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For several years, there has been increasing awareness of a novel tick-borne infection within our region: neoehrlichiosis. The causative agent, Neoehrlichia mikurensis , is an intracellular bacterium classified within the Anaplasmataceae family. Predominantly afflicting immunosuppressed individuals, its clinical presentation often involves persistent fever accompanied by vascular complications. Diagnostic protocols typically entail targeted PCR testing of blood specimens, often leading to diagnostic delays. Timely initiation of doxycycline therapy typically ensures prompt resolution of symptoms., Competing Interests: Les auteurs n’ont déclaré aucun conflit d’intérêts en relation avec cet article.

Canine tick-borne diseases, such as babesiosis, rangeliosis, hepatozoonosis, anaplasmosis and ehrlichiosis, are of veterinarian relevance, causing mild or severe clinical cases that can lead to the death of the dog. The aim of this study was detecting tick-borne protozoan and rickettsial infections in dogs with anemia and/or thrombocytopenia in Uruguay. A total of 803 domestic dogs were evaluated, and 10% were found positive (detected by PCR) at least for one hemoparasite. Sequence analysis confirmed the presence of four hemoprotozoan species: Rangelia vitalii, Babesia vogeli, Hepatozoon canis and Hepatozoon americanum, and the rickettsial Anaplasma platys. The most detected hemoparasite was R. vitalii, followed by H. canis and A. platys. This is the first report of B. vogeli in Uruguay and the second report of H. americanum in dogs from South America. The results highlight the importance for veterinarians to include hemoparasitic diseases in their differential diagnosis of agents causing anemia and thrombocytopenia., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Vector-borne diseases (VBDs) affecting dromedary camels (Camelus dromedarius) have considerable importance in the United Arab Emirates (UAE) because of the consequences associated with production decline and economic losses. Our study aimed to determine the prevalence of selected VBDs in camels in the UAE and identify risk factors. This research is currently affected by the low number of epidemiological molecular surveys addressing this issue. Blood samples were obtained from 425 dromedary camels from different locations across the UAE. Whole genomic DNA was isolated, and PCR screening was done to detect piroplasmids (Babesia/Theileria spp.), Trypanosoma spp., and Anaplasmataceae spp. (Anaplasma, Ehrlichia, Neorickettsia and Wolbachia spp.). Amplicons were sequenced, and phylogenetic trees were constructed. Trypanosoma sequences were identified as T. brucei evansi, whereas Anaplasmataceae sequences were identified as A. platys-like. All camels were negative for Babesia/Theileria spp. (0%); however, 18 camels were positive for T. b. evansi (4%) and 52 were positive for A. platys-like (12%). Mixed infection with T. b. evansi and A. platys-like was found in one camel. Statistical analyses revealed that camels with a brown coat colour were significantly more prone to acquire the A. platys-like strain compared with those having a clearer coat. A similar finding was observed when comparing urban moving camels with desert indoor and urban indoor camels. Continuous disease surveillance is required to ensure and maintain the good health status of the camels in the UAE. Nonetheless, the risk of disease outbreak remains if the misuse of drugs continues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Tick-borne diseases (TBD) are considered neglected diseases in Thailand with disease burden likely underestimated. To assess risk for emerging TBD in Thailand, the seasonality of questing tick and pathogen prevalence were studied in Khao Yai National Park, a top tourist destination., Methods: During 2019, questing ticks around tourist attractions were systematically collected bimonthly and analyzed for Rickettsia and Anaplasmataceae bacterial species by polymerase chain reaction and DNA sequencing., Results: Larvae and nymphs of questing ticks peaked in Khao Yai National Park during the late rainy-winter season, though no specific trends were observed in adult ticks. Winter (November to February) was the highest risk for human tick-bites due to higher numbers of both ticks and visitors. Of the total 5916 ticks analyzed (651 pools), Anaplasma phagocytophilum, Neoehrlichia mikurensis, Ehrlichia ewingii, and Ehrlichia chaffeensis were detected at low rates (≤0.05%). There was a higher prevalence of human rickettsioses (0.2-7%) in ticks surveyed with Rickettsia tamurae, Rickettsia raoultii, and Rickettsia montana the major species. Amblyomma ticks had the highest prevalence of Rickettsia (85%, 35/44 Amblyomma adults), in which only R. tamurae and R. raoultii were found in Amblyomma with mixed species infections common. We report the first detection of R. africae-like and N. mikurensis in Ixodes granulatus adults in Thailand, suggesting I. granulatus as a potential vector for these pathogens., Conclusion: This study demonstrated the risk of emerging TBD in Thailand and underscores the need for tick-bite prevention among tourists in Thailand., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tick-borne microorganisms in many tick species and many areas of China are still not thoroughly investigated. In this study, 224 ticks including two species (Haemaphysalis longicornis and Haemaphysalis qinghaiensis) were collected from four cities in Hebei, Shandong, and Qinghai provinces, China. Ticks were screened for the presence of tick-borne bacterial microorganisms including Rickettsia, Anaplasmataceae (Anaplasma, Ehrlichia, Neoehrlichia, etc.), Coxiella, Borrelia, and Bartonella. Two Anaplasma species (Anaplasma ovis and Anaplasma capra) were detected in H. longicornis from Xingtai City of Hebei Province, with a positive rate of 3 % and 8 %, respectively. A Coxiella species was detected in H. longicornis ticks from all three locations in Hebei and Shandong provinces, with the positive rate ranging from 30 to 75 %. All the 16S and rpoB sequences were very similar (99.77-100 % identity) to Coxiella endosymbiont of Haemaphysalis ticks. An Ehrlichia species was detected in H. qinghaiensis (6/66, 9 %) from Xining City, Qinghai Province. The 16S and groEL sequences had 100 % and 97.40-97.85 % nucleotide identities to "Candidatus Ehrlichia pampeana" strains, respectively, suggesting that it may be a variant of "Candidatus Ehrlichia pampeana". All the ticks were negative for Rickettsia, Borrelia, and Bartonella. Because all the ticks were removed from goats or humans and were partially or fully engorged, it is possible that the microorganisms were from the blood meal but not vectored by the ticks. Our results may provide some information on the diversity and distribution of tick-borne pathogens in China., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The aim of this study was to report the first detection of Candidatus Ehrlichia pampeana in Haemaphysalis juxtakochi from Argentina. Free-living ticks were collected from vegetation by drag-flag method on five sample sites in Entre Ríos Province, central Argentina, belonging to the Espinal Phytogeographic Province. Molecular detection of order Rickettsiales agents was performed using gltA (Rickettsia spp.), 16 S rRNA and groEL (Anaplasmataceae) genes as targets. A total of 67 ticks of Amblyomma aureolatum (20 nymphs and 4 adults), Amblyomma dubitatum (15 nymphs) and Haemaphysalis juxtakochi (24 nymphs and 4 adults) were collected. While all tested ticks were negative for Rickettsia spp., and Anaplasmataceae agents could neither be detected in A. aureolatum nor in A. dubitatum, Candidatus Ehrlichia pampeana was detected in one male of H. juxtakochi. DNA sequences of this microorganism (16 S rDNA and groEL) are related to sequences of Ehrlichia ewingii. The findings of the current study represent the first report of this Ehrlichia strain for Argentina., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: Competing interests: This work was supported as part of NorthTick, an Interreg project supported by the North Sea Programme of the European Regional Development Fund of the European Union (Grant number 38-2-7-19). The funding bodies had no role in writing the manuscript. Outside of the present work: MØ has received an unrestricted research grant from Rigshospitalets Forskningspuljer. AML discloses speaker honoraria and advisory board activities from Gilead, ViiV/GSK, and Pfizer. AML reports a grant from The Lundbeck Foundation (R366-2021-127) and Aase and Ejnar Danielsen’s Foundation. BG discloses research grants (paid to institution) from AbbVie, Sandoz. MLH discloses research grants (paid to institution) from AbbVie, Biogen, BMS, Celltrion, Eli Lilly, Janssen Biologics B.V, Lundbeck Foundation, MSD, Pfizer, Roche, Samsung Biopsies, Sandoz and Novartis. MLH discloses honoraria (paid to institution) from Pfizer, Medac, and Sandoz. MLH has chaired the steering committee of the Danish Rheumatology Quality Registry (DANBIO, DRQ), which receives public funding from hospital owners and funding from pharmaceutical companies. MLH co-chairs EuroSpA, which generates real-world evidence of treatment of psoriatic arthritis and axial spondyloarthritis based on secondary data and is partly funded by Novartis. The other authors declare no conflict of interest., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Bacteria of the sister genera Ehrlichia and Anaplasma (Anaplasmataceae) are obligate intracellular Alphaproteobacteria that are transmitted mostly through arthropod vectors. These agents can infect different vertebrate cells, depending on the species involved, and can cause diseases in animals and humans. In this study, we evaluated the presence of Anaplasmataceae bacteria in Amblyomma calcaratum ticks collected from a road-killed Tamandua tetradactyla in the Rainforest ecoregion in Argentina. All samples were screened for Anaplasmataceae DNA using a real-time PCR assay targeting the 16S rRNA gene. Evidence of Anaplasmataceae DNA was detected in three out of thirty-nine Am. calcaratum ticks. Phylogenetic analysis of a portion of 16S rRNA gene positioned one sample (Ehrlichia sp. strain Ac124) with Ehrlichia sequences and the other two samples with Anaplasma sequences; Anaplasma sp. strain Ac145 close to Anaplasma odocoilei and Anaplasma sp. strain Ac152 in an ancestral position to most Anaplasma species. The groEL sequence obtained showed that Ehrlichia sp. strain Ac124 was phylogenetically related to Ehrlichia sp. strain Iberá reported infecting Amblyomma tigrinum from Iberá wetlands in Argentina. Phylogenetic analysis using the rpoB sequence positioned Anaplasma sp. strain Ac145 close to the canine pathogen Anaplasma platys, while Anaplasma sp. strain Ac152 was positioned close to the bovine pathogen Anaplasma marginale. In this study, three Anaplasmataceae agents were detected in adults of Am. calcaratum associated with a T. tetradactyla. These results suggest that the number of Anaplasmataceae species, as well as their distribution, is largely unknown., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier GmbH.)

