DETECTION OF NEWCASTLE DISEASE VIRUS IN LAUGHING DOVES (STREPTOPELIA SENEGALENSIS) AND CHICKENS IN BACKYARD POULTRY FARMS AND THE LIVEBIRD MARKET IN SAMARU, ZARIA, NIGERIA
Newcastle disease (ND) is a highly infectious viral disease of birds caused by Newcastle disease virus (NDV). Doves have been incriminated in previous outbreaks of the disease in chickens in some parts of the world. Newcastle disease is the most important enzootic viral disease of chickens in Zaria with annual outbreaks that discourage backyard poultry production. The objectives of the study were to detect and characterize NDV in laughing doves and backyard chickens, and to assess biosecurity measures and farm management practices in relation to outbreak of ND as recalled by backyard poultry farmers. This cross sectional study (September 2014 to February 2015)was conducted on 184 swabs from cloacae and pharynxes of 67 trapped laughing doves and 25 backyard chickens from residential areas of Samaru-Zaria, Nigeria, by convenience sampling. Haemagglutination assay (HA) followed by haemagglutination inhibition (HI) test were performed using positive oropharyngeal and cloacal swabs as antigen and hyperimmune serum respectively. Red blood cell adsorption-de-absorption concentration of NDV followed by conventional reverse transcriptase polymerase chain reaction (RT-PCR) were conducted on the HA and HI positive samplesfor molecular detection of NDV. Biosecurity assessment of backyard poultry farms was conducted by face-to-face interview of backyard poultry farmers.Pre-tested semi-structured questionnaires and on the farm inspection were used to assess biosecurity measures, owner‟s reported outbreak ofND, and farm management practices that favour the outbreak of ND in backyard poultry. This study showed that of the 65.7% (88/134) of dove oropharyngeal and cloacal swabs positive by HA, 42.1% (37/88) were HI positive. Of the 37 HI positives, 21(56.8%) were RT-PCR positive of which eight were lentogenic, twelve were velogenic while one had both lentogenic and velogenic NDV.
While of the 50 chicken oropharyngeal and cloacal swabs screened, 46% (23/50) were HA positive, and of these 69.6% (16/23) were positive by HI. Only 25% (4/16) of the HI positives produced bands after RT-PCR and gel electrophoresis indicative of 3 lentogenic and a velogenic NDV. Also, the biosecurity assessment study revealed that sun-drying household grains and or flour, overstocking above 300 birds, the peak of the rains (August) and start of the cold dry season (November) and accessibility of laughing doves to poultry feed were statistically associated (P<0.05) with farmers‟ reported outbreaks of ND. From the study it was concluded that laughing doves were demonstrated to be infected with either lentogenic or velogenic NDV or both. Also, detection of NDV in laughing doves corresponded with detection of NDV in backyard chickens (with statistically greater detection of NDV in chickens and doves in cluster 1 than cluster 2). The use of red blood adsorption-de-adsorption concentration of NDV enhanced RT-PCR detection using fusion gene primers NDV-F 4829 and NDV-R 5031. The detection of not only lentogenic but velogenic NDV in laughing doves poses a great risk to backyard poultry production. An epizootic of velogenic ND in wild birds could lead to an epizootic in backyard poultry with accompanying economic loss. It is recommended that veterinary agencies of Government should encourage more research on the role of laughing doves in the spread of ND to poultry.
