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Background Tick-borne infections are no longer confined to rural areas, they are documented with increasing frequency in urban settlements across the world. They are known to cause diseases in humans as well as in their companion animals. Methods During a period of 2 years, from January 2013 until December 2014, ticks were collected from dogs and cats in 18 veterinary clinics in the Wrocław Agglomeration, Poland. In total, 1455 ticks were found on 931 pets: 760 domestic dogs and 171 cats. For molecular examinations 127 I. ricinus ticks (115 females and 12 males) were randomly selected, all collected I. hexagonus (n = 137, 32 females, 98 nymphs, 7 larvae) and all collected D. reticulatus (n = 46, 31 females, 15 males) were taken. Ixodes ricinus and I. hexagonus ticks were tested for Rickettsia spp., Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis and Babesia spp., while D. reticulatus ticks were investigated for Rickettsia spp. and Babesia spp. only. Results In total, 65.4 % I. ricinus ticks were infected with at least one pathogen. Over 50 % of I. ricinus were positive for Rickettsia spp. (R. helvetica and R. monacensis). The infection level with A. phagocytophilum was 21.3 %. DNA of Cand. N. mikurensis was detected in 8.1 % I. ricinus ticks. Interestingly only female ticks were infected. The prevalence of Babesia spp. was confirmed in 9.0 % of I. ricinus involving the species B. microti and B. venatorum. A total of nineteen double, one triple and two quadruple infections were found in I. ricinus ticks only. Almost 11 % of I. hexagonus ticks were positive for at least one of the tested pathogens. Rickettsia spp. infection was found in 2.2 %, while A. phagocytophilum was detected in 8.1 % of I. hexagonus ticks. Only one nymph was positive for Cand. N. mikurensis and none of I. hexagonus ticks harbored a Babesia spp. Over 60 % of D. reticulatus ticks were positive for rickettsial DNA, exclusively belonging to the species R. raoultii. Conclusion The high tick infestation rates and the prevalence of pathogens found in these ticks demonstrate a serious level of encounter to tick-borne diseases in urban dogs in the Wroclaw area, and provide evidence that dogs and cats themselves may substantially contribute to the circulation of the ticks and pathogens in the urban area.

Background Candidatus Neoehrlichia mikurensis (CNM) is an emerging tick-borne pathogen causing severe disease in immunocompromised patients. In Europe, Ixodes ricinus is the primary vector and rodents act as reservoir hosts. New data on the prevalence of CNM in ticks and rodents contribute to the knowledge on the distribution of endemic areas and circulation of the bacterium in natural foci. Methods Questing ticks were collected and rodents were trapped in urban/suburban and natural habitats in South-Western Slovakia from 2011 to 2014. DNA from questing and rodent-attached ticks and rodent tissues were screened for CNM by real-time PCR. Rodent spleen samples positive for CNM were characterised at the groEL gene locus. Spatial and temporal differences in CNM prevalence in ticks and rodents and co-infections of ticks with CNM and Anaplasma phagocytophilum were analysed. Results The presence of CNM was confirmed in questing and rodent-attached I. ricinus ticks and in rodents. Total prevalence in both ticks and rodents was significantly higher in the natural habitat (2.3 % and 10.1 %, respectively) than in the urban/suburban habitat (1.0 % and 3.3 %, respectively). No seasonal pattern in CNM prevalence in ticks was observed, but prevalence in rodents was higher in autumn than in spring. CNM was detected in Apodemus flavicollis, Myodes glareolus, Microtus arvalis and Micromys minutus, with the highest prevalence in M. arvalis (30 %). By screening CNM dissemination in rodent tissues, infection was detected in lungs of all specimens with positive spleens and in blood, kidney, liver and skin of part of those individuals. Infection with CNM was detected in 1.3 % of rodent attached I. ricinus ticks. Sequences of a fragment of the groEL gene from CNM-positive rodents showed a high degree of identity with sequences of the gene amplified from ticks and infected human blood from Europe. Only 0.1 % of CNM-positive questing ticks carried A. phagocytophilum. Ticks infected with CNM prevailed in the natural habitat (67.2 %), whereas ticks infected with A. phagocytophilum prevailed in the urban/suburban habitat (75.0 %). Conclusion The study confirmed the circulation of CNM between I. ricinus ticks and rodents in South-Western Slovakia, and indicates a potential risk of contracting human infections.

