548
15
Neoplastic Diseases
­Introduction
Venugopal Nair
Summary
Agents, Infections, and Disease.  This chapter refers to all the
diseases that have neoplasm or cancer as a common feature.
The majority of these diseases are caused by herpesviruses
or retroviruses, designated as Marek’s disease (MD),
avian leukosis, and reticuloendotheliosis. The chapter
also describes a set of tumors of a non‐infectious etiology.
Diagnosis.  Most of the neoplastic diseases can be
diagnosed by the characteristic pathological changes in
the affected tissues, but confirmed by virological,
serological, and molecular methods of diagnosis.
Intervention.  While diseases such as MD is controlled
mainly by the widespread use of vaccines, most of the
retroviral diseases are controlled by eradication of the
virus from the infected flocks, supplemented by selection
for genetic resistance.
Neoplastic diseases of poultry comprise a variety of condi-
tions possessing a single common denominator: neoplasia
involving one more of the cell types. Unlike in human med-
icine where the vast majority of cancers are of non‐infec-
tious origin (1), most of the neoplastic diseases affecting
avian health have a viral etiology. Indeed, studies on avian
oncogenic viruses have contributed immensely to our cur-
rent understanding of a number of molecular mechanisms
of cancer (2). This chapter deals primarily with the three
most economically important virus‐induced transmissible
neoplastic diseases of poultry, namely: (1) the herpesvirus‐
induced Marek’s disease (MD); (2) retrovirus‐induced
avian leukosis/sarcoma, and (3) reticuloendotheliosis. An
additional final section covering tumors of unknown etiol-
ogy is also included. Each of these neoplastic diseases or
disease complexes are described in a separate section
because of its etiologic distinctness.
Diseases of Poultry, Fourteenth Edition. Editor-in-chief David E. Swayne.
© 2020 John Wiley & Sons, Inc. Published 2020 by John Wiley & Sons, Inc.
The first section describes MD, a T‐cell lymphoma
induced in chickens by the highly cell‐associated Marek’s
disease virus (MDV). Marek’s disease lesions consist of
CD4+ T‐cell lymphomas affecting a number visceral
organs and tissues, together with lymphoid cell infiltra-
tion into the peripheral nerves resulting in paralytic
symptoms. Recent advances in MD research have pro-
vided significant insights into the molecular mechanisms
of the disease, further strengthening its significance as
an excellent biomedical model for T‐cell lymphomas
(3–6). Marek’s disease has been controlled since the early
1970s by use of conventional live attenuated antigeni-
cally related vaccines. During the last four‐and‐a‐half
decades, research on MD has provided a better under-
standing of the viral gene functions, molecular mecha-
nisms of the disease and factors affecting host genetic
resistance (7). However, despite widespread use of vac-
cines and development of new methods of vaccination,
MD still remains a major challenge to poultry health,
particularly from the continuing increase in virulence of
MDV strains (8, 9), possibly contributed by the vaccines
themselves (10). Furthermore, the incidence of MD in
other avian species such as turkeys and geese (11, 12)
demonstrate the increasing host range and economic
significance. Clearly, in the absence of control measures
including vaccine failures, MD is capable of causing dev-
astating losses in poultry (13, 14). As a disease occurring
worldwide, with reports of vaccination breaks and prob-
able emergence of more virulent pathotypes, MD contin-
ues to pose severe threats to the poultry industry, and
developing strategies for its control remains one of the
great challenges today (7). This section gives an up to
date account of the scientific understanding of MD and
its control strategies.
A second section describes a group of leukoses, sarco-
mas, and related neoplasms induced by a number of
closely related groups of avian retroviruses termed the

leukosis/sarcoma (L/S) viruses. The contributions of
these retroviruses to tumor virology, such as the land-
mark study on transmissible tumors, the discovery of the
oncogenes and the reverse transcriptase, have been hugely
significant. The term leukosis is used because a leukemic
blood picture is not always present during the course of
leukemia‐like proliferative diseases of the hemopoietic
system (15–17). The various forms of hemopoietic sys-
tem neoplasia induced by the L/S group of avian retrovi-
ruses include the lymphoid leukosis (LL), myeloid
leukosis (ML), and erythroid leukosis (EL) caused by dif-
ferent subgroups of the virus. Primarily affecting the
bursa of Fabricius and visceral organs, LL is the most
common form of leukosis although efforts in the past
have successfully eradicated these viruses from many of
the commercial breeding flocks (15, 18). Other neo-
plasms of hematopoietic origin that can also be seen in
avian leukosis virus (ALV)‐infected chickens, albeit
infrequently, include erythroblastosis, myeloblastosis,
myelocytomatosis, and certain related neoplasms such
as nephroblastoma and osteopetrosis (19). With the
emergence of a new ALV subgroup J in the late 1980s,
myelocytomatosis, became a major neoplasia particu-
larly in meat‐type chickens (16, 20). The incidence of
ALV‐J has been significantly reduced in Europe and
North America through the successful implementation
of eradication programs. However, it is alarming that sig-
nificant losses continue to occur in countries such as
China, where in addition to the commercial meat‐type
Table 15.1  Transmissible neoplasms.
Virus type Nucleic acid type Virus classification of etiological agent
Retrovirus RNA Leukosis/sarcoma group
Reticuloendotheliosis group
Herpesvirus DNA Marek’s disease virus
Chapter 15  Neoplastic Diseases 549
chickens, commercial layers and native breeds of chick-
ens are affected (21–23). The section on leukoses/sar-
coma viruses gives a comprehensive coverage of the
recent advances in our understanding of the pathogenic
mechanisms, diagnosis, and eradication methods.
Technological advances in genome editing and the
potential for inducing genetic resistance as a tool for dis-
ease control are also mentioned (24).
The third section describes reticuloendotheliosis
(RE), a group of disease syndromes caused by reticu-
loendotheliosis virus (REV), unrelated to the leukoses/
sarcoma group of viruses (25). The REV group includes
different viruses associated with a number of disease
syndromes. These consist of defective REV‐T strain
that induces rapid neoplastic transformation by virtue
of the very potent virus‐encoded oncogene v‐Rel, an
NFkB homolog (26). The most common clinical
­diseases induced by REV include chronic lymphoma
as  well as immunosuppressive runting disease.
Reticuloendotheliosis virus infects chickens, turkeys,
ducks, geese, pheasants, quail, and probably many other
avian species. However, a major economic concern of
REV arises from its potential as contaminants of live
vaccines produced in chicken embryo cells or tissues.
Reticuloendotheliosis virus could also be a barrier for
the export of breeding stock to certain countries. The
section gives very detailed account of some of patho-
genic mechanisms of REV, and the recent developments
in diagnosis and control.
Neoplastic diseases
Leukoses
Lymphoid leukosis
Erythroblastosis
Myeoloblastosis
Sarcomas and other connective tissue tumors
Fibrosarcoma, fibroma
Myxosarcoma, myxoma
Osteogenic sarcoma, osteoma
Histiocytic sarcoma
Related neoplasms
Hemangioma
Nephroblastoma
Hepatocarcinoma
Osteopetrosis
Reticuloendotheliosis
Marek’s disease
550 Section II  Viral Diseases
The fourth and final section describes tumors of
unknown etiology on the basis of morphologic charac-
teristics. Included are a wide variety of benign and malig-
nant neoplasms derived from muscle, epithelial, and
nerve tissues; serous membranes; and pigmented cells.
Lymphoproliferative disease (LPD), another neoplastic
disease of turkeys reported in Europe and Israel is also
induced by another retrovirus (27). The incidence of
LPD of turkeys has always been sporadic and hence not
included in this chapter.
Because many of the avian tumor viruses appear to
have multipotent characteristics, that is, they can some-
times induce a variety of neoplasms, classification and
nomenclature of virus‐induced neoplasms present a
­Marek’s Disease
Venugopal Nair, Isabel Gimeno, and John Dunn
Summary
Agent and Disease.  Marek’s disease (MD) is widespread
disease of chickens characterized by rapid‐onset
lymphoid tumors, immunosuppression, and paralysis
caused by the highly contagious Marek’s disease virus, an
alphaherpesvirus belonging to Mardivirus genus.
Diagnosis.  Marek’s disease virus is ubiquitous and
detecting the virus alone is not sufficient to make a
confirmatory diagnosis, unless it is associated with
characteristic clinical signs and lesions including visceral
tumors and peripheral nerve lesions. A number of
molecular diagnostic tests based on the detection of viral
nucleic acids and viral proteins.
Intervention.  Control of the disease is a challenge because
of the ubiquitous nature of the virus, latent infection, and
continuous shedding of the virus from the infected birds
and long‐term persistence of the virus outside the host in
the poultry environment. Vaccination is the cornerstone of
the control of MD along with improved biosecurity
measures.
Introduction
Marek’s disease (MD) is a common lymphoproliferative
disease of chickens, usually characterized by mononu-
clear cellular infiltrates in peripheral nerves and various
other organs and tissues including iris and skin. The dis-
ease is caused by a herpesvirus, is transmissible, and can
be distinguished etiologically from other lymphoid
­neoplasms of birds.
problem. The dilemma is largely due to the fact that cer-
tain strains of these viruses induce some pathologic
lesions difficult to distinguish from those induced by
another unrelated virus. The two major lymphoid neo-
plastic diseases MD and lymphoid leukosis are particu-
larly confusing. Although REV‐induced lymphomas are
infrequent and generally arise from contaminated vac-
cines, it could also add to the problems in differential
diagnosis. A practical and useful strategy for the differ-
ential diagnosis of viral lymphomas has been proposed
(28). The choice of terminology for the neoplasia used in
this chapter (see Table 15.1) is based on that originally
adopted by the World Veterinary Poultry Association
(29) with suitable modifications.
Because the literature on MD has greatly expanded, it
is no longer feasible to cite all relevant publications that
provide the scientific basis for our current knowledge of
the disease. In this chapter, literature is cited selectively,
and reviews are often substituted for original papers.
Readers are advised to refer to the chapter on MD from
the previous editions of this book for more details from
previous years. Useful recent books on MD are Marek’s
Disease (259) and Marek’s disease: An evolving problem
(158) and proceedings from the international symposia
on MD (1).
Definition and Synonyms
The seminal description by Jozsef Marek (360) identified
the disease as polyneuritis. Other common synonyms
included neuritis, neurolymphomatosis gallinarum, and
range paralysis. Jungherr and colleagues (294) proposed
that the term lymphomatosis be subdivided into visceral,
neural, and ocular forms. In 1961, Biggs (44, 48) p
­ roposed
the term Marek’s disease to distinguish the condition
clearly from etiologically different lymphoproliferative
diseases.
Marek’s disease has also been subdivided into acute
and classical forms, where the latter term designates
forms of the disease prevalent prior to the 1950s (44).
Marek’s disease virus (MDV) can also induce other clini-
cally distinct disease syndromes such as transient paral-
ysis, early mortality syndrome, cytolytic infection,
atherosclerosis, and persistent neurological disease.
Economic Significance
Prior to use of vaccines, MD constituted a serious eco-
nomic threat to the poultry industry causing up to 60%

mortality in layer flocks and 10% condemnations in
broiler flocks. Because vaccines are not 100% effective,
sporadic losses still occur, but they are no longer as seri-
ous a problem. In 2004, the worldwide annual losses
from MD were in the range of US$1 to 2 billion, although
the authors have indicated that these figures are impos-
sible to verify (381). The disease remains a major con-
cern for the poultry industry due to the unpredictability
of outbreaks and the possibility that vaccines may ulti-
mately fail as a consequence of the evolution of more
virulent strains of MDV.
Public Health Significance
Although there are several reports suggesting a role for
MDV in the etiology of multiple sclerosis, there has
been no evidence to suggest the MDV is infectious to
humans (496, 562) or associated with human cancer
(455).
Scientific Significance
Research on MD has contributed greatly to veterinary
medicine, basic science, and comparative oncology. The
disease is uncommonly complex, featuring an interplay
of neoplasia and inflammation expressed as several dis-
tinct clinical syndromes, each modified in important
ways by host genetic influences. Marek’s disease virus,
an alphaherpesvirus with lymphotropic properties of
gammaherpesviruses, is highly cell‐associated but read-
ily transmitted, and its virulence varies and evolves. It
has two unique sister viruses, both nononcogenic, that
naturally infect chickens and turkeys. Infection induces
complex immune responses usually resulting in high
levels of protection. Vaccination for MD constitutes an
outstanding example of successful disease control in
veterinary medicine, and MD vaccines are the first
effective vaccines against cancer in any species.
Evolution of MDV virulence has also been shown as an
example of a biological arms race of pathogens in the
face of vaccination (57).
History
The seminal report by József Marek, published in 1907
(360), of paresis in four roosters is the first account of the
disease named after him. A detailed history of MD
research can be found in the previous edition of Diseases
of Poultry (499), a paper by Professor Biggs (47), the vide-
otape Legacy of the 1960s, and the historical archives of
the American Association of Avian Pathologists. A long
view on the last 40 years of MD research has recently
been published as part of the 40th Anniversary of Avian
Pathology (51).
Chapter 15  Neoplastic Diseases 551
Etiology
Classification
Marek’s disease virus is a cell‐associated herpesvirus
(citations in [484]) with lymphotropic properties similar
to gammaherpesviruses. However, its molecular struc-
ture and genomic organization are similar to alphaher-
pesviruses (68, 335, 567). As per the recent classification
by the International Committee on Taxonomy of
Viruses (ICTV) all MDV serotypes are grouped together
in the genus Mardivirus (314). Members of the genus
Mardivirus, described previously as three serotypes are
now grouped as three species: Gallid alphaherpesvirus 2
(serotype 1), Gallid alphaherpesvirus 3 (serotype 2), and
Meleagrid alphaherpesvirus 1 (herpesvirus of turkey
[HVT], serotype 3). Serotype 1 MDV is the prototype
virus for this group of avian viruses, and except where
otherwise indicated MDV refers to serotype 1 virus. On
the basis of their virulence, serotype 1 strains are further
divided into pathotypes, which are often referred to as
mild (m)MDV, virulent (v)MDV, very virulent (vv)MDV,
and very virulent plus (vv+)MDV strains (597, 604).
Nononcogenic Gallid herpesvirus 2 isolated from chick-
ens (50, 125, 490) and HVT (307, 613) belonging to genus
Mardivirus are also included in this chapter.
Morphology
The morphology and morphogenesis of MDV have been
reviewed (142, 163, 164). Hexagonal nucleocapsids
85–100 nm in diameter and enveloped particles 150–
160 nm in diameter may be seen in thin sections of
infected cell cultures. Keratinocytes derived from
chicken embryonic stem‐cells showed capsids/virions
although extracellular viruses could not be demonstrated
(141). Enveloped virus particles appearing as irregular
amorphous structures and measuring 273–400 nm have
been demonstrated in negatively stained preparations of
feather follicle epithelium (FFE) (92). Thin‐section prep-
arations of the FFE revealed large numbers of cytoplas-
mic enveloped herpesvirus particles in keratinizing cells.
The morphology of MD virions in cell cultures and FFE
is shown in Figure 15.1A.
The morphology of serotype 2 and 3 strains resembles
that of MDV serotype 1 (393, 427, 490). In thin sections,
however, nucleocapsids of HVT commonly show a
unique crossed appearance (393).
Chemical Composition
Viral DNA
Physical Properties.  The complete sequence of several
strains belonging to the three serotypes confirmed
that  the genomes are very similar consisting of linear,
552 Section II  Viral Diseases

(A) (B)

(C)

Figure 15.1  Electron micrographs of Marek’s disease virus (MDV). (A) Thin section of cultured duck embryo fibroblasts infected with MDV
showing scattered virions in nucleus. ×38,400. (B) Thin section of cultured duck embryo fibroblast infected with MDV, showing enveloped
virions in a nuclear vesicle. ×360,000. (C) Thin section of feather follicle epithelium (FFE) of chicken infected with MDV showing enveloped
virions within the cytoplasmic inclusions. Note difference in morphology compared with (B). ×70,000. (413). (Nazerian)

double‐stranded DNA molecules of approximately 160–
180 kb with a buoyant density in neutral CsCl of 1.706 g/
mL for serotype 1 (158, 499). It is difficult to separate
viral DNA from host cell DNA because its density is
close to that of chicken DNA. Cloning of the complete
MDV genome as bacterial artificial chromosomes (BAC)
has greatly facilitated the production of MDV DNA (506,
645). Infectious BAC clones of a number of strains have
been constructed allowing rapid manipulation of MDV
genomes to identify various determinants associated
with MD biology (118, 313, 541). Similarly, the rescue of
infectious MDV from overlapping cosmid clones of the
Md5 strain of MDV has also been reported (463).
Structural Organization.  The genomic structure of the three
serotypes is typical for alphaherpesviruses as previously
suggested (114) with a unique long (UL) and a unique short
(US) sequence. These unique sequences are flanked by sets
of inverted repeat sequences: the terminal repeat long
(TRL), internal repeat long (IRL), internal repeat short
(IRS), and terminal repeat short (TRS), respectively. Alpha
(a)‐like sequences, believed to be important for the cleavage
and packaging of viral DNA into virions, are located at the
terminal ends of the TRL and IRL and between the IRL and
IRS regions (11, 282, 335, 540, 567).
The complete genome sequences of a number of sero-
type 1 MDV strains as well as other vaccine serotypes

have now been determined (499). Comprehensive analy-
sis of the sequence of the TRL/IRL regions in the
genomes of 13 strains of varying virulence has identified
several single nucleotide polymorphisms (SNPs) which
loosely partition between attenuated and non‐attenu-
ated strains (537, 539).
DNA Structure in  Infected Cells.  The structure of viral
DNA in infected cells is dependent on the virus–cell
interaction. Linear viral DNA can be found in nuclei of
cells undergoing virus replication (114). It remains
unknown how viral DNA is maintained in latently
infected, nontransformed cells (80, 376), although there
was some evidence on the role of epigenetic regulation of
the viral genome (63). The status of DNA in transformed
cells has been difficult to determine in part because a
variable percentage of the transformed cells may undergo
viral replication at any point in time, in which case linear
DNA can be detected. Initial investigations into the
status of the MDV genome have indicated an absence of
integration (553), MDV genome is now thought to exist
as a mixture of integrated and episomal DNA (499).
Marek’s disease virus can be found integrated at multiple
sites in the chromosomes of cells derived from MDV‐
related T‐cell lymphomas, suggesting a correlation
between integration and oncogenicity. The integration
sites are preferentially located near the ends of the
chromosomes within the telomeric region. The telomere‐
like sequences located at the terminal ends of the MDV
genome are thought to assist in the preferential
integration of the viral DNA (312). Interestingly, MDV
encodes an RNA telomerase subunit (viral TR [vTR])
that shares 88% sequence identity with the chicken TR
(cTR) gene (201). The role of telomeres and telomerases
in MDV integration, pathogenesis and oncogenesis has
been recently reviewed (312).
Structural Changes by Recombination and/or Mutation.  category of genes can be divided into immediate early
Serotype 1 strains quickly develop altered biologic
characteristics upon serial passage in vitro, such as loss of
oncogenicity, reduced expression of glycoprotein C (gC)
(131), and decreased replication in vivo (494) indicating
that spontaneous mutations may have occurred.
Accumulations of such mutations after continuous cell
culture passages and generations of mixed population of
viruses have been recently demonstrated by sequence
analysis (537, 539). The gradual evolution of pathotypes
toward greater virulence and the changes in biologic
properties of MDV during in vivo backpassage (591)
further support the mutability of MDV.
These biological changes are accompanied by several
molecular changes, although it is not clear which molec-
ular change correlates with a specific biological change.
An expansion was found within the Bam‐HI D and H
fragments that are commonly associated with cell ­culture
Chapter 15  Neoplastic Diseases 553
passage and attenuation in serotype 1 strains (204, 260,
528). This expansion was caused by a tandem amplifica-
tion of direct 132 bp repeat (266, 359, 476). Other
changes have also been described including the deletion
of 400 bp in the Bam‐HI A fragment of CVI988 clone C
(988C) and 988 C/R6 (266) and a deletion of 200 bp in the
BamH1 L fragment of the vvMDV strain Md11 (587).
Additional changes have been reported for CVI988 in
the meq gene (335, 538) and the ICP4 promoter/
enhancer region of CVI988 (299). It is not clear whether
these differences are a consequence of cell culture pas-
sage or reflect strain differences.
Viral Genes and Proteins
Over the last 25 years, a number of individual genes
of  MDV‐1 have been identified and sequenced, and
the  proteins have been characterized (478, 498).
Comprehensive reviews based on the complete sequences
for the three serotypes have been published including
lists of open reading frames (ORFs) and their putative
products (499). Table  15.2 summarizes the location of
the ORFs and indicates the number of ORFs with homo-
logues to HSV, the number of ORFs with homologues
shared among the three serotypes, and the number of
unique genes for each serotype. Many of the genes in the
UL and the US regions have homologues with HSV and
equine herpesvirus‐1 and 4‐, and the genome organiza-
tion is similar to these two alphaherpesviruses (354). For
this chapter, the MDV genes are grouped into two
­general categories: genes with homologues in alphaher-
pesviruses and genes unique for MDV. Only the genes
that are important for the pathogenesis and immune
responses will be reviewed briefly. The reader is referred
to the contemporary literature for additional informa-
tion (158, 499).
Genes with  Homologs in  Alphaherpesviruses.  This broad
(IE), early, and late genes, which are with few exceptions
important for virus replication.
Immediate Early and  Early Genes with  Homology to  HSV. 
The IE genes are important transcriptional regulators.
Four IE genes have been identified: intracellular protein
(ICP)4, ICP0, ICP22, and ICP27. Anderson et  al. (15)
identified ICP4 as a 4245 bp ORF, but sequence data
indicated the presence of an ORF of 6969 bp. This agrees
with the finding that two functional promoter/enhancer
regions are located upstream of the larger ORF and that
the putative promoter/enhancer region for the short
ORF was nonfunctional in vitro (299). Proof that ICP4
protein is a transactivator was provided by transfection
of the MD cell line (MDCC) MSB‐1 with the short form
of ICP4, showing increased transcription of the endoge-
nous ICP4, pp38, and pp24 genes (185, 447).
554 Section II  Viral Diseases

Table 15.2  Number of tentative genes in the three serotypes of Marek’s disease virus (MDV) in relation to other alphaherpesviruses.a

Location of expected functional open reading frames (ORFs)b

Serotype Gene classification TRL (R‐LORF) UL (L‐ORF) IRL (R‐LORF) IRS (RS) US (S‐ORF) TRS(RS) Totalc

1 HSV Homologd 0 57 0 1 7 1 65,66

MDV‐specifice 1 4 1 0 1 0 6,7
h H
Serotype‐specific 13 8 13 2 3 2 26,41
Total 14 69 14 3 11 3 97,114
2 HSV Homolog 0 59 0 1 7 1 67,68
MDV‐specificf 1 4 1 0 1 0 6,7
Serotype‐specific 9 4 9 1 4 1 17,27
Total 10 66 10 2 12 2 90,102
3 HSV Homolog 0 59 0 1 8i 1 68,69
g
MDV‐specific 0 6 0 0 1 0 7,7
Serotype‐specific 4 2 4 6 1 6 13,23
Total 4 67 4 7 10i 7 88,99
a
 For reference see (15, 279, 344, 373).
b
 Based on the location of the start codon.
c
 The italic numbers indicate the number of single genes for each serotype; the bold figures give the total number of genes including the
duplications in the repeat regions.
d
 Based on the sequence of the GA strain, nomenclature adapted from (340).
e
 Serotype‐specific genes with homologues present in serotype 2 or 3.
f
 Serotype‐specific genes with homologues present in serotype 1 or 3.
g
 Serotype‐specific genes with homologues present in serotype 1 or 2.
h
 The sequence for Md5 has minor differences compared with GA.
i
 Includes 2 copies of US8.

The MDV ICP27 phosphoprotein (465) localizes in the
nucleus, can transactivate pp38 and pp14 independently
of ICP4, and represses the early thymidine kinase gene
(466). Additionally, MDV ICP27 also interacts with SR
proteins and inhibits splicing of cellular telomerase
chTERT and viral vIL8 transcripts (14). ICP0 (LORF1)
has been identified as an ORF in the TRL and IRL, and a
recent study using proteomic approaches has demon-
strated that the ICP0 gene product is expressed in MDV‐
infected CEF (348).
Late Genes.  The late gene products include the nucle-
ocapsid proteins, the tegument proteins, including VP16,
and the glycoproteins (354). The glycoproteins (gB, gC,
gD, gE, gH, gI, gK, gL, and gM) are presumed to be
important for infection of cells, transfer of virus from cell
to cell, and immune responses.
gB, encoded by UL27, consists of a complex of three gly-
coproteins with molecular weights of 100, 60, and 49 kDa
and is important for cell attachment and/or ­penetration
based on the production of gB‐specific virus‐neutralizing
(VN) antibodies (reviewed by [354]). Deletion of gB from
MDV prevented the cell to cell spread demonstrating the
essential nature of this protein for MDV replication (506).
In a recent study adenovirus‐based expression of gB was
shown to be sufficient in inducing protection against viru-
lent MDV challenge (28). Similarly, some of the peptides
derived from gB showed antiviral properties providing
some insights into MDV entry into cells (583).
The UL44 gene encodes gC, a 57–65 kDa glycoprotein
identified in some early references as gA, which is exten-
sively synthesized in productively infected cells and is
expressed on the cell surface. In addition, gC is actively
secreted by infected cells (139, 272–274) and is one of
the major antigens to which the chicken immune system
mounts a substantial serological response. More recently,
gC and UL13 were shown to be important in horizontal
transmission (286).
The importance of gD, coded by US6, is poorly under-
stood. It is nonessential for horizontal transmission (16)
and is expressed poorly (406, 552) in vitro probably as a
consequence of no or limited transcription. Limited
expression of gD compared to pp38 and gB has been
described in FFE (398), suggesting that specific tran-
scription factors in the FFE may be needed for the pro-
duction of gD.
The functions of the other glycoproteins have not been
studied in detail. The gI and gE proteins interact with each
other based on immunoprecipitation assays (552).
Associations of mutations in the gL with MDV pathogenesis

and reduced immune responses have been reported (479,
555). Mutants constructed in a BAC clone carrying d ­ eletions
in the gM, gI, or gE gene indicate that the encoded glycopro-
teins are essential for virus replication, because the deletion
mutants are unable to transfer infectivity from infected to
uninfected cells (507, 564).
Genes Unique for  MDV.  Several genes have been
identified that are unique for MDV strains (Table 15.2).
Some of these genes are present only in serotype 1, and others
may have homologues in MDV serotype 2 and/or HVT.
Latency Associated Transcripts (LATs).  The LATs are a
group of transcripts antisense to ICP4 and have been
reviewed in detail (80, 376). These include a large 10 kb
transcript as well as several spliced transcripts referred
to as MSR (MDV small RNA) or SAR (small antisense
RNA). The importance of LATs for latency or transfor-
mation is unclear. Latency associated transcripts are
expressed in both lytically infected and transformed
cells, including the MDV positive QT35 cell line (633).
Meq (Marek’s EcoQ).  The molecular biology of Meq (R‐
LORF7) has been reviewed (324, 376, 388, 416, 551). The
Meq protein of 339 amino acids contains a basic leucine
zipper (bZIP) domain at the N terminal closely resembling
the jun/fos oncogene family. Several studies, inclusion
deletion analysis have clearly demonstrated the critical
role of Meq in oncogenesis (352). Meq gene also shows
diversity among different isolates and it has been sug-
gested that the positive selection may be driving evolution
(410, 626). Variations in the sequence of the proline‐rich
domains also showed association with virulence (512).
Meq shows differential binding to different promoters
depending on its dimerization status. As homodimers or
heterodimers with leucine zipper proteins, Meq can trans-
activate different promoters (64, 546, 547) resulting in the
upregulation of a number of genes including interleukin
(IL)‐2 and CD30, a member of the tumor necrosis factor
receptor II (TNFR‐II) family (78, 85). A number of subse-
quent studies have demonstrated efficacy of Meq‐deleted
viruses as efficient vaccines (331, 336, 526). The mecha-
nism behind such improved immunogenicity is not fully
understood, however gene expression changes in Meq‐
deleted viruses (336) and reduced immunosuppressive
effects (344) are thought to contribute to this effect.
vIL‐8.  The vIL‐8 gene (R‐LORF2) is located in the long
repeat region and originally was identified as a spliced
meq variant (432, 433). The gene consists of three exons
and is expressed late during cytolytic infection. IL‐8
attracts T cells, especially after IL‐8 receptors are upreg-
ulated by interferon‐γ (IFN‐γ) suggesting a role in the
switch of infection from B to T lymphocytes (502).
Subsequent studies have also shown that vIL‐8 promotes
Chapter 15  Neoplastic Diseases 555
lymphoma formation through targeted recruitment of B
cells and CD4+ CD25+ T cells (186).
Viral Lipase.  All the three serotypes of MDV encode the
viral lipase gene (vLIP) (11, 282, 316, 335, 567). vLIP, a
soluble, glycosylated protein, is encoded by the LORF‐2
gene consisting of two exons. The first exon codes for the
signal peptide, and the second exon codes for the lipase
activity. vLIP is probably an IE or early protein (296). The
glycosylated protein is required for the efficient lytic rep-
lication in birds (297).
pp38/pp24.  The MDV phosphorylated protein com-
plex, often referred to as pp38/pp24, is coded by two
genes located at opposite ends of the UL region (654).
Details of the previous research to understand the role of
pp38/pp24 have been reviewed in previous editions of
the book (499). Homologs for pp38 have been identified
in serotype 2 strains (282, 407) and HVT (11, 530), but
their functional relationships are not known.
The function of the pp24/pp38 complex has not been
elucidated. Originally, it had been linked to oncogenicity
because pp38 is expressed in the cytoplasm of a variable
proportion of MDV‐transformed, latently infected lym-
phocytes (146, 271). Expression of pp24/pp38 can be
enhanced by activation (447, 633) suggesting that pp38
may play a role during reactivation and subsequent virus
replication. pp38 is essential for cytolytic infection of B
cells and maintenance of transformed state (463),
although deletion did not affect the ability of the virus to
spread horizontally (228). CVI988 also expresses pp38,
but shows an amino acid mutation in an epitope defined
by monoclonal antibody (mAb) H19 (148). The demon-
stration that vvMDV5 strain expressing the pp38 protein
from CVI988 remains oncogenic indicates that the
attenuation of CVI988 is not associated with pp38 (329).
The 1.8 kb Gene Family.  The promoter/enhancer regions
of pp38 and pp24 are part of a bidirectional promoter
complex regulating the transcription of pp38/pp24, the
1.8 kb gene family and the origin of replication (165, 522).
Please refer to the chapter in the previous edition of this
book for detailed descriptions of the previous research on
this gene family (499). Several IE transcripts originate
from the 1.8 kb gene family containing three exons (376,
478). These transcripts are truncated in attenuated strains
due to an expansion of a tandem 132 bp direct repeat (DR)
(132 bp DR) (58, 476). The difference in the numbers of
copies of these repeats between virulent and vaccine
strains forms the basis of their differentiation by polymer-
ase chain reaction (PCR) assays (34, 525, 655), although
these are not directly linked to the oncogenicity (527).
Telomerase RNA (vTR).  The existence of a unique gene
encoding the RNA telomerase subunit (vTR) was identified
556 Section II  Viral Diseases
in the IRL/TRL region of the MDV genome (201). Marek’s
disease virus vTR showed nearly 88% sequence identity to
the chicken telomerase RNA (ChTR) indicating its trans-
duction from the host genome. vTR, regulated also by the
action of c‐myc (523), can constitute telomerase activity by
interacting with chicken telomerase reverse transcriptase
(ChTERT) more efficiently than ChTR (201, 202). The
direct association between MDV oncogenicity and vTR was
demonstrated using MDV lacking either one or both copies
of vTR (119, 565) that resulted in significantly impaired
ability to induce lymphomas with reduced ability for dis-
semination (303–305). The role of telomeres and telomer-
ase in MD pathogenesis has recently been reviewed (312).
MDV‐encoded microRNAs.  MicroRNAs (miRNAs) are a
distinct class of small regulatory molecules of approxi-
mately 22 nucleotides affecting gene expression in vari-
ous cell types (635). These have been identified in a large
range of organisms including several herpesviruses (389).
A number of miRNAs have been identified in MDV many
of which are expressed at very high levels (138, 561, 636,
639). Some of these miRNAs have been shown to have
direct roles in pathogenesis (556, 652, 653, 656).
Other Unique Genes.  Proteins have not been identified
for several unique ORFs that are transcribed in tumor
cells. Most of these have not been further studied with a
few exceptions. RLOR5a (400) is expressed in tumor cell
lines, although its function remains unknown. Similarly,
RLORF4 has also been shown to be associated with
tumor development (289). Marek’s disease virus also
encodes the ubiquitin‐specific protease domain within
the major tegument protein‐coding gene UL36 (285,
570). Expressed at high levels in the FFE cells, it may have
a role in virus morphogenesis in the feather (286).
Viral Vectors.  Several nonessential sites in the three
serotypes of MDV can be used for the insertion and
expression of foreign and specific MDV genes (reviewed
in [261, 349, 412]). The anticipated advantages of MDV‐
vectored vaccines are that these vaccines will protect
simultaneously against MD and other pathogens, and
reactivation from latency will reinforce immune responses
against MD and the other pathogens (18, 67, 584).
Virus Replication
Replication of the three serotypes is typical of other cell‐
associated herpesviruses and has been reviewed exten-
sively (47, 301, 473, 484, 498). For initial infection of
cultures or chickens by cell‐free virus, enveloped virions
bind to cellular receptors probably by gB perhaps in
combination with other glycoproteins. Heparan sulfate,
a member of the glycosaminoglycans, has been identified
as one of the cellular receptor molecules (338). Recent
data demonstrate the role of the US3‐encoded kinase in
the morphogenesis as well as cell to cell spread of virions
through the effect on stress fiber breakdown and polym-
erization of actin (509). The glycoproteins gE, gI, and gM
play a role in the transfer of virus from infected to unin-
fected cells (507, 564). Replication rates vary with sero-
type, passage level of the virus strain, cell type, and
temperature of incubation.
The spread of virus in vivo from cell to cell will require
intimate contact between infected and uninfected cells,
which are most often lymphocytes, although epithelial
cells also can be involved in this process. The precise
interaction between these cells remains one of the
important unsolved issues, although recent demonstra-
tion of epithelial‐specific expression of the UL47
­tegument protein (287) suggest distinct virus–host inter-
actions in these cell types.
Virus‐cell interactions
Three general types of virus–cell interactions are recog-
nized: productive, latent, and transforming.
Productive Infection.  During productive infection,
replication of viral DNA occurs; proteins are synthesized,
and in some cases, virus particles are produced.
Replication is correlated with virulence (172), but with
all serotypes the number of genome copies per cell can
increase 100‐fold and exceed 1,200 in the case of HVT
(300). Two types of productive infection exist. Fully
productive infection in the FFE of chickens results in
development of large numbers of enveloped, fully
infectious virions (92). In productive‐restrictive
infection, most of the virions are nonenveloped and
noninfectious. A variable number of the virions in
cultured cells may be enveloped, which can be recovered
as cell‐free, infectious virus by disruption of cells. In all
susceptible cells, productive infection leads to
intranuclear inclusion body formation and lysis of the
cell. A gene for the viral host shut‐off protein has been
identified, UL41 (354), that is probably responsible for
the initiation of the lytic process. Lytic infection in vivo
can cause frank necrobiotic lesion formation. Because of
this, productive infection has been termed cytolytic, and
the terms are used synonymously (89).
Latent Infection.  Latency and tumorigenesis in MD have
been reviewed (387). Latent herpesvirus infections have
been defined as the presence of viral DNA in the absence
of viral transcripts and proteins, although LATs have
been described for many herpesviruses. This definition
is appropriate for the nontransforming serotype 2 and 3
strains. For serotype 1 strains, the distinction between
latency and transformation is often problematic. In both
cases, the viral genome is present, but no information is
available on differences in transcriptional regulation

between latently infected and transformed cells,
because  it is impossible to separate latently infected,
nontransformed cells from noninfected cells. As a
consequence, studies on latency have often been done in
MD transformed cell lines.
Marek’s disease virus latency mostly is associated with
CD4+ T cells, although CD8+ T cells and B cells can also
be latently infected (105, 337, 386). Fewer than five cop-
ies of the viral genome are present in latently infected
cells (473). Marek’s disease virus latency is maintained
through various mechanisms including non‐random de
novo DNA methylation and histone modifications, with
different genomic regions separated by chromatin
boundary elements (63, 351). The MDV genome can be
reactivated from latently infected cells and tumor cells by
inoculation of susceptible chickens, co‐cultivation with
permissive cells, and in vitro cultivation of latently
infected lymphocytes. The latter approach can be used
to estimate the number of latently infected cells by enu-
meration of antigen‐positive cells at 0 hours and 48 hours
in culture (107).
Transforming Infection.  Transforming infections occur
only in cells infected with serotype 1 MDV. Selection of
transformed cells from the background of immunologically
committed and noncommitted cells (431) would facilitate
comparative studies on transformed cells in tumors and
tumor cell lines. The search for specific surface markers
associated with tumors has identified antigens, broadly
referred to as MD tumor‐associated surface antigen
(MATSA), detected on cells from MD lymphomas and
lymphoblastoid cell lines but not on the surface of
productively infected cells (443, 623). Recent studies
confirm that CD30hi expression is characteristic of MD
lymphomas suggesting that CD30 is a component of a
critical intracellular signaling pathway perturbed in
neoplastic transformation (78, 511). Both MATSA and
CD30 can be used to enrich for transformed cells in
tumor cell suspensions.
Virus Stock Production and Stability.  Productively infected
cell cultures are a common source of cell‐associated
virus stocks for all three viral serotypes and for cell‐free
HVT stocks. Techniques for the production and
cryopreservation of cell‐free and cell‐associated virus
stocks have been described (reviewed in 130). Cell‐
associated stocks of MDV or HVT are routinely stored at
−196°C. The infectivity of such stocks, however, is
directly related to viability of the cells contained in these
preparations and depends also on optimal freezing and
thawing techniques. Under ideal conditions, the half‐life
of diluted, cell‐associated virus stocks or vaccines should
be at least 2–6 hours (559).
Cell‐free serotypes 1 and 2 virus stocks are best
obtained from FFE (low‐passage virus) or infected cell
Chapter 15  Neoplastic Diseases 557
cultures (high‐passage virus). Small quantities of low‐
passage virus can be obtained from infected cell cultures
by lysing cells in SPGA (sucrose–phosphate–glutamate–
albumin) buffer (98). The production of cell‐free HVT is
best achieved by lysing heavily infected cell cultures. Cell‐
free MDV and HVT can be stored at −70°C or lyophilized
(98). Potency of both cell‐associated and cell‐free vac-
cines can be affected adversely by storage temperature,
reconstitution technique, choice of diluent, and holding
time and temperature after reconstitution (239, 423).
Susceptibility to Chemical and Physical Agents
The stability of cell‐associated MDV serotype 1 and 2
strains is completely dependent on the viability of the
cells. Any treatment affecting cell viability will impact
directly the infectivity of virus stocks.
Cell‐free MDV obtained from the skin of infected
chickens was inactivated when treated for 10 minutes
at pH 3 or 11 and stored for 2 weeks at 4°C, 4 days at
25°C, 18 hours at 37°C, 30 minutes at 56°C, or 10 min-
utes at 60°C (91). Dander, litter, and feathers from
infected chickens are infectious and presumably con-
tain cell‐free virus from the FFE bound to cellular
debris. The infectivity of such materials was retained
for 4–8 months at room temperature (263, 602) and for
at least 10 years at 4°C (87). Virus infectivity was inac-
tivated by a variety of common chemical disinfectants
within a 10‐minute treatment period (97, 262). Survival
of virus in litter may be affected adversely by increased
humidity (602).
Strain Classification
Serotypes
Following full genome sequencing, the three MDV
serotypes have now been designated as three distinct
species: Gallid alphaherpesvirus 2 (serotype 1), Gallid
alphaherpesvirus 3 (serotype 2), and HVT (serotype 3).
Von Bülow and Biggs (576, 577) originally classified the
MDV herpesvirus group into three distinct serotypes
that correlated with biologic properties. Type‐specific
mAbs (270, 332) usually are used to determine virus
serotype.
A number of biological characteristics are associated
with viral serotypes (50, 484). Low‐passage serotype 1
viruses grow best in duck embryo fibroblast (DEF) or
chicken kidney cell (CKC) cultures, grow slowly, and
produce small plaques. Serotype 2 viruses grow best in
chicken embryo fibroblasts (CEF), grow slowly, and pro-
duce medium plaques with some large syncytia.
Herpesviruses of turkey grow best in CEF, grow rapidly,
and produce large plaques. More infectious virus can be
extracted from HVT‐infected cells than from cells
infected with serotype 1 or 2 viruses.
558 Section II  Viral Diseases
Pathotypes
Virulence or oncogenicity is only associated with sero-
type 1 MDVs. Within this group, however, a wide varia-
tion in pathogenic potential is recognized and
undoubtedly represents a continuum from nearly aviru-
lent to maximally virulent. Pathotype classification
schemes have evolved over the last 30 years with the con-
tinued increase in virulence. Current classification
schemes recognize four groups of viruses. These groups
are designated as mMDV, vMDV, vvMDV, and vv+MDV
(597, 604). Pathotyping of virus isolates involves com-
parative pathogenicity tests in vaccinated and unvacci-
nated maternal antibody positive chickens with prototype
viruses as controls (592, 601). Detailed reviews on the
increasing virulence and the details of prototype viruses
have been published (211, 498, 598, 599, 601). The evolu-
tion in the virulence of MDV strains is recognized, but
the molecular basis for this evolution has not been eluci-
dated, as genome sequence analyses of a number of these
strains have not provided definite markers associated
with the virulence phenotypes.
Certain biologic characteristics are associated with
pathotypes of serotype 1 MDVs but are most pronounced
between low‐passage and high‐passage (attenuated)
strains. Serial passage in vitro (30–70 passages usually
are required) results in attenuation of virulent isolates
(470, 494, 589). Attenuated strains grow more readily in
vitro but produce lower viremia titers in vivo (609),
which may be associated with a marked decrease in their
ability to infect and/or replicate in lymphocytes and
spread between birds (160, 494, 591). There could also be
incomplete attenuation resulting in minor lesions in sus-
ceptible chickens (439, 575) or over‐attenuation result-
ing in low level in vivo replication and poor immune
response (318, 614). The in vivo growth potential of
attenuated serotype 1 isolates can be increased by back-
passage in chickens (161, 591). A recent study on the
molecular basis of attenuation identified several de novo
mutations associated with reduced virulence (258).
Laboratory Host Systems
Marek’s disease virus usually is propagated and assayed
in tissue cultures, newly hatched chicks, and embryo-
nated eggs. Lymphoblastoid cell lines from MD lympho-
mas are also an important laboratory host system.
Cell Cultures
The propagation of MDV serotypes in vitro has been
reviewed (484, 487, 498). Isolation of low‐passage MDV
in CEF or embryonal CKC cultures is far less efficient
than in CKC or DEF (487) as propagation in CEF leads to
accelerated attenuation (494). In embryonal CKC, repli-
cation of serotype 1 MDV (but not HVT) is abortive,
leading to loss of infectivity within two to three passages.
Attenuated MDV and serotype 2 and 3 viruses can be
isolated readily and propagated in CEF (490). Infected
cultures usually develop discrete focal lesions, called foci
or plaques, which consist of clusters of rounded, refrac-
tile degenerating cells when mature (Figure 15.2). Plaques
are usually less than 1 mm in diameter and of variable
cell density, although plaque size varies with viral strain,
time, and other factors. Polykaryocytosis is seen in
­cultured fibroblasts and is a major component of the
viral plaques or foci frequently used as a marker in virus
assays. Affected cells may contain two to several hun-
dred nuclei, and type A intranuclear inclusion bodies are
commonly seen. Despite release of rounded cells into the
medium as plaques mature, large areas of cell lysis are
not seen.
Serotype 1 plaques develop in 5–14 days on primary
isolation and in 3–7 days after adaptation to culture and
usually are enumerated by microscopic examination, but
different staining techniques have been developed,
allowing enumeration at a later time. Differences in
development and morphology of serotype 1 plaques in
chick and duck cells and in plaques induced by the three
viral serotypes (484) have been described. Other cell cul-
ture systems such as chick embryo skin (445), tracheal
explants (460), and embryo fibroblasts from several
avian species including Japanese quail (453) also have
been used.
A few avian cell lines such as OU2 (8, 9), quail muscle
cell line QM7 constitutively expressing MDV‐1gE (471,
508), JBJ‐1 (206), chicken embryo liver (327), and lines
derived from Muscovy duck retinal tissue (293) have also
been used for the propagation of MDV strains with vary-
ing success. Quail cell lines free of MDV can be used to
propagate serotype 1 MDV and HVT, but SB‐1 did not
replicate efficiently (343). Recently developed keratino-
cyte cell lines support MDV replication, producing ­cell‐
associated viral progeny (140).
Serotype 1, but not serotype 2, MDVs can also be
grown in chicken splenic lymphocytes in vitro (106).
Passages are made by the addition of fresh spleen cells to
the suspension cell cultures every two days, and infec-
tion is monitored by IF. Herpesvirus of turkey may be
similarly grown in turkey spleen cell cultures, but viral
antigen is rarely seen, if at all.
Chickens
Newly hatched chicks inoculated with virulent, serotype
1 MDV develop gross lesions or lesions that can be
detected histologically in ganglia, nerves, and certain
viscera after 2–4 weeks. Response is greatly dependent
on genetic susceptibility of the chicken and virulence of
the MDV isolate. Presence of virus or antibody, which
can be detected by in vitro tests, or the presence of virus‐
associated antigen detected by FA tests on tissues, are
also specific host responses of inoculated chickens to
 Chapter 15  Neoplastic Diseases 559

(A) (B)

(C) (D)

(E) (F)

Figure 15.2  Focal lesions in cultured cells infected with various Marek’s disease virus (MDV) serotypes. (A) Low‐passage serotype 1 MDV
in chicken kidney cells cultured from an infected chicken, 9 days. (B) Low‐passage serotype 1 MDV in duck embryo fibroblasts (DEF), 5
days. (C) High‐passage, attenuated serotype 1 MDV in chick embryo fibroblasts (CEF), 5 days. (D) Low‐passage serotype 2 MDV in CEF, 8
days. (E) Low‐passage HVT (serotype 3) in CEF, 4 days. (F) Low‐passage turkey herpesviruses (HVT) in DEF, 12 days. All photos unstained,
about ×40. (Witter)
560 Section II  Viral Diseases
MD infection. All these responses are markedly enhanced
in chicks lacking maternal antibodies against MDV (86).
The induction of virus‐specific lesions in the wing web
(99) or the feather pulp (377) constitutes alternate
approaches that provide direct access to the site of lesion
development.
Embryos
Virus pocks develop on the chorioallantoic membrane
(CAM) of chicken embryos following yolk sac inocula-
tion with cellular MDV preparations (49, 574). Embryos
have also been used for MD vaccine evaluation, because
in ovo vaccination is becoming increasingly common in
the field. Embryos may also be used to isolate MDV
viruses that cannot be isolated directly in cell culture for
unknown reasons. Yamaguchi et  al. (633) reported the
isolation of MDV from the QT35 cell line by using kid-
ney cell cultures prepared from 4‐ to 7‐day‐old chicks
that had been inoculated at embryonic day (ED) 8 with
QT35 cells.
Lymphoblastoid Cell Lines
Lymphoblastoid cell lines developed from MD lympho-
mas grow continuously in cell culture without attach-
ment to the culture vessel. Success rates for establishing
cell lines from MD lymphomas have improved because
of better methodology (108, 428). Many cell lines are
now available including several from MD lymphomas in
turkeys (392). The majority of the chicken cell lines
established from lymphomas are CD4+/CD8‐ T cells
expressing major histocompatibility complex (MHC)
class II and T cell receptor (TCR) 2 or 3 (386, 417, 495).
Lymphoblastoid cell lines can also be established from
lymphocytes harvested from early (4–6 days postinfec-
tion) lesions induced in the wing web or pectoral muscle
by injection of a mixture of MDV and allogeneic kidney
cells. The cell lines from early lesions may be CD4+/
CD8−, CD4−/CD8+, or CD4−/CD8− (495). Cells of the
MDCC‐RP1 line are illustrated in Figure 15.3.
Some transformed cells contain about 5–15 copies of
viral genome, although the mean number may be consid-
erably higher in different cell lines, perhaps in relation to
the proportion of productively infected cells in the
­population (376, 473). Most cell lines can be termed
“producer” lines, because a small proportion (1–2%) of
the cells enter into productive infection (108, 443). Viral
DNA can be highly methylated in cell lines although
methylation is not essential for maintaining the trans-
formed state (298, 417). Methylation profiles of the viral
genome in the cell lines are not very different from those
in the primary tumors (63).
Marek’s disease tumor-derived cell lines have been
used to analyze the potential interaction with tumor
suppressor genes and cellular oncogenes, such as Meq.
The data generated from the systematic analysis of the
Figure 15.3  Smear from the MDCC‐RP1 cell line. Note the
characteristic lymphoblastoid morphology and the mitotic
figures. Giemsa, ×1500. (Nazerian)
gene expression from a number of MDV‐induced
tumors and transformed cell lines (358, 531, 559) will
help to identify the molecular and biochemical path-
ways of transformation and the maintenance of the
transformed phenotype in these cells.
Pathobiology and Epizootiology
Multiple Syndromes
Marek’s disease consists of several distinct pathologic
syndromes (90) with apparent differences among these
syndromes typically seen in commercial flocks,  and
those induced in the laboratory. Lymphoproliferative
lesions, especially lymphomas, are most frequently
associated with MD and have the most practical
importance (Table  15.3A). However, skin leukosis
in  broilers, fowl paralysis, persistent neurological
disease, and ocular lesions are additional clinical
­
manifestations with lymphoproliferative components.
Some of the lymphoproliferative syndromes may also
have degenerative components. Several additional
clinical syndromes characterized solely by degenera-
tive and inflammatory lesions, often with accompanying
immunosuppression, are induced by experimental
infection (Table 15.3B). Non‐neoplastic brain pathol-
ogy, mainly vasogenic edema, is responsible for tran-
sient paralysis (231). Vascular lesions are manifested
as atherosclerosis (192). Under laboratory conditions,
young chicks inoculated with tumor cells may develop
transplantable tumors (267, 544, 558). Inoculation
of  MDV‐infected, allogeneic CKC in the wing web
may induce local tumor lesions (99). Some of the syn-
dromes induced under laboratory conditions are rare
or nonexistent in the field, probably because most
 Chapter 15  Neoplastic Diseases 561

Table 15.3A  Clinical and pathologic syndromes associated with Marek’s disease virus (MDV) (part A).

