Neoehrlichia mikurensis is uncommon in rheumatological patients receiving tumour necrosis factor inhibitors and in blood donors: a retrospective cohort study.

INTRODUCTION: Neoehrlichia mikurensis is a tick-borne bacterium that primarily causes disease in immunocompromised patients. The bacterium has been detected in ticks throughout Europe, with a 0%-25% prevalence. N. mikurensis infection presents unspecific symptoms, which can easily be mistaken for inflammatory disease activity. We aimed to determine the prevalence of N. mikurensis in rheumatological patients receiving tumour necrosis factor inhibitors (TNFi) and a cohort of healthy individuals. MATERIALS AND METHODS: This retrospective cohort study included 400 rheumatological patients treated with TNFi and 400 healthy blood donors. Plasma samples were retrieved from the Danish Rheumatological Biobank and the Danish Blood Donor Study between 2015 and 2022. Age, sex, diagnosis and duration of TNFi treatment were recovered from the Danish Rheumatological Database, DANBIO. Data on age and sex were available for the blood donors. One plasma sample per individual was tested for N. mikurensis DNA-specific real-time PCR targeting the groEL gene. RESULTS: In the rheumatological patients, the median age was 61 years (IQR 55-68 years), 62% were women, and 44% had a diagnosis of seropositive rheumatoid arthritis. In total, 54% of the patients were treated with infliximab. The median time from TNFi initiation to blood sampling was 20 months (IQR, 5-60 months). N. mikurensis DNA was not detected in any samples from patients or blood donors. CONCLUSION: N. mikurensis infection does not appear to represent a prevalent risk in Danish rheumatological patients receiving TNFi or in blood donors.

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).

[Neoehrlichiosis has entered the Stockholm region]. / Ovanlig fästingsjukdom har gjort sitt intåg i Stockholmsregionen.

The number of cases diagnosed with neoehrlichiosis in Stockholm has increased over the last years. PCR analysis is needed for the detection of the intracellular bacterium Neoehrlichia mikurensis. The real number of cases in the area is unknown since the specific PCR for N mikurensis is not routinely included in the workup for unknown fever in Stockholm. By describing three cases, we want to increase the awareness of neoehrlichiosis among clinicians. Symptoms of prolonged fever, myalgia and thrombosis among immunocompromised patients should raise the suspicion of neoehrlichiosis and the specific PCR analysis should be performed. The diagnosed patients were all treated with doxycycline; the fever disappeared within a few days, and clinical improvement was observed. After treatment no relapses were noticed, despite immunological deficiencies in the patients.

Neoehrlichia mikurensis in Danish immunocompromised patients: a retrospective cohort study.

BACKGROUND: The tick-borne bacterium, Neoehrlichia mikurensis (N. mikurensis) can cause severe febrile illness and thromboembolic complications in immunocompromised individuals. We investigated the presence of N. mikurensis DNA in retrospectively collected plasma from a well-characterized cohort of Danish immunocompromised patients. METHODS: Plasma samples from 239 patients with immune dysfunction related to hematological or rheumatological disease or due to immunosuppressive therapy, were retrieved from a transdisciplinary biobank (PERSIMUNE) at Rigshospitalet, Copenhagen, Denmark. Serving as immunocompetent controls, plasma samples from 192 blood donors were included. All samples were collected between 2015 and 2019. Real-time PCR targeting the groEL gene was used to detect N. mikurensis DNA. Sequencing was used for confirmation. Borrelia burgdorferi sensu lato IgG antibodies were detected by ELISA as a proxy of tick exposure. Prevalence was compared using Fisher's exact test. RESULTS: Neoehrlichia mikurensis DNA was detected in 3/239 (1.3%, 95% confidence interval (CI): 0.3 - 3.6%) patients, all of whom primarily had a hematological disease. Follow-up samples of these patients were negative. N. mikurensis DNA was not detected in any of the blood donor samples. IgG antibodies against B. burgdorferi s.l. were detected with similar prevalence in immunocompromised patients and blood donors, i.e., 18/239 (7.5%, 95% CI: 4.8-11.5%) and 11/192 (5.7%, 95%: CI 3.2-10.0%). CONCLUSION: In this study, patients with N. mikurensis were not identified by clinical indication and N. mikurensis may therefore be underdiagnosed in Danish patients. Further investigations are needed to explore the clinical significance and implications of this infection.