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.

Background: Hippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria., Methods: Louse flies were collected from naturally infested cattle (n = 25), sheep (n = 3), and red deer (n = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses., Results: A total of 282 hippoboscid flies corresponding to three species were identified: Hippobosca equina (n = 62) collected from cattle, Melophagus ovinus (n = 100) from sheep and Lipoptena cervi (n = 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina. Anaplasmataceae DNA (Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida., Conclusions: Molecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. Bartonella spp.) and the first report of Trypanosoma sp. in H. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context., (© 2023. The Author(s).)

Background: Neoehrlichia mikurensis (N. mikurensis) is a newly discovered tick-borne pathogen that can inflict life-threatening illness in immunocompromised patients. N. mikurensis infection is only detectable by polymerase chain reaction (PCR)-based methodologies. We describe three distinct clinical manifestations of N. mikurensis infection (neoehrlichiosis) in Danish patients receiving B-lymphocyte-depleting therapy, rituximab, for underlying hematological, rheumatological, or neurological disorders. All three patients went through a protracted pre-diagnostic period., Methods: N. mikurensis DNA was detected and confirmed using two methods. Blood was tested by specific real-time PCR targeting the groEL gene and by 16S and 18S profiling followed by sequencing. Bone marrow was analyzed by 16S and 18S profiling., Results: N. mikurensis was detected in blood samples in all three cases and in bone marrow from one of the three. The severity of the symptoms ranged from prolonged fever lasting more than 6 months to life-threatening hyperinflammation in the form of hemophagocytic lymphohistiocytosis (HLH). Interestingly, all patients presented with splenomegaly and two with hepatomegaly. After starting doxycycline therapy, symptoms were relieved within a few days, and biochemistry and organomegaly quickly normalized., Conclusion: We present three Danish patients recognized by the same clinician over a period of 6 months, strongly suggesting that many cases are going unrecognized. Second, we describe the first case of N. mikurensis-induced HLH and emphasize the potential severity of undetected neoehrlichiosis., (© 2023 The Authors. Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.)

Ticks play an important role in the evolution and transmission of Anaplasmataceae bacteria which are agents of emerging infectious diseases. In this study, a total of 1286 adult ticks belonging to five species were collected from cattle, goats, horses and vegetation in Harbin area, Heilongjiang province, northeastern China. The tick-borne Anaplasmataceae bacteria were identified by amplifying and sequencing the 16S rRNA (rrs) and heat shock protein-60 encoding (groEL) genes. The results showed that Ixodes persulcatus was dominant (38.8%, 499/1283) among the five tick species, and Anaplasmataceae bacteria were detected in all tick species with an overall prevalence of 7.4%. Four species of Anaplasmataceae bacteria (Anaplasma phagocytophilum, Anaplasma ovis, Anaplasma bovis, and "Candidatus Neoehrlichia mikurensis"), which are pathogenic to humans and/or animals, were identified from tick samples by phylogenetic analyzes of the rrs and groEL gene sequences. Interestingly, the cluster 1 strains were first identified in Asian, and a novel cluster was also detected in this study. These data revealed the genetic diversity of Anaplasmataceae bacteria circulating in ticks in Harbin area, highlighting the need to investigate these tick-borne pathogens and their risks to human and animal health., Competing Interests: Declaration of Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier GmbH.)

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., (© 2023. The Author(s).)

We describe a case of neoehrlichiosis in an immunocompetent child with acute febrile illness in South Africa. Neoehrlichiosis was diagnosed by PCR on 16S rDNA from bone marrow aspirate. Phylogenetic analysis indicated an organism closely related to Candidatus Neoehrlichia. Clinicians should be aware of possible ehrlichiosis even in immunocompetent patients.

