THE STATUS OF MAREKS DISEASE, VACCINATION DYNAMICS, AND MOLECULAR CHARACTERISATION OF VIRUS ISOLATES

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ABSTRACT

Mareks disease (MD) a lymphoproliferative, neoplastic disease of birds with worldwide distribution and significant economic consequences has not been studied in Abia State or its environs. In order to understudy its status, occurrence, vaccination dynamics and pathophysiological changes that can be associated with its viral etiology, structured, open and close ended questionnaires were employed in cross sectional study to obtain information on clinicopathological, post mortem, host and type factors influencing MD occurrence in the three senatorial zones. A second questionnaire was employed with oral interviews to obtain data on vaccine use and application on the same farms in addition to the hatcheries they patronize. An indirect ELISA was used to audit difference vaccination protocol adopted in the different hatcheries. These protocols were experimentally replicated in unvaccinated d.o.c. and the sera harvested for eight weeks and subjected to ELISA. Tissue samples including feather tips suspected of MD were collected within the study region. The tissues were processed for histopathological examination. Total DNA was extracted from the feather tips through PCR technique. Meq gene analysis of the DNA showed five MD positive samples. Phylogenetic analysis of three of the samples was conducted using the deduced amino acid sequences of the Meq gene. Results showed a retrieval rate of 52.1% and a positive occurrence of MD in 76% of analyzed cases. Significant occurrence of MD was recorded in all the zones with Abia Central having the highest. The classical form of MD was dominant with severe weight loss being the most observed clinical sign. Though 77.5% of farmers were aware of MD vaccination need, only 8.5% revaccinated their flock. Use of antibiotics for brooding significantly affected MD occurrence. 70% of hatcheries patronized by farmers were localized in the South West of Nigeria. Whereas 80% of the hatcheries were using Rispens alone or in combination with HVT, 20% used HVT alone and they contributed to the highest number of outbreak in farms sourcing d.o.c from them. ELISA evaluation of vaccine protocols showed sustained seroconversion in vaccinated birds but with Rispens group having better results especially form the second week. Reduction in vaccine dose significantly reduced antibody titers across the groups. Boaster administration of HVT after a week showed slight advantage over the rest of the protocols. Histopathological examination of most of the suspected cases revealed consistent pleomorphism which is diagnostic of MD. Phylogenetic analysis of the Meq gene in MD positive samples showed 5 non-synonymous and one synonymous mutations. Sequences clustered in two clads. Two sequences showed close to 91% identity to the very virulent Egyptian vv+ strain while the other that segregated with Netherland strain was of the vv strain. Diagnostic challenges and vaccination aberration appears to be the major limiting factors in MD control in the study region. Novel mutations recorded in this study can represent a drive towards higher virulence in the study region.







TABLE OF CONTENTS

                                                                                                                                   

Title page    i                                                                                                        

Certification             ii               

Declaration                                                                                                                                  iii

Acknowledgement                                                                                                                      iv

Table of contents                                                                                                                                       v

List of tables   vi                                                                                                      

List of figures vii

List of plates   viii

List of appendices                                                                                                                   ix

Abstract                                                                                                                                       x          

 

CHAPTER 1                    1                                                                            

INTRODUCTION                                                                                      1                                                                                                                   

1.1 Background of the study              1

1.2 Statement of Research Problem                                                                                                    10

1.3 Justifications of the study   10

1.4 Significance of the study   11

1.5 Aims             11

1.6   Objectives                                                                                                 11

 

CHAPTER TWO  13

LITERATURE REVIEW             13

2.1 Definition of Neoplasm             13

2.2 Marek’s Disease Definition             14

2.2 Historical account of Marek’s disease 16

2.2.2 Economic significance of Marek’s disease 19                                                                                                     

2.2.3 Public health significance of Marek’s disease             20

2.2.4 Scientific significance of Marek’s disease 21

2.2.5 Etiology of Marek’s disease             22

2.2.5.1 Classification                                                                                            22

2.2.5.2 Morphology of Marek’s Disease Virus             23

2.2.6. Composition of Marek’s disease virus             23

2.2.6.1 Physical properties of Marek’s disease virus             23

2.2.6.2 Structural organization of Marek’s disease virus             24

2.2.6.3 Marek’s disease viral deoxyribonucleic acid structure in infected cells     27                                                                                                     

2.2.6.4 Marek’s disease virus structural changes                                                                                                     27

2.2.6.5 Marek’s disease viral genes and proteins             29

2.2.6.6 Genes with homologues in alpha herpesviruses             29

2.2.6.7 Mareks disease virus genes have homology to hemorrhagic septicemia virus             29

2.2.6.8 Late genes of Marek’s disease virus             31

2.2.7 Marek’s disease oncogenic genes             33

2.2.7 Marek’s disease virus replication             34

2.2.7.1 Marek’s disease virus-cell interactions             35

2.2.7.2 Marek’s disease virus productive infection             35

2.2.7.3 Marek’s disease latent infection             37

2.2.7.4 Marek’s Disease Transforming Infection             38

2.3 Viral Replication of Other Marek’s Disease Virus Serotypes             40

2.3.1 Stock production and stability of Marek’s disease virus             40

2.3.2 Susceptibility of Marek’s disease virus to chemical and physical agents 41

2.3.3 Classification of Marek’s disease virus serotypes             42

2.3.4 Forms of Marek’s disease             42

2.3.4.2 Incidence and distribution of Marek’s disease             43

2. 3 .5 Natural and experimental hosts of Marek’s disease             44

2.3.5.1 Quails             45

2.3.5.2 Turkeys             45

2.3.5.3 Marek’s disease in other avian species             47

2.4 Transmission, Carriers and Vectors of Marek’s Disease             47

2.5. Incubation Period of Marek’s Disease             49

2.6 Pathogenesis of Marek’s Disease                                                                                                      50

