D9316.PDF

Rev. sci. tech. Off. int. cpiz., 2000,19 (2), 544-564
Neoplastic diseases:
Marek's disease, avian leukosis and
reticuloendotheliosis
L.N. Payne & K. Venugopal
Institute for Animal Health, Compton, Newbury, Berkshire RGZO 7NN, United Kingdom
Summary
The commercially important neoplastic diseases of poultry are Marek's
disease, which is caused by a herpesvirus, and the avian leukoses and
reticuloendotheliosis, which are caused by retroviruses. These diseases are
responsible for economic loss due to both mortality and depressed performance.
Marek's disease virus (MDV) and avian leukosis viruses (ALVs) are prevalent
throughout the world, and new strains which arise in particular locations may
spread across borders, thereby undermining national disease control measures.
Reticuloendotheliosis virus (REV) is also present in many countries. Marek's
disease virus is transmitted horizontally only, and international spread in hatching
eggs and day-old chicks can be prevented by appropriate hygiene precautions.
Transmission of ALV and REV occurs both horizontally and vertically (through the
egg), and measures to prevent international spread are more demanding. Marek's
disease is controlled by vaccination, whilst avian leukosis is controlled by virus
eradication programmes, mainly at the primary breeding level. Similar virus
control measures can be applied for reticuloendotheliosis if necessary. No strong
evidence exists to suggest that these avian tumour viruses constitute a danger to
public health.
Keywords
Avian leukosis - Control - Diagnosis - Epidemiology - International trade - Marek's
disease - Poultry - Public health - Reticuloendotheliosis - Surveillance - Tumour viruses
-Vaccination.
Introduction
Neoplastic diseases of poultry fall into two broad classes,
namely: those with an infectious aetiology and those which
are non-infectious. Those of the former category are of the
greater economic importance because the viruses that cause
these diseases are widely prevalent in commercial stock and
the mesenchymal neoplasms that they cause affect relatively
young birds. These infections and diseases can be enzootic
and epizootic. Neoplasms of a non-infectious aetiology occur
mostly in birds older than the usual lifespan of commercial
birds. Such tumours are often of epithelial cell origin, with
ovarian tumours being common in hens over two years of age.
Even so, tumours of the magnum region of the oviduct,
adenomas and adenocarcinomas, of non-infectious aetiology,
can occur in commercial laying hens at the end of the first
laying season. However, the non-infectious neoplasms are
generally sporadic and not of great economic significance.
Three main classes of virus cause neoplasms in poultry, as
follows:
a) Marek's disease virus (MDV), a herpesvirus
b) avian leukosis virus (ALV), a retrovirus
c) reticuloendotheliosis virus (REV), also a retrovirus.
Another retrovirus, lymphoproliferative disease virus (4), has
caused significant losses from lymphomas in turkeys in the
United Kingdom and Israel, but now appears to be rare, and is
not considered further in this paper.
Domestic chickens are the poultry species most commonly
affected by neoplasms, which may be caused by MDV, ALV or

Rev. sci. tech. Off. int. Epiz., 19 (2|
545
REV. Turkeys and quail can suffer neoplasms caused by MDV
and REV. Reticuloendotheliosis virus also causes neoplasms
in geese and Muscovy ducks, and in game birds (pheasants
and partridges). Ducks are not commonly affected by
neoplasms, although hepatocellular carcinomas have been
reported in the People's Republic of China, where mycotoxins
in feed have been regarded as a likely aetiological factor,
possibly with the involvement of duck hepatitis B virus, a
member of the family Hepadnaviridae.
Economic importance
Neoplastic diseases, and the viruses that cause them, are
important in poultry for several reasons. The presence of the
vims or the neoplasm causes economic loss from mortality
and depressed performance. For Marek's disease (MD),
additional costs arise from the development, production and
use of vaccines for disease control, and for avian leukosis,
from the implementation of virus eradication programmes,
particularly by primary breeding companies. The viruses are
prevalent throughout the world, but new strains arise
periodically in particular locations (especially strains of MDV
but also of ALV). If these spread between countries, national
disease control measures can be undermined.
Before the introduction of vaccination of commercial flocks in
1971, MD was a major global disease of chickens. Vaccination
dramatically reduced losses, but the disease remains one of
significant economic importance, particularly because of the
periodic appearance of new strains of MDV against which
existing vaccines provide suboptimal protection. This has
required the continued development of new vaccines and
vaccination strategies (7). In 1984, the total world-wide
economic loss caused by MD, including the cost of
vaccination, was estimated at US$943 million (30). The
International Animal Health Code of the Office International
des Epizooties (OIE) places MD on List B, comprised of those
diseases which have socio-economic and/or public health
importance within countries and which are significant to
international trade of animals and animal products (24).
