ABSTRACT
An experiment was conducted using a total of one hundred and ten indigenous chickens of three genotypes and their crosses, comprising of thirty from each genotype and twenty from the indigenous breeds’ crosses with NAPRIx. The experiment was to determine the susceptibility or otherwise of the various genotypes and their crosses to Newcastle disease and to determine whether the Mx gene is involved in any resistance. The genetic diversity of the various genotypes was also assessed. The birds were inoculated with the Newcastle disease virus KUDU-113 strain. They were bled on days 0, 3, 7, 14, 21, 28 and the collected blood samples were used for molecular analysis in the laboratory while data from the molecular work was analyzed using the R statistical package. Geometric mean titre of Newcastle disease anti body was highest (4.7) in Normal feathered(NF) and lowest (3.1) in the naked neck (NN) while the NAPRIx × NF had the highest titre of 1024 on day 28. Mortality and morbidity results showed that morbidity was 100% in all genotypes and mortality was highest in NF (87.5%) and lowest in NN (45%). The ELISA results showed that the naked neck (NN) had the highest percentage of antigen (21%) while the NAPRIx×FF had the least (4%). The Association of the Mx gene genotype with resistance to Newcastle disease showed that the frequency of the resistant A allele was higher than the G allele for susceptibility. Analysis of genetic diversity showed that the number of alleles ranged from 2-3 for NF; Polymorphism information content (PIC) ranged from 0.637 to 0.976. The allele number for FF ranged from 2-3 with PIC mean of 0.981. Expected (He) and observed (Ho) heterozygosity were 0.601 and 0.765 respectively. Allele number range from 2-3 for NN, mean PIC was 0.825 while Ho and He were 0.627 and 0.513, respectively. The allele number ranged from 2-3 for all crosses. Mean PIC for NAPRIx × NF was 0.838, Ho and He 0.569 and 0.520, respectively. Mean PIC for NAPRIx × NN was0.948 while Ho and He were
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0.608 and 0.575, respectively. Mean PIC forNAPRIx×FF was 0.967 while Ho and He was 0.706 and 0.592, respectively. Analysis of molecular variance (AMOVA) showed an estimated variation of 0.142 among the populations and 0.042 among individuals while variation within individuals accounted for 98% of the total variation. The paired wise population matrix of Nei’s genetic distance showed that the longest distance of 0.409 was observed between FF andNAPRIx ×NF while the shortest distance (0.177) was between FF and NAPRIx×NN.Dendogram analysis also shows that the studied chicken populations formed five clusters of genetic dissimilarity. Based on this finding, it can be concluded that the indigenous chicken populations have the ability to resist Newcastle disease and that the Mx gene is involved in that resistance and that the naked neck was more resistant to the disease. It is therefore, recommended that crosses involving the indigenous chicken and other Broiler strains should be carried out with the normal feathered. Further experiments should also be carried out, involving the naked neck and its crosses to determine to what extent the Mx gene is involved.
TABLE OF CONTENTS
DECLARATION…………………………………………………………………………………………………………… ii
CERTIFICATION ……………………………………………………………………………………………………….. iii
DEDICATION……………………………………………………………………………………………………………… iv
ACKNOWLEDGEMENTS …………………………………………………………………………………………… v
ABSTRACT ………………………………………………………………………………………………………………… vii
TABLE OF CONTENT ………………………………………………………………………………………………… ix
LIST OF TABLE ………………………………………………………………………………………………………… xii
LIST OF FIGURES ……………………………………………………………………………………………………. xiii
CHAPTER ONE …………………………………………………………………………………………………………… 1
1.0 INTRODUCTION……………………………………………………………………………………………………. 1
1.1 Background of the Study ………………………………………………………………………………………….. 1
1.2 Justification …………………………………………………………………………………………………………….. 3
1.3 Objectives………………………………………………………………………………………………………………… 4
CHAPTER TWO ………………………………………………………………………………………………………….. 5
2.0 Literature review …………………………………………………………………………………………………….. 5
2.1 Nigerian Indigenous chicken …………………………………………………………………………………….. 5
2.2 Newcastle Disease …………………………………………………………………………………………………….. 7
2.2.1 Etiology …………………………………………………………………………………………………………………. 8
2.2.2 Epidemiology …………………………………………………………………………………………………………. 8
2.2.3 Pathogenesis …………………………………………………………………………………………………………… 8
2.2.4 Diagnosis…………………………………………………………………………………………………………….. 9
2.2.5 Prevention and control ………………………………………………………………………………………… 10
2.3 Myxo virus Gene ……………………………………………………………………………………………………. 11
2.4 Genetic Diversity ……………………………………………………………………………………………………. 12
2.5 Kudu-113 Virus ……………………………………………………………………………………………………… 17
CHAPTER THREE …………………………………………………………………………………………………….. 18
3.0 MATERIALS AND METHODS …………………………………………………………………………….. 18