2.7. Clinical Signs Associated With Marek’s Disease             51

2.8 Morbidity and Mortality Due to Marek’s Disease             52

2.9 Factors that Influence Mortality and Lesions Due to Marek’s Disease             53

2.9.1 Marek’s disease virus strain             53

2.9.2 Dose and route of exposure of Marek’s disease virus             54

2.9.3 Host gender response to Marek’s disease             54

2.9.4 Maternal antibody             54

2.9.5 Host genetics and age at exposure to Marek’s disease             55

2.9.6 Impact of early natural infection             56

 2.9.7 Effects of environmental and stress factors in spread of Marek’s disease             56

2.10 Pathology Due to Marek’s Disease             57Top of Form

 

2.10.1 Gross pathology             57

2.10.1.1 Nerve involvement due to Marek’s disease             57

2.10.1.2 Visceral Organs Involvement Due to Marek’s Disease             58

2.10.1.3 Integument involvement due to Marek’s disease             59

2.10.1.4 Eyes involvement due to Marek’s disease             60

2.10.1.5 Other syndromes of Marek’s disease             60

2.10.2 Histopathologic changes due to Marek’s disease             60

2.10.2.1 Nerves lesions in Marek’s disease             60

2.10.2.2 Brain lesions in Marek’s disease             62

2.10.2.3 Marek’s disease lesions in visceral organs             63

2.10.2.4 Marek’s disease lesions in the integument             64

2.10.2.5 Marek’s disease lesions in the eyes             64

2.10.2.6 Marek’s disease lesions in the blood             65

2.11 Differential Diagnosis of Marek’s Disease             66

2.12 Diagnosis of Marek’s Disease             69

2.12.1 Marek’s disease virus isolation from chicken             70

2.12.2 Cell culture techniques for the isolation of Marek’s disease virus             70

2.12.3 Identification of Marek’s disease virus isolate             71

2.12.4 Marek’s disease virus assay and titration             72

2.12.5 Marek’s disease viral markers in tissues             73

2.12.6 Marek’s Disease Viral Antigen Detection             73

2.12.7 Polymerase chain reaction for Marek’s disease virus             74

2.12.7.1 Marek’s disease virus deoxyribonucleic acid probes             75

2.12.7.2 Electron Microscopy             75

2.12.7.3 Marek’s disease antibody detection             75

2.12.7.4 Clinical signs and gross pathology             76

2.12.7.5 Histology, cytology and histochemistry of tumour cells             76

2.12.7.6 Virology of Marek’s disease virus             77

2.12.7.7 Pathotyping of Marek’s disease virus strains             79

2.13 Control of Marek’s Disease             79

2.13.1 Vaccination against Marek’s disease             80

2.13.1.1 Types of Marek’s disease vaccine             80

2.13.1.2 Marek’s Disease Vaccine Administration             81

2.13.1.3 Marek’s disease vaccination strategies             82

2.13.1.4 Factors affecting Marek’s disease vaccines efficacy             83

2.13.1.5 Use of Genetic-Resistant Birds in Marek’s Disease Control             85

2.13.1.6 Biosecurity in farms against Marek’s disease             86

           

CHAPTER THREE                                                                                                    87

MATERIALS AND METHOD                                                                                                87

3.1. Retrospective occurrence of Marek’s disease in Abia State.   87

3.1.1 Study Area    87

3.1.2 Study design  87

3.1.3 Study population                                                                                                 87

3.1.4 Sample size determination                                                                                           88

3.1.5 Data Collection                                                                                                 89

3.1.6 Diagnosis and classification of MD      90

3.1.7 Analysis of Data             90

3.2 MD-vaccination indices by farms in Abia State and hatcheries they patronize             91

3.2.1 Study Area    91

3.2.2 Study Population                                                                                                 91

3.2.3 Data Collection.             92

3.2.3 Data Analysis             92

3.3. Antibody responses to different Marek`s disease-vaccination modules by farms in

       Abia state.                                                                                                                                93

3.3.1 Experimental Animal                                                                                                      93

3.3.2 Experimental Design 93

3.3.2.1 Determination of antibody responses by ELISA             93

3.4. Histopathological and Molecular identification and phylogenetic analysis of MDV in MD

       Outbreaks in Abia State                                                                                              94

3.4.1. Sample collection:             94

3.4.2 Histopathological Examination             95

3.4.3. Genomic DNA Extraction:             95

3.4.4. PCR amplification the Meq – gene    96

3.4.5 Electrophoresis of the PCR product                                                                                                      97

3.4.6   Sequence and Phylogenetic Analyses                                                                                                   98

 

CHAPTER FOUR 99

RESULTS

4.1. Questionnaires distributed and returned             99

4.1.1. Distribution of MD in Senatorial zones of Abia state     99

4.1.2 Clinical signs of Marek’s disease and associated mortalities in Abia state, Nigeria.                                                                                                   101

