Diagnosis of Potomac horse fever (syn. equine neorickettsiosis) in 2 foals in southwestern Ontario.

Potomac horse fever (PHF) is characterized by fever, depression, anorexia, ileus, diarrhea, and occasionally, laminitis. The disease is caused by infection with Neorickettsia risticii and/or N. findlayensis. Equids of all ages may be affected; however, the condition has not been well-characterized in foals. This report describes clinical signs, laboratory findings, and treatment of 2 foals diagnosed with PHF in southwestern Ontario. Feces submitted for an equine PCR panel tested positive for Neorickettsia spp. and were subsequently confirmed to be N. risticii (Case 1) and N. findlayensis (Case 2). Both foals recovered following hospitalization and intensive care. Key clinical message: The purpose of this report is to make veterinarians aware that foals may develop PHF. During summer (July to September), when encountering foals in endemic areas with clinical signs compatible with PHF, veterinarians should consider PHF as a diagnostic rule-out. For confirmation of the diagnosis, blood and feces should be submitted for PCR testing for Neorickettsia spp.

Diagnostic de la fièvre équine du Potomac (syn. néorickettsiose équine) chez 2 poulains dans le sud-ouest de l'Ontario. La fièvre équine du Potomac (PHF) se caractérise par de la fièvre, une dépression, de l'anorexie, un iléus, de la diarrhée et, occasionnellement, une fourbure. La maladie est causée par une infection par Neorickettsia risticii et/ou N. findlayensis. Les équidés de tous âges peuvent être atteints; cependant, cette pathologie n'a pas été bien caractérisée chez les poulains. Ce rapport décrit les signes cliniques, les résultats de laboratoire et le traitement de 2 poulains diagnostiqués avec PHF dans le sud-ouest de l'Ontario. Les matières fécales soumises à un panel PCR équin se sont révélées positives pour Neorickettsia spp. et ont ensuite été confirmées comme étant positives pour N. risticii (cas 1) et N. findlayensis (cas 2). Les deux poulains se sont rétablis après une hospitalisation et des soins intensifs.Message clinique clé :Le but de ce rapport est de sensibiliser les vétérinaires au fait que les poulains peuvent développer une PHF. Pendant l'été (juillet à septembre), lorsqu'ils rencontrent des poulains dans des zones d'endémie présentant des signes cliniques compatibles avec le PHF, les vétérinaires doivent considérer le PHF comme une exclusion diagnostique. Pour confirmer le diagnostic, du sang et des selles doivent être soumis à un test PCR pour Neorickettsia spp.(Traduit par Dr Serge Messier).

Improved molecular detection of Neorickettsia risticii with a duplex real-time PCR assay in the diagnosis of Potomac horse fever.

Neorickettsia risticii, an obligate intracellular bacterium, is the causative agent of Potomac horse fever (PHF). Diagnosis of PHF is based on demonstration of serum antibodies, isolation of N. risticii, and/or detection of nucleic acid by a PCR assay. An existing real-time PCR assay targeting the N. risticii 16S rRNA has been validated using blood samples from horses with colitis, and snails; to our knowledge, the performance of the assay for other sample types has not been reported. We describe here a modification of the 16S rRNA gene assay by the addition of a set of primers and probe targeting the N. risticii p51 gene to form a duplex assay. We validated the new assay using diagnostic specimens from 56 horses with suspected PHF. The assay consistently detected down to 5 copies of synthetic targets, and did not show any cross-reaction with common equine enteric pathogens. Although we did not establish the diagnostic sensitivity and specificity of the duplex assay, results for both gene targets were in complete agreement, with the exception of 4 fecal samples that tested positive for the 16S rRNA gene only. Further analysis indicated that testing of fecal samples using our 16S rRNA gene assay alone can produce a false-positive result.

Detection of Neorickettsia risticii, the agent of Potomac horse fever, in horses from Rio de Janeiro, Brazil.

This study aims to report the presence of Neorickettsia risticii DNA in blood samples from naturally infected horses in Rio de Janeiro, provide clinicopathological findings related to the infection, and report the phylogenetic diversity of the 16S rDNA of N. risticii in order to evaluate its heterogeneity. Real-time quantitative polymerase chain reaction (qPCR) was performed to investigate the presence of N. risticii in samples collected from horses (n = 187). Five positive samples were found in the molecular screening. Hypoalbuminemia and high levels of creatine kinase and lactate dehydrogenase were the predominant findings in the biochemical analysis. The sequences were similar to those of N. risticii. Phylogenetic analysis revealed genotype segregation based on the geographical distribution in the N. risticii sequence clade. Dendrograms constructed with five hypervariable regions revealed that V4 distinguished Neorickettsia at the species level and produced a phylogeny that best represented the phylogeny obtained with the complete 16S rDNA sequence. This is the first report of N. risticii DNA in the blood of Brazilian horses based on sequences deposited in GenBank. Further studies are necessary to clarify the epidemiological chain of this vector-borne parasite in order to determine and establish appropriate preventive measures in the equine trading market.

