100-2022

Occurrence of Marek's Disease in Backyard Chicken

Flocks in Vietnam

Authors: Viet Thu, Ho Thi, Trang, Huynh Ngoc, Phuoc Chien, Nguyen
Tran, Ngu, Nguyen Trong, and Hien, Nguyen Duc
Source: Avian Diseases, 66(2) : 1-7
Published By: American Association of Avian Pathologists
URL: https://doi.org/10.1637/aviandiseases-D-22-00009

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 AVIAN DISEASES 66:000–000, 2022
100-2022
Case Report—

Occurrence of Marek’s Disease in Backyard Chicken Flocks in Vietnam

Ho Thi Viet Thu,AC Huynh Ngoc Trang,A Nguyen Tran Phuoc Chien,A Nguyen Trong Ngu,A and Nguyen Duc HienABC
A
Can Tho University, Campus II, Area II, 3/2 Street, Xuan-Khanh Ward, Ninh-Kieu District, Can Tho City, Vietnam
B
Department of Animal Health, Can Tho City, Vietnam
Received 20 January 2022; Accepted 8 April 2022; Published ahead of print 7 July 2022

SUMMARY. Marek’s disease (MD) is a common lymphomatous and neuropathic disease of birds, especially chickens, and has
caused significant losses to chicken production as a result of high mortality and morbidity. This study aims to determine the status
of MD in backyard flocks in the Mekong Delta of Vietnam and to analyze clinical cases occurring during a year. The study was
carried out from August 2018 to July 2019, during which time 16 suspected cases of chicken flocks with MD were observed, 40
chickens were subjected to anatomopathological examination, and PCR was performed for diagnosis of MD and determination of
Marek’s disease virus (MDV) serotypes. The results showed that all of the examined flocks were confirmed as experiencing MD.
Nearly all cases were in an acute form with typical lesions of visceral lymphomas. Besides the presence of Marek’s disease virus
serotype 1 (MDV-1) from 100.0% of tested chicken flocks (16/16), nononcogenic turkey herpesvirus serotype 3 (MDV-3) was also
found from 8 of 16 (50.0%) of examined chicken flocks. Morbidity and mortality at sampling time varied from 1% to 42.11% and
0.6% to 10%, respectively. Chicken flocks with MD vaccination had lower morbidity and mortality. These first findings confirm
endemic MD in backyard chicken populations in Vietnam and confirm it continues to be a threat to chickens in backyard flocks
and poultry production in general.
RESUMEN. Presentación de la enfermedad de Marek en parvadas de pollos de traspatio en Vietnam.
La enfermedad de Marek (MD) es una enfermedad linfoproliferativa y neuropática común de las aves, especialmente de los pollos y
ha causado pérdidas significativas en la producción de pollos como resultado de la alta mortalidad y morbilidad. Este estudio tiene
como objetivo determinar el estado de la enfermedad de Marek en parvadas de traspatio en el delta del Mekong en Vietnam y analizar
los casos clı́nicos que ocurren durante un año. El estudio se llevó a cabo de agosto de 2018 a julio de 2019, tiempo durante el cual se
observaron 16 casos sospechosos de parvadas de pollos con enfermedad de Marek, 40 pollos fueron sometidos a examen
anatomopatológico y se les realizó PCR para el diagnóstico de enfermedad de Marek y determinación del serotipo del virus. Los
resultados confirmaron que todas las parvadas examinadas presentaban enfermedad de Marek y casi todos los casos presentaban una
forma aguda con lesiones tı́picas de linfomas viscerales, especialmente en el hı́gado. Además de la presencia del serotipo 1 del virus de
la enfermedad de Marek (MDV-1) en el 100% de las parvadas de pollos analizadas (16/16), también se encontró el serotipo 3 del
herpesvirus del pavo no oncogénico (MDV-3) en 8 de 16 (50.0 %) de las parvadas de pollo examinadas. La morbilidad y la
mortalidad en el momento del muestreo variaron del 1% al 42.11% y del 0.6% al 10%, respectivamente. Las parvadas de pollos con
vacunación contra la enfermedad de Marek tuvieron menor morbilidad y mortalidad. Estos primeros hallazgos confirman la presencia
endémica del virus de la enfermedad de Marek en las poblaciones de pollos de traspatio en Vietnam y confirman que continúa siendo
una amenaza para los pollos en parvadas de traspatio y para la producción avı́cola en general.
Key words: chicken, Marek’s disease, Vietnam, serotype
Abbreviations: HVT ¼ herpesvirus of turkey; MD ¼ Marek’s disease; MDV ¼ Marek’s disease virus; MDV-1 ¼ Marek’s disease
virus type 1; MDV-2 ¼ Marek’s disease virus type 2; MDV-3 ¼ Marek’s disease virus type 3; PM ¼ postmortem