|TABLE OF CONTENTS|
|Table of Contents||–||–||–||–||–||–||–||–||–||x|
|List of Figures||–||–||–||–||–||–||–||–||–||xiii|
|List of Tables||–||–||–||–||–||–||–||–||–||–||xiv|
|List of Plates||–||–||–||–||–||–||–||–||–||–||xvi|
|List of Appendices-||–||–||–||–||–||–||–||–||–||xvii|
|Abbreviations, Definitionsand Symbols||–||–||–||–||–||–||xviii|
|CHAPTER 1: INTRODUCTION||–||–||–||–||–||–||–||1|
|1.2||Statement of Research Problem||–||–||–||–||–||–||3|
|1.3||Justification of the Study||–||–||–||–||–||–||–||5|
|1.4||Aim of the Study||–||–||–||–||–||–||–||–||6|
|1.5||Objectives of the Study||–||–||–||–||–||–||–||6|
|CHAPTER 2: LITERATURE REVIEW||–||–||–||–||–||–||8|
|2.1||Aetiology of Newcastle Disease||–||–||–||–||–||–||8|
|2.2||Transmission of Newcastle Disease Virus||–||–||–||–||–||9|
|2.3||Prevention, Control and Decontamination in an Outbreak of Newcastle|
|2.4||Zoonotic Potential of Newcastle Disease Virus||–||–||–||–||11|
|2.5||VaccinationAgainst Newcastle Disease||–||–||–||–||–||11|
|2.6||Diagnosis of Newcastle Disease||–||–||–||–||–||–||12|
|2.6.1||Conventionaldiagnosis of Newcastle disease virus||–||–||–||–||12|
|2.6.2||Molecular diagnosis of Newcastle disease virus||–||–||–||–||13|
|2.6.3||Epidemiological significance of molecular diagnosis||–||–||–||–||16|
|2.6.4Conventional RT-PCR following RBC adsorption-de-adsorption|
|concentration of Newcastle disease virus||–||–||–||–||–||–||17|
|2.7||Historical Overview of Newcastle Disease in Doves||–||–||–||18|
|CHAPTER 3: MATERIALS AND METHODS||–||–||–||–||–||24|
|3.2||Study Design –||–||–||–||–||–||–||–||–||27|
|3.3||Sampling and Sample Collection||–||–||–||–||–||–||27|
|3.3.2||Trapping of laughingdoves||–||–||–||–||–||–||–||27|
|3.3.4||Preparation of washed chicken red blood cells –||–||–||–||–||29|
|3.3.6||Positive control serum||–||–||–||–||–||–||–||–||29|
|3.4||Laboratory Analyses -Detection of Newcastle Disease Virus||–||–||30|
|3.4.1||Haemagglutination assayand haemagglutination inhibition assay||–||–||30|
|3.4.2||RBC adsorption-de-adsorption concentration of Newcastle disease virus||–||30|
|3.4.3||Ribonuclease acid extraction||–||–||–||–||–||–||–||31|
|3.4.4||Reverse transcriptase –polymerase chain reaction||–||–||–||–||32|
|3.4.5 Agarose gel electrophoresis||–||–||–||–||–||–||–||33|
|3.5||Assessment of Biosecurity Measures-||–||–||–||–||–||33|
|3.7||Public Health Action –||–||–||–||–||–||–||–||35|
|CHAPTER 4: RESULTS||–||–||–||–||–||–||–||–||45|
|4.1||Detection of Newcastle Disease Virus||–||–||–||–||–||45|
|4.2||Assessment of Biosecurity Measures||–||–||–||–||–||52|
|4.3||Assessment of Newcastle Disease Outbreak||–||–||–||–||52|
|4.4||Assessment of Farm Management Practices||–||–||–||–||53|
|4.5||Monthly Distribution of Reported Newcastle DiseaseOutbreak –||–||53|
|4.6||Distribution of Reported Newcastle Disease Based on FlockSize and|
|4.7||Distribution of Poultry Farmers, Poultry Population and Newcastle|
|Disease Virus Positivity||–||–||–||–||–||–||–||54|
|4.8||Assessment of Biosecurity and Farm Management in Relation to|
|Outbreak of Newcastle Disease||–||–||–||–||–||–||54|
|4.9||Distribution of Doves Trapped and Sampled in Comparison to the Backyard|
|Poultry Houses in the Study Area||–||–||–||–||–||–||54|
|CHAPTER 5: DISCUSSION||–||–||–||–||–||–||–||66|
|CHAPTER 6: CONCLUSION AND RECOMMENDATIONS –||–||–||75|
1.1 Background Information
Velogenic Newcastle disease (ND) also known as exotic Newcastle disease (END) is a contagious and fatal viral disease affecting all species of birds. Exotic Newcastle disease is one of the most infectious diseases of poultry in the world and is so deadly that birds die without showing any signs of disease. A mortality rate of almost a hundred percent can occur in unvaccinated poultry flocks. Velogenic Newcastle diseasevirus can infect and cause death even in vaccinated birds (Margaret, 2006). Exotic Newcastle disease affects the respiratory, nervous and digestive systems of birds. The incubation period of Newcastle disease ranges from 2-15 days. Signs include sneezing, gasping for air, nasal discharge, coughing; greenish watery diarrhoea; depression, muscular tremors, drooping wings, twisting of the head and neck (torticollis), circling, complete paralysis; partial to complete drop in egg production, production of thin-shelled eggs; swelling of tissues around the eyes and in the neck; sudden death and increased deaths in a flock (USDA website, 2014). Exotic Newcastle disease is spread by direct contact between healthy birds and the bodily discharges of infected birds (droppings and nasal, oral or ocular secretions). Exotic Newcastle disease spreads rapidly among birds kept in confinement such as commercially raised chickens. Shoes, clothing and equipment of vaccination and debeaking crews, manure haulers, truck drivers, feed delivery personnel, poultry buyers, egg service people and poultry farm owners and employees all serve as agents of spread of exotic Newcastle disease virus (Margaret, 2006). Prevention of Newcastle disease is by ensuring optimum bio-security standards for poultry farms. All dead wild birds in a far should be handled as Trojan horses containing an unknown number of pathogenic agents, so treated as suspected contagion (which should be picked up as soon as they are found with gloved hands, placed in a plastic bag, sealed, sent to a reference virology laboratory for diagnosis and feedback report) (USDA website, 2014).