Lymphoproliferative syndromesab (Marek’s disease)

Situation in which syndrome Lymphomas and nerve Fowl paralysis (nerve Skin leukosis Blindness and ocular
observed lesions lesions) (integument) lesions

Experimental chickens (laboratory)

Clinical signs Depression, death, Paralysis Swollen feather Blindness, ocular
stunting, paralysis follicles lesions
Mortality 0–100%c 0–30%cd None Rare or nonec
E
Age Onset 2–8 weeks PI Growing birds Young birds 4–8 weeks PI
Organ Visceral Mostly peripheral Skin Eye (iris, cornea)
organs+peripheral nerves
nerves
Layer/breeder flocks (field)
Clinical signs Depression, death, Paralysis, death Swollen feather Blindness, gray eye
paralysis follicles
Prevalence Common Occasionald Rare or nonee Rare
Mortality 0–60% 0–20% None None
Age 4–90 weeks 8–20 weeks 4–8 weeks PI >10 weeks
Broiler flocks (field)
Clinical signs Depression, death, Paralysis, death Swollen feather Blindness, gray eye
paralysis follicles, red leg
Prevalence Common Rare or noned Commone Rare or none
Mortality Minor — None None
Age At processing — At processing —
a
 Neoplastic lesions may include inflammatory components.
b
 Severity of syndrome usually less in vaccinated flocks.
c
 Depends on experimental conditions (virus strain, dose, chicken genotype, maternal antibody status, prior vaccination, etc.).
d
 Rarely induced by contemporary MDV strains, except in conjunction with visceral neoplastic lesions.
e
 Not usually recognized except at broiler processing or after feather removal.

commercial chickens are maternal antibody positive
and vaccinated.
Subclinical syndromes may also occur but are more
difficult to define. Vaccinated flocks produced more eggs
than nonvaccinated flocks, indicating that MDV may
depress productivity in otherwise normal‐appearing,
nonvaccinated chickens (454).
Incidence and Distribution
Marek’s disease exists in all poultry‐producing countries.
Marek’s disease is generally considered ubiquitous
among vaccinated commercial flocks, although recent
evidence suggests not all flocks are necessarily infected
(56, 581). Reporting systems vary, however, and it is dif-
ficult to determine the true MD incidence (174). Even in
susceptible chickens, infection does not always induce
clinical disease and, in genetically resistant or vaccinated
chickens, infection may rarely cause overt disease.
Since 1961, the United States Department of Agri­
culture (USDA) Food Safety and Inspection Service has
collected condemnation data from processing plants,
including young broiler chickens condemned for leuko-
sis, which almost exclusively refers to MD. This data,
analyzed and reported from the USDA National
Agricultural Statistics Service, shows a gradual decrease
in leukosis condemnations of young broiler chickens
since the early 1970s, reaching an all‐time low of
0.00069% during 2016 (Figure 15.4). Regional differences
are striking, as illustrated by annual leukosis condemna-
tion rates in Delaware versus Georgia. The data has also
revealed a biphasic pattern with maximum condemna-
tion rates around April and minimum frequencies
around August, although this seasonal trend has steadily
decreased since about 1990 (309, 498, 596).
Sporadic outbreaks of MD occur on individual farms
or regions. Recent industry‐wide surveillance study in
Pennsylvania demonstrated the persistence of the virus
with fluctuation in virus loads at different farms (309).
Several reports lend credence to the implication of
exceptionally virulent MDV isolates in vaccine failures
(e.g., 35, 597), but it is important to exclude CIAV as a
562 Section II  Viral Diseases

Table 15.3B  Clinical and pathologic syndromes associated with Marek’s disease virus (MDV) (part B).

Lymphodegenerative Vascular
syndromes CNS syndromes syndromes Other syndromes

Early mortality syndrome, Transient paralysis and

Situation in which cytolytic infection, persistent neurological Local lesions;
syndrome observed immunodepression diseases Atherosclerosis transplants (transpl.)

Experimental chickens (laboratory)

Clinical signs Depression, stunting, Transient paralysis, tics, None Swelling at
death, increased disease torticollis, death inoculation site
susceptibility
Mortality 0–100%ab 0–100%ab None Yes (transpl.)
Age 9–20 days PI 9–28 days PI Adult birds Young birds
Organ Bursa, thymus, spleen Brain Blood vessels Web–local and
many–transpl.
Layer/breeder flocks (field)
Clinical signs Increased disease Transient paralysis, tics, — N/A: Only
susceptibility torticollis experimental
Prevalence Rarea Rarea Rare or none —
Mortality — Rare — —
Age — 5–12 weeks — —
Broiler flocks (field)
Clinical signs Increased disease Transient paralysis, tics, — N/A: Only
susceptibility torticollis experimental
Prevalence Rarea Occasionala None —
Mortality — Rare — —
Age — 5–7 weeks — —
a
 Not normally observed in chickens vaccinated for MD.
b
 Depends on experimental conditions (virus strain, dose, chicken genotype, maternal antibody status, prior vaccination, etc.).

10 Figure 15.4  Marek’s disease condemnations

in young broilers for the period 1961–2016
(data from National Agricultural Statistics
1 Service). (Dunn)

0.1
Percent

0.01

0.001

USA
0.0001
Delaware
Georgia

0.00001
1960 1970 1980 1990 2000 2010 2020

cofactor (370). It is usually difficult to associate increased
virulence of MDV isolates with regional fluctuations in
MD frequency, but layer outbreaks in Ohio in 1995
yielded isolates of unusual virulence (95, 597).
In recent years, MD has been reported as the most
commonly diagnosed infectious disease among backyard
chickens in multiple countries (369, 438).
Natural and Experimental Hosts
Virtually all chickens including game fowl are susceptible
to MDV infection and tumor development (498) and are
by far the most important natural host. Quail, turkeys,
partridges, pheasants, and some species of ducks and
geese are also susceptible to infection and disease (1,
499). Outbreaks in Japanese quail are most commonly
reported among quail. In turkeys, occasional outbreaks
have recently been reported (54). Vaccination with
CVI988 appears to offer protection (137), whereas HVT,
interestingly, does not (182). Marek’s disease virus can be
re‐isolated from experimentally infected ducks (39), but
development of MD has not been demonstrated. Most
other avian species including sparrows, pigeons, and pea-
fowl are probably refractory (reviewed in [499]). Recently
a herpesvirus was isolated from three species of endan-
gered pheasants causing hepatocellular necrosis. Based
on sequence analysis of two genes, this virus is related to
HVT and is proposed to be a new member of the genus
Mardivirus (510). Marek’s disease‐like lymphoid tumors
in passerines are frequently cited, but need to exclude
lymphoma‐like lesions caused by infection with Isospora,
especially in captive populations (150). Mammals, includ-
ing several species of primates, are refractory to experi-
mental inoculation (reviewed in [499]).
Transmission, Carriers, Vectors
Marek’s disease virus is transmitted readily by direct or
indirect contact between chickens by the airborne route
(citations in 46, 499). The FFE produces fully infectious
virus (92). These cells present in feathers and dander form
the major source of contamination of the environment
and infection of chickens (41, 110). Contaminated poultry
house dust remains infectious for at least several months
at 20–25°C and for years at 4°C (citations in 89, 499).
Under commercial conditions, young chickens are most
commonly exposed to MDV by contact with r­ esidual dust
and dander in the growing house or by aerosolized dust
from adjacent chicken houses, fomites, or personnel. After
the virus is introduced into a chicken flock, regardless of
vaccination status or genetic resistance, infection spreads
quickly from bird to bird. Early studies, based on contact
infection, demonstrated that virus excretion begins about
two weeks postinfection (310) and continued indefinitely
(622). The development of qPCR assays for the three sero-
Chapter 15  Neoplastic Diseases 563
types has provided measurements of virus load in feather
pulp and dust (2, 31, 276, 469). These assays may measure
cell‐associated virus in the feather pulp and therefore do
not necessarily provide a measure for the amount of infec-
tious virus shed into the environment. The latter question
is relevant because dust collected from poultry houses
containing HVT‐vaccinated flocks is positive for HVT
DNA. It is possible that this DNA is derived from cell‐
associated HVT orginally present in feather pulp cells
rather than true cell‐free infectious virus. Older literature
had suggested that horizontal spread of HVT from vacci-
nated chickens is limited at best (498). Feather dust has
recently been used as a direct source for MDV whole
genome sequencing for representing shed virus from a
flock versus a point source in one animal (413).
Vertical transmission of MDV does not occur (470, 535,
536). Transmission from dam to progeny as the result of
external egg contamination is also unlikely because of
poor virus survival at temperature and humidity levels
used during incubation (97). Passive transmission by dar-
kling beetles (Alphitobius diaperinus) has been reported,
but free‐living litter mites, mosquitoes, and coccidial
oocysts do not transmit MDV (40, 59, 60, 179).
Experimental transmission is commonly accomplished
by parenteral inoculation of susceptible chickens with
cell‐associated virus from cell cultures, tumor cell sus-
pensions, or blood cells from infected chickens. Exposure
by direct or indirect contact with infected chickens is
also effective. Cell‐free but not cell‐associated virus can
be used for intratracheal instillation or inhalation expo-
sure. An aerosol‐based exposure model was described
(6), which may facilitate studies on the early events in the
pathogenesis.
Incubation Period
The incubation period for experimentally induced MD is
well established (see reviews 27, 90). Mononuclear infil-
trations containing AV37+CD4+ lymphocytes can be
detected in nerves of maternal antibody‐negative, genet-
ically susceptible chickens as early as 5 days PI (75).
Monoclonal antibody AV37 recognizes chicken CD30hi,
which is considered a marker for transformed lympho-
cytes (78). Clinical signs and gross lesions generally do
not appear until between the third and fourth weeks.
The incubation periods can be short for several non-
lymphomatous syndromes associated with MDV infec-
tion. Cytolytic infections occur at 3–6 days PI and are
followed by degenerative lesions (atrophy) of the thymus
and bursa of Fabricius within 6–8 days PI (74). The early
mortality syndrome (EMS) is characterized by deaths at
8–14 days PI (615). The clinical expression of both acute
and classical forms of transient paralysis usually occurs
from 8–18 days PI (311, 607) and can occur when SPF
birds are challenged with vv+MDV between 30 and 102
564 Section II  Viral Diseases
weeks of age (605). Field cases of transient paralysis are
seen mainly between 6–12 weeks of age, probably reflect-
ing MDV exposure 8–10 days prior to the onset of symp-
toms. Development of atherosclerosis requires 3–7
months (191). Induction of tumors within 10–14 days
after inoculation of cellular material is suggestive of a
transplantation response (544). Local lesions in the wing
web are visible 3–4 days PI with allogeneic MDV‐infected
CKC. Cell lines have been established from these lesions
suggesting that transformed cells are present (99).
Under field conditions, MD outbreaks sometimes
occur in unvaccinated layer chickens as young as 3–4
weeks. Most of the serious cases begin after 8–9 weeks
but sometimes commence well after the onset of egg
production, especially in broiler breeders (381) or subse-
quent to molting (600). Witter (600) differentiated the
outbreaks in commercial, vaccinated chickens as “early”
or “late” breaks. Witter and Gimeno (605) suggested that
late breaks were not likely the result of recent infections
alone and that additional factors are needed to cause the
late breaks. The different manifestations of MD, includ-
ing EMS (113), may occur in backyard flocks, which are
often not vaccinated.
Clinical Signs
Signs associated with MD vary according to the specific
syndrome (Tables 15.3A and 15.3B). Chickens with MD
lymphoma or fowl paralysis syndromes may exhibit
signs, but few are specific to MD (45). In general, signs
related to peripheral nerve dysfunction are those asso-
ciated with asymmetric progressive paresis and, later,
complete spastic paralysis of one or more of the extrem-
ities. Involvement of the vagus nerve can result in
paralysis and dilation of the crop and/or gasping.
Because locomotory disturbances are easily recognized,
incoordination or stilted gait may be the first observed
sign. A particularly characteristic clinical presentation
is a bird with one leg stretched forward and the other
back as a result of unilateral paresis or paralysis of the
leg (Figure 15.5). However, chickens with MD lympho-
mas may appear clinically normal but have extensive
neoplastic involvement when euthanized, while other
birds may become depressed and comatose prior to
death. Nonspecific signs such as weight loss, paleness,
anorexia, and diarrhea may be observed, especially in
birds in which the course is prolonged. Under commer-
cial conditions, death often results from starvation and
dehydration because of the inability to reach food and
water or from trampling by flockmates. Some birds
develop nervous tics or torticollis 18–26 days PI, often
after recovery from classical transient paralysis. This
syndrome, termed persistent neurological disease
(231), can be induced by partially attenuated MDVs
that no longer induce ­transient paralysis (229). However,
Figure 15.5  Fowl paralysis. Spastic paralysis of limbs associated
with peripheral nerve involvement in Marek’s disease. (Witter)
the central nervous system (CNS) signs are difficult to
distinguish from those associated with MD nerve
lesions.
Birds with ocular involvement may show evidence of
blindness (198, 415, 543). Ficken et al.(198) isolated two
MD strains from commercial flocks with greater than
90% blindness. The blindness was reproduced in experi-
mentally‐infected commercial chickens. Gross ocular
lesions were not always present in blind birds. The blind-
ness can be unilateral or bilateral, although recognition
of clinical blindness requires careful observation.
Affected eyes gradually lose their ability to accommo-
date to light intensity.
Early mortality syndrome results in high mortality
8–16 days PI of young chickens with virulent MDV strains
(571, 615). Chickens become depressed and comatose
prior to death, which occurs within 48 hours of the onset
of signs. Some affected chickens may also exhibit flaccid
neck paralysis prior to death (607). Chickens undergoing
acute cytolytic infection at 3–6 days PI may be depressed
but rarely die during this period, although some may die
later from EMS. Immunosuppressed chickens may suc-
cumb to ancillary infections, but some chickens die 20–40
days PI with few signs.
Classical and acute transient paralysis syndromes
have been described in field flocks (643) and is associ-
ated with MDV infection (311). It has been observed
infrequently in the field since vaccination for MD has
become widespread, but may be encountered in non‐
vaccinated backyard flocks. In the classical form,
affected chickens display varying degrees of ataxia and
flaccid paralysis of the neck or limbs beginning 8–12
days PI (Figure 15.6). Signs typically last 1–2 days fol-
lowed by a rapid and complete recovery, although
recovered chickens may succumb a few weeks later
with MD lymphomas. The acute (fatal) form results in
death within 24–72 hours following the onset of para-
lytic signs (607).

Figure 15.6  Transient paralysis. Flaccid paralysis of neck of young
chicken nine days after inoculation with Marek’s disease virus.
(Courtesy of Avian Diseases.) (Witter)
Morbidity and Mortality
The incidence of MD is quite variable in commercial
flocks and in general low since the worldwide introduc-
tion of vaccines, although problems are sometimes
reported (174, 381). Most birds developing clinical dis-
ease die. A few birds may apparently recover from the
clinical disease (52, 75), but the recovery is rarely perma-
nent. Prior to the use of vaccines, losses in affected flocks
were estimated to range from a few birds to 30% and
occasionally as high as 60% (381). In broilers, MD con-
demnations averaged 1.0% in 1970 with 10% or higher in
individual flocks (452). The average condemnation rate
for MD in the United States has decreased dramatically
(see Incidence and Distribution).
Some flocks experience significant disease outbreaks
despite vaccination. After the disease appears, mortality
builds gradually and generally persists for 4–10 weeks.
Outbreaks occur and abate spontaneously in isolated
flocks or occasionally in several flocks in a region or in
succeeding flocks on a farm. The reasons for these fluc-
tuations are poorly understood.
Response rates or mortality approaching 100% for
lymphomas, EMS, acute cytolytic infection, or transient
paralysis can be achieved following inoculation or expo-
sure of unvaccinated, susceptible chickens to MDV.
Because the response frequency is influenced by many
factors (see Factors that Influence Mortality and Lesions),
laboratory experiments can be designed to produce a
wide range of specific clinical and pathologic responses.
Factors that Influence Mortality and Lesions
Virus Strain.  The virulence of MDV strains varies widely
and appears to have increased over time (597) and is
reviewed in (159, 489). Compared to the milder forms of
the disease, which caused mainly peripheral nerve
lesions, very virulent (vv) and vv+ pathotypes frequently
induce higher mortality and more visceral lymphomas,
and have the tendency to more frequently break through
genetic host resistance or immunity induced by
Chapter 15  Neoplastic Diseases 565
vaccination (408, 604). The extent of disease induced by
a given strain depends in part on the genetic constitution
of the host (493).
Virus Dose and  Route.  Dosage may influence disease
frequency under natural conditions, although the MD
response in genetically susceptible birds given virulent
virus was found to be maximal even when a limiting
dilution of virus was inoculated (532). Infection with
cell‐free MDV through intratracheal inoculation or by
aerosol may enhance early virus replication and increase
the development of lymphomas compared to parenteral
inoculation (6, 19, 84).
Host Gender.  Several studies indicated that females died
earlier and experienced higher losses than males (357,
427), but the opposite has also been reported (363). The
differences were apparently not due to sex hormones,
varied with the genetic strain, and were most pronounced
with genetically susceptible chickens and with viruses of
higher virulence. In practice, the influence of gender is
probably less important.
Maternal Antibodies.  Maternal antibodies reduce and
delay MD mortality (129), EMS (615), and transient
paralysis (311), probably by limiting, but not preventing,
the spread of virus in tissues during the first few days
post  exposure (86, 429). Thus maternal antibodies do
not  provide a sterilizing immunity. Breeder stocks are
vaccinated uniformly and exposed to virulent MDV and
virtually all chickens are hatched with maternal antibodies
against multiple serotypes. Specific pathogen free flocks
are a source of antibody‐free chicks for laboratory studies.
Host Genetics and  Age at Exposure.  Genetic factors (see
reviews [20, 71]) and age at initial exposure are important
determinants of MD susceptibility (see Chapter 2).
Age‐related resistance is an expression of genetic
resistance and develops after hatching paralleling the
development of immune competence. Newly hatched
chicks and older chickens are both susceptible to infec-
tion and cytolytic infection (90), but cytolytic infections
are resolved more rapidly in older birds (82) and virus
load is somewhat lower (156). The frequency of lympho-
mas is variable and often markedly reduced in older
chickens compared to newly hatched chicks, especially
in genetically resistant lines (498). However, non‐vacci-
nated, SPF, older chickens may develop high rates of lym-
phomas and transient paralysis following challenge with
vv and vv+ strains (269, 605). Age‐related resistance can
be abrogated by neonatal thymectomy (520) suggesting
that other immunosuppressive factors, for example,
CIAV (501), may increase the susceptibility of older
chickens to disease. Lesion regression has been linked to
age‐related resistance (75, 519).
566 Section II  Viral Diseases
Prior Infection.  Early studies had shown that mild,
serotype 1 strains can induce protective immune
responses against challenge (533). Dunn et  al. (176)
demonstrated superinfection following a short interval,
but if the second challenge was given after 14 days,
regardless of virulence, superinfection was significantly
reduced.
Environmental Factors and Stress.  Various environmental
factors and intercurrent infections appear to affect
the  incidence of MD, probably through interference
with immune responses. Gross (235) observed increased
incidence among chickens selected for high concentra­
tions of plasma corticosterone or subjected to a high
degree of social stress. The administration of cor­
ticosteroids to latently infected chickens precipitated the
appearance of clinical MD (441). Feeding of corticosteroid
inhibitors tended to increase resistance to MD (134).
Restricted feed intake delayed and reduced incidence
of  MD (240) whereas high‐protein diets (450) or the
selection for fast growth rate were associated with
increased susceptibility to MD (240).
Because MDV infection may depress host immune
responses in its own right (see Immunosuppression),
concurrent infections are often exacerbated. However,
when the concurrent infection is itself immunosuppres-
sive, the resulting immunosuppression usually will exac-
erbate both disease processes. Examples include IBDV,
REV, and CIAV (291, 578, 610, 648). Problems with CIAV
contamination invariably interfere with the evaluation of
MDV stocks for relative virulence (371).
Pathology
Gross Pathology
Pathologic changes in MD have been reviewed (425, 427)
and consist mainly of nerve lesions and visceral lympho-
mas. Macroscopic changes are not seen in the brain, but
gross enlargements can be found in spinal ganglia.
Nerves.  Severely affected peripheral nerves may show
loss of cross‐striations, gray or yellow discoloration, and
sometimes an edematous appearance. Usually, plexi of
the sciatic and brachial nerves are more enlarged than
the respective trunks. Localized or diffuse enlargement
causes the affected portion to be 2–3 times normal size,
in some cases much more. Goodchild (233) reported
that autonomic nerves and especially the celiac plexus
are affected at a higher frequency than peripheral nerves,
for example, the sciatic and brachial nerves. Witter (595)
found the cervical vagus to be of particular diagnostic
importance. Because unilateral and or minimal
enlargements may be important indicators of disease, it
is helpful to examine opposite nerves and, in experimental
infections, to compare with age‐matched normal
Figure 15.7  Enlarged sciatic plexus (left) and normal plexus
(right). (Peckham).
controls to detect changes. Careful examination of the
various nerve ramifications may be necessary to expose
gross lesions in some birds, because enlargements can
vary in both presence and degree from one portion of an
affected nerve to another. Figure 15.7 illustrates unilateral
gross enlargements in the sciatic plexus.
Visceral Organs.  Lymphomas may occur in one or more
of a variety of organs and tissues. Lymphomatous lesions
can be found in the gonad (especially the ovary), lung,
heart, mesentery, kidney, liver, spleen, bursa, thymus,
adrenal gland, pancreas, proventriculus, intestine, iris,
skeletal muscle, and skin. Probably no tissue or organ is
without occasional involvement. Both the genetic strain
of chicken and the virus strain can influence the organ
distribution of lesions. Visceral lymphomas are common
in more virulent forms of the disease (597). Visceral
tumors can occur in the absence of gross nerve lesions,
especially in certain strains of chickens. Marek’s disease
lymphomas in most viscera appear as diffuse
enlargements, sometimes to several times the normal
size, and a diffuse white or grayish discoloration is often
present (Figure  15.8B). Alternatively, lymphomas may
occur as focal, nodular growths of varying size
(Figures 15.8E and 15.8F). Nodules are white or gray in
color and are firm, and the cut surface is smooth.
Necrosis is rare but may occur in the center of rapidly
growing lesions.
Diffuse infiltration of the liver causes loss of normal
lobule architecture and often gives the surface a coarse
granular appearance. Nodular tumors may also be seen
in the liver. Lesions in the immature ovary are observed
as small to large grayish translucent areas (Figure 15.8B).
With large tumors, the normal foliated appearance of the
ovary is obliterated. Mature ovaries may retain function,
even though some follicles are tumorous. Marked

involvement is indicated by a cauliflower‐like appear-
ance. The proventriculus becomes thickened and firm as
a result of focal leukotic areas within and between the
glands, which may be seen through the serosal surface or,
if involvement is diffuse, detected by palpation. Affected
hearts are pale from diffuse infiltration or have single
or  multiple nodular tumors in the myocardium
(Figure 15.8F). Pinpoint foci may be seen in the epicar-
dium. Involvement of the lung (Figure  15.8E) may be
indicated by increased firmness of the organ upon palpa-
tion. Muscle lesions may be present in both superficial
and deep layers and are most common in the pectoral
muscle (43). Gross changes vary from tiny whitish streaks
to nodular tumors.
Integument.  Skin lesions, probably the most important
cause of condemnation in broiler chickens, usually are
associated with feather follicles. The nodular lesions may
involve few scattered follicles, or they may be numerous
and coalesce. The distinct whitish nodules (Figure 15.8A),
especially evident in dressed carcasses, may become
scablike with brownish crust formation in extreme cases
(43). Lapen and Kenzy (325) found the highest incidences
of lesions in external and internal crural and dorsal
cervical tracts. Erythematous involvement of the shank
integument is seen, especially in virulent forms of the
disease in broiler chickens (177) and is commonly known
as “Alabama redleg.” Swelling of the comb or wattles may
indicate lymphoma growth in underlying tissues (177,
180). Interestingly, FFE is not essential for virus
production or development of skin tumors, because
scaleless chickens produced cell‐free infectious virus in
epithelial cells and developed skin tumors (250).
Eye.  Gross ocular changes, including loss of
pigmentation in the iris (“gray eye”) and irregularity of
the pupil, are caused by mononuclear infiltration of the
iris (Figure  15.8C). Nearly all field isolates can induce
ocular lesions in nonvaccinated or HVT‐vaccinated
chickens (597). Conjunctivitis, occasionally with
multifocal hemorrhages and corneal edema have also
been observed (198).
Other Syndromes.  Gross lesions are associated with at
least some of the other MDV‐associated syndromes. The
lymphodegenerative syndromes, related to intense
cytolytic infections of lymphoid organs, usually are
characterized by severe bursal and thymus atrophy. The
cytolytic infection is first evident 3–6 days PI, but may
persist becoming more obvious at 8–14 days PI (95, 571,
615). After inoculation with highly virulent field strains,
some chickens may die at 20–50 days without gross lesions
except severe bursal and thymic atrophy (597). Some
chickens also develop a transient splenomegaly within
4–12 days PI (93). The splenomegaly is a non‐neoplastic
Chapter 15  Neoplastic Diseases 567
response to viral replication and is induced by all three
serotypes. Vascular syndromes are manifested principally
by occlusive atherosclerosis (192). Susceptible P‐line
chickens inoculated with the CU2 isolate of MDV
developed grossly visible fatty atheromatous lesions in
large coronary arteries, aortas, major aortic branches, and
other arteries (Figure 15.8D). Lymphoid tumor transplants
and local lesions are experimental syndromes characterized
by nodular growths at the site of inoculation, although
some transplantable tumors metastasize readily to the
liver and spleen, causing diffuse enlargements (544).
Microscopic Pathology
Histopathologic changes associated with MD lym-
phoproliferative lesions have been described by numer-
ous workers who are in general agreement about the
types of histologic lesions and the cells involved (reviewed
in 424, 425, 427, 499).
Nerves.  In peripheral nerves, two main types of
lymphoproliferative lesions are recognized, which are
referred to as type A and B, respectively. Type A lesions
consist mostly of CD30+CD4+ T cells (75) and some B
cells and is considered neoplastic. In some cases,
demyelination and Schwann cell proliferation are
associated with A‐type lesions. The second, B‐type,
lesions are essentially inflammatory and are characterized
by diffuse, light‐to‐moderate infiltration by small
lymphocytes and plasma cells, usually with edema, and
sometimes, with demyelination and Schwann cell
proliferation. A few macrophages may be found. The two
types may be observed in different nerves of the same
bird or even in different areas of the same nerve. Lawn
and Payne (326) observed cellular infiltrations as early as
five days PI, which gradually increased in intensity until
three weeks when severe proliferative, type‐A lesions
were seen in the absence of paralysis or demyelination.
Coincident with initial neurologic signs seen at four
weeks PI, areas of widespread demyelination could
be  found within the proliferative lesions. Finally,
characteristic inflammatory, type‐B lesions (edema,
sparse infiltrations) appeared, suggesting the occurrence
of regression of A type lesions. A third, C‐type, lesion
consists of light infiltration of lymphocytes and plasma
cells. The sequence of events has been reviewed in detail
by Payne et al. (427). Characteristic changes in nerves are
illustrated in Figure 15.9.
Brain.  The principal CNS lesion in MD consists of mild,
but persistent, perivascular cuffing usually accompanied
by gliosis but without primary demyelination as the
principal CNS lesion in MD (231, 427). Experimental
infection with less virulent MDV strains only resulted in
transient, moderately severe inflammatory lesions as
early as 7–10 days PI. In contrast, lesions induced by
 (B)

(A)

(C) (D)

(E) (F)

Figure 15.8  (A) Leukotic tumors involving feather follicles (skin leukosis). (Peckham) (B) Experimentally induced Marek’s disease (MD)
lymphoma in immature ovary (bottom) compared with normal ovary (top) (Witter). (C) Ocular lesions of MD. Note that the normal eye
(left) has a sharply defined pupil and well‐pigmented iris. Affected eye (right) has a discolored iris and very irregular pupil as a result of
mononuclear cell infiltration. (Peckham). (D) Gizzard from a chicken infected with CU‐2 isolate of MDV. Note the grossly obvious
atherosclerotic change in the arteries. (C. Fabricant). Microscopic changes from similar arteries are shown in Figure 15.16B. (E) Multiple
lymphomas in lungs. (F) Multiple lymphomas in heart. (Shivaprasad) (For color detail, please see the color section.)
 Chapter 15  Neoplastic Diseases 569

(A) (B)

Figure 15.9  Microscopic lesions of Marek’s disease in peripheral nerves. (A) Type A lesion characterized by marked cellular infiltration,
numerous proliferating lymphoblastic cells, and no edema. H&E, ×550. (B) Type B lesion with edema, scattered infiltrating small and
medium lymphocytes, and plasma cells. H&E, ×420. (Gimeno)

Figure 15.10  Extensive infiltration of lymphoblasts extending

into the neuropil in the cerebellum of a Marek’s disease virus‐
infected chicken three weeks postinfection (PI). H&E, ×400.
(Gimeno)

vv+MDV strains appeared earlier, were more extensive,

and were followed by proliferative lesions (227). The
initial lesions involve vascular elements; endotheliosis
occurs at 6 days PI and is followed at 8–10 days PI by a
moderate to severe infiltration of lymphocytes and
macrophages around blood vessels and scattered
throughout the neuropil (227). The vasculitis and edema Figure 15.11  Lymphoid cell infiltration of ovary. Organ is
composed largely of tumor cells, but a few ovarian follicles can be
disappear and may be followed by lymphoproliferative seen. H&E, ×116. (Witter)
infiltrations of large lymphocytes and glial cells. These
lesions tend to persist and are associated with persistent
neurological disease. Thus, as in nerves, brain lesions are Visceral Organs.  Lymphomatous lesions in visceral
both inflammatory and lymphoproliferative. Severe organs (Figures  15.11 and 15.12) are more uniformly
lymphoid infiltration in the cerebellum is shown in proliferative in nature than those in nerves and similar
Figure 15.10. in appearance to A‐type lesions consisting of diffusely
570 Section II  Viral Diseases
Figure 15.12  Higher magnification of a kidney lymphoma
showing pleomorphic tumor cells. Kidney tubules (bottom) show
degeneration caused by tumor cell pressure. H&E, ×450 (Gimeno)
proliferating small to medium T lymphocytes and
lymphoblasts, NK cells, B cells and macrophages, while
plasma cells are rarely present (reviewed in 73, 427).
The cellular composition of tumors is similar from one
organ to another, even though the gross pattern of
involvement may vary. Tumor cells are characterized by
large, pleomorphic nuclei with prominent nucleoli
(169). The majority of transformed T cells express
MHC class II and CD4 although CD4−CD8+, CD4+CD8+,
and CD4−CD8− T cells can also be transformed by
MDV (495). In addition the tumor cells express high
levels of CD30 (77) and resemble Treg cells (511).
Pradhan et  al. (444) found immune complexes in the
kidney, leading to glomerulopathy, in MDV‐infected
chickens. They suggested that these lesions might be
one of the major causes of death in MD.
Integument.  Lymphoproliferative nodules often sur­
round feather follicles that contain MDV viral antigens
and intranuclear inclusions in the FFE (92, 127). These
lesions in the skin appear largely inflammatory but may
also be lymphomatous. Massive proliferative lesions may
cause disruption of the epidermis, resulting in an ulcer.
Moriguchi et al. (377) described both inflammatory and
lymphoproliferative lesions in the feather pulp; the latter
were closely related to the incidence of MD and may be
useful for antemortem diagnosis (128).
Eye.  Smith et al. (534) and Pandiri et al. (414) examined
the histology of eye lesions after experimental infection
with the GA and Md5 strains, respectively. The former
group found that apparently transformed cells could be
detected as early as 11 days PI in the arachnoid layer of
the optic nerve and subsequently in ciliary nerves and
uvea. Pandiri et  al. (414) distinguished early and late
lesions. The early lesions, between 6–11 days PI,
consisted of hypertrophy of endothelial cells, vasculitis,
and infiltration of mostly CD8+ T cells, plasma cells,
heterophils, and macrophages. The cellular infiltration
probably represented an immune response to MDV viral
antigens such as pp38, which is recognized by CTL (404).
The late lesions appeared between 26–56 days PI and
consisted of uveitis, keratitis, and retinal necrosis.
Infiltrating cells included CD4+ and CD8+ T cells,
macrophages, plasma cells, and granulocytes. Early and
late lesions did not contain transformed lymphocytes
based on the absence of meq expression.
The unusually severe ocular lesions described by Ficken
et al. (198) include uveal changes with increased aqueous
humor protein, vascular engorgement, mild hyperemia to
severe swelling of the iris, severe inflammatory changes,
and edema of the cornea, including intranuclear ­inclusion
bodies.
Blood.  Leukemia consisting of T lymphoblasts may
occur occasionally (100, 430). Extravascular hemolytic
anemia has been reported after infection with vvMDV
strains in the absence of CIAV (209). The importance of
this finding is not clear because hematocrit values are
not routinely used as a MD parameter. Jakowski et  al.
(283) reported MDV caused anemia as a consequence of
bone marrow aplasia, but their MDV was later found to
be contaminated with CIAV (501). Blood lymphocyte
numbers may vary during the infection cycle: T cells may
be elevated while B cells are decreased (430). However,
Morimura et  al. (378) reported an initial increase of
CD4+TCRαβ1 cells around 16 days PI followed by a
decrease at 30 days PI. Infection with vv+ C12/130
caused significant increases in the absolute number of
blood monocytes around 8 days PI. B cells, CD4+ and
CD8+ T cells decreased during the early cytolytic
infection followed by an increase between 8–10 days PI,
but these changes were also seen for T cells after infection
with HPRS‐16 (37).
Lymphodegenerative Syndromes.  Marek’s disease virus
replication in the bursa of Fabricius and thymus results
in transient acute cytolytic changes accompanied by
atrophy (citations in 89, 427). In experimental infections,
bursal lesions consist of follicular degeneration,
lymphoid necrosis with depletion, and cyst formation
(Figure  15.13A). Thymic atrophy is often severe, and
lymphocytes are depleted in both cortex and medulla
(Figure 15.13B). Intranuclear inclusions can sometimes
be found in cells associated with degenerative lesions.
Viral antigens such as pp38 can be abundant during the
acute cytolytic phase in infections with vv+MDV strains,
especially in the medullary regions of the thymus and
bursal follicles (Figures 15.14A and 15.14B). Infection in
the absence of maternal antibodies may cause focal or
generalized necrosis in a variety of organs, including the
kidney (86, 199). Following the acute cytolytic phase,
 Chapter 15  Neoplastic Diseases 571

(A) (B)

Figure 15.13  Degenerative lesions in bursa and thymus of chickens inoculated with the 648A (vv1) strain of Marek’s disease virus.
(A) Bursa at 10 days postinfection (PI) shows degeneration and atrophy of follicles. (B) Thymus at 6 days PI shows necrosis and lymphoid
cell depletion. ×12 (Gimeno)

(A) (B)

Figure 15.14  Acute cytolytic infection of lymphoid tissues 6 days postinfection (PI) with the 648A (vv+) strain of Marek’s disease virus. The
pp38 viral antigen is visualized by immunohistochemical staining (black). (A) Bursa of Fabricius. (B) Thymus. ×30 (649). (Witter)

antigen positive cells disappear, and at least partial
repopulation with lymphocytes occurs. Bursal and
thymic atrophy, however, may persist for several weeks
or longer. In the bursa, some interfollicular lymphoid
infiltration with T cells may occur. Early mortality
syndrome is characterized by severe lymphoid
degeneration and death, often with enlarged, necrotic
spleens (615). Early mortality syndrome has been linked
with CNS signs and transient paralysis (342, 607).
CNS Syndromes (Transient Paralysis).  Lesions in classical
and acute transient paralysis are very similar. The critical
lesion in both types is vasculitis (Figure 15.15) leading to
vasogenic brain edema (231, 550, 607). Leakage of
albumin and IgG around affected vessels results in
vacuolization. Edema and vasculitis develop coordinately
with clinical flaccid paralysis and resolve in 2–3 days
(549). Other, apparently unrelated, brain lesions
(perivascular cuffing, lymphocytosis, and gliosis) may be
observed after clinical recovery or in infected but
clinically normal birds.
Vascular Syndromes.  Arterial lesions associated with
MDV‐induced atherosclerosis include proliferative and
fatty‐proliferative changes in aortic, coronary, celiac,
gastric, and mesenteric arteries (192, 372) (Figures 15.16A
and 15.16B) (557). Internal and medial foam cells,
extracellular lipid, cholesterol clefts, and calcium
deposits characterized the fatty‐proliferative lesions.
Marek’s disease virus antigens could be detected by IF
adjacent to the arterial lesions.
Tumor Transplants and  Local Lesions.  Tumor transplants
are composed of uniform, lymphoblastic cells with few, if any,
572 Section II  Viral Diseases

infiltrating host cells. In regressing tumor transplants small Pathogenesis

lymphocytes, heterophils, vascular invasion, and necrosis
Several reviews on the pathogenesis of MD have been
may be present (200). Local lesions induced in the wing
published (27, 90, 502), which also provide references for
web or pectoral muscle by inoculation of MDV‐infected,
older reviews. The use of BACs and overlapping cosmid
allogeneic CKC are inflammatory in nature, consisting
technologies has allowed the deletion of specific genes.
of  lymphocytes and macrophages and  sometimes
Using these technologies, the importance of several
accompanied by hemorrhages and necrosis (99).
genes for the pathogenesis has been established.
Four phases of infection in vivo can be delineated. (1)
Early productive–restrictive virus infection causing
primarily degenerative changes. The infection is
­
­productive–­restrictive because the virus remains cell‐
associated and is only transferred by cell to cell contact.
(2) Latent infection. (3) A second productive–restrictive
infection phase coincident with permanent immunosup-
pression. (4) The proliferative phase involving nonpro-
ductively infected lymphoid cells that may or may not
progress to the point of lymphoma formation
(Figure 15.17). This Division is somewhat arbitrary and
phases 2–4 can coexist in different cells in the same bird.
Infection with some of the vv+ strains may not follow
this general pattern, and mortality can occur without
even entering into the latent phase. The next section will
describe the classical pathogenesis in lymphoid tissues,
Figure 15.15  Transient paralysis lesions in the brain. Vasculitis in based mostly on studies in SPF chickens. The pathogen-
the cerebellum at 10 days postinfection (PI) showing endothelial
cell necrosis, lymphoid cell accumulations, and vacuolization.
esis of infection in the FFE involves epithelial cells and
Note intramural necrotic debris (arrow) as well as infiltration of will be discussed in the section on cytolytic infection
heterophils in the vessel wall. H&E, ×250. (Gimeno) in FFE.

(A) (B)

Figure 15.16  (A) Gastric artery of normal chicken. (B) Atherosclerotic artery in gizzard of chicken infected with CU2 isolate of Marek’s
disease virus. Lumen is occluded by thickened intima, and atheromatous changes have occurred deep in the intima and media. H&E, ×24.
(C. Fabricant)

Infection by inhalation
of poultry dust
Virus shedding
from feather
follicle epithelium
Environment
MD lymphoma
Latent phase:
lymohocytes
infiltration
of nerves
Transformation T
of lymphocytes
Latently-infected
(3/4 weeks) T-cell
Figure 15.17  Schematic diagram showing the different stages of Marek’s disease (MD) pathogenesis including the virus shedding from
the feather follicle epithelium and the transformation of T lymphocytes in susceptible birds.
Early Productive‐Restrictive Infection (Phase 1)
The virus enters the host via the respiratory tract and
cell‐free virus reaches the lymphoid organs within 24–36
hours after intratracheal inoculation (6, 482). Marek’s
disease virus is probably transferred to the lymphoid
organs by phagocytic cells (90). Based on the presence of
MDV proteins (36, 225) and transcripts (19) mac-
rophages can become infected, but because virus parti-
cles could not be demonstrated, it is not sure if this is an
abortive or productive–restrictive infection.
Shortly after infection either by inhalation of cell‐free
virus or by parenteral inoculation with cell‐associated
virus, cytolytic infection can be detected in the spleen,
bursa of Fabricius, and thymus, peaking between 3–6
days. Splenectomy (483) and embryonal bursectomy
(EBx) (491) delayed or reduced productive–restrictive
infection and the development of lymphomas suggesting
a central role for these two organs in the pathogenesis of
MD. In contrast, neonatal thymectomy enhanced the
pathogenicity of the low‐oncogenic CU‐1 and CU‐2
strains (94). The explanation for these findings was pro-
vided by Shek et  al. (521) discovering that the primary
target cells in all three organs are B cells. Recently it has
been shown that B cells are also infected in the lung two
days after intratracheal inoculation (19). Infection of T
cells requires activation based on the expression of MHC
class II antigens and activated but not resting T cells can
undergo cytolytic infection (102, 104, 105). Baigent and
Davison (29) confirmed that the early cytolytic infection
occurs mostly in B cells using dual staining techniques
with mAb specific for B‐ and T‐cell markers and MDV
pp38. In addition, they demonstrated that a small per-
Chapter 15  Neoplastic Diseases 573
Early replication
in lungs
Infection
B-cell
B
Early Cytolytic phase
“cell death”
T
Activated T-cell
“cell death”
1 week
centage of CD4+ and CD8+ T cells expressing TCRαβ can
become cytolytically infected during the early phase of
the pathogenesis. The consequence is a transient atrophy
of the lymphoid organs, especially the thymus and the
bursa. Depending on the virulence of the challenge
strain, birds may recover between 8–14 days PI, or the
atrophy may become permanent (93, 95). The cytolysis is
likely initiated by the activation of the host shut‐off pro-
tein leading to cell‐death by apoptosis (379). Although
MDV‐infected cells in the thymus are mostly B cells
(521), thymocytes undergo massive apoptosis possibly as
the consequence of viral infection (29) or virus‐induced
cytokine changes.
During phase one hyperplasia of lymphoid and reticu-
lum cells occurs (427), causing splenomegaly between
4–7 days PI, which is also observed after infection with
MDV‐2 and HVT (93). The level of infection is in general
similar in genetically resistant and susceptible strains
during phase 1 (7, 295, 642). However, genetically resist-
ant line 6 chickens have a significantly lower level of
infected lymphocytes than susceptible line 7 chickens.
Interestingly, line 6 has more B cells than line 7, thus the
difference is not caused by a lack of target cells in line 6
(29). In spleens of line 7 birds, dramatic changes occur
with irregular patches of pp38+ B cells becoming sur-
rounded by TCRαβ1+ CD4+ and CD8+ cells, thus provid-
ing optimal conditions for virus transfer from B to T cells.
These data suggest that MDV may replicate and spread
more efficiently in line 7 than line 6 chickens (29, 104).
Recently several studies have been conducted to ana-
lyze the changes in transcriptome and proteome in
­various tissues after MDV infection (152, 242). Changes
574 Section II  Viral Diseases
have been found for many genes and pathways, but addi-
tional studies will be needed before general conclusions
can be drawn for the understanding of the pathogenesis
and immune responses.
Several factors can modify the early pathogenesis.
Prior vaccination or the presence of maternal antibodies
reduce the cytolytic infection (90). The reduction in
cytolytic infection also will reduce the number of latently
infected cells and reduce or delay tumor development.
Exposure at one day of age prolongs the cytolytic infec-
tion compared to exposure at two or seven weeks of age
(82). Likewise, the pathogenicity of the virus strain may
affect the severity of early infection. The vv and vv+
strains can cause more severe lymphoid organ atrophy
than the less oncogenic strains, resulting in an early mor-
tality syndrome (95, 615).
The apoptosis of lymphocytes during the early cytol-
ytic phase (379) may cause transient or permanent
immunosuppression, depending on the virulence of the
challenge strain. In addition, a transient suppression of
mitogen stimulation has been reported, but this may
actually represent a protective response (497, 502).
Latent Infection (Phase 2)
At about 6–7 days, the infection becomes latent when
cytolytic infection can no longer be demonstrated, and
tumors are not yet detectable. The development of
latency coincides with the development of immune
responses. The integration of the virus into the host tel-
omeres is considered a key virus–host interaction for
achievement of latency (367). Impairment of cell‐medi-
ated immunity (CMI) (82) or infection with the more
virulent pathotypes delays the onset of latency (642).
Several soluble factors have been implicated in the
induction of latency including IFN‐α, IFN‐γ, latency
maintaining factor, and NO (81, 573, 631). Based on
infection of CEF with RB‐1B in the presence of IFN‐con-
taining supernatants, Levy et al. (340) suggested that IFN
may block virus replication before translation of late
genes. In one study IL‐10 expression leading to a Th2
response was the only cytokine upregulated in spleno-
cytes during latent infection (253), but this could not be
confirmed in another study (422).
Most latently infected cells are activated CD4+ T cells,
although CD8+ T cells and B cells can also be involved
(105, 337, 521). Infection in genetically resistant birds
often remains latent and can last for the lifetime of the
bird aside from a persistent low‐grade productive infec-
tion in the FFE (90). Apoptosis of T cells during latent
infection has been described (378, 380), although it can-
not be excluded that MDV was reactivated in these cells.
Susceptible birds or resistant birds infected with vv or
vv+ strains may develop a second wave of cytolytic infec-
tions after the second or third week, coincident with per-
manent immunosuppression.
The extent to which nonlymphoid cells are latently
infected is not known, although apparent latent infection
has been observed in Schwann cells and satellite cells in
spinal ganglia (434).
Second Phase of Cytolytic Infection (Phase 3)
The second cytolytic infection phase has not been stud-
ied in great detail and does not always occur depending
on genetic resistance of the host and the virulence of
MDV. If present, localized foci can be found in the lym-
phoid organs and in tissues of epithelial origin in various
visceral organs (e.g., kidney, pancreas, adrenal gland, and
proventriculus). Focal cell death and inflammatory reac-
tions develop around affected areas (10, 90). If the sec-
ond cytolytic infection occurs, many of the cytokines
upregulated during the first cytolytic phase are again
upregulated (422).
Cytolytic Infection in FFE
Starting around 14 days PI cytolytic infection occurs in
the FFE (96), which is the only known site of complete
virus replication. The replication occurs in genetically
resistant as well as susceptible birds independently of the
virulence of the MDV strain. Marek’s disease virus most
likely is transferred to the FFE by infected lymphocytes.
Viral DNA of all three serotypes can be detected in
feather tips as early as 5–7 days PI by qPCR (see
Transmission, Carriers, Vectors). It is not known if this
really represents infectious cell‐free virus or viral DNA
inside feather tips, because MDV‐positive peripheral
blood leukocytes (PBL) can be found in feather pulp as
early as 4 days PI concomitant with increased IFN‐γ
transcription (4).
Lymphocyte aggregates consisting of small lympho-
cytes with nuclear inclusions can be detected in the peri-
follicular dermis as early as 7 days PI (126). The lymphoid
aggregates can develop into either necrotic areas consist-
ing of FFE cells and degenerating lymphocytes or into
cutaneous tumors. The former is associated with strong
expression of pp38, which is the first viral protein
expressed in the FFE after reactivation followed by gB
and gD (398). In contrast, the cutaneous tumors have
only a few pp38+ cells. It is likely that virus is reactivated
from latency in the FFE, because mutant strains lacking
vIL‐8 (145) or pp38 (228) are able to produce virus in the
FFE. Detailed dynamics of MDV interaction in the
feather and its importance in spread has recently been
reviewed (249, 286).
Development of Lymphomas (Phase 4)
Lymphoproliferative changes, constituting the ultimate
response in the disease, may progress to tumor develop-
ment. Death from lymphomas may occur at any time
from about three weeks onward. Regression of lesions

