Description of the Study Area
The current study was conducted in four selected areas of central part of Oromia, Ethiopia (Bishoftu, Sebata, Holeta and Addis Ababa) from November 2018 to May 2019 (Figure 1). Fecal samples were collected from four selected of dairy farms. There are many small, medium and large-scale dairy farms in four selected area that supply milk and milk products to consumers of the town and surrounding urban areas. These dairy farms contain either local or exotic breeds depending on the scale of production.
Bishoftu town is found in east Shewa Zone, Oromia Regional State, located about 45 km South-east of the capital city, Addis Ababa. The area is located at 9°N latitude and 40°E longitude at altitude of 1850 m.above sea level. According national meterology agence (NMA) (2016), annual rain fall of 866 mm of which 84% is in the long rainy season (June to September) with annual minimum and maximum temperature of 11and 29°C, respectively. The domestic animals reared in Bishoftu town are 30887cattle, 43138 poultry, 9322 equine, 9294 sheep and 4753 goats (Bishoftu City Administration Agricultural Desk, 2014).
Sebeta town is located in the Oromia Special Zone Surrounding Finfinne (Addis Ababa) of Oromia Region. The district is located 25 km south west of Addis Ababa at an altitude of 1800-3385 m above sea level and at latitude and longitude of 8°55-8.917°N and 38°37-38.617°E respectively. It receives an average annual rainfall of 1073 ml and has temperature that ranges from 11.3-280C. It has a total area of 102,758 km (SHFDO, 2018). According to the information obtained from Sebeta Hawas district Administration Office (2018), both livestock rearing and crop production are the main economic activities of the majority of communities. The major livestock reared in the district include cattle, sheep, goats and poultry.
Holeta Town is located in the central part of the country, 31 km west of Addis Ababa in Oromia Regional state, west Shewa Administrative Region. The area is bounded between latitude 8o 53’ 75” to 9o 14’ North and longitude 38o 21’ 40” to 38o 36’ 14” East. The Town has an area of 5550 hectares. Holeta Town found at an average 2449 m above sea level. The annual mean maximum and the minimum temperatures are 25.9 and 7.20C, respectively (HTRADO, 2009).
Addis Ababa, the capital city of Ethiopia, lies at an elevation of 2300 m above sea level and is featured by a grassland biome. It is geographically located at 9°1′48″N latitude and 38°44′24″E longitude. It has a typical highland climate with temperature ranging from 11°C-24 °C. Addis Ababa has a mean annual rainfall of 1300 mm with bimodal distribution (NMA, 2016).
The study was conducted in both apparently healthy cow calves and calves having clinical sign of diarrhea namely profuse watery diarrhea, systemic dehydration and depressed during investigation. Cow calves up to 30 days of age groups, all breed and sex reared under intensive management conditions were included in the study. Diarrhea was considered if feces are semi-liquid to liquid, with or without other abnormal characteristics such as presence of blood or mucous. Any calf with feces without these characteristics was considered non-diarrheic or apparently healthy (Ammar et al., 2014).
A cross-sectional study was conducted in different dairy farms found in four selected site of (Bishuftu, Sebeta, Holeta and Addis Ababa) central part of Oromia, Ethiopia from November 2018 up to May 2019. Information about the calves was gathered by interviewing farm owners and animal health workers of selected study sites. The information collected were recorded on data collection sheet, and then calves were clinically examined for presence of diarrhea or not and fecal samples were collected for diagnostic testing as follows. At the time of sampling the name of the farm, date of sampling, consistency of feces, age, breed, and tag number was recorded for each calf on proper recording format.
