A Serological Survey for Infectious Bursal Disease Virus Antibodies in Free-range Village Chickens in Northern Tanzania

a INTRODUCTION Free-range poultry are an important constituent of Tanzanian's poultry industry , with 80 % of the country's 56 million chickens reared in traditional free-range systems in villages 20,21 Most flocks are small and of mixed age, distributed between many households and feed mainly by scavenging. This feeding habit exposes them to contact with wild birds, which might be a source of infectious bursal disease virus (IBDV) infection 16,32. The seroprevalence of IBDV has been demonstrated in free-living wild birds in Japan including migratory species 24. Very virulent IBDV has been isolated from wild birds in Korea 11. Infectious bursal disease (IBD) or Gumboro disease is a highly contagious immunosuppressive viral infection of young chickens (3–6 weeks old) causing severe economic and production losses worldwide 22. IBDV is the causative agent of IBD. IBDV is a double-stranded dsRNA virus that has a bi-segmented genome and belongs to the genus Avibirnavirus of the family Birnaviridae 26. The virus is extremely lymphocidal and infects


INTRODUCTION
Free-range poultry are an important constituent of Tanzanian's poultry industry, with 80 % of the country's 56 million chickens reared in traditional free-range systems in villages 20,21 Most flocks are small and of mixed age, distributed between many households and feed mainly by scavenging. This feeding habit exposes them to contact with wild birds, which might be a source of infectious bursal disease virus (IBDV) infection 16,32 . The seroprevalence of IBDV has been demonstrated in free-living wild birds in Japan including migratory species 24 . Very virulent IBDV has been isolated from wild birds in Korea 11 .
Infectious bursal disease (IBD) or Gumboro disease is a highly contagious immunosuppressive viral infection of young chickens (3-6 weeks old) causing severe economic and production losses worldwide 22 . IBDV is the causative agent of IBD. IBDV is a double-stranded dsRNA virus that has a bi-segmented genome and belongs to the genus Avibirnavirus of the family Birnaviridae 26 . The virus is extremely lymphocidal and infects IgM-bearing B-lymphocytes in the bursa of Fabricius, leading to immunosuppression 1 . There are two distinct serotypes of IBDV, and within each serotype antigenic variation is considerable 9 . Only serotype 1 viruses cause economically significant immunosuppressive disease in young chickens 1,30 . Serotype 2 infects chickens and turkeys but does not cause clinical disease. 'Variant' strains of IBDV, which have major antigenic differences from the 'standard' strains, cause immunosuppression but not clinical disease in older chickens 10 . Continuous presence of IBDV in village poultry populations has been reported elsewhere 13,31 . The first IBDV isolates from various locations in Tanzania were characterized as very virulent (vv) type in 2007 13 . These 'variants' were found to be widely distributed throughout Tanzania and demonstrated great similarities with isolates from western Africa and European/Asian vvIBDV variants 14,19 .
Some of the risk factors that have been associated with the maintenance of IBDV include carrier chickens, village poultry population dynamics, other poultry species, including wild birds, and heterogeneity of IBDV 13,29,30 . Transfer of the virus from house to house is through fomites. The virus is also released in the faeces, ingestion of which is the main method of bird-to-bird spread 33 .
The epidemiology of IBDV in village chickens in Tanzania is insufficiently studied but it appears that IBDV is the most important recurring disease every year 2,31 . Although IBDV represents one of the most severe poultry diseases and is responsible for marked economic losses, few studies of IBDV have been done on chickens in Tanzania, which hinders the implementation of effective diseasecontrol measures. For this reason, the aim of the present study was to determine the seroprevalence rate of IBDV and the factors that may be associated with risk of infection in free-range poultry in northern Tanzania.
The climate is sub-humid with temperatures ranging from 14°C to 23°C in high elevation areas and 30°C to 37°C along the northern coast of the Indian Ocean. The study areas experience 2 main seasons, the dry season from May to October, and the wet season from November to April. Rainfall ranges from 635 mm to 3.050 mm, with low rainfall in the low-lying areas and high rainfall on the high-altitude and plateau areas.

Household selection and data collection
Households for the study were selected based on the past experience of chicken keeping, possession of chickens and willingness to participate in the study. This sampling procedure included 40 villages and 90 households. Primary data related to chicken production were collected through consultative procedures from various sources including the respective district agriculture and livestock department offices. The secondary data, coupled with a checklist and farm inspection, were collected using semi-structured questionnaires which were completed at all selected households during a single visit. The questionnaire was administered in the national Swahili dialect by veterinary department staff members, who were trained in participatory research methodologies. Important household and flock-level data recorded included location, owner, source (brought in or homebred), history of IBD vaccination, health status at time of visit (subjectively classified as healthy or unhealthy), sex, housing (classified as permanent chicken Banda or dwelling), external parasite type and infestation were categorized as Yes or No. Birds were categorized as follows: chicks (1 to ≤3 months of age), growers (>3 to ≤9 months) and adults (≥9 months). The data were collected during November and December 2009.

Blood sampling and storage
Approximately 1.5 to 2 m of blood was collected from the humeral region of the wing vein with a 3-m syringe. The syringe was laid nearly horizontally until the blood clotted. After clotting, the syringe was returned to a vertical inverted position to permit the serum to ooze out. The sample was then kept at 37°C for several hours or left overnight before the serum was removed. The separated serum was transferred to cryovials, labelled, and stored at -20°C until the enzyme-linked immunosorbent assay (ELISA) was performed to detect antibodies against the IBD virus 26,27 .

