The study was carried out in the towns of Weliso in the South-West Shewa Zone, and Ambo and Ejaji in theWest Shewa Zone, Oromia, Ethiopia (Figure 1). Weliso district is one of the 14 districts found in the South-West Shewa Zone. Weliso town is an administrative town of Weliso district, and the South-West Shewa Zone, which is located 114 km southwest of Addis Ababa and the district has a longitude of 37° 57' 59.99'' E and latitude of 8°31' 59.99'' N with an elevation of 2063 meters above sea level (m.a.s.l). The annual rainfall and a temperature range from 950-2,718.3mm and 13.6°C-25°C respectively.
Ambo town is an administrative town of Ambo district, and the West Shewa Zone, which is located 115 km West of Addis Ababa and the town has a latitude of 8°59′N and longitude of 37°51′E and an elevation of 2101m.a.s.l. The annual rainfall and a temperature range from 800-1000mm and 15°C-29°C respectively. The mean temperature is 18.6°C. Whereas, Ejaji town is the administrative town of Elu Gelan district, which is located 90 km West of the zonal capital, Ambo, and 215 km West of Addis Ababa. Ejaji town has a longitude of 37° 9.8'E and a latitude of 8° 59.9'N and has an altitude ranging from 1565-1790 m.a.s.l. The annual rainfall and a temperature range from 2000-2300mm and 27°C and 30°C respectively.
The climatic conditions of the study areas vary from very hot (lowland) to mid-temperate (midland) and cold temperatures (highland).
The study population and study animals
The study population consists of dogs found in the study towns. Local, cross, and exotic breeds of dogs are found in the study towns. Nevertheless, local breeds are predominantly found in the study areas, followed by a cross and exotic breeds of dogs. Dogs are kept for house guard in most parts of urban areas in the country; however, in rural areas they are also used for protecting livestock from thieves in the evening and hunting. In this study, healthy dogs above six months of age, both sexes, and different kinds of breeds were included. A total population of 7,098 dogs is found in the current study towns. Of these, the highest population of dogs is found in Ejaji (2,816), followed by Ambo (2,180), and Weliso (2,102) towns. Dogs aged 6 months; 6 months to 1.5 years, 1.6 to 7 years, and above 7 years were considered as juvenile, adolescent, adult, and geriatrics respectively .
Study design and sampling methods
This cross-sectional study was carried out from October 2019 to September 2020. Dogs were brought to the selected places in the study areas arranged for sample collection. Dog owners were invited to the area through microphone announcement with the help of the kebele (the smallest formal administrative unit in a town) administration members. The study animals were selected by using a simple random sampling method. This consists of randomly drawing “n” individuals from “N” people in a sampling frame list. Dog owners were informed to handle their dogs properly up to the end of the sample collection process. Dogs that cannot get to the selected locations were sampled in their homes.
Inclusion and exclusion criteria
Agreement by the dog owner to participate in the study was an inclusion factor. Accessible dogs whose owners are willing for sampling and dogs above six months of age were included in the study while highly debilitated, sick dogs and those under treatment were excluded. Dogs known for their extreme aggressiveness were also excluded from the study.
Sample size determination
The desired sample size for the study was calculated by the formula given by Thrusfield assuming 50% expected seroprevalence of CLI in the study areas (since there was no previous study in the current study areas) and a 5% level of precision. The sample size (n) is determined by the formula:
Where, n sample size required, 1.96 = the value of Z at 95% confidence interval, Pexp= expected seroprevalence, d = desired absolute precision
Although, the calculated sample size was 384, due to kit shortage 368 dogs were tested (i.e., 146, 113, and 109 dogs from Ejaji, Ambo, and Weliso towns respectively).
Sampling procedure and techniques
Before blood collection, personal protective pieces of equipment like disposable gloves, gowns, and waste management materials were made ready and used properly. The dogs were restrained in portable dog crush, by rope, and dog catcher accordingly. Then, the site of blood collection (cephalic and lateral saphenous vein) was washed with soap and water, shaved with a sterile surgical blade, disinfected with 70% isopropyl alcohol, and tied by the tourniquet. A blood sample (5ml) was collected from each dog by intravenous cannulation with plain vacutainer tubes and needles.
Each tube was individually identified and the blood was allowed to clot and then centrifuged at 3000 revolutions per minute (rpm) for 5-10 min to obtain serum. Centrifugation took place in the field, i.e., during the evening in the town of each study area., The serum samples were then immediately transported using an icebox containing ice packs to the Ambo University Zoonosis and Food Safety Laboratory and stored at -20oC until serologically tested. Finally, the serum samples were transported using an icebox to Sebeta National Animal Health Diagnostic and Investigation Center (NAHDIC) for Indirect ELISA Serological examination.
All the collected serum samples were tested for the presence of rK39 antibodies against CLI following the protocol of the manufacturer of the indirect ELISA kit (IDvet Innovative Diagnostics, ID Screen®, Leishmania Indirect, France).
Data management and analysis
The raw field data obtained from sampled dogs and findings of laboratory examination were entered into Microsoft Excel 2019 Spreadsheet to create a database followed by refining and coding. The collected data were analyzed using the STATA version 14 software program (Stata Crop. College Station, USA). Descriptive statistics were used to summarize the data. Pearson’s Chi-square ( ) and logistic regression (univariable and multivariable) tests were used to evaluate the association between hypothesized potential risk factors like town, season, kebele, sex, age, breed, living environment, and presence of other domesticated animals in the households with CLI seropositivity. Additionally, the collinearity (confounding) of the risk factors was also analyzed and from collinear risk factors, only one independent variable with a strong biological association with the disease outcome was selected and used for the final logistic regression model. Those variables with P ≤ 0.25 in the univariable logistic regression analysis were further analyzed using multivariable logistic regression after checking for multicollinearity. In all the cases P< 0.05 was set for significance.
The study protocols were approved by the ethical review committee of the Ambo University, Mamo Mezemir Campus, School of Veterinary Medicine. Each study subject involved in the study gave oral consent to participate and only those individuals who showed their willingness were selected to participate in the study. All dogs were enrolled in this study with informed permission from their caretakers, and all dogs sampled were examined with the assistance and acceptance of their owners. Blood samples were collected by the researcher according to the good practices of veterinary medicine i.e., all animals were handled strictly by following the good animal handling practice to minimize animal sufferings during sampling.