THESIS

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thesis entitled

Influence of Marek's Disease Virus on Subgroup J
Avian Luekosis Virus Infection in
Meat-Type Chickens

presented by

Bedriye Bilge Yondem

has been accepted towards fulfillment
of the requirements for

M. S. degree in Veterinary
Pathology

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INFLUENCE OF MAREK’S DISEASE VIRUS ON SUBGROUP J AVIAN
LEUKOSIS VIRUS INFECTION IN MEAT-TYPE CHICKENS

By

Bedriye Bilge Yondem

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathobiology and Diagnostic Investigation

2002

ABSTRACT

INFLUENCE OF MAREK’S DISEASE VIRUS ON SUBGROUP J AVIAN
LEUKOSIS VIRUS INFECTION IN MEAT-TYPE CHICKENS

Bedriye Bilge Yondem

The objective of this study was to determine the influence of various
serotypes of Marek's disease viruses (MDV) on avian leukosis virus subgroup J
(ALV-J)-induced viremia, cloacal shedding and tumors, in meat-type chickens.
Chickens from two different breeders were placed in six different groups and
infected with ALV-J at hatch; chickens were also inoculated with various strains
of MDV vaccines at hatch and challenged with very virulent plus (W+) MDV at
two weeks of age.

Data from this study suggest that bivalent MD vaccines containing
serotype 2 and 3 MDV, namely turkey herpesvirus and 88-1 (HVT+SB-1) may
reduce viremia and cloacal shedding of ALV-J infection in one of the two strains
of chickens used. Chickens infected with ALV-J and bivalent MDV vaccine had
lower percentage of viremia and cloacal shedding than chickens inoculated with
serotype 1 MDV vaccine (Rispens) or w+MDV (strain 648A). In the second
experiment, commercial broiler breeder chickens infected with serotype 1 MDV
vaccine had the highest incidence of viremia and cloacal shedding. There was no

influence of strain of MDV vaccine on ALV-J tumors.

ACKNOWLEDGMENTS

I express my sincere appreciation to my research advisor Dr. Aly M. Fadly
and academic advisor Dr. Willie M. Reed, for providing guidance and
encouragement throughout this study. My appreciation is also extended to
members of the guidance committee, Dr. Margo 8. Holland and Dr. Richard M.
Fulton for their advice and help during the course of this study.

I am also grateful to Dr. R.L. Witter and Dr. Susan M. Williams, Ms. Melanie
Flesberg and all ADOL staff for their help and encouragement.
My personal thanks go to my family and my parents who have always

given me support and encouragement through out my stay in the US.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................... v
LIST OF FIGURES ............................................................................................ vi
INTRODUCTION ................................................................................................ 1
MATERIAL AND METHODS ............................................................................ 13
RESULTS ......................................................................................................... 18
DISCUSSION ................................................................................................... 31
REFERENCES ................................................................................................. 35
VITA ................................................................................................. 42

LIST OF TABLES

Table 1: Experimental design for experiments 1 and 2 ............................... 17
Table 2A: ALV-J-induced viremia, shedding and antibody in White Rock strain of
chickens (Experiment 1) ............................................................................ 20
Table 28: ALV-J-induced viremia, shedding and antibody in White Rock
chickens (Experiment 1) ............................................................................ 21
Table 3A: ALV-J-induced viremia, shedding and antibody in commercial broiler
breeder chickens (Experiment 2) ............................................................... 25
Table 3B: ALV-J-induced viremia, shedding and antibody in commercial broiler
breeder chickens (Experiment 2) ............................................................... 26
Table 4: Tumor incidence in White Rock and commercial broiler breeder
chickens ..................................................................................................... 27
Table 5: Influence of various strains of MDV ALV-J tolerant infection in 17-24-

week-old meat-type chickens .................................................................... 28

LIST OF FIGURES

Figure 1. Liver lesion from 24 weeks old White Rock chicken with ML ......... 29

Figure 2. Myelocytes with eosinophilic granules from ML ............................. 30

vi

ABBREVIATIONS

ALV-J = Subgroup J avian leukosis virus

MD = Marek's disease

MDV = Marek's disease virus

w+MDV = very vimlent plus Marek's disease virus
CPE = cytopathic effect

ELISA = enzyme-linked immunosorbant assay

VN = virus neutralization

V=Wmmm

S = cloacal shedding

vii

INTRODUCTION

Most neoplasms of lymphoid and other hematopoietic cells in commercial
poultry are caused by viruses, which belong to one of four distinct groups: a)
Marek’s disease virus (MDV), an oncogenic herpesvirus; b) avian leukosis virus
(ALV); c) reticuloendotheliosis virus (REV); and lymphoproliferative disease virus
of turkey (LPDV) (Witter, 1997b).

ALVs infect primarily chickens, whereas REV infects chickens, turkeys and
other avian species. ALVs are members of the leukosis/sarcoma (US) group of
avian retroviruses. Based on properties of viral envelope glycoproteins, members
of the LIS group including ALVs from chickens are classified into five exogenous
subgroups: A, B, C, D and J and one endogenous subgroup E (Payne and Fadly,
1997). Among the structural polypeptides (p27, p19, p15, p12 and p10) shared
by all members of US group of avian retroviruses including endogenous and
exogenous ALVs, p27 is the most abundant. Exogenous ALVs are capable of
inducing a variety of neoplasms, but endogenous ALVs are rarely oncogenic.
Under natural conditions, lymphoid leukosis, a B-cell lymphoma that originates in
the bursa of Fabricius and metastasizes to various visceral organs, is the most
common neoplasm induced by exogenous ALVs (Fadly, 1997).