The family Anaplasmataceae includes tick-borne bacteria of major public and veterinary health interest, as best illustrated by members of the genera Anaplasma and Ehrlichia. Recent epidemiological surveys have also reported on the presence of a novel putative genus in the Anaplasmataceae, Candidatus Allocryptoplasma, previously described as Candidatus Cryptoplasma in the western black-legged tick, Ixodes pacificus. However, the genetic diversity of Ca. Allocryptoplasma and its phylogenetic relationship with other Anaplasmataceae remain unclear. In this study, we developed a multi-locus sequence typing approach, examining the DNA sequence variation at five genes of Ca. Allocryptoplasma found in ticks. Combining this multi-locus sequence typing and genetic data available on public databases, we found that substantial genetic diversity of Ca. Allocryptoplasma is present in Ixodes, Amblyomma and Haemaphysalis spp. ticks on most continents. Further analyses confirmed that the Ca. Allocryptoplasma of ticks, the Ca. Allocryptoplasma of lizards and some Anaplasma-like bacteria of wild mice cluster into a monophyletic genus, divergent from all other genera of the family Anaplasmataceae. Candidatus Allocryptoplasma appears as a sister genus of Anaplasma and, with the genera Ehrlichia and Neoehrlichia, they form a monophyletic subgroup of Anaplasmataceae associated with tick-borne diseases. The detection of genetically distinct Ca. Allocryptoplasma in ticks of significant medical or veterinary interest supports the hypothesis that it is an emergent genus of tick-borne pathogens of general concern., (© S. Ouass et al., published by EDP Sciences, 2023.)

The genus Anaplasma ( Anaplasmataceae, Rickettsiales ) includes tick-transmitted bacterial species of importance to both veterinary and human medicine. Apart from the traditionally recognized six Anaplasma species ( A. phagocytophilum , A. platys , A. bovis , A. ovis, A. centrale , A. marginale ), novel strains and candidate species, also of relevance to veterinary and human medicine, are emerging worldwide. Although species related to the zoonotic A. platys and A. phagocytophilum have been reported in several African and European Mediterranean countries, data on the presence of these species in sub-Saharan countries are still lacking. This manuscript reports the investigation of Anaplasma strains related to zoonotic species in ruminants in Senegal by combining different molecular tests and phylogenetic approaches. The results demonstrated a recent introduction of Candidatus ( Ca ) Anaplasma turritanum, a species related to the pathogenic A. platys , possibly originating by founder effect. Further, novel undetected strains related to Candidatus ( Ca ) Anaplasma cinensis were detected in cattle. Based on groEL and gltA molecular comparisons, we propose including these latter strains into the Candidatus ( Ca ) Anaplasma africanum species. Finally, we also report the emergence of Candidatus ( Ca ) A. boleense in Senegal. Collectively, results confirm that Anaplasma species diversity is greater than expected and should be further investigated, and that Anaplasma routine diagnostic procedures and epidemiological surveillance should take into account specificity issues raised by the presence of these novel strains, suggesting the use of a One Health approach for the management of Anaplasmataceae in sub-Saharan Africa.

Background: Microbial communities can affect disease risk by interfering with the transmission or maintenance of pathogens in blood-feeding arthropods. Here, we investigated whether bacterial communities vary between Ixodes ricinus nymphs which were or were not infected with horizontally transmitted human pathogens., Methods: Ticks from eight forest sites were tested for the presence of Borrelia burgdorferi sensu lato, Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis by quantitative polymerase chain reaction (qPCR), and their microbiomes were determined by 16S rRNA amplicon sequencing. Tick bacterial communities clustered poorly by pathogen infection status but better by geography. As a second approach, we analysed variation in tick microorganism community structure (in terms of species co-infection) across space using hierarchical modelling of species communities. For that, we analysed almost 14,000 nymphs, which were tested for the presence of horizontally transmitted pathogens B. burgdorferi s.l., A. phagocytophilum, and N. mikurensis, and the vertically transmitted tick symbionts Rickettsia helvetica, Rickettsiella spp., Spiroplasma ixodetis, and Candidatus Midichloria mitochondrii., Results: With the exception of Rickettsiella spp., all microorganisms had either significant negative (R. helvetica and A. phagocytophilum) or positive (S. ixodetis, N. mikurensis, and B. burgdorferi s.l.) associations with M. mitochondrii. Two tick symbionts, R. helvetica and S. ixodetis, were negatively associated with each other. As expected, both B. burgdorferi s.l. and N. mikurensis had a significant positive association with each other and a negative association with A. phagocytophilum. Although these few specific associations do not appear to have a large effect on the entire microbiome composition, they can still be relevant for tick-borne pathogen dynamics., Conclusions: Based on our results, we propose that M. mitochondrii alters the propensity of ticks to acquire or maintain horizontally acquired pathogens. The underlying mechanisms for some of these remarkable interactions are discussed herein and merit further investigation. Positive and negative associations between and within horizontally and vertically transmitted symbionts., (© 2022. The Author(s).)

Background: The tick-borne bacterium Neoehrlichia mikurensis can cause persistent asymptomatic bloodstream infections, but transfusion-mediated transmission has not been reported. This study aimed to investigate the prevalence of N. mikurensis in blood donors, and recipients of blood components from N. mikurensis -positive donors were traced., Methods: In 2019 and 2021, 1007 blood donors were recruited. Participants completed a questionnaire and additional blood samples were collected during blood donation. Detection of N. mikurensis was performed by PCR followed by sequencing. Positive donors were interviewed and retested. Look-back was performed on positive donations and on all subsequent donations., Results: N. mikurensis was detected in 7/1006 (0.7%) donors. A total of 380/1005 (38%) donors reported at least one noticed tick bite during the current season. The questionnaire could not detect any differences between negative and positive N. mikurensis -donors. Two of the positive donors were still positive on days 318 and 131 after the index donation, respectively. One donor with persistent N. mikurensis in blood experienced slight fatigue. All other had no symptoms attributable to neoehrlichiosis. Look-back included ten donations and 20 blood components. Eight components were discarded, and 12 recipients of N. mikurensis -positive donations were identified. PCR was negative in seven recipients. Five recipients had died, but their medical records gave no evidence for neoehrlichiosis., Conclusions: Although N. mikurensis was found in 0.7% of blood donors, transfusion-mediated infection was not detected, despite several recipients being at high risk for severe neoehrlichiosis. The results warrant further studies as well as raised clinical awareness.

