Isolation and Molecular Analysis of a Novel Neorickettsia 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 Neorickettsia risticii, an endosymbiont of digenean trematodes. Here, we report the cell culture isolation of a new Neorickettsia species found in two locations in eastern Ontario, Canada, in 2016 and 2017 (in addition to 10 variable strains of N. risticii) from N. risticii PCR-negative horses with clinical signs of PHF. Gene sequences of 16S rRNA and the major surface antigen P51 of this new Neorickettsia species were distinct from those of all previously characterized N. risticii strains and Neorickettsia species, except for those from an uncharacterized Neorickettsia species culture isolate from a horse with PHF in northern Ohio in 1991. The new Neorickettsia species nonetheless had the characteristic intramolecular repeats within strain-specific antigen 3 (Ssa3), which were found in all sequenced Ssa3s of N. risticii strains. Experimental inoculation of two naive ponies with the new Neorickettsia 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 Neorickettsia species were higher than those against N. risticii. Whole-genome sequence analysis of the new Neorickettsia species revealed unique features of this bacterium compared with N. risticii. We propose to classify this new bacterium as Neorickettsia finleia 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 Neorickettsia biology in general. IMPORTANCE Despite the detection of Neorickettsia species DNA sequences in various trematode species and their hosts, only three Neorickettsia 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 Neorickettsia 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. Neorickettsia risticii is the only known bacterial species that causes PHF. Ingestion of insects harboring N. risticii-infected trematodes by horses leads to PHF. Our discovery of a new Neorickettsia 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 Neorickettsia ecology, pathogenesis, and biology.

Epidemiological survey of Neorickettsia risticii in equids from the State of Rio de Janeiro, Brazil

ABSTRACT: Equine neorickettsiosis (EN), also known as Potomac Horse Fever, is a non-contagious disease caused by the bacterium Neorickettsia risticii of the Anaplasmataceae family. The objectives of this study were to detect the presence of anti-N. risticii antibodies by the indirect immunofluorescence assay (IFA) and of its DNA by qPCR in equids at high and low altitude regions in the State of Rio de Janeiro, Brazil, and to identify factors associated with seropositive equids by multiple logistic regression analysis. The frequency of anti-N. risticii antibodies was 16.05% (n=113/704). The animal age and breeding region were the factors that influenced the seropositivity rate for N. risticii in the equids (p<0.05). Equids from the lowland region had higher seropositivity (p<0.05; OR=5.87) compared to those of the mountain region. The presence of snails on the farm was a factor associated with this result (p<0.05; OR=2.88). In the lowland region, age of the animal and site of breeding were protective factors for the detection of antibodies anti-N. risticii in equids, with lower frequency of seropositivity in younger animals (p<0.05; OR=0.06) and in animals raised in dry areas (p<0.05; OR=0.22). The presence of the target DNA of N. risticii by qPCR was not observed in any of the samples tested. The existence of seropositive equids for N. risticii demonstrates a possible circulation of this agent in the studied area, and that the age related characteristics and equids breeding region are important factors regarding seropositivity in the State of Rio de Janeiro.

An Ecotype of Neorickettsia risticii Causing Potomac Horse Fever in Canada

ABSTRACTNeorickettsia(formerlyEhrlichia)risticiiis 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. risticiiin 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. risticiiculture isolates, all of which are from horses within the United States, and the strain diversity and environmental spreading and distribution of pathogenicN. risticiistrains remain poorly understood. This paper reports the isolation ofN. risticiifrom the blood of a horse with acute PHF in Ontario, Canada. IntracellularN. risticiicolonies were detected in P388D1cells 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. risticiito improve culture isolation.IMPORTANCENeorickettsia risticiiis 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. risticiihas not been cultured outside the United States. This paper reports the first Canadian strain cultured and a new method to effectively culture isolateN. risticiifrom 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. risticiistrains would confirm the geographic presence of pathogenicN. risticii, help elucidateN. risticiistrain diversity and environmental spreading and distribution, and improve diagnosis and development of vaccines for this dreadful disease.