Exogenous ALVs spread by vertical and horizontal transmission, a
process that requires a fully infectious vims. In contrast, endogenous viral (ev)
genes are inherited as host genes and may or may not be expressed (Payne and

Fadly, 1997).

Chickens infected with exogenous ALV shed virus into the albumen of
eggs and vaginal and cloacal secretions and congenitally transmit virus to the
next generation. In contrast, endogenous ALVs are believed not to be shed or
shed at a very low level (Smith at al., 1986; Crittenden, 1991 ). The oviduct which
is the source of virus for shedding of ALV contains the highest viral titers,
compared to other visceral organs. Congenital transmission of ALV is more
frequent in hens with low antibody titer and can occur in chickens with no
detectable shedding of group specific (gs) antigen p27 (Payne and Fadly, 1997).

Congenital transmission from dam to progeny is the major method of
maintaining infection in flocks. Most congenitally infected chicks become
immunologically tolerant to the virus and remain viremic throughout their lifetime
without developing neutralizing antibody. Such females transmit virus to nearly
100% of their progeny. Males do not appear to transmit virus (Fadly and
Crittenden, 1987). Viremic chicks that are in close contact with other chicks early
in life transmit the infection through feces, which contaminates drinking water.
Transfer of infection can also occur via vaccination needles or by vent sexing in
the hatchery (Fadly et al., 1981 ).

The frequency of birds exposed to ALV early in life that become
permanently viremic and lack neutralizing antibody is highly variable as in the
frequency of birds that develop transient viremia with development of neutralizing
antibody. Some antibody positive hens can transmit the infection to the next
generation, but the rate is usually lower than with viremic tolerant hens. The

frequency with which horizontal transmission occurs depends on genetic

makeup, concurrent exposure to immuno-depressive agents, and the strain of
ALV (Fadly et al.,1985; Fadly et al., 1987).

The most common neoplasm induced by ALV is lymphoid leukosis. In this
disease, the c-myc gene in a bursal lymphoid cell (B cell) is activated. B cell
lymphomagenesis is a multi-stage event, with activation of other cellular
oncogenes leading to tumor progression and metastasis. The first event can be
seen in the bursa a few weeks after infection by ALV in which the normal B cell
components are replaced by proliferating transformed B lymhoblasts. Some of
the transformed follicles regress, but one or more may progress over a period of
several months to neoplasia with metastasis to other organs such as liver and
spleen, leading to death of the chicken. The cells arising from one follicle are
clonal but progression to neoplasia in several follicles leads to polyclonal tumors
(Payne and Fadly, 1997).

A less common expression of infection by ALV is erythroid leukosis
(erythroblastosis), a disease in which the c—erbB gene in an erythroid cell is
activated. The bone marrow is largely replaced by proliferating erythroblasts,
leukemia develops, and there is localization in the liver, spleen and other organs
which become enlarged due to accumulation of intravascular erythroblasts.
Erythroid leukosis has a shorter latent period than LL.

Another disease caused by ALV is myeloid leukosis (myelobastosis and
myelocytomatosis). Cases generally occur sporadically in adult birds. A myeloid

cell becomes transformed, with the development of a severe leukemia. The liver,

W‘Y‘fij.

 

 

spleen and other organs become infiltrated by intravascular and extravascular
myeloid cells.

A variety of other tumors are also induced by the various strains of ALV.
The most common are myxosarcoma, histiocytic sarcoma, osteosarcoma,
chondrosarcoma, hemangioma and osteopetrosis (Payne, 1998).

Endogenous and exogenous ALVs can be differentiated on the basis of
host range determined in gp85 by biological assays in which selective chicken
embryo fibroblasts (CEFs) are used. A number of biological assays can be used
for the detection of endogenous and exogenous ALVs. Most ALVs produce no
visible morphologic changes in culture. Thus, indirect biologic assay such as
complement fixation (COFAL), Enzyme linked immunosorbant assay (ELISA),
phenotypic mixing (PM), resistance inducing factor (RIF), and non producer (NP)
cell activation tests are used for detection. These procedures are cumbersome
and require the use of selective cell culture. The most sensitive procedure for
differentiating between endogenous and exogenous ALV is the vian isolation.
Samples are inoculated on C/E (cell line resistant to endogenous ALV) and 0/0
(cell line susceptible to both endogenous and exogenous ALVs) CEF; 7 to 9
days later cell lysates are tested for the presence of ALV gs antigen by ELISA. If
a positive ELISA is obtained from 0/0 but not C/E CEFs the sample is positive
for endogenous ALV; positive ELISA on both C/E and C/O indicates exogenous
ALV. Currently, the most specific test for detection of antibody to ALVs is the
virus neutralization test. Both virus isolation and virus neutralization tests require

genetically susceptible chicken embryo fibroblasts (Fadly and Witter, 1998).