Background: The incidence or recurrence of tick-borne diseases (TBDs) in animals and humans is increasing rapidly worldwide, but there is insufficient information about TBDs infecting dogs in Egypt. Thus, the present study was conducted to screen and genetically identify tick-borne pathogens (TBPs) in dogs and associated ticks by microscopic examination and polymerase chain reaction (PCR)., Methods: In Cairo and Giza governorates, 208 blood samples were collected from dogs of different breeds, ages, and sex. In addition, 1266 dog-associated ticks were collected (546 ticks were used to prepare hemolymph smears, and 720 ticks were kept in 70% ethanol until PCR analysis). PCR was applied to 124 dog blood samples and 144 tick pools prepared from 720 ticks., Results: All ticks collected from dogs were Rhipicephalus sanguineus (s.l.). Microscopic examination revealed that TBP prevalence among dogs was 23.56% (49/208), including Anaplasma and Ehrlichia with 11.1% (23/208) and Babesia canis with 8.2% (17/208). Hepatozoon canis was not detected in blood smears. Co-infections with two pathogens were visible in 4.33% (9/208) of examined dogs. The prevalence of TBPs in hemolymph smears was 45.97% (251/546) including 35.89% (196/546) for H. canis, 8.1% (44/546) for B. canis, and 2.01% (11/546) for Anaplasmataceae (A. phagocytophilum, A. marginale, A. platys, and E. canis). The overall molecular prevalence rate of TBPs was 25.81% and 29.17% in the blood of examined dogs and in ticks, respectively. The molecular prevalence of Anaplasmataceae family, Babesia canis, and H. canis in dog blood samples was 19.35%, 6.45%, and 0.0%, respectively, while in ticks, it was 20.83%, 5.55%, and 2.8%, respectively. A sequential analysis identified six different species of TBPs, namely B. canis vogeli, Hepatozoon canis, A. phagocytophilum, A. marginale, A. platys, and E. canis. The obtained sequences were submitted to GenBank and assigned accession numbers., Conclusions: The present study detected a wide range of TBPs (B. canis, H. canis, A. platys, A. phagocytophilum, A. marginale, and E. canis) that are considered a threat to domestic animals and humans in Egypt. Hepatozoon canis and A. marginale were reported in dogs and associated ticks for the first time in Egypt., (© 2022. The Author(s).)

Bacterial pathogens are a long-standing threat to the longevity and survival of crustacean hosts. Their presence and continuing emergence require close monitoring to understand their impact on fished, cultured, and wild crustacean populations. We describe a new bacterial pathogen belonging to the Anaplasmataceae family (Alphaproteobacteria: Rickettsiales), providing pathological, ultrastructural, phylogenetic, and genomic evidence to determine a candidate genus and species ('Candidatus Mellornella promiscua'). This bacterium was found to infect the mud crab, Eurypanopeus depressus, on the North Carolina coastline (USA) at a prevalence of 10.8%. 'Candidatus Mellornella promiscua' was often observed in co-infection with the rhizocephalan barnacle, Loxothylacus panopaei. The bacterium was only found in the hepatopancreas of the mud crab host, causing cytoplasmic hypertrophy, tubule necrosis, large plaques within the cytoplasm of the host cell, and an abundance of sex-pili. The circular genome of the bacterium is 1,013,119 bp and encodes 939 genes in total. Phylogenetically, the new bacterium branches within the Anaplasmataceae. The genome is dissimilar from other described bacteria, with 16S gene similarity observed at a maximum of 85.3% to a Wolbachia endosymbiont. We explore this novel bacterial pathogen using genomic, phylogenetic, ultrastructural, and pathological methods, discussing these results in light of current bacterial taxonomy, similarity to other bacterial pathogens, and the potential impact upon the surrounding disease ecology of the host and benthic ecosystem., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Stray dogs may be highly exposed to vector-borne pathogens (VBPs), including zoonotic agents, and therefore may pose a high risk of spreading infections to other animals and humans. Among the Anaplasmataceae, Anaplasma phagocytophilum, A. platys and Ehrlichia canis are commonly identified species in dogs in Europe; however, information on the occurrence of these pathogens in canine populations from Bosnia and Herzegovina (B&H) is still lacking. Thus, the aim of this study was to determine the seroprevalence of Anaplasma spp. and Ehrlichia spp. in stray dogs in the Sarajevo region of B&H and to identify A. phagocytophilum, A. platys, E. canis and E. ewingii by molecular techniques. A total of 903 blood samples of stray dogs were screened by SNAP 4Dx Plus Test for the presence of antibodies against A. phagocytophilum/A. platys and E. canis/E. ewingii. Real-time PCR assays were performed for the detection of Anaplasmataceae, A. phagocytophilum, A. platys, E. canis and E. ewingii in seropositive dogs. Antibodies to A. phagocytophilum/A. platys and/or E. canis/E. ewingii were detected in 187 (20.7%) samples. Seroprevalence was highest for A. phagocytophilum/A. platys (184/903, 20.4%). Two dogs had antibodies to E. canis/E. ewingii, while one dog was found to have antibodies to A. phagocytophilum/A. platys and to E. canis/E. ewingii. Forty-eight (25.7%) of the 187 seropositive dogs examined by Real-time PCR were positive for Anaplasmataceae. A. phagocytophilum was detected in 45 (24%) samples, while one sample was positive for A. phagocytophilum and A. platys. Two samples positive for Anaplasmataceae tested negative in the species-specific PCRs. E. canis or E. ewingii could not be detected in any of the Ehrlichia-seropositive dogs. These findings highlight the need for dog health monitoring, improving the health and welfare of stray dog population, and establishment of effective surveillance systems to combat VBDs., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

We amplified Ehrlichia and Anaplasma DNA from Amblyomma dubitatum tick-infested capybaras (Hydrochoerus hydrochaeris) in southern Brazil. Sequencing of 16S rRNA, sodB, and groEL indicated a novel Ehrlichia species, and sequencing of 16S rRNA from 2 capybaras indicated a novel Anaplasma species. The tick vectors remain unknown.

Species belonging to the genus Anaplasma (Rickettsiales) include bacteria of veterinary and public health importance. Beside the zoonotic Anaplasma phagocytophilum, A. platys, the etiological agent of canine cyclic thrombocytopenia, has been sporadically reported in clinically ill human patients. The ongoing emergence of novel strains related to this species in vertebrate hosts emphasises the need for genetic comparisons among strains identified in different regions of the world. In this paper we developed a PCR test suitable for amplification of the still undescribed gltA gene of Anaplasma strains related to A. platys from Mediterranean ruminants and applied on a panel of 248 samples. gltA sequencing allowed phylogenetic comparison with strains related to A. platys recently identified in China, and strains representative of the Anaplasmataceae family. Results suggest the designation of Candidatus A. turritanum, including Mediterranean A. platys - like strains, and Candidatus A. cinensis, including strains isolated in China. Data generated in this study are a solid reference for future epidemiological studies of novel unclassified strains related to A. platys and for their diagnosis and raise concern on their potential veterinary and public health implications encouraging investigating the suspected unexplored diversity within the genus Anaplasma in animals and human., (Copyright © 2021. Published by Elsevier GmbH.)