3.1 Location of the Experiment…………………………………………………………………………………….. 18
3.3 Procedure/Methodology …………………………………………………………………………………………. 18
3.3.1 Challenge test ……………………………………………………………………………………………………….. 18
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3.3.2 Blood sample collection …………………………………………………………………………………………. 19
3.4.1 Red blood cells of chicken ……………………………………………………………………………………… 19
3.4.2 Haemagglutination test (HA) ………………………………………………………………………………….. 19
3.4.3 Haemagglutination inhibition test (HI) …………………………………………………………………….. 20
3.4.4 Elisa (The enzyme-linked immunosorbent assay) ……………………………………………………… 20
3.5 Mx Gene Genotyping ……………………………………………………………………………………………… 21
3.5.1 DNA extraction …………………………………………………………………………………………………….. 21
3.5.2 PCR Amplification………………………………………………………………………………………………… 21
3.6 Data Collection and Analysis ………………………………………………………………………………….. 25
CHAPTER FOUR ……………………………………………………………………………………………………….. 26
4.0 RESULTS ……………………………………………………………………………………………………………… 26
4.1 Host response to inoculation with KUDU-113 virus of Newcastle disease …………………. 26
4.2 ELISA results of Nigerian indigenous chickens and their crosses with NAPRIx challenged with KUDU-113 virus…………………………………………………………………………..28
4.3 Mortality and Morbidity of Three Genotypes of Nigeria’s Indigenous Chickens and their crosses………………………………………………………………………………………………………….29
4.4 Mx gene Genotypes found in Nigerian Indigenous Chickens and their crosses …………. 32
4.5 Average genetic distances of Nigerian indigenous chickens ……………………………………… 34
4.6 Average genetic distances of the crosses of Nigerian indigenous chicken populations .. 36
4.7 Analysis of molecular variance (AMOVA) of Nigerian indigenous chicken and their crosses with NAPRIx……………………………………………………………………………………………. 38
4.8 Pairwise population matrix of Nei genetic distance of Nigerian indigenous chickens and their crosses with NAPRIx.40
CHAPTER FIVE ………………………………………………………………………………………………………… 48
5.0 DISCUSSION ………………………………………………………………………………………………………… 48
5.1 The Geometric mean titre (GMT) of Nigerian indigenous chickens and their crosses..48
5.2 Mx gene and its Association with Newcastle disease in Nigerian indigenous Chicken and their Crosses……………………………………………………………………………………………………48
5.3 Genetic Diversity ……………………………………………………………………………………………………. 49
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5.3.1. Number of allele (Na), observed (Ho) and Expected Heterozygosity (He), and polymorphism information content (PIC) of Normal Feathered indigenous chickens of Nigeria…………………………………………………………………………………………………………………………………….. 49
5.3.2. Number of allele (Na), observed (Ho) and Expected Heterozygosity (He), and polymorphism information content (PIC) of Frizzled feathered chicken of Nigeria……….50
5.3.3. Number of allele (Na), observed (Ho) and Expected Heterozygosity (He), and polymorphism information content (PIC) of naked neck chicken of Nigeria………………..51
5.3.4. Genetic Distance ………………………………………………………………………………………………….. 51
5.3.5. Analysis of Molecular variance ……………………………………………………………………………… 52
5.3.6. Cluster Analysis …………………………………………………………………………………………………… 52
CHAPTER SIX …………………………………………………………………………………………………………… 53
6.0 SUMMARY, CONCLUSIONS AND RECCOMENDATION ………………………………….. 53
6.1 Summary ……………………………………………………………………………………………………………….. 53
6.2 Conclusion …………………………………………………………………………………………………………….. 55
6.3 Recommendation……………………………………………………………………………………………………. 56
REFERENCES ……………………………………………………………………………………………………………. 57
APPENDIX …………………………………………………………………………………………………………………. 70
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CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the Study
Nigeria is the most populous country in Africa and is home to about 172 million domestic poultry (FAOSTAT, 2011).The livestock sector is vital to the socio-economic development of Nigeria and contributes about 9-10% of Agricultural GDP(FAO, 2006). Consequently, livestock represents an important source of high quality animal protein, providing about 36.5 percent of the total protein intake of Nigerians. It is one of the highest investments in Agriculture with net worth of ₦250 billion.The poultry sub-sector is the most commercialized of all Nigeria’s agricultural sub-sectors (AICP, 2007). Smallholder poultry production makes use of local or indigenous genetic resources, which differ from commercially bred poultry in several respects: The birds are adapted to a harsh environment where resources are often limited and where challenges imposed by climatic conditions, pathogens and predators are severe. They are often utilized for several purposes simultaneously, and therefore may outperform specialized commercial breeds when scored for multipurpose productivity.Native chicken constitutes 80% of the poultry birds in Nigeria and are sources of high quality protein, reserved for times of celebrations and a good source of income for rural families (Abubakaret al., 2008).
