Leptospira spp. culturing
Kidney samples from 20 euthanized brown rats were collected aseptically for Leptospira culturing. Isolation of leptospires was performed as previously described (Faine et al., 1999). Briefly, samples were macerated and diluted 1:10 in phosphate-buffered saline (PBS, pH 7.4; NaCl 0.137 M; KCl 0.0027 M; Na2HPO4 0.01 M; KH2PO4 0.0018 M/L), then 1 mL of the diluted samples was seeded into 9 mL of liquid EMJH medium enriched with a selective supplement (Nalidixic acid, 50 mg/L; Cycloheximide, 100 mg/L; Chloramphenicol, 5 mg/L and Neomycin, 5 mg/L). Cultures were incubated at 30 ºC in for a period of 12 to 24 h and then seeded in the semi-solid EMJH medium without the selective supplement. The tubes were incubated at 28 °C for 12 weeks and examined weekly by dark-field microscopy to evaluate the presence of spirochetes.