Prevalence and sequence analyses of Neorickettsia risticii

The presence of Neorickettsia (Ehrlichia) risticii DNA was confirmed by PCR amplification and sequence analysis from cercaria in snails collected from stream water in Chungcheong and Jeonra provinces. A total of 3,219 snails were tested for trematode cercariae and N. risticii infection. N. risticii 16S rRNA gene fragment was amplified in cercariae from Semisulcospira libertina and Radix auricularia coreana snails by nested PCR. More than four genus cercariae (Schistosomatidae, Microphallidae, Furcocercus, and Xiphidiocercaria) as well as unidentified cercariae were found from Semisulcospira libertina snails. Three species of cercariae (E. cinetorichis, E. hortense, and Fasciola sp.) were found from Radix auricularia coreana snails. The cercariae were present in 429 (13.3%) snails of 3,216 collected at the Chungcheong and Jeonra provinces. The prevalence of N. risticii in these 429 cercariae was 17.9% (77 of 429 cercariae-infected snails). The amplicons of N. risticii 16S rRNA gene fragment (527 bp) from cercariae DNA had two genotypes (NR-JA1 and NR-JA2) with an identity of 96.4% between their nucleotide sequences. However, when compared to the sequence of N. risticii Shasta strain these sequences showed a 94.3% and 96.4% homology, respectively. The comparison of N. risticii 51 kDa major antigen gene sequences (572 bp) from NR-JA1 and NR-JA2 were 100% identical to the sequence of the isolates from Juga sp., Caddisfly larvae, Shasta, Juga yrekaensis, and trematode of California. This study reports for the first time the detection of N. risticii from cercariae found in Radix auricularia coreana snail. These data also indicate that N. risticii could be widespread in Korea.