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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