Avian leukosis viruses are widespread but until recent years
have not caused serious mortality nor been considered to be
of major economic importance. Avian leukosis is placed on
List C of the Food and Agriculture Organization Animal
Health Yearbook 1995 (13), designating diseases of
socio-economic and/or public health importance at the local
level. However, the world-wide spread of the newly emergent
subgroup J strain of ALV (27, 43) and associated mortality
during the 1990s has generated serious concern and warrants
the inclusion of avian leukosis amongst diseases significant to
international trade.
Infection by REV in commercial breeding flocks appears to be
more prevalent than was once known. The infection is usually
subclinical and without serious economic impact.
Nevertheless, associated outbreaks of disease have been
reported from some countries and the infection should be
regarded as at least a potential threat, and one that is
significant in international trade.
In addition to direct effects on poultry, ALV and REV are
potential contaminants of live virus vaccines produced in
chicken cells for veterinary or human use, requiring use of
vaccine substrate cells from specific-pathogen-free (SPF)
poultry. If present as contaminants in poultry vaccines, these
viruses present a disease hazard to poultry. No direct evidence
exists to suggest that avian tumour viruses in poultry or
poultry products present a disease hazard to humans, but any
such possibility receives consideration from regulatory bodies
(19,31).
Avian tumour viruses in international trade
The importation of poultry and poultry products into a
country poses a risk of introducing infections, and veterinary
authorities of importing countries therefore lay down
procedures for minimising this risk. Exporting countries have
procedures for providing certification of the health status of
exported materials. Details vary between countries and the
specific requirements must be obtained from the countries
concerned. Within the European Union, rules exist for
movement of animals and animal products between member
countries and into member countries from third countries.
Exports to third countries are mostly subject to bilateral trade
agreements. Recommended procedures for regulating animal
health aspects of international trade are provided by the OIE
in the International Animal Health Code (24) and the Manual
of Standards for Diagnostic Jests and Vaccines (23). The OIE is
recognised by the World Trade Organization for the setting of
international standards for animal health (40).
Freedom from clinical disease and proper vaccination are
sufficient to prevent the introduction of Marek's disease
infection through hatching eggs or day-old chicks, since the
disease is not vertically transmitted. In the case of ALV and
REV, reliance on freedom from clinical disease in-imported
birds or source flocks will not prevent introduction of
infection, because of the occurrence of subclinical infection
and vertical transmission. Veterinary Administrations of
importing countries should therefore obtain from the
exporting country more specific evidence of freedom from
these infections if concerned about the introduction of ALV
and REV (as discussed below in the sections entitled 'Avian
leukosis' and 'Reticuloendotheliosis').
Marek's disease
Diseases
Marek's disease is a lymphoproliferative and neuropathic
disease of domestic chickens, and less commonly, turkeys and
quails, caused by a highly contagious, cell-associated,
oncogenic herpesvirus (7). As a disease occurring world-wide,
with increasing reports of vaccination failures and emergence

546
Rev. sci. tech Off. int. Epiz., 19 (2)
of more virulent pathotypes, MD poses a severe threat to the
poultry industry. Developing strategies for the control of MD
remains a significant challenge.
The pathogenesis of MD is complex. The infection is thought
to be transmitted by the respiratory route from the inhalation
of infected dust in poultry houses. Although the very early
events in the disease are not yet clear, the pattern of events
after infection with an oncogenic MDV in susceptible birds
can be divided into the following stages:
a) early cytolytic infection
b) latent infection
c) late cytolytic infection with immunosuppression
d) neoplastic transformation (6).
Marek's disease virus is a lymphotropic virus and targets
lymphocytes, the principal cells of the immune system.
B-lymphocytes, the cells of the antibody-forming arm of the
immune system, are first targeted by the virus in a lytic
infection. Following this, cytolytic infection occurs in the
activated T-lymphocytes that are involved in cell-mediated
immune responses. These early cytolytic events result in
atrophic changes in the bursa of Fabricius and thymus,
leading to severe debilitation of the immune system and
marked immunosuppression. The cell-associated viraemia
that develops during this period is believed to be the route by
which the virus spreads throughout the body, including the
feather follicle epithelium, the only site where a fully
productive infection occurs which allows shedding of the
virus into the environment. After the early cytolytic phase, the
infection switches to a latent phase in the infected T cells, and
the regressive changes in the lymphoid organs start resolving,
largely restoring the architecture of these lymphoid organs.
