Operculate snails (the family Pleuroceridae: Elimia livescens) were collected between June and October 1998 from a river in central Ohio where repeated cases of Potomac horse fever (PHF) have occurred. Of collected snails, consistently 50 to 80% carried a combination of cercariae and sporocysts of digenetic virgulate trematodes. The trematodes obtained from each snail were pooled and tested for Ehrlichia risticii, the agent of PHF, by nested PCR using primers specific to the 16S rRNA gene. Out of a total of 209 trematode pools, 50 pools were found to be positive by PCR. The DNA sequence of the 16S rRNA gene identified in one trematode pool was identical to that of the type strain of E. risticii, and the sequence of the gene identified in another pool differed from that of the type strain by 1 nucleotide. Comparison of the deduced amino acid sequence of the partial 51-kDa antigen gene from various sources revealed that Maryland, Ohio (except Ohio 081), and Kentucky strains are in a cluster distinct from the sequences obtained from sources in California and Oregon. Ohio 081 was shown previously by antigenic composition analysis to be distinct from other groups. However, all sequences examined were not segregated according to their sources: horse blood or infected trematodes. E. risticii was found to be transmittable from trematodes to mice and was subsequently passaged from infected mice to additional mice, as determined by PCR analysis. Our findings suggest the evolution of E. risticii in the natural reservoir in separate geographic regions and persistent infection of trematode populations with E. risticii during summer and early fall. The study also suggests that the mouse can be used to isolate E. risticii from the infected trematode.