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

Antigenic analysis of C. pecorum isolates: The antigenicity of isolates were compared by immunoblot analysis using rabbit antisera [6] to Bo/Yokohama [6] , Bo/Maeda [7] and Ov/IPA [6] strains of C. pecorum. Antisera to Bo/Maeda and Ov/IPA strains were prepared as described by Fukushi and Hirai [6] . HmLu-1 cells were infected with C. pecorum 22-58, Bo/Yokohama, Bo/Maeda and Ov/IPA strains and C. psittaci Cal10 [9] strain and then incubated at 37°C in 5% CO 2 until CPE was observed. The cells were recovered from dishes with 0.02% EDTA in phosphate-buffered saline (PBS). After centrifugation at 200 × g for 5 min at 4°C, the supernatant was removed, and the cells were resuspended in PBS. Cells were then mixed with an equal volume of 2 × concentrated sample buffer consisting of 6.25 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 20% glycerol and 0.001% bromophenol blue. Samples were boiled for 3 min, placed on ice for 3 min and centrifuged at 13,000 × g for 3 min at room temperature. Cell lysates were electrophoresed using 12% SDS-PAGE and transferred to a PVDF membrane (Immobilon-P; Millipore, Billerica, MA, U.S.A.). After blocking with Tris-buffered saline (TBS) containing 3% gelatin (EIA Grade Reagent Gelatin; Bio-Rad, CA, U.S.A.) for 45 min at 37°C, the membrane was washed three times with TBS containing 0.05% Tween 20 (T-TBS). After incubation with diluted rabbit antisera for 45 min at 37°C, the membrane was washed 3 times with T-TBS. Then, the membrane was reacted with peroxidase-conjugated purified recombinant protein A/G (Thermo Fischer Scientific, Rockford, IL, U.S.A.) for 45 min at 37°C. After washing the membrane with T-TBS and TBS three times each, specific bands were visualized using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Wako Pure Chemical Industries, Ltd., Osaka, Japan).