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).

Potomac Horse Fever.

Potomac horse fever (PHF) is a common cause of equine colitis in endemic areas. Until recently, the only causative agent known to cause PHF was Neorickettsia risticii. However, N. findlayensis has been isolated from affected horses. Horses typically become infected upon ingestion of Neorickettsia spp.-infected trematodes within aquatic insects. The most common clinical signs include diarrhea, fever, anorexia, lethargy and colic. The diagnostic test of choice for PHF is PCR of blood and feces. Tetracyclines remain an effective treatment. Supportive care, including fluid therapy, colloid administration, NSAID and anti-endotoxin medication, and digital cryotherapy, is also necessary in some cases.

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.

A diagnostic primer pair to distinguish between wMel and wAlbB Wolbachia infections.

Detection of the Wolbachia endosymbiont in Aedes aegypti mosquitoes through real-time polymerase chain reaction assays is widely used during and after Wolbachia releases in dengue reduction trials involving the wMel and wAlbB strains. Although several different primer pairs have been applied in current successful Wolbachia releases, they cannot be used in a single assay to distinguish between these strains. Here, we developed a new diagnostic primer pair, wMwA, which can detect the wMel or wAlbB infection in the same assay. We also tested current Wolbachia primers and show that there is variation in their performance when they are used to assess the relative density of Wolbachia. The new wMwA primers provide an accurate and efficient estimate of the presence and density of both Wolbachia infections, with practical implications for Wolbachia estimates in field collected Ae. aegypti where Wolbachia releases have taken place.

Potomac horse fever in Ontario: Clinical, geographic, and diagnostic aspects.

Clinical findings, geographic locations, laboratory diagnoses, and culture isolation of Neorickettsia spp. in Potomac horse fever (PHF) cases diagnosed in Ontario between 2015 and 2019 are described. Forty-six confirmed PHF cases occurred from late June to early September. Of 41 horses admitted to the Ontario Veterinary College, 28 (68%) survived and 13 (32%) were euthanized due to poor prognosis or financial constraints. Most cases were in southern Ontario along the Canada-USA border. Blood and fecal samples from 43 suspect PHF cases were submitted to 2 laboratories for polymerase chain reaction (PCR) testing for Neorickettsia risticii. Agreement between both laboratories for detection of N. risticii DNA was excellent for feces [κ = 0.932, 95% confidence interval (CI): 0.80 to 1], and fair for blood samples (κ = 0.494, 95% CI: 0.13 to 0.85). Neorickettia spp. were isolated from 16 of 41 (39%) blood samples. DNA analysis confirmed 14 isolates were N. risticii and 2 were N. findlayensis, a novel species of Neorickettsia recently demonstrated to cause PHF.

La fièvre équine du Potomac en Ontario : aspects cliniques, géographiques et diagnostiques. Les résultats cliniques, emplacements géographiques, diagnostics de laboratoire et isolement par culture de Neorickettsia spp. dans les cas de fièvre équine du Potomac (PHF) diagnostiqués en Ontario entre 2015 et 2019 sont décrits. Quarante-six cas confirmés de PHF sont survenus de la fin juin au début septembre. Sur 41 chevaux admis au Ontario Veterinary College, 28 (68%) ont survécu et 13 (32%) ont été euthanasiés en raison d'un mauvais pronostic ou de contraintes financières. La plupart des cas se trouvaient dans le sud de l'Ontario, le long de la frontière canado-américaine. Des échantillons de sang et de matières fécales provenant de 43 cas suspects de PHF ont été soumis à deux laboratoires pour des tests de réaction d'amplification en chaîne par la polymérase (PCR) pour Neorickettsia risticii. La concordance entre les deux laboratoires pour la détection de l'ADN de N. risticii était excellente pour les selles [κ = 0,932, intervalle de confiance (IC) à 95% : 0,80 à 1] et passable pour les échantillons sanguins (κ = 0,494, IC à 95% : 0,13 à 0,85). Neorickettia spp. ont été isolés à partir de 16 des 41 échantillons de sang (39%). L'analyse de l'ADN a confirmé que 14 isolats étaient N. risticii et deux étaient N. findlayensis, une nouvelle espèce de Neorickettsia récemment démontrée comme causant le PHF.(Traduit par Dr Serge Messier).

Genetic diversity of vector-borne pathogens in spotted and brown hyenas from Namibia and Tanzania relates to ecological conditions rather than host taxonomy.

