Diagnostic approaches, aetiological agents and their associations with short-term survival and laminitis in horses with acute diarrhoea admitted to referral institutions.

BACKGROUND: An international description of the diagnostic approaches used in different institutions to diagnose acute equine diarrhoea and the pathogens detected is lacking. OBJECTIVES: To describe the diagnostic approach, aetiological agents, outcome, and development of laminitis for diarrhoeic horses worldwide. STUDY DESIGN: Multicentre retrospective case series. METHODS: Information from horses with acute diarrhoea presenting to participating institutions between 2016 and 2020, including diagnostic approaches, pathogens detected and their associations with outcomes, were compared between institutions or geographic regions. RESULTS: One thousand four hundred and thirty-eight horses from 26 participating institutions from 4 continents were included. Overall, aetiological testing was limited (44% for Salmonella spp., 42% for Neorickettsia risticii [only North America], 40% for Clostridiodes difficile, and 29% for ECoV); however, 13% (81/633) of horses tested positive for Salmonella, 13% (35/262) for N. risticii, 9% (37/422) for ECoV, and 5% (27/578) for C. difficile. C. difficile positive cases had greater odds of non-survival than horses negative for C. difficile (OR: 2.69, 95%CI: 1.23-5.91). In addition, horses that were positive for N. risticii had greater odds of developing laminitis than negative horses (OR: 2.76, 95%CI: 1.12-6.81; p = 0.029). MAIN LIMITATIONS: Due to the study's retrospective nature, there are missing data. CONCLUSIONS: This study highlighted limited diagnostic investigations in cases of acute equine diarrhoea. Detection rates of pathogens are similar to previous reports. Non-survival and development of laminitis are related to certain detected pathogens.

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.

Real-Time PCR Differential Detection of Neorickettsia findlayensis and N. risticii in Cases of Potomac Horse Fever.

Potomac horse fever (PHF) is an acute and potentially fatal enterotyphlocolitis of horses with clinical signs that include anorexia, fever, diarrhea, and laminitis. Its incidence is increasing despite a commercially available vaccine. PHF is caused by Neorickettsia risticii, and the recently rediscovered and classified N. findlayensis. PHF diagnosis is currently accomplished using serology or nested PCR. However, both methods cannot distinguish the two Neorickettsia species that cause PHF. Further, the current N. risticii real-time PCR test fails to detect N. findlayensis. Thus, in this study, two Neorickettsia species-specific real-time PCR assays based on Neorickettsia ssa2 and a Neorickettsia genus-specific real-time PCR assay based on Neorickettsia 16S rRNA gene were developed. The ssa2 real-time PCR tests differentiated N. findlayensis from N. risticii in the field samples for which infection with either species had been verified using multiple other molecular tests and culture isolation, and the 16S rRNA gene real-time PCR detected both Neorickettsia species in the samples. These tests were applied to new field culture isolates from three Canadian provinces (Alberta, Quebec, Ontario) and Ohio as well as archival DNA samples from suspected PHF cases to estimate the prevalence of N. findlayensis in different geographic regions. The results suggest that N. findlayensis frequently causes PHF in horses in Alberta and Quebec. The development of these tests will allow rapid, sensitive, and specific diagnosis of horses presenting with clinical signs of PHF. These tests will also enable rapid and targeted treatment and help develop broad-spectrum vaccines for PHF.

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.

Immunogenicity of Potomac horse fever vaccine when simultaneously co-administered with rabies vaccine in a multivalent vaccine or as two monovalent vaccines at separate sites.

