doxycycline (5 mg/kg PO q12h) treatment for 3 weeks
is generally effective in eliminating clinical signs, but
titers (especially high titers) may persist indefinitely,
suggesting that many animals remain persistently
infected after treatment. Blood transfusions may be
required in severe cases. Clinical and hematologic

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improvement is generally rapid following initiation of

doxycycline therapy, except in severe cases of E. canis
infections with hypoplastic or aplastic bone marrow
where weeks or months may be required for hematologic
parameters to completely return to normal. Imidocarb
dipropionate at a dosage of 5 mg/kg IM twice 2 weeks is
also reported to be effective in treating dogs with E.
canis infections.
BABESIOSIS
B. canis is a large (3-5 m in length) pear-shaped
parasite. Three genetically distinct subspecies of this
genus (B. canis canis, B. canis vogel, and B. canis rossi)
and have been identified. In addition, a new large
Babesia has been described in a North Carolina dog that
is genetically distinct from the B. canis subspecies.
B. gibsoni is a small Babesia (1.0-2.5 m in diameter).
There has been a rapid increase in the number of cases
of B. gibsoni (Asian genotype) reported, predominantly
in Pit Bull Terriers and American Staffordshire Terriers in
various parts of the US. A second small Babesia has
been recognized in California that causes severe clinical
disease. Although classified as B. gibsoni (California
genotype) based on morphology, this California
organism is distinctively different from B. gibsoni (Asian
genotype) based on molecular biology studies and my
be assigned to the genus Theileria. A third small
piroplasm has been identified causing disease in dogs
outside the US that is closely related to B. microcoti.
A provisional name of Theileria annae has been
assigned to this organism. Finally, B. equi (Theileria
equi) has been identified in the blood of several southern
European dogs by PCR.
Clinical Findings
The subspecies of B. canis (presumably B. canis
vogel) present in the US generally cause mild or
unapparent disease in adults (unless immunosuppressed), but severe disease in pups. Small babesial
organisms cause clinical disease in some adults and
unapparent disease in other adults. Clinical signs that
may occur in dogs include fever, lethargy, anorexia, pale
mucous membranes, amber to brown urine,
splenomegaly, weight loss, rapid respiration, rapid heart
rate, and icterus. B. canis rossi in South African can
cause severe disease and death in adult dogs.
In addition to causing hemolytic anemia, some South
African dogs exhibit a hypotensive shock syndrome and
death before anemia can develop.
Laboratory Findings
Animals with babesiosis are usually anemic and a
regenerative response (reticulocytosis) is present in
most cases. Mild to severe thrombocytopenia is often
present, but hemorrhage is seldom apparent. Clinical
chemistry profiles may demonstrate bilirubinemia and
abnormalities related to anemic hypoxia, but profiles can
be normal. Bilirubinuria is common, and hemoglobinuria
is sometimes present. Infected animals may be Coombs
test positive.

Small Animal Hematology

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Diagnosis
A diagnosis of Babesia infection is made by
identification of piroplasms in stained-blood films, but
they may not always be found, and the small babesias
can be difficult to recognize. A presumptive diagnosis
with regards to babesial species may be made based on
size. Large organisms are most likely B. canis and small
organisms are most likely B gibsoni in the US.
Serological diagnosis can be made using IFA tests, but
some cross-reactivity occurs between babesial species.
High titers suggest current infection, but IFA tests may
be negative in acutely infected animals. The species of
an organism can be identified in blood using PCR and
sequencing of the 18S rRNA gene.
Therapy
Adult dogs with mild anemia and clinical signs may
not require therapy. Imidocarb dipropionate (Imizol) (6.6
mg/kg SQ or IM, single injection, repeat the dose in two
weeks) along with supportive care (transfusions or IV
fluid therapy) may be efficacious in treating B. canis. The
small babesias including B. gibsoni do not respond well
to imidocarb dipropionate therapy. Treatment with a
combination of azithromycin (10mg/kg PO q24h) and
atovaquone (13.3 mg/kg PO q8hr with a fatty meal) for
10 days is effective. Treated and untreated dogs often
remain carriers of babesial infections.
HEMOTROPHIC MYCOPLASMS
Hemotrophic mycoplasmas are gram-negative,
nonacid-fast, bacteria that attach to the external surface
of erythrocytes. They were classified as rickettsia in the
genus Haemobartonella for many years in dogs;
however, results from sequencing of the 16S rRNA gene
indicate that these parasites are mycoplasmas.
Hemoplasmas has been proposed as a trivial name for
these hemotrophic mycoplasmas.
Mycoplasma haemocanis (formerly Haemobartonella
canis) is reported to be the causative agent of
hemotrophic mycoplasmosis in dogs.
It has been
considered to be a distinctly different organism from
M. haemofelis, but the sequence of the 16S rRNA gene
of a M. haemocanis isolate from one dog was
remarkably similar (99% homology) to that of

M. haemofelis. Hemoplasmas that are closely related to

M. haemominutum have recently been identified in dogs
and one of these isolates has been classified as
"Candidatus Mycoplasma haemoparvum."
Clinical Findings
Unless other diseases are also present, clinical signs
are rarely apparent in nonsplenectomized dogs infected
with M. haemocanis. Splenectomized dogs infected with
M. haemocanis become listless and develop pale
mucous membranes as the anemia progresses, but
generally have normal rectal temperatures and
appetites.
Laboratory Findings
M. haemocanis organisms are usually present when
clinical evidence of anemia is recognized. Anemia may
be mild to severe and reticulocytosis is typically present.
Spherocytosis and positive Coombs' test results occur in
some cases. Dogs with latent infections generally have
normal hemograms.
Diagnosis
M. haemocanis can generally be identified in blood of
anemic dogs although organisms may be low in number
and difficult to find. The most useful criterion is the
tendency of M. haemocanis to form chains of organisms
across the erythrocyte surface. PCR-based assays can
be used to identify hemoplasma infections by species.
Therapy
Doxycycline (5mg/kg PO q12hr) or tetracycline (20
mg/kg PO q8hr) are effective when given for 3 weeks.
References
1.
2.
3.

Boozer AL, Macintire DK. Canine babesiosis. Vet

Clin North Am Small Anim Pract. 2003;33:885-904.
Cohn LA. Ehrlichiosis and related infections. Vet
Clin North Am Small Anim Pract. 2003;33:863-884.
Harvey JW. Hemotrophic mycoplasmosis. In:
Greene CE, 3rd ed. Infectious Diseases of the Dog
and Cat. Philadelphia: W.B. Saunders; 2005: in
press.

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