BACKGROUND: Improved knowledge on vector-borne pathogens in wildlife will help determine their effect on host species at the population and individual level and whether these are affected by anthropogenic factors such as global climate change and landscape changes. Here, samples from brown hyenas (Parahyaena brunnea) from Namibia (BHNA) and spotted hyenas (Crocuta crocuta) from Namibia (SHNA) and Tanzania (SHTZ) were screened for vector-borne pathogens to assess the frequency and genetic diversity of pathogens and the effect of ecological conditions and host taxonomy on this diversity. METHODS: Tissue samples from BHNA (n = 17), SHNA (n = 19) and SHTZ (n = 25) were analysed by PCRs targeting Anaplasmataceae, Rickettsia spp., piroplasms, specifically Babesia lengau-like piroplasms, Hepatozoidae and filarioids. After sequencing, maximum-likelihood phylogenetic analyses were conducted. RESULTS: The relative frequency of Anaplasmataceae was significantly higher in BHNA (82.4%) and SHNA (100.0%) than in SHTZ (32.0%). Only Anaplasma phagocytophilum/platys-like and Anaplasma bovis-like sequences were detected. Rickettsia raoultii was found in one BHNA and three SHTZ. This is the first report of R. raoultii from sub-Saharan Africa. Babesia lengau-like piroplasms were found in 70.6% of BHNA, 88.9% of SHNA and 32.0% of SHTZ, showing higher sequence diversity than B. lengau from South African cheetahs (Acinonyx jubatus). In one SHTZ, a Babesia vogeli-like sequence was identified. Hepatozoon felis-like parasites were identified in 64.7% of BHNA, 36.8% of SHNA and 44.0% of SHTZ. Phylogenetic analysis placed the sequences outside the major H. felis cluster originating from wild and domestic felids. Filarioids were detected in 47.1% of BHNA, 47.4% of SHNA and 36.0% of SHTZ. Phylogenetic analysis revealed high genetic diversity and suggested the presence of several undescribed species. Co-infections were frequently detected in SHNA and BHNA (BHNA median 3 pathogens, range 1-4; SHNA median 3 pathogens, range 2-4) and significantly rarer in SHTZ (median 1, range 0-4, 9 individuals uninfected). CONCLUSIONS: The frequencies of all pathogens groups were high, and except for Rickettsia, multiple species and genotypes were identified for each pathogen group. Ecological conditions explained pathogen identity and diversity better than host taxonomy.

Detection of pathogens in ixodid ticks collected from animals and vegetation in five regions of Ukraine.

The distribution and prevalence of zoonotic pathogens infecting ixodid ticks in Western Europe have been extensively examined. However, data on ticks and tick-borne pathogens in Eastern Europe, particularly Ukraine are scarce. The objective of the current study was, therefore, to investigate the prevalence of Anaplasma phagocytophilum, Anaplasmataceae, Rickettsia spp., Babesia spp., Bartonella spp., and Borrelia burgdorferi sensu lato in engorged and questing ixodid ticks collected from five administrative regions (oblasts) of Ukraine, namely Chernivtsi, Khmelnytskyi, Kyiv, Ternopil, and Vinnytsia. The ticks were collected from both wild and domestic animals and from vegetation. Of 524 ixodid ticks collected, 3, 99, and 422 ticks were identified as Ixodes hexagonus, Ixodes ricinus, and Dermacentor reticulatus, respectively. DNA samples individually extracted from 168 questing and 354 engorged adult ticks were subjected to pathogen-specific PCR analyses. The mean prevalence in I. ricinus and D. reticulatus were, respectively: 10 % (10/97) and 3 % (12/422) for A. phagocytophilum; 69 % (67/97) and 52 % (220/422) for members of the Anaplasmataceae family; 25 % (24/97) and 28 % (117/422) for Rickettsia spp.; 3 % (3/97) and 1 % (6/422) for Babesia spp.; and 9 % (9/97) and 5 % (20/422) for Bartonella spp. Overall, between the five cities, there was no significant difference in the prevalence of any of the pathogens for the respective ticks (p > 0.05). The prevalence of B. burgdorferi s. l. in the questing and engorged I. ricinus varied from 0 to 27 % and 14-44%, respectively, with no statistical significance identified between the five cities (p > 0.05). In addition to reporting the updated data for Kyiv and Ternopil, this study is the first to provide the prevalences of the tick-borne pathogens for Chernivtsi, Khmelnytskyi, and Vinnytsia. This investigation is also the first to detect Neoehrlichia mikurensis in ixodid ticks from Ukraine. These new data will be useful for medical and veterinary practitioners as well as public health officials when diagnosing infections and when implementing measures to combat tick-borne diseases in Ukraine.