AVIAN LEUKOSIS VIRUS SUBGROUP J:

Avian Leukosis Virus (ALV) subgroup J (ALV-J) was first reported in the
United Kingdom in 1991 (Payne, 1991). ALV strain HPRS-103, the prototype of
ALV-J, appears to be a recombinant between an exogenous ALV and ancient
endogeneous avian retroviral envelope (E51) sequences (Benson et al., 1998
a,b).

ALV-J was isolated from commercial meat-type chickens (Payne et al.,
1991a,b). The virus induces predominantly myelocytic myeloid leukosis and
nephromas (Arshad et al., 1999). Domestic fowl, red jungle fowl, Sonnerat's
jungle fowl and turkey are susceptible to infection (Payne et al., 1992).

The most frequent gross lesion in chickens with myeloid leukosis (ML) is
moderate to massive enlargement of the liver caused by diffuse or miliary tumor
infiltration. A common occurrence is the development of skeletal myelocytomas,
which are creamy in color and can be seen on the inner surface of the sternum,
ribs, vertebrae and synsacrum. ML tumors can also be seen in the spleen,
thymus, gonads and kidney. Bursal involvement has not been reported.
Microscopically, the presence of immature myelocytes with characteristic
eosinophilic cytoplasmic granules in the affected tissues is considered
pathogenomonic for ML (Payne et al., 1992,1993). Strain HPRS-103, the
prototype ALV-J, lacks a viral oncogene and presumably induces

myelocytomatosis by insertional mutagenesis (Bai et al., 1995).

During the last several years, several strains of ALV-J were isolated from
broiler breeder and commercial broiler flocks experiencing a relatively high
incidence of ML in the United States, and many other countries around the world
(Nakamuro, 2000). In the United States, ALV-J induced ML was diagnosed in
affected flocks at 4 weeks of age or older. Strain ADOL-Hc1 of ALV-J had been
designated as the US prototype of ALV-J (Fadly and Smith, 1999).

Field observations suggest that flocks which experience a relatively high
rate of Marek’s Disease outbreaks also develop a high incidence of ALV-J-
induced tumors. In addition, field observations suggest that ALV-J is gaining
virulence, as losses in affected flocks are occurring earlier and at a higher
incidence. Most recently, ALV-J infection and tumors have been diagnosed on
two occasions in flocks other than meat-type chickens (Fadly, 1998).

The current program for control of ALV-J infection in broiler breeder
chickens is based on the elimination of dams that test positive for ALV-J by virus
isolation tests or by direct ELISA, a test that presently does not discriminate
between endogenous and exogenous ALV (Fadly and Witter, 1998).

The ALV infection status of birds is determined by the presence (+) or
absence (-) of viremia (V), viral shedding (S) and serum antibodies (A). In most
cases horizontal transmission of ALV, occurs through close contact with infected
chicks leads to immune non-shedders infection status (V-S-A+). However,
ALV-J infection status in meat-type chickens, post-hatch infected can result
either in tolerant viremic infection (V+S+A—) similar to congenitally infected

chickens, or in an immune status after showing a transient viremia. A proportion

of the immune chickens may also shed (V-S+A+) the virus. Chickens exposed to
the virus at an early age after hatch develop tolerant viremia (V+S+A-), whereas
chickens exposed to the virus at an older age develop immunity and may or may

not shed the virus (Venugopal, 1999).

MAREK'S DISEASE

Marek's Disease (MD) is the most common virus induced
Iymphoproliferative disease of chickens, characterized by mononuclear cell
infiltration of peripheral nerves, gonad, iris, various viscera, muscle and skin
(Payne, 1985). The disease was first described in 1907 in roasters with paresis
due to mononuclear infiltration of peripheral nerves and spinal roots (Marek,
1907)

MD is caused by a cell-associated herpesvirus, Marek's disease virus
(MDV). Molecular structure and genomic organization of MDV are similar to
those of alpha herpesviruses. The DNA of MDV is linear, double stranded, and
166-184 kb in size. Strains of MDV that induce disease in chickens are classified
as serotype 1. However, two additional classes of MDV, serotype 2 and 3, occur
naturally, in chickens and turkeys, respectively. Both are non-oncogenic. but
share antigenic determinants as determined by immunological assays and cross
protection tests (Schat, 1985). Because of considerable variation in disease
induction among isolates of pathogenic serotype 1 MDV, isolates are further

classified as mild (m), virulent (v), and very virulent (w) and very virulent plus

(w+) (Witter, 1997). This classification is based on ability of serotype 1 MDV to
induce lesions in vaccinated chickens (Witter, 1998).

MDV spreads by direct or indirect contact between birds, primarily by the
airborne route. Epithelial cells in the keratinizing layer of the feather follicle is the
site of MDV replication. Fully infectious vims in these cells serve as a source of
MDV in the environment. Many apparently normal birds are carriers that can
transmit the infection. Continual shedding of virus by infected birds and
hardiness of the virus results in a high prevalence of infection. There is no
vertical transmission of MDV (Calnek and Witter, 1997).