Newcastle disease (NCD) is a major problem of poultry in many parts of the world (Alexander and Senne 2008; Muniret al., 2012; Luoet al., 2013). The causative agent of the disease is Newcastle Disease virus (NDV) which is the prototype virus of the avian Paramyxovirus type 1 in the family Paramyxoviridae (Lamb et al., 2005; Alexander and Senne, 2008). The Paramyxoviruses isolated from avian species have been classified into 9 serotypes designated as APMV-1 to APMV-9 (Alexander, 2003). While the disease is enzootic in Africa and Asia (Mathivananet al., 2004), it is exotic in Europe and North America. Despite the advances made
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so far in the control of the disease by vaccination and bio security, devastating outbreaks of NCD still occur in many parts of the world (Capua et al., 2002). This is because there are many factors that make the control of NCD very difficult. NDV has a very wide host range, affecting at least 241 avian species (Alexander and Senne, 2008). Newcastle disease virus (NDV) has been classified into velogenic, mesogenic and lentogenic strains on the basis of their pathogenesis and virulence. Chickens with Newcastle disease have severe neurological and respiratory signs and show decreased egg quality and production (Islam and Nishibori, 2009). The Myxovirus (Mx) gene codes for a protein that has direct antiviral activity and inhibits a wide range of viruses by blocking an early stage of the viral replication cycle. The chicken Mx protein has been reported to exhibit antiviral activity against the influenza virus and the vesicular stomatitic virus (Koet al., 2002). Mx plays a major role in IFN-induced host defence. Extensive research about resistance genes in poultry have been undertaken, the major included histocompatibility complex MHC genes, which relates with the resistance to diseases such as the Marek’s disease and Rous sarcoma (Niikuraet al., 2004; Nikolichet al., 2004; Xuet al., 2007). Mx proteins are key components of the innate antiviral state induced in many species such as human, mouse and chicken among other organisms (Haller et al., 2007) and belongs to the super family of GTPases (Luan et al., 2010).
Genetic diversity within a livestock species is reflected in the range of types and breeds that exist and in the variation that is present within each genotype. It has been shown that differences among breeds substantially exceed those within breed, suggesting that geneticvariation among breeds is a major component of the readily accessible livestock diversity.Losses of unique types and breeds compromise access to their unique genes and gene combinations, (Adebambo, 2003).If genetic diversity is to decline, then selection of stock for commercially desired traits
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will beunproductive for developing improved breeds. Given this prospect then,maintenance of genetic diversity must be given a major consideration in Nigeria’s livestock improvement programmes in this millennium (Adebambo, 2003).Ohwojakporet al. (2012) recommended a further study of the level of genetic diversity in different populations so as to bring about improvement in the performance of chicken in the developing countries.
1.2 Justification
There is no effective treatment for Newcastle disease; however, the use of prophylactic vaccines and maintenance of strict bio-security measures can reduce the likelihood of outbreaks. Thus, the ability of chickens to mount an antibody response to NDV plays a key role in controlling Newcastle disease outbreaks, while understanding the molecular basis of immune response to NDV is important for the control of avian Newcastle disease. The antibody response to the same virus differs between chicken breeds , and selection for an antibody response may improve disease resistance in chickens (Luoet al., 2013). Despite the advances made in the diagnosis of and vaccination for Newcastle disease since it was first described in 1926, the disease continues to negatively impact on poultry producers by infecting birds worldwide (Alexander et al., 2012). From 2006 to 2009, the most widespread animal diseases in terms of the number of countries affected, were rabies, Newcastle disease (NCD) and Bovine tuberculosis (Anonymous, 2011). Newcastle Disease ranked as the fourth most important disease in terms of the number of livestock units lost for poultry species, behind highly pathogenic avian influenza, infectious bronchitis, and lowly pathogenic avian influenza (Kapczynskiet al., 2012; Anonymous, 2011).
Genetic resistance to a disease is an attractive solution because it is reliable, long lasting, and environmentally sound. Also, chicken lines selected for Mareks Disease (MD) resistance have been shown to have greater vicinal immunity and higher egg production than susceptible lines (Liu et al., 2003).
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This study was therefore designed to test the following hypothesis: H0: That the Mx gene is not involved in immunity in Nigerian indigenous chicken populations and their crosses against Newcastle disease. HA: That the Mx gene is involved in immunity in Nigerianindigenous chicken populations and their crosses against Newcastle disease.
1.3 Objectives of this study is to determine
1. The host response to NCD infection in different Nigerian indigenous chicken Populations and their crosses
2. The genetic basis for NCD host relationship between Nigerian indigenous Chicken Populations and their crosses
3. The various genetic diversity in the Nigerian indigenous chicken populations and their crosses
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