has been reported after infection with vMDV strains and
depends on the genetic resistance of the bird and the age
at infection (75, 323, 519). However, it is not clear how
important tumor regression is in commercial poultry.
The transformed T cells are mostly CD4+ cells expressing
TCRαβ1 or TCRαβ2 and MHC‐II (495) with limited clonal-
ity (386). Other subsets (e.g., CD8+CD4−, CD3−CD4−CD8−
and CD3+CD4−CD8−) can be transformed under special
conditions (99, 402, 495). Burgess and Davison (77) further
characterized tumor cells using ex vivo lymphoma cells and
tumor cell lines as MHC‐Ihi, MHC‐IIhi, CD4+, TCRαβ1+ or
TCRαβ2+, CD25+, CD28−, CD30hi. This profile is compati-
ble with Treg cells (420, 511). The expression of CD30hi sug-
gests that MD could be a natural model for Hodgkin’s
disease (78). Marek’s disease tumor cells may also express
poorly characterized MATSA (623) and fetal antigens (442).
In addition to tumor cells CD8+ T cells are present and are
consisting of oligoclonal expansions with public and private
CDR3 sequences (386).
The infection in transformed cells is nonproductive in
vivo and in vitro. CD4+ and CD30hi express Meq and
SAR (see Viral Proteins), but are negative for pp38 and
gB (77, 477). Meq is the key protein involved in the trans-
formation of lymphocytes probably in conjunction with
viral telomerase (see Genes Unique for MDV) (388, 565).
The possibility that MD tumors are of clonal origin has
been proposed based on observations of random MDV
DNA integration into the genomes of lymphoma cells
(162). Analyzing the TCRβ repertoire of tumors, Mwangi
et al. (386) concluded that most of the tumors are clonal
in origin, but that birds could have tumors originating
from different clones. In addition, PBL can be positive
for these clonal tumor cells a few weeks before the birds
succomb. Earlier studies by Schat et al. (495) showed that
different lymphomas in the same bird could yield cell
lines representing different T‐cell phenotypes.
One of the major problems in understanding the
molecular basis for transformation has been the lack of a
reliable method for in vitro transformation of T cells,
however such a model has been published which will
allow analysis of interaction between virus and target
cells in an easily accessible system (503).
Factors Influencing Pathogenesis
The pathogenesis of infection with oncogenic MDV,
which has become attenuated by passage in vitro, has
been studied by Bradley et al. (quoted in 427, 499) and
Schat et al. (494). Attenuated viruses failed to cause cyto-
lytic infection and cell‐associated viremia levels were
low. Moreover, attenuated virus was not infectious for
lymphocytes in vitro, perhaps explaining the in vivo
observations.
Virus strains differ in oncogenicity, but the molecular
basis for differences in pathogenesis are not well defined.
All cause similar early cytolytic infections, although the
Chapter 15  Neoplastic Diseases 575
vv+ strains may cause a prolonged and more severe cyto-
lytic infection (642). Some of the new strains are capable
of infecting macrophages leading to increased death of
macrophages (38). Murata et  al. (385) suggested that
point mutations in meq resulting in amino acid substitu-
tions in the protein could change transactivation and
hence change the pathogenicity.
The immune response itself may be responsible for
some lesions characteristic of MD. Nerve lesions have
some characteristics suggestive of an autoimmune dis-
ease (449, 505), although the presence of MDV was not
positively shown. Additional evidence supporting an
autoimmune component for MD comes from studies
showing immune complexes in the kidneys of MDV‐
infected chickens and quail (306, 444).
The primary cytolytic infection is not an absolute pre-
requisite for tumor development. Schat et al. (491) found
that MDV infection in EBx chickens resulted in the
development of tumors in the absence of the primary
cytolytic infection, which also is the case in vaccinated
chickens. However, stress and immunosuppressive infec-
tions may induce secondary cytolytic infections, reduc-
ing the benefits provided by vaccination.
Pathogenesis of Non‐Tumor Diseases
Marek’s disease virus infection can cause several non‐
neoplastic disease syndromes (Table 15.3B). The patho-
genesis of MDV‐induced atherosclerosis has not been
elucidated. Microscopic lesions consisting of fatty prolif-
erative lesions with alterations in lipid metabolism in
arterial smooth muscle cells could be detected as early as
one month after infection (191, 237). In contrast to the
original work by Fabricant et al. (192, 193), Njenga and
Dangler (399) were unable to demonstrate arterial lipid
accumulation without cholesterol supplementation.
Cellular infiltrates were detected in the intima and serum
cholesterol was increased significantly compared to non-
infected control chickens.
The pathogenesis of the neurological lesion complex
consisting of classical transient paralysis (TP), acute TP,
persistent neurological syndrome (PND), and late paral-
ysis (LP) (231) is not fully understood. The difference
between classical and acute TP is somewhat arbitrary
(607) and the early pathogenesis is probably similar. The
development of both types of TP is influenced by the
MHC and the virulence of the MDV strain with the more
virulent strains causing acute rather than classical TP
(504, 607). B cells are required for the induction of tran-
sient TP (418), probably because these cells are essential
for the early cytolytic infection. The brain lesions start
with vasculitis at 6–8 days followed by leakage of albu-
min from blood vessels into vacuoles (550). This vaso-
genic edema is transient and correlates with classical TP
symptoms (548). Jarosinski et  al. (288) noted that the
development of neurological symptoms induced by
576 Section II  Viral Diseases
vv+RK‐1 correlated with increased levels of iNOS mRNA
in the cerebellum and NO in blood serum (see Nitric
Oxide). Nitric oxide can cause vasodilation and could be
the cause of the edema. Chickens inoculated with the
vMDV JM‐16 strain did not show neurological signs or
iNOS mRNA in the cerebellum.
The degree of virus replication in the brain may be
related to the severity of the disease. The absence or low
levels of virus replication correlated with the absence of
neurological symptoms (3, 229), while high levels of rep-
lication in MHC resistant and susceptible chickens
resulted in neural lesions (288). Attenuation of the
vv+648A strain resulted in reduced induction of TP
coordinately with a reduction in viral replication in lym-
phoid organs and FFE (229).
The clinical signs of PND are associated with a strong
infiltration of lymphoblasts in the neuropil, many of
which express Meq protein. The later occurrence of PND
after 3 weeks PI suggests that its pathogenesis may paral-
lel that of lymphoma induction in other tissues.
Moreover, PND was shown to be closely related with the
onset of lymphoproliferative lesions in peripheral nerves
and visceral organs (229).
Immunity
Infection with pathogenic MDV or vaccine strains not
only results in the activation of innate, nonspecific and
acquired, or specific immune responses but may also
cause immunosuppressive effects especially after infec-
tion with pathogenic serotype 1 strains. The importance
of the interactions between immune responses and
immunosuppression for the pathogenesis of MD cannot
be overemphasized. A distortion in the balance toward
immunosuppression will lead to disease. Immune
responses and immunosuppressive features of MD have
been extensively reviewed (56, 157, 245, 497, 502).
Immune Responses
The immune responses developing during the early
cytolytic phase of infection are crucial for the outcome of
infection. Impairment of immune responses during this
phase delays establishment of latency resulting in pro-
longing the lytic infection and the subsequent continued
destruction of immune cells by virus‐induced apoptosis.
Impairments include infection at one day of age when
the immune responses are not yet fully developed or
treatment with cyclosporin or neonatal thymectomy
combined with cyclophosphamide treatment (82). The
importance of immune responses during latency is rele-
vant for protection against the second cytolytic phase
and is dependent on CMI. It has often been suggested
that vaccine‐induced immunity is an antitumor immune
response because vaccination does not prevent superin-
fection with wild‐type virus but does prevent tumor
development. However, vaccination clearly reduces the
early cytolytic infection (101, 252, 492), thus preventing
extensive damage to the immune system and reducing
the number of latently infected T cells. Lesion regres-
sion, however, has been described (75, 519) suggesting
that immune responses against tumor cells may occur.
Initiation of  Immune Responses.  Professional antigen‐
processing cells (APC), such as dendritic cells, encountering
pathogens are activated by interactions between the PAMP
and the pattern recognition receptors (PRR), for example,
TLR, on the APC. These interactions result in the activation
of cytokines which direct both the innate and acquired
immune responses. There is currently no information on
PAMPs associated with any of the three MDV serotypes.
Although the distinction between innate and acquired
immune responses has become less defined over the last
few years, the innate and acquired responses will be dis-
cussed in separate sections.
Innate Immune Responses.  Innate immune responses
include changes in cytokine expression, natural killer
(NK) cells, and macrophages.
Cytokine Responses.  Infection with MDV results in the
upregulation of a number of proinflammatory cytokines
(see Early Productive‐Restrictive Infection for details)
driving a TH1 immune response.
IFN‐γ is an important pleiotropic cytokine with many
functions in antiviral immune responses, but few studies
have been performed on the roles of IFN‐γ in protective
immunity to MD. In vitro studies indicate that IFN‐γ is
able to inhibit virus replication directly or indirectly
through the induction of NO production and reactive
oxygen intermediates (166, 631).
Detailed reviews on immunity to MD have been
described elsewhere (56, 245, 246).
Nitric Oxide.  Nitric oxide is synthesized by three iso-
forms of NOS with iNOS (NOS II) being inducible in
macrophages, glial cells, astrocytes, and perhaps other
cells as well. The induction of iNOS occurs as part of the
nonspecific inflammatory immune response to micro-
organisms. Nitric oxide and other reactive nitrogen spe-
cies are very versatile molecules with many functions.
Nitric oxide has been linked to beneficial affects by kill-
ing pathogens but also to neurodegenerative processes
in humans (65, 171).
Nitric oxide can inhibit MDV replication in vitro (166,
631). Increased transcription of iNOS has been reported
between 6–12 days PI with MDV (630) resulting in
increased levels of NO in the plasma of genetically resist-
ant but not in genetically susceptible chickens (167, 290).
Nitric oxide may be beneficial, because it inhibited MDV
replication in vivo when genetically resistant chickens

were challenged with vMDV (631). However, pathology
may be associated with very high levels of NO produc-
tion especially in genetically resistant birds challenged
with vv+MDV (288).
NK Cells.  Natural killer cells are the first line of defense
because these cells can lyse virus‐infected and tumor
cells without prior exposure to the pathogen. Natural
killer cells are also potent inducers of IFN‐γ. In order to
lyse target cells, NK cells must recognize the target cells
as foreign (e.g., the MHC‐I has been altered or down-
regulated). Thus the downregulation of MHC‐I during
the lytic infection (268) supports a potential role for NK
cells. Thus far functional NK cell assays have used total
spleen cell populations and LSCC‐RP9 as target cells in
chromium release assays (CRA). The recent develop-
ment of NK cell‐specific mAb (284) will facilitate further
examination of the importance of NK cells in MD
immunity.
Macrophages. Activated macrophages can restrict
MDV replication and perhaps as a consequence reduce
tumor incidence. These effects are probably the result of
NO production (497, 502) or by reactive oxygen inter-
mediates (166). Increased numbers of macrophages have
been noted shortly after infection in the lung in conjunc-
tion with increased transcription of iNOS. Macrophages
harvested shortly after MDV infection can inhibit prolif-
eration of MD cell lines in vitro, which was considered to
be a transient immunosuppressive effect (334, 513). This
inhibition may actually be a protective response, because
it may limit the number of activated T cells during the
critical switch of MDV from B to T cells (497, 502).
Acquired Immunity
Humoral Immunity.  Chickens infected with MDV
develop precipitating and VN antibodies within 1–2
weeks; a transient IgM response is replaced by IgY (256).
Due to the cell‐associated nature of MDV, antibodies are
of limited importance in MD immunity. Virus‐
neutralizing antibodies are important only when cell‐
free virus infects chickens or when MDV proteins are
expressed on the surface of cells. In the latter case,
antibodies plus complement or antibody‐dependent,
cell‐mediated cytotoxicity (ADCC) can lyse infected
cells (317, 472). However, the target antigens and the
effector cells involved in ADCC were not identified. In
vivo VN has indeed been demonstrated using cell‐free
and cell‐associated virus (79). Maternal antibodies
reduce the cytolytic infection (86) and can reduce the
efficacy of cell‐associated vaccines with low titers or if
cell‐free HVT is used (111, 315).
The possibility that surface antigens found on MDV‐
transformed cells could be involved in immunity was
suggested by protection against challenge after immuni-
Chapter 15  Neoplastic Diseases 577
zation anti‐idiotype antibodies against MATSA (151).
Similarly, antibodies against CD30 may provide protec-
tion against tumor cells (78).
Cell‐Mediated Immunity.  Cytotoxic T lymphocytes (CTL)
recognize small peptide fragments of 8–12 amino acids
presented in the context of MHC‐I antigens. These
peptides are generated from de novo synthesized proteins
through a complex process involving the proteasome
and transporters associated with antigen‐processing
(TAP) 1 and 2. In vitro demonstration of antigen‐specific
CTL requires effector and target cells expressing the
same MHC‐I antigens (515).
Recently, Mwangi et al. (386) found a limited number of
CD8+ clonal cell populations, which were generated
shortly after infection suggesting that MDV infection
results in a limited number of antigen‐specific clones.
Earlier, Pratt et al. (448) stably transfected and expressed
MDV genes in REV‐transformed cell lines with known
MHC antigens. These cell lines were used to show that
CTL from infected or vaccinated chickens recognize pep-
tides derived from pp38, Meq, ICP4, ICP27, gB, gC, gH,
gI, and gE (361, 404, 502). The effector cells developed
around 7 days PI and were characterized as typical CTL
expressing CD3, CD8, and TCRαβ1 but not CD4 (405).
Important differences were noted in the recognition of
proteins by CTL from resistant and susceptible chicken
lines. Cytotoxic T lymphocytes from resistant N2a (MHC:
B21B21) but not from susceptible P2a (MHC: B19B19)
chickens recognized ICP4 (404). Effective killing of
infected cells as soon as ICP4 is expressed, for example,
when latently infected cells are reactivated, and before
virus replication is completed could be one of the con-
tributing factors to MHC‐based genetic resistance.
Cytotoxic T lymphocyte‐specific responses to gB, gI and
pp38 also appear important to MD protection. (328, 394).
Cytotoxic T lymphocytes and NK cells can lyse target
cells through the perforin/granzyme pathway. Increased
transcription rates of Granzyme A occur between 4–15
days PI (251, 480, 481).
Vaccinal Immunity.  Herpesvirus of turkey, attenuated
MDV, and serotype 2 MDV protect against early
replication of virulent viruses in the lymphoid organs of
challenged birds, reduce the level of latent infection, but
do not prevent infection (reviewed in 211, 497, 600).
Based on current knowledge, the following sequence of
events is proposed to explain vaccine‐induced immunity
with challenge occurring within three days after hatch as
is typical in the field (Figure 15.14).
Vaccination with CVI988 results in upregulation of
IFN‐γ, IL‐8, IL‐18, and iNOS in lung and spleen as early
as 3 days PI (213), which is probably also the case after
vaccination with HVT and SB‐1. IFN‐γ can reduce virus
replication and stimulate macrophages to initiate the
578 Section II  Viral Diseases
transcription of iNOS, producing NO between 3 and 7
days post vaccination, thus limiting the replication of
challenge virus. Shortly afterwards, NK cells are acti-
vated (254, 514), probably producing more IFN‐γ and
killing virus‐infected B cells.
Feather pulp can be used to examine temporal changes
in immune responses in individual, vaccinated chickens.
This will facilitate monitoring cytokine responses
induced by different vaccines (5, 244). Vaccination with
CVI988 showed a down regulation of RB‐1B in the
feather pulp (244). Challenge did not increase replication
of CVI988 (34, 244) in contrast to the observation that
HVT and SB‐1 shedding increased after challenge with
MDV (278).
Immunoevasion, caused by MDV, can interfere with
vaccine‐induced immunity. MHC‐I downregulation is
one strategy to reduce the host defenses, as reviewed
elsewhere (245). The MDV gene MDV012 was recently
demonstrated as capable of reducing surface expression
of MHC‐I on chicken cells (248). The host response can
also be altered by differences between vaccines from dif-
ferent manufacturers, which probably relates to passage
levels (331). Concurrent infections with immunosup-
pressive viruses, for example, CIAV (362) or stress may
interfere with vaccine‐induced CMI responses. Deletion
of humoral immunity by bursectomy and X‐irradiation
does not seem to have a major effect on protection con-
ferred by attenuated MDV (183), although similar treat-
ment partially impairs HVT vaccine immunity (467).
Immunosuppression
Suppression of the immune response by MDV infection
is a critical feature of the disease, contributing to the
virulence of MDV isolates and altering susceptibility of
the host to other pathogens (reviewed in 486, 500). Initial
impairment of the immune response is the result of the
lytic infection of lymphocytes during the first cytolytic
infection (see Pathogenesis) (citations in 486, 500). The
onset of immunosuppression later in life appears to be a
unique feature of vv+MDV strains and is not eliminated
by vaccination (196, 197). It is difficult to distinguish
between cause and effect because tumor cells might have
suppressor activity (69, 456). Because immunocompe-
tence is required for the maintenance of latency (82) it
might be that immunosuppression associated with the
appearance of transformed lymphoblasts results in addi-
tional reactivation of the lytic infection. This, in turn,
will cause the loss of additional B and T cells, thus com-
pounding the situation and resulting in the bursal and
thymic atrophy seen in birds destined to succumb to
MD. However, immunosuppression may not be a prereq-
uisite for the development of tumors, as observed with
several experimental vaccine candidates (175, 611).
Although virus‐induced immunosuppression and onco-
genicity are not invariably linked, they are often expressed
concurrently and, in such cases, immunosuppression
may serve to augment oncogenic potential.
Humoral and CMI can be suppressed by MDV infec-
tion leading to reduced antibody responses to a variety of
antigens and alterations in T cell functions, such as skin
graft rejection, mitogen stimulation of lymphocytes,
delayed hypersensitivity, reduced NK cell activity, pri-
mary and secondary infections with coccidia, and
impaired Rous sarcoma regression (citations in 427, 499).
Diagnosis
Techniques for diagnosis of infection with MDV are dif-
ferent from those needed for differential diagnosis of the
disease. The infection is ubiquitous, but the disease is
not. The principal methods to identify the presence of
infection are isolation of the virus, demonstration of viral
DNA or antigens in tissues, and detection of antibody.
The applications of different diagnostic procedures have
been recently reviewed (223, 401, 606).
Virus Isolation
Virus isolation is performed to confirm its presence for
diagnostic purposes and to secure the infectious virus
for further study. Techniques for isolation of all sero-
types have been reviewed (223, 487).
Source of Virus
Marek’s disease virus can be isolated as early as 1 or 2 days
PI or 5 days after contact exposure and throughout the life
of the chicken (reviewed in 499). Intact viable cells are the
preferred inoculum because, in most cases, infectivity is
avidly cell‐associated, although cell‐free preparations from
skin, dander, or feather tips of infected chickens may con-
tain the virus (92). Inocula may consist of blood lympho-
cytes, heparinized whole blood, splenocytes, or tumor cells.
Marek’s disease virus can often be recovered from infected
cell suspensions following s­ torage for 24 hours at 4°C, thus
facilitating transport of samples (reviewed in 499).
Cell Culture Techniques
Probably, the most widely used method for primary iso-
lation of MDV is inoculation of susceptible cell cultures
with blood lymphocytes or single‐cell suspensions from
lymphoid tissues of infected chickens. Chicken kidney
cell and DEF cultures are preferred substrates for ­primary
isolation of serotype 1 MDV; whereas CEF normally are
used for isolation of viruses of serotypes 2 and 3 as well
as for attenuated serotype 1 vaccine strains. Although
CEF are less permissive for growth of low passage
­serotype 1 virus (reviewed in 487), some contemporary
isolates may grow well in CEF, even on primary isolation.
Cultures are inoculated with 1–2 × 106 cells, although

some inhibition of viral plaque formation may be
encountered with doses greater than 8 × 106 cells for
some viruses (109).
Development of typical plaques (Figure 15.3) in inocu-
lated cultures within 3–12 days and the absence of such
changes in comparable uninoculated (or sham inoculated)
control cultures are evidence for isolation of MDV. The
plaques induced by serotype 1, 2, and 3 viruses can be dis-
tinguished, with practice, by morphologic criteria (484,
590), but IF staining with serotype‐specific mAbs provides
a more accurate differentiation. Optimal time for observa-
tion of plaques varies with the cell substrate and serotype
of the virus. Marek’s disease virus also has been isolated by
direct culture of kidney cells from infected chickens or by
inoculation of normal kidney cultures with trypsinized
kidney cells from infected chickens (621).
Isolate Identification
Marek’s disease virus serotype 1 isolates should be free of
contaminating MD vaccine strains. It normally is useful
for the isolate to be plaque purified or cloned at the earli-
est possible passage. Serotype identity and purity can be
confirmed using staining techniques with serotype‐spe-
cific mAbs (332). Freedom from extraneous viruses is
critical, because contamination with passenger viruses
may alter the apparent pathogenicity of the isolate (291,
409). Propagation of MDV isolates for up to six passages
in CEFs or CKC cultures appears to exclude contaminants
such as CIAV (641) and permits preparation of seed and
working stocks, which can be more easily standardized
and titrated. To preserve virulence, some workers have
preferred to propagate serotype 1 viruses in vivo, prepar-
ing stocks of cryopreserved spleen or buffy coat cells from
infected chickens. Pathotyping of serotype 1 MDVs,
although not routine, may be accomplished by compari-
son of pathogenicity with that of prototype strains by
inoculation of non‐vaccinated chickens as well as chickens
vaccinated with HVT or bivalent vaccines (604).
Virus Assay and Titration
Viruses of serotypes 1, 2, and 3 can be assayed by in vitro
techniques similar to those described for virus isolation.
Methods differ for different serotypes, but most rely on
plaque induction in susceptible cell cultures. Enumeration
should be done as soon as plaques become mature (time
varies with isolate), because secondary plaques may
occur when cultures are maintained with liquid medium.
Procedures for titration of vaccine viruses have been
reviewed (559) and are not fundamentally different from
those for pathogenic isolates.
Viral Markers in Tissues
It is often desirable to detect the presence of viral infec-
tion in chickens without isolating the virus in culture.
Chapter 15  Neoplastic Diseases 579
Such infection markers also have value for the identifica-
tion of putative MDV isolates in cell cultures. However,
only detection of oncogene meq and high load of MDV
DNA in tumors can be used as diagnostic criteria for the
disease (226).
Viral Antigen Detection
Monoclonal antibodies prepared against type‐common
and type‐specific epitopes of all three MDV serotypes
(332) are now used in preference to polyclonal antibodies
for the detection of antigens in tissues. Monoclonal anti-
bodies H19 and T65 can be used to differentiate CVI988
from other serotype 1 MDVs strains based on differences
in the amino acid sequence of protein pp38 (147, 220).
Viral antigens can be detected in feather tips and FFE,
cytolytically infected lymphoid tissues, brain, or infected
cell cultures with appropriate antibodies by fluorescent
antibody tests, immunohistochemistry, dot‐ELISA (322),
agar gel precipitin (AGP) tests, and immunoassay
(reviewed in 223, 499). In MD lymphomas, oncogene meq
is the only antigen that is consistently expressed (226, 477)
but pp38‐positive cells occasionally are observed (226).
Viral Nucleic Acid Detection
Polymerase Chain Reaction (PCR) Assay.  The availability of
the nucleotide sequences of different genes from a large
number of viruses including the complete genome
sequence of the three serotypes of MDV allows the use of
PCR‐based methods of specific detection of MDV.
Primers designed to amplify sequences specific for each
serotype (277, 464) as well as to differentiate between
serotype 1 oncogenic and attenuated strains based on the
132 bp (42, 525, 655), and between CVI988 and other
serotype 1 MDV strains (30, 220, 468) have been
described. However, PCR may not always be sensitive
enough to detect latent infection due to the lower
frequency of positive cells and the lower number of viral
genomes per cell. Furthermore, detection of MDV DNA,
even from an oncogenic MDV, does not have any
diagnostic value as chickens get exposed to oncogenic
MDVs, if properly immunized, they never develop MD.
Quantitative PCR (qPCR) assays using various primer
sequences have been used to assay viral load in tissues
from infected chickens (30, 31, 72, 76, 220, 277, 464) and
are essential tools for diagnosis and epidemiological
studies of MD. Unlike conventional PCRs, qPCR has
been shown to be very valuable in the diagnosis of MD
(218, 220, 226, 368, 582), for monitoring MD vaccination
(136, 214, 216, 217, 220, 241, 276, 279, 469), and for stud-
ying different aspects of MD biology including replica-
tion kinetics of MDV in blood, feathers, lymphoid
tissues, and dust (34, 136, 244, 276, 279, 280, 457–459).
DNA Probes.  Methods using DNA–DNA dot‐blot
hybridization with DNA probes for the detection of
580 Section II  Viral Diseases
MDV DNA in feather tip extracts (154) have been used
for detection of MDV (382–384). Furthermore,
localization of virus‐infected cells has been accomplished
by in situ hybridization for both MDV (185, 434, 475)
and HVT (265) and by fluorescence in situ hybridization
(FISH) for MDV (302).
Loop‐Mediated Isothermal Amplification.  Loop‐mediated
isothermal amplification (LAMP) methods for the rapid
detection of MDV‐1 genome at high sensitivity were
developed for detection of MDV in less‐equipped
laboratories as well as under field conditions (17, 175,
625, 627, 628).
Antibody Detection
Tests for identifying the presence of specific antibodies
in chicken sera are useful in studies of viral pathogenesis
and for monitoring SPF flocks. However, it does not have
any value in the diagnosis of MD in commercial flocks. A
number of procedures including AGP, fluorescent anti-
body ELISAs (373, 646), and VN tests are in common use
(review in 223).
Diagnosis of the Disease
Despite long‐established guidelines for the pathologic
diagnosis of MD (524), diagnosis of the clinical disease
remains difficult in practice because of the absence of
truly pathognomonic gross lesions and the widespread
nature, often with coinfection, of other pathogens such
as ALV and REV (149, 153), complicating diagnostic
efforts that depend on virological methods.
Diagnosis of MD must primarily be addressed by con-
sideration of the characteristics of the proliferating cell
populations that constitute the disease. Other disease‐
specific criteria, such as epidemiological factors, are also
valuable. Detection of virus, viral DNA, or viral antigens
(with the exception of oncogene meq) does not have any
diagnostic value since infection is ubiquitous and vacci-
nation does not prevent superinfection (226). However,
quantification of MDV DNA load in tumors is a diagnos-
tic criterion that can confirm the diagnosis of MD (218,
220, 226). The process commences with the acquisition of
a flock history and a sufficient number (5 to 10) of repre-
sentative sick and dead chickens showing the lesions of
the disease and proceeds as a series of steps (606).
Step 1—Clinical Data and Gross Pathology
Although enlarged peripheral nerves and visceral lym-
phomas are common in MD and one or both are invari-
ably present, neither lesion occurs consistently nor is
pathognomonic. Thus, other criteria, such as age and
lesion distribution, must be considered in the postmor-
tem diagnosis of MD. Chickens may be diagnosed
­ rovisionally as MD if at least one of the following con-
p
ditions is met: (1) leukotic enlargement of peripheral
nerves; (2) lymphoid tumors in various tissues (liver,
heart, gonad, skin, muscle, and proventriculus) in birds
under 16 weeks of age; (3) visceral lymphoid tumors in
birds 16 weeks or older that lack neoplastic involvement
of the bursa of Fabricius; or (4) iris discoloration and
pupil irregularity, as in Figure  15.8C. Proper examina-
tion of the bursa is particularly important and requires
incision of the organ with close inspection of the epithe-
lial surface. However, diagnoses based only on gross
pathologic criteria are not definitive and additional
steps are required.
Step 2—Histology, Cytology, and Histochemistry
of Tumor Cells
Affected tissues, fixed in formalin or fresh‐frozen, are
used to prepare paraffin and cryostat sections, respec-
tively. Impression smears of tumors may also be used
(524). Histopathology can be very useful to confirm the
diagnosis of lymphoma and to evaluate the morphology
and distribution of tumor cells. Marek’s disease tumors
consist of a mixed population of small to large lympho-
cytes, lymphoblasts, plasma cells, and macrophages
(222, 426). The presence of such infiltrates in the nerves
(lesion type A) is the only pathognomonic lesion of MD
(326, 426). However, MDV is also able to induce inflam-
matory lesions characterized by edema, demyelination,
and plasma cell infiltration (lesion type B) that are not
necessarily related with MD lymphoma and can be con-
fused with peripheral neuropathy (25, 228, 326, 605).
Likewise, minor infiltration of lymphocytes in peripheral
nerves might be indicative of infection with MDV but
not of MD lymphoma (review in 606).
Characterization of the cell phenotype by immunohis-
tochemistry can aid in the differential diagnosis of MD
lymphomas. Marek’s disease tumor cells express MHC
class II antigen and T‐cell markers, especially CD4 (495).
CD30 (477) and MATSA (623) are commonly found in
MD tumor cells although they are also present on retro-
virus‐induced B cell lymphoma (226) and activated T
cells (226, 365).
Step 3—Virologic Criteria
For tumors that satisfy MD criteria listed in steps 1 and 2
or for atypical tumors, the association of MDV with the
tumor cell is a useful confirmation. Viral antigen Meq
can be consistently detected in tumor cells by in situ
hybridization, immunohistochemistry, or fluorescent
antibody tests and can be used as diagnosis criterion to
confirm MD tumors (226, 477). Detection of other viral
antigens such as pp38 has been described in tumors
(390) but expression is sporadic, it seems to be related to
reactivation of virus in neoplastic cell, and it is not an
adequate diagnostic criterion for MD tumors (226).

Polymerase chain reaction assays and virus isolation
from buffy coat, spleen cells, or feather pulp samples as
well as detection of viral proteins demonstrate the virus
in the bird but do little to associate the virus with the
tumor cells. However, there is a quantitative association
between virus load and MD tumors. Low levels of virus
or viral DNA may be detected in lymphocytes from
nontumor‐bearing chickens, but most tumor‐bearing
­ induced by REV but, thus far, have only been observed
chickens have high viremia titers (622) and high load of
MDV DNA measured by qPCR (33, 218, 220, 226, 459).
Thus, the demonstration of high load of MDV DNA in
tumor cells and the absence of other tumor viruses, along
with the criteria in steps 1 and 2, is sufficient to establish
MD diagnosis. Evaluation of MDV DNA load in blood of
feather pulp of chickens as early as 21 days of age can also
be used to predict the MD outcome on those flocks (218,
220, 459). Proper collection of samples for MD diagnosis
using qPCR has been recently reviewed (223).
Pathotyping of MDV Strains
The concept of MDV pathotypes has arisen from the
recognition of the existence of strains that are associated
with increased virulence that show correlation with
breaking of vaccinal immunity in the field (604). The
ADOL (Avian Disease & Oncology Laboratory) method
of pathotyping, based on induction of lymphoprolifera-
tive lesions in chickens vaccinated with different vacci-
nation regimes is the most widely used. This method was
used to characterize more than 45 isolates into distinct
vMDV, vvMDV, or vv+MDV pathotype groups (597).
Even though the ADOL method stipulates the use of line
15x7 chickens for pathotyping, experiments with other
lines of birds have given similar results (83, 170, 604).
The ADOL pathotyping assays are cumbersome and
require infrastructure that is lacking in most laborato-
ries. Therefore, different methods to differentiate
between classical strains of MDV and the more virulent
pathotypes have been examined including neuropatho-
typing (230), virus replication (172), lymphoid organ
atrophy and immunosuppression (95, 196), and sequence
of various genes (512, 555). However, classification
obtained by the alternative methods do not always ren-
der the same results as ADOL pathotyping and most of
these techniques are considered an adjunct to pathotyp-
ing more than substitutes of the “gold standard” ADOL
pathotyping assay.
Differential Diagnosis
Details on how to do differential diagnosis of MD with
other poultry tumor diseases has been reviewed (606).
The major differential diagnosis for MD is lymphoid leu-
kosis (LL). Lymphoid leukosis is a clonal, bursal lym-
phoma induced by ALV and, under some conditions, by
Chapter 15  Neoplastic Diseases 581
REV in chickens older than 16 weeks of age. Chickens
usually have gross tumors in the bursa of Fabricius, and
tumor cells are uniform, blast‐like, pyroninophilic, and
express B‐cell markers and IgM. Also, the tumor cells
have clonal insertions of proviral DNA near the c‐myc
gene (see Leukosis/Sarcoma Group). Nerve enlarge-
ment, runting, and nonbursal T‐cell lymphomas can be
under experimental conditions or where chickens
have  been inoculated with contaminated vaccines.
Lymphocytes obtained from REV‐induced nerve lesions
or tumors do not express Meq and have low MDV DNA
load, if any (226). Cells from nonbursal RE lymphomas
are negative for MHC class II and predominantly stain
for CD8 antigen (606) (see Reticuloendotheliosis).
Exclusion of ALVs or REVs, where possible, through neg-
ative PCR, histochemical assays on tumors, or antibody
tests may provide strong support for a diagnosis of MD
when other MD‐related criteria are positive. However, it
is possible to have mixed infections and in those cases
further diagnostic assays (qPCR for MD and southern
blot for retroviruses) are needed (190, 226).
Peripheral neuropathy is a neurological disease of
uncertain etiology that causes paralysis and nerve
enlargement in a low proportion of commercial chickens
6–12 weeks of age (25, 53) and is characterized by a Th1‐
to‐Th2 shift (26). Affected chickens lack visceral lym-
phomas; the nerve lesions are uniformly B‐type; and
MDV is rarely, if ever, demonstrated. If MDV is present
on those chickens, MDV DNA load is low (606). Other
diseases that may present confusing gross lesions or par-
alytic signs are myelocytomatosis (myeloid leukosis),
myeloblastosis, erythroblastosis, histiocytic sarcomas,
carcinoma of the ovary, various other nonviral neo-
plasms, riboflavin deficiency, tuberculosis, histomonia-
sis, genetic gray eye, Newcastle disease, hepatitis E, and
joint infections or injuries. Myeloid leukosis is a com-
mon tumor in broiler breeder flocks that superficially
resembles MD but can be differentiated histologically.
The tumor cells are myeloid in nature and lack T cell and
MD viral markers.
Diagnosis of Other MD Syndromes
Transient paralysis occasionally is observed in the field,
especially in chickens not vaccinated against MD once
maternal antibodies have waned between 30–40 days of
age (reviewed in 222). Development of TP in vaccinated
commercial flocks would indicate failures on the vacci-
nation process as proper vaccination against MD pro-
tects against the development of TP (reviewed in 222).
Diagnosis of TP is done based on the history (sudden
onset of flaccid paralysis that last 24–48 hours and can
lead to death or total recovery), lack of gross lesions in
nerves or viscera, and histopathology (brain edema and
582 Section II  Viral Diseases
vasculitis). There are three diseases that need to be
included in the differential diagnosis with TP: botulism,
neurological form of MD (fowl paralysis), and peripheral
neuropathy. Both TP and botulism cause flaccid paraly-
sis of the neck progressing to the limbs and are not asso-
ciated with gross lesions in the nerves or viscera.
Confirmation of TP can be done by histopathological
examination of the brain as botulism does not produce
brain edema and vasculitis characteristic of TP. Unlike
TP, the neurological form of MD appears as permanent
spastic paralysis with enlargement of peripheral nerves
and pathognomonic type A lesions in the nerves.
Peripheral neuropathy tend to last longer than TP and is
also characterized by peripheral nerve enlargement with
lesions that resemble MD B‐type lesions. Davidson et al.
(155) differentiated transient paralysis from peripheral
neuropathy on the basis of PCR tests for MDV on brain
tissue. However, brains from MDV‐infected chickens
without transient paralysis may also be detected as posi-
tive by PCR assays (607). In contrast, detection of viral
antigens in the brain appeared to correlate with the onset
of paralytic signs (227).
Skin leukosis (the skin form of MD) can be differenti-
ated from dermal squamous cell carcinoma, which is
commonly observed in defeathered broiler chickens at
processing (236, 391). Marek’s disease lesions are nodu-
lar and contain lymphoid cells, whereas squamous cell
carcinomas have a craterlike gross appearance and are
composed of squamous epithelial cells.
Due to its complexity, the most difficult syndrome to
diagnose is MDV‐induced immunosuppression (MDV‐IS).
Early MDV‐IS is associated with replication of MDV in the
lymphoid organs of unvaccinated chickens lacking mater-
nal antibodies and it is characterized by atrophy of the bursa
and thymus. However, under field conditions lymphoid
organ atrophy could be due to several infectious and nonin-
fectious diseases. Experimentally it is possible to detect
MDV antigens in the lymphoid organs before the atrophy
occurs, however such expression occur for a short period
(1–2) days and is not associated with clinical signs or gross
lesions. Late MDV‐IS is even more complicated to diagnose
than early MDV‐IS as it is not associated with lymphoid
organ atrophy and/or tumors (195–197). Because late‐
MDV‐IS cannot be p ­ rotected by currently vaccination pro-
tocols (197), it is very likely causing problems in commercial
flocks and should be considered in the differential diagnosis
when immunosuppression is suspected.
Intervention Strategies
The development of successful vaccines for control of
MD (132, 403, 470, 488) was a significant achievement.
Vaccination represents, for now and the foreseeable
future, the central strategy for the prevention and ­control
of MD. Genetic resistance and biosecurity, however, are
critical adjuncts to properly executed vaccination proce-
dures. No effective practical treatment exists for the
­disease in individual chickens or infected flocks. An inte-
grated strategy to prevent early infection, to slow the
acquisition of virulence of field strains, and to provide
superior immune responses seems most likely to suc-
ceed. Various reviews on MD vaccines and control
­procedures are available (67, 211, 462, 489).
Vaccination
Types of Vaccines
Several different types of MD vaccines are in common
use, both individually and in various combinations. The
most widely‐used products are low pathogenic serotype
1 MDV attenuated in cell culture (470) and naturally
nononcogenic serotype 3 (HVT) (403), and serotype 2
viruses (490, 618). The latter usually are combined with
HVT to take advantage of the synergistic activity
­documented between serotypes 2 and 3 (492, 589). All
vaccine types are protective but to varying degrees.
Herpesvirus of turkey virus, mainly strain FC126 (613)
is extensively used because it is effective and economical
to produce and combines well with other products.
Although both cell‐free and cell‐associated forms of
HVT are available, the latter has been most widely used
because it is more effective than cell‐free virus in the
presence of maternal antibodies (603). Bivalent vaccines
consisting of HVT and SB‐1 (490) or 301B/1 (618)
strains of serotype 2 MDV were introduced in the mid‐
1980s. The CVI988 strain (470) used in the Netherlands
(356) and other countries since the early 1970s, was
introduced to the United States in the early 1990s.
Serotype 1 and 2 vaccines are available only as cell‐­
associated products.
Besides the traditional vaccines, in the recent years a
number of recombinant vaccines have been developed,
licensed, and are currently commercialized (reviewed in
462). Efforts to develop recombinant MD vaccines started
in the early 1990s using either fowlpox virus (394) or
HVT (374, 474) as vectors. However, it has been in the
last few years that several products using HVT as a vector
for various diseases (infectious laryngotracheitis, infec-
tious bursal disease, Newcastle disease, and avian influ-
enza) have been licensed and widespread used; either
alone or in combination with MD vaccines of other sero-
types (188, 189, 205, 208, 232, 292, 339, 411, 435, 568). In
addition to HVT vector vaccines, recombinant vaccines
based on serotype 1 MDV strains have been developed
either by using attenuated serotype 1 MDV strains as vec-
tor (275, 349, 566, 647), by modifying or deleting genes
(62, 144, 333), by insertional mutation of the long termi-
nal repeat (LTR) region of the REV in various MDV sero-
type 1 strains (149, 313, 353, 364, 611), or by combining

various techniques (i.e., REV LTR insertion and deletion
of meq, meq‐deleted vaccines as vectors) (215, 545). To
date, the only recombinant vaccine based on serotype 1
MDV strains that is licensed and commercialized in some
countries is a recombinant CVI988 vaccine with an inser-
tion of the REV LTR (66, 353). This vaccine has been
shown to replicate and protect better than the parental
CVI988 and does not induce lymphoid organ atrophy in
susceptible chickens lacking maternal antibodies (66).
However, experimental vaccine based on vvMDV strain
Md5 with deletion of both copies of the oncogene meq
(352, 526) has been shown to be the most protective vac-
cine against early challenge with vv+MDV (115, 331).
This vaccine protects not only against the development of
tumors but also against the development of late MDV‐IS
in commercial meat type chickens (197). Furthermore, it
can be successfully used as a vector vaccine for other
poultry diseases (215, 651). Unfortunately, the deletion
mutant is causing severe thymus and bursa atrophy in
maternal antibody‐negative, one‐day‐old chicks (175)
preventing licensing in the United States under current
regulations. The negative effect on the lymphoid organ in
chickens lacking maternal antibodies can be reversed by
attenuation in cell culture or by adding a UL5 helicase‐
primase subunit point mutation; albeit at a cost of reduc-
ing protection (257, 330).
Other strategies to improve vaccines through recom-
binant DNA approaches that might have potential use in
the future includes DNA vaccines (563), incorporation of
cytokines (168, 243, 554), and Toll‐like receptor‐based
adjuvants (419, 421).
Vaccine Administration
Marek’s disease vaccines are administered to chicks at
hatch by subcutaneous or intramuscular inoculation or
in ovo at ED18 (67, 516) In ovo vaccination is now per-
formed by automated technology and is used in more
than 90% of commercial broiler chickens in the United
States (586). In ovo vaccination not only reduces labor
costs and has greater precision of vaccine administration
but also confers better protection against early challenge
with MDV than one‐day‐old vaccination (649).
Furthermore, it has been recently shown that in ovo vac-
cination with HVT hastens the maturation of the
immune system of the chicken embryo rendering chicks
more immunocompetent at hatch (221). Deposition of
the vaccine by the amniotic or intraembryonic route is
essential for optimal protection (580, 585). Proper han-
dling of vaccine during thawing and reconstitution is
crucial to ensure that adequate doses are administered
(207, 239).
Factors Affecting Efficacy
Vaccines typically are given at doses of 2,000–6,000
plaque forming units (PFU) per chick, but these are often
Chapter 15  Neoplastic Diseases 583
significantly reduced in broilers. The older literature
suggests that increased doses of HVT above a threshold
level of around 400 PFU did offer little improvement
(178). However, recent studies indicate that a substan-
tially higher threshold is needed to protect against chal-
lenge with vv and vv+ strains (214, 216, 217).
Revaccination has been recently shown to increase
protection against early challenge with MDV (219, 224,
629). The benefits of double vaccination has been repro-
duced under laboratory conditions (224) when the sec-
ond vaccine is more protective than the first vaccine
administered and both vaccinations occur before chal-
lenge with oncogenic MDV (224). It has been suggested
that the best revaccination program will include a low
protective vaccine in ovo (i.e., HVT) followed by a high
protective vaccine at day of age (i.e., HVT+SB‐1 or/and
CVI988) (219).
Several other factors may influence vaccine efficacy
such as the passage level of the vaccine viruses. Increased
passage level may cause a decrease in vaccine replication
resulting in decreased immunity (330, 614). Unfortunately,
passage levels of commercial vaccines are in general not
available. Maternal antibodies against the three serotypes
are typically present in commercial one‐day‐old chicks.
These antibodies may reduce the effectiveness of cell‐
associated vaccines but do not abrogate the protective
effect (315), if sufficient PFU are administered.
In addition to improper vaccination techniques (381),
early exposure is undoubtedly one of the most important
causes of excessive MD in vaccinated flocks because field
exposure usually occurs very soon after placement of
chickens (612) and because at least seven days is required
to establish solid immunity after vaccination (281)
(reviewed in 502). The vaccine strain of virus also has a
major influence on vaccine efficacy. Immunity induced
by weaker vaccines such as HVT may be excellent against
low virulence challenge but can be completely over-
whelmed by early challenge with highly virulent strains
(597). Protection conferred by CVI988, however, does
not seem to be as affected by the pathotype of the
­challenge strain (459) as vaccines of other serotypes.
Although high virulence strains commonly are invoked
to explain field outbreaks of disease, many alternate
causes should be considered.
Stress appears to interfere with the maintenance of
vaccinal immunity. In chickens properly vaccinated at
hatch and well‐protected following challenge, Powell
and Davison (441) induced MD lesions and mortality
by immunosuppressive treatment at 10 weeks of age. The
onset of egg production deserves consideration as a stress
factor precipitating vaccine breaks (600). Infection with
other pathogens such as IBDV, REV, CIAV, and r­ eoviruses
have been reported to interfere with the induction of vac-
cinal immunity (reviewed in 500), although very specific
conditions are sometimes required. T2‐toxin has been
584 Section II  Viral Diseases
recently shown to have detrimental effect on the MD
outcome of nonvaccinated chickens but it did not affect
protection conferred by HVT (321).
The strain of chicken is also an important determinant
of vaccine efficacy. Schat et  al. (492) found that HVT
vaccine in genetically resistant chickens resulted in a
stronger immunity than did the bivalent (HVT+SB‐1)
vaccine in susceptible chickens. Chang et  al. recently
demonstrated that in genetically resistant lines of chick-
ens, HVT can induce as much protection as some of the
commercial CVI988 strains (117). In commercial chick-
ens, protective immunity conferred by vaccines is influ-
enced by the B‐haplotype (22) as well as by non‐MHC
genetic variation (116).
Vaccination Strategies
Marek’s disease vaccines as a class are unusually effec-
tive, often achieving greater than 90% protection under
commercial conditions (600). However, attention is often
focused on flocks in which MD losses are perceived to be
excessive (381). Causes for such vaccine failures are dif-
ficult to ascertain by retrospective analysis (381, 600).
However, there are several checkpoints that could be
evaluated in the event of an immunization failure (212).
Auditing at the hatchery to evaluate improper vaccina-
tion techniques, titration of the batch of vaccines used,
replication of the vaccine in the chickens, early exposure
to oncogenic MDV, coinfection with other immunosup-
pression agents (especially with CIAV infection), and the
emergence of new MDV strains with increased virulence
need to be considered (212).
Efficacy data comparing certain groups of vaccines are
available (citations in 485, 600). Bivalent serotype 2+3
vaccines are clearly more effective than HVT, but the
original CVI988 vaccine seems to be the most effective
(593, 608), a result consistent with earlier reports from
Europe (572). However, differences in the level of protec-
tion conferred by CVI988 from different sources have
been documented (214, 608). Recent studies demon-
strate that a recombinant vaccines with the deletion of
both copies of the oncogene meq provides even better
protection than the most protective CVI988 strain (115).
The propensity of MDVs to evolve to greater virulence
is critical to the strategic use of vaccines for MD control
(210). Vaccination itself no doubt contributes to this vir-
ulence increase, which, in turn, tends to make earlier
vaccines obsolete. Kreager (320) noted that the useful life
of a MD vaccine has been about 10 years under current
management conditions. Although this is perhaps an
overstatement, the implications are serious. Since
CVI988 was introduced in the United States, some evi-
dence already suggests that contemporary strains have
increased their virulence in CVI988‐vaccinated chickens
(599). In a recent study, Dunn et al. demonstrated that in
those farms where vv+MDV were isolated in late 1990s
current MDV isolates are still vv+MDV strains suggest-
ing that once vv+MDV infection gets established in a
farm it will stay (173).
The emergence of increasingly virulent viral strains,
coupled with an apparent reduction of vaccine efficacy
during the past 20 years, has prompted justifiable con-
cern. This suggests that vaccination by itself does not
provide a complete control program and is not the ulti-
mate solution for MD. In fact, recent evidence also points
towards the potential contribution of the early genera-
tions of vaccines themselves driving virulence and selec-
tion of more virulent MDV pathotypes (461). Strict
biosecurity procedures to reduce early exposure and the
presence of genetic resistance are essential adjuncts to a
successful vaccination program. Furthermore other than
increased protection against tumors vaccine research
should focus on protecting against other aspects of MDV
infection such as infection, transmission, and MDV‐IS.
Genetic Resistance
The well‐known variation in susceptibility of different
lines of chickens to MD challenge is determined by
genetic factors (20, 71, 120) and provides a unique
opportunity to include genetic approaches to control
MD. Indeed, poultry breeders have included resistance
to MD in selection programs for many years (366).
However, genetic resistance can be overcome by chal-
lenge with highly virulent MDVs and is best applied in
concert with vaccination and biosecurity to achieve opti-
mal control. Genetics influences virtually every aspect of
host response to MD. However, only those issues ger-
mane to disease control programs are considered here.
For successful incorporation of selection for resistance
in breeding programs several conditions need to be met
(reviewed in 20). The heritability of resistance is rela-
tively large, thus selection has a considerable impact.
Selection for resistance is at least neutral for production
traits or is correlated positively with production traits
(13). Sufficient heterogeneity exists in single‐sire families
to warrant selection for resistance in commercial chick-
ens, which is still the case in recent commercial genetic
stocks (184).
Selection Methods
Selection programs for resistance historically have been
based on progeny testing or family selection (133) or
reproduction from survivors of exposed nonvaccinated
breeding flocks through mass selection (355). Bacon and
Witter (21) found that resistance may better be deter-
mined in vaccinated stocks. Under certain conditions
acquisition of resistance can be obtained within four to
six generations (133, 355). Family selection may be more
appropriate than mass selection for commercial breeders
in order to avoid high loss of genetic material on initial
challenge exposure (20). Resistance has been considered

dominant, although this varies to some extent (255). In
most cases, resistance of crosses has been intermediate
to that of the parent strains (55).
In addition to selection based on challenge with MDV,
blood typing for MHC can be used based on the close
relationship between MD resistance and certain alleles
of the B‐F region of the MHC, especially B21 (61, 71).
However, the value of selection for MHC‐associated
markers may vary considerably among commercial lines
and crosses (55, 247).
Several non‐MHC genes may also be involved in resist-
ance. For example, line 6 and line 7 chickens differ mark-
edly in MD susceptibility, but both lines are both
homozygous for the MHC B2 allele (143). Because non‐
MHC effects were considered more important than
MHC effects in several commercial lines (234) the need
to identify genetic markers associated with MD resist-
ance became priority in all genomic studies. Various
strategies have been used to identify genetic markers of
MD resistance including identification and mapping of
map quantitative trait loci (QTL), RNA expression
­profiling, expression quantitative trait loci, and allele‐
specific expression (ASE) screens (reviewed in 120).
Genome‐wide QTL scans with microsatellite markers
have identified 14 or more putative QTL associated with
MD resistance (70, 569, 632). To complement the QTL
scans, gene expression profiling using microarray
­technology has been integrated. Gene profiling has been
conducted to identify differentially expressed genes
between MD‐resistant and MD‐susceptible lines after
MDV challenge (346); among MHC‐congenic lines of
chickens following inoculation with different MD vac-
cines(375); and in CEF transformed with Meq (341).
Marek’s disease virus‐chicken protein–protein interac-
tions have been very useful to confirm genes associated
with MD resistance(397) such as growth hormone (GH1)
(346), stem cell antigen 2 (SCA2) (347), and MHC class II
β chain (B‐LB)(396). However, purely genetic‐driven
approaches to identify high‐confidence candidate genes
have not been as successful as first thought. A combina-
tion of genetic and functional genomic approaches such
as ASE, epigenetics, and RNA expression profiling seems
to be more useful in identifying candidate genes (121,
308, 350, 358, 436, 634, 640).
Applications to Control
The knowledge that genetically resistant chickens are
protected by vaccination to a greater extent than more
susceptible strains (542) has fueled interest by commer-
cial breeders to emphasize MD resistance in selection
programs. However, synergy between host genetics and
vaccines is complex. Some resistant B‐haplotypes were
demonstrated only by challenge of previously vaccinated
chickens (21). However, the relative efficacy of MD vac-
cines is also influenced by B‐haplotype, leading to the
Chapter 15  Neoplastic Diseases 585
concept to select the most appropriate vaccine based on
the predominant B‐haplotypes in a particular strain (23).
In practice, this issue has been either ignored or
addressed through the use of vaccines containing multi-
ple serotypes. Recently, vaccine efficacy has been shown
to be influenced by non‐MCH genetic variation (116).
In light of the selection tools available, the absence of
negative correlations, and the major benefits to be
derived, it is not surprising that breeders place a high
priority on this approach. Although selection for B‐hap-
lotype has been practiced with variable success and has
proven to be complex, especially in meat strains (366),
breeders acknowledge the value of improved genetic
resistance to offset virulence increase by viral strains and
the limitations of current vaccines (319, 624).
Management Procedures
Strict biosecurity practices to limit the extent of early
MDV exposure, although impractical as a primary con-
trol procedure, are a crucial and cost‐effective adjunct to
vaccination. Marek’s disease control is compromised
because modern poultry management decisions are
often linked to cost analyses. As a consequence replace-
ment flocks of different ages are placed in close proxim-
ity to each other, vaccine doses are reduced and/or litter
from a previous broiler flocks is used (489). The failure
to prevent early exposure is perhaps the most important
single cause of vaccine failures. Improved hygiene has
often appeared to play a key and cost‐effective role in the
elimination of excessive MD losses in vaccinated flocks.
Relevant sanitation principles have been reviewed (423).
For SPF flocks, higher standards of biosecurity are
required and become cost effective. Most SPF operations
use filtered‐air, positive‐pressure houses, which, along
with strict biosecurity measures, successfully can main-
tain large flocks free of MDV infection. In this case, bios-
ecurity becomes a substitute for vaccination and provides
a practical demonstration that MDV infection can be
prevented, at least under specialized conditions.
Nononcogenic Avian Herpesviruses
As mentioned earlier, two additional groups of nononco-
genic herpesviruses, MDV‐2 (Gallid herpesvirus 3) and
HVT (Meleagrid herpesvirus 1), isolated from chickens
(50, 125) and turkeys (307, 613), respectively, are consid-
ered part of the Mardivirus genus. The classification of
these two viruses as two distinct serotypes, originally
based on the recognition of common and distinct anti-
genic epitopes (576, 577), has been justified from the
data on the complete sequence for serotype 2 strains
HPRS‐24 and SB‐1 (282, 540), and serotype 3 FC126
virus strains (11, 316).
586 Section II  Viral Diseases
Interest in MDV‐2 and HVT derives mainly from their
use as live vaccines against MD. However, both viruses
occur in nature independent of vaccination, and it seems
appropriate to also consider some aspects of their epizoo-
tiology and pathogenesis in their natural or alternate avian
hosts. Reviews providing additional details on pathogen-
esis of these infections may be consulted (88, 90).
Turkey Herpesvirus
Herpesvirus of turkey is endemic and ubiquitous in
domestic turkeys (619) and has also been isolated from
wild turkeys (135). In chickens, the virus has also become
ubiquitous because of its widespread usage as vaccines
(67). Increasingly, HVT is used successfully as recombi-
nant live virus vectored vaccines against a number of
­diseases (review in 462).
The 160 kb‐long HVT genome encodes nearly 100
functional genes that include sets of homologous and
unique genes (11, 316), as well as novel microRNAs (579,
637). The function of HVT genes in relation to their dis-
tinct properties of high immunogenicity, lack of onco-
genicity, and ability to produce cell‐free virus are not
fully understood. The availability of infectious BAC
clones of HVT (32) will be helpful in gaining further
insights into HVT gene functions.
In turkeys, HVT spreads rapidly through exposed
flocks by contact exposure as no evidence of vertical
transmission has been demonstrated (620). Virtually all
individual turkeys become viremic and develop antibod-
ies within a few weeks (619). The virus appears to mature
in the FFE, because cell‐free skin extracts are infectious
(620) although viral antigen was found only infrequently
and at low levels in the FFE (194). Herpesvirus of turkey
may be transmitted from turkeys to chickens under
experimental conditions (620), although this is consid-
ered extremely rare. Only limited contact spread occurs
among chickens, but transmission could not be demon-
strated by the airborne route (122). Replication of virus
in the FFE of infected chickens appears limited and tran-
sient (122, 446), although increased levels of HVT DNA
were observed in FFE of HVT‐vaccinated chickens after
MDV challenge (278).
Fabricant et al. (194) studied the early pathogenesis of
HVT infection in chickens and turkeys. Chickens had no
cytolytic infections in any lymphoid organ. Turkeys
infected with HVT had some viral antigen‐positive cells
at 4–14 days PI in the spleen, but no cytolytic infections
in bursa or thymus were seen. In chickens, there was no
depression of bursa or thymus size, although a transient
splenomegaly was variably present (93, 194). Expression
of gB was detected in spleen, with limited levels in thy-
mus and bursa of Fabricius (264). B cells are rarely
infected, but latent infection is probably established in
MHC class II‐positive T cells (88). Natural killer cell
activity was stimulated through at least 8 week PI (517).
Herpesvirus of turkey can be recovered from infected
chickens for long periods, and antibodies persist for life
(451, 617). The virus is apparently nononcogenic in tur-
keys (588), but the possibility of fertility problems in
HVT‐infected toms has been raised (560). The virus
generally causes no clinical disease in intact or immuno-
suppressed chickens (518, 613). However transient B‐
lymphocyte dysfunction (203), atrophy of the bursa and
thymus with high doses (238), and minor cellular infiltra-
tions in nerves (194, 617) have been reported. In con-
trast, up to 19% of S‐line chickens infected with HVT in
ovo (ED 8) were reported to develop clinical paralysis
and gross nerve enlargement due to inflammatory type
lesions (101). Chickens exposed to HVT at ED 14 or ear-
lier showed higher incidence of immunological tolerance
resulting in a persistent HVT viremia (650). A possible
role for HVT as a predisposing factor in autoimmune
disease is suggested by its implied involvement in auto-
immune vitiligo in certain strains of chickens (187).
Herpesvirus of turkeys seems to have an adjuvant
effect when administered with other vaccines such as
serotype 2 MDV strains (103, 609) and fowlpox virus
(395). Furthermore, recently it has been shown that
administration of HVT to chicken embryos at 18 ED has-
tens the development of the immune system (221).
Serotype 2 Marek’s Disease Virus
Apathogenic strains isolated from clinically normal
chickens (50, 125) subsequently were determined to be a
separate serotype based on antigenic properties (577)
and genome sequences. The 166 kb‐long MDV‐2 genome
encodes several homologous and unique genes (282,
540) as well as novel microRNAs (638). Availability of
infectious BAC clones of SB‐1 virus (437, 529) could
facilitate functional analysis of MDV‐2 genes.
Serotype 2 viruses are widespread, although not uni-
versal in commercial chicken flocks (50). The epizootiol-
ogy has been complicated by distribution of the virus
through a seeder chick program (644) and widespread
use of MDV‐2 vaccines (103, 616). Other unique features
of this virus group were further elucidated following the
isolation of the SB‐1 strain (490).
Serotype 2 viruses replicate in the FFE (124) and spread
readily by contact (490, 618). Following inoculation of
day‐old chickens, the virus can be first isolated 5–6 days
PI, reaching peak titers at 2–4 weeks, persisting thereafter
for long periods (93, 618). A transient splenomegaly was
induced between 4–12 days PI with SB‐1, but no bursal
atrophy and only occasional thymic atrophy was seen and
cytolytic infection of lymphoid organs was not observed
(93). In contrast, Lin et  al. (345) found viral antigens in
spleen and bursal tissues 5–14 days PI, primarily in B cells,
but no gross or microscopic changes were observed.