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.

Infection with Neoehrlichia mikurensis promotes the development of malignant B-cell lymphomas.

The tick-borne pathogen Neoehrlichia (N.) mikurensis is implicated in persistent infection of the vascular endothelium. B cells are crucial for the host defence to this infection. Chronic stimulation of B cells may result in B-cell transformation and lymphoma. Five patients with malignant B-cell lymphoma and concomitant N. mikurensis infection were investigated regarding clinical picture, lymphoma subtype, B-cell lymphoma immunophenotype and IGHV (variable region of the immunoglobulin heavy) gene repertoire. Three of the five patients improved markedly and ceased lymphoma treatment after doxycycline treatment to eliminate N. mikurensis. Sequencing the B-cell lymphoma IGHV genes revealed preferred usage of the IGHV1 (IGHV1-2, and -69) and IGHV3 (IGHV3-15, -21, -23) families. In conclusion, N. mikurensis infection may drive the development of malignant B-cell lymphomas. Eradication of the pathogen appears to induce remission with apparent curing of the lymphoma 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.

Cytokine responses of immunosuppressed and immunocompetent patients with Neoehrlichia mikurensis infection.

PURPOSE: The tick-borne bacterium Neoehrlichia mikurensis causes the infectious disease neoehrlichiosis in humans. Vascular endothelium is one of the target cells of the infection. Neoehrlichiosis patients with compromised B cell immunity present with more severe inflammation than immunocompetent patients. The aim of this study was to compare the cytokine profiles of immunocompetent and immunosuppressed patients with neoehrlichiosis. METHODS: Blood samples from Swedish and Norwegian immunosuppressed (N = 30) and immunocompetent (N = 16) patients with neoehrlichiosis were analyzed for the levels of 30 cytokines, using a multiplex cytokine assay and ELISA. A gender-matched healthy control group (N = 14) was analyzed in parallel. Data were analyzed using the multivariate method OPLS-DA. RESULTS: The multiplex cytokine analyses generated more cytokine results than did the uniplex ELISA analyses. Multivariate analysis of the multiplex cytokine results established that increased levels of FGF2, GM-CSF, CXCL10, and IFN-γ were associated with immunosuppressed patients, whereas increased levels of IL-15 and VEGF were associated with immunocompetent neoehrlichiosis patients. When multivariate analysis findings were confirmed with uniplex ELISA, it was found that both groups of patients had similarly elevated levels of VEGF, FGF2 and IFN-γ. In contrast, the immunosuppressed patients had clearly elevated levels of CXCL10, CXCL13 and BAFF, whereas the immunocompetent patients had the same levels as healthy controls. CONCLUSION: Pro-angiogenic and type 1 cytokines were produced as part of the host response of neoehrlichiosis independent of immune status, whereas immunosuppressed neoehrlichiosis patients produced cytokines required for B cell-mediated defense.

[Neoehrlichiosis as a cause of feaver of unknown origin]. / Neoehrlichiose als Ursache eines Fiebers unklarer Genese.

Establishing a diagnosis in cases of fever of unknown origin (FUO) in immunocompromised patients can be difficult. In 25-35% infectious diseases are the underlying cause. This article reports the case of a 74-year-old woman with a 5-month history of fever. Through open biopsy of the femoral shaft and microbiological analysis, a diagnosis of neoehrlichiosis could be established. After initiation of treatment with doxycycline, the symptoms quickly resolved resulting in a complete recovery.

Aedes albopictus Strain and Dengue Virus Serotype in the Dengue Fever Outbreaks in Japan: Implications of Wolbachia Infection.