Marek’s disease (MD) is a common lymphomatous and
neuropathic disease of chickens caused by an alphaherpesvirus
called the Marek’s disease virus (MDV). The disease was first
reported by Jozséf Marek (1). Before the use of vaccines, loss of the
MDV-infected flocks was estimated to range from 25% to 30%,
and occasionally as high as 60% (2). After the first vaccine was
introduced, vaccination has been the best choice for the prevention
and control of this disease (3). MD morbidity and mortality are
reduced, but vaccines now seem to be less effective due to
continuing evolution of virulence and the emergence of more
virulent pathotypes (4). In Vietnam, the majority of farm
operations have used vaccines for prevention, but MD outbreaks
have still occurred and caused a great impact on chicken production
due to high morbidity and mortality (5), and many backyard
chicken flocks have been reported carrying oncogenic Marek’s
C
Corresponding authors. E-mail: htvthu@ctu.edu.vn; ndhien@ctu.edu.vn
disease virus serotype 1 (MDV-1) (6). Although MD is a major
concern of industrial chicken production, many backyard chicken
raisers and extension workers are unaware of MD, and there are rare
reports on this disease, in particular, no literature in English about
MD in our country. Therefore, this study aims to investigate the
status of MD in chicken backyard flocks in the Mekong Delta,
Vietnam and to determine the types of MDVs circulating in
diseased chicken flocks.
MATERIALS AND METHODS
Study time and site. The study was conducted over a year, during
the period from August 2018 to July 2019, by examination of 16
chicken flocks that were suspected of being ill with MD by clinical signs
and macrolesions from five provinces (Tra Vinh, Hau Giang, Vinh
Long, Long An, and Can Tho) in the Mekong Delta. This study used
many examination methods to diagnose MD from the chicken flocks,

1
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 2 H. T. V. Thu et al.

Fig. 1. Liver with multisized (arrow) tumorous nodules (A) or enlargement whitish appearance with many times bigger than normal occupied
whole abdominal cavity (B), lung (C), and spleen (D) with multisized (circle) tumorous nodules; kidney (E) and heart (F) with lymphocyte tumors
(circle) which made the architecture deformed; multiple lymphomas in proventriculus (G), intestine (H), and hemorrhage at the tumors of
proventriculus and intestines (arrow).

including postmortem, histopathology examination, and conventional
PCR for viral DNA confirmation.
Postmortem. All diseased chickens and chicken carcasses from 16
MD suspicious flocks at visitations were subjected to postmortem (PM)
examination. The chicken’s carcasses were carefully observed, and gross
lesions were identified and recorded. Visceral organs with lymphomas of
40 chickens of these flocks were collected for further testing. The
samples collected included one or more varieties of tumorous tissues
(heart, liver, spleen, kidney, proventriculus, and intestine). Each sample
was divided into two parts; one was placed on ice, and the other was put
into a 10% formalin solution bottle and transported to the laboratory of
the Veterinary Medicine Department, College of Agriculture, Can Tho
University, for preparation of histopathological sections and PCR tests.
Information on 16 diseased chicken flocks was also surveyed in detail
using a questionnaire for collecting epidemiological data such as flock
population, chicken age, number of birds infected and died, vaccination
status (with or without MD vaccination, and type of MD vaccine), and
bird breeds. The number of birds infected and deceased was calculated
based on necropsy and carefully interviewing chicken owners to
minimize bias.
Histopathological examination. Samples of different organs of MD
suspected chickens were kept in 10% formalin until fixation,
dehydrated in ethanol (70%–100%), cleared in xylene, and embedded
in paraffin. The 5 lm thickness of the paraffin section was prepared and
labeled appropriately and thereafter deparaffinized and stained with
hematoxylin and eosin dyes. Finally, histopathological sections were
examined under a light microscope at 1003, 2003, and 4003 high-
powered fields.
Detection of MDVs with PCR. The tumors from the visceral organs
of each suspicious MD chicken were pooled into one for preparing a

