An Ecotype of Neorickettsia risticii Causing Potomac Horse Fever in Canada

Neorickettsia (formerlyEhrlichia )risticii is an obligatory intracellular bacterium of digenetic trematodes. When a horse accidentally ingests aquatic insects containing encysted trematodes infected withN. risticii , the bacterium is transmitted from trematodes to horse cells and causes an acute and often fatal disease called Potomac horse fever (PHF). Since the discovery ofN. risticii in the United States in 1984, using immunofluorescence and PCR assays, PHF has been increasingly recognized throughout North America and South America. However, so far, there exist only a few stableN. risticii culture isolates, all of which are from horses within the United States, and the strain diversity and environmental spreading and distribution of pathogenicN. risticii strains remain poorly understood. This paper reports the isolation ofN. risticii from the blood of a horse with acute PHF in Ontario, Canada. IntracellularN. risticii colonies were detected in P388D1 cells after 47 days of culturing and 8 days after the addition of rapamycin. Molecular phylogenetic analysis based on amino acid sequences of major surface proteins P51 and Ssa1 showed that this isolate is distinct from any previously sequenced strains but closely related to midwestern U.S. strains. This is the first Canadian strain cultured, and a new method was developed to reactivate dormantN. risticii to improve culture isolation.IMPORTANCE Neorickettsia risticii is an environmental bacterium that lives inside flukes that are parasitic to aquatic snails, insects, and bats. When a horse accidentally ingests insects harboring flukes infected withN. risticii , the bacterium is transmitted to the horse and causes an acute and often fatal disease called Potomac horse fever. Although the disease has been increasingly recognized throughout North and South America,N. risticii has not been cultured outside the United States. This paper reports the first Canadian strain cultured and a new method to effectively culture isolateN. risticii from the horse blood sample. Molecular analysis showed that the genotype of this Canadian strain is distinct from previously sequenced strains but closely related to midwestern U.S. strains. Culture isolation ofN. risticii strains would confirm the geographic presence of pathogenicN. risticii , help elucidateN. risticii strain diversity and environmental spreading and distribution, and improve diagnosis and development of vaccines for this dreadful disease.