Isolation of virulent Rhodococcus equi from native Japanese horses

R. equi was isolated from soil samples obtained from the environment of seven native Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, Miyako and Yonaguni) and from fecal samples collected from three native horse breeds (Hokkaido, Kiso and Misaki). Virulent R. equi at various levels (ranging from 0.5 to 12.9%) was isolated from the feces or soil environment of Hokkaido, Kiso and Misaki horses. Isolates were investigated both for the presence of 15- to 17-kDa antigens (virulence-associated protein antigens; VapA) by colony blotting, using the monoclonal antibody 10G5, and the gene of VapA by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases, and the digestion patterns of the plasmids of virulent isolates were divided into three types. Two of the three types (87-kb type II and 90-kb type I) had already been reported in Japanese isolates, and a new type (tentatively designated as 90-kb type II) had been found in isolates from Kiso horses. Six virulent R. equi isolates from the Hokkaido horses contained an 87-kb type II plasmid. Eight of 24 isolates from the Kiso horses contained an 87-kb type II plasmid, and the remaining 16 contained a 90-kb type II (a new type) plasmid. Two isolates from the Misaki horses contained a 90-kb type I plasmid. These results demonstrate the geographic difference in the distribution of virulence plasmids in R. equi isolates among native Japanese horses.