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 Marek’s disease in backyard chicken flocks in Vietnam
Fig. 1. Continued.
homogenate. DNA was extracted from homogenates of the tissue
samples mixed using DNA TopPUREt Tissue viral extraction (ABT
Biomedical Solution Company, Vietnam). In total, 40 processed
samples were subjected to PCR, using serotype 1, 2, and 3 specific
primers. Commercial vaccines (CVI-998, SB-1, and HVT) were used as
positive for the MDV serotypes and synthetic DNA fragments,
sequences of primer pairs for detecting three serotypes (1, 2, and 3)
of MDVs, and PCR procedures performed following the research of
López-Osorio et al. (7). Conventional PCR was used to detect specific
MDV genes. Sets of specific oligonucleotide primers (Gallid herpesvirus
2 Meq, Gallid herpesvirus 3 gD, and Meleagrid herpesvirus 1 sORF 1
genes) were used to detect each of the MDV serotypes (Marek’s disease
virus type 1 [MDV-1], Marek’s disease virus type 2 [MDV-2], and
herpesvirus of turkey [HVT]) and their locations as in Supplemental
Table S1. Samples were considered positive for MDV-1, MDV-2, and
HVT by amplification of products of 1148, 1040, and 350 base pairs,
respectively.
RESULTS
Postmortem examination. In this present study, PM examination
of suspicious chickens revealed visceral lymphomas in one or more
of a variety of chicken internal organs. The most frequently reported
was from the liver with multisized tumorous nodules or enlargement
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3
with a grayish-whitish appearance and many times bigger than
normal (Fig. 1A–E), and hemorrhages occasionally were seen at the
tumorous proventriculus (Fig. 1G) and intestine (Fig. 1H). There
were only a few chickens that were collected from the flocks at the
ending outbreak periods that had grayish mild enlargement of the
sciatic nerve.
The histopathological examination. The histopathological chang-
es observed from tissue sections of internal organs (liver, lung,
kidney, heart, gizzard, and intestine) obtained from infected
chickens included infiltration and aggregation of lymphocytes at
different levels from mild to severe. They were gathered in a mass
(circle) or dispersed (arrow) among parenchyma or tissue of internal
organ cells (Fig. 2). Under the microscope, sections of internal
organs at 2003 magnification presented extensive infiltration round
neoplastic cells (Fig. 2A, B); at higher magnification (4003), there
was a proliferation of pleomorphic tumor cells (Fig. 2C, D) which
replaced and compressed the parenchyma or tissue cells leading to
distortion of tissue architecture.
Detection of MDVs by the polymerase chain reaction. In the
current study, 40 pooled internal organ samples of 40 chickens
suspected of having MD from 16 chicken flocks were subjected to
screening of MDV-1, MDV-2, and HVT by conventional PCR.
The results revealed that the MDV-1 oncogenic strain (Fig. 3A) and
 4 H. T. V. Thu et al.