4.1.3. Distribution of outbreaks of Marek’s disease in Abia state, Nigeria                                                                                                    101

4.1.4. Some epidemiological characteristics of MD in Abia State Nigeria,                                                                                                   103

4.2   MD-vaccination indices by farms in Abia State and hatcheries they patronize                                                                                                 110

4.3   Evaluation of MDV antibody response of different vaccination practices in

        broiler chickens                                                                                                                           114

4.4. Results of some suspected field outbreaks of MD      119

 

CHAPTER FIVE   132

5.1       DISCUSSION                                                                            132

5.2 Discussion on Second Study   135

5.3 Discussion on Third Study   139

5.4 Discussion on Fourth Study             143

5.4 Conclusions and Recommendations                                                                                  145

5.3 Recommendations                                                                                  149

                                                 

 

 

 

 

 

 

 

 

                                                       LIST OF TABLES

 

Table 2.1:        Differential diagnosis of Marek’s disease. Feature                                         67

Table 4.1.1      Clinical manifestations according to the different zones                                        103

Table 4.1.2      Annual retrospective occurrence of Marek’ Disease in poultry

                        farms in Abia State, Nigeria                                                                           105

Table 4.1.3      Monthly occurrence of Marek’s disease in Abia State, Nigeria                        106  

Table 4.1.4      Seasonal occurrence of Marek`s disease in Abia State, Nigeria                        106

Table 4.1.5      Occurrence of Marek`s disease in senatorial zones of Abia State,              108

                        Nigeria.

Table 4.1.6      Occurrence of Marek`s disease in Abia State, Nigeria among                         108

different chicken   age-groups

Table 4.1.7      Occurrence of Marek`s disease in Abia State, Nigeria among                         109

 types of chicken

Table 4.1.8      Occurrence of Marek`s disease in the study zones in chickens                      109

                        under different management-systems

Table 4.1.9      Mortality due to MD in poultry farms in Abia                                               109

Table 4.2.1      Association between occurrence of MD and location of poultry

                         farms in Abia State.                                                                                       110

Table 4.2.2      Association between occurrence of MD and Awareness of MD

                        vaccination need in poultry farms in Abia State.                                           110

Table 4.2.3      Association between occurrence of MD and administration of

                        vaccine with antibiotics in hatcheries patronized by poultry

                       farms in Abia State.                                                                                         110

Table 4.2.4:   Association between occurrence outbreak of MD and revaccination

                      in poultry farms in Abia State.                                                                         111

Table 4.2.5:  Association between occurrence of MD and administration of

                      antibiotics during brooding in poultry farms in Abia State.                                    111

Table 4.2.6:   Association between occurrence of MD and breeds of birds in

                      poultry farms in Abia State.                                                                             111

Table 4.2.7    MD vaccine characteristics from hatcheries patronized by farms

                       in Abia state                                                                                                     113

Table 4.3.1      Weekly antibody titers of Marek’s disease in chicks                                               117  

vaccinated in Abia state Nigeria

Table 4.4.1      Nucleotide and amino acid substitutions in partial meq                                     131

                        gene sequence of MDV from Abia state, Nigeria

                


 

                                                 LIST OF FIGURES

Figure 1. Genetic organization of GA strain of Marek’s disease virus          26

Figure 2: showing the17 Local Government Areas of Abia state              88

Figure 3: Pie chart showing retrospective occurrence of MD in poultry farms in Abia state, Nigeria                    100                                           

Figure 4: Forms of suspected cases of Marek’s disease (%) in poultry farms in Abia state, Nigeria        100

Figure 5: Pie chart showing percentage distribution of clinical signs (diagnostic parameters) of Marek’s disease                         102

Figure 6: weekly mean ELISA values of MDV antibody under different vaccination programme      118

Figure 7 Phylogenetic tree based on the alignment of the partial Meq gene (266 nt) of Marek’s disease virus (MDV) sequences detected in this study and other MDV retrieved from the GenBank                 130

 

 

  

 

 

 


                                                        LIST OF PLATES

                                                                

Plate A; Wrongful administration of HVT vaccine by marketers before sales                 183

Plate: B & C Marek’s affected pullet showing typical demeanor with marked         

                cachexia.                                                                                                             120

Plate D; Marek’s affected pullet showing paresis of the right leg with a

               characteristic posture of one leg pointing backwards                                          121                                  

Plate E; Marek’s affected broiler chicken showing gross skin leukotic nodules                  121

Plate F; 15 weeks old de-feathered broiler with advanced cutaneous lesions of MD          122

Plate G; 26 weeks old layer with knife edge keel in suspected MD                                     122

Plate H; Typical cachexia induced by MD                                                                            123

Plate I; Multiple nodule formations in A...Heart, B... Lung, C liver, D...Spleen,                 124

Plate J; Multiple nodules in the intestines and mesentery of a 30-week old layer                124

Plate K; Immature ovarian follicles in 26weeks old layer                                                               125

Plate L; Marek`s disease affected heart showing widespread multifocal                          

               infiltration of a pleomorphic population of lymphoblastic cells                                      127

Plate M; Marek`s disease affected liver showing Section of the liver with

                multifocal nodular aggregation of infiltrating pleomorphic population               127