Nerve lesions is a frequent finding in affected birds. Macroscopic changes
are found in one or more peripheral nerves and in spinal roots and ganglia.
Celiac, cranial, mesenteric, brachial and sciatic plexuses can be affected and
characterized by loss of cross-striations, gray or yellow discoloration and
sometimes with an edematous appearance (Payne, 1985; Calnek and Witter,
1997)

MDV-induced lymphoid tumors may occur in one or more of a variety of
organs. Lymphomatous lesions can be found in the gonad, lung, heart,
mesentery, kidney, liver, spleen, bursa, thymus, adrenal gland, pancreas,
proventriculus, intestine, iris, skeletal muscle and skin. Visceral tumors are
especially common in more acute forms of the disease, and may be found in the
absence of gross nerve lesions. Severe outbreaks induce a high prevalence of
visceral lesions (62-89%), while in others the prevalence is low (5-7%) (Calnek

and Witter, 1997).

Diffuse infiltration of the liver causes loss of normal lobule architecture
and often gives the surface a course granular appearance. Nodular tumors can
be seen in the liver. Also, the proventriculus becomes thickened and firm, with
small to large focal areas of infiltration within and between glands. Muscle
lesions may be both superficial and in deep layers, and are most common in
pectoral muscles. Gross changes vary from tiny whitish streaks to nodular
tumors.

Microscopically there are two types of lesions that can be seen in
peripheral nerves (Calnek and Witter, 1997). Type A lesions consist of masses
of proliferating lymphoblastic cells; demyelination and Shwann cell proliferation.
Type B lesions 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 can be found.

Visceral tumors in MD often appear to be infiltrating tissue and consist of
a pleomorphic population of small to medium lymphocytes, lymphoblasts, large
reticular cells, large basophilic cells with large, dark nuclei. Plasma cells can be
seen.(Pope, .1996; Calnek and Witter, 1997). lrregularity of the pupil and loss of
pigmentation is the result of mononuclear cell infiltration of the iris.

Very virulent plus (w+) MDV causes early mortality with 77-100% of the
affected birds showing bursal and thymic atrophy. Visceral lesions with heart
affected more often than other organs, and a high percentage of ocular lesion

can be seen in chickens infected with w+ MDV (Witter, 1997a).

Lymphoid organs from chickens infected with w+MDV show little
evidence of recovery between 8 and 14 days post infection (DPl), whereas the
chickens infected with vMDV have a significant return towards normal by 14 DPI.
Chickens infected with w MDV had evidence of intermediate degrees of bursal
and thymic atrophy and recovery (Calnek et al., 1998).

MDV can be isolated from blood lymphocytes, heparinized whole blood,
and isolated tumor cells or feather tips. Chicken kidney cell and duck embryo
fibroblast cultures are preferred for isolation of serotype 1 MDV, whereas CEF
are used for isolation of serotype 2 and 3 MDV viruses. On the basis of plague
Serotype 1, 2 and 3 MDV can be distinguished. But lmmunofluorescent (IFA)
staining with using specific monoclonal antibodies provides a more accurate
differentiation. Both viral antigens and antibodies can be detected by IFA,
immunoperoxidase, agar gel precipitation (AGP), and ELISA tests (Calnek and
Witter, 1997).

Epidemiological, pathological, molecular and immunohistochemical
assays can be used in differential diagnosis of MDV induced lymphomas, and
those induced by ALV and REV. Microscopically, LL tumor cells are primarily
immature lymphocytes, which are characterized by poorly defined cytoplasmic
membranes, abundant cytoplasm and vesicular nuclei. If typical LL bursal lesions
are not present, LL and MD can not be distinguished by gross examination. In
such cases, the history, symptoms, gross and microscopic lesions, and cytology
should all be considered before a diagnosis of LL or MD can be made. The

demonstration by immunofluorescence of specific cell surface antigenic markers

10

on the tumor cells (MATSA and T-cell for MD and surface IgM for LL) is the best
way to establish a definite diagnosis of field cases that can not be readily
diagnosed on the basis of clinical signs and gross and microscopic lesions
(Fadly and Witter,1998).

MD has been well controlled in most flocks by the use of live vaccine
viruses. Commercial vaccines have been prepared with all three serotypes of
MDV. Serotype 1 MD vaccines are attenuated mild MDV, represented by the
Rispens strain (CVl-988) and MD11/75 (Witter, 1991). Serotype 2 MD vaccines
are derived from naturally occurring non-virulent chicken herpesvirus,
represented by 83-1 and 301B/1. Serotype 3 MD vaccines are derived from non-
virulent Turkey herpesvirus (HVT). There is a synergetic effect when viruses from
two or more types are combined together, especially between serotypes 2 and 3.
(Calnek et al., 1983; Calnek and Witter, 1997).

Losses have decreased dramatically since the advent of vaccination in the
early 19708 to eariy 1980s due to efficient vaccination with HVT. However,
because of the emergence of wMDV, HVT alone did not provide adequate
protection. In late 1980s, bivalent vaccines were developed and provided good
protection for over ten years before more virulent forms of field virus emerged
(Witter, 1997b). More virulent field MDV, now termed w+MDV, emerged in the
early 19905 and is not well protected against by bivalent HVT and SB-1
vaccines. Results of four consecutive trials by Witter (1996) and Kreager (1998)
indicated that Rispens, whether given alone or in bivalent or trivalent

combinations, provides better protection against these w+MDV than other

11

licensed vaccine strains. Vaccines are administered by inoculation of newly
hatched chicks or 18-day-old embryos.