Fig. 2. Microscopic lesions of internal organs; intensive infiltration of lymphocytic cells which focused (circle) or dispersed (arrow) among
hepatic parenchyma (A), and submucosa of the gizzard (B) at 2003 magnification; at higher magnification (4003), heart section (C) showing
infiltration of pleomorphic lymphocytes in the myocardium, and lung section (D) showing the proliferation of pleomorphic lymphocytes in the
alveoli (arrow) and around bronchiole (circle).

the HVT nononcogenic strain (Fig. 3B) were amplified from these MDV-1 from samples of internal organs having tumors. There were
pooled samples. 32/40 (80%) chickens that were positive with MDV-1. In addition,
The results of the PCR assays in Table 1 confirmed that all 16 HVT was also detected from 8 of 16 examined flocks; MDV-2 was
suspected flocks (100%) were involved with MD by detecting not detected.

Fig. 3. (A) Agar gel electrophoresis of PCR products of MDV-1; M: Marker 100 bp, well 1, 2, 3, and 4: positive samples, (): negative control,
(þ): positive control. (B) Agar gel electrophoresis of PCR products of Marek’s disease virus type 3 (MDV-3); M: Marker 100 bp, well 1, 2, 3, 4, 5, 6:
positive samples, (): negative control, (þ): positive control.

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 Marek’s disease in backyard chicken flocks in Vietnam 5
Table 1. Results confirming MD diagnosis by PCR and distribution of MD outbreaks by place.

No. No. No. No. No.

MDV-1–infected MDV-2–infected MDV-2–infected MDV-3–infected infected
Place birds (%) flocks (%) birds (%) flocks (%) birds (%)
Tra Vinh 10/11 (90.9) 0/4 (0) 0/11 (0) 2/4 (50) 4/11 (36.4)
Hau Giang 3/3 (100) 0/3 (0) 0/4 (0) 2/3 (75) 2/3 (75)
Vinh Long 6/6 (100) 0/2 (0) 0/4 (0) 0/2 (0) 0/6 (0)
LongAn 7/12 (58.3) 0/3 (0) 0/13 (0) 3/3 (100) 7/12 (58.3)
Can Tho 6/8 (80) 0/4 (0) 0/8 (0) 1/4 (25) 2/8 (25)
Total 32/40 (80) 0/16 (0) 0/40 (0) 8/16 (50) 15/40 (37.5)

Vaccination status and other epidemiological data collected from
16 flocks in this study are shown in Table 2. The data from Table 2
show that the age of affected birds ranged from 29 to 210 days, and
morbidity and mortality rates varied from 1% to 42.1% and 0.6%
to 20%, respectively. However, cumulative morbidity and mortality
would be higher due to these data being collected only one time at
sampling. Such studies of surveillance need to be conducted on a
regular and continuous basis to assess a more precise measure of the
economic losses from actual MD.
DISCUSSION
PM and histopathological examination. The results of PM
examination (Fig. 1) of diseased chickens showed that chickens had
lymphomatous tumors in visceral organs from every flock, and a few
chickens had lesions of neuritis at the end of the outbreak. In
addition, the best method for confirming the clinical cases of MD is
histopathological observation (8), which was also realized in this
study, and the specific microlesions of MD also were found in
tumorous visceral organs (Fig. 2) of diseased chickens by infiltration
and aggregation of lymphocytes. These results suggest that all of
these examined flocks were affected by ‘‘visceral lymphomatosis’’ or
acute form.
Detecting MDVs with the polymerase chain reaction. Although
specific macro and microlesions are quite enough to confirm MD
clinical cases (8). In some circumstances, MD can be misjudged with
reticuloendotheliosis and lymphoid leucosis, or other cancer
diseases. PCR is a quickly performed and accurate assay that can
demonstrate the presence of virus in the tumor that is sufficient to
make a positive diagnosis. In addition, PCR tests enable
differentiation of oncogenic (MDV-1) and nononcogenic strains
(MDV-2 and HVT) (9,10,11,12). MDV-1 is capable only of
inducing tumors. So the presence of MDV-1 from chickens bearing
tumors can lead to the conclusion that it is indeed MD in the
chickens. As the data in Table 1 show, MDV-1 positive chickens
were found in all 16 flocks (100.0%). All of the chickens from the
flocks in Hau Giang and Vinh Long were positive, but some in Long
An, Tra Vinh, and Can Tho were negative. This might suggest that
there were other cancer diseases such as conditions caused by
retroviruses, reticuloendotheliosis virus, and avian leucosis virus that
concurrently affected these flocks (3,13); other studies should be
conducted to clarify this situation. The presence of HVT may be a
natural infection, such as a latent infection from the vaccine virus
(14,15,16,17).
In the Mekong Delta, chicken production is mainly based on
middle- and small-scale operations with indigenous chicken breeds
for broilers and exotic breeds for layers. MD in chickens might be
due to lack of vaccination because the necessity is not commonly
realized by small chicken flock holders, but MD outbreaks have not
been rare in vaccinated flocks. The data in Table 2 show that 6 of 16
MD-positive flocks were vaccinated earlier. Vaccinations of birds