The “gold standard” for identification of Newcastle disease virus is isolation and cultivation in embryonated chicken eggs followed by haemagglutination test (HA), haemagglutination inhibition test (HI) and pathotyping of the virus. Pathogenicity traditionally is determined by the intracerebral pathogenicity index. These assays are labour intensive and time-consuming, requiring up to ten days in completing and avoidable suffering to embryos and destruction of animal life. This hinders the authorities in undertaking adequate measures in a timely manner to limit the spread and eradicate the infection. The development and implementation of rapid, reliable, and high-throughput diagnostic methods for detection of the virus could provide a valuable contribution to controlling the disease. The real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) is a test that satisfies the requirements for high sensitivity and specificity coupled with a short turnaround time (Dimitrov et al., 2014). The F gene of virulent NDV (END) has a unique pair of dibasic amino acids at the cleavage site (115 and 116 with a phenylalanine at 117) and a basic amino acid at 113 while lentogenic strains have monobasic amino acids and lack dibasic amino acids (Naresh et al., 2009). Bruce et al. (1995) using Reverse Transcription Polymerase Chain Reaction (RT-PCR) coupled with Direct Nucleotide Sequencing and Sequence Database for NDV pathotype prediction, showed that lentogenic vaccine isolates LaSota, England/F, B1, and Queensland/V4 and the lentogenic field isolate 91/33 all have the sequence 109-SGGGRQGRLIG-119 at the Fusion Cleavage site, while the mesogenic and velogenic viruses have the sequence 109-SGGRRQR(K)RFIG-119 containing the two diagnostic pairs of dibasic amino acids associated with virulence. Furthermore, virulent isolates have the RR sequence instead of the GR sequence at positions 112 and 113 as well as RR or RK pair rather than the GR sequence at positions 115 and 116. Real Time RT-PCR however is quite expensive for routine diagnostic screening of backyard poultry flocks. Using haemagglutination test (HA), haemagglutination inhibition (HI) and conventional reverse transcription-polymerase chain reaction (RT-PCR) assays would be affordable, reliable and give similar results to real time RT-PCR, enabling effective prevention and control by the veterinary professionals (Bruce et al., 1995).
1.2 Statement of Research Problem
Backyard poultry production has come to stay in Nigeria. In Zaria, many residential areas have residents who raise broilers and layers to meet the local demand for chicken meat and eggs (personal communication with respondents). The manure derived as a by-product is often bagged and sold to manure haulers and hawkers for additional revenue generation. This is a profitable business and is mostly managed by the housewives especially in the staff quarters of the Ahmadu Bello University Zaria and other residential areas in Zaria (personal communication with respondents). There is ready and easy access to avian veterinarians and vaccines and poultry feed can readily be obtained in Zaria as well. Most poultry farmers have a vaccination schedule and sometimes employ the services of professional veterinarians or veterinary technicians/attendants in drafting one and carrying out vaccinations. In spite of these prophylactic vaccinations against endemic poultry diseases such as Newcastle disease, Marek‟s disease and fowl pox, farmers still suffer outbreaks of Newcastle disease that sometimes cripples production to zero (personal communication with respondents). This may be attributed to various reasons which may not be limited to source of birds, transportation, biosecurity measures in the farm, vaccine break and vaccine failure, incursion of poultry houses with wild birds like doves, pigeons, and finches. Oladele et al.(1996) and Sa‟idu et al. (2004) established through serological techniques (HA and HI) that pigeons and doves in Zaria were infected with Newcastle disease virus, just as Wambura (2010) detected antibodies to Newcastle disease virus in guinea fowls and pigeons in Tanzania.