Plate N; Marek`s disease affected kidney showing                                                              127

Plate O; Marek’s disease affected skin showing                                                                  128

Plate P; Marek`s disease affected spleen showing                                                               128

 Plate Q: Some of the PCR positive bands at 2kb base pairs (bp).                                       129   






                                                           

LIST OF APPENDICES

 

I. Questionnaire on Marek’s disease cases and occurrence in poultry farms in Abia state      181                                                                                                                                         

II. Questionnaire (study 2) on MD-vaccination indices by farms in Abia State and hatcheries they patronize                              182                                                                                                                             

III. Wrongful administration of HVT vaccine by marketers before sales                               183

IV. Nanodrop results of MD suspected samples collected from Abia State                           184        

V.  FAST file MD sequences of Meq positive samples                                                          185

 

 

 

 

 

 

                          

                             LIST OF ABBREVIATIONS AND ACRONYMS

 

A: Antigen (glycoprotein designated C (gC) Soluble A antigen and cell-bound B antigen are known as gC and gB respectively

ADOL: Avian Disease and Oncology Laboratory

ALV: Avian Leukosis Virus

AGPT: Agar Gel Precipitation Test

BAC: Bacterial Artificial Chromosome

BCtP: Biological Critical Thresh Point

B-locus: linked to genetic resistance of chickens against MD

Bp: base pairs

BSA: Bovine Serum Albumen

C12/130: A hypervirulent strain of Marek’s Disease Virus

CD: Cluster of differentiation (of cellular antigenic marker)

CD4+ T-cells: Cells associated with Marek’s Disease Virus latency, although

CD8+ T-cells: and B cells can be latently infected

CD30+: A second antigen detected by Marek’s Disease Antibodies in CD4+ T-cell and Marek’s Disease Virus

CEF: Chicken Embryo Fibroblast

CIAV: Chicken Infectious Anaemia Virus

CTL: Cytotoxic T lymphocytes

CFT: Complement Fixation Test

CI: Confidence Interval

CV1988: Rispens Marek’s Disease Vaccines Serotype 1

DOC: Day old chick

FFE: Feather Follicle Epithelium

UL: Unique Long

US: Unique Short

TRL: Terminal Repeat Long

H2SO4: Sulphoric Acid to stop reaction in ELISA

HSV: Heamorrhagic Septicemia Virus

HVT: Herpes Turkey Virus Vaccines Serotype 3

EL: Erythroid Leukosis.

EDTA Ethylene Diamine Tetra-Acetic Acid

FFE: Feather Follicle Epithelium

Fc126: ` Vaccine Serotype 3 (HVT)

FOA: Food and Agricultural Organization

g Glycoproteins (g) of gB, gC, gD, gH, gI, gE, gL and gM

gB: GlycoproteinB

gD: Gene of Marek’s Disease Virus

G+C Guanine plus cytosine (G+C) ratio is different for the 3 serotypes and

ranges from 43.9-53.6% in serotype 1 and 2, respectively, and 47.6% for HVT

G-HRP: Protein G-Horse Radish Peroxidase

GS: Glycine Saline

Gs: Group-Specific Antigen

gp85: Envelope of the Marek’s disease virion (contains a glycoprotein encoded by the env- gene which determine the subgroup specificity of the virus)

gag Gene which is common to all viruses of the group and important in certain diagnostic tests

gag, pol and env genes which form the Marek’s disease virion.

IBD: Infectious Bursal Disease

ICFU: International Complement Fixation Units

ICTV: International Committee on Taxonomy of Viruses

ICTVdB: International Committee on Taxonomy of Viruses Data Base

ICP4, 0, 22, 27 Infected Cell Proteins 4, 0, 22, 27

ELISA: Enzyme Linked Immunosorbent Assay

IE: Immediate Early Genes

IgM: Immunoglobulin M (the tumour cells in LL have morphology of large lymphocytes or lymphoblasts, they have B-cell markers and carry surface IgM).

IFN: Gamma Interferon Test

IRL: Internal Repeat Long

IRS: Internal Repeat Short

LATs: Latency-Associated Transcripts

LPS: Lipopolysacharide

LTRs: Long Terminal Repeat (RNA copies in the virion are flanked by sequences of nucleotides of LTRs which act as promoters controlling transcription of proviral DNA to viral RNA)

MAB: Monoclonal Antibody

Mab Marek’s Disease Antibody

MD: Marek’s Disease

MDV: Marek’s Disease Virus

MATSA: Marek’s Disease Tumor Associated Surface Antigen

MATSA and CD30: Can be used to enrich the transformed cells in tumors cells suspensions

MDCC: Marek’s Disease Virus Transformed Chicken Cell-line (MSB-1)

mRNA: Messenger Ribonucleic acid

MHC: Major histocompatibility complex

MHC: genetic resistance was linked to the B-locus or major histocompatibility complex