Following this, some of the latently infected T cells become
targets for neoplastic transformation resulting in
lymphomatous lesions in various visceral organs. Due to the
complex nature of the pathogenesis with varying periods of
latency, the incubation period of MD from the point of
infection to the onset of clinical disease can vary from a few
weeks to several months.
Generally, four different clinical forms of the disease are
recognised in flocks infected with MDV, as follows:
a) the classical or neural form, where a large proportion of
the birds show signs of paresis or paralysis involving the legs,
wings and sometimes the neck. These cases, also referred to as
'fowl paralysis' or 'range paralysis', are usually seen in birds of
two to twelve months of age;
b) the acute form, a more virulent form of the disease where
lymphomatous lesions of various organs develop and high
mortality occurs in the affected flocks. Birds as young as six
weeks can be affected, with losses commonly occurring
between three and six months. Involvement of the eyes and
nerves as well as lymphomatous lesion of the skin is also
evident in some cases. Visceral and skin lesions due to MD
are important causes of carcass condemnation in
slaughterhouses;
c) transient paralysis, an uncommon condition in flocks
infected with MDV, usually occurring between five and
eighteen weeks of age. This is an encephalitic expression of
infection characterised by sudden onset of paralytic
symptoms that often only last for approximately 24 h to 48 h,
although in some instances death can occur;
d) acute mortality syndrome, a form of the disease observed
more recently, where the affected birds die with an early acute
cytolytic disease well before the onset of lymphomas. The
affected birds show characteristic atrophy of the bursa of
Fabricius and thymus. This form of the disease is thought to
be due to infection with highly virulent pathotypes of the
virus.
Aetiology
Virus structure and replication
The isolation of cell-associated herpesvirus from MD tumours
in the late 1960s (8) was an important historical landmark
which led to an improved understanding of the disease and
development of effective vaccines. Due to the lymphotropic
nature of the virus, MDV was originally classified as a
gammaherpesvirus together with Epstein-Barr virus, and the
oncogenic herpesviruses of non-human primates, herpesvirus
saimirí and herpesvirus ateles. However, on the basis of the
genomic organisation, MDV is currently classified together
with alphaherpesviruses such as the herpes simplex vims
(HSV) in the family Herpesviridae. The deoxyribonucleic acid
(DNA) of MDV is a linear double-stranded molecule of
approximately 170 kilobases, consisting of a unique long
region (UL) flanked by a set of inverted repeat (TRL and IRL)
regions and a unique short region (US) flanked by another set
of inverted repeat regions (IRS and TRS) (34). Thus, the
genomic structure of MDV from left to right can be described
as TRL-UL-IRL-IRS-US-TRS and is similar to that of other
alphaherpesviruses. The viral genomes in the infected cells are
maintained either as circular episomes or as integrated forms.
The viral genome has the capacity to encode at least seventy
proteins, sixty of which have counterparts in HSV, including
structural proteins, metabolic enzymes and transactivating
proteins such as VP 16 and ICP4 (34). However, at least ten
MDV genes have no homologues in other herpesviruses.
Similar to those of other herpesviruses, MDV genes also
belong to three kinetic classes of immediate early, early and
late genes, based on the requirements for viral protein
synthesis and DNA replication. Compared to the extensive
expression of genes, during the lytic infection, the
transcription in latently infected and transformed cells has
been largely restricted to the repeat regions of the MDV
genome. Some of the important genes recognised within the
repeat regions that could potentially be associated with
transformation include the BamHI-H family transcripts, pp38,
ICP4 sense and antisense ribonucleic acid (RNA) transcripts,
and the meq gene.

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The main types of interaction that occur between the virus
and the cell in MDV infection include the following:
a) a semiproductive infection similar to that which occurs in
vivo in lymphoid organs and in vitro in cultured cells which
results in the production of non-enveloped virions
b) a fully productive infection which occurs in the feather
follicle epithelium with production of infectious enveloped
virions
c) a non-productive infection which is commonly seen in
tumours and lymphoblastoid cell lines where the viral
genome is not expressed or is expressed to a limited extent
only.
Marek's disease virus serotypes and pathotypes
The first isolation of MDV in 1967 was followed by the
description of a number of isolates from different parts of the
world. Subsequently, these isolates where divided into three
distinct serotypes, as follows:
a) serotype 1, which includes all the pathogenic or oncogenic
strains of MDV in addition to the attenuated strains of these
viruses
b) serotype 2, which includes naturally non-pathogenic
strains of MDV
c) serotype 3, which includes the herpesvirus of turkeys
(HVT), the non-oncogenic MDV-related virus, isolated from
turkeys.