Genetic variability of Anaplasmataceae circulating in small mammals and ticks in an Ixodes persulcatus/Ixodes trianguliceps sympatric area in Russian Siberia.

A total of 705 rodents from Myodes, Microtus, and Apodemus genera, 396 adult questing Ixodes persulcatus, and 115 Ixodes larvae and nymphs taken from rodents (and then molted under laboratory conditions to nymphs and adults) were collected in 2013-2018 in Omsk province, Russian Siberia, and examined for the presence of Anaplasmataceae. DNA of Anaplasma phagocytophilum was detected in 29.5 % rodents, 3.8 % questing I. persulcatus, two molted adult I. persulcatus, and one molted adult Ixodes trianguliceps. Ehrlichia muris DNA was found in specimens from 12.1 % rodents, 3.0 % questing I. persulcatus, 14 % molted adult I. persulcatus, and one molted adult I. trianguliceps. Neoehrlichia mikurensis DNA was found in 0.6 % blood samples. It was suggested that in the studied area A. phagocytophilum and E. muris are mainly transmitted to small rodents by I. trianguliceps and I. persulcatus, respectively. Based on groEL gene sequence analysis, three phylogenetic clusters of A. phagocytophilum (clusters 4, 5, 6, according to Jaarsma et al., 2019) were identified. Most of genotyped A. phagocytophilum isolates obtained from rodents (87.6 %) and a single isolate found in a molted adult I. trianguliceps belonged to cluster 5. Cluster 6 contained 11.8 % genotyped specimens from rodents, and one questing and two molted adult I. persulcatus, while cluster 4 included specimens from 93 % genotyped questing I. persulcatus and one vole. The finding of A. phagocytophilum from clusters 5 and 6 in voles from the same sampling area indicated that clusters 5 and 6 segregate according to the tick-carriers, but not the geography. Most of the genotyped specimens of E. muris and N. mikurensis corresponded to typical genotypes detected in Asian Russia previously. In addition, new genetic variants of E. muris and N. mikurensis, which significantly differed from other known isolates and formed separate branches on phylogenetic trees, were identified in several voles.

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.

A Novel Neorickettsial Infection in 3 Dogs in the Pacific Northwest.

The genus Neorickettsia includes obligate, intracellular bacteria responsible for diseases including Potomac horse fever caused by Neorickettsia risticii and salmon poisoning disease (SPD) caused by Neorickettsia helminthoeca. The Stellanchasmus falcatus (SF) agent is a member of this genus previously associated only with mild clinical signs in dogs. Between 2013 and 2016, 3 dogs in Washington State (USA) presented with disease suggestive of SPD, but N. helminthoeca was not detected by molecular techniques. Clinical signs included depression, anorexia, and diarrhea. Cytologic examination of aspirates supported a diagnosis of granulomatous lymphadenitis with organisms suggestive of Neorickettsia. Dogs either died or were humanely euthanized due to poor response to therapy. Necropsy findings included lymphadenomegaly and hepatomegaly. Histopathology identified granulomatous and lymphoplasmacytic splenitis, lymphadenitis, enteritis, and hepatitis with extensive necrosis. Neorickettsia DNA was detected using genus-specific primers and direct sequencing showed 100% sequence identity to the SF agent in all 3 dogs. This is the first clinicopathologic description of severe disease in dogs attributed to the SF agent. These findings may suggest the emergence of a novel neorickettsial disease in the Pacific Northwest.

Neorickettsia helminthoeca associated lymphoid, enteric, and pulmonary lesions in dogs from Southern Brazil: An immunohistochemical study.

Neorickettsia helminthoeca (NH), the agent of salmon poisoning disease or canine neorickettiosis (CN), is a bacterial endosymbiont of the nematode Nanophyetus salmincola, and infections are spreading among specific fish-eating mammalians. This article describes the pathologic and immunohistochemical findings associated with spontaneous NH-induced infections in dogs from Southern Brazil. The principal pathologic findings were hypertrophy of Peyer patches and lymphadenopathy with lymphocytic proliferation, chronic interstitial pneumonia, and chronic enteritis associated with positive intralesional immunoreactivity to antigens of NH within macrophages and histiocytes. Positive immunoreactivity against canine parvovirus-2 (CPV-2) or/and canine distemper virus was not detected in the evaluated intestinal segments or in the samples from the cerebellum and lungs, respectively, from the dogs evaluated. These findings demonstrated that NH was involved in the enteric, pulmonary, and lymphoid lesions herein described, and provide additional information to confirm the occurrence of this bacterial endosymbiont within this geographical location. It is proposed that chronic pneumonia should be considered as a pathologic manifestation of NH-induced infections. Additionally, our results show that the occurrences of CN seem to be underdiagnosed in Southern Brazil due to the confusion with the incidence of CPV-2.