NDV: Newcastle Disease Virus

NDVL: Newcastle Disease Virus La Sota

OD: Optical Density

OR: Odd Ratio

OIE: Office International des Epizooties

OPD: O-phenylenediamine Dihydrochloride Substrate

POL: Point of lay

ORF2: Open Reading Frame 2

OU2: Cell Line 2

P27: Structural Protein 27

PBS: Phosphate Buffered Saline

PCR: Polymerase Chain Reaction

PP14: Phosphoprotein14

PP38: Phosphoprotein38

PI Post Inoculation

qPCR: Quantitative Polymerase Chain Reaction

R-LORF1: Right Long Open Region Flank 1

RK-1: A vv+ strain of Marek’s Disease Virus

RT-PCR: Real Time Polymerase Chain Reaction

rCh: Recombinant Chicken

rChIFN-α Alpha Recombinant Chicken Interferon

rChIFN-γ: Gama Recombinant Chicken Interferon

rFPV: Recombinant Fowl Pox Virus Vaccines

R2/23 Attenuated Serotype 1 MDV strain

REV: Reticuloendotheliosis Virus

RNA: Ribonucleic Acid (Dependent DNA polymerase reverse transcriptase and envelop glycoprotein: genetic makeup associated with slow cell transformation and tumour development over several months)

SPF: Specific Pathogen Free

SPGA: Sucrose-Phosphate-Glutamate-Albumin

SORF1: Short Open Reading Frame 1

SORF2: Short Open Reading Frame 2

SB-1: Strain of chickens in B house on the poultry farm of the first clone MDV vaccine

SNPs: Single Nucleotide Polymorphisms (which loosely partition between attenuate and non-attenuated strains)

TRS: Terminal Repeat Short

LRT: Long Terminal Repeats

LLV: Lymphoid Leukosis Virus

ICP: Intracellular Protein

SORF: Short Open Reading Frame

Taq DNA polymerase: A heat-stable DNA polymerase from Thermus aquaticus.

V-erbB gene: Viral Oncogen of Transforming ALVs (can cause erythroid leukosis)

vMDV (GA): Virulent Marek’s Disease Virus

vvMDV(Md5): Very Virulent Marek’s Disease Virus Strain

vv+MDV(584A): Very Virulent Plus Marek’s Disease Virus Strain

VN: Virus Neutralizing Antibodies

WHO: World Health Organization

 

                                                            




 

 

CHAPTER 1

INTRODUCTION

1.1 Background of the study

The high population growth in Africa and growing income significantly exerts a high demand for eggs and poultry meat, across large parts of the continent (World Health Organization 2010). According to estimates by the United States Agency for International Development (USAID), this trend is very likely to continue over the next few years (Heise, 2015). The Nigerian poultry industry which has the second largest chicken population in Africa after South Africa comprises about 180 million birds, (SAHEL, 2015).It had a production of over 300 000 tons of poultry meat in 2013 and 650 000 tons of eggs, (FAOSTAT, 2017). The emergence and sustenance of this large-scale intensive poultry husbandry is dependent on reducing or eliminating diseases, which are mainly achieved through the use of vaccines for disease control. Marek’s disease (MD), which was first described by Josef Marek in Hungary in 1907, is an economically important poultry disease throughout the world. Although Marek`s disease, is not one of the notifiable diseases according to the World Organization for Animal Health (OIE), the disease distribution has been acknowledged as worldwide, (Boodhoo et al.2016).  It is among the the diseases with highest economic impact in modern poultry production, worldwide, although precise estimates of morbidity, annual economic losses, and reports of disease distribution on each continent are lacking, (Payne, and Venugopal, 2000).  A rough estimation puts losses due to Marek`s disease in excess of $2 billion to the industry annually (Marek’s 1907; Morrow and Fehler, 2004). As a result of the difficulty associated with diagnosis of the disease, this estimate may be far lower than the actual losses. There is in addition to this direct loss, increased mortality and reduced growth, as well as subclinical immunosuppression, leading to the exacerbation of other diseases and decreased vaccinal immunity (Schat and Nair, 2013). Effective global surveillance for Marek`s disease virus (MDV) requires accuracy of reporting source and comprehensiveness. With an increasing demand in the global requirement for poultry products our dependence on intensive poultry production facilities has been on the rise. Controlling MDV infection in such a situation is very challenging as a result of its ubiquitous presence at the expense of already pre-established biosecurity programs, (Boodhoo et al.2016).Marek’s disease is a lymphoproliferative and neuropathic disease of domestic chickens, and less commonly, turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpes virus and characterized by neurological disorders and neoplastic transformation of CD4+ T cells and immunosuppression (Hennig et al.2003; Morrow and Fehler, 2004; Davison and Kaiser, 2004; Schat and Nair, 2008).It has attracted several names and nomenclatures as a result of its various manifestations, presentations and evolution. Some of the old names include, Neuritis,Polyneuritis, Neurolymphomatosis gallinarium, and Range paralysis. The MD virus (MDV) belongs to the family Herpesviridae, subfamily Alphaherpesvirinae and genus Mardivirus. The genus Mardivirus consists of five species of viruses, including Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The early classification of MDV into three serotypes, known as serotypes 1, 2 and 3 (HVT or herpesvirus of turkeys), was based on variations in antigenic determinants that correspond to the different species (Zhang et. al., 2017).  MDV strains of serotype 1 belong to GaHV-2 species, serotype 2 belongs to GaHV-3 and serotype 3 belongs to MeHV-1. Serotype 1 MDV (GaHV-2) is pathogenic and causes tumors in chickens (Witter, 2001), whereas serotype 2 (GaHV-3) from chickens and serotype 3 (MeHV-1) from turkeys are non-oncogenic, (Calnek and Witter, 1985). Based on the lesions, mortality rates, and protection offered by the vaccines (GaHV-2). Strains can be classified into 4 pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+) (Davison and Nair, 2004; Witter et al.,   2005). From the early 1990s, the vv+ MDV strains have been the predominant pathotype isolated, worldwide, from vaccinated chickens, for which vaccines do not appear to generate a very strong protection (Gimeno, 2008; Zhang et al., 2011).