In addition to differences in biological properties, these
serotypes can also be distinguished by serological methods
using polyclonal and monoclonal antibodies, DNA and
polypeptide analysis as well as by the polymerase chain
reaction (PCR).
A large diversity exists within serotype 1 concerning the
oncogenic potential, with strains varying from highly to
mildly oncogenic. Recently, serotype 1 MDV strains have
been further classified on the basis of the ability to induce MD
lesions in vaccinated chickens. Strains are classified as mild
(mMDV), virulent (vMDV), very virulent (vvMDV) and very
virulent + (w+MDV) pathotypes (Table I). The emergence of
these pathotypes is thought to represent a continuous
evolution of MDV towards greater virulence. Some of these
pathotypes show an increased propensity to produce acute
Table I
Serotypes and pathotypes of Marek's disease virus
Serotype Pathotype Representative strains
Mild (mMDV) HPRS-B14, CU2, Conn-A
Virulent (vMDV) HPRS-16, JM, GA
Very virulent (wMDV)
RB1B, ALA-8, Md5, Md11
Very virulent + (w+MDV)
61 OA,648A
Non-oncogenic
SB-1, HPRS-24.301B/1
Non-oncogenic HVT-FC126, HPRS-26
cytolytic disease with atrophic changes in the lymphoid
organs, the severity of which directly correlates with the
virulence.
Epidemiology
Prevalence of infection and disease
Marek's disease virus infection mainly occurs in domestic
chickens and is ubiquitous among poultry populations
throughout the world. The infection in other species is rare,
but occasionally the disease occurs in turkeys and quails. In
commercial chicken houses, where the infection is rampant,
virtually all birds become infected, commonly within the first
few weeks of life, although on occasions infection may be
delayed. Because of the prevalence of serotype 1 viruses of
varying pathogenicity and of non-pathogenic serotype 2 in the
poultry house environment, birds can be infected with more
than one MDV strain. Evidence suggests that the frequency of
isolation of non-pathogenic viruses becomes higher as the age
of the birds increases. Natural infection by non-pathogenic
strains of MDV can provide immunity to subsequent infecüon
by a virulent strain.
Transmission of infection
The transmission of MDV occurs by direct or indirect contact,
apparently by the airborne route. The epithelial cells in the
keratinising layer of the feather follicle replicate fully
infectious virus, and serve as a source of contamination of the
environment. The shedding of the infected material occurs
approximately two to four weeks after infection, prior to the
appearance of the clinical disease, and can continue
throughout the life of the bird. The virus associated with
feather debris and dander found in dust in the contaminated
poultry house can remain infectious for several months.
Although the inhalation of infected dust from poultry houses
remains the most common route of disease spread, other less
common mechanisms of indirect transmission, such as those
involving darkling beetles (Alphitobius diaperinus), could also
play minor roles in transmission. However, no evidence exists
to suggest that vertical transmission of MDV occurs through
the egg.
Flock infection
Because of the ubiquitous nature of the infection and the
ability to survive for long periods outside the host, flock
infections usually occur early in the life of a bird. In addition,
in most flocks, the hatched chicks usually have
maternally-derived antibodies. This antibody disappears in
most chickens by three to four weeks of age. The rate of the
spread of MD within a flock can vary greatly and depends on,
among several factors, the level of initial exposure and the
concentration of susceptible birds. A number of stress factors,
including those from handling, change of housing and
vaccination, are thought to increase the disease incidence. The
existence of genetic resistance to MD among chickens has
long been recognised and the genetic constitution of the flock
influences the outcome of MDV infection. The outcome of
infection is also influenced by sex, as females are usually more
susceptible to the development of tumours.

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Diagnostic methods
Diagnostic procedures for avian tumour viruses include both
pathological and virological methods. Pathological diagnosis
identifies the nature of the tumour that is causing mortality,
whereas virological diagnosis identifies viruses that are
present in a bird or flock. As MDV, ALV and REV occur
commonly, virological diagnosis does not necessarily establish
the cause of the tumour. Nevertheless, histopathological
identification of a tumour often determines the likely cause.
However, in some cases, the presence of an infection in a flock
may need to be established in the absence of tumour
mortality.