Sampling Technique and Sample Size Determination
Before the commencement of the actual study, preliminary data were sourced from the respective District Agricultural Office and dairy cooperatives to document the lists of dairy farm in to large scale, medium scale, and small scale dairy farm to estimate the size of study population. The Study areas were purposively selected and identified based on transport accessibility, geographical location and on the abundance of dairy farms to get more calves. Clinically diseased and non-diseased calves were sampled for isolation and characterization of rotavirus. The calves from the seven large scale dairy farms namely; Genesis farm, Asterwaqu dairy farm, Mama dairy Farm, Sisay dimma dairy farm, Haddish dairy Farm, Fantu dairy farm, Holeta agricultural research center dairy farm and a representative random sample of calves from 680 medium and small scale dairy farm were selected for the study. The sampling units were both local and crossbred dairy calves aged between birth and 1 month. Farms were categorized in to small, medium and large based on the herd size of (5-20), (21-50), and greater than 50 heads of cattle, respectively. In larger farms, a minimum of 10% of the all calves in the farm were sampled.
Considering individual members of dairy cooperatives in each study location as a cluster, cluster sampling method was used to select calves from medium and small scale dairy farm. In this study, sampling frame for study herds was taken from the dairy cooperatives located in Bishuftu, Sebeta, Addis Ababa and Holeta. A total of 680 medium and small scale dairy producers were registered in the dairy cooperatives of study areas. Accordingly, 170 dairy producer were sampled by using systematic random sampling technique (every 4nd dairy producer) from the documented sampling frame. When a selected dairy farmer did not have calf or no pregnant cows with due calving date in the six month cohort period, it was then replaced by another dairy farmer mostly from the nearby area. Sample size for cluster sampling was determined by adjusting the sample size calculated for simple random sampling. The adjustment is the function of average cluster size and intraculster correlation, and mathematically expressed as follows;
n’ = n[(1+ (( m -1)*ρ)]
Where; n’ = sample size for cluster sampling
n = sample size calculated for simple random sampling
m = average cluster size
ρ = intracluster correlation
However, in the present study the average herd (cluster) size (calves per each dairy farm) was 1.6. As clustering was found small, the effect of intracluster correlation would be small and n’ would approximate n. So the sample size calculated for random sampling was taken directly to be the sample size for this study. To estimate the prevalence of bovine rotavirus in calves, sample size was determined by using simple random sampling method (Martin et al., 1987; Thrusfield and Christley, 2018).
p= Expected prevalence
n= 1.962(p) (1-p) d= Desired level of precision (5%)
d2 n= Sample size
Using expected rotavirus prevalence 16.7% in central Ethiopia (Abraham et al., 1992), confidence level of 95% and required absolute precision of 5%; a total of 214 sample size was determined for medium and small scale dairy farm of selected study area. However, a total of 245 calves were enrolled during the study period to enhance precision and to compare prevalence across different herd sizes. Of which, 214 calves were from 680 medium and small scale dairy farm and 31 calves from seven large scale dairy farms.
Collection of fecal samples
Fecal samples were collected in sterile tube after cleaning of the anal area with a paper towel and beats by rectal stimulation with the index finger using disposable sterile plastic gloves (Ammar et al., 2014). Approximately 30 grams of fecal material was collected directly from the rectum of calves using disposable latex glove. Collected samples were placed into universal ice box containing ice packs and transported to the virology laboratory at National Animal Health Diagnostic and Investigation Center (NAHDIC), Sebeta and were stored at -80 °C until processing.
Fecal Sample Processing
Two hundred forty five fecal specimens obtained from calves with diarrheic and non-diarrheic were submitted to the National Animal Health Diagnostic and Investigation Center (NAHDIC), Sebeta, from November 2018 to May 2019. Fecal samples were prepared as a 10% (wt/vol) suspension of feces in 0.01 M phosphate buffered saline (PBS; pH 7). All samples were centrifuged at 1,500 x g, and the supernatants were tested and then stored in sterile vials at -80°C for further study.
Detection of bovine coronavirus and bovine rotavirus antigen by ELISA
Multiscreen Ag ELISA Calf digestive (BIO K 314/1, Belgium) is the type a sandwich ELISA capturing mixture of monoclonal antibodies (MAbs) against bovine coronavirus (BCoV) and bovine rotavirus (BRoV) was used to detect BCoV and BRoV Ag in the fecal suspensions. These antibodies capture the corresponding Ag in the fecal samples. The sandwich ELISA procedure was performed according to the manufacturer instruction (Kit reference BIO K 314/1, Belgium).