Neoehrlichia mikurensis is an intracellular bacterium transmitted in Europe and Asia by ticks of the Ixodes ricinus complex. Interest in this bacterium has increased since it was demonstrated to be responsible for febrile syndromes in patients. To date, most clinical cases have been reported in northern Europe, but case series have also been described in central Europe and China. Notably, thrombotic events occurred during the course of the disease. We investigated the presence of N. mikurensis in 10,885 I. ricinus nymphs in two regions of France (Alsace and Brittany) collected between 2013 and 2020 and in 934 patients suspected of human granulocytic anaplasmosis in Alsace, an endemic area for Lyme borreliosis, using a specific PCR assay. N. mikurensis was detected in 5.42% of the ticks from Alsace, whereas only one (0.03%) tick was found to be positive in Brittany. Spatiotemporal disparities were also noticed within the Alsace region over the four collection sites investigated, and a significant increase in the prevalence of nymphs carrying N. mikurensis was also observed in the last three years of collection. Four out of 934 screened patients were found to be positive for N. mikurensis . Two had malignancies, and the other two were apparently immunocompetent. Superficial thrombosis was noticed in one patient, and long-lasting bacteremia was noted in another patient. These four patients are the first clinical cases of neoehrlichiosis described in France. We suggest including N. mikurensis in the differential diagnosis of post-tick bite febrile syndromes to treat patients and prevent the occurrence of thrombotic complications.

Background: Anaplasmosis and ehrlichiosis are tick-borne diseases affecting humans and livestock, particularly in tropical and subtropical regions. Animal husbandry is the main activity of people on the borders of Iran and Pakistan, with thousands of cattle crossing the border each week., Methods: PCR and sequencing of the 16S rRNA gene was used to determine the percentage and geographical distribution of the pathogens carried by Hyalomma spp. (n = 306) collected from 126 goats, cattle and camels in the region between November 2017 and late March 2018., Results: In total, 1124 hard ticks including 1020 Hyalomma spp. ticks belonging to six species (Hyalomma anatolicum, Hyalomma asiaticum, Hyalomma marginatum, Hyalomma dromedarii, Hyalomma schulzei, and Hyalomma detritum) were found on the borders of Iran and Pakistan, with H. anatolicum being the most prevalent tick species. Anaplasma spp. and/or Ehrlichia spp. DNA was found in 68.3% of the engorged tick specimens (n = 256). Sequencing of a subset (12.6%) of PCR-positive samples revealed Anaplasma ovis, Anaplasma marginale, and Ehrlichia ewingii DNA in 81.8%, 9.1%, and 9.1% of the ticks, respectively. To our knowledge, this is the first report of E. ewingii, an important human pathogen, in Iran., Conclusions: Based on molecular analysis, three pathogenic Anaplasmataceae were detected in six Hyalomma spp. parasitizing cattle, goats and camels, confirming the presence of these pathogens along the Iran-Pakistan border., (© 2021. The Author(s).)

Anaplasma platys and Ehrlichia canis are members of the Anaplasmataceae family that cause disease in dogs and are mainly transmitted by Rhipicephalus sanguineus species group ticks. We performed a cross-sectional study on these pathogens across six bioclimatic regions of Chile, including 719 free-ranging rural dogs, 132 Andean foxes (Lycalopex culpaeus), and 82 South American gray foxes (Lycalopex griseus). Dog and fox blood samples were first screened for DNA of Anaplasmataceae followed by two Ehrlichia-specific protocols. Antibodies against Anaplasma sp. and E. canis were assessed by immunofluorescence in dogs. Ectoparasites were collected and identified, with the determination of the lineages of the Rhipicephalus sanguineus species group by molecular and phylogenetic analyses. Finally, potential risk factors for infection were investigated across the different bioclimatic regions and host species. All DNA amplicons obtained from the screening protocol corresponded to Anaplasma platys. The occurrence of both A. platys DNA and antibodies was confirmed in all six bioclimatic regions, except for regions at high altitude and/or without either R. sanguineus species group lineage present. Dogs infested with R. sanguineus ticks were significantly more prone to be infected and exposed to Anaplasma spp. Prevalence of DNA was significantly higher in juvenile (19%) than in adult dogs (9%), whereas the opposite was found for seroprevalence (19% versus 35%, respectively). Overall prevalence of A. platys DNA was higher in dogs (11%) than in foxes (4%), probably owing to markedly lower tick infestations in the foxes. Ehrlichia canis DNA was not detected in any sample, and antibodies against this pathogen were detected only in four dogs, in areas with both R. sanguineus lineages present. Free-ranging dogs in Chile could be favoring the maintenance of A. platys in all areas suitable for its tick vector. Although apparently infrequent, spillovers from dogs to foxes may be taking place and should be considered in management plans in Chile., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

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.

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.

We assessed the presence of Rickettsia spp., Coxiella burnetii and Anaplasma phagocytophilum in reptiles, their ectoparasites and in questing ticks collected in a nature preserve park in southern Italy, as well as in a peri-urban area in another region. We also investigated the exposure to these pathogens in forestry workers, farmers and livestock breeders living or working in the nature preserve park given the report of anecdotal cases of spotted fever rickettsioses. Rickettsia spp. were molecularly detected in Podarcis muralis and Podarcis siculus lizards (i.e., 3.1%), in Ixodes ricinus (up to 87.5%) and in Neotrombicula autumnalis (up to 8.3%) collected from them as well as in I. ricinus collected from the environment (up to 28.4%). Rickettsia monacensis was the most prevalent species followed by Rickettsia helvetica. An undescribed member of the family Anaplasmataceae was detected in 2.4% and 0.8% of the reptiles and ectoparasites, respectively. Sera from human subjects (n = 50) were serologically screened and antibodies to Rickettsia spp. (n = 4; 8%), C. burnetti (n = 8; 16%) and A. phagocytophilum (n = 11; 22%) were detected. Two ticks collected from two forestry workers were positive for spotted fever group (SFG) rickettsiae. Ixodes ricinus is involved in the transmission of SFG rickettsiae (R. monacensis and R. helvetica) in southern Europe and lizards could play a role in the sylvatic cycle of R. monacensis, as amplifying hosts. Meanwhile, N. autumnalis could be involved in the enzootic cycle of some SFG rickettsiae among these animals. People living or working in the southern Italian nature preserve park investigated are exposed to SFG rickettsiae, C. burnetii and A. phagocytophilum., Competing Interests: The authors have declared that no competing interests exist.

Background: Anaplasmosis, an animal disease caused by rickettsial bacteria in the genus Anaplasma, is of considerable economic importance in livestock animals in many countries worldwide. The objectives of this study were to determine the identity, prevalence, and geographic distribution of Ehrlichia and Anaplasma in naturally infected water buffalo in Thailand using PCR amplification and sequencing of the 16S ribosomal RNA and heat shock protein groEL genes. A total of 456 buffalo blood samples from Thailand were investigated. Species identification and genetic differentiation of intra-population and inter-population with the global isolates were conducted based on nucleotide sequences. Interplay between the infection and host factors was also assessed., Results: Overall, 41% of water buffalo were found to be infected with rickettsial organisms in the family Anaplasmataceae, but Ehrlichia spp., Neorickettsia spp., and Wolbachia spp. were not found in any of the sequenced samples in this study. Female buffalo were more frequently infected with bacteria in the family Anaplasmataceae than males [71 out of 176 females (40.3%) versus 11 out of 47 males (23.4%)]. The Odds Ratio value indicated that the risk of infection for female buffalo was 2.2-fold higher than that for males (p < 0.05). We detected three haplotypes of A. marginale 16S rRNA gene and they were placed in a clade that was closely related to the A. marginale in buffalo in China; and cattle in Thailand, Uganda, and China. Homology searching of groEL sequences against the GenBank™ database using the BLASTn algorithm revealed that the obtained sequences had a high percentage similarity (98.36-99.62%) to A. platys sequences. The groEL sequences of three A. platys-like isolates were clustered in the same clade as the A. platys from the tick Rhipicephalus microplus in China., Conclusions: Our data showed that the apparently healthy buffalo were naturally infected by bacteria in the family Anaplasmataceae at a relatively high prevalence. We also report the finding of A. platys-like infections in water buffalo in Thailand for the first time. Water buffalo serving as the reservoir host of anaplasmosis is of concern for managing the disease control and prevention in ruminants.

