Tóm tắt luận Án tiếng anh nghiên cứu sự lưu hành và Đặc Điểm di truyền của virus gây bệnh marek trên gà Ở một số tỉnh thuộc Đồng bằng sông cửu long

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Major: Animal pathology and disease treatment Major code: 9640102 HUYNH NGOC TRANG STUDY ON CIRCULATI

MINISTRY OF EDUCATION AND TRAINING

CAN THO UNIVERSITY

SUMMARY OF DOCTORAL THESIS

Major: Animal pathology and disease treatment Major code: 9640102

HUYNH NGOC TRANG

STUDY ON CIRCULATION AND GENETIC CHARACTERISTICS OF MAREK’S DISEASE VIRUS ON CHICKENS IN SOME PROVINCES

OF THE MEKONG DELTA

Can Tho, 2024

THE THESIS WAS COMPLETED AT

CAN THO UNIVERSITY

Supervisor: Assoc Prof Dr Ho Thi Viet Thu

The thesis was defended before the Committee evaluating the institutional level doctoral thesis

Confirmed review of Chairman

The thesis can be found at the library:

Learning resource center, Can Tho University

National Library of Vietnam

LIST OF PUBLICATIONS

International journal

1 Huynh Ngọc Trang, Nguyen Tran Phuoc Chien, Ho Thi Viet Thu

(2022) Prevalence of Marek’s disease virus in unvaccinated healthy backyard chickens in Cantho city, Vietnam and genetic

characterization of meq gene, Veterinary Integrative Sciences, 20(3),

709-718

2 Huynh Ngoc Trang, Nguyen Le Hung Phong, Nguyen Tran Phuoc Chien, Ho Thi Viet Thu (2024) The survey on the circulation of

Marek’s disease virus in local chickens in Tra Vinh province,

VietnamVeterinary Integrative Sciences, 22(3), 823-830

National journal

1 Huynh Ngoc Trang, Đinh Thi Ngoc Lien, Nguyen Tran Phuoc Chien,

Ho Thi Viet Thu (2022) State of Marek’s disease virus infection

in unvaccinated chickens for Marek’s disease in households in Dong Thap province Journal of Veterinary Science and

Technology, 29(9), 10-14

2 Huynh Ngoc Trang, Nguyen Tran Phuoc Chien, Ho Thi Viet Thu

(2023) Survey on Marek’s disease in chickens in An Giang and Tien Giang provinces Journal of Veterinary Science and

Technology, 30(6), 40-44

CHAPTER 1 INTRODUCTION 1.1 The necessity of the thesis

Marek’s disease (MD) is a contagious oncogenetic disease of

chickens caused by Gallid alherpesvirus 2 Marek’s disease virus (MDV)

has also induced immunosuppression, increasing the susceptibility to

secondary infections (Gimeno & Schat, 2018; Rozins et al., 2019)

Marek’s disease has caused economic losses due to lower feed conversion, weight loss, decreased egg production, and high morbidity

and mortality (Payne & Venugopal, 2000)

Chickens are the most important natural host of MDV The virus has remained in the infected chickens for a long time and the increase of virus shedding to the environment, horizontal transmission, and incidence Besides chicken, some species like quails, turkeys, partridges, pheasants, ducks, and geese are infected and diseased (Schat & Nair, 2013)

Currently, there have been vaccines to prevent Marek’s disease, but outbreaks have occurred in vaccinated chicken flocks and to threaten the chicken industry due to the occurrence of many mutations of virus strains

tending to virulent increase (Dunn et al., 2019) According to Lee et al (2008), the meq gene has been important to the virulent properties of Marek’s disease virus serotype 1 (MDV-1) Point mutations in meq gene

are related to changes in the virulence of the virus

In recent years, the indigenous chicken flocks in the Mekong Delta have shown symptoms suspected to be of Marek´s disease, but the studies on Marek’s disease have been limited Meanwhile, the presence

of Marek's disease virus in other domestic poultry such as ducks, and quails has not been thoroughly studied yet To prevent Marek's disease, it

is essential to conduct researches on the disease's prevalence, virulence characteristics of the causative agent, as well as the circulation of the virus among poultry On that practical basis, this study was conducted

1.2 The aims of the study

Determination of the presence of MDV-1 in indigenous chickens,

ducks, and quails in some provinces in the Mekong Delta

Determination of the disease and pathological characteristics of Marek’s disease in chickens

Determination of characteristics of genetic meq gene of MDV-1

strains

1.3 Practical significance

The results of the thesis determined characteristics of epidemiology, typical pathology of Marek’s disease, virulence of the virus, and determination of the disease by inoculation in the experimental indigenous chickens This provides a basis for diagnosing and preventive measures against Marek's disease in chicken flocks in the Mekong

Delta

1.4 New contributions

The first study detected the carry of MDV-1 in ducks in the Mekong Delta

The first comprehensive and systematic study about Marek´s disease

in indigenous chicken flocks in the Mekong Delta including characteristics of epidemiology, pathology and genetics

CHAPTER 2 RESEARCH METHODS 2.1 The contents of the study

Content 1: Survey on circulation of MDV-1 in the indigenous chickens, ducks, and quails