Although the virus is highly cell-associated, it has been found to be cell-free and fully infectious in the feather follicles, explaining its highly contagious nature (Calnek et al., 1970). It is relatively stable in the poultry house environment; hence dust and dander are vehicles for natural transmission (Nazarian and Witter, 1970).

 The virus replicates in B and T-lymphocytes during the early cytolytic infection and subsequently establishes a latent infection in T-lymphocytes, which may become transformed and form lymphomatous lesions in visceral organs, peripheral nerves, and skin (Calnek, 2001).It has been associated with many disease syndromes in chickens such as lymphomatosis in nerves, skin, eyes, and visceral organs; lymphoid degeneration in the immune system; transient paralysis in the central nervous system; and atherosclerosis in blood vessels (Witter and Schat, 2003). These lesions can be present even in vaccinated birds ((Buscaglia et al.,   2004; Witter et al.,   2005). MD can manifest in affected chickens as early mortality with the absence of gross or microscopic lesions; depression, pale crest, reduced feed intake and weight gain, ataxia, and paralysis (Buscaglia et al.,   2004).

Clinical manifestations of MD and the presence of lymphomas are influenced by the immune response, which may be influenced by genetic factors. Resistant chicken strains tend to maintain the virus latency, while in susceptible chickens the infection causes lymphomas (Burgess et al.,   2001; Kaiser et al.,   2003).

At necropsy, MD gross lesions are characterized by diffuse enlargement of the liver and the spleen, presence of lymphomas in the liver, kidney, ovary, proventriculus, spleen, lungs, nerves, heart, skin, as well as atrophy of the bursa of Fabricius and of thymus.  Histopathology of affected organs shows marked cellular polymorphism, with the presence of lymphocytes, lymphoblasts, fibroblasts, and infiltration of tumor cells arranged in circumscribed or diffused form (Okonkwo, 2015). In the liver, these lesions are accompanied by degeneration and necrosis of parenchymal liver cells, atrophy of the hepatic ducts, and vacuolization while in the thymus and bursa of Fabricius necrosis and destruction of lymphoid cells have been described(Buscaglia et al.,   2004; Witter et al.,   2005; Fodor et al.,   2011). Lymphomas can be found in the absence of any nerve injury or clinical signs; therefore, they may go unnoticed during rearing or even at processing. Gross lesions may also be unspecific, and do not confirm the diagnosis of MD, demanding microscopic examination of the lesions for their proper characterization (Vieira-Pinto et al.,   2003), as well as for the differential diagnosis with other neoplasms or diseases that cause enlargement of the peripheral nerves (Schat and Nair, 2008).

There are no methods of treatment of MD and control is based on management methods that isolate growing chickens from sources of infection, the use of genetically resistant stock, and vaccination. Because vertical transmission of infection does not occur, chickens hatched and reared in isolation will be free of MD virus. Owing to the highly infectious nature of the disease and the ubiquity of the virus, this is not easy. Chickens free from infection have been produced in isolators and houses maintained under positive pressure with filtered air. These procedures are not normally economical for the management of commercial poultry. They may be used for the housing of fowls kept for experimental purposes or for providing tissues or eggs for vaccine production. Although it is unlikely that farmers will be able to keep their flocks free of MD virus, management measures can be used to reduce or delay infection and lessen the chance of serious disease. Young chicks should be reared in isolation from older stock, and an all-in-all-out policy should be adopted within a building and preferably for a whole site. In this way, it should be possible to break the infection cycle by disinfection when the houses are empty. Construction of the houses should be such as to allow thorough disinfection. Because insects may act as reservoirs of infection treatment of premises with insecticides is desirable.

 Selection for resistance to MD by poultry breeders would increase the genetically controlled resistance of commercial poultry to the disease and thus reduce the incidence of MD. If a reasonably heavy selection pressure is used, evidence suggests that a rapid increase in resistance of poultry to MD would result. For example, in three generations, a resistant line with 7.3% susceptibility and a susceptible line with 94.4% susceptibility were derived from random-bred breeding stock with 51.1% susceptibility by using virus inoculation of progeny in an eight-week test period to select breeders (Cole, 1968). Similar selection procedures have been used by a number of breeders and there has been some progress, but because adequate selection pressure was not possible, progress has been disappointingly slow.