Background: Species of the Tabanidae are potent vectors of human and animal diseases, but they have not been thoroughly investigated to date. In Senegal (West Africa), little information is available on these dipterans. Our objective in this study was to investigate Senegalese tabanids and their diversity by using molecular and proteomics approaches, as well as their associated pathogens., Methods: A total of 171 female tabanids were collected, including 143 from Casamance and 28 from Niokolo-Koba. The samples were identified morphologically by PCR sequencing and by MALDI-TOF MS, and PCR analysis was employed for pathogen detection and blood-meal characterization., Results: The morphological identification revealed four species concordantly with the molecular identification: Atylotus fuscipes (79.5%), Tabanus guineensis (16.4%), Chrysops distinctipennis (3.5%) and Tabanus taeniola (0.6%) (not identified by PCR). The molecular investigation of pathogens revealed the presence of Trypanosoma theileri (6.6%), Leishmania donovani (6.6%), Setaria digitata (1.5%), Rickettsia spp. (5.1%) and Anaplasmataceae bacteria (0.7%) in A. fuscipes. Tabanus guineensis was positive for L. donovani (35.7%), S. digitata (3.6%) and Anaplasmataceae (17.8%). Leishmania donovani has been detected in 50% of C. distinctipennis specimens and the only T. taeniola specimen. No Piroplasmida, Mansonella spp. or Coxeilla burnetii DNA was detected. In addition to humans (96.43%), Chlorocebus sabeus, a non-human primate, has been identified as a host of (3.57%) analysed tabanids. MALDI-TOF MS enabled us to correctly identify all tabanid species that had good quality spectra and to create a database for future identification., Conclusions: Tabanids in Senegal could be vectors of several pathogens threatening animal and public health. To fully characterize these dipterans, it is therefore necessary that researchers in entomology and infectiology employ molecular characterization and mass spectrometric techniques such as MALDI-TOF MS to analyse these dipterans in Senegal and West Africa.

Neoehrlichia mikurensis is an emerging tick-borne intracellular pathogen causing neoehrlichiosis. Its putative morphology was described in mammalian, but not in tick cells. In this study, we aim to show the presumptive morphology of N. mikurensis in salivary glands of engorged females of Ixodes ricinus. To accomplish this, we collected I. ricinus ticks in a locality with a high N. mikurensis prevalence, allowed them to feed in the artificial in vitro feeding system, dissected salivary glands and screened them by PCR for N. mikurensis and related bacteria. Ultrathin sections of salivary glands positive for N. mikurensis but negative for other pathogens were prepared and examined by transmission electron microscopy. We observed two individual organisms strongly resembling N. mikurensis in mammalian cells as described previously. Both bacteria were of ovoid shape between 0.5-0.8 μm surrounded by the inner cytoplasmic and the rippled outer membrane separated by an irregular electron-lucent periplasmic space. Detection of N. mikurensis in salivary glands of I. ricinus suggests that this bacterium uses the "salivary pathway of transmission" to infect mammals.

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.

A 77-year-old woman had a history of mantle cell lymphoma, splenechtomy and rituximab-treatment. For six months she had fever, night sweats and weight loss. Thorough investigations did not reveal the cause of the fever, and empiric antibiotics had no effect. Eventually she developed an erythema nodosum-like rash on both legs. A biopsy was sent for 16S rRNA PCR, which was positive for Candidatus Neoehrlichia mikurensis. She was treated with doxycycline with resolution of all symptoms. This is the first case report of neoehrlichiosis in Denmark, and the first case diagnosed on a skin biopsy.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Recent studies reported the detection of DNA from tick-borne pathogens (TBPs) of veterinary relevance such as Anaplasma marginale, Babesia bigemina, Babesia bovis and Theileria orientalis in bovine blood samples from Mongolia. These findings were unexpected, as the known tick vectors of these pathogens are not known to occur in Mongolia. We therefore conducted a study in May and June 2013 in six districts of Khentii province where DNA of the said TBPs was previously found. Ticks collected from the vegetation and rodents, as well as blood samples from cattle, were screened for the presence of TBPs by reverse line blot (RLB) hybridization. Tick larvae collected from rodents were pooled. A total of 310 adult ticks were collected from the vegetation, and 249 tick larvae were collected from 24 rodents. Adult ticks (n = 2,318) and blood samples were collected from 481 heads of cattle. All adult ticks were identified as Dermacentor nuttalli. DNA from Rickettsia raoultii (252/310; 81.3%), an uncharacterized Anaplasma species preliminary named Anaplasma sp. Mongolia (26/310; 8.4%), Candidatus Midichloria sp. (18/310; 5.8%), Theileria equi (16/310; 5.2%), Babesia caballi (5/310; 1.6%), T. orientalis (1/310; 0.3%), Borrelia afzelii (1/310; 0.3%) and Candidatus Neoehrlichia mikurensis (1/310; 0.3%) was detected in ticks collected from the vegetation. DNA of R. raoultii (27/28; 96.4%) and Midichloria sp. (2/28; 7.1%) was detected in the pooled tick larvae. Anaplasma sp. Mongolia, a species related to Anaplasma ovis based on a multi-locus analysis, was also detected in 153/481 (31.8%) of the bovine blood samples. DNA of B. bovis, B. bigemina and A. marginale was not detected in the ticks or bovine blood samples from Khentii district., (© 2019 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

Increasing amount of data attest that (in the context of vector-borne infections) birds are not only important as hosts of blood-sucking arthropod vectors, but also as reservoirs of vector-borne pathogens. From 2015 to 2019 cadavers of 100 birds (from 45 species, nine orders) were collected in Hungary, and their organs were screened for DNA from a broad range of vector-borne bacteria with PCR and sequencing. Molecular analyses revealed the presence of Anaplasmataceae, and sequencing identified bacteria closely related to Neorickettsia helminthoeca and Ehrlichia chaffeensis in a Eurasian teal (Anas crecca) and a song thrush (Turdus philomelos), respectively. All samples were PCR negative for rickettsiae, borreliae, Francisella and Coxiella spp., as well as for piroplasms. To our knowledge, this is the first report of a Neorickettsia and an Ehrlichia sp., which belong to the phylogenetic groups of N. helminthoeca and E. chaffeensis, respectively, from Europe. The potential presence of these two vector-borne bacteria needs to be taken into account during future studies on the eco-epidemiology of Anaplasmataceae in Europe.

