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

Identification of isolates by PCR, sequence and phylogenetic analysis of C. pecorum omp1 gene: C. pecoruminfected cells and the supernatants from organs described above were used for DNA extraction. Total DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN, Hiden, Germany). PCR was carried out with TaKaRa Ex Taq Hot Start Version (TaKaRa Bio Inc., Otsu, Japan). Primer pairs targeting fragments of genus-specific and species-specific Chlamydia omp1 genes were used for PCR as described by Kaltenböck et al. [13] . PCR products were electrophoresed on 2.0% agarose gel and visualized using ethidium bromide staining. PCR products were purified using Min-iElute PCR Purification Kit (QIAGEN, Germantown, MD, U.S.A.) and directly sequenced using a BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, Austin, TX, U.S.A.). Phylogenetic analysis was performed using the MEGA 5 program. Sequence data were aligned using ClustalW method [23] . Genetic distances were calculated using the Tamura-Nei model [22] . Phylogenetic trees were constructed using neighbor-joining methods [19] , and the reliability of the branch was evaluated by bootstrapping with 1,000 replicates.