Identification of Trematode Cercariae Carrying Neorickettsia risticii in Freshwater Stream Snails

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Abstract
We provide evidence of Neorickettsia (Ehrlichia) risticii Holland, the agent of Potomac horse fever, in trematode larval stages found in aquatic snails and insects collected from a stream in Korea, using nested polymerase chain reaction (PCR) and sequence analysis of the 16S rRNA gene fragment amplified from several cercaria species. It was observed that of 423 (13.1%) of 3,219 snails infected with cercariae, 77 (20.8% of the 371) were infected with N. risticii. Five families of trematode cercariae, Schistosomatidae, Echimostomatidae, Heterophyidae, Microphallidae, and Acanthocopidae were identified morphologically within Semisulcospira libertina, Radix auricularia coreana, and S. gottschei snails. Echinostoma cinetorchis, E. hortense, and Metagonimus sp. were identified based on both the cercarial morphology as well as by phylogenetic analysis of the amplified 18S rRNA gene sequences. Adult aquatic insects were also collected from the same sites and were sorted into five species, Ischnura asiatica in Coenagrionidae and Calopteryx japonica, Sympetrum darwinianum, Symptrum eroticum, and Symptrum parvulum in Calopterygoidae. One thousand and two hundred eighty five metacercariae (classified into groups A through F) were isolated from 310 adult aquatic insects, and the average number of metacercariae per aquatic insect was 4.1. However, there was no amplification of N. risticii from these metacercariae.