Description of Study Area
The study was conducted from February to April, 2020 in two districts namely (Asayita and Mille), which are located in the administrative zone one of afar region, Ethiopia. The afar pastoral region is located in northeast of Ethiopia between 39°34’ to 42°28’E longitude and 8°49’ to 14° 30’ N latitude (Figure 1). The region shares common international boundaries with Eritrea in the northeast and Djibouti in the east and it is characterized by an arid and semi-arid climate with low and erratic rainfall. Rainfall is bi-modal throughout the region, with a mean annual rainfall below 500 mm in the semi-arid western escarpments and decreasing to 150 mm in the arid zones to the east. The altitude of the Region ranges from 120 m below sea level in Danakil depression to 1500 m above sea level. Temperatures vary from 20°C in higher elevations to 48°C in lower elevations. The human population of Afar region is 1.5 million in which the majority are pastoralists who largely depend on livestock production for their livelihood. The study populations were managed under pastoral husbandry which allows high mobility of animals and these animals are usually mixed with other animal species. Sheep and goats that were kept under the extensive farming system [1].
Study Population
The study populations were sheep and goats aged greater than 6 months were considered for estimation of seroprevalence of PPR virus infection in the study areas. Blood samples were collected from non-vaccinated sheep and goats and age group of the study population was classified as young (6 months to 1 year); Adult (1 year ≤ 1.5years) and old age (>1.5 years) [26].
Study design and sampling strategy
A cross-sectional study design was employed to estimate the seroprevalence of PPR and to assess associated risk factors from study districts of Afar region as of February to April, 2020. There is no serological test available to differentiate animals vaccinated with PPR vaccine from animals that had recovered from a natural PPR in Ethiopia. Therefore, questionnaire was deemed to gather the best source of information regarding vaccination status of sheep and goats to aid in sampling. The sampling method was three stage random sampling to collect the sample since the study districts were purposively selected based on higher study population, access to transportation, history of no vaccination for the last six months, absence of outbreak cases and willingness of pastoralists to participate in this research work. Then, the first stage was pastoral association, the second stage was the herd and the third stage was animals (target population). Finally, simple random sampling technique was employed to select individual animals during sample collection.
Sample size determination
Although sheep and goats are two species, they can be considered as one study population due to the management practices and the similar course of the diseases in both species. So, the sample size was determined according to the formula given by [27], using 50% expected prevalence (since there is no previous seroprevalence report of PPR infection in the study areas), 5% desired absolute precision and 95% confidence interval as below:
Where: n = required sampling units
Z = Multiplier from normal distribution at 95% Confidence interval (1.96)
Pexp = Estimated (expected) prevalence 50% (0.5)
(1-P) = Probability of having no disease 50% (0.5)
D = Desired absolute precision 5% (0.05)
Sampling was proportionally distributed based on the total small ruminants’ population in the study districts’ kebeles. The number of sheep and goats sampled is proportional to the herd sizes as well as within the district. Accordingly, a total of 384 sheep and goats from twenty four herds and eight kebeles or peasant associations and two districts were included in this study.
Sample Collection and transportation
Whole blood samples approximately 6-8ml was collected from the jugular vein of non-vaccinated sheep and goats using plain 10 ml vacutainer tubes and 19 gauge sterile needles. The samples were labelled to allow identification of each animal. The associated risk factors (such as species, age, sex, herd size, body condition score and study areas) were recorded during sampling. Collected samples were kept in slant position overnight at room temperature to allow serum separation. Then, serum was decanted and aliquoted into cryovials and stored in a freezer (-20°C) at microbiology laboratory of Samara University, and transported to National Veterinary Institute (NVI) in order to detect for antibodies against natural PPR infection exposure using serological analysis. All sera samples were transported to NVI laboratory in icebox and stored at -20°C until processed.
Serological analysis
Antibody detection against PPR Infection
Serum samples were analyzed at the National veterinary Institute (NVI, Debre Zeit, Ethiopia) using a competitive ELISA kit (c-ELISA kit) according to the instructions of the manufacturer (Institute for Animal Health, Pirbright Laboratory, UK) [28] . The ELISA micro-plates were read with an immunosbent reader with an inference filter of 492 nm. The reader was connected to a computer loaded with ELISA Data Information (EDI) software, which is used to automate the reading and calculation of the competitive percentage values (S/N%). The samples result with competitive percentage (S/N%) ≤50% was considered as positive. (Sample/Negative control×100) S/N%=OD sample/ODNc×100. Then, according to this calculation samples presenting a S/N%:
- Less than or equal to 50% are considered positive result.
- Less than and greater or equal to 60% is doubtful and
- Greater than or equal to 60% are considered as negative result.
Administration of Questionnaire Survey
A structured questionnaire format was prepared to interview individual sheep and goat owners. Respondents from each district were randomly selected and interviewed to assess associated factors of PPR disease such as; species, sex, age, herd size and study areas. All necessary epidemiological information was collected on individual animal bases.
Data Management and StatisticalAnalysis
All collected data generated from field and laboratory analysis was entered in to the Microsoft excel sheet data management and analysis Window® 2007 and then it was analyzed using Stata version 14 software. Descriptive statistics was employed to quantify the results of seroprevalence of PPR antibodies. The seroprevalence of PPR virus infections was calculated as the number of PPR positive animals divide to the total population at risk of acquiring the disease [29] . The association of associated factors such as different location, species, sex, herd size and age to the results of seroprevalence of PPR infection was analyzed using Univariable and multivariable logistic regression model. A statistically significant association between variables was said to exist if the calculated P-value is less than 0.05 at 95% confidence interval (CI).