BACKGROUND: Potomac horse fever (PHF) is a potentially fatal enterocolitis of horses caused by Neorickettsia risticii. The disease was originally recognised almost 40 years ago in the state of Maryland in the US. It is now known to occur in many areas of North America, as well as having been described in South America and Europe. Monocomponent PHF vaccines are available, but clinical protection with vaccination has been reported to be inconsistent. OBJECTIVES: This study was designed to assess the immunogenicity of a commercially available Potomac Horse Fever (PHF) vaccine when administered as either a monovalent PHF vaccine simultaneously co-administered with a separate monovalent Rabies vaccine or as a multivalent PHF/Rabies vaccine in horses. STUDY DESIGN: Randomised parallel group trial. METHODS: Ninety-one client or University owned horses participated in this open-label randomised study, with 45 horses receiving the monovalent vaccines at separate sites and 46 receiving the multivalent vaccine at a single site. Serum PHF IFA titres were determined twice prior to vaccination and at 1, 2 and 3 months after vaccination. RESULTS: Both vaccination protocols exhibited poor immunogenicity, with only one-third of all the animals demonstrating seroconversion, defined as an increase in titre of greater than 400 over baseline, at any time point after vaccination. The monovalent PHF vaccine exhibited significantly greater immunogenicity in terms of the number of horses exhibiting seroconversion, as compared to the multivalent vaccine, at one (20 vs. 11, P = 0.03) and two (18 vs. 9, p = 0.02) months post vaccination. The monovalent PHF vaccine also exhibited significantly greater immunogenicity in terms of the median (interquartile range) IFA titres, as compared to the multivalent vaccine, at one (800 [200-1600] vs. 400 [200-800], P = 0.009) and 2 months (400 [200-1600] vs. 400 [100-800], P = 0.02) post vaccination. There was no significant difference between groups at 3 months in either seroconversion rate or median IFA titers. MAIN LIMITATIONS: This study did not assess the actual protective effects of PHF vaccination but rather used the serologic response to vaccination as a surrogate biomarker of immunity. CONCLUSIONS: The multivalent PHF/Rabies vaccine exhibited lower immunogenicity as compared to the monovalent PHF vaccine co-administered with a separate Rabies vaccine.

Frequency of Equine Monocytic Ehrlichiosis (EME) in Brazil / Relato epidemiológico da Erlichiose monocítica equina (EME) no Brasil

From a cross-sectional observational study with convenience samples, 347 blood samples from horses were collected from different physiographic regions, as follows: Santa Catarina Plateau (Santa Catarina State - SC), Médio Paraíba do Sul (São Paulo State - SP and Rio de Janeiro State RJ), Mountainous and Metropolitan regions (Rio de Janeiro State - RJ). Samples were tested for the presence of antibodies (IgG) anti Neorickettsia risticii by indirect immunofluorescence assay (IFA). The frequency obtained in this study corroborates with the ones obtained in the U.S.A., which refers to endemic regions. Fisher's exact test showed significant differences in the number of positive animals between regions, indicating that the probability of an animal becoming infected varies depending on the area. The CI 95% revealed no association between infection and geopolitical space. Moreover, Odds ratio test showed differences of an animal getting infected in different regions. This event could be influenced by the type of treatment used in each area, as the seasonal frequency of injury or even potential vectors. Therefore, there are seropositive animals for N. risticii in the studied areas, suggesting that this agent may be circulating in those regions. Future studies mainly based on molecular analyzes are needed to confirm these serological findings.

A partir de um delineamento observacional transversal com amostras de conveniência, 347 amostras de sangue foram coletadas de diferentes regiões fisiográficas: Planalto de Santa Catarina (Estado de Santa Catarina - SC), Região do Médio Paraíba do Sul (Estados de São Paulo - SP e Rio de Janeiro - RJ), Região Serrana e Metropolitana (ambas do Estado do Rio de Janeiro - RJ). As amostras foram testadas para a presença de anticorpos (IgG) anti-Neorickettsia risticii por imunofluorescência indireta (IFI). A prevalência obtida no presente estudo corrobora com demais resultados obtidos nos Estados Unidos da América. O Teste Exato de Fisher demonstrou diferença significativa no número de animais positivos entre as regiões, indicando assim que a probabilidade de um animal se infectar varia dependendo da região. O intervalo de confiança (IC 95%) revelou não haver associação entre a infecção e o espaço geopolítico, este evento pode ser influenciado pelo tipo de tratamento em cada área, como sazonalidade do agravo ou frequência de potenciais vetores. Assim, a soropositividade ora encontrada sugere a circulação de N. risticii nas áreas estudadas. Estudos futuros baseados, principalmente, em análises moleculares serão importantes para a confirmação dos achados sorológicos no presente trabalho.

Molecular detection of Neorickettsia risticii in Brazilian free-tailed bats (Tadarida brasiliensis) from Buenos Aires, Argentina

Neorickettsia risticii is the causative agent of Potomac Horse Fever, a severe febrile disease affecting horses, transmitted by trematodes species with a complex life cycle. A total of 30 insectivorous bats (Brazilian free-tailed bat Tadarida brasiliensis) were analyzed by PCR for presence of genus Anaplasma, Ehrlichia, Neorickettsia and Rickettsia. Three samples showed positive reactions for genus Anaplasma, Ehrlichia and Neorickettsia, and the sequences were 99.67% identical to Neorickettsia risticii. The role of bats in the life cycle of N. risticii has yet to be elucidated; however bats may be reservoirs for this bacterium. To our knowledge, this is the first evidence of N. risticii in Argentina.

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.