Content 2: Survey on the status of Marek’s disease in the indigenous chicken flocks

Content 3: Genetic characteristic analysis of the meq gene of MDV-1

Content 4: Survey on pathological characteristics of Marek’s disease

in the experimentally infected chickens

5 day-old crossed Noi chickens Meq gene of MDV-1 in healthy

chickens, healthy ducks and diseased chickens

2.3 Materials of research

2.3.1 Research time and location

Time: The study was carried out from 12/2020 - 10/2023

Location: The feather follicle samples of healthy chickens, ducks, and

quails and tissue samples of diseased chickens were collected from households and farms in some provinces in the Mekong Delta

Sample examination by PCR and Nested PCR were performed at the laboratory, raising the experimental chickens in the laboratory animal room of the Veterinary Medicine Department, College of Agriculture, Can Tho University

Histopathological specimen examination was performed at Regional Animal Health No 6 in Ho Chi Minh City Sequencing samples were performed at Nam Khoa Company (Ho Chi Minh City)

2.3.2 Materials, equipment, and biological products of research

Materials and equipment needed for sampling feather follicle samples, dissection, tissue sample collection, sample preservation, biosafety cabinet, PCR machine, and micropipettes

DNA extraction kit (TopPURE® Tissue viral extraction), biological products for PCR, and Nested PCR

2.4 Methods of research

2.4.1 Survey on circulation of MDV-1 in the indigenous chickens, ducks, and quails

2.4.1.1 Survey on circulation of MDV-1 in the indigenous chickens

a The object of research

Healthy indigenous chicken breeds including crossed Noi, crossed Tau, and crossed Tre had been unvaccinated for Marek’s disease They are approximately 1 month and are raised in households in Can Tho City, Dong Thap, and Tra Vinh provinces

b Methods

Investigation: The study was carried out by a cross-sectional survey

Information on the size of the population, chicken breed, age, raising method, and vaccination status was collected on sampled chicken flocks

Sample collection: The sample size was estimated according to the

formula from Thrusfield (2007) The number of samples was collected from each flock based on an adjusted formula The flock size was more than 30 heads sampled from 7-8 chickens Each chicken obtained 5 feather follicles from its wings, and they were pooled into a sample

(López Osorio et al., 2019)

The formula for estimating sample size according to Thrusfield

(2007)

Z2 (1 - p) p

n =

d2

n: sample size collection

Z: normal distribution value at 95% confidence level (Z = 1.96) d: difference of no more than 5% (d = 0.05)

p: estimated prevalence

The P value was based on the research of Ho Thi Viet Thu et al

(2021) with p=27.27%

Table 2.1: Number of samples collected from healthy chickens

Sample test: PCR technique was used to identify the infected

chickens with MDV-1 by detecting the meq gene in feather follicles

TopPURE® Tissue viral extraction kit was used to extract the DNA of MDV-1 The primers, product size, and thermal cycle were used to detect

the meq gene of MDV-1 according to López-Osorio et al (2017)

c Parameters of the survey

- Prevalence of MDV-1 in chickens

- Risk factors related to the prevalence of MDV-1 status in chickens

2.4.1.2 Survey on circulation of MDV-1 in ducks and quails

a The object of research

Muscovy duck, Co duck, Super meat duck and Japanese quail from 1 month old were raised at households and farms in Can Tho City, Dong Thap, and Tra Vinh provinces

b Methods

Investigation: The study was carried out by a cross-sectional survey

Information on the size of the population, age, and breed was collected on sampled flocks

Sample collection: The sample size was estimated according to the

formula from Thrusfield (2007) The prevalence of ducks was based on survey results of 70 samples with an MDV-1 prevalence of 8.5% The number of quail flocks was limited and only 10 flocks sampling Seven birds was sampled from each duck and quail flock Five feather follicles collected from each birds were pooled into 1 sample

Table 2.2: Number of samples collected from ducks and quails

Sample test: A Nested PCR technique was used to increase the

sensitivity of detecting the meq gene of MDV-1 from duck and quail

feather follicles The primers and thermal cycle of the first and the second

PCR were based on the research from Chang et al (2002)

c Parameters of the survey

Prevalence of MDV-1 in ducks and quails

2.4.2 Survey on Marek’s disease status in indigenous chicken flocks

2.4.2.1 The object of research

48 indigenous chicken flocks in the provinces of Ben Tre, Tra Vinh,

Tien Giang, Vinh Long, An Giang, Dong Thap, and Can Tho City had shown symptoms and lesions of Marek´s disease

2.4.2.2 Methods

a Investigation

The study was carried out using a cross-sectional survey Information

on the flock size, breed, number of diseased and dead chickens, and

vaccination for Marek’s disease was collected on sampled flocks

b Sample collection

Sampling on diseased chicken flocks was carried out according to National Standard 8400-30, 2005 (TCVN 8400-30, 2005) Symptoms and lesions on postmortem chickens were recorded and sampled liver, spleen, lung, kidney and proventriculus

