Study area
The study was conducted in North, Western and South Kordofan States. Kordofan covers an area of 376,145 km² (146,932 miles²), with an estimated population of 3.6 million people in the 2000 Census (Figure 1). It is largely an undulating plain, with the Nuba Mountains in the southeast quarter. During the rainy season, the area is fertile, but in the dry season it is virtually desert (22).
Study population
The study focused on sheep herds as reference target population. The total population of sheep in Kordofan region is 2,759,124 heads, 30% are Hamari sheep and 70% other breeds of local Sudanese sheep (23).
Sample size determination
A cross-sectional study design was carried out from March 2011 to September 2011 to determine the sero-prevalence of sheep pox in Sudanese sheep flocks in Kordofan. A multistage sampling technique was carried out. Starting from the region and the 2 states, localities were purposively selected, then flocks within a locality were purposively selected and individual animals within the flocks were sampled.
The sample size was calculated for an expected prevalence of 63.5% in a locality, according to a previous study by Eshafi and Ali (21), using a 5% desired absolute precision and a 95% confidence level. Farmer’s permission to use their animals was clarified by communicating to local leaders before approaching farmers. A total of 1500 animals were sampled in the 6 localities in North and South Kordofan States, due to lack of stock of ELISA test at time of the study only 850 samples were evaluated by ELISA. While, 260 samples were shipped to National Centre for Foreign Animal Disease, Winnipeg, MB, Canada, and assessed using virus neutralization. Risk factors were identified as individual animal risk factors including: sex, age group, weight, species and breed; other potential risk factors were location, herd size, movement pattern, mixed herd, agri-ecologic zone, temperature and relative humidity.
Serum sample collection
Only non-vaccinated sheep flocks were used to collect blood. Blood samples were collected from sheep using plain 10 ml vacutainers and 19 gauge needles (Bectin Dickinson, UK). The collected blood was allowed to clot for up to 24 hours in the shade. Two aliquots of sera were transferred into cryovials labeled and were kept in a -20°C freezer and transferred to the laboratory in cooled containers with ice bags. Serological diagnosis was carried out on the collected specimens at the Veterinary Research Institute (VRI), Department of Virology, and Department of Viral Vaccine Production, Sudan and the National Center for Animal Disease (NCFAD) in Canada.
Virus neutralization test
Sheeppox virus specific antibodies were determined using a virus neutralization test (VNT). The assay was performed using negative and positive control sera as well as the test sera which were heat inactivated at 56°C for 30 minutes prior to performing the assay. Sera were diluted starting at 1:10 in DMEM and then serially diluted by a factor of 2. After dilution, the 125 µl of diluted sera was added to a 96 well microtitre plates containing 125 µl of 100 TCID50 per 100 µl of Kenya sheep pox virus. The plates were incubated at 37°C for 1 h. After this period media was removed from confluent OA3.Ts cells where obtained at Canadian Food Inspection Agency, National Centre For Foreign Animal Disease, Winnipeg, MB, Canada, which had been seeded 24 h previously on 96 well microtitre plates, 200 µl of the virus/sera mixture was added and incubation was continued for 6 days. Development of CPE was then scored using an inverted microscope. Neutralization was considered positive if greater than a 50% reduction in CPE was observed at a dilution of 1:10 or greater (24).
Capripox double antigen ELISA
Ovine samples were examined to detect antibodies against sheeppox using a capripox double antigen ELISA according to the manufacturer instructions (ID-VET, France).
Producer questionnaire
Data from North and South Kordofan States were collected by questionnaires filled out by farmers. Other information and secondary data for the questionnaire was taken from General Planning and Animal Resources Economics Administration, Federal Ministry of Animal Resources and Fisheries of the Sudan. The method of the questionnaire were used according to guideline and regulation of Federal Ministry of Animal Resources and Fisheries, Sudan and consent of respondent to participate in the current study. All experiments were carried out according to regulation and guidance of Central Veterinary Research Laboratory, (CVRL), Canadian Food Inspection Agency (CFIA), and Australian Animal Health Laboratory (AAHL). Also, information was obtained from respondents or and/ informants under 18 with their parents or and/guardian.
Statistical analysis
Collected data were organized and managed in a Microsoft Excel spreadsheet for analysis by the Statistical Package for Social Sciences (SPSS) at Central Veterinary Research Laboratory, Sudan. The results of statistical analysis was extrapolated for the sample size difference. The risk factors associated with sheeppox seropositivity, were analyzed using descriptive statistics and univariate analysis chi-square test.