From several reports, it is known that there are interactions between
Retrovirus and MDV. Bacon et al. (1989) Reported serotype 2 MDV vaccine
increased ALV-A-induced LL in certain lines of chickens and serotype 3 MDV
vaccine did not augment LL incidence. Pulaski et al. (1992) reported that SB—1
has been increasing the production of ALV virus in vitro. Marsh et al. (1995)
reported chickens inoculated with serotype 2 MDV and subgroup A avian
leukosis virus had higher ALV-induced hyperplastic follicles in bursa. Aly et al.
(1996) reported that serotype 2 MDV also augmented both ALV- and REV-
induced bursal lymphoma. Salter et al. (1999) reported that serotype 2 MDV
vaccine virus, strain SB-1, a component of the bivalent MD vaccine increased
the bursal lymphoma incidence in chickens carrying ALVA6 (subgroup A ALV
envelope glycoprotein).

Clearly, interactions between subgroup A ALV and serotype 2 MDV is well
documented, but it is not known whether various strains of MDV influence
subgroup J ALV-induced infection.

The objective of this study was to determine the influence of various MDV
serotypes on ALV-J-induced infection and tumors in two different lines of

chickens.

12

MATERIAL AND METHODS

VIRUSES:

5.38

Strain ADOL- Hc1, of ALV-J; stock titer of 10 tissue culture infectious units

(TCIU)/ml

Strain RAV-1, of ALV-A

Serotype 1 w+ MDV, strain 648A

Serotype 2 MDV, strain SB-1

Serotype 3 MDV, strain HVT (FC-126)

Dr. R.L. Witter, Avian Disease and Oncology Laboratory (ADOL) East Lansing,
Michigan kindly provided all Marek's disease viruses.

CHICKENS: Fertile eggs were obtained from: 1) White Rock breeder flock
maintained at USDA-ARS Southeast Poultry Research Laboratory, in Athens,
Georgia, and 2) commercial meat-type broiler breeder flock. The White Rock
breeders are known to be free from infection with ALV subgroups A, B, J as well
as MDV.

All embryonated eggs were hatched at ADOL. In both experiments,
chickens were maintained in isolators provided with filtered air under positive
pressure from the day of hatch until the end of the experiments. Chickens in

each treatment group were housed in separate isolators.

VIROLOGIC ASSAYS FOR ALV-J: At various ages, samples of blood and
cloacal swabs were collected from chickens in all groups. Blood was obtained

from jugular or radial vein and collected into sterile tubes containing heparin as

13

anticoagulant. Samples of blood and cloacal swabs were centrifuged at 1500
rpm for 10 minutes at 4 C and tested for ALV as previously described (Fadly and
Witter, 1998). Briefly, samples were inoculated on Line 0 CEF in 35 mm plates;
7-9 days later cell lysates were tested for the presence of ALV group specific
antigen (p27) by an enzyme-linked immunosorbent assay (ELISA) (Smith et al.,
1979)

SEROLOGIC ASSAYS FOR ALV-J: The vims neutralization (VN) test
was used to test samples for antibody to ALV-J as described by Fadly and Witter
(1998). Plasma samples were inactivated at 56 C for 30 minutes and diluted 1:5

in tissue culture medium without serum. Fifty microliter of strain ADOL-Hc1 of

ALV-J, with a titer of 103 infectious unit/ml was added to each well of a 96 well

Dynatech lmmulon l U-bottom microtiter plate. Fifty microliter of plasma samples

were added to each well. After incubation for 45 minutes at 370, 5x104 of line
0 CEF cells (150 microliter) were added to each well. Plates were incubated for
7-9 days. After freezing and thawing for two times, samples were tested for p27
with ELISA. Samples that tested negative by ELISA were considered positives
for antibody. Positive and negative controls were included for each test.
VIROLOGIC ASSAYS FOR MDV: Whole blood was collected from the
jugular vein into sterile tubes using heparin as anticoagulant. Samples were
centrifuged at low (700) rpm. Peripheral blood monocytes were collected and
inoculated on line 0 CEF cell culture. Cultures were incubated for 5—7 days and
examined for MDV-induced plaque. Samples with plaques were scored positive

for MD infection.

14

EXPERIMENTAL DESIGN:

The experimental design for both experiment 1 and 2 is shown in Table 1.

EXPERIMENT 1: Day-old progeny chicks of White Rock breeders were
divided into 8 groups; 20 chicks in each group. Chicks in groups 1, 2, 3, 4, 5, 6

were inoculated with 2X 10 3 infectious unit of strain ADOL-H01 at one day of

age. Chicks in groups 7 and 8 were inoculated with 2x103 infectious unit of

strain RAV-1 of ALV subgroup A. Also at hatch, chicks in groups 1, 4, and 7

_were inoculated with a mixture of HVT, 1500 PFU/0.1ml, and SB-1, 1500 PFU/

0.1 ml. Chicks in groups 2 and 5 were inoculated with 500 PFU/ 0.2ml of Rispens,
strain of MDV. At 2 weeks of age, chickens in groups 1, 2, 3 were challenged
with 500 PFU/ 0.1ml of w+ MDV strain 648A. Chickens in all treatment groups
were inoculated with respective inoculum via intra-abdominal route.

All chickens in experiments 1 and 2 were tested for ALV-J at hatch. In
addition, at two weeks of age, immediately before challenge with w+ MDV
chickens in groups 1, 2 and 3 were tested for the presence of various strains of
MDV vaccines, HVT, SB-1 and Rispens.