Isolation and Molecular Analysis of a Novel Neorickettsia Species That Causes Potomac Horse Fever.

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.

Neorickettsia risticii, Rickettsia sp. and Bartonella sp. in Tadarida brasiliensis bats from Buenos Aires, Argentina.

Bats are potential reservoirs of many vector-borne bacterial pathogens. The aim of the present study was to detect species of Anaplasma, Ehrlichia, Neorickettsia, Rickettsia, Borrelia and Bartonella in Brazilian free-tailed bats (Tadarida brasiliensis, Molossidae) from Buenos Aires city, Argentina. Between 2012 and 2013, 61 T. brasiliensis from urban areas of Buenos Aires city were studied. The samples were molecularly screened by PCR and sequencing. Five bats (8.2%) were positive to Neorickettsia risticii, one (1.6%) was positive to Rickettsia sp. and three bats (4.9%) to Bartonella sp. For molecular characterization, the positive samples were subjected to amplification and sequencing of a fragment of p51 gene for N. risticii, a fragment of citrate synthase gene (gltA) for Rickettsia genus and a fragment of gltA for Bartonella genus. Phylogenetic tree was constructed using the maximum-likelihood method. Phylogenetic analysis of N. risticii detect in our study revealed that it relates to findings in the USA West Coast; Rickettsia sp. detected is phylogenetically within R. bellii group, which also includes many other Rickettsia endosymbionts of insects; and Bartonella sp. found is related to various Bartonella spp. described in Vespertilionidae bats, which are phylogenetically related to Molossidae. Our results are in accordance to previous findings, which demonstrate that insectivorous bats could be infected with vector-borne bacteria representing a potential risk to public health. Future research is necessary to clarify the circulation of these pathogens in bats from Buenos Aires.

An Ecotype of Neorickettsia risticii Causing Potomac Horse Fever in Canada.

UNLABELLED: Neorickettsia (formerly Ehrlichia) risticii is an obligatory intracellular bacterium of digenetic trematodes. When a horse accidentally ingests aquatic insects containing encysted trematodes infected with N. 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 of N. 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 stable N. risticii culture isolates, all of which are from horses within the United States, and the strain diversity and environmental spreading and distribution of pathogenic N. risticii strains remain poorly understood. This paper reports the isolation of N. risticii from the blood of a horse with acute PHF in Ontario, Canada. Intracellular N. 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 dormant N. 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 with N. 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 isolate N. 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 of N. risticii strains would confirm the geographic presence of pathogenic N. risticii, help elucidate N. risticii strain diversity and environmental spreading and distribution, and improve diagnosis and development of vaccines for this dreadful disease.

Detection of Neorickettsia risticii from various freshwater snail species collected from a district irrigation canal in Nevada County, California.

This study investigated the role of a district irrigation canal in Nevada County, California, USA, as the point source of infection for Neorickettsia risticii, causative agent of equine neorickettsiosis (EN). A total of 568 freshwater snails comprising Juga spp., Planorbella subcrenata (Carpenter, 1857) (Rough Rams-horn), Physella virgata (Gould, 1855) (Protean Physa) and feces from three horses with EN were collected and tested for N. risticii by real-time PCR. A total of four freshwater snails tested PCR positive for N. risticii. Phylogenetic analysis showed 99.8-100% homology between the different snail and horse N. risticii isolates. This study represents the first report of infection with N. risticii in Planorbella subcrenata and suggests that the irrigation canal was the aquatic environment responsible for the spread of N. risticii.

Neorickettsia risticii surface-exposed proteins: proteomics identification, recognition by naturally-infected horses, and strain variations.

Neorickettsia risticii is the Gram-negative, obligate, and intracellular bacterial pathogen responsible for Potomac horse fever (PHF): an important acute systemic disease of horses. N. risticii surface proteins, critical for immune recognition, have not been thoroughly characterized. In this paper, we identified the 51-kDa antigen (P51) as a major surface-exposed outer membrane protein of older and contemporary strains of N. risticii through mass spectrometry of streptavidin-purified biotinylated surface-labeled proteins. Western blot analysis of sera from naturally-infected horses demonstrated universal and strong recognition of recombinant P51 over other Neorickettsia recombinant proteins. Comparisons of amino acid sequences for predicted secondary structures of P51, as well as Neorickettsia surface proteins 2 (Nsp2) and 3 (Nsp3) among N. risticii strains from horses with PHF during a 26-year period throughout the United States revealed that the majority of variations among strains were concentrated in regions predicted to be external loops of their ß-barrel structures. Large insertions or deletions occurred within a tandem-repeat region in Ssa3. These data demonstrate patterns of geographical association for P51 and temporal associations for Nsp2, Nsp3, and Ssa3, indicating evolutionary trends for these Neorickettsia surface antigen genes. This study showed N. risticii surface protein population dynamics, providing groundwork for designing immunodiagnostic targets for PHF.

Analysis of complete genome sequence of Neorickettsia risticii: causative agent of Potomac horse fever.

Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. sennetsu showed that 758 (88.2%) of protein-coding genes are conserved between N. risticii and N. sennetsu. Four-way comparison of genes among N. risticii and other Anaplasmataceae showed that most genes are either shared among Anaplasmataceae (525 orthologs that generally associated with housekeeping functions), or specific to each genome (>200 genes that are mostly hypothetical proteins). Genes potentially involved in the pathogenesis of N. risticii were identified, including those encoding putative outer membrane proteins, two-component systems and a type IV secretion system (T4SS). The bipolar localization of T4SS pilus protein VirB2 on the bacterial surface was demonstrated for the first time in obligate intracellular bacteria. These data provide insights toward genomic potential of N. risticii and intracellular parasitism, and facilitate our understanding of PHF pathogenesis.

Abortion in a horse following Neorickettsia risticii infection.

A pregnant 18-year-old Quarterhorse mare presented with fever, anorexia, tachycardia, tachypnea, and gastrointestinal hypermotility at day 68 of gestation. Potomac horse fever was diagnosed based on polymerase chain reaction (PCR) analysis of whole blood and a high antibody titer to Neorickettsia risticii. The mare made a rapid clinical recovery following antibiotic therapy, but aborted 98 days later. Necropsy on the aborted fetus revealed lymphohistiocytic colitis, lymphadenitis, myocarditis, and hepatitis. The placenta was grossly and histologically normal. Formalin-fixed lymph node, thymus, liver, and colon taken from the aborted fetus were positive by PCR for N. risticii DNA. Potomac horse fever is a common disease in horses that may result in delayed abortion. The microscopic lesions in the fetus are characteristic, and the diagnosis can be confirmed by PCR on formalin-fixed tissues.

Cloning and expression of 51-kDa antigenic protein of Neorickettsia risticii NR-JA1.

Neorickettsia (Ehrlichia) risticii is a causative agent of acute diarrheal syndrome in horses, commonly known as Potomac horse fever. Korean isolate of N. risticii NR-JA1 was cultivated in mouse macrophage cell line P388D1. A complete ORF of p51 antigenic protein gene was amplified and cloned into pQE32 and pcDNA3.1 vectors and the resultant clones were named as pQE32/Nr-51 and pcDNA3.1/Nr-51, respectively. Recombinant p51 (rp51) protein antigen was expressed in E. coli (pQE32/Nr-51) and cos-7 cell line (pcDNA3.1/Nr-51). The rp51 protein showed immunoreactivity with anti- mouse p51 antibodies. BALB/c mice were inoculated with recombinant plasmid DNA (pcDNA3.1/Nr-51). The serum samples collected from these BALB/c mice showed IgG ELISA titers of 1:128. In a Western immunoblot assay, these serum samples showed a strong reactivity to rp51 expressed in cos-7 cell line transfected with pcDNA3.1/Nr-51. The results of this preliminary indicate that N. risticii p51 protein is an immmuno-dominant antigen and may be a good target for the development of serological or a molecular diagnostic test and possibly an improved recombinant DNA based vaccine against Potomac horse fever.

Two cases of Neorickettsia (Ehrlichia) risticii infection in horses from Nova Scotia.

Two horses from Nova Scotia were diagnosed with Potomac horse fever (PHF). Polymerase chain reaction analysis was performed on formalin-fixed colon tissue or whole blood to show the presence of Neorickettsia risticii DNA, the causative agent of PHF. These are the first reported cases of PHF in the Maritime Provinces.

Prevalence and sequence analyses of Neorickettsia risticii.

The presence of Neorickettsia (Ehrlichia) risticii DNA was confirmed by PCR amplification and sequence analysis from cercaria in snails collected from stream water in Chungcheong and Jeonra provinces. A total of 3,219 snails were tested for trematode cercariae and N. risticii infection. N. risticii 16S rRNA gene fragment was amplified in cercariae from Semisulcospira libertina and Radix auricularia coreana snails by nested PCR. More than four genus cercariae (Schistosomatidae, Microphallidae, Furcocercus, and Xiphidiocercaria) as well as unidentified cercariae were found from Semisulcospira libertina snails. Three species of cercariae (E. cinetorichis, E. hortense, and Fasciola sp.) were found from Radix auricularia coreana snails. The cercariae were present in 429 (13.3%) snails of 3,216 collected at the Chungcheong and Jeonra provinces. The prevalence of N. risticii in these 429 cercariae was 17.9% (77 of 429 cercariae-infected snails). The amplicons of N. risticii 16S rRNA gene fragment (527 bp) from cercariae DNA had two genotypes (NR-JA1 and NR-JA2) with an identity of 96.4% between their nucleotide sequences. However, when compared to the sequence of N. risticii Shasta strain these sequences showed a 94.3% and 96.4% homology, respectively. The comparison of N. risticii 51 kDa major antigen gene sequences (572 bp) from NR-JA1 and NR-JA2 were 100% identical to the sequence of the isolates from Juga sp., Caddisfly larvae, Shasta, Juga yrekaensis, and trematode of California. This study reports for the first time the detection of N. risticii from cercariae found in Radix auricularia coreana snail. These data also indicate that N. risticii could be widespread in Korea.