Calnek (88) considered B cells and macrophages relatively
refractory to infection, and the cells supporting latent
infection lacked MHC class II antigens, thus differing
from those in HVT infections. T cells also may not be very
susceptible because of the poor re‐isolation rates of SB‐1
from CD4+ and CD8+ cells of infected chickens (337),
although MDV‐transformed MSB‐1 cells can be dually
infected with MDV‐1 and MDV‐2 viruses (638). SB‐1 is
not normally considered immunosuppressive (181)
although a diminished response to a B lymphocyte‐­
specific mitogen and decreased antibody responses to
bovine serum albumen have been reported in chickens
vaccinated with SB‐1 strain in combination with HVT.
However, as it has not been associated with neoplastic
lesions in chickens or embryos, SB‐1 is designated nonon-
cogenic rather than apathogenic (490). The absence of
lymphoma was confirmed by other workers (50, 123, 618),
although there was one report of visceral lymphomas in 2
of 48 chickens inoculated with the HPRS‐24 strain (440).
­Leukosis/Sarcoma Group
Venugopal Nair
Summary
Agent and  Disease.  Avian leukosis sarcoma group of
pathogens are retroviruses associated with a number of
neoplastic diseases in poultry. These viruses are grouped
into different envelope subgroups and induce diseases
such as lymphoid leukosis (LL) and myeloid leukosis
(ML) that are widespread in many countries causing
major economic losses and animal welfare issues.
Diagnosis.  Major challenge in the clinical diagnosis of
the disease is because of the difficulty in differentiating
from other avian neoplastic diseases. Flock‐level
monitoring for the presence of viral antigens, combined
with serological and molecular diagnostic tests are
important disease diagnosis and eradication.
Intervention.  Eradication of the pathogen by application
of flock monitoring tests to eliminate infected birds at the
pedigree breeding flock level is the best intervention
strategy. Selection of birds for genetic resistance to
infection will also be a useful adjunct in the control strategy.
Introduction
Definition and Synonyms
The leukosis/sarcoma (L/S) group of diseases designates
a variety of transmissible benign and malignant neoplasms
Chapter 15  Neoplastic Diseases 587
Vaccination with serotype 2 viruses causes a pro-
nounced enhancement of B‐cell lymphomas in certain
genetic strains of chickens exposed at an early age to sub-
group A ALV (24) or REV (12) (see Leukosis/Sarcoma
Group). The ability of serotype 2 virus to enhance LL was
attenuated without abrogation of its protective proper-
ties against MD challenge (594). Avian leukosis virus‐A
has now been eradicated from most of the chicken lines
susceptible to serotype 2 enhancement and field prob-
lems due to serotype 2 enhancement of LL are rare (see
Leukosis/Sarcoma Group). Recently, it has been shown
that serotype 2 MDV can enhance the development of
spontaneous ALV‐like bursal lymphomas in ALVA6
transgenic chickens (resistant to infection with sub-
groups A and E of ALV) (112). Such enhancement is
independent of age of vaccination (in ovo vs hatch) and
might explain some of the spontaneous lymphomas
observed in commercial flocks that are not related to
ALV or REV (112).
of chickens caused by members that belong to the family
Retroviridae (201). Because of the expansion of the lit-
erature on this disease, it is no longer feasible to cite all
relevant publications that provide the scientific basis for
our current knowledge of the disease. Hence the litera-
ture is cited selectively, with reviews often used instead
of original papers. For more detailed references to the
literature, readers are advised to refer to the chapter in
the previous edition (266).
Lymphoid leukosis (LL) has been the most common
form of L/S group of diseases seen in field flocks,
although myeloid leukosis (ML) has also become preva-
lent. The neoplasms and their synonyms are listed in
Table  15.4. Members of this group of avian viruses are
characterized, as are all members of the Retroviridae, by
possession of an enzyme reverse transcriptase, which
directs the synthesis of the proviral DNA form of the
RNA virus that forms part of the retroviral life cycle and
from which the family name is derived.
Sections reflecting the host response (Pathology and
Pathogenesis of the Infectious Process) are discussed
under the pathologic entities without regard for the
properties of the inducing virus(es) other than their
inclusion in the L/S group.
Economic Significance
Infection of chickens with avian leukosis virus (ALV) is
the most common L/S virus infection encountered in
field flocks and is known to be of significant economic
588 Section II  Viral Diseases

Table 15.4  Neoplasms caused by viruses of the leukosis/sarcoma group.

Neoplasm Synonyms

Leukoses
Lymphoid leukosis Big liver disease, lymphatic leukosis, visceral lymphoma, lymphocytoma,
lymphomatosis, visceral lymphomatosis, lymphoid leukosis
Erythroblastosis Leukemia, intravascular lymphoid leukosis, erythroleukosis,
erythromyelosis, erythroblastosis, erythroid leukosis
Myeloblastosis Leukemic myeloid leukosis, leukomyelosis, myelomatosis, myeloblastosis,
granuloblastosis, myeloid leukosis
Myelocytoma(tosis) Myelocytoma, aleukemic myeloid leukosis, leukochloroma, myelomatosis
Connective tissue tumors
Fibroma and fibrosarcoma
Myxoma and myxosarcoma
Histiocytic sarcoma
Chondroma
Osteoma and osteogenic sarcoma
Epithelial tumors
Nephroblastoma Embryonal nephroma, renal adenocarcinoma, adenosarcoma,
nephroblastoma, cystadenoma
Nephroma Papillary cystadenoma, carcinoma of the kidney
Hepatocarcinoma
Adenocarcinoma of the pancreas
Thecoma
Granulosa cell carcinoma
Seminoma Adenocarcinoma of the testis
Squamous cell carcinoma
Endothelial tumors
Hemangioma Hemangiomatosis, endothelioma, hemangioblastomas,
hemangioendotheliomas
Angiosarcoma
Endothelioma
Mesothelioma
Related tumors
Osteopetrosis Marble bone, thick leg disease, sporadic diffuse osteoporostitis,
osteopetrosis gallinarum
Meningioma
Glioma

importance. Economic losses from ALV‐induced dis-

eases are attributed to two sources. First, tumor mortal-
ity commonly amounts to around 1–2% of birds, with
occasional losses of up to 20% or more. Second, subclini-
cal infection by ALV, to which most flocks are subject,
produces a depressive effect on a number of important
performance traits, including egg production and quality
(152). Economic losses due to ALV tumor mortality and
reduced productivity are estimated to be in millions of US
dollars each year (266). Currently the virus is causing huge
economic losses to the poultry industry in China (229).
Public Health Significance
Recent studies have addressed the relationship between
avian tumor viruses, particularly ALVs, and human
health. Evidence for the presence of antibodies to ALVs in
humans usually has been lacking or at best is presumptive
(192). In a serological survey that included 549 human
subjects, including groups exposed and not exposed to
chickens, significant differences between men and
women were found for the prevalence of a­ ntibodies to
ALV but were not related to exposure to chickens (70).
 Chapter 15  Neoplastic Diseases 589

Detection of reverse transcriptase activity as a sensitive TM SU

assay for the presence of ALV in human v­ accines derived
from chicken cells was known to be of public health sig-
nificance (332). While the test is of great value in detect- MA
NC
ing contaminating viruses in vaccines (35), no evidence of
antibodies or proviral sequences of ALV was found in the IN RT
vaccines or in the sera of vaccine recipients (187, 352).

History
The earliest reports of leukotic diseases in fowl are those
of Roloff (334), who described a case of “lymphosar-
comatosis” in 1868, and of Caparini (58), who in 1896
described cases of “fowl leukemia.” Observations of
osteopetrosis lesions in the bones of chicken recovered
from ancient Roman burial sites were probably associ-
ated with avian leukosis (42).
Viral oncology was initiated as a discipline from the work
with the demonstration of transmission of erythroleukemia
and myelogenous leukemia by inoculating cell‐free filtrates
(116). Peyton Rous received the Nobel Prize in 1966 for his
seminal work on transplantable tumor that could be trans-
mitted by cell‐free filtrates (336). Detailed reviews of the
history of avian retrovirus research are available elsewhere
(107, 266, 298, 306, 338, 389–391, 409, 415, 427).
RNA
CA
Lipid bilayer PR
Figure 15.18  Schematic diagram of avian leukosis virus particle.
The viral envelope is a lipid bilayer in which the gp37
transmembrane (TM) and the gp85 surface (SU) proteins, encoded
by the env gene, are inserted. Internal components encoded by
the gag/pro gene are p19 matrix (MA) protein, p27 capsid (CA)
protein, p12 nucleocapsid (NC) protein, and p15 protease (PR). The
pol gene encodes the reverse transcriptase (RT) and p32 integrase
(IN). The core of the particle contains two viral RNA strands.
RNA VIRUS

R U5 gag/pro pol env U3 R

Etiology 5′ CAP AAA 3′
Controlling Coding sequences Controlling
Classification sequences sequences

As per the latest classification of viruses (1), avian L/S Reverse transcription
& integration
group are placed in the Alpharetrovirus genus of the
family Retroviridae. Members of this family are RNA DNA PROVIRUS
viruses characterized by the possession of the enzyme 5′ LTR 3′ LTR
reverse transcriptase, which is necessary for the forma- U3 R U5 gag/pro pol env U3 R U5Host DNA
tion of a DNA provirus that is integrated in the host
genome during virus replication. Avian leukosis virus is Controlling Coding sequences Controlling
the type species of the genus (Figures 15.18 and 15.19). sequences Transcription sequences
Rous sarcoma virus (RSV) and a number of replication‐
defective, acutely transforming viruses such as MC29
and MH2 that carry oncogenes are also in the genera.

Translation Messenger RNAs Progeny viral

Morphology genomic RNA

Ultrastructure
In thin‐section electron microscopy, avian leukosis/sar- Structural core Reverse Envelope
proteins: transcriptase (RT); proteins:
coma viruses (ALSV) have an inner, centrally located, p19 (MA), p10, integrase (IN) gp85 (SU),
electron‐dense core about 35–45 nm in diameter, an p27 (CA), gp37 (TM)
p12 (NC);
intermediate membrane, and an outer membrane. This p15 protease (PR)
appearance typifies the C‐type retroviral morphology.
The overall diameter of the virus particle is 80–120 nm, Figure 15.19  Key features of the viral RNA and proviral DNA forms
of the genome of avian leukosis virus. CAP, 5’ end structure; AAA,
with an average of 90 nm. Immature virions budding polyadenylation of 3’ end; R, repeat sequence; U5, unique 5’ end
from the cell membrane can be visualized (Figure 15.20). sequence; U3, unique 3’ end sequence; LTR, long terminal repeat.
Characteristic knobbed spikes about 8 nm in diameter For other abbreviations, see Figure 15.18 and the text.
590 Section II  Viral Diseases

(A) (B)

(C) (D)

Figure 15.20  Ultrastructure of leukosis/sarcoma viruses. (A) BAI‐A of avian myeloblastosis virus (AMV), unfixed, and negatively stained
with neutralized phosphotungstic acid. Peripheral fringe about particles is resolved in some places into discrete “knobs.” ×150,000. (B)
Ultrastructure of leukosis/sarcoma virus release. Virus budding at cell membrane of a leukemic myeloblast. Surface of buds and particles
peripheral to outer membrane is irregular and indistinct (pnu, dense prenucleoid). ×215,000. (C) Thin section of BAI‐A of AMV
sedimented from plasma, fixed in osmium tetroxide, and stained with lead subacetate. Inner and outer membranes and granular
character of nucleoid can be seen. Impression of granules might be derived from sectioning of filaments. Some granules appear to be
hollow. ×510,000. (D) Purified BAI‐A of AMV fixed and shadowed with chromium. ×50,000. (Bonar and de Thé).

are present on the surface of the particles and comprise
the viral envelope glycoproteins. These projections can
also be seen in thin sections. Significant advances have
recently been made in ultrastructural studies of retrovi-
ral capsids (311, 356).
Size and Density
By filtration through membranes of graded pore size,
ultracentrifugation, and electron microscopy, viruses
have a diameter of 80–145 nm. The value of 1.15–1.17 g/
mL for the buoyant density in sucrose is characteristic
for C‐type retroviruses (266).
Chemical Composition
The overall composition of avian myeloblastosis virus
(AMV), which has been studied extensively, is 30–35%
lipid and 60–65% protein, of which 5–7% is glycopro-
tein, 2.2% is RNA, and small amounts of DNA are pre-
sent, apparently of cellular origin (266). Quantitative

proteomics analysis of virus‐infected cells using
improved mass spectrometric methods has identified
a number of host proteins associated with these
viruses (225).
Viral Nucleic Acids
The major class sizes of RNA sediment at 60–70 S, which
is the viral genome, and at 4–5 S, most of which is host
tRNA, are thought to be accidentally included in the
virion. A tRNA is also associated with the 70 S RNA and
serves as a primer for the DNA polymerase during tran-
scription of viral RNA to DNA. Small amounts of 18 and
28 S RNA, viral and cellular mRNA, and DNA are also
present. The 60–70 S genomic RNA is a dimer and can
be split into two subunits of about 34–38 S, which are
believed to represent the diploid genome. These subu-
nits of genomic RNA are mRNAs, and their genes have
been mapped for a number of avian retroviruses.
The sequence of the structural genes of ALV, from the 5’
end to the 3’ end of the RNA molecule, is gag/pro‐pol‐env;
these genes encode, respectively, the proteins of the virion
group‐specific (gs) antigens and protease, RNA‐depend-
ent DNA polymerase (reverse transcriptase, or RT), and
envelope glycoproteins (Figure  15.19). The structural
genes are flanked by terminal genomic sequences with
gene promoter and enhancer activities, and that, in the
DNA provirus, form the long terminal repeat (LTR)
regions. The viral genome is about 7.3 kb in size.
Acutely transforming viruses possess additional trans-
duced oncogene sequences that initiate neoplastic
transformation. Acquisition of a viral oncogene usually
is accompanied by genetic defects elsewhere in the viral
genome (see Pathogenicity). Non‐defective RSV has the
genetic composition gag/pro‐pol‐env‐src. The additional
gene, src, responsible for sarcomatous transformation,
evidently was acquired originally from a normal cellular
oncogene, cellular src. The gene cellular src is an exam-
ple of a number of host cell genes, termed proto‐onco-
genes or onc genes, concerned with acute transformation
(211, 424). Viral and cellular versions of onc genes, and
of the specific varieties such as src, are distinguished by
the prefixes v‐ and c‐. Specific v‐onc genes, with c‐onc
counterparts in normal cells (Table 15.5).
Viral Proteins
The nature, location, and synthesis of proteins that con-
stitute avian retroviruses have been extensively studied
(394) (Figures 15.18 and 15.19). The virion core contains
five non‐glycosylated proteins encoded by the gag/pro
gene: MA (matrix, p19); p10; CA (capsid, p27), which is
the major gs antigen (Gag) in the core shell; NC (nucle-
ocapsid, p12), involved in RNA processing and packag-
ing; and PR (protease, p15), involved in cleavage of
protein precursors. Other minor polypeptides have been
reported.
Chapter 15  Neoplastic Diseases 591
The pol gene encodes the enzyme reverse transcriptase
(RT) present in the core. It is a complex consisting of the
b subunit (95 kDa) and the a subunit (68 kDa) derived
from it and has RNA‐ and DNA‐dependent polymerase
and DNA–RNA hybrid‐specific ribonuclease H activi-
ties. The b subunit also contains the IN domain (inte-
grase, p32), the enzyme necessary for integration of viral
DNA into the host genome. Recent structural studies on
the catalytic core domain of the ALSV integrase sug-
gested that it can dimerize in more than one state allow-
ing the flexibility for multifunctionality during different
steps of retroviral life cycle (19).
The virion envelope contains two glycoproteins
encoded by the env gene: SU (surface, gp85), the viral
surface knob‐like structures that determine viral enve-
lope subgroup specificity of the ALSV; and TM (trans-
membrane, gp37), representing the transmembrane
structure that attaches the knobs to the envelope. These
two envelope (Env) proteins are linked to form a dimer,
termed virion glycoprotein (VGP).
Enzymes and other proteins are found in virions and
are considered to be cellular components incorporated
during virus maturation (393). Of practical value is the
presence in AMV obtained from blood of infected chick-
ens, or from myeloblast cultures, of adenosine triphos-
phatase derived from the cell membrane and incorporated
into the virus particle during maturation.
Virus Replication
As with other retroviruses, replication of ALSV is char-
acterized by the formation, under the direction of reverse
transcriptase, of a DNA provirus that becomes linearly
integrated into the host cell genome (Figure  15.19).
Subsequently, the proviral genes are transcribed into
viral RNAs, which are translated to produce precursor
and mature proteins that constitute the virion.
Penetration of the Host Cell
Detailed reviews describing the recent understanding of
the early ALV interactions with the host cells are available
(266, 393). Although adsorption of the virion to the cell
membrane is nonspecific, occurring even in cells resist-
ant to infection, penetration of cells is dependent on the
presence, in the cell membrane, of host gene‐encoded
receptors specific for particular virus envelope subgroups
and on fusion of viral and cell membranes. The receptor
for subgroup A ALV, designated TVA, is related to the
human low‐density lipoprotein receptor (167, 244).
Decreased susceptibility subgroup A ASLV in vitro and in
vivo from intronic deletions in close‐bred line of domes-
tic chickens resulting in inefficient splicing of the tva
mRNA has been reported (63, 328). The receptors for
ALV subgroups B, D, and E, designated TVBs3 and TVBs1,
resemble a receptor for cytokines of the tumor necrosis
592 Section II  Viral Diseases

Table 15.5  Acutely transforming avian sarcoma and leukemia viruses classified according to viral oncogene.

Oncogene(s) Oncogene Predominant

Virus strain carried product neoplasm(s) Cells transformed in vitro

RSV, B77, S1, S2 src Nr ptk Sarcoma Fibroblast

FuSV, UR1, PCR II, PCR IV fps Nr ptk Sarcoma Fibroblast
Y73, ESV yes Nr ptk Sarcoma Fibroblast
UR2 ros R ptk Sarcoma Fibroblast
RPL30 eyk R ptk Sarcoma Fibroblast
ASV‐17 jun Tf Sarcoma Fibroblast
ASV‐31 qin Tf Sarcoma Fibroblast
AS42 maf Tf Sarcoma Fibroblast
ASV‐1 crk Ap Sarcoma Fibroblast
AEV‐ES4, erbA, erbB Tf, R ptk Erythroblastosis, Erythroblast, fibroblast
sarcoma
AEV‐R erbA, erbB Tf, R ptk Erythroblastosis Erythroblast
AEV‐H erbB R ptk Erythroblastosis, Erythroblast, fibroblast
sarcoma
S13 sea R ptk Erythroblastosis, Erythroblast, fibroblast
sarcoma
E26 myb, ets Tf Myeloblastosis, Myeloblast, erythroblast
erythroblastosis
AMV myb Tf Myeloblastosis Myeloblast
MC29 myc Tf Myelocytoma, Immature macrophage, fibroblast
endothelioma
CMII myc Tf Myelocytoma Immature macrophage, fibroblast
966 ALV‐J myc Tf Myelocytoma Immature macrophage
OK10 myc Tf Endothelioma Immature macrophage, fibroblast
MH2 myc, mil Tf, S/tk Endothelioma Immature macrophage, fibroblast
Note: Ap 5 Adaptor protein; Nr ptk 5 Nonreceptor protein tyrosine kinase; R ptk 5 Receptor protein tyrosine kinase; S/tk 5 Serine/threonine
kinase; and Tf 5 Transcription factor.

factor family (2–4, 205, 329). Polymorphisms within the
TVA and TVB receptors have been reported among
chicken populations (228, 450). Editing of TVB receptor
sequences in DF1 cells can induce resistance to ALV B
infections (218). The receptor for the subgroup C avian
sarcoma and leukosis viruses, Tvc, is related to mamma-
lian butyrophilins, members of the immunoglobulin
superfamily (115, 263). The host cell receptor used by the
ALV subgroup J, which has a distinct envelope with lim-
ited homology to those of other subgroups, has been
identified as the chicken Na(+)/H(+) exchanger type 1
(chNHE1) protein (59, 61, 138, 279), with the noncon-
served tryptophan 38 residue critical in discriminating
resistant and susceptible avian species (206, 327). While
Japanese quail was shown to be resistant to ALV‐J, some
of the new world quail species with tryptophan 38 residue
are susceptible and could potentially serve as reservoirs
of infection (315). Recent study also showed that genetic
resistance to ALV‐J could be engineered in DF‐1 cells by
genome editing of the receptor sequence (217).
Synthesis and Integration of Viral DNA
Detailed reviews on the synthesis and integration of viral
DNA have been provided elsewhere (71, 117, 166, 222,
383). Major stages in formation of retroviral DNA are:
(1) synthesis of the first (minus) strand of viral DNA by
reverse transcription of viral RNA by reverse tran-
scriptase, forming an RNA : DNA hybrid; (2) removal of
RNA from the hybrid by RNase‐H and formation on the
template of minus‐strand DNA of second (plus) strands
of viral DNA, giving rise to linear DNA duplexes; and (3)
migration of linear DNA to the cell nucleus. Linear viral
DNA becomes linearly integrated into the host DNA
under the influence of the enzyme integrase. This inte-
gration can occur at many sites, and infected cells can
contain up to 20 copies of viral DNA. Recent studies
have demonstrated the importance of host factors such
as the SSRP1 and Spt16 of the FACT protein complex as
a principal cellular binding partner of ALV integrase
(435). The proviral genes occur in the same order as their
RNA copies occur in the virion, and they are flanked on

either side by identical sequences of nucleotides—the
long terminal repeats (LTRs) (Figure 15.19).
Transcription
Formation of new virions in the infected cell is the result
of transcription and translation of proviral DNA involv-
ing multiple steps (reviewed in 266, 417). Viral RNA
molecules give rise to mRNA in association with polyri-
bosomes, and they also serve as genomic RNA in newly
formed virions. The mRNA species are translated to
form the gag, pol, and env gene‐coded proteins that
­compose the virion. The env gene product is a precursor
protein gPr92 (92 kDa) from which the viral envelope
proteins SU (gp85) and TM (gp37) are derived.
Translation of env is from a spliced subgenomic RNA.
The viral proteins localize at the plasma membrane of
the cell, where crescent‐shaped structures develop and
virions that bud off from the cell may be visualized.
Defectiveness and Helper Viruses
A number of avian retroviruses (Table  15.6) have been
shown to have defective genomes and arise either sponta-
neously or as a result of experimental mutagenesis (177,
238). They will transform cells but require the presence of
a helper leukosis virus to enable them to replicate (e.g.,
BH‐RSV and AMV lack the env gene, and AEV and MC29
lack the pol and env genes). Other acutely transforming
viruses, such as certain strains of RSV, have lost their
v‐onc gene and ability to transform rapidly. They are
called transformation defective (td) mutants and have an
oncogenic potential similar to that of nondefective ALVs.
Stocks of rd mutant RSV must by their existence contain
helper viruses; these originally were referred to as Rous‐
associated viruses (RAVs). Infectious RSVs formed in
these circumstances are called pseudotypes, and their
designation includes the helper virus when this is identi-
fied (e.g., BH‐RSV(RAV‐1) when RAV‐1 strain ALV
helper virus is used). Use of defective strains of RSV
­enables tailor‐made RSV to be produced with envelope
properties of the helper ALV. Determinations of host
Table 15.6  Phenotypic expression of representative endogenous
avian leukosis viral (ev) genes in normal chicken cells.
Phenotype Symbol ev locus
No detectable viral product gs2chf2 1, 4, 5
2 1
Expression of subgroup E envelope gs chf 9
antigen
Coordinate expression of group‐specific gs1chf1 3 15I5
and envelope antigens
Spontaneous production of subgroup E V‐E1 2 Figure 15.21  Endogenous viral (ev) loci detected in six inbred lines
virus
Source: Adapted from Smith (464).
Chapter 15  Neoplastic Diseases 593
range, interference pattern, and neutralization can be
performed more easily with the appropriate RSV pseudo-
type than with the ALV, because the former can be readily
quantified in cell culture.
Endogenous Leukosis Viruses
Avian leukosis viruses that are transmitted as infectious
virus particles are termed exogenous viruses. The nor-
mal chicken genome also contains several classes or fam-
ilies of avian retrovirus‐like elements that are transmitted
genetically and are termed endogenous viruses. Extensive
literature on earlier studies on these elements can be
accessed in earlier editions of this book (266). Certain
retroelements are believed to represent stages in the evo-
lution of retroviruses from cellular movable genetic ele-
ments (transposons); whereas others are thought to be
degenerate proviral forms of exogenous retroviruses that
have lost the ability to produce infectious virus due to
mutations.
The genetic sequences of the ev loci are related to sub-
group E ALVs and are present as either complete or
defective genomes in almost all normal chickens
(Figure 15.21). The chromosomal locations of a number
of ev loci have been determined using new sequence
analysis pipelines on the chicken genome (32, 342–344).
The phenotypic expressions of these loci vary, depend-
ing on the viral genes present and on poorly understood
control mechanisms (Tables 15.6 and 15.7). More recent
studies have demonstrated the role of endogenous retro-
viruses in generating genomic variations (219) and that
the PIWI‐interacting RNAs (piRNAs) can protect germ
line from targeting endogenous retroviruses (385). The
expression of endogenous ev genes is thought to be
KB EV
21 6,10
13 7
9.5 1
12
6.3 3
2
72 151 15I4 15B 63
of White Leghorns by restriction fragment polymorphisms
generated after Sac‐1 endonuclease digestion of red blood cell DNA
and hybridized to 32P‐labeled RAV‐2 genomic sequences (469).
594 Section II  Viral Diseases

responsible for a dominant form of genetic resistance of
chicken cells to infection by subgroup E ALV (ALV E)
from a block of virus receptors by envelope protein (239).
Endogenous viruses have either beneficial or detri-
mental effects, perhaps by their induction of immunity
or tolerance to tumor virus antigens, depending on when
they are expressed. Long‐term selection studies showed
that only a few of the integrations contributed to the high
ALV E expression which seemed to correlate with lower
body weights for the females indicating potential linkage
to the loci regulating growth (197). Endogenous viruses
of the ev family are not essential, because it has been pos-
sible to produce chickens free of ev genes (8). A line of
such chickens has been produced, designated line 0 (81),
that is of value in research studies and certain diagnostic
tests in which birds or cells free from ev loci are needed.
Of particular importance is the ev21 locus, which is
linked tightly in White Leghorn stock to the dominant
sex‐linked gene, K, on the Z chromosome (13), which reg-
ulates slow feathering (79). The ev21 gene is expressed as
an infectious endogenous ALV, EV21, in the dam, which is
transmitted congenitally to the progeny, inducing immu-
nological tolerance and, consequently, increased suscepti-
bility to infection by exogenous ALV (13, 175, 363, 365).
The biologic functions, if any, of the other endogenous
elements such as endogenous avian retrovirus (EAV),
ART‐CH, and CR1 are not fully understood. EAV family
are not expressed as infectious virus, but RT activity can
be expressed and has been found in live virus vaccines
(404, 428). A member of the EAV family, EAV‐HP (also
termed ev/J), is believed to be the origin of the env gene of
subgroup J ALV (17). Strongest evidence of the role of
EAV‐HP in the emergence of ALV‐J by recombination was
obtained from the identification of an intact chicken EAV‐
HP locus showing a uniquely close relationship to the
ALV‐J prototype clone HPRS‐103 env region (345, 346).

Table 15.7  Phenotypes of endogenous avian leukosis (ev) genes

Susceptibility to Chemical and 
in inbred and commercial lines of White Leghorn chickens. Physical Agents
Details of the susceptibility to chemical agents, condi-
ev Phenotype Line or Sourcea tions of thermal inactivation, pH stability and suscepti-
bility to ultraviolet radiation can be found in the same
1 gs2chf2 Most lines
chapter of the previous editions of this book (266).
1
2 V‐E RPRL72
3 gs1chf1 RPRL63
2 2
Strain Classification
4 gs chf SPAFAS
5 gs2chf2 SPAFAS Antigenicity
Avian leukosis/sarcoma viruses that occur in chickens
6 gs2chf1 RPRL151
have been divided into six envelope subgroups, A, B, C,
7 V‐E1 RPRL15B
D, E, and J, on the basis of differences in their viral enve-
2 2
8 gs chf K18 lope glycoproteins, which determine antigenicity, viral
9 gs2chf1 K18 interference patterns with members of the same and dif-
10 V‐E 1
RPRL 15I4 ferent subgroups, and host range in chicken embryo
11 V‐E1 RPRL 15I4 fibroblasts (CEF) of different phenotypes (266, 306). The
1 other subgroups, F, G, H, and I, represent endogenous
12 V‐E RPRL 151
ALVs occurring in pheasants, partridge, and quail (266).
14 V‐E1 H&N
Occurrence of new ALV subgroup K (ALV‐K) has been
15 (C) None K28 3K16 reported in China based on sequence analysis (107, 224),
16 (D) None K28 3K16 although additional criteria based on the results of
17 2
gs chf 2
RC‐P ­subgroup‐specific neutralization, interference, and host‐
18 V‐E1 RI range analysis will be required for absolute confirmation
1 b of its status as a new subgroup.
19 V‐E (?) RW
Viral interference patterns (Table 15.8) and host range
20 V‐E1(?)b RW
patterns (Tables  15.9 and 15.10) are the most reliable
1
21 V‐E Hyline FP methods for subgroup classification. Antigenicity, as
2 2
Note: Ev13 is associated with the gs chf phenotype, but restriction determined by the production of neutralizing antibodies
fragments have not been characterized. or neutralization by known subgroup‐specific antibod-
a
 Not exclusive to line or source. K, Kimber; R, Reaseheath; H & N, ies, can also be used for strain classification, but is less
Heisdorf and Nelson; for references see Smith (464). dependable. Viruses within a subgroup usually cross‐
b
 The presence of 5 ev loci in Reaseheath line w birds precludes
definitive assignment with the V‐E1 phenotype. Definitive association
neutralize to varying extents, but with the exception of
requires further segregation of ev genes. Hyline FP birds also carry partial cross‐neutralization between subgroup B and D
ev1, ev3, and ev6. viruses, viruses of different subgroups do not.
 Chapter 15  Neoplastic Diseases 595

Table 15.8  Interference patterns between avian leukosis virus Molecular Characteristics
(ALV) and Rous sarcoma virus (RSV) of subgroups A–E and J. Sequence analysis of the gp85 encoding sequences of the
env genes of ALVs of subgroups A–E have identified two
Subgroup of challenge RSV hypervariable regions, hr1 and hr2, and three less varia-
Subgroup of ble regions, vr1, vr2, and vr3, in which differences
interfering ALV A B C D E J between the subgroups are present (37, 38, 109). Studies
of recombinants indicated that hr1 and hr2, and to a
A 1 2 2 2 2 2
lesser extent vr3, play the major role in determining
B 2 1 2 1 1 2 receptor tropism (108). However, the exact locations and
C 2 2 1 2 2 2 nature of the differences that determine host range and
D 2 2 2 1 2 2 antigenicity have not yet been identified. The gp85
E 2 2 2 2 1 2 sequence of the env gene of subgroup J ALVs differ more
extensively from those of the other five subgroups, nota-
J 2 2 2 2 2 1
bly at hr1, hr2, vr2, and vr3, and to a lesser extent also
Note: Susceptible avian embryo fibroblast cultures are infected with between these regions (17, 18). Different subgroup J
ALV of each subgroup and challenged several days later with RSV of ­isolates also vary at particular hypervariable regions of
each subgroup. Reduction in RSV foci in infected cultures compared
with uninfected controls is indicative of viral interference. 1,
gp85 (227, 245).
interference; 2, no interference.
Pathogenicity
Table 15.9  Examples of host range of subgroup A–E and J avian Numerous strains of ALSVs exist, many of which were
leukosis/sarcoma viruses in chicken embryo cells of different isolated from naturally occurring or experimentally‐
phenotypes.
induced neoplasms over many years. Many induce a
predominant type of neoplasm and can be named
­
Examples Subgroup of virus accordingly; for example, lymphoid leukosis virus (LLV),
Phenotype (chicken or although ALV is more commonly used than LLV; avian
of cells cell lines) A B C D E J erythroblastosis virus (AEV); avian myeloblastosis virus
(AMV); and avian sarcoma virus (ASV) (Table  15.11).
C/0 15B1 S S S S S S
RPL12 strain of ALV induces lymphoid leukosis, eryth-
C/AE C, alv6 R S S S R S
roblastosis, osteopetrosis, hemangiomas, and sarcomas;
C/A,B,D,E 72 R R S R R S the BAI A strain of AMV induces myeloblastosis,
C/E 0, 15l, BrL S S S S R S lymphoid leukosis, osteopetrosis, nephroblastomas,
­
C/EJ DF‐1/Ja S S S S R R sarcomas, hemangiomas, thecomas, granulosa cell
­
tumors, and epitheliomas (266).
Note: S, susceptible; R, resistant. The cell phenotype designation
denotes chicken (C) cells resistant to (/) the specified subgroup (0, no
Strains of ALSV can also be placed into two major
subgroup; AE, subgroups A and E; etc.). classes in respect of rapidity of induction of tumors:
a
 Cell line, Hunt et al. (265).
1) Acutely transforming viruses. These viruses can
induce neoplastic transformation, in vivo or in vitro,
Table 15.10  Host range of different subgroups of Rous sarcoma
virus (RSV) in embryo fibroblasts of various avian species.
within a few days or weeks. They cause various types
of acute leukemia (leukosis) or solid tumors (usually
sarcomas) (211, 266). The acutely transforming
Subgroup of RSV
viruses are those that carry viral oncogenes in their
genome (Table  15.5). Details of viral oncogenes and
Avian species A B C D E J
the biochemical functions of their products have been
Red jungle fowl S S R R R S reviewed elsewhere (211, 242).
Common pheasant S R R R S R
2) Slowly transforming viruses. These ALVs do not carry
viral oncogenes. They induce tumors by a “promoter
Japanese quail S R R R S R
insertion” or a related mechanism that activates a cel-
Guinea fowl S S S S S R lular oncogene to bring about neoplastic transforma-
Turkey S S S S S S tion, with the development of tumors taking several
Peking duck R R S R R R weeks or months (211, 242).
Goose R R S R R R
Nomenclature
Note: Embryo fibroblast cultures from avian species are challenged
with RSV and susceptibility to RSV focus formation determined. S, A variety of conventions, which reflect the classification
susceptible; R, resistant. Data from Payne et al. (389). methods outlined previously, are used in designating
596 Section II  Viral Diseases

ALSVs; many of these are illustrated in Table  15.11.
They are given a full and an abbreviated designation
based on the predominant neoplasm they induce, with
an affix to indicate their origin with an individual (e.g.,
Rous sarcoma virus [RSV]) or a location (e.g., Regional
Poultry Research Laboratory isolate 12 [RPL12 of ALV).
Substrains of RSV are designated according to individu-
als who worked with them (e.g., Bryan’s high‐titer strain
[BH‐RSV]) or to location (e.g., Prague [PR‐RSV]).
Subgroups (e.g., subgroup A) may be designated also:
PR‐RSV‐A. The general terms avian leukosis (or leuke-
mia) virus (ALV) and avian sarcoma virus (ASV) are
used widely to ­designate members of the group.
Helper viruses isolated from stocks of defective viruses
are named, for example, as Rous‐associated virus (RAV)
or myeloblastosis‐associated virus (MAV), and isolates

Table 15.11  Laboratory strains of avian leukosis/sarcoma viruses of the chicken classified according to predominant neoplasm induced
and virus envelope subgroup.

Virus class according to envelope subgroup

Virus class according to No subgroup

neoplasm A B C D E J (defective virus)a

Lymphoid leukosis RAV‐1 RAV‐2 RAV‐7 RAV‐50 RAV‐60

virus (LLV)
RIF‐1 RAV‐6 RAV‐49 CZAV
MAV‐1 MAV‐2
RPL12
HPRS‐F42
Avian erythroblastosis AEV‐ES4
virus (AEV)
AEV‐R
AEV‐H
Avian myeloblastosis AMV‐BAI‐A
virus (AMV)
Avian sarcoma virus SR‐RSV‐A SR‐RSV‐B B77 SR‐RSV‐D SR‐RSV‐E BH‐RSV
(ASV)
PR‐RSV‐A PR‐RSV‐B PR‐RSV‐C CZ‐RSV PR‐RSV‐E BS‐RSV
EH‐RSV HA‐RSV FuSV
RSV29 PRCII
PRCIV
ESV
Y73
UR1
UR2
S1
S2
Myelocytoma and HPRS‐103 MC29
endothelioma
viruses ADOL‐Hc1 966
MH2
CMII
OK10
RAV‐0
Endogenous virus (EV) EV21
(no neoplasm) ILV‐E
a
 Defective viruses have the envelope subgroup of their helper virus.
 Chapter 15  Neoplastic Diseases 597

are numbered (RAV‐1, MAV‐1, etc.). Where a helper

virus is used for replication of a defective virus, this is
indicated. Thus, BH‐RSV grown with RAV‐1 as a helper
is designated BH‐RSV(RAV‐1). Endogenous ALV is
abbreviated EV (e.g., EV21). Strains of ALV that act as
resistance‐inducing factors (see Diagnosis) were desig-
nated RIFs, but this term is now rarely used. Further
details of the origins of the abbreviations (297) are given
in Table 15.11.