Candidatus Neoehrlichia mikurensis, the causative agent of tick-borne "neoehrlichiosis" has recently been reported in humans, mammals and ticks in Europe. The aim of this study was to map the distribution of this bacterium in questing ticks in the Czech Republic. A total of 13,325 Ixodes ricinus including 445 larvae, 5270 nymphs and 7610 adults were collected from vegetation by flagging in 140 Czech towns and villages from every region of the Czech Republic. The ticks were pooled into 2665 groups of 5 individuals respecting life stage or sex and tested for the presence of Ca. Neoehrlichia mikurensis by conventional PCR targeting of the groEL gene. The bacterium was detected in 533/2665 pools and 125/140 areas screened, showing an overall estimated prevalence of 4.4 % in ticks of all life stages. Phylogenetic analysis revealed only small genetic diversity among the strains found. Two pools of questing larvae tested positive, suggesting transovarial transmission. According to this study, Ca. Neoehrlichia mikurensis is another tick-borne pathogen widespread in I. ricinus ticks in the Czech Republic., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Birds are important hosts for the first life stages of the Ixodes ricinus tick and they can transport their parasites over long distances. The aim of this study was to investigate the prevalence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Neoehrlichia mikurensis and Rickettsia helvetica in ticks collected from migratory birds in Norway. A total of 815 Ixodes ricinus ticks from 216 birds trapped at Lista Bird Observatory in southern Norway during spring and autumn migration in 2008 were analysed by real-time PCR. B. burgdorferi s. l. was the most prevalent pathogen, detected in 6.1% of the ticks. The prevalence of N. mikurensis, A. phagocytophilum and R. helvetica was 1.2%, 0.9% and 0.4% respectively. In addition, one sample (0.1%) was positive for B. miyamotoi. In total, 8.2% of the ticks were infected with at least one pathogen. Co-infection with B. burgdorferi s. l. and N. mikurensis or A. phagocytophilum was found in 6.0% of the infected ticks. Our results show that all the known major tick-borne bacterial pathogens in Norway are subject to transport by migratory birds, potentially allowing spread to new areas. Our study showed a surprisingly high number of samples with PCR inhibition (57%). These samples had been extracted using standard methodology (phenol-chloroform extraction). This illustrates the need for inhibition controls to determine true prevalence rates., Competing Interests: The authors have declared that no competing interests exist.

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.

Small non-volant mammals (marsupials and small rodents) were captured at three different timepoints from 23 forest fragments across three municipalities (Alta Floresta, Sinop and Cláudia) covering the Amazonian biome of the Mato Grosso State in Midwestern Brazil. The animal tissues (liver and spleen) and blood were screened using molecular tools for the detection of Babesia, Coxiella, Cytauxzoon, Hepatozoon, Theileria, and Anaplasmataceae agents. A total of 230 specimens (78 rodents and 152 marsupials) were trapped. Hepatozoon and Piroplasmorida agents were detected in the common opossums (Didelphis marsupialis). In turn, all samples (blood, liver, or spleen) collected from the small mammals were negative for the genus Coxiella and the family Anaplasmataceae, as detected by polymerase chain reaction (PCR). Phylogenetic analyses inferred from partial sequences of the 18S rRNA gene highlighted the occurrence of new Hepatozoon and Piroplasmorida haplotypes. Future studies determining the role of common opossum (D. marsupialis) in the epidemiological cycles of Hepatozoon and Babesia under natural conditions in the Amazonian biome are necessary.

The sheep ked, Melophagus ovinus, and the forest fly, Hippobosca equina, are parasitic dipteran insects of veterinary importance. As hematophagous insects, they might be considered as potential vectors of diseases which may be transmissible to humans and animals. The purpose of this study was to present initial primary data about these two species in Algeria. To do so, we conducted a molecular survey to detect the presence of bacterial DNA in flies collected in Algeria. A total of 712 flies including, 683 Melophagus ovinus and 29 Hippobosca equina were collected from two regions in northeastern Algeria. Monitoring the monthly kinetics of M. ovinus infestations showed something resembling annual activity, with a high prevalence in January (21.67%) and May (20.94%). Real-time quantitative PCR assays showed that for 311 tested flies, 126 were positive for the Bartonella spp. rRNA intergenic spacer gene and 77 were positive for Anaplasmataceae. A random selection of positive samples was submitted for sequencing. The DNA of Bartonella chomelii and Bartonella melophagi were amplified in, respectively, five and four H. equina. 25 M. ovinus positive samples were infected by Bartonella melophagi. Amplification and sequencing of the Anaplasma spp. 23S rRNA gene revealed that both species were infected by Wolbachia sp. which had previously been detected in Cimex lectularius bed bugs. Overall, this study expanded knowledge about bacteria present in parasitic flies of domestic animals in Algeria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Infectious haemolytic anaemia (IHA) in dogs share similar clinical signs including fever, lethargy, icterus, paleness of mucous membranes and splenomegaly. Postmortal findings are similar and, without additional diagnostic methods, an accurate aetiological diagnosis is difficult to achieve. In order to investigate causes of lethal IHA in Croatian dogs, we performed a retrospective study on archived formalin-fixed, paraffin-embedded tissue blocks (FFPEB) from dogs that died due to haemolytic crisis, using microscopic and molecular diagnostic tools to determine the aetiological cause of disease. Molecular analysis was performed on kidney, lung, myocardium and spleen on FFPEB from all dogs. The originally stated aetiological diagnosis of B. canis or leptospirosis was confirmed in only 53% of the dogs. PCR and sequencing revealed that, in addition to the expected pathogens, B. canis and Leptospira interrogans, the presence of previously undiagnosed "new" pathogens causing anaemia including Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum. Furthermore, Theileria capreoli was detected for the first time in a dog with postmortal descriptions of lesions. Intensive extravascular hemolysis was noticeable as jaundice of the mucosa, subcutis and fat tissue, green or yellow discoloration of renal parenchyma caused by bilirubin excretion in the renal tubules and bile accumulation within the liver in 90% of the dogs. This work highlights the value of molecular diagnostics to complement traditional ante-mortem and post-mortem diagnostic protocols for the aetiological diagnosis of pathogens associated with IHA., (Copyright © 2019. Published by Elsevier Ltd.)

