2.5 Samples collection and antimicrobial screening
Urine samples were aseptically collected from at least a cattle that voided urine during the visits to the farms. Collections were done with sterile sample collection bottles and transported to the laboratory in cool icebox at 4 oC. Approximately, each urine sample collected weighted 4 ml.
Disc Diffusion Test was used for the screening. It was performed using Mueller-Hinton Agar (MHA), a medium for susceptibility tests. It has low sulfonamide, trimethoprim, and tetracycline inhibitors, and good reproducibility with satisfactory growth of most pathogens (www.eucast.org). Lyophilised cultures ofBacillus subtilis and MHA (Merck, Darmstadt, Germany) were used. Pour plates of MHA were prepared and 1 ml of B. subtilis broth culture was inoculated into each agar plate. A 1 cm diameter sterile Whatman filter paper discs were dipped into each urine sample for 15 min. It was then taken out and excess fluid removed by expressing against inner side of the sample container, and placed on the MHA plate. The plate was incubated at 37 °C for 24 hours. Thereafter, it was examined for inhibition zone of B. subtilis growth around the discs in the plate. Antimicrobial susceptibility was assessed by measuring the diameter of inhibition zones around the paper disks with scientific ruler. An inhibition zone ≥ 2mm was considered positive, while that less than 2 mm was considered negative (Shahbazi et al., 2015).