Laboratory Host Systems

Chick Inoculation
Rous sarcoma and other sarcoma viruses produce tumors
when injected by the subcutaneous (SC), intramuscular
(IM), or intra‐abdominal (IA) routes and at times by con-
tact with inoculated chickens. Subcutaneous injection
into the wing web or IM injection can be used for sar-
coma virus isolation and propagation (Reviewed in 266).
In susceptible chickens, these may grow rapidly, ulcerate,
and metastasize; in resistant chickens, the sarcomas may
regress. Virus isolates that caused LL also caused eryth-
roblastosis. Osteopetrosis, hemangiomas, and fibrosar-
comas were also observed in chickens of certain strains
and passages (see 266).
Avian myeloblastosis virus can be titrated in suscepti-
ble chicks by IV inoculation at 1–3 days of age. Avian
myeloblastosis virus in chicken plasma can be assayed by Figure 15.22  Pocks induced by BH‐RSV on the chorioallantoic
its adenosine triphosphatase activity, a method useful for membrane (CAM) of a chicken embryo. (Piraino).
routine and large‐scale studies. Osteopetrosis‐inducing
activity of virus strains can be examined by IV or IM
inoculation of day‐old chicks. Guinea fowl are particu- susceptible embryos (20–22). When the HPRS‐103
larly susceptible to osteopetrosis induced by MAV‐2(O) strain of subgroup J ALV was inoculated by IV into 11‐
virus (see 266). day‐old embryos, first death from tumor (myelocytoma)
was not until 9 weeks of age, and median tumor mortality
Embryo Inoculation was at 20 weeks (301).
When RSV and other sarcoma viruses are inoculated
onto the chorioallantoic membrane (CAM) of 11‐day‐ Cell Culture
old susceptible embryos, tumor pocks develop Rous sarcoma virus and other sarcoma viruses induce
(Figure 15.22), which can be counted eight days later and rapid neoplastic transformation of cells when inoculated
are related linearly to virus dose (112). This technique is onto monolayer cultures of chicken embryo fibroblasts
also useful for detecting genetic resistance to infection. (323). The transformed cells proliferate to produce
Avian leukosis viruses have been quantitated by IV within a few days discrete colonies or foci of transformed
inoculation into 11‐day‐old susceptible chicken embryos. cells (Figure  15.23), which under agar can be used for
Depending on the virus, within two weeks of hatching, a quantitative assay of virus (400).
high incidence of neoplasms (mainly erythroblastosis) Most leukosis viruses replicate in fibroblast cultures
can occur, although hemorrhages and solid tumors can without producing any obvious cytopathic effect. Their
develop including fibrosarcomas, endotheliomas, presence can be detected by a variety of tests (see
nephroblastomas, and chondromas. When chicks are Diagnosis). Avian leukosis viruses of subgroups B and D
held for a postinoculation period of 46 days, responses may induce cytopathic plaques that may be used for virus
are higher by 1–2 log10 dilutions than those following assay (163). The cytopathic effect of these two subgroups
chicken inoculation. Most chickens that survive the is explained by their use of the death receptor of the
acute neoplasms develop LL after 100 days PI (312). tumor necrosis factor receptor family (41, 69, 100, 101).
Avian myeloblastosis virus produced a myeloblastosis Acutely transforming ALVs will transform hematopoi-
response within a few weeks when injected by IV into etic cells in vitro (257). Yolk sac and bone marrow cells in
598 Section II  Viral Diseases

(A) (B) (C)

Figure 15.23  Foci induced by Rous sarcoma virus (RSV) in cell culture. (A) Unstained focus of transformed spherical, refractile chicken
embryo cells infected six days previously with Bryan’s standard strain of RSV. ×100. (B) Unstained focus of transformed, polygonal, opaque
Rous sarcoma cells infected six days previously with Bryan’s high‐titer strain. ×100. (C) Unstained focus of transformed round and fusiform
cells infected six days previously with Popken’s preparation of RSV. ×100.

culture are transformed to neoplastic myeloblasts on
infection with AMV (258), and bone marrow cells trans-
form to erythroblasts with AEV (164). Transformation of
hematopoietic cells by MH2, MV29, and OK10 viruses
was observed by Graf and Beug (165). Acutely trans-
forming variants of subgroup J ALV can also transform
bone marrow cells and blood monocytes in vitro (66,
302). In vitro transformation of B‐lymphocytes by non-
defective ALV has not been reported, and bone marrow
cultures were not transformed by nondefective subgroup
J ALV (302).
The properties of ALSVs in cell culture are described
in more detail under Diagnosis.
Pathogenicity
As discussed previously (see Strain Classification),
strains of ALVs may produce more than one type of neo-
plasm, and the oncogenic spectrum of each strain tends
to be characteristic but often overlaps with responses to
other strains. Viral factors including the origin and dose,
and host factors such as route of inoculation, age, geno-
type, and sex influence the oncogenic patterns of differ-
ent virus strains.
Origin of Virus
Differences in tumor spectrum may be seen in virus
strains newly isolated from the field, as exemplified by
tumor spectrums of RPL26, RPL27, and RPL28 isolates
of ALV (143). Please refer to the chapter in the previous
edition for earlier studies in this area (266).
Virus Subgroup
Usually no relationship has been observed between virus
subgroup and oncogenicity except for endogenous E
subgroup ALVs, such as RAV‐0, which have little or no
oncogenicity (261). However, the low oncogenicity of
RAV‐0 is believed to be related to the weak promoter
activity of the subgroup E LTR and not to the env gene.
Subgroup J ALV‐induced myelocytomatosis (301), and
the env as well as other elements are thought to be asso-
ciated with the unique oncogenicity (66–68, 234, 235).
Virus Dose
High doses of RPL12 ALV mainly induced erythroblasto-
sis; whereas doses close to the endpoint predominantly
induced LL (50). Sarcomas, endotheliomas, and hemor-
rhages were also more common with high virus doses.
Occurrence of osteopetrosis showed no dependency on
dose (144).
Route of Inoculation
Responses obtained after virus administration by less
efficient portals of entry into the host apparently reflect
the decreased effective dose. Thus, exposure of suscepti-
ble birds by contact with birds inoculated with a high

dose of strain RPL12 of ALV resulted in a LL response
similar to that expected with 1/1000 of the inoculated
dose (see 266). Intramuscular inoculation of strain
RPL26 of ALV favored sarcoma induction; whereas IV
inoculation mainly produced erythroblastosis and hem-
orrhages (see 266). These differences may reflect varia-
tions in amounts of virus that reach the target cells by
different routes.
Age of Host
In general, resistance of birds to the development of neo-
plasms of all types increases with age, the rate varying
with route of inoculation. Resistance increases rapidly
between 1 and 21 days of age with oral or nasal adminis-
tration but relatively slowly when virus is inoculated
intravenously (48).
Genotype and Sex of Host
The genetic constitution of the host has a strong influ-
ence on response to ALSVs (see Pathobiology and
Epizootiology). Females are more susceptible to LL than
males. Castration increases the incidence of disease, and
treatment with testosterone increases resistance of males
and capons (51). These effects are probably a conse-
quence of hormonal effects influencing regression and,
hence, target cell numbers in the bursa of Fabricius.
Pathobiology and Epidemiology
Incidence and Distribution
Notwithstanding the eradication programs instituted by
many primary breeding companies, ALV infections still
occur in many flocks. The incidence of subgroup A ALV‐
induced LL, the most common neoplasm observed in
infected flocks, is usually low, in the order of 1 or 2%,
although losses of up to 20% can occur. Diseases associ-
ated with subgroup J continue to be a problem in many
parts of China (306).
Incidence of Disease
Earlier reports on the incidence of ALV‐associated dis-
eases can be seen in this chapter in the previous edition
of this book (266). Although sporadic cases of ALV‐
induced neoplasia occur in most flocks, it is only occa-
sionally that even the most common neoplasm, LL,
produces heavy losses. Lymphoid leukosis mortality in
the Netherlands during 1973–1979 was 2.18% of 11,220
white layers and 0.57% of 7,920 brown layers (96, 97),
although the incidence has been reduced significantly
since these studies. Serotype 2 MDV was found to
enhance the development of LL in certain lines of chick-
ens following exposure to ALV after hatch (16, 124, 128,
Chapter 15  Neoplastic Diseases 599
133). Molecular and in situ hybridization analysis of the
bursa from chickens coinfected with ALV and serotype
2  MDV proved that MDV was closely associated with
transformed, but not with nontransformed, bursa cells
(148, 240).
Until recently and before the recognition of subgroup J
ALV (299, 308), myelocytomatosis was mainly a sporadic
disease seen among young and adult birds (321). An over-
all incidence of 27% myelocytomatosis was reported in
meat‐type chickens inoculated with strain HPRS‐103 of
ALV‐J (301). High incidence of ALV‐J tumors have been
reported in many countries with mortalities up to 1.5% in
excess of normal levels per week in some commercial
broiler breeder flocks (306). Avian leukosis virus‐J‐
induced disease in China is characterized by the occur-
rence of the disease in meat‐type as well as layer population
including the native breeds (229, 246) and is characterized
by a high incidence of hemangiomas (215, 451).
Connective tissue tumors, which are often not the
­primary cause of death, make up about 20% of nonlym-
phoid tumors in broilers (57). The incidence of
connective tissue tumors in chickens is probably less
than 1 in 1,000 (321), but epizootics have occurred. An
outbreak of histiocytic sarcomas was reported in a flock
of 600 1‐year‐old hens, during which tumors were found
in 90% of 400 birds examined during a 4‐month period
(310). Low incidence of histiocytic sarcomatosis associ-
ated with ALV‐J has also been reported (5).
Osteopetrosis occurs much less frequently than LL,
and epizootics occur sporadically in broilers. In all types
of chicken, males are more frequently affected than
females. Examination of a 1986 outbreak of osteopetrosis
revealed ALV sequences (23).
Incidence of Virus Infection
Subgroup A ALV is the most common subgroup of L/S
viruses isolated from field outbreaks of LL; it is encoun-
tered more frequently than subgroup B. In general, fewer
studies of the prevalence of ALV in meat lines have been
made compared with those in egg lines. Antibodies to the
novel subgroup J ALV were found in three of five meat‐
type chicken lines, but not in seven layer lines examined in
the United Kingdom (305). Using virological and serologi-
cal assays, the incidence of ALV‐J infection in affected
broiler breeder flocks was reported to be as high as 87%
(132). Similarly high levels of ALV‐J infection have been
reported in other countries also (139, 226, 229, 246). The
incidence of infection with subgroup ALV‐J was also influ-
enced by other factors such as age at exposure (433, 434).
Subgroups A, B, C, and D of ALV have been isolated
from commercial flocks in Finland; 5 of 10 flocks
­surveyed had antibody to all 4 subgroups (348).
Antibodies to subgroups A and B are common among
wildfowl and domestic chickens in Kenya and Malaysia,
and some evidence exists of antibody to subgroup
600 Section II  Viral Diseases
D viruses in Kenya. Subgroup F viruses have been found
in ring‐necked and green pheasants, and subgroup
G  viruses in Ghinghi, silver, and golden pheasants.
Subgroup H virus has been isolated from Hungarian
­partridges and subgroup I virus from Gambel’s quail (see
Virus Subgroups). Viruses that do not fit within known
subgroups have been isolated from Mongolian and
Swinhoe pheasants, Chinese quail, and chickens.
However, none were found in Japanese quail, pigeons,
geese, and Pekin and Muscovy ducks (64, 315).
In most vertebrate species, endogenous retroviral
genomes are inherited in a Mendelian fashion and occur
at distinct chromosomal loci. DNA sequences related to
RAV‐0, the endogenous avian retrovirus, occur in the
germ lines of most domestic chickens and several species
of galliform birds. For example, partridges, true pheas-
ants, grouse, and jungle fowl contain sequences comple-
mentary to RAV‐0; whereas guinea fowl, quail, peafowl,
ruffed pheasants, gallo‐pheasants, and turkeys do not
(145). The structure, function, and regulation of endog-
enous retroviruses in the genome of the chicken have
been reviewed (79, 219, 344, 385).
Natural and Experimental Hosts
Chickens are the natural hosts for all viruses of L/S group
(296); these viruses have not been isolated from other
avian species except pheasants, partridges, and quail (see
Virus Subgroups). Experimentally, however, some mem-
bers of the L/S group of avian retroviruses have a wide
host range and can be adapted to grow in unusual hosts by
passage in very young animals or through the induction of
immunologic tolerance prior to inoculation of the virus.
Rous sarcoma virus has the widest host range; it will cause
tumors in chickens, pheasants, guinea fowl, ducks,
pigeons, Japanese quail, turkeys, and rock partridges.
Ducks were shown to be an ideal experimental system for
studying persistence of ALV as the virus appeared to
­persist even up to three years with no viremia or neutral-
izing antibodies after embryonic infection (272). However
ducks infected as embryos with subgroup C showed wast-
ing disease soon after hatching (382, 392, 403). There has
been one report of lymphoid leukosis in ostriches (150).
Some strains of RSV induce tumors in mammals (414),
including monkeys (207–210). Osteopetrosis can be
­produced in turkeys by inoculation of fresh whole blood
from affected chickens (181). Also, turkeys were suscepti-
ble to ALV‐J infection and tumors induced by the acute
form of strain HPRS‐103 of ALV‐J (412).
Transmission
Exogenous ALVs are transmitted in two ways: vertically
from hen to progeny through the egg and horizontally from
bird to bird by direct or indirect contact (Figure  15.24)
(reviewed by 266, 306). Although usually only a small
­percentage of chicks are infected vertically, this route of
transmission is important epizootiologically because it
affords a means of maintaining the infection from one gen-
eration to the next. Most chickens become infected by close
contact with congenitally infected birds. Although vertical
transmission is important in the maintenance of the infec-
tion, horizontal infection may also be necessary to maintain
a rate of vertical transmission sufficient to prevent the
infection from dying out (300). The infection does not
spread readily from infected birds to birds in indirect con-
tact (in separate pens or cages), probably because of the
relatively short life of the virus outside the birds (see
Thermal Inactivation). However, contact exposure at hatch
was shown to be an effective method of spread of ALV‐J
among broiler breeder chickens (132, 436, 437) and was
prevented by small group rearing (440).
Four classes of ALV infection are recognized in mature
chickens: (1) no viremia, no antibody (V−A−‐); (2) no
viremia, with antibody (V−A+); (3) with viremia, with
antibody (V+A+); and (4) with viremia, no antibody
(V+A−) (reviewed by 266, 306). Birds in an infection‐free
flock and genetically resistant birds in a susceptible flock
fall into the category V−A−‐. Genetically susceptible
birds in an infected flock fall into one of the other three
categories. Most are V−A+, and a minority, usually less
than 10%, are V+A−. Most V+A− hens transmit ALV to a
varying but relatively high proportion of their progeny
(266, 306). A small proportion of V−A+ hens transmit
the virus congenitally and do so more intermittently; the
tendency for congenital transmission of ALV in this cat-
egory was found to be more frequent in hens with low
antibody titer (406). Congenitally infected embryos
develop immunologic tolerance to the virus and after
hatching make up the V+A− class, with high levels of
virus in the blood and tissues and an absence of antibod-
ies. By 22 weeks of age, up to 25% of meat‐type chickens
exposed to ALV‐J at hatch were found to be V+A−,
although this could be affected by a number of factors
including the virus strain (288, 291).
The role of males in the transmission of ALV is at best
equivocal. Infection of the cock apparently does not influ-
ence the rate of congenital infection of progeny (339, 376).
The genetics of the host and the strain of ALV influence
shedding and congenital transmission after horizontal
infection (90). With electron microscopy, virus budding
has been seen on all structures of reproductive organs of
cocks except germinal cells (99), indicating that the virus
does not multiply in germ cells. The cock, therefore, acts
only as a virus carrier and source of contact of venereal
infection to other birds or through semen (226, 366).
Congenital infection of embryos is strongly associated
with shedding by the hen of ALV into egg albumen and
with presence of virus in the vagina of hens (304, 375).
These traits are also highly correlated with viremia.
 Chapter 15  Neoplastic Diseases 601

Shedding of ALV into egg albumen and transmission
to the embryo is a consequence of virus production by
albumen‐secreting glands of the oviduct. In most hens
congenitally transmitting ALV, the highest titers of
virus were found in the ampulla of the oviducts, sug-
gesting that embryo infection is closely related with
ALV produced at the oviduct but not with ALV trans-
ferred from other parts of the body (405). Electron
microscopy studies have revealed a high degree of virus
replication in the magnum of the oviduct (102). Virus
budding also occurs in various cell types in the ovary
but not in the follicular cells or ovum, and transovarial
infection does not seem to be important (304). Not all
eggs that have ALV in the albumen give rise to infected
embryos or chicks; in the studies of Spencer et al. (375),
Payne et  al. (304), and Tsukamoto et  al. (406), only
about one‐half to one‐eighth of embryos were infected
from eggs with virus in the albumen. This intermittent
congenital transmission may be a consequence of neu-
tralization of virus by antibody in the yolk and of loss
because of thermal inactivation.
In flocks infected with subgroup A ALV, only a
minority of ALV‐infected birds develop LL; the others
remain as carriers and shedders. Viremic‐tolerant
(V+A−) birds are reported to be several times more
likely to die of LL than those with antibody (V−A+)
(339). Incidence of leukosis decreases rapidly if infec-
tion by natural routes occurs after the first few weeks
of age (48); there are well‐established genetic differ-
ences in susceptibility to LL development in chickens
that are equally susceptible to virus infection (88).
Endogenous ALVs (see Etiology) usually are transmit-
ted genetically in germ cells of both sexes (Figure 15.24).
Many are genetically defective and incapable of giving
rise to infectious virions, but some are not and may be
expressed in an infectious form in either embryos or
hatched birds. In this form, they then are transmitted
similarly to exogenous viruses, although most chickens
are genetically resistant to such exogenous infection.
Endogenous viruses have little or no oncogenicity
but  may influence response of the bird to infection
by  ­exogenous ALV. Immunodepression induced by

HORIZONTAL

Infectious virus RNA

genome subgroups A & B
or Transient viremia immunity
leukemia rare

VERTICAL

Congenital Genetic
infection transmission

or

Infectious virus RNA Viral DNA genome integrated

genome subgroups A & B in gamete DNA subgroup E

Chronic viremia tolerance Usually latent no viremia

leukemia common no leukemia

Figure 15.24  Horizontal and vertical transmission of exogenous avian leukosis virus (ALV) (e.g., subgroups A and B) and genetic
transmission of endogenous (subgroup E) virus with potential outcomes (115).
602 Section II  Viral Diseases
i­nfectious bursal disease virus increased the rate of
­shedding of ALV (135); also strain of virus can influence
the incidence of tolerantly infected chickens (127).
Incubation Period
Members of the L/S group of viruses are multipotent
viruses capable of inducing a variety of neoplastic dis-
eases. The incubation period for these diseases is
dependent on strain and dose of virus, route of infection,
age at exposure, and genetic constitution of the host.
Susceptible chicks inoculated as embryos or at 1–14‐
days‐of‐age with a standard strain of ALV‐RPL12 (50),
B15, F42 (34), or RAV‐1, developed LL between weeks 14
and 30 weeks of age. It is very seldom that LL cases occur
in chickens under 14 weeks. Certain laboratory recombi-
nant viruses have been shown to cause LL within 5–7
weeks (198), although such short incubation periods are
not found in field outbreaks.
Another determining factor is whether the virus
strain lacks or possesses a viral oncogene. For exam-
ple, diseases such as erythroblastosis, induced by
slowly transforming viruses lacking a viral oncogene,
usually develop after a long latent period (67, 146,
211, 422), as in such cases transformation is induced
by promoter insertion activation of the cellular onco-
gene c‐erbB. After IA inoculation of the slowly trans-
forming RPL12 strain virus into susceptible day‐old
chicks, the incubation period varies from 21–110
days (50). On IV inoculation of 11‐day‐old embryos,
chicks occasionally have been found to have erythro-
blastosis on hatching. Field strains and viruses pas-
saged in cell culture induce erythroblastosis after a
longer incubation period (49). Passage from donors
with erythroblastosis greatly shortens the incubation
period (143).
Other strains of virus including F42 (34), ES4, and
strain 13 (28) also produce erythroblastosis. Field cases
usually occur in birds older than three months of age.
Viruses such as RPL12 and F42 are non‐defective and
slowly transforming; whereas ES4 and R are defective
and acutely transforming (165).
Strain BAI‐A of ALV predominantly induces myelo-
blastosis. Virus stocks are defective and contain helper
viruses of both A and B subgroups (191). After inocula-
tion of susceptible day‐old chicks with large doses of
virus, changes in the blood can be observed in 10 days,
and birds die a few days thereafter. Mortality continues
for about one month, and only a few deaths occur after
this (53, 114). The virus E26 (165) also predominantly
induces myeloblastosis.
Virus‐induced myelocytomatosis generally has a
longer incubation period than erythroblastosis and
myeloblastosis induced by the acutely transforming
virus strains, but shorter than LL. On IV injection of
MC29 into young chicks, myelocytomas were obtained
in 3–11 weeks (253). The incubation period in field
cases is unknown, but most cases are observed in
immature birds. The virus CMII also induces myelocy-
tomas (165). Myelocytomatosis induced by the
HPRS‐103 strain of ALV, which lacks a viral oncogene,
had a long latent period (median time to death was 20
weeks) (301). However, median time to death with the
acutely transforming 879‐strain variant of HPRS‐103,
believed to carry a viral oncogene, was nine weeks
(302). Field cases of subgroup J‐induced myelocytoma-
tosis were reported in broiler breeder chickens as young
as four weeks of age (132).
Most strains of ALV have been found to cause heman-
giomas (45, 144). These tumors can be found in birds of
various ages. In naturally occurring outbreaks, most
mortality from hemangiosarcomas occurred at 6–9
months (54, 55). Induction of lung angiosarcomas by
subgroup F ALVs is reported (362). After experimental
inoculation of young chicks with field strains of virus
(143), hemangiomas appeared in three weeks to four
months.
In field cases, ALV‐induced renal tumors are rarely
seen in chickens younger than five weeks; most cases are
seen in birds between two and six months of age.
Nephroblastomas induced by strain BAI‐A may reach an
incidence of 60–85% in birds not dying of myeloblastosis
(53). Renal carcinomatous lesions induced by strain
MC29 are found as soon as 18 days or as late as 7 weeks
after virus inoculation.
Osteopetrosis may develop any time after one month
following experimental inoculation of day‐old chicks
with strain RPL12‐L29 of ALV (349) or other viruses
(179, 181, 320); it is most commonly seen in birds 8–12
weeks of age. The disease probably has a similar incu-
bation period in the field. MAV‐2(O) virus will induce
palpable osteopetrosis 7–10 days after hatching in
chicks inoculated at 1 day of age or as 11–12‐day‐old
embryos (142).
Sarcomas may occur any time after inoculation with
ALVs but are most frequently observed in the first 2–3
months (52). In field flocks, connective tissue tumors
may occur in birds at any age (448). Sarcomas also
develop readily and are palpable within three days after
inoculation of chicks with high doses of acutely trans-
forming RSV.
Clinical Signs
Outward signs of the leukotic diseases are mostly non-
specific. They include inappetence, weakness, diarrhea,
dehydration, and emaciation. In LL especially, there may
be abdominal enlargement. The comb may be pale,
shriveled, or occasionally cyanotic. In erythroblastosis
and myeloblastosis, hemorrhage from feather follicles

also may occur. After clinical signs develop, the course is
usually rapid, and birds die within a few weeks. Other
affected birds may die without showing obvious signs.
In myelocytomatosis, skeletal myelocytomas may
cause protuberances on the head, thorax, and shanks.
Myelocytomas may occur in the orbit of the eye, causing
hemorrhage and blindness. Hemangiomas may occur in
the skin, appearing as “blood blisters,” and these may
rupture causing hemorrhage (136, 196). Renal tumors
may cause paralysis due to pressure on the sciatic nerve.
Sarcomas and other connective tissue tumors may be
seen in the skin and musculature. When advanced, these
various other tumors may be accompanied by the non-
specific signs given previously. Benign tumors may
­follow a long course, malignant tumors a rapid one.
In osteopetrosis, the long bones of the limbs are com-
monly affected. Uniform or irregular thickening of the
diaphyseal or metaphyseal regions can be detected by
inspection or palpation. The affected areas are often
unusually warm. Birds with advanced lesions have char-
acteristic “bootlike” shanks. Affected birds usually are
stunted and pale and walk with a stilted gait or limp.
Avian leukosis virus also has been shown to be associ-
ated with the “so called fowl glioma” (281), associated
with cerebellar hypoplasia and myocarditis (269, 270).
Pathology
Introduction
One or more specific neoplasms induced by ALSVs may
occur in a given flock of chickens, and more than one
type of neoplasm may occur in an individual bird. This is
particularly true in flocks infected with subgroup J ALV.
The presence of a tumor similar to that produced experi-
mentally is only provisional evidence that a bird was
infected with a virus of this group. Firmer evidence is
provided by ALSV detection or isolation, and if appro-
priate by experimental reproduction of tumors by a virus
isolate.
In this section, the pathology of the different neoplasms
is discussed without regard for virological properties of
the inducing agent(s). Only entities that have been repro-
duced with ALSVs are described.
Nonneoplastic Conditions
The clinical consequences of infection of chickens with
exogenous ALV vary. Some chickens, principally those
with a tolerant viremic infection (arising from congenital
or early neonatal infection, see Immunity), may show a
variety of clinical signs, as detailed later in this chapter,
including depression of body weight and of other pro-
duction traits. Birds with tolerant viremic infections are
also those most likely to develop neoplasia. Ultrastructural
and virological studies of congenitally or neonatally
infected birds have shown the virus to be widespread in
Chapter 15  Neoplastic Diseases 603
most tissues and organs of the body (6, 7, 104, 111, 223,
333, 381, 402). DiStefano and Dougherty (103, 111)
observed virus budding in cells of every type of tissue
examined, except germ cells and neurons.
Avian leukosis virus infection in the absence of overt
disease can adversely affect the productivity of egg‐lay-
ing chickens. Compared with nonshedders, hens that
shed virus produced 20–35 fewer eggs per hen housed to
497 days of age; matured later sexually (i.e., age at first
egg); and produced smaller eggs (374), at a lower rate,
and with thinner shells. Mortality from causes other than
neoplasms was 5–15% higher, fertility was 2.4% lower,
and hatchability was 12.4% lower in shedders than non-
shedders (154). In this study, shedding refers to the
transfer of ALV to egg albumen and nonshedders, as well
as shedders, would likely be ALV‐infected; shedders are
mostly viremic birds; whereas nonshedders are immune
with ALV antibody. Avian leukosis virus infection has
similar effects on broiler breeders and causes a consist-
ent, although often small, reduction in broiler growth
rate (87, 153). However, these effects are more marked in
meat‐type chickens infected with subgroup J ALV (377,
378, 380, 381). In broiler breeder flocks affected by mye-
loid leukosis caused by subgroup J ALV, smaller eggs
were associated with the presence of gs antigen in allan-
toic fluid and virus in the embryo (374). The most criti-
cal time for the immunosuppressive effects of ALV‐J
infection in experimental studies was the 3–4 week
period after infection (420). Other studies on reduced
productivity in chickens with ALV infections and the
genetic consequences have been reviewed (152, 189,
373). The presence of ALV in semen was not associated
with reduced semen production, but some evidence sug-
gested an effect on semen quality and fertility (357). The
physiological bases for these effects have not been
studied.
A number of other nonneoplastic effects of ALV infection
have been observed, mostly in experimental infections.
Chickens, turkeys, and jungle fowl exposed when
young to certain ALVs (RAV‐1, RAV‐60, MAV‐2(O)),
and ALVs of subgroup B and D develop anemia, hepati-
tis, immunodepression, and wasting; some may die (82,
371). A myocarditis and chronic circulatory syndrome
was reported in chickens inoculated with RAV‐1 ALV
(158). Intracytoplasmic viral matrix inclusion bodies
have been observed in the myocardium of adult ALV
infected chickens (156, 267). Chickens inoculated with
RAV‐7 develop neurologic signs including ataxia, leth-
argy, and imbalance resulting from a nonsuppurative
meningoencephalomyelitis (429). A persistent infection
of the central nervous system (CNS), with inflamma-
tory lesions and clinical signs, followed in ovo infection
with RAV‐1 (120). Fowl glioma‐associated virus also
shows involvement of brain with cerebellar hypoplasia
(270, 402).
604 Section II  Viral Diseases

Figure 15.25  Nodular lesions in liver and spleen of bird with lymphoma leukosis (LL) inoculated at one day of age with RPL12 virus. Bursa
also has a small tumor.

In MAV‐2(O) infection, anemia occurs due to an aplas-

tic crisis in the bone marrow in which erythrocytes fail to
incorporate iron into hemoglobin and exhibit a decreased
survival time (93). Administration of antiviral antibody
will prevent anemia (320). The immunodepression may
involve atrophy or aplasia of lymphoid organs, hypergam-
maglobulinemia, decreased mitogen‐induced blastogen-
esis, and decreased antibody response (371). The changes
in the immune system are likely a result of cessation of
B‐cell maturation and a block in the development of T‐
suppressor cells, possibly due to interference with the
synthesis of functional interleukin‐2 (179, 214).
In addition to stunting and atrophy of the lymphoid
organs, RAV‐7 caused obesity, high triglyceride and
­cholesterol levels, reduced thyroxine levels (hypothy-
roidism), and increased insulin levels. The frequent
occurrence of stunting may relate to the virus’s suppres-
sion of thyroid function. Stunting of chicks with congen-
ital subgroup J ALV infection was also associated with
hypothyroidism, possibly mediated via effects on the
pituitary (44) or other effects (378).

Lymphoid Leukosis
Gross.  Fully developed LL occurs in chickens of about
four months of age and older. Grossly visible tumors
almost invariably involve the liver (Figures  15.25 and
15.30A), spleen, and bursa of Fabricius (Figures  15.26
and 15.30G). Other organs often grossly involved include
kidney, lung, gonad, heart, bone marrow, and mesentery.
Tumors are soft, smooth, and glistening; a cut surface Figure 15.26  Large tumor of bursa of Fabricius (B) and kidneys
appears grayish to creamy white and seldom has areas of (arrow) in a naturally occurring case of lymphoid leukosis (LL) in
necrosis. Tumor growth may be nodular (Figure 15.25), the adult hen.

miliary, diffuse (Figure  15.30A), or a combination of
these forms. In the nodular form, the lymphoid tumors
vary from 0.5–5 cm in diameter and may occur singly or
in large numbers. They are usually spherical but may be
flattened when they are close to the surface of an organ.
The miliary form, which is most obvious in the liver,
­consists of numerous small nodules less than 2 mm in
diameter uniformly distributed throughout the paren-
chyma. In the diffuse form, the organ is uniformly
enlarged, slightly grayish in color, and usually very
­friable. Occasionally, the liver is firm, fibrous, and almost
gritty.
Microscopic.  All tumors are focal and multicentric in
origin. Even in organs appearing diffusely involved when
examined grossly, the microscopic pattern is one of
coalescing foci. As tumor cells proliferate, they displace
and compress cells of the organ rather than infiltrate
between them (Figure 15.27). Nodules in the liver usually
are surrounded by a band of fibroblast‐like cells that have
Figure 15.27  Liver tumor from a 20‐week‐old chicken inoculated
at one day of age with RAV‐1 ALV. Note displacement and
compression of hepatic parenchyma. ×700 (179).
Chapter 15  Neoplastic Diseases 605
been shown to be remnants of sinusoidal endothelial
cells (168). In the bursa, a follicular pattern of tumor
growth usually can be seen.
Tumors consist of aggregates of large lymphoid cells
(lymphoblasts) that may vary slightly in size but are all at
the same early developmental stage. They have a poorly
defined cytoplasmic membrane, much basophilic cyto-
plasm, and a vesicular nucleus in which there are mar-
gination and clumping of the chromatin and one or more
conspicuous acidophilic nucleoli (297).
The cytoplasm of most tumor cells contains a large
amount of RNA, which stains red with methyl green
pyronin, indicating that the cells are immature and rap-
idly dividing see (266). Characteristic features of the cell
can best be seen in wet‐fixed impression smears that
have been stained with May–Grunwald–Giemsa, methyl
green pyronin, or other cytological stains. The tumor
cells have B‐cell antigen markers and produce and carry
IgM on their surface (77, 307).
Ultrastructural.  Vacuoles are found infrequently in
lymphoid cells of birds with LL, but some virus particles
have been observed budding from the plasma membranes
of lymphoblasts (for details of references to this work, see
266). Inclusion bodies in enteric smooth muscle and
virions resembling avian retroviruses in the adjacent
intercellular spaces of a bird positive for ALV‐J PCR (171).
Pathogenesis.  Avian leukosis viruses multiply in most
tissues and organs of the body (111). Transitory lymphoid
foci may occur in various tissues and are considered to be
inflammatory in nature (Figure  15.28). The infection
persists longer in bursal lymphocytes than in other
hematopoietic tissues (9, 10), and cells of the bursa of
Fabricius are the target cells that neoplastically transform.
The target cells must be resident in the bursa, because
surgical bursectomy up to five months of age and other
treatments that destroy the bursa of Fabricius will eliminate
the disease. (For details of references, please refer to 266).
Medullary macrophages appear to be the principal bursal
cells for virus replication and may be important in
transmitting infection to the lymphoid cells (157). At a
variable time after infection, which can be as short as four
weeks in experimental studies, a proliferation of
lymphoblasts occurs in one or more lymphoid follicles in
the bursa. These altered bursal follicles are termed
transformed follicles (9, 275, 321), and the change is
regarded as a focal preneoplastic hyperplasia (182, 183)
(Figure 15.29). The transformed follicle is a consequence of
activation of the c‐myc gene by nearby insertion of ALV.
This places the c‐myc gene under the control of the
enhancers of the viral LTR, resulting in over‐expression of
myc, causing a maturation arrest and proliferation of bursal
stem cells, associated with changes in global gene expression
profiles and genomic instability (276). Arrest of maturation
606 Section II  Viral Diseases
Figure 15.28  Lesions in young chickens induced by leukosis virus.
Heart from a 4‐week‐old RIF3‐infected chick showing diffuse
accumu­lations of lymphoid cells among myocardial fibers ×430.
(Calnek).
Figure 15.29  Lymphoblastic transformation in single bursal
follicle in chicken with lymphoma leukosis (LL). All surrounding
follicles are histologically normal in this and other sections from a
16‐day‐old chicken infected with RPL12 virus at hatching. Methyl
green pyronin, ×40. (Dent).
of the transformed B cells results in interference of the
normal intraclonal switch of immunoglobulin production
from IgM to IgG, hence the surface IgM that characterizes
LL cells. The cells grow within the confines of the bursal
follicle and are not neoplastic. Sometimes, many follicles
are transformed, but the majority of these appear to regress,
and only a few continue to grow to give rise to nodular
tumors in the bursa, which are visible grossly from about 14
weeks of age (76, 275). Progression of the transformed
follicle to the fully neoplastic state requires additional
genetic changes, and other putative oncogenes, Blym‐1
(162, 273) Mtd/Bok (43) and c‐bic (72, 395), have been
implicated. Studies have suggested that the oncogenicity
associated with the non‐coding c‐bic transcript is due to a
novel microRNA designated miR‐155 (396). Oncogenicity
assays demonstrated that bic can cooperate with c‐myc in
lymphomagenesis and erythroleukemogenesis, providing
direct evidence for the involvement of untranslated RNAs
in oncogenesis (397). Genome‐wide analysis of palindrome
formation has shown that myc‐induced genomic instability
from palindrome formation in many sites including the
c‐bic/miR‐155 locus is a major factor triggering the bursal
lymphoma (274). Analysis of the proviral integration sites in
B‐cell lymphomas showed that the telomerase reverse
transcriptase (TERT) promoter/enhancer region was a
common integration site, suggesting that upregulation of
cellular TERT by insertional activation is a factor initiating/
enhancing B‐cell lymphomas (445). Recent studies have
also identified other integration sites that contribute to the
development of tumors (195, 196, 309). Evidence also
suggests that apoptosis of neoplastic bursal cells is inhibited
by an antagonist of apoptotic cell death, NR‐13, related to
the Bcl‐2 proto‐oncogene (221, 280). Induction of
angiogenic factors from the transformed cells also
contributes to the generation of myc‐induced lymphomas
(40). From about 12 weeks of age, cells in the clonal bursal
tumors metastasize to other organs and tissues and result in
the terminal disease. Metastatic tumors in the viscera
usually have the same DNA fragments as bursal tumors
from the same birds, supporting their clonal origin (83), but
multiple bursal tumors can give rise to polyclonal metastatic
disease (368).
Experimentally, B‐cell lymphomas also have been
induced by c‐myb activation, following embryonic infec-
tion with ALV (198, 313). The tumors were unusual in
that metastatic disease occurred within seven weeks of
infection, and preneoplastic and primary bursal neo-
plasms were not detected. Spontaneous bursal lympho-
mas of unknown etiology have also been observed in
lines of chickens free of exogenous ALV and including
line 0 free of ev loci (91, 92). Reticuloendotheliosis virus
(see Reticuloendotheliosis) can also induce LL associ-
ated with c‐myc activation.
Transplantable LL tumors can be developed from
ALV‐induced tumors. The RPL12 transplantable tumor,

from which the RPL12 strain of ALV was isolated, is a
well‐known example. Several other new LL transplanta-
ble tumors have been described (284). Transplantable LL
tumors grow to a palpable size within 5–10 days and
become widely disseminated, inducing rapid mortality.
Erythroblastosis
Gross.  Natural cases of erythroblastosis (erythroid
leukosis) usually occur in birds between three and six
months of age. The liver and kidney are moderately
swollen, and the spleen often is greatly enlarged. The
enlarged organs are usually cherry red to dark mahogany
(Figure 15.30B) and are soft and friable. The marrow is
hyperplastic, semi‐liquid, and red in color. Petechial
hemorrhages occur in various organs such as muscles,
subcutis, and viscera. Thrombosis, infarction, and
rupture of the liver or spleen may be observed. Edema of
the lungs, hydropericardium, and a fibrinous clot on the
liver may occur.
With severe anemia, atrophy usually is seen in visceral
and lymphoid organs, particularly the spleen.
Changes in the blood reflect those in other organs,
such as liver, spleen, and bone marrow, and depend
largely on the extent of anemia or leukemia. When severe
anemia exists, the blood is watery and light red and clots
slowly. In contrast, acute cases may show no grossly
apparent changes, although usually the blood appears
dark red with a smoky overcast.
Microscopic.  Examination of the marrow in early cases
reveals blood sinusoids filled with rapidly proliferating
erythroblasts that fail to mature. In advanced cases,
marrow consists of sheets of homogeneous erythroblasts
with small islands of myelopoietic activity and little or no
adipose tissue. With concurrent anemia, the number of
erythropoietic cells may be reduced.
Alterations in visceral organs are primarily due to
hemostasis, resulting in an accumulation of erythro-
blasts in the blood sinusoids and capillaries (Figure 15.31).
The liver sinusoids, splenic red pulp, bone marrow, and
sinusoids of other organs are filled with proliferating
erythroblasts.
The sinusoids become greatly distended, resulting in
pressure atrophy of the parenchyma. Although accumu-
lations of erythroblasts may be extensive, they always
remain intravascular, unlike those in LL and
myeloblastosis.
Varying degrees of anemia may occur. Sometimes
erythroblastosis occurs, and there may be only severe
anemia. Extramedullary erythropoiesis is common.
The primary cell involved is the erythroblast. The cell
has a large round nucleus with very fine chromatin, one
or two nucleoli, and a large amount of cytoplasm that is
basophilic. A perinuclear halo, vacuoles, and occasion-
ally fine granules are present. The cell is irregular in
Chapter 15  Neoplastic Diseases 607
shape and often has pseudopodia. Erythroblasts have cell
markers that identify them as members of the erythro-
cytic series.
Stained blood smears reveal a variable number of
erythroblasts (Figure  15.30D). These vary in maturity
from the early erythroblast, which is the dominant cell,
to the various stages of polychrome erythrocytes. The
more mature cells often appear early in the course of the
disease or during remission, if it occurs. The thrombo-
cytic series of cells may be somewhat increased in num-
ber and immaturity. Similarly, in most naturally occurring
cases, immature cells of the myelocytic series appear in
the peripheral circulation. Occasionally, they are as
prominent as the erythroblasts. Cases of mixed erythro-
blastosis and myelocytomatosis may occur.
Ultrastructural.  Numerous studies have been made of the
primitive cells in erythroblastosis induced by different
strains of ALV and RPL12 (for details of these references
please refer to 266). Neoplastic erythroblasts are for the
most part indistinguishable from corresponding cells in
the normal bird, except that virus particles may be present
in extracellular spaces and within vacuoles inside cells. In
erythroblasts in the circulating blood, as in cell culture,
there is a great increase in membrane activity, with
vacuolization of the cytoplasm and budding of virus
particles from the cell membrane.
Pathogenesis.  Inoculation of slowly transforming strains
(i.e., those lacking a viral oncogene) of ALV such as
RPL12 into 11‐day‐old chick embryos induces
erythroblastosis from the first week of age (312). When
day‐old chicks are inoculated, the incubation period
varies from 21 to more than 100 days (50). Induction of
erythroblastosis by slowly transforming ALV involves
activation of the cellular oncogene c‐erbB by LTR
insertion (67, 147, 213), and new acutely transforming
AEV strains with transduced c‐erbB genes may arise (for
references to these, please refer to (266). Whether such
acutely transforming viruses spread naturally and induce
more erythroblastosis is not clear.
Experimentally, acutely transforming AEV strains,
such as ES4 and R, cause mortality from erythroblastosis
7–14 days after inoculation (165). ES4 carries the gene
v‐erbA, which blocks erythroid precursor cell differenti-
ation, in addition to the v‐erbB oncogene (for details of
these references, please refer to 266). Two subgroup J
ALV isolates, 1B and 4B, have been shown to be acutely
transforming and to induce erythroblastosis as well as
myelocytomatosis and other tumors (411). Their viral
oncogenes have not yet been identified. More recently
ALV‐J‐associated outbreak of erythroblastosis has also
been reported (421).
When birds are exposed to an acutely transforming
AEV, the first alterations are found in three days as foci of
(A) (B) (C)

(D) (E) (F)

(G) (H)

Figure 15.30  Comparison of leukosis. (A) Lymphoid leukosis (LL): Diffuse form affecting the liver. Lesion is grossly indistinguishable from
those in Marek’s disease (MD); (B) Erythroblastosis: enlarged cherry red liver and spleen, note the fibrinous exudates; (C) Myeloblastosis,
with enlarged gray‐red liver; (D) Erythroblastosis, note the basophilic cytoplasm and perinuclear halo, blood smear, Giemsa stain, ×975;
(E) Myeloblastosis: myeloblasts are slightly smaller than erythroblasts; cytoplasm is not as basophilic, nucleus is less vesicular, and nucleoli
are as not frequent or conspicuous, blood smear, Giemsa stain, ×975; (F) Myelocytomatosis, note myelocytes are packed with acidophilic
granules, section of tumor, Giemsa, ×975 (Beard); (G) LL tumors in the bursa of Fabricius (from the same bird with the liver tumor shown in
(A); (H) Myeloid leukosis tumor on the surface of the skull (Peckham). (For color detail, please see the color section.)

Figure 15.31  Erythroblastosis. Liver sinuses permeated with
erythroblasts in bird 40 days after inoculation with strain MC29
leukosis virus. ×280. (Beard).
proliferating erythroblasts in bone marrow sinusoids. By
day seven, the primitive cells reach the circulating blood,
and some foci of erythroid proliferation are present in
sinusoids of the liver and spleen. Erythroblasts continue
to accumulate in hepatic sinusoids and elsewhere until
death of the host and transplantable erythroblastosis
tumors can be developed (316).
Myeloblastosis
Gross.  Natural cases of myeloblastosis (myeloblastic
myeloid leukosis) are uncommon and usually occur in
adult chickens. The liver is greatly enlarged and firm
with diffuse, grayish tumor infiltrates, which give a
mottled or granular (“Morocco leather”) appearance
(Figure  15.30C). The spleen and kidneys are also
diffusely infiltrated and moderately enlarged. The bone
marrow is replaced by a solid, yellowish‐gray tumor cell
infiltration.
A severe leukemia exists, with myeloblasts compris-
ing up to 75% of peripheral blood cells and forming
a  thick buffy coat and usually an anemia and
thrombocytopenia.
Microscopic.  Parenchymatous organs, notably the liver,
show a massive intravascular and extravascular accu­
mulation of myeloblasts with a variable proportion of
promyelocytes (Figure 15.32). In the spleen, these tumor
cells accumulate in the red pulp. In the bone marrow,
myeloblastic activity is confined to extrasinusoidal areas.
Chapter 15  Neoplastic Diseases 609
Figure 15.32  Myeloblastosis. Distribution of myeloblasts in liver
of bird with myeloblastic leukemia 19 days after inoculation with
BAI‐A virus. ×280. (Langlois).
Myeloblasts in leukemic blood smears are large cells
with slightly basophilic clear cytoplasm and a large
nucleus containing 1–4 acidophilic nucleoli, which do
not stain prominently (Figure 15.30E). Often, promyelo-
cytes and myelocytes are also present; they easily can be
identified by their specific granulation, which in the early
forms is primarily basophilic. The disease may result in a
secondary anemia, with the presence of polychrome
erythrocytes and reticulocytes. Such a secondary anemia
is distinguished easily from the conditions in which
erythroblastosis and myeloblastosis occur together,
because then blast cells of both cell series are present in
the circulating blood.
Ultrastructural.  In circulating myeloblasts from birds
with myeloblastosis induced by BAI‐A AMV, virus
particles are only rarely found and then in small numbers
in clear vacuoles (28, 105, 172, 173). However, reticular
and phagocytic elements of the spleen and bone marrow
frequently are packed with virus particles. When
myeloblasts are transferred to cell culture, large numbers
of lysosomes appear in the cytoplasm. After some time
in cell culture, virus particles can be seen in lysosomes,
in vacuoles, and budding at the cell membrane. No other
changes are observed in these cells.
Pathogenesis.  The v‐myb gene of AMV is responsible
for neoplastic transformation of the target myeloblasts
(118). Experimental infection is followed within a few
days by the appearance of multiple foci of proliferating
myeloblasts in the extrasinusoidal areas of the bone
marrow, followed rapidly by leukemia and infiltration of
the liver, spleen, and other organs (for details of these
610 Section II  Viral Diseases

references, please see 266). The v‐myb oncogene acts as

a transcription factor that transforms myelomonocytic
cells by deregulating the expression of specific target
genes as well as through alterations of the nucleosomal
organization (430).

Myelocytomatosis
Gross.  Tumors of myelocytomatosis (myelocytic myeloid
leukosis) are distinctive and can be recognized on
gross  examination with some degree of certainty.
Characteristically, they occur on the surface of bones in
association with the periosteum and near cartilage,
although any tissue or organ can be affected. Myelocytomas
often develop at the costochondral junctions of the ribs,
on the inner sternum, pelvis, and on the cartilaginous
bones of the mandible and nares. Flat bones of the skull
are also commonly affected (Figure 15.30H). Tumors may
also be seen in the oral cavity, trachea, and in and around
the eye (317). The tumors are usually nodular and multiple,
with a soft, friable consistency and of creamy color. In the
disease caused by subgroup J ALV, myelocytomatous
infiltration often causes enlargement of the liver and
spleen and other organs, in addition to skeletal tumors
(432). Myelocytic leukemia may also occur (299).

Microscopic.  Tumors consist of masses of uniform,

usually well‐differentiated, myelocytes. Their nuclei are
large, vesicular, and usually eccentrically located, and a
distinct nucleolus is usually present. The cytoplasm is
usually tightly packed with acidophilic granules, which
are usually spherical. When imprint preparations of
fresh tumors are stained with May–Grunwald–Giemsa,
granules appear brilliant red (Figure  15.30F). Areas of
less well‐differentiated myelocytes are not uncommon
within the myelocytomas, and areas of undifferentiated
cells, which may be stem cells of the myelocyte–
monocyte series, may also be found. In the liver,
accumulations of neoplastic myelocytes occur around
blood vessels and in the parenchyma. In the spleen,
tumor cells are present in the red pulp. In the marrow,
the extrasinusoidal myelopoietic areas are greatly
expanded by uniform neoplastic myelocytes. A detailed
description of bone and bone marrow lesions in
myelocytomatosis apparently caused by subgroup J ALV
is provided by Nakamura et al. (268).
Although the naturally occurring disease has been
stated to be usually aleukemic, myelocytomatosis
induced by subgroup J ALV frequently is accompanied
by a marked leukemia of myeloid cells. Laboratory strains
of myelocytomatosis‐inducing virus, such as MC29, also
cause leukemia (Figure 15.33).
Ultrastructure.  Ultrastructural features of myelocytoma
cells vary from those of well‐differentiated myelocytes to
those of undifferentiated, nongranulated myeloid cells (253).
Figure 15.33  Myelocytomatosis. Granulated myelocytes in blood
smear from bird 23 days after inoculation with strain MC29
leukosis virus. ×750. (Beard).
Pathogenesis.  Acutely transforming strains of ALV that
induce myelocytomatosis, such as MC29 and CMII,
carry the v‐myc oncogene (118, 257). Slowly transform­
ing strains of subgroup J ALV that also induce
myelocytomatosis, such as HPRS‐103 and ADOL‐Hc1,
do not carry an oncogene, but molecular studies of
HPRS‐103‐induced myelocytomatosis indicate that
c‐myc is activated (66–68). The acutely transforming
strain 966 ALV, derived from myelocytoma and induced
by strain HPRS‐103 of subgroup J ALV, has been shown
to carry v‐myc (66, 302). Studies on HPRS‐103 and 966
showed that they have a tropism for the myelomonocytic
cell lineage, which may relate to their ability to cause
myelocytomas (6, 7). A recombinant ALV with envelope
of subgroup B and LTR of subgroup J (ALV‐B/J) was
isolated from a field outbreak of myeloid leukosis in
commercial layers has been reported (159). However,
inoculation of experimental and commercial strains of
White Leghorn chickens with this recombinant ALV‐B/J
resulted in primarily LL, but not myeloid leukosis,
suggesting that differences in the genetic makeup of the

commercial layers from which ALV‐B/J was originally
isolated and lines of chickens used in experimental
inoculations studies may be responsible for the
differences in pathogenicity observed (233).
The earliest alterations occur in bone marrow in which
there is crowding of intrasinusoidal spaces, principally
by myelocytes, and destruction of sinusoid walls. The
spaces may contain two types of cell—the primitive
hemocytoblast‐like cell (myeloid stem cell) and the neo-
plastic myelocyte. The latter appears to arise directly
from the stem cell, and differentiation is arrested both at
the nongranulated and granulated myelocyte level (253).
Myelocytes proliferate and soon overgrow the bone mar-
row. Tumors form by expansion of marrow growth and
may crowd through the bone and periosteum.
Extramedullary tumors may also arise by blood‐borne
metastasis.
Hemangioma
Gross.  This tumor is found in the skin or in visceral
organs in chickens of various ages. They appear as blood‐
filled cystic masses (blood blisters) (Figure  15.34) or
more solid proliferative, lesions (56). They are often
Figure 15.34  Hemangioma of gizzard serosa of RPL12 virus‐
inoculated bird. Note the dark circumscribed and raised tumor
nodules (236).
Chapter 15  Neoplastic Diseases 611
multiple and may rupture, causing fatal hemorrhage
(372). More recently, many workers have reported the
incidence hemangiomas in layer chickens infected with
ALV‐J in China (215, 287, 454).
Microscopic.  The cavernous form is characterized by
greatly distended blood spaces with thin walls composed
of endothelial cells (Figure 15.35). Capillary hemangiomas
are solid masses in which endothelium may proliferate
into dense masses (hemangioendothelioma), leaving
mere clefts for blood channels (Figure  15.36); develop
into a lattice with capillary spaces; or grow into collagen‐
supported cords with larger interspersed blood spaces.
Solid and papillary forms have also been described (264).
Ultrastructural.  Hemangiomas consisted mainly of
undifferentiated mesenchymal cells and had an alveolar
structure (241).
Pathogenesis.  Sequence analysis of an avian
hemangioma‐inducing virus isolated from layer hens
revealed unique elements in both env gene and LTR
Figure 15.35  Cavernous hemangioendothelioma of mesentery.
(Feldman and Olson).
612 Section II  Viral Diseases

Figure 15.36  Endothelioma in liver of bird inoculated with RPL30

leukosis virus. Occlusion of portal vein by inward‐growing spindle
cells from blood vessel ×250. (Fredrickson).

that were thought probably responsible for its biologic

and pathogenic characteristics (55). This virus was
cytocidal and had an affinity for endothelial cells
(330, 331). Although several sequence changes have
been reported in the genomes of hemangioma‐
inducing ALV‐J strains isolated from layers (287,
443), the precise molecular mechanisms remain
unknown. Recent study has suggested MET gene as a
common integration site in cases of hemangiomas
induced by ALV‐J (196).