Medfly-Wolbachia symbiosis: genotype x genotype interactions determine host's life history traits under mass rearing conditions.

BACKGROUND: Wolbachia pipientis is a widespread, obligatory intracellular and maternally inherited bacterium, that induces a wide range of reproductive alterations to its hosts. Cytoplasmic Incompatibility (CI) is causing embryonic lethality, the most common of them. Despite that Wolbachia-borne sterility has been proposed as an environmental friendly pest control method (Incompatible Insect Technique, IIT) since 1970s, the fact that Wolbachia modifies important fitness components of its hosts sets severe barriers to IIT implementation. Mass rearing of Mediterranean fruit fly, Ceratitis capitata (medfly), is highly optimized given that this pest is a model species regarding the implementation of another sterility based pest control method, the Sterile Insect Technique (SIT). We used the medfly-Wolbachia symbiotic association, as a model system, to study the effect of two different Wolbachia strains, on the life history traits of 2 C. capitata lines with different genomic background. RESULTS: Wolbachia effects are regulated by both C. capitata genetic background and the Wolbachia strain. Wolbachia infection reduces fertility rates in both C. capitata genetic backgrounds and shortens the pre-pupa developmental duration in the GSS strain. On the other hand, regardless of the strain of Wolbachia (wCer2, wCer4) infection does not affect either the sex ratio or the longevity of adults. wCer4 infection imposed a reduction in females' fecundity but wCer2 did not. Male mating competitiveness, adults flight ability and longevity under water and food deprivation were affected by both the genetic background of medfly and the strain of Wolbachia (genotype by genotype interaction). CONCLUSION: Wolbachia infection could alter important life history traits of mass-reared C. capitata lines and therefore the response of each genotype on the Wolbachia infection should be considered toward ensuring the productivity of the Wolbachia-infected insects under mass-rearing conditions.

Molecular investigation and phylogeny of species of the Anaplasmataceae infecting animals and ticks in Senegal.

BACKGROUND: Our study aimed to assess the diversity of the species of Anaplasmataceae in Senegal that infect animals and ticks in three areas: near Keur Momar Sarr (northern region), Dielmo and Diop (Sine Saloum, central region of Senegal), and in Casamance (southern region of Senegal). METHODS: A total of 204 ticks and 433 blood samples were collected from ruminants, horses, donkeys and dogs. Ticks were identified morphologically and by molecular characterization targeting the 12S rRNA gene. Molecular characterization of species of Anaplasmataceae infecting Senegalese ticks and animals was conducted using the 23S rRNA, 16S rRNA, rpoB and groEL genes. RESULTS: Ticks were identified as Rhipicephalus evertsi evertsi (84.3%), Hyalomma rufipes (8.3%), Hyalomma impeltatum (4.9%), R. bursa (1.5%) and R. muhsamae (0.9%). The overall prevalence of Anaplasmataceae infection in ticks was 0.9%, whereas 41.1% of the sampled animals were found infected by one of the species belonging to this family. We identified the pathogen Anaplasma ovis in 55.9% of sheep, A. marginale and A. centrale in 19.4% and 8.1%, respectively, of cattle, as well as a putative new species of Anaplasmataceae. Two Anaplasma species commonly infecting ruminants were identified. Anaplasma cf. platys, closely related to A. platys was identified in 19.8% of sheep, 27.7% of goats and 22.6% of cattle, whereas a putative new species, named here provisionally "Candidatus Anaplasma africae", was identified in 3.7% of sheep, 10.3% of goats and 8.1% of cattle. Ehrlichia canis and Anaplasma platys were identified only from dogs sampled in the Keur Momar Sarr area. Ehrlichia canis was identified in 18.8% of dogs and two R. e. evertsi ticks removed from the same sheep. Anaplasma platys was identified in 15.6% of dogs. Neither of the dogs sampled from Casamance region nor the horses and donkeys sampled from Keur Momar Sarr area were found infected by an Anaplasmataceae species. CONCLUSIONS: This study presents a summary of Anaplasmataceae species that infect animals and ticks in three areas from the northern, central and southern regions of Senegal. To our knowledge, our findings demonstrate for the first time the presence of multiple Anaplasmataceae species that infect ticks and domestic animals in Senegal. We recorded two potentially new species commonly infecting ruminants named here provisionally as Anaplasma cf. platys and "Candidatus Anaplasma africae". However, E. canis was the only species identified and amplified from ticks. None of the other Anaplasmataceae species identified in animals were identified in the tick species collected from animals.

