Isolation and Molecular Analysis of a Novel<i>Neorickettsia</i>Species That Causes Potomac Horse Fever

Abstract

Potomac horse fever (PHF), a severe and frequently fatal febrile diarrheal disease, has been known to be caused only by <i>Neorickettsia risticii</i>, an endosymbiont of digenean trematodes. Here, we report the cell culture isolation of a new <i>Neorickettsia</i> species found in two locations in eastern Ontario, Canada, in 2016 and 2017 (in addition to 10 variable strains of <i>N. risticii</i>) from <i>N. risticii</i> PCR-negative horses with clinical signs of PHF. Gene sequences of 16S rRNA and the major surface antigen P51 of this new <i>Neorickettsia</i> species were distinct from those of all previously characterized <i>N. risticii</i> strains and <i>Neorickettsia</i> species, except for those from an uncharacterized <i>Neorickettsia</i> species culture isolate from a horse with PHF in northern Ohio in 1991. The new <i>Neorickettsia</i> species nonetheless had the characteristic intramolecular repeats within strain-specific antigen 3 (Ssa3), which were found in all sequenced Ssa3s of <i>N. risticii</i> strains. Experimental inoculation of two naive ponies with the new <i>Neorickettsia</i> species produced severe and subclinical PHF, respectively, and the bacteria were reisolated from both of them, fulfilling Koch's postulates. Serological assay titers against the new <i>Neorickettsia</i> species were higher than those against <i>N. risticii</i> Whole-genome sequence analysis of the new <i>Neorickettsia</i> species revealed unique features of this bacterium compared with <i>N. risticii</i> We propose to classify this new bacterium as <i>Neorickettsia finleia</i> sp. nov. This finding will improve the laboratory diagnosis of and vaccine for PHF, environmental risk assessment of PHF, and understanding of PHF pathogenesis and <i>Neorickettsia</i> biology in general.<b>IMPORTANCE</b> Despite the detection of <i>Neorickettsia</i> species DNA sequences in various trematode species and their hosts, only three <i>Neorickettsia</i> species have been cell culture isolated and whole-genome sequenced and are known to infect mammals and/or cause disease. The molecular mechanisms that enable the obligatory intracellular bacterium <i>Neorickettsia</i> to colonize trematodes and to horizontally transmit from trematodes to mammals, as well as the virulence factors associated with specific mammalian hosts, are unknown. Potomac horse fever (PHF) is a severe and acute systemic infectious disease of horses, with clinical signs that include diarrhea. <i>Neorickettsia risticii</i> is the only known bacterial species that causes PHF. Ingestion of insects harboring <i>N. risticii</i>-infected trematodes by horses leads to PHF. Our discovery of a new <i>Neorickettsia</i> species that causes PHF and whole-genome sequence analysis of this bacterium will improve laboratory diagnosis and vaccine development for PHF and will contribute to our understanding of <i>Neorickettsia</i> ecology, pathogenesis, and biology.