Nephroma and Nephroblastoma
Gross.  Two types of renal tumor occur: nephroblastomas
(Wilms’ tumor) and adenomas and carcinomas.
Nephroblastomas vary from small, pinkish‐gray nodules
embedded in the kidney parenchyma to large, yellowish‐
gray, lobulated masses that replace most of the kidney
tissue (Figure 15.37). Tumors may be pedunculated and
connected to the kidney by a thin fibrous vascular stalk.
Large tumors are often cystic and may involve both
kidneys. Adenomas and carcinomas vary in size and
appearance, similar to nephroblastomas. They are often
multiple and within cysts.
Microscopic.  In nephroblastomas, the histologic
variation between different tumors or areas of the same
tumor is striking. There is usually neoplastic proliferation
of both epithelial and mesenchymal elements, although
their proportion and differentiation vary widely.
Epithelial structures vary from enlarged tubules with
invaginated epithelium and malformed glomeruli;
through irregular masses of distorted tubules; to groups
of large, irregular, cuboidal, undifferentiated cells with
little tubular organization (Figure  15.38). The growth
may be embedded in a loose mesenchymal or sarcomatous
stroma. There may be islands of keratinizing stratified
Figure 15.37  Nephroblastoma. Bird was inoculated at one day of
age with avian myeloblastosis virus BAI‐A strain.
squamous epithelial structures (epithelial pearls),
cartilage, or bone (98, 178, 190). Primary multiplicity of
tumors may occur, but metastases are rare.
Adenomatous or carcinomatous growths also vary
greatly in microscopic appearance. In tubular adenocar-
cinomas, primitive abnormal glomeruli frequently occur
in large numbers among abnormal tubules. Papillary cyst
adenocarcinomas are frequent. At times, solid carcino-
mas with little evidence of renal tubules develop (30,
253). Rarely is there cartilage and never other mesenchy-
mal tumor tissues. A trabecular fibrous tissue stroma
may separate masses of epithelial tumor tissue.
Ultrastructural.  In the epithelial nephronic elements of
nephroblastomas induced by strain BAI‐A of ALV (28),
cytoplasmic aberrant structures occasionally are seen in
large or small aggregates. Virus particles bud from cell
membranes of epithelial cells, fibroblastic elements of
the stroma, and chondrocytes. Sarcomatous elements
consist of cells similar in morphology to those in other
avian sarcomas. Virus particles have been observed

Figure 15.38  Nephroblastoma. Bird was inoculated at one day of
age with cloned preparation of avian myeloblastosis virus BAI‐A
strain. Note primary multiplicity of tumors of two distinct types in
different areas (arrows) ×20.
budding from epithelial cells in cystadenomas and
adenocarcinomas induced by strain MC29 myelocy­
tomatosis virus (255). Large accumulations of particles
in spaces in the cysts and tubules probably were related
to a lack of tubule and glomerular drainage.
Pathogenesis.  A target oncogene for ALV‐induced
nephroblastomas was not consistently identified (74).
More recently, a new proto‐oncogenes such as nov, (194)
and twist (285) were identified as common integration
sites in nephroblastomas (286).
Nephroblastomas originate from nephrogenic blas-
tema (embryonic nephrons and embryonal rests) (56,
190). This blastema tissue is present in the metanephros
(functional kidney) at hatching until at least six weeks of
age and appears as wedge‐shaped foci of immature renal
tissue particularly beneath the capsule. These epithelial
structures enlarge and become neoplastic. The support-
ing stroma of mesenchymal elements also proliferates
and, in turn, may be altered. There is extensive multipli-
cation of tumor cells (usually convoluted tubules and/or
stroma) and varying degrees of differentiation, some
abnormal. In the most differentiated form, nephrogenic
Chapter 15  Neoplastic Diseases 613
cells form glomeruli, tubules, or keratinized epithelium;
whereas cells of the stroma form sarcomas, cartilage, and
bone. Anaplasia of kidney cells can result in sheets of
large epithelioid cells with almost no tubular organiza-
tion. Malformed and blocked tubules result in cysts.
Nephroblastomas have been induced by BAI‐A strain
AMV (29, 53, 419), MAV‐2(N) (445), MAV‐2‐O (36),
and subgroup J‐related strain 1911 (301). Transplantable
nephroblastomas have been developed (419).
Carcinomatous growths originate only from the epi-
thelial part of the embryonal blastema and not from
mesenchymal elements. Depending on the degree of
anaplasia of epithelial elements, tumors formed may be
adenomas, adenocarcinomas, or solid carcinomas. These
tumors have been induced by MC29, ES4, and MH2
virus strains and by various field isolates. Renal adeno-
mas and carcinomas can be caused by slowly and acutely
transforming subgroup J ALV (for details of these refer-
ences, please see 266).
Fibrosarcoma and Other Connective Tissue Tumors
Gross.  A variety of benign and malignant connective
tissue tumors occur naturally, usually sporadically, in
young and mature chickens, and transmission of many of
these by cell‐free filtrates has been demonstrated. These
tumors include fibromas and fibrosarcomas, myxomas
and myxosarcomas, histiocytic sarcomas, osteomas and
osteosarcomas, and chondromas and chondrosarcomas.
The benign tumors grow slowly, are localized, and are
noninfiltrative. The malignant counterparts grow more
rapidly, infiltrate surrounding tissue, and may metastasize.
Fibromas arise as firm fibrous lumps attached to the
skin, subcutaneous tissues, muscles, and occasionally
other organs; fibrosarcomas are of a softer consistency.
In the skin, they may ulcerate. Myxomas and myxosarco-
mas are softer and contain tenacious slimy material.
They occur mainly in the skin and muscles. Histiocytic
sarcomas are firm, fleshy tumors occurring mainly in the
viscera. Osteomas and osteosarcomas are uncommon
and occur as hard tumors that may arise from the perios-
teum of any bone. Chondromas and chondrosarcomas
are rare. They occur where cartilage is present and some-
times within fibrosarcomas and myxosarcomas.
Ganglioneurosarcoma was reported associated with sub-
group J ALV infection (161).
Microscopic.  Fibromas in their simplest forms consist of
mature fibroblasts interspersed with collagen fibers
arranged in wavy parallel bands or whorls. Slowly
growing tumors are more differentiated and contain
more collagen and fewer cells than those growing more
rapidly. Some fibromas may have edematous areas and
should not be confused with myxomas and
myxosarcomas. If necrosis, ulceration, and secondary
infection have occurred, various inflammatory and
614 Section II  Viral Diseases

necrotic alterations may be observed in the tumor. Histiocytic sarcomas are derived from cells of the
Inflammatory changes may be so prominent that the monocyte and macrophage lineage, and the cellular con-
tumor may be confused with a granuloma. stituents are highly variable both within a tumor and
Aggressive and destructive growth, their cellular com- between tumors (Figure 15.41). They have been reported
position, and the immaturity of constituent cells associated with infection by subgroup J ALV (5, 169).
(Figure 15.39) characterize fibrosarcomas. Large irregu- Further studies have shown that ALV‐J‐induced histio-
lar and hyperchromic fibroblasts are abundant, and cytic sarcomatosis occurred in persistently viremic but
mitosis is common. Tumors contain less collagen than not immunotolerized meat‐type chickens (289).
fibromas, and this is concentrated in and near irregular
septa that subdivide the tumor. Regions of necrosis often
occur in rapidly growing tumors. Edema is sometimes
present. Multiple undifferentiated pulmonary sarcomas
associated with subgroup J ALV infection have been
reported (170).
Myxomas consist of stellate or spindle‐shaped cells
surrounded by a homogeneous, slightly basophilic,
mucinous matrix. In the malignant form (myxosarcoma),
the mucinous matrix is less abundant, and fibroblasts are
proportionally more numerous and immature than in
myxomas (Figure 15.40). Myxosarcomas associated with
ALV‐A infection have been reported in fancy breed
chickens (431).

Figure 15.40  Myxosarcoma induced by Rous sarcoma virus ×240.

(Helmboldt).

Figure 15.39  Fibrosarcoma in musculature of breast ×120. Figure 15.41  Histiocytic sarcoma of the heart. Note the varied
(Feldman and Olson). character of cellular components ×240. (Helmboldt).
 Chapter 15  Neoplastic Diseases 615

The cells may be spindle‐shaped, usually appearing Osteopetrosis

in groups or bundles as in fibrosarcomas; stellate
reticulum‐producing elements; and/or large phago-
cytic cells or macrophages. Tumors apparently derived
from stem cells of the myelomonocytic lineage may
also be considered to be histiocytic sarcomas. The so‐
called endotheliomas induced by MH2 and MC29 may
be tumors of this lineage (119). In primary tumors,
spindle‐shaped cells usually predominate; whereas in
metastatic foci, primitive histiocytic forms are more
numerous.
Osteomas are structurally similar to bone except that
much of the inner histologic detail is lacking. They con-
sist of a homogenous acidophilic matrix of osseomucin
containing collections of osteoblasts at irregular inter-
vals. Osteosarcomas are usually very cellular infiltrative
growths that invade and destroy surrounding tissues.
The cells are spindle‐shaped, ovoid, or polyhedral, and
many are in mitosis. Nuclei are prominent, and cyto-
plasm is basophilic. Multinucleated giant cells may be
quite numerous. Although very cellular and rapid grow-
ing, some areas usually have sufficient differentiation for
the production of osseomucin, which is usually sufficient
to identify these tumors.
Chondromas have a typical and unique structure (i.e.,
groups of two or more chondrocytes lying in a matrix of
chondromucin). In chondrosarcomas, considerable cel-
lular variation exists, ranging from the most immature to
the fully mature chondrocyte.
Gross.  The first grossly visible changes occur in the
diaphysis of the tibia and/or tarsometatarsus. Alterations
soon are seen in other long bones and bones of the
pelvis, shoulder girdle, and ribs but not the digits.
Lesions are usually bilaterally symmetric; they first
appear as distinct, pale yellow foci against the gray‐
white, translucent, normal bone. The periosteum is
thickened, and the abnormal bone is spongy and at first
easily cut. The lesion is commonly circumferential and
advances to the metaphysis, giving the bone a fusiform
appearance (Figures 15.42 and 15.43). Occasionally, the
lesion remains focal or is eccentric. Severity of the lesion
varies from a slight exostosis to a massive asymmetric
enlargement with almost complete obliteration of the
marrow cavity. In long‐standing cases, the periosteum is
not as thickened as it was earlier; when it is removed,
the porous irregular surface of the very hard
osteopetrotic bone is revealed.

Ultrastructural.  Only the sarcomas produced by RSV

have been examined in detail. The morphology of
fibroblasts, macrophage‐like cells, and mast cells, found
in Rous sarcomas, have been described (28, 172).

Pathogenesis.  Induction of sarcomas and other

connective tissue tumors in the field is likely to be by
activation of a cellular oncogene by a slowly transforming
ALV, occurring up to several months after infection (388).
Avian leukosis virus related to MAV‐1 has been implicated
in a field outbreak of sarcomas in commercial layers;
inoculation of susceptible White Leghorn chickens with
new isolate resulted sarcomas and myelocytomas (448).
Viral oncogenes that have been associated with sar-
coma induction include src, fps, yes, ros, eyk, jun, qin,
maf, crk, sea, and erbB (73, 118, 309, 416) (Table 15.5).
These viral oncogenes reflect the cellular oncogenes
that are activated by insertional mutagenesis and that
may undergo mutation. These cellular oncogenes con-
trol a variety of functions in the cell (their products are
generally growth factors, growth factor receptors, signal
transducers, or DNA transcription factors), and it is
their altered expression that results in the loss of regula- Figure 15.42  Osteopetrosis. A 24‐week‐old chicken, injected with
tion of cell proliferation or differentiation that causes RPL12 at one day of age, with advanced osteopetrotic lesions of
neoplasia. the shanks. (Sanger).
616 Section II  Viral Diseases

(A) (B)

Figure 15.43  Osteopetrosis of tibia in 10‐week‐old chicken. (A) Shorter length of bone is due to reduced growth. Lower tibia is from
control bird of same age. (B) Cross‐section of middle of shaft of bones in A (451).

Early in the disease, the spleen may be slightly enlarged.

Later, severe splenic atrophy occurs as well as premature
bursal and thymic atrophy. Lymphoid leukosis often
occurs in individual birds with osteopetrosis.

Microscopic Lesions.  The periosteum over the lesion is

greatly thickened from an increase in number and size
of basophilic osteoblasts. The number of osteoclasts
per tibia increases, but the density of osteoclasts (i.e.,
the number per unit volume of bone) decreases (354).
Affected bones differ from normal bones in the
following ways. Spongy bone converges centripetally
toward the center of the shaft (Figure  15.44). An
increase occurs in size and irregularity of the haversian
canals, as well as an increase in number and size, and an
alteration in position, of lacunae. Osteocytes are more
numerous, large, and eosinophilic; the new bone is
basophilic and fibrous.
The blood picture is ordinarily aleukemic, and a sec-
ondary anemia often exists. There may be active erythro-
poiesis in remaining bone marrow and sometimes in
focal areas in the liver. Experimentally, viruses that cause
osteopetrosis can induce an aplastic anemia and an
increased corpuscular fragility (179, 320).

Ultrastructural.  Virus particles bud transiently from

osteoblasts and continuously from osteocytes and
accumulate in the periosteocytic space. With calcification
of the bone, the particles become incorporated in the
bone trabeculae. No virus production is observed from
osteoclasts (141).
Pathogenesis.  Osteopetrosis is a polyclonal disease of
the bone and is thought to be caused by high levels of
virus infection perturbing the growth and differentiation
of osteoblasts. Much higher levels of virus infection were
found in diseased bones than in cultured osteoblasts
infected with the Br21 strain of an osteopetrosis‐inducing
Figure 15.44  Osteopetrosis. Cross‐section of humerus from
8‐week‐old chicken. Six separate osteopetrotic foci are present,
two of which extend from endosteum to periosteum ×18 (451).
ALV (140). Severe cases of osteopetrosis contained 10
times more viral DNA, 30 times more gag precursor
protein, and 2–3 times more env protein than the infected
osteoblast cultures. The osteopetrotic lesion is basically
proliferative or hypertrophic (57, 349) and may be
neoplastic (39, 354). Lesions of the lymphoid organs and
bone marrow are degenerative or anaplastic (179).

The propensity for certain ALVs to induce osteopetrosis
depends on sequences in the gag‐pol‐5’env region of the
viral genome (358). Env proteins have also been implicated
in osteopetrosis induction (193). Detection of exogenous
ALV sequences most similar to the envelope of
myeloblastosis associated virus type 1 (MAV‐1) was
identified from the archived bones of an outbreak of
osteopetrosis in commercial birds have been reported (23).
Other Tumors
Apart from renal tumors, epithelial tumors caused by ALV
are uncommon. They mainly have been reported follow-
ing experimental infections with acutely transforming
viruses, although some have occurred in natural and
experimental infections with subgroup J ALV. Strains
BAI‐A (29) and HPRS‐103 (301) of ALV have induced the-
comas and granulosa cell tumors of the ovary. A semi-
noma in the testis occurred in a bird inoculated with strain
MH2 (29) and possibly in birds inoculated with subgroup
J ALV isolates (308). Adenocarcinomas of the pancreas
have been induced in chickens by strains MC29, MH2,
and HPRS‐103 of ALV (29, 254, 301). The Pts‐56 strain of
osteopetrosis virus produced pancreatic adenomas and
adenocarcinomas and duodenal papillomas in guinea fowl
(202–204). Squamous cell carcinomas have been observed
in a few chicks with strains MC29 and MH2 (29). The
MC29 and MH2 strains have induced hepatocarcinomas
(29, 216). Other epithelial tumors induced by subgroup J
ALV include cholangioma and ovarian carcinoma (301).
Strains MC29 (29) and HPRS‐103 of ALV (301) have
been shown to induce mesotheliomas.
Immunity
Active Immunity
Immune responses to oncogenic viruses including ALV
have been reviewed (106, 137, 262, 265, 425). Under
natural conditions, most chicks become infected by
­ weeks‐of‐age, but after 4‐weeks‐of‐age, the response
exogenous ALV from penmates or their surroundings
and, after a transient viremia, develop virus‐neutralizing
antibodies directed against virus envelope antigens that
rise to a high titer and persist throughout the life of the
bird. The virus‐neutralizing antibodies serve to restrict
the amount of virus in the bird, which in turn, will limit
neoplasia, but they generally are considered to have little
direct influence on tumor growth. After inoculation of
birds with ALV at four weeks of age or older, transient
viremia was detectable at one week and was followed by
antibodies at three weeks and later (237). In a study of
birds naturally infected after hatching, antibodies were
first detected at 9 weeks of age, with a marked increase in
the proportion with antibodies between 14 and 18 weeks,
when 80% were positive (340).
Antibodies against gs‐antigen may also occur in ALV‐
infected birds, but these apparently have no influence on
Chapter 15  Neoplastic Diseases 617
tumor growth (335, 359). The presence of cytotoxic lym-
phocytes against viral envelope antigens has been shown
in birds immunized with ALV or RSV (26, 27, 212), and
cell‐mediated immunity and the major histocompatibil-
ity complex (MHC) complex are clearly implicated in the
regression of Rous sarcomas (350, 351). Viral proteins
expressed on the surface of tumor cells appear to be
important targets for the cell‐mediated immunity, and
nonvirion transformation‐specific cell surface antigens
may also be implicated.
Chickens that are infected congenitally by ALV do
not develop immune responses to the virus. Instead,
they become immunologically tolerant to the virus
and develop a persistent viremia in the absence of
neutralizing antibodies (248, 340). Inoculating chick-
ens up to two weeks of age with ALV may also induce
tolerant infection. Early infection with subgroup J
ALV is particularly likely to induce a tolerant infection
(132, 436, 438). Birds with a tolerant viremic infection
are more likely to develop neoplasms than are
immune‐infected birds, because of the greater virus
load in viremics.
Infection by ALV can depress primary and secondary
antibody responses and cell‐mediated immunity (341)
to unrelated antigens, although these effects have been
variable in different studies. Fadly et  al. (129), in a
study of congenital infection with an A subgroup ALV,
RAV‐1, failed to detect effects on B‐ and T‐cell func-
tion during the early and late stages of infection, and
they reported no histological damage to the bursa, thy-
mus, or spleen. In contrast, subgroup B ALVs have
been reported to induce a marked suppression of the
humoral immune response to several antigens and
decreased responsiveness to several mitogens (426).
Evidence that subgroup J ALV is immunosuppressive
appears to be equivocal (136, 230, 231, 379, 380). ALV‐J
infection induced a strong immune response at 2‐
decreased quickly suggesting that 3–4 weeks postin-
fection is the critical time at which the ALV‐J virus
exerts its immunosuppressive effects on the host (420).
Nucleotide sequence analysis of consecutive isolates
from V+A+ infection profile suggested viral evolution
to escape the host immune response thereby contrib-
uting to ALV J persistence (290).
Passive immunity
Serum antibodies, which are mainly in the IgG fraction
(249), are passed on by the hen to her progeny via the egg
yolk and provide a passive immunity that lasts 3–4
weeks. Passive antibody delays infection by ALV (439),
reduces the incidence of viremia and shedding of ALV
(122) and reduces the incidence of tumors (46). Level
and persistence of antibody in the chick is related to the
titer of antibody in the dam’s serum.
618 Section II  Viral Diseases

Genetic Resistance (i.e., susceptible to A, B, C, D, E, and J). Recent studies

have shown that precise editing of receptor sequences
Two levels of genetic resistance to leukosis or sarcoma
could be used to induce resistance to infection by ALV
virus‐induced tumors are recognized: cellular resistance
(217, 218).
to virus infection and resistance to tumor development
Chickens with genetic resistance to infection and
(for references to these, please see 266).
tumor induction by different subgroups of ALSVs usu-
Inheritance of cellular resistance to infection is of a
ally fail to develop antibodies (78, 85). Genetic resistance
simple Mendelian type (Table 15.12). Independent auto-
to tumor development has been studied mainly with the
somal loci control responses to infection by ALSVs of
Rous sarcoma (355, 398), regression of which is deter-
subgroups A, B, and C and are designated tva (tumor
mined by a dominant gene, R‐RS‐1, that lies within the
virus A subgroup), tvb, and tvc respectively (79). At each
MHC locus of the chicken and located in the BBL region
tv locus, alleles for susceptibility and resistance exist that
(75, 155, 319, 353). Conserved peptide motifs of the RSV
are designated tvas, tvar; tvbs, tvbr; and tvcs tvcr, respec-
proteins that bind to the MHC have been identified and
tively, and the susceptibility alleles are dominant over the
shown to be immunoprotective against Rous sarcoma
resistance alleles. These genes usually are abbreviated to
growth in chickens with Class I allele B‐F12 (180) and
as, ar, etc. It is probable that multiple alleles occur at each
peptide motifs of the single dominantly expressed class I
locus, encoding different levels of susceptibility (see 232,
molecule explain the MHC‐determined responses to
266, 393).
RSV (418). The MHC (Ea‐B) locus also influences inci-
Mutations in the tvb receptor gene account for the
dence of erythroblastosis and, to a lesser extent, LL (15).
resistance to subgroups B, D, or E infections (25, 184,
Some influence of the lymphocyte antigen Bu‐1 locus on
205, 329). Similarly, intronic deletions that disrupt
Rous sarcoma regression and of the Th‐1 locus on LL is
mRNA splicing of the tva receptor gene was shown to
reported (12). Structural analysis of the MHC alleles in
result in decreased susceptibility to infection by sub-
RSV tumor regression has ruled out the DMA1, DMA2,
group A ALV (328).
BRD2, TAPBP, and BLB2 genes in regression in B6 hap-
Genetic resistance to infection by subgroup J virus has
lotype (386, 387).
not been recognized in chickens, although a number of
other avian species are resistant (306). There is no evi-
dence of the segregation of the ALV‐J cell receptor, Na+/
H+ exchange type I molecule (59), in the chicken popula-
tion (279). However differences between avian species in
Diagnosis
susceptibility to ALV‐J were dependent on the polymor-
Isolation and Identification
phism in this receptor (206, 315, 327).
of Causative Agent
Cellular susceptibility phenotypes associated with
these genes are designated according to a convention Because ALV is widespread among chickens, virus isola-
that recognizes the virus subgroups to which the chicken tion and the demonstration of antigen or antibody have
(C) cell is resistant (/) (e.g., C/AE denotes a cell resistant limited or no value in diagnosing field cases of lympho-
to A and E subgroups but susceptible to B, C, D, and J mas. However, assays for the detection of ALV are very
subgroups); C/0 denotes a cell resistant to no subgroup useful in identification and classification of new isolates,

Table 15.12  Genes controlling cellular susceptibility to leukosis and sarcoma viruses.

Locus Alleles

Virus subgroup Old New Old New Dominant trait

A tva TVA tvas tvar TVA*S TVA*R Susceptibility

s1 r
B and D tvb TBV tvb tvb TVS*S1, S3 TVB*R Susceptibility
C tvc TVC tvcs tvcr TVC*S TVC*R Susceptibility
s r
E tved TVE tve tve TVE*S TVC*R Susceptibility
ie Ie ie Resistance

Note: The locus designation is adapted from Crittenden (117). The new locus designation is that agreed by the
Poultry Committee of the USDA National Animal Genome Research Program, 1994. The allele previously
designated tvbs2 now is considered to be identical to tvbS1. The existence of a tve locus is not settled. The ie locus
is now considered to be an ev locus, with blocking of the subgroup E virus receptor by endogenous virus ENV
glycoprotein expression.

safety testing of vaccines, and in testing pathogen‐free
and other breeder flocks for freedom from virus infec-
tion. Samples most commonly used for detection of ALV
include blood, plasma, serum, meconium, cloacal and
vaginal swabs, oral washings, egg albumen, embryos,
and tumors (265, 266). Virus also can be isolated from
albumen of newly laid eggs or the 10‐day‐old embryo of
eggs laid by hens that are transmitting virus vertically,
from feather pulp, and from semen. All ALSVs are very
thermolabile and can be preserved for long periods only
at temperatures below −60°C. Thus, materials used for
biological assays for infectious virus should be collected
and placed on melting ice or stored at −70°C until
assayed. In contrast, samples for detection of ALV gs
antigens by direct assays can be stored at −20°C; reviewed
in (134, 266).
Because most strains of ALV produce no visible mor-
phologic changes in cell culture, assays for ALV are based
on the following: (1) detection of specific proteins or
­glycoproteins coded for by one or more of the three
major genes of ALV, namely gag, pol, and env genes
(Figure 15.18), or (2) detection of specific proviral DNA
or viral RNA sequences of ALV by the polymerase chain
reaction (PCR) and reverse transcription (RT)‐PCR,
respectively.
The presence of virus is determined by the detection of
ALV p27 by indirect biologic assays, such as complement
fixation (CF) for avian leukosis (COFAL), ELISA for ALV,
phenotypic mixing, resistance‐inducing factor, and non‐
producer cell activation. The biological assays require
CEF with specific host range (Table  15.9). Chicken
embryo fibroblasts that are resistant to infection with
endogenous ALV (C/E) are desirable to use in tests for
detection and isolation of exogenous ALV. Other cells,
such as those resistant to subgroup A (C/A) and resistant
to subgroup J ALV (C/J) (186), can also be used to con-
firm the subgroup of isolated ALV. Testing samples on
CEFs that are susceptible to all subgroups of ALV (C/O),
and those that are resistant to subgroup E (C/E) can be
used in differentiating exogenous and endogenous ALV.
If a positive test is obtained from using C/O but not C/E
CEFs, the sample is positive for endogenous ALV. Positive
tests using both C/E and C/O indicates the presence of
exogenous ALV. Recently, a flow cytometry method
using a highly specific alloantibody termed R2 has been
described for detection of endogenous ALV envelope in
chicken plasma (11, 14). It should be noted that some
tests such as CF and ELISA and possibly non‐producer
(NP), phenotypic mixing (PM), R(‐)Q cell, and FA can be
suitable for all leukosis and sarcoma viruses. The resist-
ance‐inducing factor (RIF) test can be performed only on
ALVs that are not rapidly cytopathogenic. Other tests are
specific for certain virus strains. Rapid transformation of
fibroblast cultures is produced only by certain RSV and
of hematopoietic cell cultures only by defective ALV. The
Chapter 15  Neoplastic Diseases 619
test for adenosine triphosphatase activity is specific for
avian myeloblastosis virus. The procedures that are most
widely used have been reviewed extensively in the previ-
ous edition of this book as they are not covered here
(126, 134, 266). These include the RIF test (337), the
COFAL test used to detect the group‐specific antigens of
ALV (123, 266) and tests based on phenotypic mixing of
viruses (80, 266, 282, 322).
Tests for Viral‐Internal, Group‐Specific (gs) Antigens
Detection of the major antigen (p27) forms the basis of
several diagnostic tests for virus. Highly sensitive
ELISA tests for gs antigens are widely used directly for
the assay of test material or indirectly using cell cul-
tures inoculated with test material. These antigens
may also be detected in cells by FA techniques (200,
294). Using indirect FA tests, monoclonal antibodies to
ALV‐J proved useful in the detection of ALV‐J infected
cell cultures (131, 324, 384, 410). A variety of samples
can be tested by ELISA for the presence of ALV; how-
ever, serum has been shown to be unsuitable for the
detection of exogenous ALV by direct ELISA (303). For
the detection of exogenous ALV, samples are inocu-
lated on CEFs that are genetically resistant to subgroup
E ALV. Seven to nine days later, cell lysates are tested
for the presence of ALV gs antigen by ELISA (123, 134,
364). Rabbit anti‐p27 antibody, which is used to coat
ELISA plates and rabbit anti‐p27 conjugate, as well as
complete kits for running ELISA for detection of ALV
gs antigen, are available commercially. An indirect
ELISA using a recombinant capsid protein has also
been reported (326).
Comparison of Tests
In vivo and in vitro cell culture tests for detection or assay
of exogenous ALVs are compared in Table 15.13.
All the in vitro tests require a standard source of
chicken embryos free from exogenous ALSVs and of
known phenotype for use in cell culture. The following
reagents are also required: for the RIF test, stocks of
challenge RSV of each subgroup; for the COFAL and
ELISA tests, specific antiserum; for the NP test, quanti-
ties of NP cells; and for the PM test, stocks of RSV with
endogenous helper, RSV(RAV‐0). Cells obtained from
embryos of unknown genetic origin should not be used
in RIF, COFAL, and indirect ELISA tests because the
results may be confused by genetic resistance. Both
COFAL and indirect ELISA tests require either pro-
longed maintenance of culture or several subcultures to
propagate the virus sufficiently; therefore, much more
work is involved than in the NP or PM tests.
The subgroup of an infecting ALV can be determined
by any of the tests. In the RIF test, only RSV belonging to
the same subgroup as the ALV is subjected to interfer-
ence. In COFAL and ELISA tests, genetically resistant
620 Section II  Viral Diseases

Table 15.13  Comparison of methods for assaying exogenous avian leukosis viruses (ALVs).

Response Additional requirements for subgroup Time required

Method Requirements measured determination (days)c

In vivo
Chick inoc 1 day IA LL susceptiblea LL Genetically resistant chickens 270
Chick inoc 1 day IA Erythro susceptibleb Erythro Genetically resistant chickens 63
Embryo inoc 11 days IV Erythro susceptible Erythro Genetically resistant chickens 43
Cell culture RSV pseudotypes, Resistance to Challenge virus of known subgroup 12 + 6
RIF C/E cells formation of
RSV foci in CCd
COFAL Hamster antiserum, Complement Genetically resistant cells 14 + 1
C/E cells fixation
ELISA Enzyme‐linked antisera Color change of Genetically resistant cells 14 + 1
C/E cells substrate
NP NP cells (chicken or RSV foci in CC Genetically resistant cells of RIF test 8+6
quail) with leukosis virus of known
subgroup
PM RSV (RAV‐0), C/O, RSV foci in CC Genetically resistant cells of RIF test 5+6
and C/E cells with leukosis virus of known
subgroup

Note: C/E, cells, genetically resistant to infection with viruses of E subgroup, but susceptible to viruses of other subgroups; C/O, cells
phenotypically susceptible to infection by viruses of all subgroups; COFAL, complement fixation for avian leukosis viruses; CC, cell culture;
ELISA, enzyme‐linked immunosorbent assay; erythro, erythroblastosis; IA, intraabdominal; IV, intravenous; LL, lymphoid leukosis; NP,
nonproducer; RIF, resistance‐inducing factor; RSV, Rous sarcoma virus; RSV (RAV‐0), Rous sarcoma virus with endogenous helper.
a
 Chickens susceptible to LL tumor formation (e.g., line 15I chickens).
b
 Chickens susceptible to virus infection and to development of erythroblastosis (or myeloblastosis).
c
 Approximate number of days necessary to cultivate the virus plus the number of days to indicate the presence of virus.
d
 Cell culture.

cells can be used; thus, an ALV of subgroup A will not
produce CF antigens in cells of the C/A phenotype
(resistant to subgroup A viruses). In the NP test, geneti-
cally resistant NP cells can be prepared, and in the PM
test, genetically resistant cells can be used in the mixing
phase. In NP and PM tests, supernatant from the activa-
tion or mixing phase, which contains RSV of the same
subgroup as the ALV, can be placed on genetically resist-
ant cells or embryos or used in an interference test with
an ALV of known subgroup.
Immunohistochemical Tests
Direct (200) and indirect (294) FA tests as well as flow
cytometry (185, 186) have been used to detect viral anti-
gen in CEF cultures; flow cytometry has also been shown
to be a very useful tool in identifying the subgroup of
ALV strains contaminating commercial MD vaccines
(24, 121, 361).
Enzyme Assays
Avian myeloblastosis virus has on its surface an enzyme
(ATPase) that dephosphorylates adenosine triphosphate.
This activity can be used as a quantitative assay to deter-
mine the amount of virus present in the plasma of
infected chickens or in supernatants of myeloblast
­cultures (31).
Assays for RT activities have been used for the detec-
tion of oncogenic RNA viruses including all ALSVs (399).
Detection of this enzyme, either directly when the cor-
rect template is used (199, 401) or indirectly when the
radioimmunoassay is used (292), is an indication of pres-
ence of virus. Most recently, a highly sensitive PCR‐
based RT assay has been used to screen human vaccines
that are produced in CEF or embryonated eggs for free-
dom from avian retroviruses (188, 236, 404, 413).
Detection of Viral Nucleic Acids
The PCR is the most common DNA‐based test used for
detection and identification of ALV including subgroup
E viruses (Figure 15.45). Reverse transcriptase‐PCR has
also been used to detect several subgroups of ALV (176,
454). A specific PCR for ALV subgroup A can be used to
detect proviral DNA and viral RNA in various tissues
from ALV‐infected chickens (408). Reverse tran-
scriptase‐nested PCR (RT‐nested PCR) test that ampli-
fies a fragment of the LTR of exogenous ALV subgroups
A, B, C, D, and J, but not endogenous retroviral sequences
has been described (151). Several primers specific for the
 Chapter 15  Neoplastic Diseases 621

(A) the detection of ALV (444). As further modification of

M 1 2 3 4 5 6 7 8 9 10 PCR, loop‐mediated isothermal amplification (LAMP)
method for ALV subgroups have also been developed
(423, 453).

Hematopoietic Transformation
Avian myeloblastosis virus, an acutely transforming
0.3 Kb strain of ALV, harboring an oncogene, can infect and
transform cultures of avian myeloblasts. Assays usually
are based on a quantal response in which individual cul-
tures are scored as positive or negative (21, 256). Focus
(B) assays for myeloblastosis, erythroblastosis, and other
M 1 2 3 4 5 6 7 8 9 10 defective ALVs have been developed (164, 165, 258).
Cultured chicken bone‐marrow cells and blood mono-
cytes are useful in isolation and propagation of acutely
2.3 Kb transforming viruses recovered from cases of myeloid
leukosis induced by strain HPRS‐103 ALV‐J (302).

Transformation of Fibroblasts and Cytopathology

Avian sarcoma viruses transform spindle‐shaped flat
CEFs into spherical and refractile foci (323, 400) that can
be seen microscopically after 4–5 days (Figure  15.23).
(C) Genetically susceptible cultures are inoculated with test
M 1 2 3 4 5 6 7 8 9 10
material. The next day, medium is decanted and is
replaced with an agar overlay (134). Inoculated cultures
should be examined daily for RSV‐induced foci, which
2.3 Kb usually develop within 4–7 days PI.

Figure 15.45  Polymerase chain reaction (PCR) analysis of DNA
isolated from line 0 CEF uninfected and infected with RAV‐1
(ALV‐A), RAV‐2 (ALV‐B), RAV‐49 (ALV‐C), RAV‐50 (ALV‐D), ADOL‐
HC‐1 (ALV‐J), and ADOL‐R5‐4 (ALV‐J), and 15B1 cells uninfected
and infected with RAV‐0 (ALV‐E) and EV21 (ALV‐E). (A). PCR analysis
using primers specific for ALV‐A‐E. (B). PCR analysis using primers
specific for ALV‐E. (C). PCR analysis using primers specific for ALV‐J
Lanes: M, 1 kb plus DNA ladder; 1, RAV‐1; 2, RAV‐2; 3, RAV‐49; 4,
RAV‐50; 5, RAV‐0; 6, EV21; 7, ADOL‐HC‐1; 8, ADOL‐R5‐4; 9 line 0
CEF; 10, 15B1 CEF (481, 482). (B. Lupiani).
detection of the most commonly isolated ALVs, particu-
larly subgroup A (234), and the new subgroup ALV‐J
(360, 369, 370) have been developed. Other primers spe-
cific for endogenous, subgroup E ALV can also be used
to detect cell culture infected with endogenous ALV‐E,
but not those infected with exogenous ALV of subgroups
A, B, C, D, and J (134). Recently, a sensitive and specific
multiplex PCR for detecting subgroups ALV‐A, ALV‐B,
and ALV‐J has been reported (149). Taqman‐based or
SYBR Green‐based real‐time PCR tests for detection of
ALV have also been used for diagnosis of ALV (94, 325).
Use of a proximity ligation technique combined with PCR
was used to develop a novel immune‐PCR (Im‐PCR) for
Serology
Plasma, serum, and egg yolk are suitable samples for the
detection of antibodies to ALSVs.
Tests
Antibody to ALV can be measured by its reaction with
RSV or ALV; a virus of one subgroup will not be neutral-
ized by antibodies provoked by a virus of a different sub-
group. Usually, a 1 : 5 dilution of heat‐inactivated (56°C
for 30 minutes) serum is mixed with an equal quantity of
a standard preparation of RSV of a known pseudotype;
after incubation, the residual virus is quantitated by any
one of many procedures, the cell culture assay being
most commonly used. A microneutralization test to
assay for residual virus can be used for detection of ALV
antibody (134). The test can be conducted in 96‐well
microtiter plates, and the neutralization of the virus is
determined by an ELISA on culture fluids (16).
An indirect immunoperoxidase absorbance test (250,
251), ELISA tests (252, 367, 405, 407), and flow cytometry
(185, 186) have been described for the detection of antibod-
ies. ELISA kits for the detection of antibodies to ALV
­subgroups A and B are available commercially. Also, molec-
ularly cloned, baculovirus‐expressed, envelope glycopro-
teins of ALV‐J now are being used in commercial ELISA kits
specific for the detection of antibody to ALV‐J (220, 410).
622 Section II  Viral Diseases
Serotypes
Based on host range, interference spectrum, and viral
envelope antigens, viruses of L/S group occurring in
chickens are divided into six subgroups A, B, C, D, E, and
J. Viruses of different subgroups can be distinguished by
the ability of monovalent antiserums to neutralize them.
Even though some cross‐neutralization usually exists
between viruses belonging to the same subgroup, the
kinetics of neutralization vary, and slopes of curves for
heterologous systems differ from those of homologous
systems. No common neutralization antigens are among
the viruses of different subgroups, except for a relation-
ship between subgroups B and D. The diagnosis of infec-
tion by serologic means requires that representatives of
all serotypes be employed. Avian leukosis viruses them-
selves may be used, but more commonly, RSV pseudo-
types are employed in the neutralization tests (134).
Differential Diagnosis
Lymphoid Leukosis
Differential diagnosis of lymphomas in chickens can be
difficult. The two most common lymphoid neoplasms,
namely MD and LL are particularly confusing (441).
Lymphoid tumors observed in REV‐infected chickens,
although only infrequently in cases of use of REV‐con-
taminated vaccines, or under experimental conditions
(see Reticuloendotheliosis), may also add to the confu-
sion. Lymphoid leukosis cannot be differentiated from
REV‐induced bursal lymphomas on the basis of pathol-
ogy, immunohistochemistry, and molecular changes in
the c‐myc region. Virologic, serologic, or PCR tests may
be helpful in establishing infection for one virus and
exclusion for the other. However, such assays are not par-
ticularly helpful in the diagnosis of virus‐induced lym-
phomas of chickens including LL, as avian oncogenic
viruses are widespread, and infection in the absence of
tumor formation is common. Detection of proviral DNA
and integration junctions by PCR assays (67, 160, 314)
has been shown to be useful for tumor diagnosis.
Because LL tumors should contain ALV proviral DNA
sequences inserted near the c‐myc gene, differentiation
between LL and REV‐induced bursal lymphomas can be
made by southern blots and hybridization analysis of tumor
DNA for clonal insertion of ALV (see previous discussion).
Lymphomas in which bursal tumors are lacking or in
which the latent period is too short for that of LL can be
confused primarily with MD; however, under certain cir-
cumstances, REV‐induced lymphoma should also be
ruled out (see Reticuloendotheliosis). In cases in which
bursal tumors are lacking, LL and MD can be differenti-
ated only with difficulty, because similar lymphoid
tumors may occur in both diseases in the same visceral
organs during the same age period. Visceral lesions of
these two diseases cannot be distinguished by gross
examination. Diagnosis is possible in most instances on
careful microscopic examination; however, considerable
experience is necessary. In coming to a decision, history,
signs, gross and microscopic lesions, and cytology should
all be considered. Ordinarily, LL does not occur before
14 weeks of age, and most of the mortality occurs
between 24 and 40 weeks. Marek’s disease, however, may
occur as early as 4 weeks, and the mortality peak varies
from 10–20 weeks. Occasionally, losses continue and
may reach a peak after 20 weeks.
Nodular tumors of the bursa can often be palpated
through the cloaca in birds infected with ALV. Paralysis
associated with gross lesions in autonomic and periph-
eral nervous systems and gross lesions of the iris (“gray
eye”) are specific for MD.
As stated previously, the bursa of Fabricius plays a cen-
tral role in development of LL. When distinct focal or
nodular lymphoid tumors are present in the bursa, a
diagnosis of LL can be made; however, REV‐induced
bursal lymphomas must be ruled out. Such tumors are
sometimes quite small and may be overlooked. In some
birds, MD induces a premature atrophy of the bursa. In
others, the bursa may be tumorous, in which case the
walls and the plica may be thickened from interfollicular
infiltration with pleomorphic lymphocytes. In contrast,
intrafollicular tumors of the bursa consisting of uniform
large lymphocytes are usual with LL.
Microscopic lymphoid infiltration in nerves, cuffing
around small arterioles in the white matter of the cere-
bellum, and the feather follicular pattern of lymphoid cell
infiltration in the skin, which are characteristic of MD,
are not seen with LL.
Cytologically, LL tumors generally are composed of a
homogeneous population of lymphoblasts (Figure 15.27).
In contrast, tumors of MD usually contain lymphoid
cells varying in size and maturity from lymphoblasts to
small lymphocytes, and plasma cells may also be present.
Special stains such as methyl green pyronin are helpful
for cytology. Immature lymphoblasts characteristic of LL
tumors are highly pyroninophilic; whereas the medium
and small lymphocytes that predominate in tumors of
MD do not stain with pyronin.
Lymphoid leukosis tumors are composed almost
entirely of B cells and have surface IgM markers; whereas
60–90% of MD tumor cells are T cells that lack IgM
markers and only about 3–25% are B cells. In addition,
from 0.5–35% of MD tumor cells have a tumor‐associ-
ated cell surface antigen (MATSA), which is absent from
LL tumor cells (113, 277, 278, 318). Recently, Witter et al.
(441) introduced a diagnostic strategy for the differential
diagnosis of viral lymphomas in chickens.
Other diseases that may be confused with LL are eryth-
roblastosis, myeloblastosis, myelocytomatosis, ­pullorum
disease, tuberculosis, enterohepatitis, Hjarre’s disease,
and fatty degeneration of the liver.

Erythroblastosis
Although gross lesions of liver, spleen, and bone marrow
provide the basis for a presumptive diagnosis, a firm diag-
nosis must be based on finding large numbers of erythro-
blasts by microscopic examination of a blood smear and
sections or smears of liver and bone marrow. Chickens in
early stages of disease or without obvious signs may be
missed easily unless microscopic examination is made.
Erythroblastosis with concurrent anemia is often dif-
ficult to differentiate from anemia resulting from non‐
neoplastic causes. In erythroblastosis, there is usually a
defect in maturation of erythroblasts, resulting in the
presence of large numbers of them and very few poly-
chrome erythrocytes. In anemia, the reverse usually
occurs. Extramedullary erythropoiesis and stasis of
erythroblasts in the sinusoids are usually more promi-
nent in erythroblastosis than in anemia.
Erythroblastosis can be distinguished from myeloblas-
tosis on the following grounds. In myeloblastosis, the
liver is usually pale red and the marrow is whitish;
whereas in erythroblastosis, the liver and marrow are
usually cherry red (Figures 15.30B and 15.30C). In mye-
loblastosis, the cells accumulate intravascularly and
extravascularly, whereas in erythroblastosis they are
always intravascular. The erythroblast and myeloblast
may be difficult to distinguish. Erythroblasts have a
basophilic cytoplasm and perinuclear halo; myeloblasts
often have some granules (Figures 15.30D and 15.30E).
Erythroblasts are cells of the erythropoietic system and
can be differentiated from cells of the myelopoietic system
on the basis of the presence of certain markers. Thus,
erythroblasts have erythroid markers including hemo-
globin, chicken erythrocyte‐specific histone H5, and
chicken erythrocyte‐specific cell surface antigens detected
by immunofluorescence. Myeloblasts and myelocytes have
myeloid markers including adherence and phagocytic
capacity, Fc receptors as determined by rosette formation,
macrophage‐ and granulocyte‐specific cell surface antigen
as detected by immunofluorescence, and dependence of
colony formation on colony‐stimulating factor (165, 259).
Erythroblastosis can be distinguished from LL by the
nature and distribution of lesions. Microscopically, the
cytoplasm of lymphoblasts is somewhat less basophilic
than that of erythroblasts, and there is also a larger
nuclear<thin: cytoplasmic ratio than in the latter cells.
Lymphoblasts are more variable in size and shape than
erythroblasts, but they are all at the same primitive
developmental stage. Lymphoblasts tend to have an
ovoid rather than spherical nucleus and a finer, more
delicate‐looking chromatin network. Myelocytomas and
erythroblastosis can be distinguished histologically.
Myeloblastosis
As in erythroblastosis, a tentative diagnosis may be based
on gross lesions; however, these are often so similar to
Chapter 15  Neoplastic Diseases 623
those of LL that specific diagnosis cannot be made with-
out examination of a blood smear. Examination of liver
or bone marrow sections is helpful when identity of the
cell type is in doubt. The myeloblast is, on the average,
smaller than the erythroblast or lymphoblast; its cyto-
plasm is more acidophilic and is polygonal or angular.
The nucleus is less vesicular; the nucleolus, while pre-
sent, is not nearly so frequently seen or conspicuous as in
the other two leukoses. Myeloblasts also have physio-
logic markers that identify them as members of the
­myeloid series.
Myelocytomatosis
The distinctive character and location of tumor (see the
previous discussion) provide the basis for diagnosis,
which can be verified by examination of a stained smear
or tumor section. Gross tumors must be differentiated
from myeloblastosis, LL, osteopetrosis, and necrotic
and/or purulent processes occurring in tuberculosis,
pullorum disease, and mycotic infections. In recent out-
breaks of ALV‐J‐induced tumors, myelocytomatosis was
diagnosed primarily on the basis of presence of charac-
teristic microscopic feature of tumor cells (see 266).
Hemangioma
Hemangiomas on the skin should be differentiated from
wounds, bleeding feather follicles, and cannibalism.
Those in the visceral organs should be differentiated
from hemorrhages and sarcomas.
Renal Tumors
Renal tumors should be suspected when tumor nodules
or large masses are found only in the kidney or are
encountered suspended from the lumbar region.
Diagnosis can be verified by microscopic examination.
Tumors should be differentiated from other causes of
kidney enlargement including hematomata, LL, and
accumulation of urates.
Osteopetrosis
Bone lesions of advanced cases are sufficiently distinc-
tive to present no difficulty in diagnosis. Cross‐ and
­longitudinal‐sectioning of long bones is helpful in detect-
ing slight exostoses and endostoses, particularly in early
stages.
Intervention Strategies
Vaccination
No commercial vaccine is available for the protection of
chickens from infection with ALV. However, the idea of
using vaccines to increase host resistance to ALV infec-
tion is very attractive (347). In a series of attempts to
624 Section II  Viral Diseases
inactivate ALV by various means, however, Burmester
(47) demonstrated that ability of these virus preparations
to induce antibody was destroyed almost concurrently
with inactivation. Attempts to produce attenuated
strains of ALV that do not induce disease have also failed
(283). Results of experimental vaccination with live ALV
on shedding and congenital transmission of the virus are
equivocal. Some success has been obtained in attempts
to increase the resistance of the host to RSV by immuni-
zation with viral or cellular antigens (33, 295).
Recombinant ALV‐J gp85 protein vaccine with either a
liposomal, a cytosine–phosphate–guanine oligodeoxy-
nucleotide (CpG‐ODN), or silica nanoparticles adjuvant
provided partial protection and elicited high antibody
titers (65, 110, 449, 452). Recombinant ALVs expressing
subgroup A (60, 125, 243, 442) and ALV‐J (220, 410)
envelope glycoproteins have been produced that could
have potential as vaccines to protect against horizontal
transmission. It is worth noting that congenitally infected
chicks are immunologically tolerant and, thus, cannot be
immunized even if a suitable vaccine was available. These
chickens constitute the major source of ALV transmis-
sion and are the most likely to develop neoplasms.
Treatment
No practical measures have been found for treatment of
the various forms of the avian leukosis complex. In
­general, all attempts to treat virus‐induced neoplasia
have resulted in negative or non‐reproducible results.
RNA interference (RNAi)‐based methods of inhibiting
ALV replication have been demonstrated experimentally
(62, 247) although its value in future treatment cannot be
predicted. Recombinant chicken interferon‐alpha can
inhibit ALV replication in DF‐1 cells and could be useful
for antiviral approaches (95).
Prevention and Control Procedures
Eradication
Eradication of ALV from primary breeding stocks is the
most effective means for controlling ALV infection in
chickens. Primary breeding companies of layer‐type and
meat‐type stock have made significant progress in reduc-
ing or eradicating ALV of subgroups A, B, and J from
their elite breeding lines (306).
Programs for eradication of ALV infection depends on
breaking the vertical transmission of virus from dam to
progeny. Breeder hens are tested by various methods for
the presence of ALV, and those that test positive are dis-
carded. In order to establish an ALV‐free flock, it is nec-
essary to hatch, rear, and maintain in isolation a group of
chickens free from congenital infection. To achieve this,
embryos must be obtained from dams that are not
­transmitting virus to their progeny. In earlier work on
development of ALV‐free flocks, several methods for
selecting dams were used. The dams selected to produce
the next generation and hoped to be a virus‐free genera-
tion were:
1) immune, non‐virus shedders. Hens with antibody
were selected on the assumption that they were less
likely to shed virus than hens without antibody.
Chicks were hatched from those that did not transmit
virus to their embryos, based on tests on at least three
embryos per hen.
2) Nonimmune, nonvirus shedders. Hens without anti-
body were selected on the assumption that they never
been infected and were less likely to become intermit-
tent shedders.
3) Nonviremic hens regardless of immune status. These
were used to provide replacements; however, up to
four generations of testing were needed to establish
freedom, and even then infection could not be ruled
out (446).
Application of eradication programs of ALV to com-
mercial flocks has depended on associations between
virus infections in hens, eggs, embryos, and chicks (375):
(1) egg albumen may contain exogenous ALV and gs
antigen, and both are usually present together; (2) a
strong association exists between ALV or gs antigen in
egg albumen and ALV in vaginal swabs; (3) an associa-
tion exists between ALV in vaginal swabs or egg albumen
and ALV in chicken embryos and newly hatched chicks.
Consequently, hens with a low probability of producing
infected embryos are hens negative for virus (or gs anti-
gen) by the vaginal swab test, or hens that produce eggs
with albumen free from virus or gs antigen. Commonly,
virus in vaginal or cloacal swabs may be detected by
ELISA, NP, or PM tests and in egg albumen by ELISA or
direct COFAL tests. It is unlikely that a single test will
detect all potential shedder hens. A problem that arises
in applying the ELISA test to albumen or swabs is the
need to differentiate positive reactions due to the pres-
ence of gs antigen derived from endogenous ALV or loci
from the reactions due to the presence of exogenous
ALV infection. Reactions due to the latter are usually
markedly higher, but the setting of the boundary between
endogenous and exogenous virus infections is some-
times difficult and somewhat arbitrary. High reactions
due to exogenous virus are clearer with albumen samples
than with swabs (89).
A procedure for eradication of ALV involves: (1) selec-
tion of fertile eggs from hens negative in the egg albumen
or vaginal swab test (reviewed in 266); (2) hatching of
chicks in isolation in small groups (25–50) in wire‐
floored cages, avoidance of manual vent sexing and vac-
cination with a common needle to prevent mechanical
spread of any residual infection; (3) testing of chicks for
ALV by a biologic assay or PCR on blood, discarding