The ELISA was performed to detect coronavirus and rotavirus Ag in the fecal samples. The 96 well plates provided in the kit contained two different capture antibodies. Rows C, E, D, F, H and G were coated with coronavirus and rotavirus specific capture antibodies and rows A and B coated with non-specific antibodies, which acted as controls (positive and negative control). These control rows allow the differentiation between specific immunological reaction and non-specific bindings so as to eliminate false positives.
Feces were diluted in the dilution buffer provided in the kit. A volume of 100µl of diluted sample was added to corresponding wells of specific and non-specific antibody coated rows, respectively. Similarly both the positive and negative controls were added to their respective well per plate. The plate was incubated at 25°C for 0.5 hour and washed 3 times with washing solution (diluted in the ratio 1:20 with distilled water) provided in the kit. Ready to use conjugate of coronavirus and rotavirus specific monoclonal antibody labelled peroxidase was used as such and poured in 100µl quantities per well. The plates were covered with a lid and incubated at 25°C for 0.5 hour in a dark room and washed three times with the provided washing buffer. Then 100 µl of the chromogen (tetramehtyl benzidine) solution added to each well on the plate. The plates were then incubated for 10 minutes at 25°C without covering and away from direct light.
Finally the reaction was stopped by adding stop solution (1M phosphoric acid) provided in the kit. The optical density was measured at 450nm after stopping the reaction with 50µl per well of stop solution. The optical density was measured at 450 nm using an ELISA plate reader at 450nm immediately after stopping the reaction with the stop solution. The test was validated using the positive control and data sheet provided by the kit. The net optical density of each sample was calculated by subtracting the reading for each sample well from corresponding negative control. Net optical density (O.D.) = (O.D. of specific binding -O.D. of non-specific binding). The ELISA reader was used to transfer optical density values to excel spread sheet of computer connected to the reader. Even positive and negative reaction results in ELISA was decided based on color changes after adding stop solution as well as calculating the optical density value. Blue color changed to yellow after adding stop solution was recognized as positive and optical density value was > 0.15 Elisa units (EU) for positive and < 0.15 Elisa units (EU) for negative to both bovine coronavirus and rotavirus.
Extraction of bovine coronavirus and rotavirus RNA
Coronavirus and Rotavirus RNA were extracted from the fecal suspension using QIAamp viral RNA mini kit (Qiagen, Crawley, West Sussex, UK) following the manufacturer’s instructions. About 1g of fecal sample was added to 1ml of phosphate buffer saline (PBS). The mixture was vortexed vigorously for 40 seconds followed by centrifugation at 10,000 rpm for 5 minutes. All the supernatant (about 500µl) was transferred to new tubes.
Briefly, 140 µl of original fecal supernatant was added in to 560 µl buffer AVL-carrier RNA in the microcentrifuge tube, vortexed for 15 seconds to ensured efficient lysis and homogeneous solution, then the incubated at room temperature (15-25°C) for 10 minutes to lysis viral particle. The solution was centrifuged to remove drops from the inside of the lid then 560 µl ethanol (96%) was added in to the sample, mixed by vortexing for 15 seconds and again centrifuged the tube to remove drops from inside the lid. Then 630 µl from the solution was taken and pipetted in to the QIAamp Mini column and centrifuged at 8000 rpm for 1 minutes and the filtered was discarded. This action was repeated twice.
Then 500 µl buffer AW1 was added to QIAamp Mini column and centrifuged at 8000 rpm for 1 min. The filtrate was discarded and the column was placed in a fresh 2ml collection tube .Then 500μl of buffer AW2 were added to the column then centrifuged at 14,000 rpm for 3 min and the filtrate was discarded. Carefully the QIAamp mini column was opened and added 60 µl Buffer AVE equilibrated to room temperature. Then 65μl of Buffer AVE was added to the column, equilibrated at room temperature for 1 minute then centrifuged at 8000 rpm for 1 minute. Finally, the nucleic acid of the virus was obtained. A double elution using 2 x 40 µl Buffer AVE was performed to increased yield. Final extracted viral RNA was stored in -800C for further processing.