Vaccination of newly hatched chicks with live vaccines has been widely used to control MD successfully since the early 1970s. This is the first effective use of an antiviral vaccination to prevent a naturally occurring cancer in any species, (Davison and Nair, 2005). Despite this success, infection with the virulent MDV strains and subsequent vaccine breaks still occur. The vaccine breaks may be caused by many factors such as wrong handling of vaccines or increased virulence of MDV strains (Witter, 1997) over the last four decades. Moreover, infection with any immunosuppressive agent or the difficulties associated with the vaccine handling due to its cell-associated form may also cause vaccine breaks (Jarosinski et al.,   2006). The vaccine that currently offers the highest level of protection against MD in long-lived layer and breeder chickens is the Rispens CVI988 vaccine (Davison and Nair, 2005). Rispens CVI988 is an attenuated vaccine strain of a serotype 1 MDV first isolated in the Netherlands and found to be protective in both laboratory and field trials (Davison and Nair, 2014). Rispens has since proven to offer superior protection against clinical MD and is administered worldwide, particularly to breeder and layer chickens (Ralapanawe et al., 2016). Although the Rispens vaccine provides superior protection against MD, like other MD vaccines it does not prevent infection with wild-type MDV (Rispens et al.,1972a). Such vaccines are regarded as ‘imperfect’ (Gadson et al.,2001) hence they allow both vaccinal and wild-type viruses to replicate in the host. This potentially drives MDV towards higher virulence (Atkins et al.,2013).Although MD vaccines have been very successful at protecting chickens against tumors and mortality, they do not provide sterilizing immunity and vaccinated chickens still support the replication and shedding of virulent field stains (Davidson and Nair, 2005; Gandon et al.,2001; Gimeno,2008). This is very important for diagnosis. Finding an MDV strain in a chicken therefore has no diagnostic value as most chickens will be infected without developing the disease (Gimeno, 2017). Indeed, recent outbreaks in both unvaccinated and vaccinated birds caused by more virulent strains of MDV have prompted concerns that current vaccines may be rendered ineffective with the emergence and spread of more virulent strains (Nair, 2005). However, in-ovo vaccination of any of the strain of the MD vaccine provides a better protection (Gimeno et al.,2011; Gimeno et al.,2012) as it accelerates maturation of the chicken embryo immune system resulting in chicks that are capable of responding to an early challenge with MDV and also to non-related antigen. The process and technique used to administer vaccines in-ovo is critical as the delivery must be made to precise locations within the egg and with the highest hygiene levels possible. For optimal performance, vaccine inoculation must be done between 18 and 19 days of incubation either through the amniotic or the intra-embryonic route (Gimeno, 2017).

Diagnoses of MD is done, taking into consideration epidemiological information such as age, clinical signs and gross lesions. Often times these are enough to make proper diagnoses (Gimeno, 2017). Features such as age of birds, unilateral paralysis in birds, enlargement of the brachial, or coeliac plexus, involvement of the bursa of Fabricious, nature, consistency, and distribution of visceral tumors, skin tumors or muscle tumors, and ocular involvement are important diagnostic tools. The histopathology of MD tumors consist of  highly pleomorphic lymphoid cells comprising of lymphoblasts, small, medium and large lymphocytes and reticular cells as against the lymphoblasts primarily seen in the lymphoid leucosis. However, confirmation of MD is done using histopathology, real time PCR and immunohistochemistry. The MDV genome encodes more than 200 genes, and several genes unique for oncogenic MDVs have been identified presently. (Lee e.t al., 2000; and Lupiani et. al., 2004). The gene encoding a major lytic and transformation maintaining phosphoprotein antigen (pp38) together with the Marek’s EcoRI-Q-encoded protein (meq) and virus-induced IL-8 homology (vIL-8) genes were reported to play roles in viral oncogenicity and pathogenicity (Tian et al.,   2011). The Meq protein is one of the most important MDV proteins that is only present in MDV-1 strains. It is highly expressed in MDV-1-transformed cell lines and tumor samples (Jones et al.,   1992). Meq gene mutation has been incriminated as a possible cause for the increased oncogenicity (Shamblin et al.,   2004; Wozniakowski et al.,   2010; Woźniakowski et al.,2014).Many other genes also play important roles in the development of lymphomas (Jarosinski et al.,2006). The phosphorylated protein complex (pp38) is required for the induction of cytolytic infection in B lymphocytes and for the production of adequate levels of latently infected T-lymphocytes in the lymphoid organs. In addition, pp38 has been shown to play a role in maintaining the transformation of T-lymphocytes by preventing their apoptosis (Gimeno et al.,2005). The vIL-8 gene is involved in early cytolytic infections in lymphoid organs, presumably the recruitment of B or T-lymphocytes in vivo. Deletion of vIL-8 leads to weak activation of T-cells, resulting in reduced numbers of target cells for transformation and significantly decreased pathogenicity and tumors` incidence (Cui et al.,   2004).The nucleotide and amino acid changes in the main oncoproteins Meq, pp38, and vIL-8 could be a criterion in  differentiation and determination of  pathogenicity and oncogenicity of MDV strains as these changes have been incriminated in the emergence of more virulent MDV strains in different parts of the world (Cui et al., 1991;Parcells et al., 2001and Hoda et al., 2018)