reactors and contact chicks; and (4) rearing ALV‐free
groups in isolation (130, 440). In practice, selection of
hens with a low shedding rate is a simpler requirement to
fulfil than the subsequent chick testing and isolation
rearing needed to achieve complete eradication.
Consequently, some commercial breeder organizations
are concentrating only on reduction of infection rate by
hen testing. Small group hatching and rearing proce-
dures allowed identification and removal of groups con-
taining chickens infected prior to hatching and prevented
horizontal transmission of ALV‐A in egg‐type chickens
and ALV‐J in meat‐type chickens (440).
Chicks are most susceptible to contact infection by
ALVs during the period immediately after hatching.
Although congenitally infected hatchmates are likely to
be the main source of such infection, several procedures
can reduce or eliminate infection remaining from previ-
ous populations. Incubators, hatchers, brooding houses,
and all equipment should be thoroughly cleaned and dis-
infected between each use. Chick boxes should not be
reused, and each farm ideally should have only one age
group of chickens. Demonstration of natural infection
and transmission of MAV‐1 in egg‐type chickens stress
the importance of testing of birds to prevent introduc-
tion of infection (447). The danger of introducing strains
of virus not already present in the population can be
eliminated if eggs or chicks from different sources are
not mixed, and if chicks are reared under isolation, con-
ditions that will prevent cross‐contamination of flocks.
Selection for Genetic Resistance
The frequencies of the alleles that encode cellular sus-
ceptibility and resistance to infection by exogenous
ALSVs (see Genetic Resistance) vary greatly among
­Reticuloendotheliosis
Guillermo Zavala and Venugopal Nair
Summary
Agent and Disease.  Reticuloendotheliosis (RE) represents a
group of syndromes associated with a common, but not
ubiquitous retrovirus designated reticuloendotheliosis
virus (REV). The syndromes include chronic lymphoid
neoplasia, runting disease syndrome or acute reticulum cell
neoplasia. Reticuloendotheliosis virus also poses significant
danger as a contaminating pathogen in avian vaccines.
Diagnosis.  Clinical diagnosis of the disease is challenging
because of the difficulty in differentiating from
other  avian neoplastic diseases. Hence virological,
molecular, and serological diagnostic tests are needed
for confirmation.
Chapter 15  Neoplastic Diseases 625
commercial lines of chickens (84, 260). In some lines,
high frequencies of a resistant allele may be found natu-
rally. In others, frequencies of the resistant alleles can be
increased by artificial selection.
In artificial selection, genotypes of unknown parents
may be determined in a progeny test by mating them to
recessive tester birds of the subgroup in question (e.g.,
arar for A subgroup virus) (293). Depending on the seg-
regation of susceptible and resistant progeny in a par-
ticular mating, the genotype of the unknown parent may
be determined. The phenotypic identification of progeny
in the test may be determined by inoculation of RSV
onto the CAM, the embryo being scored as susceptible
or resistant on the basis of pock count (86) or intracra-
nial inoculation of RSV into hatched chicks, chicks being
scored on the basis of death or survival. The former
method is preferable and has many advantages.
There are concerns whether genetic selection approach
could cause problems with generation of mutant viruses
that may overcome the restriction imposed by this selec-
tion (78). However, this type of resistance is poorly
defined but may be controlled by a number of genes and
is, consequently, more difficult to overcome by viral
mutation.
Recent technological advances in genome editing have
already demonstrated the feasibility of this approach in
generating cell lines resistant to infections with different
ALV subgroups (217, 218). Success in the application of
new methodologies of avian transgenesis using geneti-
cally modified primordial germ cells (174, 271) point
toward the feasibility of using these approaches for
experimental generation of resistant stock as another
tool for the control of avian retroviral diseases in
poultry.
Intervention.  Eradication of the pathogen by testing and
eliminating infected birds is the best intervention
strategy. However, vaccination has very little value,
except perhaps in endangered avian species.
Introduction
Definition and Synonyms
Reticuloendotheliosis (RE) designates a group of syn-
dromes associated with chronic and acute neoplasia,
immunosuppression, runting disease, and acute death in
several avian species caused by retroviruses of the reticu-
loendotheliosis virus (REV) group. Clinical disease is
infrequent but infection appears to be widespread.
626 Section II  Viral Diseases
The REV group includes the laboratory strain T
(REV‐T), chick syncytial virus (52), duck infectious ane-
mia virus (140), and spleen necrosis virus (230). Many
other nondefective strains have been isolated from avian
species including turkeys, chickens, ducks, pheasants,
partridges, geese, prairie chickens, sparrows, and pigeons
(87, 166, 231, 271). Nondefective strains associated with
runting disease and chronic neoplasia belong to a single
serotype, but three antigenic subtypes have been identi-
fied (46). The acute reticulum cell neoplasia is induced
only by the laboratory‐derived defective strain T (REV‐T)
not known to occur in nature. REV‐T carries a unique
oncogene of cellular origin (v‐rel) that is responsible for
its acute oncogenicity (101, 102). Stocks of REV‐T also
contain a nondefective helper REV designated as REV‐A
that replicates in chicken fibroblasts but lacks acute
oncogenic properties (101).
Economic Significance
Severe runting syndrome, feathering abnormalities,
chronic neoplasia, or immunosuppression have occurred
when REV‐contaminated vaccines were administered to
embryos or very young chickens (85, 112, 127–129, 267).
Clinical disease from natural infection is relatively rare,
but exports of seropositive breeding stock may be pro-
hibited. Vaccine and specific pathogen free companies
must routinely monitor their products for REV contami-
nation. Significant immunosuppression may result from
environmental exposure, administration of contami-
nated vaccines, or congenital transmission (250, 267).
Public Health Significance
The extended host range of REVs, which includes certain
mammalian cells (2, 171, 243) and other characteristics
of REV suggesting an evolutionary linkage with mamma-
lian retroviruses (15, 126, 171, 189), raised the possibility
of human infection (113). Reticuloendotheliosis virus
antibodies have been detected in human sera (49, 114,
115), but such findings have been considered insufficient
to warrant concern (68, 70, 71, 94, 200).
Scientific Significance
Reticuloendotheliosis virus has received considerable
attention as an oncogenic and immunosuppressive virus
with a wide host range (171). Reticuloendotheliosis virus
can infect or transform various cell types (2, 15) and has
been used in comparative retrovirology models. Some
REV subgenomic sequences display high similarity to
those of some mammalian retroviruses (17, 171).
Reticuloendotheliosis virus can integrate into the
genome of cells and of large DNA viruses, including
Marek’s disease (MD) and fowl pox viruses (100, 110).
Recent historical, phylogenetic, and paleovirological
­evidence suggest the origin of REV as a mammalian ret-
rovirus that was iatrogenically introduced into the avian
hosts which subsequently spread through herpesviruses
and poxviruses (77, 171).
History
The the defective strain T (REV‐T), was obtained in 1957
from turkey visceral lymphomas (191). REV‐T is acutely
oncogenic, causing death of young chicks 6–21 days
postinoculation (207). Theilen et al. confirmed the acute
oncogenicity of REV‐T for young chickens, turkeys, and
Japanese quail; and designated the disease as reticuloen-
dotheliosis on the basis of the prominent cell type in
tumors (227), now termed acute reticulum cell neopla-
sia. Importantly, Theilen et  al. (227) propagated the
strain in cell culture, determined it to be a retrovirus
unrelated to avian leukosis virus, and named it “reticu-
loendotheliosis” virus (strain T).
Purchase (182, 183) recognized the antigenic relation-
ships between REV‐T, the nononcogenic chick syncytial
spleen necrosis and duck infectious anemia viruses, all
within the reticuloendotheliosis virus group. Thus,
nomenclature for the disease and the virus originated
from the atypical pathology induced by REV‐T and was
extended to all viruses in the group.
Additional reviews on the history of REV are available
for consultation (147, 166, 171, 177, 183, 249).
Etiology
Classification
Reticuloendotheliosis viruses are retroviruses that are anti-
genically, morphologically, and structurally distinct from
the leukosis/sarcoma (L/S) group of avian retroviruses
(ALSV) (183). Lack of nucleic acid sequence homology
between REV and members of the ALSV group has been
long recognized (118). The International Committee on
Taxonomy of Viruses (ICTV 9th Report, 2011) has classi-
fied REVs within the family Retroviridae, subfamily
Orthoretrovirinae, genus Gammaretrovirus, with no
endogenous counterpart. Reticuloendotheliosis virus is
phylogenetically related to mammalian C‐type retroviruses
based on virion morphology, nucleic acid and major poly-
peptide amino acid sequences (171), as well as immuno-
logic determinants and receptor interference patterns (122).
Morphology
Viral particles are typical of retroviruses, with about
100 nm in diameter (270); display surface projections

about 6 nm long and 10 nm in diameter (116). Virions
have a density of 1.16–1.18 g/mL in sucrose density
­gradients (23), and can be differentiated from avian L/S
viruses by morphology in thin sections (153, 272).
The  morphology of the viral particles is shown in
Figure 15.46.
Chemical Composition
Nucleic Acid
The genomic single‐stranded, positive sense RNA con-
sists of a 60–70 S complex containing two 30–40 S RNA
subunits, each having a size of about 3.9 × 106 d (25, 142).
The nondefective REV has a genome of about 9.0 kb
(17), while the replication‐defective REV‐T genome is
only about 5.7 kb due principally to a large deletion in
the gag‐pol region and a smaller deletion in the env
region (50). Moreover, the REV‐T genome contains a
substitution of 0.8–1.5 kb in the env region that repre-
sents the transforming gene v‐rel (44, 51, 261), which is
not present in nondefective REVs or other avian or
mammalian retroviruses. Related sequences (c‐rel
proto‐oncogene) are present in the DNA of normal
avian cells, including turkey cells from where the onco-
gene was most likely transduced (44, 45, 247, 248). No
endogenous REV sequences in host DNA have been rec-
ognized. The long terminal repeats (LTRs), 569 base
pairs (bp) in length (17) are efficient promoters in a vari-
ety of cell types (14, 17, 138). Several complete genome
sequences of various field and vaccine‐contaminant iso-
lates from China and the United States have been
resolved (14, 17, 138).
(A)
Figure 15.46  Electron micrographs of thin sections of chicken embryo fibroblasts infected with reticuloendotheliosis virus (REV). (A)
Typical virus particles in the extracellular spaces ×40,000. (B) REV particles budding from the plasma membrane of infected cells (arrow).
×60,000. (Nazerian).
Chapter 15  Neoplastic Diseases 627
Oncogene
The v‐rel oncogene is transcribed in REV‐T‐transformed
lymphoid cells and produces a phosphoprotein product
identified as pp59v‐rel. The v‐rel protein is a member of the
rel/dorsal family of proteins, related to nuclear factor kappa
B, which function as DNA‐binding transcription factors (26,
194). It differs from the c‐rel protein both in structure and
transforming ability and, unlike most other oncogene prod-
ucts, can be detected in both the cytoplasm and nucleus of
transformed cells (29). The v‐rel protein is usually com-
plexed with cellular proteins (124, 136, 216) and is responsi-
ble for the acute oncogenicity of REV‐T (29). REV‐T‐induced
transformation is associated with several changes in gene
expression, including induction of miRNAs (264). However,
REV isolates other than REV‐T have induced neoplastic dis-
ease within very short latent periods (18, 72, 73, 184, 186).
Proteins
Reticuloendotheliosis virus genes encode various struc-
tural proteins, a protease, a polymerase and an integrase
(17). The protein encoded by the v‐rel gene is only pre-
sent in the defective REV‐T. The RNA‐dependent DNA
polymerase (reverse transcriptase) differs structurally
and immunologically from comparable enzymes of
l­eukosis/sarcoma viruses (22, 153). The preference of the
REV polymerase for Mn2+ ions is a differentiating factor
from enzymes of other avian retroviruses (153, 203, 263).
The envelope protein is composed of two peptides, the
gp90 surface unit (SU) and the gp20 transmembrane unit
(TU) (233, 234). These glycoproteins are located on the
surface of the virions (156). The C‐terminal epitope
of  gp90 locates on the surface of infected cells (234).
(B)
628 Section II  Viral Diseases
The  gp90 protein is considered the immunodominant
protein of the virus (64). The receptor binding regions
have been mapped and display structural differences
relative to other retroviruses (144).
There are five gag gene‐encoded structural proteins,
p12, pp18, pp20, p30, and p10 (232). Antiserum to the
30 kDa (p30) protein cross‐reacted with p30 of several
other REVs, thus establishing this protein as a group‐
specific antigen (143) that also plays a role in viral parti-
cle assembly and encapsidation (244).
Replication
Non‐Defective Strains
In vitro virus replication is similar to that of other retro-
viruses (70). The virus envelope glycoprotein binds to an
unidentified cell surface receptor, resulting in interfer-
ence with superinfection (86). Viral entry, RNA reverse
transcription and proviral DNA integration into the cel-
lular chromosome proceed through mechanisms typical
of simple retroviruses. Viral RNA transcription and
translation are initiated through promoter and enhancer
sequences in the LTR. Two polyproteins are encoded,
gag‐pro‐pol and env; the gag precursor protein is myri-
stylated. The encapsidation sequence is located in the
gag gene. The final stage is budding of viral particles
from the cell membrane. Virus particle production is
first noted at 24 hours (116), and peaks 2–4 (101) days
after infection in chicken cells (30, 90, 223, 224).
Defective Strain
The defective REV‐T requires a nondefective RE helper
virus for replication (101). Oncogenicity of this strain is
maintained during passage in vivo (191) or during cul-
ture of infected hematopoietic cells (101), but is rapidly
lost during passage in fibroblast (227, 257) and dog
­thymus cell cultures (2), possibly due to the loss of the
replication‐defective, acutely oncogenic REV after serial
passages (31).
Cytopathology
Replication of REV in avian fibroblasts may induce sub-
tle cytopathic changes (227), such as syncytia (52) but
degenerative changes are more commonly seen (223,
225). Accumulation of unintegrated viral linear DNA in
infected cells possibly causes cell death. Cells that are
able to prevent early superinfection have few copies of
unintegrated viral DNA and survive (225).
The acute cell death phase (Figures 15.47A and 15.47B)
lasts 2–10 days postinfection and is followed by a chronic
infection state without cytopathology but with contin-
ued virus production (Figure  15.47C) (223, 224). This
cytopathology is the basis of plaque assays (35, 47, 48,
155, 223), but the methods have not been widely used
due to inconsistency in cytopathology.
Host Range
Cell cultures from many avian species and certain mam-
malian cells are susceptible to infection and at least limited
viral replication. Nondefective REV has been grown in D17
dog sarcoma cells (15, 242, 243), Cf2th dog thymus cells (2,
209), normal rat kidney cells (121), mink lung cells (2), and
other mammalian cells. Rat and mouse cells were only
semi‐permissive for replication of REV (75, 76). Chimeric
vector particles containing the REV‐A matrix protein
infected mammalian cells more efficiently than those con-
taining the matrix protein of spleen necrosis virus (43). A
wide range of avian species support REV replication in vivo
but there is little evidence for in vivo replication of REV in
non‐avian species. Presence of REV antibodies in humans
has been reported (114) but the significance is unknown at
best (200). Although REV‐A‐based vectors could infect
human cells (125), REVs did not infect human cells due to
inability to bind to specific receptor (94).
Pseudotypes
The envelope of nondefective REV forms pseudotypes
with Rous sarcoma virus (198, 235) and with vesicular
stomatitis virus (117). Pseudotypes can be neutralized by
REV antiserum, and thus this assay was once used for
antibody detection (56).
Insertional Mutagenesis
Replication of REV requires integration of proviral DNA
into the host cell genome. REV proviral DNA can also
integrate into the genomes of high molecular weight
DNA viruses including MD virus (111) and fowl poxvirus
(92, 100). Insertions occur both in vitro and in vivo (60)
and may result from coinfections of REV and a recipient
DNA virus. Most insertions consist of a solitary, partially‐
deleted LTR (60, 154). However, full‐length, infectious
REV genomes have been detected in turkey herpesvirus
(110). A nearly full‐length, infectious REV provirus has
been detected (92, 100, 123, 210) in certain strains of fowl
poxvirus, even in fowl poxvirus stocks lyophilized for
over 50 years (123). Such insertions could alter the
­biological properties of the recipient organism and also
represent a distinct mechanism of infection.
Strain Classification
The antigenic properties of different REV isolates are
remarkably uniform (33, 182) and, except for defective
REV‐T, REV isolates have similar genetic, structural, and
chemical properties (22, 119). Although REVs belong to
a single serotype (46), three subtypes were identified on
the basis of neutralization tests and differential reactivity
with monoclonal antibodies (46, 57), although subtypes
1 and 2 could not be differentiated by receptor interfer-
ence (86), confirming the absence of major differences
between subtypes. Reticuloendotheliosis virus isolates
 Chapter 15  Neoplastic Diseases 629

differ also in certain biologic properties, including path- Cell Lines

ogenicity (183) and replication in vivo.
Laboratory Host Systems
Cell Cultures
Fibroblasts from several avian species and certain cell
lines, such as QT35 quail sarcoma cells (48, 54) and D17
dog osteosarcoma cells (15, 243), are susceptible to infec-
tion with nondefective REVs. In infected cultures, anti-
gens (Figures 15.47B and 15.47C), virus particles, proviral
DNA, cytopathology, and reverse transcriptase may be
detected and serve as criteria for virus assays. A quanti-
tative fluorescent focus assay has been developed for
infected cultured cells overlaid with agar (182). Duck
embryo fibroblasts are preferred for demonstration of
cytopathic effects (13). However, virus cultures in
chicken embryo fibroblasts or DF‐1 cells combined with
molecular detection of REV or identification of viral pro-
teins by direct or indirect immunofluorescence appear
to be most practical and efficient (17, 18).
Embryos and Birds
Laboratory host systems for REV that are now seldom
used include chicken embryos (208) and a variety of
avian species including young chickens, Japanese quail,
ducks, geese, turkeys, pheasants, and guinea fowl (18,
186, 227).
Hematopoietic cells transformed in vivo or in vitro by
REV‐T have been developed into continuous cell lines;
the cell types and surface markers vary based on the
strain of helper virus and on whether transformation
occurred in vivo or in vitro (29, 105). A line of trans-
formed chicken embryo fibroblasts has also been devel-
oped (89). Cell lines have also been derived from chronic
lymphomas induced by nondefective REV strains (168,
184). Cell lines induced by in vitro transformation of
spleen cells with defective REV are useful expression
­systems for transfected foreign genes (181, 202) or as
substrates for the propagation of other viruses (187).
Some of these transformed cell lines produce growth
­factors or cytokines (69, 93, 95).
Pathobiology and Epidemiology
Incidence and Distribution
Reticuloendotheliosis virus infection is common but not
ubiquitous in turkeys, ducks, and chickens. The preva-
lence of seropositive flocks and of seropositive birds
within an infected flock both increase with the age of the
flock. A high prevalence of virus or antibodies has been
detected in the United States, Japan, Korea, and Egypt (4,
7, 13, 206, 237, 262). Seropositive commercial chicken

(A) (B) (C)

Figure 15.47  Acute (cytopathic) and chronic (noncytopathic) infection of chicken embryo fibroblasts inoculated with nondefective
reticuloendotheliosis virus (REV) strain. (A) Mild cytopathic changes 13 days after infection. Unstained, ×55. (B) Cytopathic changes and
viral antigens 13 days after infection demonstrated by indirect immunofluorescent staining. (C) Chronically infected cultures 48 days after
infection showing relatively normal‐appearing cells, most of which contain cytoplasmic viral antigens. ×360.
630 Section II  Viral Diseases
flocks are still common in the United States, with sporadic
to negligible clinical disease. Reticuloendotheliosis virus
infection is a significant clinical problem in wild endan-
gered avian species (17, 74, 87, 268). Reticuloendotheliosis
virus is frequently detected alone or in combination with
fowl poxvirus or lymphoproliferative disease virus in wild
turkeys of the United States (1, 3).
Runting disease syndrome and chronic neoplasia
have occurred following vaccination with REV‐contam-
inated vaccines (13, 85, 112, 120, 185, 265, 266).
Immunosuppressive disease has been identified after
natural infection in Korea (206). Field cases of RE‐
related lymphomas in turkeys have been described in
the United States (55, 175, 215, 252, 258, 259), England
(147), and Israel (107). Losses from mortality and con-
demnation at slaughter in affected flocks could be as
high as 16–20% (146, 175). Lymphomas associated with
natural REV infection are sometimes reported in wild
turkeys (99, 132) and less often in chickens (61, 107, 150,
173). Chronic REV‐induced neoplasia has also been
occasionally observed in ducks (97, 176), quail (39, 201),
pheasants (73), geese (72), peafowl (152), and prairie
chickens (74, 268).
Natural and Experimental Hosts
Natural hosts for REV infection include turkeys, chick-
ens, ducks, geese, pheasants, Japanese quail, peafowl,
and prairie chickens. Experimental hosts include most of
the above species, as well as guinea fowl, chickens,
­turkeys, and Japanese quail (18).
Transmission, Vectors, Carriers
Horizontal Transmission
Experimentally, REV can be transmitted by close contact
with infected chickens, turkeys, and ducks (130, 158, 174).
Horizontal transmission may be influenced by the
host  species (182, 254) and the virus strain (254, 266).
Reticuloendotheliosis virus transmission was not detected
when chickens were separated by wire mesh (12).
Many flocks become infected at older ages (254) pre-
sumably via contaminated environment, insects, and
other biological reservoirs. Reticuloendotheliosis virus
has been detected in feces and cloacal swabs (10, 179,
260, 266), body fluids (12), and litter (241). Horizontal
transmission was limited amongst experimentally
infected Japanese quail housed on litter (18). Flocks
infected experimentally (Zavala, unpublished) or natu-
rally at later ages seroconvert, making virus detection
difficult (255). Furthermore, REVs are quickly degraded
outside the host at ambient temperatures (37).
Insect transmission represents another form of hori-
zontal spread. Virus could be recovered from Triatoma
infestans and Ornithodoros moubata after feeding on
infected chickens (62, 228, 229) and for up to five hours
post‐feeding on contaminated blood in some species of
mosquitoes (62). Reticuloendotheliosis virus has also
been isolated from mosquitoes (Culex annulirostris)
(164) in contact with viremic chickens, demonstrating
the possibility of mechanical transmission by insects,
which may explain seasonal seroconversion (63, 164) and
a higher prevalence of infection in Southern states (254,
256). Fowl poxvirus, which is also transmitted by mos-
quitoes, may function as a vector containing infectious
clones of REV (92, 100, 211).
Vertical Transmission
Chickens, turkeys, ducks, and quail with persistent
viremia may transmit infectious REV to progeny,
although usually at low frequency or not at all (10, 12, 18,
148, 236, 260). However, some studies have reported
experimental vertical transmission to over 50% of chicks
from infected dams (163). Albumen samples from toler-
ant hens frequently contained RE viral gs antigen,
although infectious virus was rarely isolated (260).
Vertical transmission may occur at higher rates in ducks,
since virus was isolated from 87% of embryos derived
from tolerant females (158). Individual antibody‐­
positive, virus‐positive turkey hens may still transmit
virus to progeny at a high rate (258).
Semen from tolerant turkeys contains infectious virus
(149, 257). Reticuloendotheliosis virus‐free turkey hens
inseminated with contaminated semen have produced
infected progeny (148). There is evidence of limited
vertical transmission after mating infected males with
noninfected female chickens (195). Genetic transmis-
sion is unlikely based on lack of clonal insertions of pro-
viral DNA in infected hens or their progeny (258).
Iatrogenic infection is possible by using accidentally
contaminated needles or vaccines for embryos or very
young chickens (13).
Contaminated Biological Materials
Accidental use of REV‐contaminated fowl pox (85, 138)
or MD (112, 120, 134, 135, 138, 245, 266) vaccines
has  been documented. Reticuloendotheliosis virus has
also been detected in contaminated vaccines against
Newcastle disease and infectious bronchitis (245).
Certain stocks of avian myeloblastosis virus, for many
years distributed as a source of reverse transcriptase for
biochemical purposes, contained a low level of REV
(256). Quality control procedures to exclude REV from
licensed poultry biologics have not always been uni-
formly effective for detection of REV contamination in
vaccines such as fowl pox (78, 84). Subgenomic REV
sequences continue to be found in some (67, 92, 100,
210), but not all (154), commercially produced fowl pox
virus vaccines. Reticuloendotheliosis virus has also been

detected in stocks of Plasmodium lophurae (140, 230),
further illustrating the diversity of possible transmission
mechanisms for REV.
Incubation Period
The runting disease syndrome represents a non‐neo-
plastic disease process with an outcome depending on
virus strain and other factors. Atrophic changes in the
bursa and thymus can be seen as early as three days
postinfection (259). Persistent weight depression in
infected chicks can be detected as early as six days of age
(159). By the second week postinoculation, chickens
developed microscopic nerve lesions (257) and had
depressed immune responses (255). Japanese quail
infected experimentally displayed severe weight depres-
sion as early as 14 days of age and lymphomas were
detected as early as 35 days of age (16).
Chronic neoplastic responses occur after moderate or
long incubation periods. Chickens developed bursal‐
derived B‐cell lymphomas 17–43 weeks after inoculation
(260). Reticuloendotheliosis virus‐associated lymphomas
in turkeys occurred between 15 and 20 weeks of age in
some trials (146, 175) and as early as 9 weeks post‐infection
in SPF turkeys (Zavala, unpublished). In transmission stud-
ies, lymphomas were induced after 8–11 weeks (174) or
11–12 weeks (146). Chronic lymphomas occur between 20
and 30 weeks in the domestic goose (72), and at 4–24 weeks
in ducks (97, 176, 178). Experimental inoculation of newly
hatched ducks induced lymphomas and other neoplasms
between 8 and 30 weeks (133, 158).
For acute reticulum cell neoplasia, the incubation
period can be as short as 3 days, but death occurs more
commonly 6–21 days after inoculation (207). Because of
the short latent period and high mortality induced by
REV‐T, this virus has been regarded as the most virulent
of all retroviruses (29).
Clinical Signs
Chickens and quail with runting disease syndrome may be
notably stunted and pale (16, 165). Weights of infected
chickens and quail may be 20–50% lower than controls by
3–5 weeks after infection (16, 255, 257). Weight depression
has also been seen in infected ducks (182). Some chickens
may display abnormal feather development, termed
Nakanuke, that is, wing feathers with focal adhesion of the
barbs to the shaft (127, 128). Lameness or paralysis is rare
even in birds with gross nerve lesions. Mortality might be
rare in chickens (255) but affected birds in commercial
flocks may be culled prior to death; a culling loss of over 50%
between 5 and 8 weeks was described in one flock iatrogeni-
cally infected with a ­contaminated MD vaccine (221).
Birds developing chronic lymphomas or acute reticu-
lum cell neoplasia after REV‐T infection show few
Chapter 15  Neoplastic Diseases 631
c­ linical signs due to the rapid onset of the disease, and
mortality rates often reach 100% (208, 227).
Pathology
Runting Disease Syndrome
In chickens, the principal changes include runting (165,
257), thymic and bursal atrophy (165), enlarged peripheral
nerves (257), abnormal feathering (112, 127–129), proven-
triculitis (112), enteritis (146), anemia (120, 140), and
hepatic and splenic necrosis (183, 230), often accompa-
nied by depression of cellular and humoral immune
responses (17, 32, 40–42, 107, 120, 255). The acute hem-
orrhagic or chronic ulcerative proventriculitis observed in
field cases (112) could not be reproduced (11) with a simi-
lar isolate. The proliferative lesions in enlarged peripheral
nerves often occur in the absence of other neoplasms and
it is not known whether the changes are neoplastic or
inflammatory (257). The infiltrating cells, which include
lymphocytes and plasma cells, are shown in Figure 15.48.
Ducks inoculated with the spleen necrosis or duck
infectious anemia strains of REV may display some fea-
tures of the runting disease syndrome (140, 230).
Hematocrit values in ducks inoculated with spleen
necrosis strain can be as low as 20%, compared to 35%
for control ducks (230). Enlarged nerves (174, 175) or
enteritis (146) have been observed in turkeys with RE‐
related chronic lymphomas.
Genetic differences in susceptibility have not yet been
described; chicks from lines of different susceptibility to
MD were equally susceptible to the development of
nerve lesions following inoculation with REV (257).
Most nondefective REV strains, when inoculated at
hatch, induce high frequencies of gross lesions (182, 255)
Figure 15.48  Microscopic lesions in a peripheral nerve of a
chicken inoculated with nondefective reticuloendotheliosis virus
(REV) strain. Infiltrating cells consist of mature and immature
lymphocytes and plasma cells.
632 Section II  Viral Diseases
but others, such as chick syncytial strain, may induce
few, if any, lesions (255).
Chicken Bursal Lymphoma
Chickens inoculated with the nondefective chick syncyt-
ial or T strains developed B‐cell lymphomas, involving
principally the liver and bursa of Fabricius (251, 260).
The gross lesions were nodular or diffuse lymphoid
lesions in the liver, other visceral organs and the bursa of
Fabricius, all indistinguishable from lymphoid leukosis
(Figure  15.49). A few birds may develop sarcomas or
adenocarcinomas. The frequency of lymphomas was
­ spleen necrosis or chick syncytial strains of REV (259).
influenced by virus strain and whether a tolerant infec-
tion had been induced (260). Interestingly, coinfection of
chickens with serotype 2 MDV enhanced the incidence
of REV bursal lymphomas (6) as had also been reported
for lymphoid leukosis (8).
The tumor cells, which are uniform populations of
lymphoblasts, were identified as B cells by IgM and
other  B‐cell specific markers (168, 260). The bursal
dependency of this tumor was confirmed in chemically
or surgically bursectomized chickens, which were refrac-
tory to tumor development (82). Bursal lymphomas may
Figure 15.49  Bursal lymphoma in a chicken. Gross lymphomas in
the liver and bursa of a chicken 25 weeks after inoculation with
the nondefective chick syncytial strain of REV.
not always be present in field cases. Grimes et  al. (96)
observed what may be lymphomas in two chickens at 22
and 24 week after inoculation with a field strain of REV,
but no bursal involvement was reported. However, typi-
cal bursal lymphomas were observed in two chicken
flocks following administration of a REV‐contaminated
fowl pox vaccine (85).
Chicken Non‐Bursal Lymphoma
Chronic non‐bursal lymphomas have been described in
chickens following experimental infection with the
Grossly, these lymphomas are focal or diffuse lymphoid
infiltrations, with enlargements of the thymus, liver, and
spleen or focal lesions of the myocardium (Figure 15.50).
The bursa of Fabricius is not involved. Nerve enlarge-
ments may be seen. Histologically, the tumors appear to
be a uniform, immature lymphoreticular cell that lacked
B‐cell markers and did not express MATSA, a cellular
antigen associated with MD tumors (259) and also
expressed on activated T lymphocytes (145). The princi-
pal tumor cell type is a CD8+ T cell but Ia antigens are
not expressed (53).
Turkey Lymphoma
Chronic lymphomas in turkeys and other avian species
consist of gross lymphoid infiltrations in the liver, intes-
tine, spleen, and other viscera. Lymphomatous lesions in
the bursa have been reported (146, 174), but this lesion
was not common. Histologically, the lesions were com-
posed of uniform populations of lymphoreticular cells
Figure 15.50  Nonbursal lymphoma in a chicken 48 days
postinoculation with the nondefective spleen necrosis strain of
reticuloendotheliosis virus (REV). Note enlargement of spleen,
nodular lymphomas on heart, and bursal atrophy of infected
chicken (top row). Organs from age‐matched controls in the
bottom row (262).

(146, 174). Crespo et al. (55) described T‐cell lymphomas
associated with a natural outbreak of reticuloendothelio-
sis in turkeys.
Lymphomas of Other Species
Various other species develop chronic gross lympho-
matous lesions that are similar to those described for
chickens and turkeys. Lesions reported in ducks
include enlarged livers and spleens with diffuse or
focal involvement, intestinal lesions, and infiltrations
in skeletal muscle, pancreas, kidneys, heart, and other
tissues (97, 133, 158). Generalized leukemia in addi-
tion to visceral lymphomas in ducks has been described
(178). Similar tumors were described in geese, with
occasional lymphoproliferative lesions in the bursa of
Fabricius (72). Pheasants and prairie chickens dis-
played proliferative cutaneous lesions on the head and
mouth in addition to nodular visceral lymphomas (73,
74, 268). Outbreaks in quail were characterized by liver
and spleen enlargements (16, 18, 39, 201, 268) or intes-
tinal lesions (268). Histologically, tumors from all these
species generally resembled those described for chick-
ens and turkeys.
Acute Reticulum Cell Neoplasia
The pathology of acute reticulum cell neoplasia has been
well described (191, 227). Grossly, affected birds develop
infiltrative focal or diffuse lesions in the livers, spleens,
pancreas, gonads, heart, and kidney. The blood shows a
decrease in heterophils and an increase in lymphocytes
(222), leading to leukemia a few hours before death.
Histologically there is infiltration and proliferation of
cells described either as large mononuclear cells of the
reticuloendothelial system (227) or primitive mesenchy-
mal cells (191, 208). Some lesions are composed almost
solely of such cells, whereas others include also smaller
lymphoid elements, probably indicating a host immuno-
logic response. Areas of necrosis are also frequent.
A typical liver lesion is shown in Figure 15.51.
Multiple Syndromes
Lesions of different types can be observed in the
same  flock, experiment, or even in individual birds.
Nondefective REV strains may first induce runting
­disease syndrome and lymphomas may occur later in the
survivors, sometimes accompanied by peripheral nerve
enlargement.
Pathogenesis
Virus Infection
Tolerant infection with persistent viremia and absence
of antibodies is readily induced in turkeys (148, 258)
and chickens by embryo inoculation (107, 260); and
by  vertical transmission from infected dams (12).
Chapter 15  Neoplastic Diseases 633
Figure 15.51  Microscopic lesions of acute reticulum cell neoplasia
(reticuloendotheliosis) in the liver of a chicken inoculated with
replication‐defective, acutely transforming strain T REV. The liver is
infiltrated with large primitive reticular cells (arrow).
Persistent infections occur rarely following inoculation
at hatch (10, 148, 260) depending on the strain of
chicken (83); and are unlikely to occur if exposure
occurs at later ages. Some birds with persistent infec-
tions develop antibody responses. Tolerant infection is
associated with higher rates of vertical transmission
and tumor development, and birds are typically stunted
and immunodepressed. Birds exposed after hatch most
commonly develop transient viremia followed by anti-
body production (10, 255). Persistence of noninfectious
RE viral antigens in the blood for several weeks follow-
ing the disappearance of infectious virus has been
reported (10). Transient infection rarely results in
­vertical transmission, immunosuppression, or tumor
development. Infection of older birds rarely results in
clinical disease (179, 182, 254, 266) except, perhaps, in
turkeys in which lymphomas have been observed
­following contact exposure (148, 149, 174).
Various other factors influence susceptibility to infec-
tion or disease. No genetic cellular resistance has been
recognized. However, some differences in the pathologic
response of genetic lines or families has been recognized
in chickens (83, 205, 259) and quail (226). Nevertheless,
differences were not apparent when two different chicken
lines were challenged with serial dilutions of REV‐T
(207). Maternal antibodies appear to limit susceptibility
to infection (213).
Runting Disease Syndrome
The pathogenesis of runting disease syndrome has not
been elucidated. Stunted chickens did not consume less
feed, but had marked reduction of phosphoenolpyruvate
carboxykinase, a key gluconeogenic enzyme in the liver
(93). The adherence of feather barbules to the shaft
634 Section II  Viral Diseases
(Nakanuke) is apparently due to REV‐induced necrosis
of feather‐forming cells (219). The microscopic lesions
of chicks with runting syndrome resemble a graft versus
host reaction (165), but a specific autoimmune compo-
nent has not been identified.
Chronic Lymphomas
The integration site for the REV DNA provirus in the
cellular genome in bursal lymphomas is located adjacent
to c‐myc, a cellular oncogene important in the induction
of lymphoid leukosis by avian leukosis virus (ALV) (172).
The molecular mechanism by which c‐myc is activated
by insertion of REV proviral DNA has been described
(91, 190, 218). The proviral insert often contains major
deletions that prevent the expression of infectious virus
(217). Based on pathology, proviral insertional activation
of c‐myc, and enhancement by serotype 2 MDVs, bursal
lymphomas induced by REV, and ALV appear indistin-
guishable. However, some subtle differences have been
noted. For example, chickens of lines resistant and
­susceptible to lymphoid leukosis were uniformly less
susceptible to lymphoma induction by REV than by ALV
(83), and the REV lymphomas frequently require longer
latent periods than those induced by ALV.
For nonbursal lymphomas, the molecular mechanism
of oncogenesis also involves insertional activation of
c‐myc, but the strong tendency for the provirus to be ori-
ented in the same direction as c‐myc in bursal lympho-
mas was not observed in nonbursal lymphomas (109).
It is not known whether a common mechanism exists
for oncogenesis in chronic lymphomas of chickens and
turkeys. In ducks, the frequency of experimentally‐
induced REV lymphomas was not affected by embryonal
bursectomy (133), indicating that these tumors may not
necessarily be of B‐cell origin.
Acute Reticulum Cell Neoplasia
The target cell transformed in vivo by replication‐­
defective REV‐T (with REV‐A helper virus) expresses T
lymphoid and myeloid markers (19). These cells also
express surface MHC class I and II antigens, as well as
interleukin‐2 receptor (103), and are immunoglobulin M
(IgM) negative but vary in expression of CT3 (19).
Similar tumors were induced in chemically bursecto-
mized chickens (19). Inoculation directly into the thy-
mus induced thymomas composed of T and B cells (29).
On the other hand, REV‐T, when associated with chick
syncytial virus helper virus instead of REV‐A, induces
IgM‐positive B‐cell lymphomas with rearrangements of
the heavy‐ and light‐chain immunoglobulin loci (20, 21).
Thus, cell tropism appears to be determined, in part, by
differential effects of the helper viruses on lymphoid
populations.
Neoplastic transformation in acute reticulum cell neo-
plasia is mediated by the oncogene v‐rel, present in
REV‐T. Transformation does not require the presence of
a helper virus (131). Lymphoid cells transformed by
REV‐T in vitro, but which produce no infectious virus,
will produce typical RE when transplanted into synge-
neic recipients (131, 193).
Immunity
Humoral Responses
Birds with nontolerant infections develop robust anti-
body responses, detected as early as 16–21 days after
inoculation in chickens (32, 157), but 6–10 weeks may be
required in contact‐exposed birds (107, 130, 148).
Antibody titers may decline with age (10, 32, 260), but
neutralizing antibodies have been detected in experi-
mentally‐infected turkeys through 40 weeks (148). Most
birds that develop tolerant infections do not develop
humoral immune responses, although a few tolerant
chickens ultimately develop antibodies (159). The pres-
ence of antibodies may influence tumor susceptibility as
chemically bursectomized quail were more susceptible
to tumors than controls (186).
Cellular Responses
Major histocompatibility complex‐restricted cytotoxic-
ity against lymphoblastoid cell lines transformed with
defective REV has been described in chickens within
seven days after inoculation with defective or nondefec-
tive RE viral strains (141, 246). This response appears to
be mediated by activated (MHC class II+) CD8+ T cells
(126). However, natural killer (NK) cells were not acti-
vated (199). The induction of cytotoxic T cells by REV
has been used as a general indicator of immune response
in the study of other avian viruses (187).
Immunodepression
Humoral and cellular immune responses are frequently
depressed in chickens infected with nondefective REV
strains. Depressed antibody responses to MDC and tur-
key herpesvirus (HVT) (32, 120), Newcastle disease
virus (107, 265), sheep erythrocytes, and Brucella abor-
tus (255) are documented. The magnitude of antibody
depression is influenced by the dose and strain of virus,
and primary responses are more severely affected than
secondary (255). Depressed responses against Pasteurella
multocida in turkeys infected with nondefective REV
isolated from Attwater’s prairie chickens have been
­documented (17).
Different strains of nondefective REV varied in their
ability to induce bursal atrophy and suppression of B cell
populations available for transformation by v‐rel (20).
Studies on chimeric viruses derived from REV‐A and
chick syncytial virus showed that regions in both gag and
env genes were associated with the strong immunode-
pressive ability of REV‐A (88).

Spleen cells from chickens infected with REV‐T dis-
played suppressed responses to the mitogen phytohe-
magglutinin (40, 204). This effect is associated with the
nondefective helper virus in strain T stocks (34, 42) and
is mediated through a population of suppressor cells (41,
192), which could be demonstrated only through the
third week after infection (193). Other cellular immune
responses inhibited by REV infection include mixed
­lymphocyte reaction and allograft rejection (240).
Depression of humoral responses and a transient
mitogen responsiveness have been identified following
infection with the chick syncytial strain, but persisted
through 10–19 weeks in tolerant chickens infected with
nondefective REV (255, 260). Infected chickens were
more susceptible to the development of a MD tumor
transplant (34); to reactions from infectious laryngotra-
cheitis vaccine (160, 212); to natural fowl pox virus
infection (162); to infectious bronchitis virus (212); to
mortality induced by Eimeria tenella (161); and to
Salmonella typhimurium infection (160). No increase in
susceptibility to MDV was noted (33), but there was
interference by REV infection with immunity induced
by turkey herpesvirus against MD in chickens (255).
Humoral immunodepression was also seen in ducks
infected with a field isolate of REV (133). In the field,
immunodepression is probably the most important con-
sequence of embryo‐ or vaccine‐derived REV infections
but is less likely to result from contact infection (254)
and has not commonly been associated with ­seropositive
flocks. Reticuloendotheliosis virus‐induced immuno-
suppression has been reviewed (267).
Tumor Immunity
Regression of strain T‐induced wing‐web tumors was
partially abrogated by bursectomy, thymectomy, and
bursectomy–thymectomy (137). Serum from hyperim-
munized (104) chickens was protective against tumor
development even after absorption to remove antiviral
antibodies (104), suggesting the existence of tumor‐­
specific transplantation antigens on RE tumor cells.
Chickens immunized with purified or inactivated prepa-
rations of nondefective strain T helper virus were resist-
ant to challenge with acutely transforming REV‐T
preparations (24). However, immunization with empty
virions (151) did not provide protection.
Diagnosis
A diagnosis of RE requires not only the presence of typi-
cal gross and microscopic lesions, but also the demon-
stration of REV, REV antibodies and the exclusion of
other oncogenic agents. Because REV, unlike avian leu-
kosis and MD viruses, is not yet as ubiquitous, the dem-
onstration of infectious virus, viral antigens, and proviral
Chapter 15  Neoplastic Diseases 635
DNA in tumor cells has diagnostic value. Diagnostic
techniques have been reviewed by Zavala et al. (269).
Isolation and Identification
Reticuloendotheliosis virus viremia is typically low titered
and transient, except in tolerant birds. Birds with lesions
are normally a good source of virus, which may be isolated
by inoculation of susceptible tissue cultures with tissue
suspensions, blood, plasma, splenocytes, white blood
cells, or other inocula. Cellular inocula are preferred over
cell‐free inocula, because of higher titers. Cytopathic
effects in cell cultures may not be evident, thus, cultures
should be maintained through at least two blind 4–7‐day
passages of infected cells. Replication of REV may be con-
firmed by demonstration of viral antigen in cell cultures
using fluorescent polyclonal or monoclonal antibodies
(17, 18, 57), immunoperoxidase (35), complement fixation
(214), enzyme immunoassay (58, 108, 170), or molecular
detection of RNA or DNA (5, 17, 38, 74, 78, 87, 92, 196,
268). In comparative studies, enzyme immunoassays were
more sensitive than complement fixation tests (58) and
indirect immunofluorescence was more sensitive than
indirect immunoperoxidase or immunoelectron micros-
copy (169). A convenient and sensitive indirect immuno-
fluorescent assay conducted in 96‐well plates (46) has
been used for virus isolation from field samples (258).
Virus isolated by any of these procedures may be used
for reproduction of the disease or for further tests. Yolk
inoculations in 5–7‐day‐old chicken or quail embryos
are useful for disease reproduction and virus replica-
tion (16, 18). Virus isolates may be assigned to antigenic
subtypes by the differential reactivity to fluorescent
mAbs (46).
Detection of proviral DNA by PCR assays has been
shown to be a sensitive and specific method for detec-
tion of REV in chicken embryo fibroblasts or DF‐1 cells,
paraffin‐embedded tissues, as well as in blood and
tumors of infected birds (5, 17, 38, 74, 78, 87, 92, 196,
268). Polymerase chain reaction is useful for tumor
diagnosis (59, 61, 63, 65) and for evaluating vaccines for
possible REV contamination (78, 84, 85, 100, 139, 220).
Veterinary Services Memorandum 800.88 by USDA/
APHIS describes PCR, in vivo virus amplification, and
serology as suitable direct or indirect tests for detection
of REV in master seed viruses for the production of
commercial poultry vaccines. Assays amplifying REV
envelope and REV 3’ LTR sequences provided a more
accurate assessment of REV provirus than PCR assays
that amplify the REV 5’ LTR region (92). Although PCR
assays are sensitive and specific they may not be as well
suited as enzyme immunoassays for large‐scale testing.
A loop‐mediated isothermal amplification (LAMP)
method for rapid detection of REV with high sensitivity
and specificity has also been reported (66).
636 Section II  Viral Diseases
Serology
Serological confirmation of REV exposure involves the
detection of antibodies in sera from experimentally inoc-
ulated chickens or from clinically affected chickens.
The  most sensitive test for detection of antibodies to
REV is virus neutralization, although ELISA is the
most  ­commonly used serological assay worldwide.
Immunoperoxidase plaque assay (35) was once shown to
be a sensitive and reliable method for detection of REV
antibody. Antibodies may be detected in serum or egg
yolk from suspect birds by indirect immunofluorescence
(7, 257) or virus neutralization (148, 182). The agar gel
precipitin test may detect viral antigen as well as anti-
body in sera (106, 107). Reticuloendotheliosis virus
ELISA antibody kits are commercially available.
Antibody tests are particularly useful in ascertaining lack
of exposure in specific pathogen free flocks or breeding
flocks producing progeny for export. Complementary
assays that are now very seldom used are reviewed in
previous editions of this book.
Differential Diagnosis
The pathology of REV‐induced lymphoproliferative
tumors can be confused with that of tumors seen in MD
and lymphoid leukosis (81, 238, 253). Thus, neoplastic
diseases must be diagnosed by exclusion; that is, by con-
firming specific agents involved and excluding other
possible etiologies. Because avian tumor viruses are
­ processing, has been confused with RE (98, 239), but can
widespread and infection in the absence of tumor forma-
tion is common, in many cases virological and serological
criteria do not always provide a definitive diagnosis.
However, diagnosis of RE should be supported by viro-
logical evidence of REV infection, as REV is not as ubiq-
uitous as MDVs and ALVs. Techniques based on
immunocytochemistry with mAbs to cellular, tumor, and
viral antigens, or molecular hybridization can be used in
the differential diagnosis of avian viral lymphomas includ-
ing RE, but are seldom used in diagnostic laboratories.
Retroviral lymphomas in chickens originate from
either B cells (RE, lymphoid leukosis) or T cells (RE),
whereas MD lymphomas are of T‐cell origin. The char-
acteristics of target cells provide the basis for tests that
distinguish among B‐ and T‐cell lymphomas using mAbs
specific for cell surface antigens of B‐ and T‐lympho-
cytes. Nondefective strains of REV have been shown to
transform chicken B or T cells (109, 218).
The PCR assays for RE, MD, and exogenous ALV can
be helpful in the differential diagnosis of RE. For instance,
because MD lymphomas should contain a significant
proportion of MD virus‐infected cells, compared to
latently infected tissues, MD lymphomas should have
more infected cells, each with greater number of viral
copies thus resulting in higher total estimates of viral
load by quantitative PCR analysis (188) or real‐time
PCR. Non‐quantitative PCR assays are probably of little
value for diagnosis of MD because of the potential to
detect MDV DNA in the absence of lymphomas.
Polymerase chain reaction has been shown to detect
REV‐LTR sequences from lymphomas and brains of
REV‐infected chickens, but not from DNA from MD or
lymphoid leukosis lymphomas (5).
Chronic neoplasia in the chicken where the tumors are
of bursal origin cannot easily be differentiated from lym-
phoid leukosis on pathologic criteria (251). Virological,
serological, or PCR tests should be performed to confirm
one oncogenic virus and exclude another.
Chronic neoplasia in the chicken in which bursal
tumors are lacking or in which the latent period is too
short for lymphoid leukosis must be differentiated from
MD. Here too, pathological criteria are insufficient and
virological assays (including PCR) may be helpful. The
pp38 antigen of MD virus, occasionally expressed in MD
lymphomas, is not present in RE lymphomas. Also, MHC
class II (Ia) antigens are reported to be present on MD
lymphoma cells (199) but absent on RE nonbursal lym-
phoma cells (53). A comprehensive diagnostic strategy
for the differential diagnosis of virus‐induced lympho-
mas in chickens has been introduced (253).
The acute reticulum cell neoplasia syndrome is not
known to occur in the field. A syndrome of broiler chickens
characterized by reticuloendothelial proliferation in the
spleen and liver, and resulting in condemnation losses at
be distinguished by the absence of RE antigens and proviral
DNA in the lesions. Virus detection tests including PCR
have been used to detect REV in a lymphosarcoma in an
Indian peafowl (152) and lymphomas in captive greater
and Attwater’s prairie chickens (74, 80, 268).
The runting disease syndrome must be distinguished
from MD, especially when nerve lesions are present.
Differences between REV‐induced and MDV‐induced
nerve lesions have been discussed (255, 257), but are not
consistent. Both types of nerve lesions must be distin-
guished from spontaneous neuropathy, possibly an auto-
immune lesion of peripheral nerves (9).
Lymphoproliferative disease (LPD) of turkeys can be
confirmed or excluded by a combination of histopathol-
ogy and molecular detection methods for REV and
LPDV (1, 3). The PCR assays for LPD (1, 3, 197) and RE
(5, 17, 38, 74, 78, 87, 92, 196, 268) should be useful, albeit
wild turkeys could bear dual infections with both viruses.
Marek’s disease has been diagnosed in turkeys in France,
Israel, Germany, and Ukraine and should be ruled out in
the differential diagnosis.
In summary, naturally occurring RE lesions can be
confused in the chicken with MD, LL, and various other
lymphoproliferative or immunodepressive conditions,
and in the turkey, with LPD and MD.