Table 2. Epidemiological measures of disease occurrence in MD-affected flocks.

Type of Chicken Population No. affected No. died Deaths/diseased

Flock no. Place chickens age (days) Vaccination status (birds) (%)A (%)A (%)
1 Tra Vinh Noi-Binhdinh 65 Cell-free HVT 1000 100 (10) 20 (2.0) 20
2 Tra Vinh Noi-Binhdinh 72 Cell-free HVT 2000 70 (3.5) 40 (2.0) 57.1
3 Tra vinh Noi-Binhdinh 75 None 2000 200 (10) 150 (7.5) 75
4 Tra vinh Noi 170 None 700 200 (28.6) 50 (7.14) 25
5 Hau giang Noi 45 None 300 49 (16.3) 10 (3.3) 20.4
6 Hau giang Noi 35 None 46 4 (8.7) 2 (4.4) 50
7 Hau giang Noi 105 None 500 100 (20) 50 (10) 50
8 Vinh Long Noi 200 None 1000 60 (6) 10 (1) 16.7
9 Vinh Long Noi 120 None 10 1 (10) 1 (10) 100
10 Long An Noi 65 None 1000 100 (10) 100 (10) 100
11 Long An IsaBrown 180 HVTþCVI-998/Rispens 50,000 1000 (2) 500 (1) 50
12 Long An IsaBrown 210 HVTþCVI-998/Rispens 49,600 500 (1) 300 (0.6) 60
13 Can Tho Noi-Binhdinh 90 Cell free HVT 2000 20 (1) 20 (1) 100
14 Can Tho Noi-Binhdinh 29 HVTþCVI-998/Rispens 3000 50 (1.7) 30 (1) 60
15 Can Tho Noi 40 None 38 16 (42.1) 3 (7.9) 18.8
16 Can Tho Noi 40 None 30 4 (13.3) 3 (10) 75
Total 113,224 2474 (2.19) 1289 (1.14) 52.1
A
The number of affected and dead chickens was achieved by interviewing the chicken raisers, observations, and necropsy of chickens during the
site visit.