Background Neoehrlichia mikurensis s an emerging and vector-borne zoonosis: The first human disease cases were reported in 2010. Limited information is available about the prevalence and distribution of Neoehrlichia mikurensis in Europe, its natural life cycle and reservoir hosts. An Ehrlichia-like schotti variant has been described in questing Ixodes ricinus ticks, which could be identical to Neoehrlichia mikurensis. Methods Three genetic markers, 16S rDNA, gltA and GroEL, of Ehrlichia schotti-positive tick lysates were amplified, sequenced and compared to sequences from Neoehrlichia mikurensis. Based on these DNA sequences, a multiplex real-time PCR was developed to specifically detect Neoehrlichia mikurensis in combination with Anaplasma phagocytophilum in tick lysates. Various tick species from different life-stages, particularly Ixodes ricinus nymphs, were collected from the vegetation or wildlife. Tick lysates and DNA derived from organs of wild rodents were tested by PCR-based methods for the presence of Neoehrlichia mikurensis. Prevalence of Neoehrlichia mikurensis was calculated together with confidence intervals using Fisher's exact test. Results The three genetic markers of Ehrlichia schotti-positive field isolates were similar or identical to Neoehrlichia mikurensis. Neoehrlichia mikurensis was found to be ubiquitously spread in the Netherlands and Belgium, but was not detected in the 401 tick samples from the UK. Neoehrlichia mikurensis was found in nymphs and adult Ixodes ricinus ticks, but neither in their larvae, nor in any other tick species tested. Neoehrlichia mikurensis was detected in diverse organs of some rodent species. Engorging ticks from red deer, European mouflon, wild boar and sheep were found positive for Neoehrlichia mikurensis. Conclusions Ehrlichia schotti is similar, if not identical, to Neoehrlichia mikurensis. Neoehrlichia mikurensis is present in questing Ixodes ricinus ticks throughout the Netherlands and Belgium. We propose that Ixodes ricinus can transstadially, but not transovarially, transmit this microorganism, and that different rodent species may act as reservoir hosts. These data further imply that wildlife and humans are frequently exposed to Neoehrlichia mikurensis-infected ticks through tick bites. Future studies should aim to investigate to what extent Neoehrlichia mikurensis poses a risk to public health.

Candidatus Neoehrlichia mikurensis is an emerging tick-borne pathogen. It is widely distributed in Ixodes ricinus ticks in Europe, but knowledge of its distribution in Norway, where I. ricinus reaches its northern limit, is limited. In this study we have developed a real time PCR test for Ca. N. mikurensis and used it to investigate the distribution of Ca. N. mikurensis in Norway.Real time PCR targeting the groEL gene was developed and shown to be highly sensitive. It was used to detect Ca. N. mikurensis in 1651 I. ricinus nymphs and adults collected from twelve locations in Norway, from the eastern Oslo Fjord in the south to near the Arctic Circle in the north. The overall prevalence was 6.5% and varied locally between 0 and 16%. Prevalence in adults and nymphs was similar, suggesting that ticks acquire Ca. N. mikurensis predominantly during their first blood meal. In addition, 123 larvae were investigated; Ca. N. mikurensis was not found in larvae, suggesting that transovarial transmission is rare or absent. Sequence analysis suggests that a single variant dominates in Norway.Ca. N. mikurensis is widespread and common in ticks in Norway and reaches up to their northern limit near the Arctic Circle. Ticks appear to acquire Ca. N. mikurensis during their first blood meal. No evidence for transovarial transmission was found.

Background Since 1997, human-biting ticks submitted to the Department of Defense Human Tick Test Kit Program (HTTKP) of the US Army Public Health Command have been tested for pathogens by PCR. We noted differences in the phenology and infection prevalence among Ixodes scapularis ticks submitted from military installations in different geographic regions. The aim of this study was to characterize these observed differences, comparing the phenology and pathogen infection rates of I. scapularis submitted from soldiers at two sites in the upper Midwest (Camp Ripley, MN, and Ft. McCoy, WI) and one site in the northeastern US (Ft. Indiantown Gap, PA). Methods From 1997 through 2012, the HTTKP received 1,981 I. scapularis from the three installations and tested them for Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi and the Ehrlichiamuris-like (EML) agent using PCR; pathogen presence was confirmed via sequencing or amplification of a second gene target. Pathogen and co-infection prevalence, tick engorgement status, and phenology were compared among installations. Results Greater rates of A. phagocytophilum and Ba. microti infections were detected in ticks submitted from installations in Minnesota than in Wisconsin or Pennsylvania, and the EML agent was only detected in ticks from Minnesota and Wisconsin. Midwestern ticks were also more likely to be co-infected than those from Pennsylvania. Both adult and nymphal ticks showed evidence of feeding on people, although nymphs were more often submitted engorged. Adult I. scapularis were received more frequently in June from Minnesota than from either of the other sites. Minnesota adult and nymphal peaks overlapped in June, and submissions of adults exceeded nymphs in that month. Conclusions There were clear differences in I. scapularis phenology, pathogen prevalence and rates of co-infection among the three military installations. Seasonal and temperature differences between the three sites and length of time a population had been established in each region may contribute to the observed differences. The synchrony of adults and nymphs observed in the upper Midwest has implications for pathogen infection prevalence. The EML agent was only detected in Minnesota and Wisconsin, supporting the previous assertion that this pathogen is currently limited to the upper Midwest.