At 4, 8, 12 and 24 weeks of age, all chickens were tested for ALV-J
induced viremia, cloacal shedding and antibody (Fadly and Witter, 1998).

EXPERIMENT 2: Day-old progeny chicks from a commercial broiler flock
were divided into 8 groups; 20 chicks in each group. The experimental design for
experiment 2 was the same as that in experiment 1, except the experiment was

terminated at 17 weeks of age.

15

PATHOLOGY: In both experiments, chickens that died and those that
survived the experiment (24 weeks, experiment 1; 17 weeks, experiment 2) were
necropsied. Chickens were examined for ALV-J- and MDV-induced tumors.
Tissues were fixed in 10% formalin buffer and paraffin embedded, sections were
stained with hematoxilyn and eosin.

STATISTICS: The SAS Institute Inc. (1999), SAS/STAT Software NC:
SAS Institute Inc. was used in chi-square tests to analyze the differences
between chickens in groups for viremia, cloacal shedding and antibody levels.

Significance was assumed at the 0.05 level of probability (Allison, 1999).

16

Table 1: Experimental design for exp_eriments 1 and 2

 

 

 

Treatment
1“ day of age 2 weeks of age
Group ALV-J ALV-A HVT SB-1 Rispens w+MDV
1 + - + + - +
2 - - - + +
3 + - - - - +
4 + - + + - -
5 + - — - + -
6 + - - - - -
7 - + + + - -
8 - + - - - -

 

Strain ADOL-H01 of Subgroup J ALV
Strain RAV-1 of Subgroup A ALV

Serotype 3 MDV vaccine, strain FC—126
Serotype 2 MDV vaccine, strain SB—1
Serotype 1 MDV vaccine, strain Rispens
Serotype 1 w+MDV challenge ,strain 648A

17

RESULTS

Experiment 1:

Tables 2A and 2B show the incidence and percentage of ALV-J-induced
viremia, antibody and cloacal shedding in White Rock chickens used in
experiment 1. At 4 weeks of age, there was no significant difference in
incidences of viremia among chickens in various groups. At 8 weeks of age, the
incidence of viremia in chickens in groups 1 and 4 that had been inoculated
with bivalent vaccine (HVT+SB-1) were significantly lower than that in other
groups. At 12 weeks of age, chickens in group 1 that received HVT+ SB-1 at
hatch had the lowest level of viremia, and chickens in group 2 that received
Rispens at hatch and w+ MDV at 2 weeks of age had the highest level of
viremia. There was no significant difference among chickens in groups 5 and 6.

At 24 weeks of age, chickens in groups 1 and 4 had no or little evidence
of viremia. Again the highest level of viremia was noted in chickens in group 2
that had been vaccinated with Serotype 1 vaccine at hatch and challenged with
w+MDV at 2 weeks of age.

As viremia levels decreased in chickens in groups infected with ALV-J and
vaccinated with bivalent MDV vaccine (from 4 weeks through 24 weeks), the
antibody levels were increasing. Chickens in groups 1 and 4 had the highest
antibody levels at 24 weeks of age.

Cloacal shedding was detected at 4, 8, 12 and 24 weeks of age. At 4

weeks and 8 weeks of age, the incidence of cloacal shedding in chickens in all

18

groups was very low if any. At 12 weeks of age the incidence of shedding in
chickens in groups 2 and 6 was higher than that in chickens in other groups. At
24 weeks of age, the highest shedding incidence was detected in chickens in
group 2, whereas, the lowest incidence was in chickens in groups 1 and 4. Due
to MDV-induced mortality, only one chicken survived to 24 week in group 3.

The very low incidence of viremia, shedding and antibody of ALV-A in
White Rock chickens suggest that the breeder flock is genetically resistant to

infection with ALV-A.

19

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Experiment 2:

Table 3A and 3B shows the incidence and percentage of ALV-J-induced
viremia, antibody and cloacal shedding, respectively in commercial broiler
breeder chickens used in experiment 2. In this experiment commercial broiler
breeders were tested for viremia, shedding and antibody at 4, 8 and 12 weeks of
age. The experiment was terminated at 17 weeks of age.

At 4 weeks of age, there was no significant difference in incidences of
viremia among chickens in various groups. At 8 weeks of age, the incidence of
viremia for chickens in groups 1 and 4, inoculated with bivalent vaccine
(HVT+SB-1) was significantly lower than that in any of the other groups.

At 12 weeks of age, except for chickens in group 2 that were inoculated
with Rispens/ w+ MDV there was no statistically significant difference in ALV-J
viremia among chickens In various groups. Chickens in group 2 had the highest
frequency of viremia. At 17 weeks of age, chickens in groups 1 and 4 that were
inoculated with bivalent vaccine had the lowest frequency of viremia. Although
there was no statistical difference among chickens in groups inoculated with
ALV-J/ Rispens/w+MDV and only ALV-J, the viremia levels were highest in the
former group.

At 17 weeks of age, cloacal shedding was significantly higher in chickens
in groups inoculated with ALV-J/Rispens/w+MDV compared to with chickens in
other groups.

