Description of the Study Area
The study was conducted in southernand centralareas.The study areas are shown in Figure 1. From southern area,three different zones were sampled, with several districts in each. Specifically, in Wolayita Zone, the districts of Bolso Sore, Damote Sore, Damote Gale, and Soddo were studied. In South Omo Zone, the districts of Nyangatom, Hammer, Benatsemay, and Male were sampled. And in Borana Zone, the districts of Surupa, Arero, and Elowayu were sampled. All of these formed what is termed “southern area” in this paper. For the more central part of Ethiopia, West Shewa was sampled (Ambo district), and in East Shewa, four districts were sampled, including Adama, Lume, Batu, and Dugda. In addition,in the special zone of Oromia surrounding Finfine, the districts of Holeta, Sululta, and Sebeta were sampled. These are considered “central area”.
The study population included cattle, small ruminants and camels over six months of age. From the southern areas, all the target populations were managed under extensive pastoral systems. In Walaita and Central Ethiopia, study populations were primarily dairy, and all were managed under intensive production systems.
Sample Size Determination and Sampling Methodology
The sample size was calculated separately for bovine, small ruminants and camels based on the previous reports of seroprevalence for the species in the study areas according to the following formula:
n= 1.962*Pexp (1-Pexp)
In this formula, n equals required sample size,Pexpequals expected prevalence, and d equals desired absolute precision. The desired precision was 5%, which allows for a 95% confidence level. Accordingly, the sample size for cattle sampling for the nine districts of the Central regions was calculated as 153 for each district based on previous reports of 11.2% seroprevalence and for the Borana were calculated as 115 based on previous reports of 8.2%[13, 52]. As there were no published reports from intensive dairy farms from Wolaita zone, the sample size was calculated by considering the expected prevalence of 50% and hence the calculated sample size was 384. Small ruminant sample size was calculated based on previous reports of 8.1% and 4.2% for Borana (230) and SouthOmo (124), respectively [5, 50]. Sample size for camel was calculated as 47 based on a report of 3.1% seroprevalence.
Based on the above calculation, 2144 animals from all districts of the study areas were determined to be included into the study. However, sample size was increased approximately two-fold so as to increase precision and reduce standard error. Accordingly, in the present study a total of 4274 animals from all districts were selected to investigate brucellosis for this study purpose, as shown in Table 1.
Multistage sampling was used to get the required animal samples for the southern areas. Zones were selected purposively and districts (woreda) from the selected zones were randomly selected and in turn kebeles and villages were selected randomly from the districts. Accordingly, from each selected district four villages and from the selected villages, households/herd/flocks were selected by simple random sampling. All animals within the selected herds were sampled.Total number of samples required was distributed according to the animal population proportionally for each administrative category. The milk producing districts from the central part were selected purposively and the farms from selected districts (towns) were selected randomly and all cattle from the selected farms were sampled.
Blood Sample Collection
For serological and molecular analysis, blood samples were collected aseptically from the jugular vein of individual animals. Approximately 5-7ml of blood was collected from each study animal using new plain vacuum tubes and then the blood samples were kept overnight at room temperature to allow clotting. The separated sera were then carefully movedinto cryovials. Harvested sera and blood clots were transported to the National Animal Health Diagnostic and Investigation Center (NAHDIC) serology and molecular laboratory in icebox with ice packs. All were stored at -20°C in the laboratory until processing.
Bacteriological sample collection
Vaginal swab samples were collected using Stuart transport medium from animals that had a history of recent abortion. This included two cows and 11 small ruminants. Swabs were transported under cool conditions to the bacteriology laboratory of the National Animal Health Diagnostic and Investigation Center (NAHDIC) and stored at -20°C until processed.
Commercial brucellosis serum indirect multi-species ELISA Kit (BRUS-MS-5P ID Screen Brucellosis Serum Indirect, Multispecies, lot number C35) was used to detect antibodies directed against B. abortus,B. melitensisand B. suisfrom 4274 sera samples and performed as per manufacturer’s instructions. This commercial test is not yet validated for use in camels, and currently there are no serologic tests fully validated for use in camels (48). However, the OIE supports the ELISA for screening of flocks/herds and individual animals in all livestock species, including camels . Optical density was measured at 450nm. The kit was verified as per kit instructions and the positive cut-off point was calculated as:
Sample positivity percentage (S/p%) = ODsample-ODNTC X 100
Accordingly, samples with a s/p% less than or equal to110% were considered negative, greater than 110% and less than 120 % were considered doubtful, and greater than or equal to 120% were considered positive.
Media preparation and culturing: Brucella selective media was prepared by suspending the required amount of brucella medium base (CONDA Cat. 1374, Spain), in sterile 5% V/V inactivated horse serum (i.e., horse serum held at 56°C for 30 minutes). Rehydrated contents of Brucella selective supplement (SR083A) were aseptically added to the sterilized brucella basal medium and homogenized before plating and then 15 to 20 ml of the medium was poured into the Petri dish and allowed to solidify . The plates were incubated at 37oC for 48 hrs for sterility check up and no bacterial colony growths were considered as sterile and used for culture.
Thirteen vaginal swab samples were streaked directly from Stuart transport medium to the plate under Biosafety Level three (BSL3) facilities with proper personal protections. Inoculated plates were incubated at 37oC aerobically. Duplicate samples were also incubated in the presence of 5%CO2 (using anaerobic candle jar) for up to two weeks. The colonies were checked every 24 hrs for Brucellaspecies growth. Brucella-suspected colonies were characterized by their typical round, glistening, pinpoint and honey drop-like appearance according to standard methods .
Microscopic examination: Brucella suspected colonies were selected using a sterile plastic loop and mixed with a drop of sterile distilled water and smeared on a clean glass slide. The smear was heat fixed on the slide and air-dried and identification of the organism was done by gram staining technique and Modified Ziehl-Neelsen staining technique .
Biochemical test: Subsequent biochemical tests, including urea testing and lack of growth on MacConkey agar were also done.
Genomic DNA extraction from blood clots of seropositive animals: Following the result of serological test using ELISA kit, 169 blood clot samples from the seropositive animals were used for detection of Brucella nucleic acid using real time PCR. Genomic DNA was extracted using QIAMP DNA Mini Kit (QIAGEN GmbH strasse 1.40724 Hilden GERMANY) as per manufacturer’s instructions.
Genomic DNA extraction from culture:DNA was extracted from solid media colonies by simple boiling method as described . Few colonies were removed and suspended in 500μl of sterile double distilled water in a 1.5ml micro-centrifuge tube and kept in a boiling water bath for 10 minutes. Five microliters of the supernatant were used for the PCR after centrifugation at 12000g for 3 minutes and the rest of the DNA sample was stored at -20oC.
NanodropDNA examination:The extracted DNA was checked using Nanodrop spectrophotometer (THERMO, USA), which checks and measures the purity of DNA by reading the absorbance at (260/280 nm) and ng/μl concentration was calculated before PCR was performed using real time PCR.
Real-Time PCR: Real Time PCR was performed for detection of Brucella spp. DNA from blood clot and culture samples by using the specific primers and TaqMan probe for IS711, B.abortus and B.melitensis sequence of forward and reverse as described in Table 2.
The thermocycler was run 95oC for 10minutes to denature double-stranded DNA, then amplification/extension occurred at 95oC for 15 second and 60oC for 1 minute for final extension. This process adjusted to run for 45 cycles. Finally, Brucella species was detected using species specific primers of B. abortus and B. melitensis. When the cycle threshold (CT) value of the samples were<45, it was considered and evaluated as positive. If greater than 45, it was considered as negative.