Background Candidatus Neoehrlichia came under the focus of recent research in terms of human and pet relevance. Candidatus Neoehrlichia mikurensis seems to be relatively abundant in animals and humans from Central European countries, whereas Candidatus Neoehrlichia lotoris was found solely in raccoons from the USA. Findings Spleen samples from a total of 164 red foxes, originating from two western provinces in Austria (Tyrol and Vorarlberg), were collected and examined for the presence of tick-borne bacteria of the family Anaplasmataceae by PCR and sequencing. In a fox sample originating from Vorarlberg Candidatus Neoehrlichia sp. was found, which is genetically (16S rRNA, groEL) closely related to Candidatus Neoehrlichia lotoris but clearly distinct from Candidatus Neoehrlichia mikurensis. Conclusions The present study revealed, for the first time, the occurrence of Candidatus Neoehrlichia sp. in a red fox worldwide. A continuing screening of wild carnivores, especially foxes, and ticks for this potential pathogen is required to evaluate the actual occurrence and distribution of these bacteria. Further research is needed to elucidate the relationships of Neoehrlichia, as well as their reservoir and impact on wildlife, pets and humans.

[Background]: The tick-borne pathogen Anaplasma marginale, which is endemic worldwide, is the type species of the genus Anaplasma (Rickettsiales: Anaplasmataceae). Rhipicephalus (Boophilus) microplus is the most important tick vector of A. marginale in tropical and subtropical regions of the world. Despite extensive characterization of the genetic diversity in A. marginale geographic strains using major surface protein sequences, little is known about the biogeography and evolution of A. marginale and other Anaplasma species. For A. marginale, MSP1a was shown to be involved in vectorpathogen and host-pathogen interactions and to have evolved under positive selection pressure. The MSP1a of A. marginale strains differs in molecular weight because of a variable number of tandem 23-31 amino acid repeats and has proven to be a stable marker of strain identity. While phylogenetic studies of MSP1a repeat sequences have shown evidence of A. marginale-tick co-evolution, these studies have not provided phylogeographic information on a global scale because of the high level of MSP1a genetic diversity among geographic strains., [Results]: In this study we showed that the phylogeography of A. marginale MSP1a sequences is associated with world ecological regions (ecoregions) resulting in different evolutionary pressures and thence MSP1a sequences. The results demonstrated that the MSP1a first (R1) and last (RL) repeats and microsatellite sequences were associated with world ecoregion clusters with specific and different environmental envelopes. The evolution of R1 repeat sequences was found to be under positive selection. It is hypothesized that the driving environmental factors regulating tick populations could act on the selection of different A. marginale MSP1a sequence lineages, associated to each ecoregion., [Conclusion]: The results reported herein provided the first evidence that the evolution of A. marginale was linked to ecological traits affecting tick vector performance. These results suggested that some A. marginale strains have evolved under conditions that support pathogen biological transmission by R. microplus, under different ecological traits which affect performance of R. microplus populations. The evolution of other A. marginale strains may be linked to transmission by other tick species or to mechanical transmission in regions where R. microplus is currently eradicated. The information derived from this study is fundamental toward understanding the evolution of other vector-borne pathogens., This research was supported by grants from the Ministerio de Ciencia e Innovación, Spain (project BFU2008-01244/BMC), the CSIC intramural project 200830I249 to JF and the Oklahoma Agricultural Experiment Station (project 1669) and was facilitated through the Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3), financed by the International Cooperation Program of the European Union, coordination action project No. 510561. VN was funded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007-2013), Spain.

Altogether 2004 Ixodes ricinus ticks, from 37 places in Hungary, were analysed in pools with a recently developed multiplex real-time PCR for the presence of Candidatus Neoehrlichia mikurensis and for other representatives of the genus. Ca. Neoehrlichia mikurensis was identified in nine sampling sites, indicating three separated endemic regions along the borders of Hungary. In addition, results of samples from seven places (except for the western part of the country) were positive in the genus-specific (Ca. Neoehrlichia sp.) PCR, but were negative for Ca. Neoehrlichia mikurensis.