Table 2.3: Number of samples collected from flocks suspected of Marek´s disease

Sample test by PCR: PCR was performed similarly on the feather

follicles test Each chicken was examined pool sample Each flock was determined MD when at least one chicken was positive PCR and typical gross and microscopic lesion of MD

Histopathological examination: The method of histopathological performance was according to National Standard TCVN 8400-30:2005

Each flock was chosen lesion specimen from 1-2 chickens, and only a specimen type had gross lesion suspect MD Histopathological specimens were looked at optical microscopes at 10X, 20X, and 40X

Table 2.4: The number of visceral organs histopathological examination

Morbidity and mortality of MD chicken flocks

Frequency of symptoms and lesions in MD chickens

Frequency of microscopic lesions in visceral organs of MD chickens

2.4.3 The survey on characteristics of the genetic meq gene of

MDV-1

2.4.3.1 Sequencing of the meq gene

Sequencing was performed by Sanger on ABI 3130 (USA) and analyzing obtained sequences was realized by BioEdit 7.0

Table 2.5: The strains for sequencing

7 MDV/Muscovy/CT/VT Can Tho Duck Feather follicle

2.4.3.2 Phylogenetic analysis

Similarity analysis of nucleotides, and amino acid as well as the construction of a phylogenetic tree of the MDV-1 strains in provinces of Mekong Delta and reference strains based on Mega X Maximum likelihood with 1,000 bootstrap replications was used to analyze the phylogenetic tree of representative strains circulating in provinces of Mekong Delta and reference strains The reference strain selections were based on serotype 1, virulence, nation, and time of detection

2.4.3.3 Parameters of survey

Virulence grouping of virus strains in the study strains

Genetic relationship of the study strains and reference strains

Proline ratio and number of motif PPPP of the study strains

Number of amino acid substitution positions in Meq protein of the study strains

2.4.4 The survey on MD on the experimental chickens

2.4.4.1 The object of research

Crossed Noi chickens 5 days old were hatched from the hens that were

unvaccinated for MD and negative with MDV-1 through meq gene

determination in feather follicles by PCR

2.4.4.2 Experimental inoculation

The virus suspension was made from livers of diseased chicken flocks The liver samples were ground and mixed with PBS 0.9% generating the suspension of 20.0% for inoculation It was treated with antibiotics (1,000 UI penicillin và 1 mg streptomycin per 1 ml suspension) The suspension was inoculated for the embryos to determine

a lethal dose of 50% (ELD50%) before inoculating for chickens In this study, each chicken was inoculated with 2.5 ELD50

Table 2.6: Experimental design in inoculation 5 day- old chickens

Virus strains Inoculation route Dose (ELD50) No of chickens Total

Male Female

2.4.4.3 The observational chickens, sampling and testing

The symptoms of experimental chickens were observed and recorded until 60 days post-inoculation Feather follicles of all chickens were sampled at 2 days post-inoculation and repeated every 2 days until they were positive for MDV-1 Every time, 2 feather follicles were collected from each chicken The dead chickens during experimental time or alive chickens at the end of the experiment were postmortem for gross lesion examination and their liver, spleen, kidney, and proventriculus were sampled Particularly, the first dead chicken was also collected feather follicles to evaluate persistent MDV-1 in feather follicles Feather follicles and tissue samples were used as pooled samples for PCR testing

to meq gene examination determining the presence of MDV-1

2.4.4.4 Parameters of survey

Morbidity and mortality

Rate of the chickens with present MDV-1 in feather follicles by time Frequency of symptoms and lesions of experimental MD chickens

2.5 Data analysis

Raw data were analyzed by Microsoft Excel 2010

The Chi-square test of Minitab version 16 and Yatest correction in

Microsoft Excel 2010 were used to compare percentage values

Mega X was used to analyze the phylogenetic tree of the survey strains and reference strains

CHAPTER 3 RESULTS AND DISCUSSION 3.1 Results of the survey on MDV-1 circulation in the indigenous chickens, ducks, and quails

3.1.1 Results of survey on MDV-1 circulation in the indigenous

chickens

Table 3.1: Prevalence of MDV-1 in the chickens by location

Location No of samples (bird) No of positive

Values in the same column with different superscripts are different (P≤0.05)

The results in Table 3.1 showed that the prevalence of MDV-1 in chickens in Tra Vinh and Dong Thap was higher than that of chickens in Can Tho with statistical significance (P=0.021) Those differences could

be due to different chicken breeds with different genetic characteristics According to Bumstead & Kaufman (2004), chicken genetics were related to resistance or susceptibility to infection of Marek’s disease virus

Table 3.2: Prevalence of MDV-1 in the chickens by breed

Breeds No of samples (bird) No.of positive

Values in the same column with different superscripts are different (P≤0.05)

The prevalence of MDV-1 in crossed Tre (35.71%) was higher than that of crossed Tau (8.79%) and crossed Noi (10.66%), and the

differences were statistically significant (P=0.001) Bacon et al (2001)

assumed that chicken breeds with different genetic characteristics had different susceptibility or resistance to MDV The research by Hartawan

& Dharmayanti (2016) on the status of MDV-1 infection in chickens in