Pathological diagnosis
The main gross and histopathological features of the
neoplasms under review are summarised in Table II. In
general, while gross appearance can provide indications of the
nature of the neoplasm, histopathological diagnosis is
essential for accurate diagnosis. Histopathological diagnosis
requires tumour material from birds which have died very
recently and from several cases from an affected flock; this
material should be placed in fixative. For the diagnosis of MD,
the most useful set of tissues to collect include the liver,
spleen, bursa of Fabricius, thymus, heart, proventriculus,
kidney, gonads, nerves, skin and other gross tumour tissues.
Although clinical signs associated with MD can occur in
chickens from four weeks of age, signs are most frequently
seen between twelve and twenty-four weeks of age, and
sometimes later. Significant diagnostic features of the classical
and acute forms of MD are described below.
Classical form
In the classical form of the disease, with mainly neural
involvement, mortality rarely exceeds 10%-15%, occurring
over a few weeks or many months. The most common clinical
sign is partial or complete paralysis of the legs and wings. The
characteristic pathological lesion is the enlargement of one or
more of the peripheral nerves. The most commonly affected
nerves that are easily seen on post-mortem examination are
the brachial and sciatic plexus and nerve trunks, coeliac
plexus, abdominal vagus and intercostal nerves. The affected
nerves are grossly enlarged, and often two or three times the
normal thickness. The normal cross-striated and glistening
appearance of the nerves is lost, instead, the nerves have a
greyish or yellowish appearance and are oedematous.
Lymphomas are sometimes present in this form of the disease,
most frequently as small, soft grey tumours in the ovary,
kidney, heart, liver and other tissues.
Acute form
In the acute form of the disease, where formation of
lymphomas in the visceral organs usually occurs, the
incidence of the disease is frequently between 10% and 30%,
and in major outbreaks can reach 70%. Mortality can increase
rapidly over a few weeks and then cease, or can continue at a
steady or falling rate over several months. The typical lesion in
this form of the disease is the widespread, diffuse
lymphomatous involvement of visceral organs such as the
liver, spleen, ovary, kidney, heart and proventriculus.
Lymphomas are also occasionally seen in the skin around the
feather follicles and in the skeletal muscles. Affected birds may
also show involvement of the peripheral nerves similar to that
seen in the classical form. The liver enlargement in younger
Table II
Principal gross and microscopic features of importance in the differential diagnosis of the leukoses and Marek's disease lymphoma
Feature Lymphoid leukosis Erythroid leukosis Myeloid leukosis
(myeloblastosis)
liver
Spleen
Bursa of Fabricius
Bone marrow
Blood
Cytology and
histopathology
Other organs and
tissues often grossly
involved
Greatly enlarged; diffuse,
miliary or nodular tumours;
moderately firm
Usually enlarged; diffuse,
miliary or nodular tumours;
soft
Usually enlarged; nodular
tumours
Often tumorous; diffuse or
focal
Occasionally lymphoblastic
leukaemia
lymphoblasts; mainly
extravascular infiltrations
Moderately enlarged;
diffuse infiltration;
cherry-red colour; soft
Often enlarged; cherry-red;
smooth; very soft
No changes
Semi-liquid; cherry-red
Kidneys, ovary
Erythroblastic leukaemia;
immature erythrocytes;
anaemia; thin buffy coat
Erythroblasts;
intravascular
Kidneys; may be
haemorrhages in muscles
Myeloid leukosis
(myelocytomatosis) Marek's disease
lymphoma
Greatly enlarged; diffuse
infiltration; mottled;
granular surface; firm
Often enlarged; diffuse
tumour; mottled; smooth;
soft
Sometimes tumorous
Diffuse, reddish-grey
tumour infiltration
Myeloblastic leukaemia;
thick buffy coat
Myeloblasts in
intravascular and
extravascular locations
Kidneys, ovary
Often yellowish white
nodular or diffuse tumours
Often nodular or diffuse
tumours
No changes
Usually diffuse
yellowish-grey tumour
infiltration
Myelocytic leukaemia;
thick buffy coat
Myelocytes in
intravascular and
extravascular locations
Kidneys, ovary, thymus,
surface of bones (sternum,
ribs, skull)
May be moderately to
greatly enlarged; miliary
or nodular tumours; firm
Often atrophic; may be
enlarged; usually diffuse
tumours
May be diffusely
enlarged
No changes
May be lymphocytosis or
lymphocytic leukaemia
Pleomorphic, sometimes
blastic, lymphoid cells in
perivascular locations
Nerves, kidneys, ovary,
proventriculus, heart,
muscle, skin, iris