From August 27 to October 15, 2014, a dengue fever outbreak with 158 autochthonous cases occurred after nearly 70 years of no reports of autochthonous cases in Japan. The most competent mosquito vector for dengue virus (DENV) transmission in Japan is Aedes albopictus. Since A. albopictus is widely distributed throughout Japan, we examined the susceptibility of this species to infection by DENV and the relationship of the endosymbiont Wolbachia (wAlbA and wAlbB) with susceptibility to DENV. The A. albopictus YYG strain, collected from the Yoyogi Park in 2014, the epicenter of the dengue fever outbreak, was found to have lower susceptibility to DENV 1 and 3 than that of the indigenous Japanese strains A. albopictus EBN 201808 (F1 from the field) and A. albopictus ISG 201603. Furthermore, the A. albopictus EBN 201808 strain showed the same susceptibility to DENV3 as the A. albopictus ISG 201603tet strain (Wolbachia-free). Susceptibility to DENV3 was not related to Wolbachia strains wAlbA or wAlbB in the A. albopictus ISG 201603 strain.

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.

Case report: first symptomatic Candidatus Neoehrlichia mikurensis infection in Slovenia.

BACKGROUND: Candidatus Neoehrlichia mikurensis (CNM) is an emerging tick-born pathogen and usually causes symptomatic infection only in immunocompromised patients. Apart from one described case found in the literature where cultivation was successful, all cases so far were diagnosed by using broad-range 16S rDNA PCR. CASE PRESENTATION: Our patient presented with a prolonged febrile state of unknown origin. Clinical presentation, extensive medical workup and classic microbiologic testing were non-conclusive. Several infectious agents and other causes for the febrile state were excluded. In the end, a broad-range 16S rDNA PCR was to be performed to confirm the diagnosis of CNM infection. Treatment was successful with doxycycline. CONCLUSIONS: Due to the obscurity of the pathogen, diagnostic workup in CNM is prolonged and challenging. More awareness is need about this emerging infectious disease in countries with high prevalence of tick-borne diseases as standard microbiological methods are not successful in confirming the diagnosis.

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.

Wolbachia infection in wild mosquitoes (Diptera: Culicidae): implications for transmission modes and host-endosymbiont associations in Singapore.

BACKGROUND: Wolbachia are intracellular bacterial endosymbionts found in most insect lineages. In mosquitoes, the influence of these endosymbionts on host reproduction and arboviral transmission has spurred numerous studies aimed at using Wolbachia infection as a vector control technique. However, there are several knowledge gaps in the literature and little is known about natural Wolbachia infection across species, their transmission modes, or associations between various Wolbachia lineages and their hosts. This study aims to address these gaps by exploring mosquito-Wolbachia associations and their evolutionary implications. METHODS: We conducted tissue-specific polymerase chain reaction screening for Wolbachia infection in the leg, gut and reproductive tissues of wild mosquitoes from Singapore using the Wolbachia surface protein gene (wsp) molecular marker. Mosquito-Wolbachia associations were explored using three methods-tanglegram, distance-based, and event-based methods-and by inferred instances of vertical transmission and host shifts. RESULTS: Adult mosquitoes (271 specimens) representing 14 genera and 40 species were screened for Wolbachia. Overall, 21 species (51.2%) were found positive for Wolbachia, including five in the genus Aedes and five in the genus Culex. To our knowledge, Wolbachia infections have not been previously reported in seven of these 21 species: Aedes nr. fumidus, Aedes annandalei, Uranotaenia obscura, Uranotaenia trilineata, Verrallina butleri, Verrallina sp. and Zeugnomyia gracilis. Wolbachia were predominantly detected in the reproductive tissues, which is an indication of vertical transmission. However, Wolbachia infection rates varied widely within a mosquito host species. There was no clear signal of cophylogeny between the mosquito hosts and the 12 putative Wolbachia strains observed in this study. Host shift events were also observed. CONCLUSIONS: Our results suggest that the mosquito-Wolbachia relationship is complex and that combinations of transmission modes and multiple evolutionary events likely explain the observed distribution of Wolbachia diversity across mosquito hosts. These findings have implications for a better understanding of the diversity and ecology of Wolbachia and for their utility as biocontrol agents.