Zaria is within the guinea savanna zone of Nigeria, and there are established populations of doves which inhabit the tall trees present in the Ahmadu Bello University Staff quarters and other residential areas in Zaria with tall trees (personal communication). These doves in foraging for food, visit households to pick up grains, flour, chaff, and chicken feed spilled on the ground around backyard poultry houses, especially when feeding their nestlings (squabs). This increases the chances of dove droppings contaminating the premises of households with backyard poultry. The soles of footwear of poultry farmers may also be contaminated with these wild bird droppings and be transferred into the backyard poultry houses. With broken chicken wire fencing of poultry houses, doves may actually invade such pens and contaminate poultry feed and water with oro-nasal discharges and faeces.
1.3 Justification of the Study
Exotic Newcastle disease, previously known as velogenic viscerotropic Newcastle disease, is so virulent that many birds die before showing any clinical signs (Margaret, 2006). A death rate of one hundred percent can occur in exposed flocks (Margaret, 2006). An END outbreak can jeopardize a state’s poultry production and limit its international trading opportunities (Margaret, 2006). Backyard poultry farming contributes to the financial income of families that run them, and ensures that broilers and eggs are readily available to the residents of the A.B.U. staff quarters and environs (personal communication).
This study sought to go a step beyond serological evidence of NDV from previous studies done in Zaria (Oladele et al., 1996), to the detection (HA and HI tests), pathotyping (RT-PCR) and electrophoretyping (agarose gel electrophoreses) of NDV isolated from doves in Zaria, and compared with NDV in chickens. This would prove for certain whether pigeon NDV plays an epidemiological role in the outbreak of ND in chickens.
Using the three diagnostic techniques HA, HI, RT-PCR and visualizing via gel electrophoresis to detect, pathotype and classify NDV over the serological HA/HI, viral isolation via embryonated eggs, MDT, and other laborious time consuming procedures is justified if the results are similar. NDV spreads rapidly, and cutting down the number of diagnostic procedures is of the greatest importance to the economy, the poultry sector and the health of wild birds.
The definition of the Office International des Epizooties (2001) reflects the current understanding of the molecular basis for virulence: Newcastle disease is defined as an infection of birds caused by a virus of avian paramyxovirus serotype 1 (APMV–1) that meets one of the following criteria for virulence: (a) The virus has an intracerebral pathogenicity index (ICPI) in day-old chicks (Gallus gallus) of 0.7 or greater. Or (b) Multiple basic amino acids have been demonstrated in the virus (either directly or by deduction) at the C-terminus of the F2 protein and phenylalanine at residue 117, which is the N-terminus of the F1 protein. The term „multiple basic amino acids‟ refers to at least three arginine or lysine residues between residues 113 to 116. Failure to demonstrate the characteristic pattern of amino acid residues as described above would require characterisation of the isolated virus by an ICPI test (Aldous and Alexander, 2001).
1.4 Aim of the Study
The aim of the study was to detect and characterize the strains of Newcastle disease viruses (NDV) in laughing doves in Zaria and assess their potential to cause exotic Newcastle disease in domestic poultry.
1.5 Objectives of the Study
The objectives of the study were to:
- determine the prevalence and pathotype of NDV in laughing doves trapped near backyard poultry farms in Zaria.
- assess biosecurity measures of backyard poultry farms against laughing doves that prevents the spread of NDV to backyard poultry in Zaria.
- assess the outbreak of Newcastle disease in chickens of backyard poultry farmers in Zaria.
- assess backyard poultry farm management practices that affect the spread of NDV between backyard poultry and doves.
1.6 Research Questions
- Do laughing-doves in Samaru-Zaria harbour Newcastle disease virus (NDV) and are the NDV isolates from laughing-doves velogenic, mesogenic or lentogenic?
- Does the use of biosecurity measures prevent the spread of ENDV from laughing doves to backyard poultry?
- Does the history of ND outbreak and signs observed in affected sick chickens of backyard poultry farmers indicate exotic Newcastle disease (END)?
- Does farm management practice affect the outbreak of END in backyard poultry?
DETECTION OF NEWCASTLE DISEASE VIRUS IN LAUGHING DOVES (STREPTOPELIA SENEGALENSIS) AND CHICKENS IN BACKYARD POULTRY FARMS AND THE LIVEBIRD MARKET IN SAMARU, ZARIA, NIGERIA