Intervention Strategies
Vaccination
Reticuloendotheliosis in commercial poultry is typically
controlled by testing and elimination of infected shedder
breeding stock and thus vaccination is never considered.
However, vaccines could be of use for immunization of
endangered avian species. Vaccination of chickens with a
recombinant fowl pox virus expressing the env gene of
REV (36, 167), or empty REV particles (151), provided
some protection against REV infection. Defective REV
particles (243) have been shown to induce neutralizing
antibodies (79). A baculovirus construct expressing the
env gene of REV has also induced REV antibody in chick-
ens (241).
Treatment
No treatment for RE is known. Since immune responses
are mounted to infection, it is possible that some affected
birds may recover.
­Other Tumors
Susan M. Williams, Rodney L. Reece, and Scott Hafner
Summary
Agent, Infection, and  Disease.  Other tumors are in the
category where a known etiology does not exist or is
uncertain. The prevalence of these types of tumors will
depend on species, breed, age, and sex along with various
intrinsic and extrinsic factors.
Diagnosis.  Avian tumors often exhibit distinctive micros­
copic appearances that may be difficult to extrapolate from
similar mammalian tumors. Information on the classification
and histologic appearance of some of the less common
tumors of poultry is available from detailed poultry
pathologist reports, zoo surveys, and noncommercial poultry
reports. Immunohistochemistry can aid in identification of
tumor cells in poultry and other avian species with careful
interpretation of the positive and negative controls.
Intervention.  There is no intervention strategy due to
nature of these types of tumors.
Introduction
Other tumors refers to tumors of poultry (chickens,
turkeys, quail, pigeons, ducks, geese, guinea fowl,
­ and examinations of the tumors of noncommercial birds.
Chapter 15  Neoplastic Diseases 637
Prevention and Control Procedures
Prevention of RE is currently accomplished through quality
assurance of poultry biologics and, in SPF flocks, by strict
biosecurity (250). It is desirable to prevent environmental
exposure and seroconversion of breeder flocks where prog-
eny is destined for export, but this is difficult to accomplish
because it may be impractical to truly prevent exposure in
the field. Control of insect vectors and fowl poxvirus infec-
tion could be important in prevention programs (250).
Procedures for the control of RE have rarely been applied
in commercial practice, mainly because the disease has
been sporadic and self‐limiting. Enzyme immunoassay to
detect RE viral antigen in albumen samples seems to be the
procedure of choice in commercial situations (108, 258).
Presumably, it would be necessary to eliminate vertical
transmission through removal of potential transmitter
hens, and to rear progeny under isolated conditions whereby
horizontal infection could be precluded, as it has been done
with ALV in chickens. Such control procedures could be
considered if REV infection becomes endemic in especially
valuable breeding stock, as is the case with the endangered
Attwater’s prairie chickens (27, 28, 74, 80, 87, 180, 250, 268).
­ heasants, and ratites) in which a known etiology does
p
not exist or is uncertain. Avian tumors often exhibit dis-
tinctive microscopic appearances that may be difficult to
extrapolate from similar mammalian tumors. The most
detailed examination of chicken tumors remains that of
Campbell (20) whose definition of a tumor as “an abnor-
mal tissue mass … which usually persists independent of
initiating factors … whose excessive, often uncoordi-
nated growth threatens the host through compression,
infiltration, or remote spread” still suits our purposes.
Detailed information on the prevalence of different
tumor types in poultry exists, but earliest reports are
skewed by a large number of virally‐induced “leukotic”
tumors and the reproductive tumors that are common in
adult hens. Tumor prevalence in poultry not only
depends on species, breed, age, and sex, but also various
intrinsic (hormonal, genetic/developmental) and extrin-
sic (viral, chemical, and other environmental) factors
(20). Some indications of the proportion of different
tumors to be expected in aged chickens unaffected by
exogenous viruses are available in the surveys of tumors
in specific pathogen free (SPF) flocks (38, 99). Information
on the classification and histologic appearance of some
of the less common tumors of poultry is available from
detailed reports by poultry pathologists, zoo surveys,
638 Section II  Viral Diseases
Examinations of tumors present in poultry at slaughter
are particularly useful as the sheer numbers of poultry
slaughtered may provide multiple examples of even very
rare tumors, and slaughtered birds are conveniently sep-
arated by age and species. Radical deviations from the
“normal” prevalence, distribution, and/or type of these
tumors may indicate changes in etiology or other factors
that require further elucidation. Immunohistochemical
techniques that utilize antibodies to lineage‐specific cell
markers have found increasing applications as an aid to
identification of tumor cells in both poultry and other
avian species. Extended formalin fixation may reduce the
avidity of some antibodies for their designated tissue
epitopes; this at times may be reversed by heated acid
solutions or applications of proteases. Some studies have
utilized antibodies to vimentin for detecting mesenchy-
mal cells, cytokeratin for epithelial cells, actin for smooth
and skeletal muscles, and neurofilament for nerves.
Some other antibodies may exhibit different avidity in
avian tumors compared to that in mammalian species,
for instance S100 proteins may stain normal nerves, but
not label tumor cells derived from those tissues (72).
Neuron‐specific enolase often will intensely stain neural
tissues, but may also stain other tissues, particularly
muscle. Careful evaluation of the particular antibody in
the both normal and neoplastic tissues of the affected
species including both positive and negative controls is
important for accurate interpretation of immunohisto-
chemical findings.
Public Health Significance
There is no known public health significance.
Urogenital System
Ovary
Despite the considerable differences between avian and
mammalian ovaries, particularly with regard to histo-
logic appearance and physiology, attempts are made to
characterize avian ovarian tumors using criteria devel-
oped for mammals, particularly humans. Even with the
difficulty of categorizing avian neoplasms by mamma-
lian criteria, classifying ovarian tumors as derived from
surface mesothelium (adenocarcinomas), sex cord tis-
sues (granulosa cell tumors and arrhenoblastomas),
germ cells (teratomas and dysgerminomas), or other
supportive tissues provides clues to both expected
behavior and possible etiology, in addition to differenti-
ating these tumors from metastatic disease. In particular,
ovarian adenocarcinomas of White Leghorn hens have
recently been investigated as an animal model for ovar-
ian cancer of women, with potential for this research to
yield insight into the development of this neoplasm in
both species (7, 13, 57, 60).
Adenocarcinoma
Early tumors are small, firm, white nodules that occur on
the surface of the ovary; these may be mistaken for atretic
follicles, but are less symmetric (37). Nodules coalesce
into a gray‐white, cauliflower‐like mass that commonly
seeds serosal surfaces with transcoelomic metastases.
Ascitic fluid often develops with growth of the metasta-
ses and terminally affected hens may assume an upright,
penguin‐like posture. Ovarian adenocarcinomas must
be differentiated from oviductal adenocarcinomas as
both may be widely metastatic and oviductal tumors may
metastasize to the ovary. Failure to detect tumors in the
lining of the oviduct suggests tumors are of ovarian
rather than oviductal origin. Tumors are assumed to
arise from the mesothelial covering of the ovary (germi-
nal epithelium), including its invaginations into the
ovary, but other suggested tissues include sex cord rem-
nants, thecal cells, interstitial cells, or even the mesone-
phros. Small tumors appear to be located in the theca
externa of small follicles (Figure 15.52) or in the ovarian
stroma; a few may originate in the ovarian stalk. Multiple
tumors may be present. Smaller tumors contain predom-
inantly epithelial cells and little stroma, while larger
tumors are often scirrhous. In advanced cases, ovarian
follicles fail to mature and oviducts are often atrophied.
Ovarian adenocarcinomas are not associated with
increased production of steroid hormones (37, 38).
Occasionally, ovaries affected by adenocarcinomas are
covered with small cysts filled with yellow fluid; these
may be dilated lymphatics or ovarian stromal lacunae
(83). A survey of 400 hens revealed approximate 2% inci-
dence rate of tumor restricted to ovary (60).
Histologically, ovarian adenocarcinomas are often
composed of acini lined by cuboidal to low columnar,
non‐ciliated, epithelial cells. These epithelial cells are
often arranged around lumina that are at times filled
with intensely eosinophilic periodic acid‐Schiff (PAS)
positive material (Figure 15.53). In other, more densely
cellular tumors, acini may be compressed into cords or
islands of epithelial cells or papillary proliferations of the
lining epithelium may be invaginated into dilated acini
(Figure 15.54). The mitotic rate is variable, and in many
cases mitoses are not prominent. In larger tumors, acini
are often surrounded by a marked scirrhous response
(Figure 15.55), and in some serosal implants there may
be a proliferative response of the underlying muscularis
(84). Similar ovarian adenocarcinomas have been
described in turkey hens (119).
Granulosa‐Theca Cell Tumor
Granulosa‐theca cell tumors are irregularly round to
multilobular and encapsulated by a smooth, glistening

Figure 15.52  Ovarian adenocarcinoma in the theca region
demonstrating delicate trabeculae and round nuclei; note the
granulosa cells and yolk of the developing ova (top). H&E, ×360.
Figure 15.53  Acinar structures, typical of ovarian
adenocarcinoma filled with eosinophilic material and lined by
cuboidal cells containing round nuclei with condensed chromatin
and sparse eosinophilic cytoplasm. H&E, ×600.
Chapter 15  Neoplastic Diseases 639
Figure 15.54  Ovarian adenocarcinoma with papillary structures
projecting into dilated acini. H&E, ×160.
Figure 15.55  Another form of ovarian adenocarcinoma with
dense bands of stromal cells enclosing clusters of neoplastic
acinar cells with intensely basophilic nuclei. H&E, ×160.
membrane. Tumors occasionally exhibit peripheral thin‐
walled cysts filled with fluid; sectioned tumors are yellow
and friable and some contain hemorrhages. These tumors
enlarge while remaining attached by the thin ovarian
pedicle. Tumors may become quite large without metas-
tasizing, but metastases to visceral organs and serosal
surfaces can occur (20, 37). Granulosa‐theca cell tumors
are by far the most common ovarian tumor of the young
broiler chicken. In these birds, there is often marked
precocious glandular hyperplasia of the oviduct (50).
­
Histologically tumor cells are polyhedral to fusiform with
640 Section II  Viral Diseases

pale eosinophilic to vacuolated cytoplasm (Figure 15.56). that he designated a thecoma. A metastatic granulosa‐
Nuclei are generally irregularly round to ovoid. The theca cell tumor has been described in a duck (16). A very
arrangement of tumor cells varies widely from the forma- low incidence of  granulosa‐theca cell tumors has been
tion of follicular structures to solid sheets composed of induced in ­meat‐type chickens inoculated as embryos with
tightly packed thin cords (Figure  15.57) separated by a ALV‐J (92).
fine fibrovascular stroma, more elaborate gyriform
arrangements (Figure 15.58), or rosettes arranged around
small central spaces. The mitotic rate is variable but often
low, and the stroma may be prominent.
Ultrastructurally, tumor cells are identified by tran-
sosomes, a structure specific to avian granulosa cells and
those of some other lower vertebrates (37). Transosomes
(lining bodies) are dense structures of the lateral and apical
plasma membranes of granulosa cells that are taken up by
the oocyte becoming associated with primordial yolk gran-
ules. These structures are involved in the transport of vitel-
logenin (a yolk precursor) into the oocyte (76). Although
granulosa cells from normal avian follicles produce proges-
terone, theca interna cells produce testosterone, and theca
externa cells estrogen (94): hens with large granulosa‐theca
cell tumors exhibit markedly elevated plasma levels of
estrogen (37). Oviducts of affected hens are the same size Figure 15.57  Granulosa‐theca cell tumor composed of a uniform
as hens in lay, but eggs are not produced. A significant the- population of tightly packed tumor cells with plentiful, pale
cal cell component may be present in granulosa‐theca cell eosinophilic cytoplasm and uniform, round vesicular nuclei. Note
tumors; the presence of numerous vacuolated theca‐like the mitotic figure (arrow). H&E, ×600.
cells may lead to characterization of a granulosa cell tumor
as “luteinized” or even as a “luteoma” (20). Campbell (20)
also includes a tumor composed primarily of thecal cells

Figure 15.56  Lobules of vacuolated cells separated by moderate Figure 15.58  Gyriform arrangements of cells in one area of an
trabeculae in a granulosa‐theca cell tumor. H&E, ×200. ovary with a granulosa‐theca cell tumor. H&E, ×400.

Arrhenoblastoma (Arrhenoma)
These tumors are associated with formation of seminifer-
ous tubules within the ovarian stroma and are accompanied
by evidence of sex reversal (virilism). Sex reversal in domes-
tic fowl was well recognized in ancient times. Most cases of
sex reversal in poultry are due to destruction of the func-
tional left ovary and formation of an ovotestis in the rem-
nants of the rudimentary right gonad rather than hormone
production by ovarian tumors (20, 36). In birds, the male is
the neutral sex and the young female is demasculinized by
the production of her ovarian hormones (88). Well‐differ-
entiated arrhenoblastomas are histologically composed of
branching cords of columnar epithelium that are often two
cells thick; these resemble primitive seminiferous tubules.
There is little, if any spermatogenesis. In less differentiated
tumors, epithelial cells may be arranged in cords, nests, and
rosettes that form incomplete tubules (Figure 15.59) sepa-
rated by a prominent interstitium that may contain intersti-
tial (Leydig) cells (98). Large tumors may be hemorrhagic.
Arrhenoblastomas have been induced by the injection of
radioactive isotopes into the left ovary (121).
Ovarian Sertoli Cell Tumors
Ovarian Sertoli cell tumors in the chicken seem to be
limited to the five cases described by Fredrickson (38).
Figure 15.59  Arrhenoma from a hen that showed sex reversal.
Network of epithelial cells arranged as ill‐defined cords and
tubules. H&E, ×350. (C.J Randall).
Chapter 15  Neoplastic Diseases 641
These tumors were composed of compact masses of
seminiferous tubules lying under the ovarian capsule.
The tubules were lined by a single, radially arranged layer
of tall columnar Sertoli cells (Figure  15.60); variable
numbers of interstitial cells were present between
tubules. There was no obvious sex reversal or alteration
in circulating hormones.
Dysgerminoma
Dysgerminomas are the equivalent of mammalian ovar-
ian seminomas, and have been detected in a few intersex
(pseudohermaphroditic) pullets (123, 124). There was no
overt sex reversal, but there was masculinization of the
comb and plumage of the head. Tumor cells were round
to polygonal and contained round to occasionally reni-
form nuclei. Some tumors had metastasized to the liver
or peritoneum.
Mesosalpinx
Leiomyoma
Leiomyomas (at times termed fibroleiomyomas or even
fibroids) are common in the ventral ligament of the ovi-
duct and the oviductal wall in domestic fowl, including
SPF hens (56). The prevalence of these tumors is varia-
ble, but may affect up to 60% of hens in the first egg lay-
ing cycle (6). Similar tumors were present in the oviduct
ligaments in some rapidly growing Japanese quail (35).
Leiomyomas of the ventral ligament of the oviduct are
usually solitary. Unless tumors are quite large, there is
little effect on oviductal function, but there may be
increased egg yolk peritonitis (77). In hens affected
by these tumors, there were elevated serum concentra-
tions of estradiol, and oviductal leiomyomas were
induced in hens treated with both diethylstilbestrol and
Figure 15.60  Ovarian Sertoli cell tumor composed of well‐defined
seminiferous‐like tubules lined by Sertoli cells. Stroma contains
interstitial cells. H&E, ×600.
642 Section II  Viral Diseases
progesterone (5, 6). Some reports (17) have also demon-
strated that the tumor cells contain receptors for both
estrogen and progesterone, suggesting steroid hormones
are likely to be involved in the etiology of these tumors.
Many tumors are discrete, pale, solid masses less than
1 cm in diameter, but some may be several centimeters in
diameter and heavily vascularized (77, 98). Tumor cells
are well‐differentiated, interlaced, smooth muscle cells
bundled by variable amounts of connective tissue
(Figure 15.61). Mitotic figures are rare. These tumor cells
are immunolabeled by α‐smooth muscle actin, desmin,
and vimentin (77).
Oviduct
Adenocarcinoma
Adenocarcinomas originating in the oviduct most com-
monly arise in the upper magnum, with occasional
tumors seen in the uterus (shell gland) or infundibulum.
These tumors, like ovarian carcinomas, are often meta-
static to the mesentery and serous surfaces of abdominal
organs, especially to the pancreas and duodenum, but
also to the ovary and occasionally to the surface of the
liver and spleen. Tumor cells may be spread transcoe-
lomically in the ascitic fluid accumulating in the intesti-
nal peritoneal cavity. In contrast, metastases to the lung
may be hematogenous (2, 67). The prevalence of ovi-
ductal adenocarcinomas as determined by examining
the oviductal mucosa for tumors varied widely (5 to 81%)
in one survey (44) and in another survey only 4% were
restricted to the oviduct (60). In some studies (4), a posi-
tive correlation existed between egg weight and body
weight, and prevalence of tumors.
Oviductal adenocarcinomas in both chickens and tur-
keys may begin as small (2–10 mm) nodular areas of
Figure 15.61  Leiomyoma of mesosalpinx composed of smooth
muscle fibers arranged in compact whorls. Mitoses are absent from
this field, and the nuclear/cytoplasmic ratio is low. H&E, ×600.
mucosal dysplasia, typically discovered during close
examination of the mucosa of the oviduct (15).
Histologically, these nodules are well‐demarcated from
adjacent glands and are composed of concentrically
arranged columnar epithelial cells with acidophilic cyto-
plasmic secretory granules and pale enlarged nuclei
(Figure 15.62). Adenocarcinomas are grossly evident as
small pink to grey, firm masses that protrude into the
lumen of the oviduct and may invade through the mus-
cularis. There is often a distinct border between the nor-
mal magnal mucosa and tumor cells (Figure  15.63).
Metastases are often well‐encapsulated (Figure  15.64)
and may be composed of well‐differentiated cells.
Implants on the enteric serosa are often anaplastic cells
embedded in dense connective tissue (Figure  15.65)
while implants on the oviduct serosa lack this intense
scirrhous response (Figure 15.66). The metastases of ovi-
ductal adenocarcinomas may in some cases be histologi-
cally quite similar to those of ovarian adenocarcinomas
and since the ovary is also a common site for metastasis,
differentiating the tumors may be difficult. The ultras-
tructural characteristics of oviductal adenocarcinomas
have been described (64). Immunohistochemical studies
have demonstrated that the cells of oviductal adenocar-
cinomas contain ovalbumin (59), but more recent stud-
ies (42) have also identified ovalbumin in ovarian
Figure 15.62  Dysplastic adenomatous focus in a fold in the
magnum showing clear demarcation from surrounding normal
glands. The columnar epithelial cells are densely packed and
oriented concentrically. H&E, ×400.

Figure 15.63  Magnal adenocarcinoma showing the well‐defined
margin between normal secretory tissue with cellular cytoplasm
containing eosinophilic granules of ovalbumin (above) and very
lightly granular tumor cells (below). H&E, ×400.
adenocarcinomas; the latter finding was attributed to
cellular dedifferentiation or return of the cell to a less dif-
ferentiated state. Cells of oviductal adenocarcinomas
retain receptors for estrogen and progesterone (5) and
the growth of these cells in vitro is stimulated by estro-
gen (4). In a recent study, few oviductal adenocarcino-
mas displayed immunostaining for v‐erbB, the chicken
oncogenic form of the epidermal growth factor homolo-
gous to human HER‐2/neu, but it was present in many
ovarian adenocarcinomas, especially those which were
large (57). The organ or tissue of origin of many meta-
static adenocarcinomas observed in the abdomens of
poultry cannot be determined and these are best
described as “of undetermined origin”.
Testis
Sertoli Cell Tumor
Sertoli cell tumors are rare in chickens (20) and a few
have been reported in Japanese quail (46), a duck (24),
pigeon (96), and a goose (118). There is a single case
report of a tumor arising in the testicular remnants
of  an  incompletely surgically‐caponized chicken (111).
Elongated Sertoli cells are commonly intratubular
(Figure  15.67) and arranged at right angles to tubular
basement membranes, while in other cases there may be
Chapter 15  Neoplastic Diseases 643
Figure 15.64  Implant of magnal adenocarcinoma deep in the
ovary showing capsule around adenocarcinomatous cells. Despite
the apparent aggressiveness of this tumor, mitotic figures are not
prominent. H&E, ×200.
areas with solid sheets of cells. Nuclei are hyperchro-
matic and there often is a high mitotic rate.
Seminoma
Seminomas have been reported in chickens, ducks, quail,
guinea fowl, and pigeons (56). These tumors are com-
monly unilateral and consist of intratubular or broad
sheets of round to polyhedral cells (Figure  15.68) with
eosinophilic cytoplasm and irregularly round, eccentri-
cally placed nuclei. Multinucleated syncytia may be pre-
sent. Some tumors have been metastatic.
Interstitial (Leydig) Cell Tumors
Interstitial cell proliferations may be present in semino-
mas of chickens (20), but interstitial cell tumors have not
been described in poultry. Interstitial cells were a com-
ponent of a mixed cell tumor affecting both testes of a
duck, but only the Sertoli cell component was present in
a metastatic site (73).
Renal
Renal nephroblastomas and adenocarcinomas occur in
chickens. Some are associated with infections with avian
leukosis/sarcoma viruses (see Leukosis/Sarcoma Group).
644 Section II  Viral Diseases

Figure 15.66  Magnal adenocarcinoma implanted on the serosa of

the isthmus is surrounded by little fibrous tissue. The dilated
acinar lumina are lined by cuboidal epithelium. H&E, ×200.

Figure 15.65  Compacted acini lined by cuboidal epithelium

surrounded by dense drifts of fibrous tissue in this cirrhotic implant
in duodenal serosa of a magnal adenocarcinoma. H&E, ×175.

Digestive System
Alimentary Tract
Squamous cell carcinomas have been most commonly
reported from the oropharynx, esophagus, and crop of
chickens (56). Both humans and chickens from certain
regions of northern China exhibit an increased preva-
lence of esophageal squamous cell carcinoma (108, 120),
suggesting that common etiologic factors could play a
role as chickens and humans may have common expo-
sures to drinking water, food, and other environmental
factors. However, detailed epidemiologic studies have
not identified a specific cause (120, 129). Such tumors
were often superficially ulcerated and composed of cords
and islands of anaplastic squamous epithelial cells, some Figure 15.67  Sertoli cell tumor in a quail. The tubule‐like
of which surrounded keratin (Figure 15.69). structures are lined by cells two layers deep. H&E, ×360.

Figure 15.68  Seminoma in a duck. Lobules of pleomorphic
polyhedral cells with finely granular cytoplasm; some
multinucleated cells. Delicate stroma. H&E, ×180.
Figure 15.69  Oropharyngeal squamous cell carcinoma in an adult
noncommercial chicken. Cords and islands of epithelial cells with
some central keratin pearls (arrow). Mucosal gland is shown at top
left. H&E, ×200.
Multiple papillomas have been described in the esoph-
agus and crop of chickens, but that report lacks histo-
logical characterization (87).
In chickens, proventricular adenomas (21) and adeno-
carcinomas (20, 99) have been described, along with
adenocarcinomas (20, 21, 98) of the gizzard (Figure 15.70).
A proventricular adenoma was described in a duck (10).
Chapter 15  Neoplastic Diseases 645
Figure 15.70  Adenocarcinoma of gizzard with growth of darkly
staining cuboidal tumor cells downward into muscularis. The
keratinous product of these cells is shown at lower right. H&E,
×200. (K. Langheinrich)
Adenocarcinomas of the small intestine have been
described in chickens (56) and ducks (104); some of these
involved the ileocecal junction. Primary adenocarcino-
mas affecting the intestinal tract must be differentiated
from metastatic adenocarcinomas of the reproductive
tract, which readily and rapidly metastasize to involve
the intestine (20).
Enterogenous cysts that are lined by mucosa derived
from the gastrointestinal tract have been described in
young chickens. These usually present as fluid‐filled
cysts partially replacing the spleen and the lining mucosa
often resembles that of the ventriculus (69).
Leiomyomas and leiomyosarcomas have been described
affecting the gastrointestinal tract (see Musculoskeletal
System). Pheasants are affected by a nodular typhlitis
caused by infection with Heterakis isolonche with larval
migration producing neoplastic submucosal nodules that
are neurofibroblastic (63) or smooth muscle in origin (79)
with possible metastasis to the liver.
Liver
Hepatocellular Tumors
Hepatocellular adenomas (hepatomas) and hepatocellular
carcinomas are uncommonly reported in chickens (56).
Adenomas consist of masses of large, polyclonal, well‐dif-
ferentiated hepatocytes that are compactly arranged in
several cell thick cords separated by connective tissue septa
(Figure 15.71); portal triads and central veins are, however,
lacking in these masses and there is a low mitotic rate.
Hepatocellular carcinomas often multifocally replace the
liver parenchyma and there may be metastasis to the lung.
In carcinomas, the tumor cells are less well differentiated,
often exhibit mitoses, and may be multinucleated (20, 95).
646 Section II  Viral Diseases

Hepatocellular tumors in chickens have been induced with Peritoneum

some oncogenic avian retroviruses (14) or by administra-
Mesothelioma
tion of diethyl‐nitrosamine (68). These tumors appear to
Mesotheliomas have been reported in chickens (56) and
be not uncommon in ducks (15, 104) and some have been
ducks (74). Multiple papilliform abdominal nodules in
induced by feeding aflatoxin (23). Duck hepatitis B virus
which mesothelial cells and anaplastic fibrocytes were
has been suggested to be involved in the development of
seen have been described in an aged female ostrich (87).
hepatocellular carcinomas in Chinese ducks (31, 127), but
There often is ascites, and tumors invest serosal surfaces
aflatoxins were also suspected.
of abdominal organs. Histologically, papillae are covered
by cuboidal mesothelial cells supported on delicate stalks
Cholangiocellular Tumors
(Figure 15.76).
Cholangiomas and cholangiocellular carcinomas are
uncommon in poultry, with only a few cases being described
in chickens, ducks, and pigeons (56). Tumors of bile ducts
must be differentiated from the proliferation that accompa-
nies chronic toxic damage to the liver due to the ingestion of
hepatotoxins. Cholangiomas consist of clusters of dilated
and often distorted ducts that are lined by well‐differenti-
ated epithelial cells; these ducts are separated by fibrous
connective tissue (Figure  15.72). In cholangiocarcinomas,
epithelial cells less commonly form defined ducts and are
separated by fibroblasts (Figure 15.73).

Pancreas
Adenocarcinoma
Tumors of pancreatic epithelial cells are often difficult to
distinguish from metastases of ovarian or oviductal
tumors, as those tumors commonly implant upon the
duodenal serosa and surface of the pancreas. Pancreatic
tumors may arise from ducts (86, 97) or exocrine cells
(38). Most probably arise from ductal epithelium and are
composed of columnar epithelial cells with lightly Figure 15.72  Cholangioma composed of dilated ducts in a loose
basophilic cytoplasm (Figure  15.74). Tumors derived
­ fibrocytic stroma. H&E, ×75.
from  exocrine cells morphologically resemble acini
(Figure  15.75) and may or may not have cytoplasmic
bodies resembling zymogen granules (38).

Figure 15.71  Hepatoma composed of large eosinophilic neoplastic Figure 15.73  Cholangiocarcinoma composed of small clusters of
cells, some in mitosis, forming irregular plates. H&E, ×600. epithelial cells in a fibroblastic stroma. H&E, ×190.

Figure 15.74  Pancreatic adenocarcinoma, probably of ductule
cell origin, composed of columnar cells forming tubular structures
among a few remnant acinar cells (arrow) ×160.
Figure 15.75  Pancreatic acinar cell adenocarcinoma with
normal exocrine tissue (right) and agranular neoplastic cells (left).
H&E, ×600.
Respiratory System
Infraorbital Sinus
Reece (98) reported two chickens with pea‐sized cystic
adenomas that extended into the infraorbital sinuses and
were filled with mucin.
Chapter 15  Neoplastic Diseases 647
Figure 15.76  Mesothelioma with prominent neoplastic epithelial
cells supported on a delicate stalk. H&E, ×600.
Figure 15.77  Adenocarcinoma of lung composed of a papillary
growth of epithelial cells that have replaced most of the normal
lung. H&E, ×200.
Lung
Adenocarcinoma
In chickens, primary adenocarcinomas of the lung
appear to be extremely rare (8, 20). Campbell described
three cases characterized by small nodules located near
the primary bronchi and Fredrickson and Helmboldt
described a papillary adenocarcinoma arising multifo-
cally from parabronchi (38). In that case, cuboidal
­epithelial cells replaced most of the lung (Figure 15.77)
and there were metastases in the thorax and abdomen.
Primary tumors of the lung, especially in mature
­chickens, need to be ­differentiated from metastases of
ovarian or oviductal adenocarcinomas, hepatocellular
carcinomas, or ­ metastases of other tumors (20, 99).
648 Section II  Viral Diseases
Reports of pulmonary adenocarcinomas are more
­common in ducks (75, 113, 128), particularly those main-
tained in zoos, than in other avian species. There are rare
descriptions in pigeons (113).
Nervous System
Central Nervous System
Astrocytoma
Both sporadic and epizootic cases of astrocytoma (fowl
glioma) have been described, commonly as multiple
tumors in aged hens (56), and one case has been
described in a domestic duck (110). These small masses
are often near the thalamus or the base of the cerebellum
(38). There frequently are perivascular accumulations of
lymphocytes adjacent to the tumor, but no hemorrhage,
giant cells, or areas of pressure necrosis (Figure 15.78).
Masses are composed of astrocytes with extended cyto-
plasmic fibrillar processes (Figure 15.79). Similar masses
Figure 15.78  One of several clearly demarcated, but
unencapsulated astrocytomas composed of fibrillar astrocytes, in
anterior brain stem. There is a significant lymphocytic infiltrate
around the blood vessels within the tumor and the adjacent
tissue. H&E, ×100.
Figure 15.79  Astrocytoma composed of uniform astrocytes with
extended cytoplasmic processes. H&E, ×190.
in some breeds of chickens have been shown to be caused
by infections with a specific subgroup A avian leukosis
virus (61, 85, 116).
The small ependymoma described by Wight and
Campbell (125) was discovered in the right cerebral
hemisphere of a 60‐day‐old chicken affected by MD;
the mass was within or adjacent to the lateral ventricle
and consisted of vacuolated cells that occasionally
formed palisades or rosettes. In the same report, two
subdural angioblastic meningiomas were described in
pullets displaying neurological signs: both were located
near the cerebellum and consisted of multiple con-
gested vascular sinuses that were lined by enlarged
endothelial cells and separated by a prominent reticulin
network.
Pineal Body Tumor
There are a few reports of pineal body tumors in chick-
ens (20, 99, 115). In the young hen described by Swayne
et al. (115), the mass was somewhat similar to a normal
pineal gland, but was greatly enlarged (3×), displaced
adjacent cerebellar tissue, and cells exhibited an
increased mitotic rate. These characteristics, in addi-
tion to a relatively decreased number of follicular cells,
identified this as a tumor rather than pineal gland
hyperplasia. In the cases reported by Reece (99), there
were clinical histories that included neurologic symp-
toms such as fine tremors and head pressing. In each
bird, there was a large mass between the cerebrum and
cerebellum that extended into or impinged upon the
cerebellum. Tumors were composed of lobular masses
of low columnar epithelial cells (Figure  15.80) sur-
rounding a central lumen. These cells were surrounded
by parafollicular cells.

Figure 15.80  Lobules of a pineal body tumor separated by fine
trabeculae. Palisaded low columnar epithelial cells with large vesicular
nuclei are surrounded by smaller parafollicular cells. H&E, ×200.
Peripheral Nervous System
Neurofibromas
The term neurofibroma is often used interchangeably with
Schwannoma (neurilemmoma), however, Schwannomas
are relatively homogenous proliferations of Schwann
cells, while neurofibromas contain a mixture of nerve
elements including Schwann cells. Separation of these
tumors is not always possible in poultry and similar
tumors have been referred to by some authors as neuro-
genic sarcomas (87). Tumors of the nerve sheath are well
recognized in young broiler chickens (21) but also occur
in older birds, often originating in major nerves or near
dorsal root ganglia. Neurofibromas are often solitary
masses, but multiple tumors have been described (3).
Tumors are composed of fibroblast‐like spindle cells that
may form concentric whorls resembling nerve sheaths
(Figure 15.81). In tumors with ­predominantly Schwann
cell proliferation, there may be nuclear palisading or
structures resembling sensory nerve endings (Wagner–
Meissner corpuscles or Pacinian corpuscles) (20).
Neuromas
Post‐traumatic neuromas commonly occur when afferent
nerves attempting to regenerate become obstructed by,
Chapter 15  Neoplastic Diseases 649
Figure 15.81  Schwannoma of the sciatic plexus showing a whorling
pattern of spindle‐shaped cells with central nuclei. H&E, ×400.
then entrapped within, densely collagenous scar tissue,
resulting in tangled masses of poorly myelinated axons,
Schwann cells, and perineurial cells separated by bands of
connective tissue (Figure 15.82). These neuromas are not
true neoplasms and are a well‐recognized although rela-
tively rare, sequela to beak‐trimming (Figure 15.83) or toe
amputation (39, 40). When young chicks or turkey poults
are partially beak‐trimmed, the resultant dermal scar tis-
sue is less dense which may p ­ artially explain the relative
absence of neuroma development in birds treated when
they were young (28, 41).
Melanoma
Melanomas, including some described as originating in
the ovary with metastasis to other organs have been
described in the chicken (20). Multifocal melanomas with
the appearance of malignancy have been seen affecting
the skin, visceral organs, musculature, and other tissues in
young broiler chickens and a 16‐week‐old pullet (99).
Some authors described similar tumors as malignant mel-
anomas and demonstrated that unbleached tumor cells
and tumor cells bleached by potassium permanganate or
hydrogen peroxide, were not immunoreactive with
­antibodies specific for S100 proteins, but did react with
antibodies to neuron specific enolase, vimentin, and
Melan‐A (126). Melanocytes in avian melanocytic tumors
often do not react with antibodies to S100 proteins even
650 Section II  Viral Diseases

Figure 15.83  Lower beak neuroma due to beak trimming. The mass

can range in size and may hinder food intake if large enough.

Figure 15.82  Post‐traumatic neuroma in beak tip showing

dense collagenous scar tissue and multiple whorls of nervous
tissue composed of poorly myelinated axons, Schwann cells,
and associated connective tissue. Martius scarlet blue, ×360.
(C.J Randall)

though normal nerves are S100 immunopositive (55, 72).

These cells may be spindloid or there may be closely
packed masses of polyhedral melanocytes reminiscent of
epithelial tissue (Figure  15.84). Malignant melanomas
have also been described in ducks and a pheasant and
melanomas, some of which were amelanotic, have been
seen in the skin of pigeons (56). Benign melanomas have
been seen within the eye of chickens (12, 32, 107). A small
number of cases of multifocal melanomas were noted in
young adult Japanese quail from one inbreed commercial
strain (101).
Special Senses
Eye
Intraorbital rhabdomyosarcomas, thought to originate
from the striated muscle of the iris, have been described
in chickens (32). Retinoblastomas (20, 26) and melano-
mas involving the eye, have been described.
Figure 15.84  Subcutaneous melanoma of the wing of a pigeon.
There is melanin pigment in many polyhedral cells. Mitotic figures
are rare. The cells are densely packed, clearly demarcated from the
surrounding tissue, and penetrate between muscle fibers and
adipocytes (lower right). H&E, ×200.
Endocrine System
Adrenal Gland
The avian adrenal gland is composed of intermingled
cords of cortical cells and enterochromaffin (medullary)
cells rather than displaying a defined cortex and medulla.
Two adrenal cortical adenomas have been described, one
in a young chicken and one in an SPF hen (21, 99). The
well‐differentiated cells comprising these neoplasms
contained abundant eosinophilic cytoplasm that was
 Chapter 15  Neoplastic Diseases 651

occasionally vacuolated. The close anatomic relationship Hemangiopericytoma

between the ovary and adrenal glands makes it impera-
tive to eliminate markedly luteinized ovarian ­carcinomas
when the ovary is also involved (20). A pheochromocy-
toma has been described in a 14‐week‐old pullet (20).
Pituitary Gland
Pituitary tumors of poultry appear to be limited to the
two chickens reported by Campbell (20). One was
described as a tumor of chromophobe cells that com-
pressed the brain stem, and the second was an infiltrative
acidophil adenoma.
Thymus
Thymomas have been most commonly described in
chickens and one has been described in a duck (56). These
have primarily been masses in the neck that are histologi-
cally characterized by sheets of epithelial cells lacking
­distinct cytoplasmic borders. Immunostaining of tumor
cells with cytokeratin has been utilized to verify epithelial
origin (11). There may be accompanying myoid cells (20).
A few hemangiopericytomas have been described in
chickens (38, 112), they were all benign and occurred
as  subcutaneous nodules in the cervical region.
Histologically, they were arranged as concentric rings
around blood vessels, and were composed of uniform
spindle‐shaped cells with a fusiform nucleus. The inter-
vening reticulin fibers could be demonstrated by a silver
stain (Figure 15.85).
Lipoma and Liposarcoma
Subcutaneous lipomas are not common in chickens (20,
97). They are generally benign, encapsulated and deli-
cately trabeculated; and may exhibit necrosis and/or
hemorrhage. These tumors are composed of large mature
vacuolated adipocytes with a pale displaced nucleus:
mitotic figures are rare. Liposarcomas of chickens are
rare and may be locally invasive or metastasize (100); in
some cases, the predominant cells are elongate and may
be ­mistaken for fibrocytes except for the presence of
small cytoplasmic fat vacuoles, others are composed of
more typical immature adipocytes. Myelolipomas and
erythrolipomas have not been reported thus far in
chickens.

Thyroid and Parathyroid Glands

Adenomas of the thyroid have been described in a 24‐
week‐old pullet (87) and a 2‐year‐old hen (20). Both
affected the left thyroid glands, contained multiple cysts
and were described as containing relatively undifferenti-
ated epithelium resembling embryonic thyroid.
Microscopic cysts lined by ciliated epithelium are occa-
sionally seen in the thyroid and are thought to be derived
from thyroglossal duct remnants (20). Tumors of the
parathyroid gland of chickens seem to be limited to an
adenoma and a parathyroid carcinoma (48).

Integument
Subcutis
Of the various sarcomas, fibromas, and myxomas that are
encountered in subcutaneous tissues of chickens, many
may be induced by avian leukosis viruses (see Leukosis/
Sarcoma Group), although some of these tumors are
reported from SPF hens and other poultry species.
Chickens systemically infected by some retroviruses may
produce tumors in response to the inflammation
­associated with wounds, as postulated to be the cause of
some mesenchymal tumors observed near the trimmed
beak of hens (99). There is a report of a fibroma in a wild,
hunter‐killed, ring‐necked duck with a large mass on the
side of the head from the commissure of the beak to ven- Figure 15.85  Hemangiopericytoma with concentric rings of
tral of the ear (19). pericytes clearly defined. Silver, ×90.
652 Section II  Viral Diseases

Cutis i­dentified (109), but in other studies, viral particles were

not seen ultrastructurally and evidence of avian leukosis
Squamous Cell Carcinoma
viruses was not detected (29, 52, 81). Carcasses most
The true squamous cell carcinoma of the skin of birds
commonly exhibit crater‐shaped ulcers with raised
originates from the surface epidermis, forming infiltrat-
­margins within feather tracts (Figure  15.86), but both
ing cords of anaplastic keratinocytes that invade down-
nodular and ulcerative lesions may be present. Smaller
ward into the underlying dermis. Epithelial cells are
circular ulcers average 5 mm in diameter, but larger
separated by intercellular bridges and resemble cells of
irregular, coalescing ulcers are present on some ­carcasses.
the stratum spinosum. Since it is likely that some of these
In live chickens, these ulcers are filled with keratin and
tumors, like those of other species, are induced by years
cell debris (Figure 15.87). Nodules are generally smaller
of sun exposure, many have occurred on the feet and
(averaging 3 mm in one study) and appear grossly as
shanks of aged birds (20, 22). Although these tumors are
locally invasive, only a few have been metastatic (1). The
tumors originally described as dermal squamous cell
carcinomas in young broiler chickens, have been desig-
nated avian keratoacanthomas, due in part due to origin
from feather follicle epithelium rather than surface epi-
dermis (109). Oropharyngeal, esophageal, and ingluvial
squamous cell carcinomas also have been recognized,
especially in chickens (see Digestive System).

Avian Keratoacanthoma
These tumors are most commonly found in the skin of
carcasses of young chickens at slaughter (56), although
some have been identified in live broiler chickens (52, 99)
and a few have been seen in older chickens (66, 114).
These tumors were formerly designated as “dermal squa-
mous cell carcinoma”, but avian keratoacanthoma was
selected as a more descriptive term since tumors unal-
tered by the processing artifacts of de‐feathering and
Figure 15.86  Typical carcass lesions of avian keratoacanthomas
scalding have the distinctive tissue architecture of kera- are craterous ulcers within feather tracts.
toacanthomas, originate from feather follicle epithelium
rather than the surface epidermis, are not metastatic,
and, in live birds, regress (52). Carcasses with extensive
lesions are condemned at slaughter, while less affected
carcasses undergo trimming. The prevalence of broiler
carcasses with multiple lesions commonly varies from
0.01% to 0.05%, but may be 0.09% or higher in individual
flocks (53, 117, 122). In some studies, chickens slaugh-
tered at less than 48 days of age exhibited more tumors
than older birds and, tumor prevalence was cyclic, being
lowest in summer months (53, 122). In other surveys, an
increased number of condemnations due to these tumors
were associated with dusty houses, birds placed in new
houses, or certain farms (43). One report has commer-
cial adult layer hens (55,000 hens) affected starting at 30
weeks of age and continuing for 28 weeks and regressing.
Approximately 0.1% of the flock was affected in the
region of the legs and toes (29).
The etiology of this condition remains unknown. Some
authors have suggested an association with fowl pox
because some studies by nested PCR detected DNA
sequences specific for fowlpox virus in lesions (33), but Figure 15.87  In live chickens with keratoacanthomas, ulcerative
other studies failed to detect evidence of fowlpox (81). In lesions contain central masses that are mixtures of keratin, cell
one study of these lesions, type C retroviruses were debris, and bacteria.

enlarged feather follicles (Figure 15.88). Microscopically,
nodular lesions appear as proliferative outgrowths of
feather follicle epithelium (Figure 15.89), cysts originat-
ing from dysplastic feather follicles, or hyperplastic
feather follicles that contain hyperkeratotic feathers.
Figure 15.88  Early keratoacanthoma lesions in the skin of live
chickens are nodules in the base of feather follicles.
Figure 15.89  Microscopically, nodular lesions of avian
keratoacanthoma are expansions of feather follicle epithelium.
H&E, ×40.
Chapter 15  Neoplastic Diseases 653
Ulcers are composed of a central cup‐shaped cavity lined
by epithelium and filled with keratin, bacteria, and cell
debris. Epithelial lips overhang this central mass of kera-
tin and cell debris. The lining epithelium keratinizes
toward the central cavity and extends thin strands of
keratinocytes into the surrounding dermal fibroplasia
(Figure  15.90). Carcass lesions are often extensively
altered by post‐slaughter de‐feathering with loss of the
central keratin core and much of the lining epithelium. In
affected live chickens, nodules progressed to ulcers and
all lesions eventually regressed (52).
Feather Folliculoma
Multiple cystic structures found on the medial surfaces
of the wings of adult hens have been described (99).
These cysts were filled with keratin and feather rem-
nants, and were lined by squamous epithelial cells. In
some areas, there was abrupt keratinization of the lining
epithelium, while in other areas, there was disorganized
feather follicle epithelium (Figure  15.91). There was an
intense inflammatory reaction in the adjacent dermis.
Similar cysts have been described in turkeys (27).
Intracutaneous Keratinizing Epithelioma
These benign masses were characterized by multiple
nodules in the facial skin of adult hens, each marked by a
central pore. These cysts were lined by stratified squa-
mous epithelium with orderly maturation from a periph-
eral basal layer. The cyst lumen contained lamellated
keratin (Figure  15.92) but no feather remnants. Little
Figure 15.90  A section through the epithelial lip of an ulcerative
keratoacanthoma from a live chicken shows the centrally
keratinizing and peripherally invasive lining epithelium (arrows)
adjacent to the central keratin and cell debris. H&E, ×100.
654 Section II  Viral Diseases
Figure 15.91  Edge of a feather folliculoma showing dysplastic
specialized feather‐forming epithelium and an adjacent cord of
basal cells. The lumen was lined by stratified cuboidal to
squamous epithelium with abrupt keratinization and contained
keratin and feather remnants. H&E, ×180.
inflammation surrounded these cysts unless there was
rupture of the cyst wall (99).
Other Tumors of the Cutis
Acanthomas are firm, often conical masses that pro-
trude from the scaled epithelium on the plantar surface
of the tarsometatarsus. Tumors are composed of keratin
whorls on a base of fibrous connective tissue that con-
tains islets of squamous epithelium with central basal
epithelial cells (20).
Mast cell tumors are extremely rare in poultry, but
have been described in a few adult chickens (54, 58, 91).
In one, there was a solitary lesion of the eyelid, while in
the other two birds, there were multiple dermal tumors
composed of solid sheets of round to ovoid mast cells. In
one hen, there was metastasis to the lung (54).
Xanthomas are not true neoplasms, but are masses of
foamy macrophages and multinucleated giant cells that
Figure 15.92  The lumen of this intracutaneous keratinizing
epithelioma contains lamellated keratin. The epithelium shows basal
cells aligned on a distinct basal lamina and progression to polyhedral
cells with abrupt keratinization. Note the small intraepithelial bulla.
H&E, ×190. (Reece (99). Courtesy of Taylor and Francis, Ltd, www.
tandfonline.com, on behalf of Houghton Trust, Ltd.).
surround cholesterol clefts. The multiple xanthomas
seen in chickens in the 1950s were attributed to chlorin-
ated hydrocarbons contaminating the fat added to
rations. The masses were considered to possibly be a
reaction to metabolites of this material accumulating in
dermal fat (106).
Musculoskeletal
Leiomyoma and Leiomyosarcoma
Leiomyomas of the ventral ligament of the oviduct are
common in domestic fowl and Japanese quail (see
Urogenital System), other smooth muscle tumors are
more rarely reported. Leiomyomas have been described
in the intestine of ducks (98), the trachea of broiler
­chickens (21, 98, 102) (Figure  15.93), attached to the
­pancreas (97) or in the liver of pigeons (25), and in the
 Chapter 15  Neoplastic Diseases 655

Figure 15.93  Leiomyoma of the trachea of a broiler chicken.

Bundles and whorls of smooth muscle fibers in the lamina propria
(upper portion) penetrate between the cartilaginous rings and
extend into the adjacent adventitia (lower right). H&E, ×100.

muscularis of chicken proventriculus, ventriculus (giz- Figure 15.94  Rhabdomyosarcoma. Some cells are strap‐like,
whereas others are large and polyhedral. The cytoplasm is
zard), and duodenum (21, 99). Leiomyosarcomas have eosinophilic and some cells contain multiple nuclei. H&E, ×400.
been described in chicken tracheal muscle (20), chicken
ventriculus (gizzard) (105), hen ovary (65), chicken
intestine (3), and thigh skeletal muscle of a hen with
metastasis (70). Leiomyosarcomas have also been
described in the lung and skin of pigeons (80, 82).

Rhabdomyoma and Rhabdomyosarcoma
Rhabdomyomas and rhabdomyosarcomas have been pri-
marily seen affecting the skeletal muscle of the breast and
legs and less commonly the musculature of the hearts of
young chickens (20, 21, 87), and a rhabdomyoma has also
been described in the eyelid of a racing pigeon (97). A
rhabdomyosarcoma originating in cranial muscle and
invading the brain occurred in a 7‐month old hen (47),
and rhabdomyosarcomas metastatic to the lung of chick-
ens have also been described (71, 99). Two intraocular
rhabdomyosarcomas have been described in chickens
(32). These muscle cell tumors are generally histologically
characterized by spindle, strap, flame, or racquet cells and
multinucleated cells (Figure 15.94). Cross striations may
be present only in rare cells; even examinations with
polarized light or phosphotungstic acid‐stained sections Figure 15.95  Osteoma showing thick irregular trabeculae. H&E, ×100.
may not detect striations in the intensely eosinophilic
cytoplasm of anaplastic myoblasts. Immunohistochemical
examinations for vimentin, myoglobin, muscle specific These osteomas were well circumscribed and were com-
actin, and desmin have been useful in some avian posed of disorganized bone trabeculae (Figure  15.95).
rhabdomyosarcomas (34). Several osteosarcomas, some of which were metastatic,
were described affecting long bones, ribs, and vertebrae
of young chickens (21). Another osteosarcoma affecting
Osteoma and Osteosarcoma
an aged hen was also metastatic (110). A report describ-
Osteomas have been described in the feet of ducks (97) ing osteosarcoma in free range aged hen had involve-
and affecting various sites in a few chickens (20, 21, 99). ment of the vertebrae with metastasis to the liver (30).
656 Section II  Viral Diseases
An osteosarcoma has also been described affecting the
tibiotarsus of a Japanese quail (97) and the foot of a
goose (78).
Chondroma and Chondrosarcomas
A single chondroma has been described as originating at
a costochondral junction in a young chicken (20).
Multifocal chondromas have been described in the foot-
pads of geese and ducks (97). These tumors were charac-
terized by lobules of chondrocytes separated by trabeculae
(Figure 15.96). Dittmer et al. described a chondrosarcoma
in an aged free range hen that involved the sternum and
extended into muscle (30). Chondrosarcomas in poultry
are exceedingly rare.
Other Tumors
Teratoma
Teratomas are composed of tissues arising from more
than one germinative layer and commonly contain a dis-
organized mixture of variously differentiated epithelial
cells, bone, cartilage, smooth muscle, fat, or other tissues
including nervous tissue, melanocytes, or cardiac mus-
cle. Often, ciliated columnar epithelial cells with goblet
cell differentiation line tubules or cysts. Other epithelial
islands may be composed of squamous epithelial cells
Figure 15.96  Multifocal chondroma of the footpad of a goose
showing lobules of cartilage that are separated by fibrovascular
trabeculae. H&E ×100.
that surround keratin (resembling primitive feather fol-
licles). Some tumors are described simply as abdominal
masses (62). In chickens, teratomas are reported to arise
more commonly in the testis than ovary (20, 21, 62).
Teratomas may also arise at other sites (20, 21, 48, 93).
Teratomas have also been induced in chickens by the
injection of various metallic ions into the testis (20, 49).
Teratomas have been observed in several ducks and a
goose (18, 89, 103).
In contrast to teratomas, hamartomas are a focal over-
growth of mature tissue indigenous to the organ or
­location in which it is found; these have not been reported
in poultry.
Multicentric Histiocytosis
Multicentric histiocytosis (histiocytic sarcomatosis, sys-
temic spindle‐cell proliferative disease) primarily affects
young broiler chickens, producing hepatomegaly and
splenomegaly. Numerous small (0.5 to 2 mm) white
masses are grossly evident in the spleen, liver, and kid-
neys. Some diseased birds are pale (anemic) and smaller
than flock mates. Microscopically, nodules of spindle‐
shaped cells replace the spleen (Figure 15.97), liver, kid-
neys, and other organs most commonly including bone
marrow, pancreas, intestine, proventriculus, and lungs
(9, 45, 51, 90, 91). Germinal centers may be present
within these nodules, especially in the spleen; liver nod-
ules may contain a more heterogenous population of
cells, including plasma cells and some typically bulge
into portal vessels. Lesions are not accompanied by mye-
loid leukosis (myelocytomatosis). Spindle cells contain
abundant eosinophilic cytoplasm and elongated ovoid to
more pleomorphic nuclei (Figure  15.98). Mitoses are
Figure 15.97  In multicentric histiocytosis, nodular masses of
spindle‐shaped cells multifocally replace the spleen. H&E, ×100.
 Chapter 15  Neoplastic Diseases 657

common, but there are no multinucleated cells. Spindle

cells are immunohistochemically positive for markers
characteristic of antigen‐presenting tissue macrophages
or dendritic cells (9, 90). Infection of meat‐type chickens
at day of hatch with strains of subgroup J avian leukosis
virus have reproduced these lesions, but only in birds
that are persistently viremic with an ineffective antibody
response (90).

Figure 15.98  Histiocytic spindle‐shaped cells in the spleen of a
chicken with multicentric histiocytosis contain elongated and
pleomorphic nuclei. H&E, ×400.
­Acknowledgement
The authors would like to acknowledge the contribu-
tions of Drs. Bruce W. Calnek, Aly M. Fadly, Karel A.
Schat, and Richard L. Witter for their contributions
to  subchapters in Neoplastic Diseases in previous
editions.

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