Description of the study area
This study was conducted in Amibara district of Gabirasu zone (Zone 3 of Afar region) located in the Middle Awash Valley of Ethiopia (Figure 1). Amibara district is about 270km to the North East of Addis Ababa and has 19 kebeles with total population of ~63,378, of which 35,374 were men and 28,004 women. The altitude of Amibara district is 740m above sea level. Fourteen years climatic data on monthly basis showed that the average maximum and minimum temperature of the area is 34°C and 19°C, respectively, and its annual total rainfall is about 571 mm (Chekol and Mnalku 2012). The livestock population of the Amibara district is composed of 103, 959 cattle, 122, 526 goats, 48,043 sheep, 3,888 donkeys and 39,995 camels (CSA 2007).
Study Population
In the present study the target study population were cattle owned by pastoralists. Only indigenous local breed of cattle with no history of vaccination and older than six months of age were recruited into the study. During sampling, cattle’s were classified into three age groups (<2 years, 2-5years and >5 years) as young ,adult and old respectively (Ibrahim et al. 2011).
Study Design and sampling techniques
A cross-sectional study design was employed from October 2019 to May 2020 in order to determine seroprevalence and associated risk factors of cattle brucellosis. Study kebeles were selected by simple random sampling technique. To select cattle herds in the proposed kebeles, purposive sampling technique was employed base accessibility and willingness of the herd owners to cooperate. Then each herd were stratified into subgroup based on age and sex to ensure equal representation of all subgroup. From each subgroup, individual animals were selected by systematic sampling technique and information related to environmental and study animals were also accessed. The sample size for serological study was calculated by Thrusfield formula using previous study result by(Asgedom et al. 2016) who reported 2.4% in Alage district . However, in order to increase precision and reduce standard error, the minimum sample size obtained by calculation was increased by four-fold and 181 cattle were sampled.
Blood sample collection
After disinfecting the site of jugular vein, about 8ml of blood sample was collected into sterile plain vacutainer tube and labeled with code describing each study animal. Then; the samples were taken to Werer Agricultural Research center, animal health research laboratory and kept overnight at room temperature to separate the serum and the clotted red blood cells according to (OIE 2018). Then the serum was gently decanted into sterile cryovials (1.8ml), labeled and stored at -200c
Laboratory Diagnosis
A) Rose Bengal Plate Test (RBPT)
All serum samples were screened using RBPT at National Veterinary Institute according to the procedure described by the World Organization for Animal Health (OIE 2018). In the laboratory, serum samples were kept at room temperature for 30 minutes and then, screened for anti-Brucella antibodies using commercial kits of the standard Rose Bengal Plate Test (RBPT). B. abortus antigens (Lillidale Diagnostics, Holt wimborne, Dorset, BH21 7DG, United Kingdom) and their positive and negative control sera were used. The detailed procedures for the RBPT were that, 30μl of cattle serum and 30μl of antigen was mixed on a test plate and rocked for 4 minutes. After four minutes of rocking, visible agglutination was considered as positive. Agglutinations were recorded as 0, +, ++ and +++, according to the degree of agglutination (Nielsen 2002). A score of 0 indicates the absence of agglutination; + indicates barely visible agglutination; ++ indicates fine agglutination, and +++ indicates coarse clumping. The presence of agglutination at any degree was considered as positive reaction while the absence of agglutination was considered as negative.
B) Complement Fixation Test (CFT)
Serum samples that reacted positively to RBPT were further retested by CFT for confirmation using standard B. abortus antigen S99 (New Haw, Addlestone, Surrey, KTI5, 3NB-UK). Preparation of the reagent is evaluated by titration and was performed according to protocols recommended by World Organization for Animal Health(OIE 2018). Sera with strong reaction, more than 75% fixation of complement (3+) at a dilution of 1:5 or at least with 50% fixation of complement (2+) at a dilution of 1:10 and above was considered as positive and lack of fixation/complete hemolysis was considered as negative result. Only samples that gave signals for both RBPT and CFT were considered positive since no single test is appropriate in all epidemiological situations due to problems of sensitivity and or specificity of the tests as recommended by OIE and other reports(Tumwine et al. 2015).
Data Analysis
Risk factors and serological results were recorded into microsoft Excel® Spread Sheet and analysis was done using R-Software version 4.0.3. Prevalence was calculated by dividing the number of positive animals to the total number of animals tested. Fisher’s exact test was used to calculate associations of risk factors with brucella seropositivity. Since the number of outcomes of interest is less than 10%, firth’s bias reduced logistic regression model was used to measure the association of potential risk factors with brucella seropositivity (Puhr et al. 2017). Odds ratio (OR) was used to indicate the relationship between risk factors with animal level seroprevalence of brucellosis. P-value less than 0.05 were considered statistically significant in all analysis.
Ethical consideration
Ethical approval for collection and analysis of animal materials was offered by animal research ethical review committee of the College of Veterinary Medicine and Agriculture (CVMA) with certificate ref. number of VM/ERC/03/01/12/2020.