Genetic and antigenic analysis of Chlamydia pecorum strains isolated from calves with diarrhea

OHTANI, Akifumi; KUBO, Masahito; SHIMODA, Hiroshi; OHYA, Kenji; IRIBE, Tadashi; OHISHI, Daiki; ENDOH, Daiji; OMATSU, Tsutomu; MIZUTANI, Tetsuya; FUKUSHI, Hideto; MAEDA, Ken

Isolation of microorganisms: Tissue specimens obtained from major organs including cerebrum, jejunum, ileum, cecum, colon and ideal contents of the dead calf (case 1) and feces of the second calf (case 2) were minced and homogenized in serum-free Eagle's minimum essential medium with kanamycin (MEM) (Nissui Pharmaceutical Co., Tokyo, Japan). After centrifugation of the 10% homogenate, the supernatant was filtered through a 450 nm membrane (Merck Millipore Ltd., Carrigtwohill, Ireland), and 0.15 ml of the supernatants were inoculated onto hamster lung (HmLu-1) cells, Mardin-Darby bovine kidney (MDBK) cells, bovine testicular (BT) cells, human rectal adenocarcinoma (HRT-18) cells and Vero cells in 24-well plates. After adsorption for 60 min at 37°C, the cells were washed with MEM, and then, 0.5 ml of MEM containing 0.1% bovine serum albumin (Bovogen Biologicals, Williams Avenue, Australia) was added to each well. The cells were incubated at 37°C, and cytopathic effects (CPE) were observed. After incubation for 10 days, cells were frozen and thawed once and then centrifuged. Subsequent passages were carried out at least twice in the same manner with 0.15 ml of the supernatant. Gimenez stain was used to identify CPE. DHL Agar, Columbia agar with 5% sheep blood and GAM agar with 5% yolk and 0.1% cysteine were used for bacterial isolation. The gene of Clostridium perfringens toxin isolated from small-intestinal contents was identified by PCR [2] .