All ELISA positive fecal samples were taken forward for virus isolation. Approximately 1 gram fecal sample was mixed with 9 ml sterile PBS containing antibiotic. The fecal suspension was then centrifuged at 800 rpm for 15 minutes. The supernatant fluids contain coronavirus and rotavirus positive were filtered through 0.45 µm membrane syringe filter and filtrates were mixed with an equal volume of Dulbecco's Modified Eagle Medium (DMEM) containing 5% fetal calf serum (FCS) and 10 µg/ml crystalline trypsin and incubated at 37°C for 60 minutes. After incubation, one ml of the mixture was inoculated into the culture flasks with confluent monolayer of Madin Darby bovine kidney (MDBK) cell lines and kept for 1hour incubation to adsorption virus. After the adsorption at 37°C for 1 hour, the cells were washed three time with plain of DMEM maintenance media and incubated at 37°C in a humidified incubator having 5% CO2. Monolayers were observed daily for development CPE for five days and viruses were sub-cultured blindly every two days after being subjected to 3 cycle of freezing and thawing. CPE was observed after 48 hours in positive case and it was characterized by a destruction of the monolayer cell, cell rounding and infected cells were disrupted and detached from the flask. Cells showing characteristic CPE were harvested by freezing and thawing thrice and centrifuged at 16,000 rpm for 20 minutes at 4°C for the removal of cell debris. The supernatant containing the virus was collected and stored at -80°C for further passages. If no CPE was observed, the sample was considered as ‘no virus detected’ (NVD) and the culture was frozen at -80°C, then thawed and centrifuged at 3,000 rpm for 10 minutes to collect supernatant for second blind passage (P2). This was repeated for third passage (P3); and if no CPE was observed on the third passage after 48 hours inoculation, then the sample was considered negative for both coronavirus and rotavirus.
Reverse transcription polymerase chain reaction (RT- PCR)
The cDNA synthesis were performed with a RT-PCR Kit (QIAGEN), according to manufacturer's instructions, for the confirmation of bovine coronavirus and rotavirus A and random primers in a 25 uL final reaction volume. The cDNA of each sample was screened separately for the BCV and BRV genome using the primers described in Table 1 based on the previous study (Park et al., 2006; Tsunemitsu et al., 1999). PCR reactions were performed according to manufacturer instructions.
Optimized reaction mixture for RT-PCR was dsRNA 2.5 µl, PCR buffer 2.5 µl, dNTPs 2.5 µl, MgC2 2.5 µl, Forward Primer (10 pmol) 3.0 µl, Reverse Primer 10µM (6µl), DNase/RNase free water 6 µl. 2.5µl of viral dsRNA were denatured at 95°C for 5 minutes and chilled immediately for 5 minutes. Then, reaction was carried out under the following conditions. RT-PCR was carried out with an initial reverse transcription step of 60 minutes at 420C, followed by PCR for activation at 940C for 15 minutes, 40 cycles of amplification (30 seconds at 940C, 45 seconds at 550C (annealing), and 45 seconds at 720C (extension)), with a final extension of 7 minutes at 720C. To analyze the PCR product, agarose gel electrophoresis was performed. For this, 1.5% gel was prepared and 1 µl of 100 base pair (bp) ladder along with the PCR product was run at 110 volts for 45 minutes. The size of the PCR product for gene segment was illuminated in a gel documentation system and a photograph was taken.
The collected data were entered in Microsoft Excel. The contingency table was used at 5% significance to assess the differences among the proportions of fecal samples positive to coronavirus and rotavirus variables such as age group and sex of the animals studied by using Chi Square. Quantitative data was coded and entered in a computer spread sheets and the R software was used for the data analysis.