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 6 H. T. V. Thu et al.
were mostly done by administering the vaccine to day-old birds
immediately after hatching with cell-free HVT or HVT combined
with MDV-1 (CVI-998/Rispens) vaccines and no booster was given.
However, the reason why the chickens were not protected against
MD although they were vaccinated was not investigated in this
study. Similar findings were reported by many researchers, who
reported that vaccination with either the MDV-1 or HVT vaccine
did not totally protect the chicken flocks against MD
(11,18,19,20,21). Failure of vaccination can come from mistakes
in the preparation, storage handling, and administration of the
vaccine. Besides, mistakes in poultry management can be made, such
as heavy and very early exposure to infection, chicken houses not
being well disinfected, or birds being reared for many cycles in the
same place without cleaning or disinfecting, etc. It was also reported
that vaccinated chickens may shed virulent virus into the
environment because of its immunosuppressive abilities, and
MDV-1 has evolved to become more competent in immune system
depression or evasion (22,23,24,25), and the evolution of MDV
virulence remains the major challenge for the control of the disease
(18,26).
Higher morbidity and mortality were found in unvaccinated
flocks (Nos. 10, 15, and 16). Shortly after the isolation of MDV in
the late 1960s vaccines were developed in many countries, and the
HVT vaccine was widely used in industrial poultry production (27).
The vaccination reduced the incidence of MD by 99% and was the
first successful vaccine against naturally occurring virus-induced
cancer (28), but in the late 1970s, HVT showed a limited effect on
viral infection and transmission (29). Currently, only the attenuated
MDV strain CVI-998-Rispens is effective in protecting against the
very virulent MDV.
In this study, the high mortality of MD chickens might be due to
the more virulent emerging MDVs (29); experimented infection of
MDV 6.13 strain in multiage chickens showed that mortality ranged
from 6.67% to 40%; the highest rate was in chickens that were
inoculated at 1 day old (5). In addition, MD chickens were found
with lesions of coccidiosis, colibacillosis, and roundworms. Infection
with pathogenic (MDV-1), nonpathogenic (MDV-2, HVT), or
vaccine strains not only results in activation of specific immunity but
may also cause immunosuppressive effects (30).
In this study, the morbidity and mortality of chickens in flocks
vaccinated with the bivalent vaccine (flocks No. 11, 12, and 14)
were lower than the others. This suggests there were very virulent
(vvMDV) and very virulent plus (vvþMDV) strains in the poultry
farm environment. Our data also provided evidence that MD has
caused large losses for chicken production in the Mekong Delta, and
vaccination alone cannot totally protect chickens from MDV. Most
of the chickens were raised in backyards or on a medium scale, and
chicken raisers had limited knowledge about disease prevention and
control, especially biosecurity. These matters lead to heavy
environmental pollution and bird immune suppression, which favor
the development of exposure to infectious pathogens. We also
witnessed MD happening in flocks with the subclinical infectious
bursal disease in the past, and all birds in flocks gradually died due to
MDV and other pathogens (unpublished data).
In this study, we also found that losses from MD of cross-breed
(from indigenous chicken lines, Noi-Binh Dinh) flocks were less
than those of pure Noi chicken flocks. The considerable variability
in the susceptibility to MD of different genetic strains of chickens
affecting losses has also been reported by many other researchers
(31,32,33). Nowadays, the increasing virulence of MDV may pose a
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threat to the standard MD prevention strategy, progressively
reducing the success of vaccine protection. The genetic resistance
to MD should be again considered, especially on small and medium
scales of chicken production in the Mekong Delta. As well,
revaccination should be realized; some studies reported that
revaccination (prime-boost), particularly with the cell-associated
MDV vaccine (CVI-998/Rispens) improved protection against the
disease and increased the magnitude of anti-MDV T cell responses
as demonstrated by enhancement of anti-MDV-neutralizing
antibodies and proliferation of CD4þ, CD8þ, and CD3þ T cells
(24,34,30).
CONCLUSION
These data are the first findings to confirm MD in the Mekong
Delta and show that it continues to be a threat to chicken
production in general. Thus, it is essential to develop sustainable
vaccine strategies and strict biosecurity practices. This has been
achieved by adopting all-in–all-out methods of production, high
standards of husbandry, good sanitation and biosecurity, especially
the prevention of early MDV exposure.
Supplemental data associated with this article can be found at
https://docs.google.com/document/d/1-2w0vwx3XSaOTdMhb
TryoumPu2nREfC0/edit.
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ACKNOWLEDGMENTS
This study is funded in part by the Can Tho University
Improvement Project VN14-P6 supported by a Japanese ODA loan,
Program A-10. The authors declare that there are no conflicts of
interest. Ho Thi Viet Thu and Nguyen Duc Hien designed and
wrote the manuscript. Nguyen Trong Ngu, Huynh Ngoc Trang and
Nguyen Tran Phuoc Chien collected samples and analyzed and
interpreted data. All authors approved the final manuscript.