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.

IMMUNOHISTOCHEMICAL AND MOLECULAR EVIDENCE OF PUTATIVE NEORICKETTSIA INFECTION IN COATIS ( NASUA NASUA) FROM SOUTHERN BRAZIL.

The pathologic, molecular, and immunohistochemical findings associated with Neorickettsia helminthoeca are described in coatis ( Nasua nasua). Tissue sections (small intestine, lungs, kidney, liver, and spleen) of coatis ( n = 3) that died at the Bela Vista Biological Refuge, Foz do Iguaçu, Paraná, southern Brazil were routinely processed from histopathology. Selected formalin-fixed paraffin-embedded (FFPE) tissue sections of the small intestine, lungs, and spleen were used in an immunohistochemical (IHC) assay designed to identify the antigens of N. helminthoeca. Additionally, FFPE tissue sections of the small intestine were used to demonstrate antigens of canine parvovirus-2 (CPV-2) by IHC. Histopathology revealed chronic enteritis in all coatis. Parasitic enteritis was diagnosed in two coatis; one of these contained examples of a trematode within the lumen of the small intestine and the ovum of a trematode encysted in the intestinal mucosa. Other significant pathologic findings included interstitial pneumonia ( n = 2) and pyogranulomatous splenitis ( n = 1). Positive immunolabeling for N. helminthoeca was identified within macrophages of the small intestine and reticuloendothelial cells within the germinal centers of the spleen of all coatis; the intestinal trematode was N. helminthoeca IHC-positive. All pulmonary sections revealed negative immunolabeling for N. helminthoeca. Furthermore, the antigens of CPV-2 were not identified in the intestine of any coati. These findings indicate that these coatis were infected by N. helminthoeca, but since clinical and gross pathological findings were not recorded, it is uncertain if this pathogen produced clinical disease in this canid host; therefore, coatis may be asymptomatic or dead-end hosts for this organism.

Detection of Anaplasmataceae agents and co-infection with other tick-borne protozoa in dogs and Rhipicephalus sanguineus sensu lato ticks.

Anaplasmosis and ehrlichiosis are of serious health concern worldwide for animals and humans. In the present study, we report the occurrence of Anaplasma platys and Ehrlichia canis in dogs and Rhipicephalus sanguineus sensu lato (s.l.) ticks from Peninsular Malaysia using a nested polymerase chain reaction assay based on amplification of the 16S rRNA gene. Anaplasma platys was detected from dogs and ticks with prevalence rates of 3.3% (8/240) and 2.9% (4/140), respectively. On the other hand, 12.9% (31/240) of the dogs and 0.7% (1/140) of the ticks were tested positive for E. canis. Additionally, co-infections of A. platys and E. canis with Babesia or Hepatozoon protozoa were also noted in this study. Double infection (E. canis + B. gibsoni) was observed in tick, whereas triple infections (E. canis + A. platys + B. vogeli and E. canis + A. platys + H. canis) were found in dogs. This study represents the first evidence of A. platys DNA in R. sanguineus s.l. in Malaysia.

Molecular evidence of a badger-associated Ehrlichia sp., a Candidatus Neoehrlichia lotoris-like genotype and Anaplasma marginale in dogs.

The family Anaplasmataceae contains pathogenic and endosymbiotic bacteria of veterinary-medical importance. In this study, 90 blood samples from rural dogs, five blood samples from road-killed European badgers and 34 ticks, i.e. 27 Ixodes (Pholeoixodes) canisuga, six I. (Ph.) hexagonus and one Haemaphysalis concinna collected from the badgers were molecularly analysed for members of Anaplasmataceae. Apart from the molecular evidence of Anaplasma phagocytophilum in one dog and Wolbachia sp. associated with Dirofilaria repens in five dogs, four species/genotypes not yet known to occur in canine hosts have also been found. These included A. marginale in two dogs, a badger-associated Ehrlichia sp. in one dog, a Candidatus Neoehrlichia lotoris-like genotype in six dogs and the DNA of arthropod-associated wolbachiae in three dogs. In two badgers the DNA from the Candidatus N. lotoris-like genotype was identified. Among ticks, four I. canisuga carried the DNA of the above badger-associated Ehrlichia sp., one I. canisuga contained the Candidatus N. lotoris-like genotype, and in H. concinna Wolbachia DNA was present. In conclusion, results shown here should be interpreted as the first molecular evidence for exposure of dogs to three members of Anaplasmataceae, i.e. A. marginale, a badger-associated Ehrlichia sp. and a Candidatus N. lotoris-like agent. The presence of DNA in the blood of relevant animals may also indicate susceptibility to these bacteria, but in support of this, further studies are needed.