Worldwide, data on the endemicity of MD within farms is not widely available due to the fact that hygiene and infection data are not made public by poultry farms and that MDV is not a notifiable disease (Morrow and Fehler, 2004). There is scanty information on MDV prevalence and severity around the world.  Only a limited number of field studies have evaluated either within-flock MD prevalence or mortality (Biggs et al.,1972; Jackson et al.,1976; Heier et al., 1999; Karpathy et al., 2003). Indeed, only three of these studies have collected data within the past 35 years (Atkins et al.,2013). A recent study conducted between 2005-2011in Australia reported that on the average 26% of unvaccinated farms and 16% of vaccinated farms tested PCR-positive in dust samples for MDV-1 which is the pathogenic strain of MDV (Walkden-Brown et al., 2013). Since diagnosis of MD was made in Nigeria (Hill and Davis, 1962;Adene, 1975), records from veterinary hospitals and clinics indicate that there have been rapid and regular reports of clinically suspected cases of the disease across the country by many poultry farmers (Jwander et al.,2012a).. A nine year retrospective study between 2001-2010 on avian neoplastic diseases in Zaria, Northern Nigeria showed an increasing emergence of such diseases with MD contributing about 85% of the reported cases (Sani et al.,2017) and 5% of the total poultry diseases (Musa et el.,2013).Another study on the molecular detection of Marek’s disease virus in birds from north central Nigeria revealed  presence of the pathogenic MDV strain in exotic and local pullets (Jwander et al., 2012b).Although outbreaks have been recorded in the South eastern part of Nigeria (Okwor and Eze, 2011; Okonkwo, 2015), there appears to be no coordinated effort at either taking a stock of occurrences by way of retrospective studies or investigations of the molecular composition of MDV strains circulating in Abia state, and their phylogenetic relationship with other strains in the database. Therefore taking into cognizance the ability of this virus for continued mutation into more virulent strains and clinical presentations, there is a need to do a study of the disease dynamics within the state.

Marek’s disease virus has already mutated several times in its history: once in the 1960s, then after the introduction of HVT, and after HVT+ SB-1. In Europe and the US, MD is being presently controlled by the CVI988/Rispens vaccine strain. There is no proven link between introduction of the vaccines and appearance of the mutant strains but the timing suggests a connection (Komments, 2010).The virulence and MD-associated losses in vaccinated flocks have been on the increase (Witter, 1983) despite an intensive vaccination policy using available vaccines. Emergence of hyper-virulent strains with a further increase in the virulence of field virus strains (Wozniakowski et al.,   2010; Gong et al., 2013; Hassanin, et al., 2013) has been suggested as the main cause of this vaccination failure. In fact, MDV strains that are able to circumvent protection provided by CVI988/Rispens already have occurred in some parts of the world (Barrow and Venugopal, 1999; Schumacher et al.,   2002; Tischer et al.,   2002).

Due to the cell associated nature of the virus, management of the vaccine (CV1988 Rispens) is complex and requires a lot of training, proper storage (at a temperature of -196 C) and administration. The HVT and SB-1 that come in lyophilized forms still need to be maintained in a cold chain. So, in a country like Nigeria where constant power supply is a problem, there is also a problem of vaccine storage. Underutilization of the vaccine means that there will be a reduced plaque forming unit (PFU) of the antigen hence a lowered antigenic stimulation. There is therefore a need to investigate the level of adherence to these precautions and implications of non-adherence. This is important because despite claims by hatchery operators of the administration of MD vaccine at day old, MD still occurs in various farms across the country. Therefore factors that could predispose farms in Abia State to the disease outbreak need to be studied. This will involve the auditing of vaccination process, assessing field challenges, and measuring level of protection.


1.2 Statement of Research Problem

MD is ubiquitous and it occurs in poultry-producing countries throughout the world, (Gimeno, 2008). Chickens raised under intensive production systems usually suffer variable levels of losses from MD. MD is one of the most economically important and devastating diseases of poultry (Hassanin et al.,   2013). So far there, is little or no ongoing research on MD in Nigeria, (Jwander, 2005).

In spite of intensive vaccination policy with the widely used CVI988 vaccine (Rispens et al.,   1972), infection with virulent MDV strains  still occur.


1.3 Justifications of the study

A study of MD and factors affecting its outbreaks in Abia state will inform stakeholders of the disease and its importance. The study will also provide baseline data on the disease for researchers and highlight status of the disease in the South East region. Such data will also reveal performance of vaccinations as control for the disease in the state.

Investigations of the disease control mechanism, specifically deficiencies and aberrations in the vaccination protocols adopted by hatchery operators and farmers with evaluation of   vaccine-practices could reveal causes of outbreaks experienced in some farms even after vaccinations.


1.4 Significance of the study

The study will provide information for monitoring epidemiologic and pathophysiologic changes (mutation) in MD. It will bring to knowledge, the molecular composition of MDV strains circulating in Abia state, Nigeria, and may thus reveal if there had been any antigenic drift with the virus.

Auditing the different vaccination protocols adopted by hatcheries and farms would assess contributions of these procedures to MD-control.

 

1.5 Aims

The aim of this study is to investigate occurrence of MD in Abia state, Nigeria, Identify strains of MDV circulating in Abia state, Nigeria and to investigate the role of vaccination in its occurrence in Abia state, Nigeria.

 

1.6   Objectives

1. To determine the occurrence of MD in Abia state by the administration of questionnaires to veterinary clinics/hospitals and farms in Abia state, Nigeria on clinical signs, suggestive of MD and on vaccination against the disease.

2. To evaluate vaccination practices in farms located in Abia state suspected of MD and in the hatcheries supplying their flock through questionnaires and hatchery visitations

3. To vaccinate chicks in Abia state with the different MD-vaccines modules and assess antibody response to each

4. To collect samples from suspected cases of MD in Abia state for confirmation of the diagnosis.

5.To conduct molecular characterization of any MDV isolated from poultry in Abia state.

 


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