100 2022 Anh

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 disease virus serotype 1 (MDV-1) (6). Although MD is a major
neuropathic disease of chickens caused by an alphaherpesvirus concern of industrial chicken production, many backyard chicken
called the Marek’s disease virus (MDV). The disease was first raisers and extension workers are unaware of MD, and there are rare
reported by Jozséf Marek (1). Before the use of vaccines, loss of the reports on this disease, in particular, no literature in English about
MDV-infected flocks was estimated to range from 25% to 30%, MD in our country. Therefore, this study aims to investigate the
and occasionally as high as 60% (2). After the first vaccine was status of MD in chicken backyard flocks in the Mekong Delta,
introduced, vaccination has been the best choice for the prevention Vietnam and to determine the types of MDVs circulating in
and control of this disease (3). MD morbidity and mortality are diseased chicken flocks.
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 MATERIALS AND METHODS
operations have used vaccines for prevention, but MD outbreaks
Study time and site. The study was conducted over a year, during
have still occurred and caused a great impact on chicken production
the period from August 2018 to July 2019, by examination of 16
due to high morbidity and mortality (5), and many backyard
chicken flocks that were suspected of being ill with MD by clinical signs
chicken flocks have been reported carrying oncogenic Marek’s
and macrolesions from five provinces (Tra Vinh, Hau Giang, Vinh
Long, Long An, and Can Tho) in the Mekong Delta. This study used
C
Corresponding authors. E-mail: htvthu@ctu.edu.vn; ndhien@ctu.edu.vn many examination methods to diagnose MD from the chicken flocks,
1
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 population, chicken age, number of birds infected and died, vaccination
PCR for viral DNA confirmation. status (with or without MD vaccination, and type of MD vaccine), and
Postmortem. All diseased chickens and chicken carcasses from 16 bird breeds. The number of birds infected and deceased was calculated
MD suspicious flocks at visitations were subjected to postmortem (PM) based on necropsy and carefully interviewing chicken owners to
examination. The chicken’s carcasses were carefully observed, and gross minimize bias.
lesions were identified and recorded. Visceral organs with lymphomas of Histopathological examination. Samples of different organs of MD
40 chickens of these flocks were collected for further testing. The suspected chickens were kept in 10% formalin until fixation,
samples collected included one or more varieties of tumorous tissues dehydrated in ethanol (70%–100%), cleared in xylene, and embedded
(heart, liver, spleen, kidney, proventriculus, and intestine). Each sample in paraffin. The 5 lm thickness of the paraffin section was prepared and
was divided into two parts; one was placed on ice, and the other was put labeled appropriately and thereafter deparaffinized and stained with
into a 10% formalin solution bottle and transported to the laboratory of hematoxylin and eosin dyes. Finally, histopathological sections were
the Veterinary Medicine Department, College of Agriculture, Can Tho examined under a light microscope at 1003, 2003, and 4003 high-
University, for preparation of histopathological sections and PCR tests. powered fields.
Information on 16 diseased chicken flocks was also surveyed in detail Detection of MDVs with PCR. The tumors from the visceral organs
using a questionnaire for collecting epidemiological data such as flock of each suspicious MD chicken were pooled into one for preparing a