Reliable data on distributional ranges of soft ticks (Argasidae) and assessments of putative tick-borne agents enhance the understanding on tick-associated microorganisms. A total of 96 ticks morphologicaly and molecularly identified as Ornithodoros rietcorreai were collected in Tocantins State, Brazil, using Noireau traps with living bait as CO2 source. Ninety-six ticks (54 nymphs, 32 males, 10 females) with different engorgement degrees were collected. Fourty-seven (48.9%) of them were individually screened by PCR for detecting bacteria of Anaplasmataceae family and genera Rickettsia, and Borrelia. The presence of protozoans of the genus Babesia was assessed as well. Fourty seven ticks were submitted to analysis. Nine ticks (19.1%) yielded sequences for gltA and htrA genes most identical with a series of endosymbiont rickettsiae and Rickettsia bellii, respectively. Upon two ticks (4.2%) we retrieved DNA of a potential new Wolbachia sp., and DNA of a putative novel Hepatozoon was characterized from three (6.4%) specimens. No DNA of Babesia or Borrelia was detected. Remarkably, amplicons of unidentified eukaryotic organisms, most closely related with apicomplexans but also with dinoflagellates (91% of identity after BLAST analyses), were recovered from two ticks (4.2%) using primers designed for Babesia 18S rRNA gene. Our records expand the distribution of O. rietcorreai into Brazilian Cerrado biome and introduce the occurrence of microorganisms in this tick species., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The European polecat, Mustela putorius, occurs almost throughout Europe. However, there is a lack of data on the ectoparasite fauna and the potential role in the circulation of tick-borne pathogens (TBP) of this mustelid species. The aim of this study was to investigate whether M. putorius contributes to the maintenance of TBP in Germany. DNA samples extracted from spleen tissue of 117 M. putorius, which had been collected mainly in North-western Germany from 2012 to 2015, were tested by real-time and conventional PCRs for Anaplasma phagocytophilum, Babesia spp., Bartonella spp., Candidatus Neoehrlichia mikurensis (CNM) and Hepatozoon spp. In addition, 200 randomly selected engorged Ixodes hexagonus ticks (100 females and 100 nymphs) collected from 39 of the 88 M. putorius were tested for these TBPs, except for Hepatozoon spp., and additionally for Borrelia spp. and Rickettsia spp. Three of six pathogens were detected in the spleen tissue of the 117 M. putorius: A. phagocytophilum (n = 5; 4.3%), Babesia cf. odocoilei (n = 1; 0.9%) and CNM (n = 1; 0.9%), including one case of co-infection (A. phagocytophilum and CNM). Ixodes hexagonus ticks tested positive only for Bartonella spp. (26/200 ticks; 13.0%) which were detected exclusively in adult female ticks. Sequencing revealed the presence of Bartonella taylorii and uncultured Bartonella spp. The results suggest that M. putorius neither seems to serve as the main reservoir nor plays an important role in maintaining TBPs in Germany but may rather contribute to the dilution of these pathogens. However, M. putorius contributes to the maintenance of tick populations, especially of I. hexagonus. The high prevalence of Bartonella spp. in I. hexagonus ticks may suggest a certain importance of this tick species in the maintenance of these bacteria in nature., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Although livestock farming (sheep, goats, pigs, and cattle) is an important economic activity in Corsica, a French Mediterranean island, knowledge about the tick fauna and microorganisms carried by them remains scarce. This study aimed to investigate the presence and perform molecular characterization of Anaplasmataceae, Rickettsia spp., and Borrelia burgdorferi sensu lato (sl) in tick species collected in Corsica. Ticks from cattle (Bos taurus), sheep (Ovis aries), and rodents (Rattus rattus) were collected from May to September 2016. DNA was purified from ticks, submitted to quantitative real-time polymerase chain reaction (qPCR) and sequenced for phylogenetic analysis. In total, 660 ticks were collected from 111 animals during the study. The most abundant collected tick species from cattle was Rhipicephalus bursa (n = 495; 84.5%), followed by Hyalomma marginatum (n = 91; 15.5%). Rhipicephalus bursa and Ixodes ricinus were the only tick species collected from sheep and rodents, respectively. Overall, Rickettsia was the most common pathogen group (n = 48; 24%) detected in ticks. Sequence analysis of partial gltA and ompA genes revealed the presence of Ri. aeschlimannii and Candidatus Ri. barbariae. Anaplasmataceae DNA was detected in eight (6%) of the 127 cattle pools and in one (2%) of the 61 R. bursa specimens collected from sheep. Sequence analysis of the rpoB gene revealed the presence of one Anaplasma species, A. marginale. Borrelia burgdorferi sl DNA was detected in one pool of H. marginatum collected from cattle and in two (15%) of the 13 I. ricinus pools collected from nine black rats. To our knowledge, this is the first report of the occurrence and molecular characterization of Candidatus Ri. barbariae, an emerging member of the Rickettsia group causing spotted fever, in Corsica. The detection of B. burgdorferi sl DNA, which was previously believed to be rare in Corsica, confirms the presence of this agent on the island., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Microorganisms harbored by Chilean autochthonous ticks have been scarcely studied and current knowledge is restricted to three species of hard ticks only. The current study aimed to assess the presence of Anaplasmataceae, Borrelia and Hepatozoon agents in ticks collected directly from the environment, on reptiles, birds and mammals in twelve localities from northern, central and southern regions of the country and Antarctica. Ticks were identified by means of a morphological and molecular approach. PCR detections point the occurrence of an Anaplasma-like agent and a relapsing fever Borrelia sp. in Ornithodoros spheniscus; an Ehrlichia sp. and a Hepatozoon sp. in Ornithodoros atacamensis; "Candidatus Neoehrlichia chilensis", Borrelia burgdorferi sensu lato (s. l.), and Hepatozoon in Ixodes ticks morphologically related to the Ixodes sigelos group; and B. burgdorferi s. l. in Ixodes auritulus. Supported by phylogenetic analyses of characterized microorganisms, this study introduces putative vector roles and initial evidence on possible new agents detected in Chilean ticks., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mutational analysis is an efficient approach to identifying microbial gene function. Until recently, lack of an effective tool for Anaplasmataceae yielding reproducible results has created an obstacle to functional genomics, because surrogate systems, e.g., ectopic gene expression and analysis in E. coli, may not provide accurate answers. We chose to focus on a method for high-throughput generation of mutants via random mutagenesis as opposed to targeted gene inactivation. In our search for a suitable mutagenesis tool, we considered attributes of the Himar1 transposase system, i.e., random insertion into AT dinucleotide sites, which are abundant in Anaplasmataceae, and lack of requirement for specific host factors. We chose the Anaplasma marginale tr promoter, and the clinically irrelevant antibiotic spectinomycin for selection, and in addition successfully implemented non-antibiotic selection using an herbicide resistance gene. These constructs function reasonably well in Anaplasma phagocytophilum harvested from human promyelocyte HL-60 cells or Ixodes scapularis tick cells. We describe protocols developed in our laboratory, and discuss what likely makes them successful. What makes Anaplasmataceae electroporation competent is unknown and manipulating electroporation conditions has not improved mutational efficiency. A concerted effort is needed to resolve remaining problems that are inherent to the obligate intracellular bacteria. Finally, using this approach, we describe the discovery and characterization of a putative secreted effector necessary for Ap survival in HL-60 cells., (Copyright © 2018 Elsevier GmbH. All rights reserved.)