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Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), a febrile colitis of horses reported in North America and Europe (17, 20). The mode of transmission of E. risticii infection and the reservoir of infection have remained unknown. Other ehrlichial agents are tick transmitted; however, evidence of tick transmission has not been found for E. risticii (4, 8, 19). Recent identification of a close genetic relationship between E. risticii and Neorickettsia helminthoeca isolated from a fluke (Nanophyetus salmincola) suggested that the vector of E. risticii might be a trematode (12, 15). We demonstrated the presence of E. risticii genes in virgulate cercariae from operculate freshwater snails from an area of northern California where PHF is prevalent (2, 13). On the basis of these findings, we formed the hypothesis that snails and their trematodes are involved in the life cycle of E. risticii. Here we report the biological, ultrastructural, and genetic identification of E. risticii in trematode stages collected from freshwater snails and helminthic transmission of E. risticii to horses.

Collection and processing of the snails. Freshwater snails were collected in October 1998 from a stream that flows through a pasture in Weed, California (Siskiyou County), with a history of PHF in some resident horses. Eighty collected snails, of the species Juga yrekaensis, were extracted from their shells by using sterile scissors and tweezers and kept in phosphate-buffered saline containing antibiotics (10,000 U of penicillin G per ml and 10,000 µg of streptomycin per ml). The mixture was briefly vortexed to allow disruption of the fragile snail tissue and release of the trematode stages. A 50-µl volume of each mixture containing trematode stages and snail tissue was processed for DNA extraction by the use of a previously described alkaline extraction protocol for genomic DNA (18). The templates were tested by a nested PCR that amplifies a 527-bp segment of the 16S rRNA gene of E. risticii as described elsewhere (1). Of the 80 processed freshwater snails, 10 (12.5%) were positive in the nested PCR. The infectious material was prepared as follows: the trematode stages (sporocyst and cercaria) from the PCR-positive snails were removed from the initial mixture by the use of a pipette until no trematode stages were released from the snail tissue by vortexing. The snail-tissue-free infectious material contained a total of 9,600 sporocysts and 208,000 cercariae.

Horses. Seven healthy 5- to 20-year-old horses, five mares and two geldings, were used as experimental animals (Table 1). The horses were housed in a vector-proof facility at the Equine Research Laboratory, University of California, Davis. Prior to inoculation, the horses tested negative for antibodies to E. risticii by indirect fluorescent antibody assay. The procedures for inoculation and care of the horses were approved by the Animal Care and Use Administrative Committee at the University of California, Davis, and the animal holding facilities are accredited by the American Association for the Accreditation of Laboratory Animal Care. Two horses (no. 1 and 2) were each inoculated subcutaneously with 4,800 sporocysts and 104,000 cercariae. We transfused 100 ml of citrate-treated whole blood collected from horse 1 on day 18 postexposure (fourth day of illness) into two horses (no. 3 and 4) and 100 ml of citrate-treated whole blood collected from horse 2 by day 21 postexposure (second day of illness) into horse 5. Horse 6 was inoculated intravenously with 10⁸ mouse macrophages infected to 80% with E. risticii collected from horse 5 during the acute stage of disease. One additional horse (no. 7), inoculated with uninfected mouse macrophages, served as a control. The inoculated animals were monitored daily for evidence of clinical abnormalities.

Laboratory examinations. Blood was collected from all horses into 10-ml evacuated glass tubes with and without anticoagulant (Vacutainer; Becton Dickinson, Franklin Lakes, N.J.) beginning on the day of experimental infection (day zero) and daily thereafter for 30 days. The leukocyte, erythrocyte, and platelet counts were determined. E. risticii antibodies were detected by the indirect fluorescent antibody assay procedure of Madigan et al. (9). DNA was extracted from peripheral blood leukocytes and from 1 g of feces by using a QIAamp Blood DNA Extraction Kit (Qiagen, Valencia, Calif.). The extracted DNA was subjected to nested PCR as described by Barlough et al. (1). E. risticii was isolated in mouse macrophage cell line P388D1 (American Type Culture Collection, Manassas, Va.) from the buffy coat cells of citrate-treated whole blood of horses 5 and 6 on the second day of fever, using a method previously described for E. risticii (6), and ultrastructurally characterized by light microscopy and transmission electron microscopy.

Sequencing of amplified PCR products. Nested-PCR products of the 16S rRNA gene from the in vitro-grown E. risticii strain, blood, and fecal samples from horses 1 to 6 were selected for sequencing. Segments of two additional ehrlichial genes, the groEL heat shock protein gene and the 51-kDa major antigen gene, of the in vitro-grown E. risticii strain were amplified essentially as described elsewhere (2, 13). The fragments were purified by the use of a gel extraction kit (QIAquick Gel Extraction Kit; Qiagen). DNA sequencing of both strands of each purified PCR product was performed with a fluorescence-based sequencing system (Applied Biosystems, Foster City, Calif.).

Results. Horse 1 developed a fever on day 15 after inoculation; this was followed by depression, anorexia, decrease in borborygmal sounds, production of cow-like feces, ventral edema, and leukopenia. The horse recovered after 8 days of sickness without treatment. The nested PCR of the blood buffy coat was positive on day 11 and remained positive through day 25, while that of the feces was positive on day 17 and remained positive through day 28. The serum antibody titer increased from 20 on day 21 to 160 by day 30.

View larger version (152164K): [in this window] [in a new window]   FIG. 1.   (A) Transmission electron micrograph of ehrlichial organisms (arrows) in cytoplasmic vacuoles of mouse macrophage culture. Bar, 1 µm. (B) Transmission electron micrograph, at a higher magnification, showing ultrastructure of the culture-derived causative agent of equine monocytic ehrlichiosis. Single organisms are surrounded by a distinct cytoplasmic vacuolar membrane. Note the rippled outer membrane of the organism. Bar, 0.2 µm.

Discussion. Despite intensive research efforts for over 20 years, PHF has remained a mystery because the life cycle of the agent and the mode of transmission are unknown. The involvement of an aquatic environment in the life cycle of E. risticii has been recently demonstrated through the detection of this organism in trematodes that use operculate freshwater snails as intermediate hosts (2, 13). In this study, we uncovered a part of the mystery, and here we describe the first helminth-associated transmission of E. risticii in horses.

Nucleotide sequence accession numbers. The sequences for the E. risticii agent newly reported in this paper have been assigned the following GenBank accession numbers: AF179351 for the 16S rRNA gene fragment, AF179349 for the groEL heat shock protein gene fragment, and AF179350 for the 51-kDa major antigen gene fragment.