Serum analysis
The ELISA test was done following procedures outlined by the infectious bursal disease virus antibody test kit (ProFLOK Plus, Synbiotics Corporation, San Diego, CA, item no: 96-6500) and Snyder et al. 28 All serum samples tested in the present study were processed in a single well each, according to the standard protocol. Briefly, the ELISA plates (Co-star) were supplied pre-coated with IBDV antigen. The samples were added and the plates subsequently washed and soaked, followed by the addition of 100 µ of goat anti-chicken IgG conjugate [HRP/ IgG (H+L) PO] diluted at 1:100 into all wells and incubation at room temperature for 30 min. Following incubation, the plates were soaked, washed in PBS-T and developed by adding 100 µ of ABTS-Hydrogen Peroxide/Substrate solution per well and incubated at room temperature for 15 min. Absorbance was measured using an ELISA reader (Multiscan Ex., Lab systems, Finland) at 405 nm. The reader was connected to a computer loaded with ELISA Data Interchange (EDI) software. Calculation of the sample to positive (sP) values was done manually using the following equation: [sP = (sample absorbance) -average normal control absorbance)/corrected positive control absorbance]. Sp values were manually converted to percentage positivity (PP) by using the following equation: [PP = Sp value of the sample/Sp value of the corrected positive control] × 100. Samples with Sp values of 0.299 and higher or equivalent to PP ≥30 % (cut-off) were considered positive for IBDV infection.

Data analysis
Collected data sets (birds, flock and laboratory) were entered, managed and analysed using Epi-Info, Version 6.04b 6 (Centres for Disease Control, Atlanta, USA). Descriptive statistics generated included counts, frequencies and proportions. Chi-square analysis was used to compare the association between dependent (seroconversion status: positive or negative) and independent variables (location, sex, age category, source, health status, level of ectoparasites infestation). In all chi-square tests a probability level of P < 0.05 was considered statistically significant.

Study flock characteristics
All the selected farms/flocks were visited, owner(s) interviewed and chickens sampled. A voluntary participating rate of 100 % was thus achieved. Overall, 362 birds of mixed ages and sex from 90 flocks/farms were examined. The number of birds examined and sampled per village varied from 2 to 29. No history of IBD vaccination was recorded in any of the surveyed sites. Thirty-two (8.8 %) birds had signs of illness (ruffled feathers, lacrimation) and drooping wings during sampling. The proportions of birds in each category of each variable investigated during the study are shown in Table 1.

DISCUSSION
There was evidence that exposure of indigenous free-range chickens and infection with IBDV was widespread in the 7 studied districts. Seroprevalence was significantly higher in Tanga and Kilimanjaro (P < 0.05) compared with Arusha and Manyara. This may suggest that free-range chickens in Kilimanjaro and Tanga are more at risk of IBD than in the Arusha and Manyara regions. In the Tanga region, seroprevalence to IBDV was highest in the Mkinga and Pangani districts, which are amongst the hottest, most humid and wettest districts in the region. Several studies have shown that IBDV is stable in the environment and capable of surviving for extended periods in contaminated farms 33 . The warm, humid coastal environment of the Tanga region may also favour the survival and spread of viruses. This observation warrants further investigation to elucidate the role of these climatic factors in virus survival and persistence.
The relatively higher overall seroprevalence rate of IBD virus antibodies in indigenous chickens may be attributed to a number of factors. The management system in traditional poultry production may favour widespread infection. Poor sanitary conditions, continuous exposure of chickens to range conditions and wild birds, nutritional deficiencies, the absence of vaccination in traditionally managed chickens, and contact of chickens of 1 village with those in other villages may facilitate the spread of IBDV. This is in agreement with a previous report 27 . The ease of contact at local open-air markets between chickens from different areas, which are then taken back to various localities, can undoubtedly facilitate the rapid spread and persistence of IBD among indigenous chickens.
The prevalence of IBDV antibody in birds reared under a free-range village management system in this study agrees with reports from other countries with similar chicken husbandry systems 5,25 . The overall individual bird-level seroprevalence of IBD in free-range village chickens in this study was higher than the reports of 30.7 % 17 and 30 % 23 in Sudan and Botswana respectively. Lower prevalence rates were also reported in indigenous village chickens in Cameroon (33.9 %) 5 and in backyard chickens in Zimbabwe (55 %) 15 . Similarly, the mean seroprevalence obtained was comparable to the reported prevalence of 60 % in village chickens in Sahel zone of Nigeria 8 . Similar studies revealed antibodies to IBDV to be distributed in poultry worldwide 7,22 . The positive samples found in unvaccinated flocks indicated that IBD virus was circulating in those farms. Source, housing system, age and sex had no significant effect on IBD seroprevalence. This finding agrees with reports from other areas of the country and countries with similar back yard chicken production systems 15,18,32 . In contrast to our finding, significant effects of age (chicks) were reported by authors who detected higher seroprevalence in semi-scavenging reared chickens in Bangladesh. 3 This study indicated that chickens infested with ectoparasites (lice and ticks) were all associated with a lower probability of infection with IBDV. It is not clear, however, why the detected probability of infection was low, considering the number and level of ectoparasite infestation recorded during our survey. This observation may warrant further investigation.
Poultry diseases such as IBD were shown to be the most important constraints for commercial and local chicken production in rural settings of Tanzania 12,18 . Vaccination of village chickens against diseases such as IBDV is rarely undertaken, therefore the antibodies detected are most likely due to natural infection 8 . It is therefore recommended that IBD antibodies in other domestic birds such as ducks and turkeys be investigated. Preventive measures such as regular vaccination of young chicks and parent stocks should be instituted. Consistently, good husbandry practices such as good housing should be encouraged as stress is a major predisposing factor of the disease. It is vitally important that further detailed studies focus on the seasonality of IBD virus infection and strain identification so that preventive and control programmes can be designed.