Prevalence of Anaplasmataceae and Filariidae species in unowned and military dogs in New Caledonia.

Dogs are competent reservoir hosts of several zoonotic agents, including Filariidae nematodes and Anaplasmataceae family bacteria. The latter family unites human and veterinary pathogens (Anaplasma, Ehrlichia and Neorickettsia bacteria) with Wolbachia, some of which are obligatory endosymbionts of pathogenic filarial nematodes. The epidemiology of Anaplasmataceae and Filariidae species infecting dogs living in kennels in New Caledonia was studied. 64 EDTA blood samples were screened for the presence of Anaplasmataceae and filarial nematodes. Molecular study was conducted using primers and probe targeting the of 23S rRNA long fragment of Anaplasmataceae species. Next, all blood sample was screened for the presence of Filariidae species targeting the primers and probe targeting the COI gene, as well as primers targeting the COI and 5S rRNA genes of all filarial worms. Anaplasma platys was identified in 8/64 (12.5, 95% confidence interval [CI]: 4.4-20.6%) and Wolbachia endosymbiont of Dirofilaria immitis in 8/64 (12.5%, CI: 4.4-20.6%). Filariidae species investigation was performed and showed that 11/64 (17.2%, CI: 7.9-26.4%) dogs were infected with D. immitis, whereas, 2/64 (3.1%, CI: 0.0-7.3%) were infected with Acanthocheilonema reconditum. Finally, we checked the occurrence of co-infection between Anaplasmataceae and Filariidae species. Co-occurrence with Wolbachia endosymbiont of D. immitis was observed in seven dogs, one dog was co-infected with A. platys and A. reconditum and another was co-infected with Wolbachia endosymbiont of D. immitis and A. reconditum. These results are the first report of Anaplasmataceae and Filariidae occurring in dogs in New Caledonia.

Tick-borne haemoparasites and Anaplasmataceae in domestic dogs in Zambia.

Tick-borne diseases (TBDs), including emerging and re-emerging infectious diseases, are important threats to human and animal health worldwide. Indeed, the number of reported human and animal infectious cases of novel TBD agents has increased in recent decades. However, TBDs tend to be neglected, especially in resource-limited countries that often have limited diagnostic capacity. The aim of this molecular survey was to detect and characterise tick-borne pathogens (Babesia, Theileria, and Hepatozoon parasites and Anaplasmataceae bacteria) in domestic dogs in Zambia. In total, 247 canine peripheral blood samples were collected in Lusaka, Mazabuka, Monze, and Shangombo. Conventional PCR to detect the selected pathogens was performed using DNA extracted from canine blood. One hundred eleven samples were positive for protozoa and 5 were positive for Anaplasmataceae. Sequencing of thirty-five randomly selected protozoa-positive samples revealed the presence of Babesia rossi, Babesia vogeli, and Hepatozoon canis 18S rDNA. Based on these sequences, a multiplex PCR system was developed to yield PCR products with different amplicons, the size of which depended on the parasite species; thus, each species could be identified without the need for sequence analysis. Approximately 40% of dogs were positive for H. canis. In particular, the positive rate (75.2%) of H. canis infection was significantly higher in Shangombo than in other sampling sites. Multiplex PCR assay detected B. rossi and B. vogeli infections in five and seven dogs, respectively, indicating that this approach is useful for detecting parasites with low prevalence. Sequencing analysis of gltA and groEL genes of Anaplasmataceae revealed that two and one dogs in Lusaka were infected with Anaplasma platys and Ehrlichia canis, respectively. The data indicated that Zambian dogs were infected with multiple tick-borne pathogens such as H. canis, B. rossi, B. vogeli, A. platys, E. canis and uncharacterized Ehrlichia sp. Since some of these parasites are zoonotic, concerted efforts are needed to raise awareness of, and control, these tick-borne pathogens.