Epidemiology and genotyping of Anaplasma marginale and co-infection with piroplasms and other Anaplasmataceae in cattle and buffaloes from Egypt.

BACKGROUND: Anaplasma marginale is an obligate intracellular bacterium and the main cause of bovine anaplasmosis in tropical and subtropical regions. In Egypt, data regarding the prevalence of A. marginale in ruminant hosts and of the circulating genotypes is lacking. This study therefore aimed to (i) investigate the presence, epidemiology and genotypes of A. marginale in cattle and buffaloes in Egypt, (ii) to evaluate suitable diagnostic tools and (iii) to identify co-infections of A. marginale with other selected tick-borne pathogens. METHODS: Blood samples were collected from 394 animals (309 cattle and 85 buffaloes) from three different areas in Egypt. For the detection of A. marginale infection, several tests were compared for their sensitivity and specificity: blood smear analysis, enzyme-linked immunosorbent assay (ELISA), PCR, real-time PCR and reverse line blot (RLB) assay. Co-infections with A. marginale, piroplasms and other Anaplasmataceae were surveyed by RLB while A. marginale genotypes were identified by amplifying and sequencing the partial msp1α gene. RESULTS: Anaplasma marginale DNA was amplified by qPCR in 68.3% of cattle and 29.4% of buffaloes. RLB showed infection with A. marginale in 50.2% of cattle and 42.5% of buffaloes. Blood smear analysis detected this agent in 16.2% of cattle and 2.4% of buffaloes. ELISA showed specific antibodies against A. marginale in 54.9% of cattle. Anaplasma marginale was associated, in cattle and buffaloes, with several tick-borne pathogens (Theileria annulata, Babesia bovis, Babesia bigemina, Babesia occultans and Anaplasma platys). A significant difference of A. marginale infection level was noticed in cattle, where animals between 3-5-years-old had a higher prevalence (79.2%) compared to those older than 5 years (36.4%) and younger than 3 years (59.7%) and one year (64.5%), respectively (P = 0.002281). Microsatellite analysis identified 15 different genotypes. CONCLUSIONS: The epidemiological findings revealed high prevalence of A. marginale in cattle and buffaloes in all the investigated areas. The circulation of diverse genotypes was observed, most of these A. marginale genotypes being specific for Egypt. The qPCR assay was confirmed to be the most sensitive tool for detection of A. marginale in cattle and buffaloes even in the carrier state, highlighting the importance of using suitable diagnostic tests.

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.

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.

Quantitative analysis of Anaplasma marginale acquisition and transmission by Dermacentor andersoni fed in vitro.

In this study, we describe a new in vitro tick feeding system that facilitates the study of ticks and tick-borne pathogens. To optimize the system, we used Dermacentor andersoni and Anaplasma marginale as a tick-pathogen interaction model. Ticks were fed on bovine blood containing 10-fold dilutions of the pathogen to determine the effect of dose on tick infection rate. After feeding on infected blood, ticks were transferred to uninfected blood to stimulate bacterial replication within the tick vector. During stimulation feeding, blood samples were collected daily to determine if infected ticks secreted viable A. marginale. The results demonstrated similar attachment rates between the first and second tick feeding. Tick midgut and salivary glands were infected with A. marginale. However, salivary gland infection rates decreased as the percentage of parasitized erythrocytes decreased during tick acquisition feeding. Bacteria recovered from the in vitro system were able to infect a naïve bovine host. Using the highly transmissible A. marginale St. Maries strain, we demonstrated that the artificial tick feeding system is a suitable tool to study tick-pathogen interactions and that A. marginale tick salivary gland infection is dose dependent. This work demonstrates the utility of an artificial tick feeding system to directly study the association between the number of acquired pathogens and transmissibility by ticks.