At 4 weeks of age, antibody was not detected in chickens in any group,

but there was a low level of shedding and the incidence of antibody positive birds

22

was increasing in all groups. Antibody was not detected in chickens used in
experiment 2. At 17 weeks of age, there was no significant difference in antibody
among chickens in all groups. Also, at 17 weeks of age, cloacal shedding was
highest in chickens in group 2, and there was difference in antibody frequency
between chickens in group 2 and chickens in groups 1, 4, 5, and 6. Due to MDV-
induced mortality in group 3 only one chicken survived to 17 weeks of age.
Commercial broiler breeder chickens used in this experiment were also found to
be resistant to infection with subgroup A -ALV.

Tumor incidence in experiments 1 and 2:
Table 4 shows the incidence of ALV-J and MD-induced tumors in MD vaccinated
and unvaccinated chickens. In both experiments chickens in groups infected with
only ALV-J or ALV-J and Rispens MD vaccine developed the highest frequency
of ALV-J -induced tumors. Only 3 birds developed ML in White Rock chickens.
Commercial broiler breeder chickens most of the ALV-J-induced tumors were LL.
Several other type of tumors such as myxosarcoma, erythroblastosis and
hemangioma were detected in both experiments. In White Rock chickens
although the groups infected with ALV-J/Rispens/w+MDV and ALV-J/Rispens
had the highest tumor incidence, there was no significant difference among other
groups.

In commercial broiler breeder chickens there was significant difference
between group 5 and groups 1, 2, 4. Although there was no significant

difference, the ALV-J-induced tumor incidence was highest in chickens infected

23

with ALV-J and vaccinated with Rispens compared with all other groups in broiler

breeder progeny.

Influence of MDV on ALV-J- induced tolerant infection:

Table 5 shows the influence of various strains of MDV on the incidence of
ALV-J tolerant infection in meat-type chickens. Because the results from
experiments, 1 and 2 were similar therefore, the data were pooled. Chickens
inoculated with HVT + SB-1 had the highest incidence of immunity against ALV-J
infection. Whereas chickens inoculated with serotype 1 MDV had the highest

incidence of tolerantly infected chickens.

24

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Table 5: Influence of virulent and vaccine strains of MDV and ALV-J tolerant
infection in

17-24 week- old meat-type chickensA

 

 

 

 

Lot # Treatment Status of ALV-J infection
ALV-J HVT SB-1 Rispens w+MDV V+s+A-l3 v-s-A+°

1 + + + - + 6/27 (22) a 16/27 (59) a

2 + - - + + 16/30 (53) b 2/30 (6) b

3 + - - - + NA 0 NA

4 + + + - - 5/26 (19) a 22/26 (85) c

5 + - - + - 14/25 (56) b 7/25 (28) b

6 + - - — - 7/21 (33) ab 7/21 (33) ab

 

AAt hatch chickens were inoculated with strain ADOL-H61 of ALV-J and with
MDV vaccines (HVT, 83-1, or Rispens) at hatch; chickens were challenged with
strain 648A of MDV at 2 weeks of age. Because virological and serological
results at 24 weeks of age (experiment 1) and at 17 weeks of age (experiment 2)
were similar, the data were pooled.

BTolerantly infected chickens (viremic, shed virus in cloacal sections and lacked
antibody).

CImmune chickens (non-viremic, non-shedder and antibody positive).

DNA = Not applicable, all chickens died from MDV within 8 weeks of age.
For each age at testing percentages within a column followed by different letters
are significantly different (P<0.05)

28

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3O

DISCUSSION

Data from experiments 1 and 2 demonstrate that chickens from both
White Rock and commercial broiler breeder flocks are susceptible to infection
with strain ADOL-H01 of ALV-J. The data from this study indicate that bivalent
MDV vaccines containing serotype 2 and 3 (HVT/SB-1) may influence the
response of chickens to infection with ALV-J. Viremia results from experiment 1
suggest that the bivalent MDV vaccine used in this study significantly reduced
the incidence ALV-J viremia in White Rock chickens. At 8 and 24 weeks of age,
the incidence of chickens with ALV-J viremia was the lowest in groups inoculated
with bivalent MDV vaccine (groups 1 and 4), regardless of challenge with
w+MDV suggesting that bivalent vaccine can also reduce ALV-J viremia in
chickens exposed to virulent virus. Chickens in group 1 that developed viremia
cleared the virus by 24-weeks of age when antibody levels were detected in
100% of the chickens.

In both experiments chickens received ALV-J/Rispens and challenged
with 648A strain of w+MDV had the highest incidence of cloacal shedding.
Cloacal shedding rates of ALV-J in chickens in groups infected with ALV-J and
vaccinated with bivalent MDV vaccine, was very low, compared to the other
groups, suggesting that there is also some influence of bivalent vaccine on ALV-
J shedding. In contrast the chickens in groups infected with ALV-J and
vaccinated with serotype 1 MD had the highest frequency of cloacal shedding

whether challenged or not with w+MDV.

31

Bacon et. al. (1989) reported that serotype 2 MDV, but not HVT, may
augment the development of subgroup A ALV-induced lymphoma in certain lines
of chickens. Pulaski et. al. (1992), Coussens (1994), reported that there is a
direct relationship between serotype 2 MDV and subgroup A-ALV, which results
in enhanced retroviral gene transcription. In the present study, bivalent vaccine
containing serotype 2 and 3 did not enhance the viremia of ALV-J, but appeared
to enhance the clearance of viremia of ALV-J in birds inoculated with these two
serotypes in the White Rock strain.