Marek’s disease in backyard chicken flocks in Vietnam 3
Fig. 1. Continued.
homogenate. DNA was extracted from homogenates of the tissue with a grayish-whitish appearance and many times bigger than
samples mixed using DNA TopPUREt Tissue viral extraction (ABT normal (Fig. 1A–E), and hemorrhages occasionally were seen at the
Biomedical Solution Company, Vietnam). In total, 40 processed tumorous proventriculus (Fig. 1G) and intestine (Fig. 1H). There
samples were subjected to PCR, using serotype 1, 2, and 3 specific were only a few chickens that were collected from the flocks at the
primers. Commercial vaccines (CVI-998, SB-1, and HVT) were used as ending outbreak periods that had grayish mild enlargement of the
positive for the MDV serotypes and synthetic DNA fragments,
sciatic nerve.
sequences of primer pairs for detecting three serotypes (1, 2, and 3)
The histopathological examination. The histopathological chang-
of MDVs, and PCR procedures performed following the research of
López-Osorio et al. (7). Conventional PCR was used to detect specific es observed from tissue sections of internal organs (liver, lung,
MDV genes. Sets of specific oligonucleotide primers (Gallid herpesvirus kidney, heart, gizzard, and intestine) obtained from infected
2 Meq, Gallid herpesvirus 3 gD, and Meleagrid herpesvirus 1 sORF 1 chickens included infiltration and aggregation of lymphocytes at
genes) were used to detect each of the MDV serotypes (Marek’s disease different levels from mild to severe. They were gathered in a mass
virus type 1 [MDV-1], Marek’s disease virus type 2 [MDV-2], and (circle) or dispersed (arrow) among parenchyma or tissue of internal
herpesvirus of turkey [HVT]) and their locations as in Supplemental organ cells (Fig. 2). Under the microscope, sections of internal
Table S1. Samples were considered positive for MDV-1, MDV-2, and organs at 2003 magnification presented extensive infiltration round
HVT by amplification of products of 1148, 1040, and 350 base pairs, neoplastic cells (Fig. 2A, B); at higher magnification (4003), there
respectively. 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.
RESULTS
Detection of MDVs by the polymerase chain reaction. In the
Postmortem examination. In this present study, PM examination current study, 40 pooled internal organ samples of 40 chickens
of suspicious chickens revealed visceral lymphomas in one or more suspected of having MD from 16 chicken flocks were subjected to
of a variety of chicken internal organs. The most frequently reported screening of MDV-1, MDV-2, and HVT by conventional PCR.
was from the liver with multisized tumorous nodules or enlargement The results revealed that the MDV-1 oncogenic strain (Fig. 3A) and

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.

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

were mostly done by administering the vaccine to day-old birds threat to the standard MD prevention strategy, progressively
immediately after hatching with cell-free HVT or HVT combined reducing the success of vaccine protection. The genetic resistance
with MDV-1 (CVI-998/Rispens) vaccines and no booster was given. to MD should be again considered, especially on small and medium
However, the reason why the chickens were not protected against scales of chicken production in the Mekong Delta. As well,
MD although they were vaccinated was not investigated in this revaccination should be realized; some studies reported that
study. Similar findings were reported by many researchers, who revaccination (prime-boost), particularly with the cell-associated
reported that vaccination with either the MDV-1 or HVT vaccine MDV vaccine (CVI-998/Rispens) improved protection against the
did not totally protect the chicken flocks against MD disease and increased the magnitude of anti-MDV T cell responses
(11,18,19,20,21). Failure of vaccination can come from mistakes as demonstrated by enhancement of anti-MDV-neutralizing
in the preparation, storage handling, and administration of the antibodies and proliferation of CD4þ, CD8þ, and CD3þ T cells
vaccine. Besides, mistakes in poultry management can be made, such (24,34,30).
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 CONCLUSION
that vaccinated chickens may shed virulent virus into the These data are the first findings to confirm MD in the Mekong
environment because of its immunosuppressive abilities, and Delta and show that it continues to be a threat to chicken
MDV-1 has evolved to become more competent in immune system production in general. Thus, it is essential to develop sustainable
depression or evasion (22,23,24,25), and the evolution of MDV vaccine strategies and strict biosecurity practices. This has been
virulence remains the major challenge for the control of the disease achieved by adopting all-in–all-out methods of production, high
(18,26). standards of husbandry, good sanitation and biosecurity, especially
Higher morbidity and mortality were found in unvaccinated the prevention of early MDV exposure.
flocks (Nos. 10, 15, and 16). Shortly after the isolation of MDV in Supplemental data associated with this article can be found at
the late 1960s vaccines were developed in many countries, and the https://docs.google.com/document/d/1-2w0vwx3XSaOTdMhb
HVT vaccine was widely used in industrial poultry production (27). TryoumPu2nREfC0/edit.
The vaccination reduced the incidence of MD by 99% and was the
first successful vaccine against naturally occurring virus-induced
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Occurrence of Marek's Disease in Backyard Chicken

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

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No. No. No. No. No.

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

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

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