Data from experiment 2 confirmed data from experiment 1, indicating that
serotype 1 MD vaccine with w+MDV increased susceptibility of chickens to ALV-
J-induced infection. There had been an enhancement of ALV-J-induced viremia
levels in chickens, which received ALV-J and serotype 1, Rispens strain of MD
vaccine and challenged with 648A strain of w+MDV. Although there was no
statistically significant difference between chickens in group 1 and chickens in
groups 4, 5 and 6 in experiment 2, viremia levels were low (35%) at 17 weeks of
age. These birds in both groups were inoculated with bivalent serotype 2 and 3
MDV vaccine and whether challenged or not with w+MDV.

There are reports that the incidence of tumor-associated mortality is
widely variable between ALV-J infected flocks, and may involve additional factors
such as the presence of immunosuppressive agents, concurrent infections, and
vaccination against other diseases (Venugopal, 1999). In the present study, it
was demonstrated that serotype1 of MDV may enhance the strain ADOL-H01 of

ALV-J infection, but not tumor development in commercial broiler breeder

32

chickens used in Experiment 2. In the same experiment there was no difference
between chickens infected with only ALV-J or ALV-J/Rlspens strain of MDV for
ALV-J induced viremia. The enhancement of viremia probably due to 648A
strain of w+MDV.

ALV-J induced tumors in meat-type chickens are primarily ML (Arshad et
al.,1999). In our study only 3 ML were seen in White Rock chickens. These were
in groups infected with ALV-J alone and various vaccine strains of MD. The
tumors in all groups in both experiments were mainly LL. This difference in type
of tumors can be probably explained by strain of chickens and virus used in the
current experiments. Myeloid tumors were same as described by Payne et al.,
(1992, 1993).

Payne et al. ( 1992), reported that there were type B like MDV nerve lesions
in chickens infected with ALV-J. In both experiments in this study, the type B
nerve lesions were seen only in chickens that were infected with 648A strain of
w+MDV, isolated from layers which causes 100% MD and bursal thymic atrophy
in non-vaccinated chickens and 98% nerve lesions in bivalent MD vaccinated
chickens (Witter, 1996). Strain HPRS-103 of ALV-J showes a lower propensity to
replicate in the medullary region of the lymphoid follicles of the bursa of
Fabricius, in comparison to subgroup A (Venugopal, 1999). Supporting the same
suggestions in our study in both experiments bursal tumors were not seen in
chickens which were infected with strain ADOL-Hc1 of ALV-J. Due to w+MDV
strain 648A there were bursal thymic atrophy in groups infected with 648A

strain.

33

In meat-type chickens, post-hatch infection of chickens with ALV-J can
result either in tolerant viremic infection (V+S+A-), similar to congenitally infected
chickens, or in an immune status after showing a transient viremia. A proportion
of the immune chickens may also shed (V-S+A+) the virus. Chickens exposed to
the virus shortly after hatch tend to develop tolerant viremia (V+S+ A-), while
exposure at an older age leads to immune chickens that may or may not shed
the virus (Venugopal, 1999.). In the current study, chickens infected with strain
ADOL-H01 of ALV-J were mostly tolerant viremic and immune shedders; these
results are in agreement with those reported by Venugopal (1999). Chickens in
groups infected with ALV-J/Rispens and challenged with w+MDV were mostly
tolerant viremic (V+S+A—), in contrast chickens infected with bivalent vaccine
(HVT/SB-1) which were mostly non-viremic, non-shedder and antibody positive
(V-S-A+).

Chickens infected with ALV-J/Rispenslw+MDV were tolerant viremic
(V+S+A-) and chickens infected with ALV-J/Rispens were both tolerant viremic
(V+S+A-) and immune non-shedders (V-S-A+), suggesting that w+MDV may
influence the infection status of ALV-J infected chickens. Chickens infected with
ALV-J/Rispens were both V+S+A- and V-S-A+.

MD-induced tumors were more frequently encountered in chickens
infected with ALV-J/Rispens/w-I-MDV than in those infected with ALV-J/HVT+SB-
1/w+MDV, regardless of strain of chickens used. Results reported in this study

are interesting and warrant further investigations.

34

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VITA

The author was born in Ankara, Turkey in 1961 and completed
Elementary, Middle and High schools in same city until 1978. She then attended
the Faculty of Veterinary Medicine at Istanbul University from 1978-1984 and
graduated with a DVM degree in 1984. Between 1986 and 1989 worked in City
Control Laboratory in Amasya City as a veterinarian. The author moved to
Manisa, Poultry Diseases Research and Vaccine Production Institute, Manisa in
1989 for Avian Diseases Specialist residency program. She received her
specialist degree in 1992. In 1998 author received scholarship from Turkish
Ministry of Agriculture to study in the US. From 1998 to 2000, the author was a
graduate student under the direction of Dr. Willie Reed at the Department of
Pathobiology and Diagnostic Investigation at Michigan State University. She
performed her MS research at the Avian Disease and Oncology Laboratory, East
Lansing, Michigan under direction of Dr. Aly Fadly on the topic of the influence of
Marek's disease virus on infection with Avian leukosis virus subgroup J in

chickens. She is married and has one son.

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