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Background: Borrelia miyamotoi and Borrelia burgdorferi sensu lato (s.l.) are important zoonotic agents transmitted by Ixodes ricinus ticks, which are widely distributed across Central Europe. Understanding the spatial distribution of these pathogens' prevalence will help identify areas with increased infection risk and facilitate the implementation of effective preventive measures. Methods: We analysed 12,955 I. ricinus ticks collected from 142 towns in the Czech Republic between 2016 and 2018. The ticks were pooled into 2591 groups of five and tested using duplex quantitative polymerase chain reaction (qPCR) for the presence of B. burgdorferi s.l. and B. miyamotoi. For each location, we estimated the overall prevalence of both agents using the EpiTools Epidemiological Calculator for pooled samples and calculated the minimum infection rate (MIR). To assess the potential risk of infection, we combined data on the abundance of nymphs and females with pathogen prevalence at each sampled site. Using a geographic information system (GIS), we mapped the MIR and infection risk of both Borrelia species across all 142 sampled locations and employed a geostatistical method (ordinary kriging) to predict MIR values and infection risk as continuous surfaces across the entire country. Results: We detected B. miyamotoi in 110 localities and B. burgdorferi s.l. in all 142 localities. The estimated prevalence of B. miyamotoi and B. burgdorferi s.l. in the collected ticks was 2.1% (95% confidence interval [CI] 1.8–2.3) and 27.1% (95% CI 26.0–28.3), respectively. For B. miyamotoi, we identified previously unknown, geographically distinct hotspots of MIR up to 8.3%, with MIR slightly higher in females (2.3%) than in males (1.9%) and nymphs (1.8%), though the difference was not statistically significant. In contrast, B. burgdorferi s.l. exhibited ubiquitous presence, with consistently high prevalence nationwide, showing similar MIRs in females (16.2%) and males (16.1%), and slightly lower in nymphs (15.6%). The highest infection risk for B. miyamotoi was 12.4 infected vectors per hour in southeastern Moravia, while the highest risk for B. burgdorferi s.l. reached 78.6 infected vectors per hour in the Bohemian-Moravian Highlands. Conclusions: Borrelia miyamotoi is widespread, forming distinct high-prevalence areas in certain regions. Borrelia burgdorferi s.l. demonstrates consistently high prevalence across most of the country, except for a few localized areas such as southwestern Czechia. Both pathogens exhibit natural nidality, forming regions with elevated prevalence and infection risk. Long-term time-series data are needed to confirm the spatio-temporal stability of these hotspots. [ABSTRACT FROM AUTHOR]

Abstract Background Bacteria of the genus Borrelia are agents of disease in both domestic animals and humans and pose a significant public health risk. Borrelia species have complex transmission cycles, often using rodents as vertebrate reservoir hosts. These bacteria are classified into three well-defined monophyletic groups: Borrelia burgdorferi sensu lato (Bbsl) complex, the relapsing fever (RF) group, and a third group associated with reptiles and echidnas. Moreover, a new group of Borrelia associated with rodents has recently been proposed, as these bacteria form a phylogenetic group separated from the previously mentioned groups. This study aimed to investigate the presence of DNA of Borrelia spirochetes in rodents in specific areas of the Colombian Caribbean. Methods A total of 155 rodent spleen samples were selected from the tissue bank. These samples were obtained in the departments of La Guajira and Córdoba (Northern Colombia). DNA extraction and specific real-time polymerase chain reaction (PCR) targeting Borrelia 16S ribosomal RNA (rRNA) gene were performed, followed by nested PCR (nPCR) on positive samples to obtain larger fragments of the 16S rRNA gene and characterize the flaB gene. Alignments of generated sequences and ortholog sequences downloaded from Genbank were performed in Clustal Omega. A phylogenetic tree was built with the maximum likelihood method in IQTREE. Results Spleen samples from rodents of the genera Heteromys, Mus, Necromys, Olygoryzomys, Proechymis, Rattus, Sigmodon, and Zygodontomys were processed. Overall, 6.5% (4/162) of the animals tested positive for Borrelia by real-time PCR. All quantitative PCR (qPCR)-positive samples were also positive for nPCR targeting the 16S rRNA gene, yielding fragments of 344–408 bp and 603–673 bp from two Sigmodon rodents and two Zygodontomys rodents from La Guajira and Córdoba. All samples were negative for the flaB gene. Only samples from Zygodontomys rodents presented good quality sequences. A BLASTn analysis showed a percentage of identity ranging between 98.16 and 96.06% with Borrelia sp. R57. Phylogenetic analysis revealed that sequences of the present study clustered with species of the recently proposed Borrelia “rodent group.” Conclusions This is the first detection of borreliae of the “rodent group” in South America. Our results reaffirm the occurrence of a group of spirochetes associated with rodents, extending its geographic distribution to the Colombian Caribbean. Graphical abstract

Background: Bacteria of the genus Borrelia are agents of disease in both domestic animals and humans and pose a significant public health risk. Borrelia species have complex transmission cycles, often using rodents as vertebrate reservoir hosts. These bacteria are classified into three well-defined monophyletic groups: Borrelia burgdorferi sensu lato (Bbsl) complex, the relapsing fever (RF) group, and a third group associated with reptiles and echidnas. Moreover, a new group of Borrelia associated with rodents has recently been proposed, as these bacteria form a phylogenetic group separated from the previously mentioned groups. This study aimed to investigate the presence of DNA of Borrelia spirochetes in rodents in specific areas of the Colombian Caribbean. Methods: A total of 155 rodent spleen samples were selected from the tissue bank. These samples were obtained in the departments of La Guajira and Córdoba (Northern Colombia). DNA extraction and specific real-time polymerase chain reaction (PCR) targeting Borrelia 16S ribosomal RNA (rRNA) gene were performed, followed by nested PCR (nPCR) on positive samples to obtain larger fragments of the 16S rRNA gene and characterize the flaB gene. Alignments of generated sequences and ortholog sequences downloaded from Genbank were performed in Clustal Omega. A phylogenetic tree was built with the maximum likelihood method in IQTREE. Results: Spleen samples from rodents of the genera Heteromys, Mus, Necromys, Olygoryzomys, Proechymis, Rattus, Sigmodon, and Zygodontomys were processed. Overall, 6.5% (4/162) of the animals tested positive for Borrelia by real-time PCR. All quantitative PCR (qPCR)-positive samples were also positive for nPCR targeting the 16S rRNA gene, yielding fragments of 344–408 bp and 603–673 bp from two Sigmodon rodents and two Zygodontomys rodents from La Guajira and Córdoba. All samples were negative for the flaB gene. Only samples from Zygodontomys rodents presented good quality sequences. A BLASTn analysis showed a percentage of identity ranging between 98.16 and 96.06% with Borrelia sp. R57. Phylogenetic analysis revealed that sequences of the present study clustered with species of the recently proposed Borrelia "rodent group." Conclusions: This is the first detection of borreliae of the "rodent group" in South America. Our results reaffirm the occurrence of a group of spirochetes associated with rodents, extending its geographic distribution to the Colombian Caribbean. [ABSTRACT FROM AUTHOR]

Background: Lyme borreliosis is the most common tick-borne disease in Europe and is often caused by Borrelia afzelii, which is transmitted by Ixodes ricinus ticks. The prevalence and abundance of infected ticks fluctuate in time and space, influencing human infection risk. Rodents are reservoir hosts for B. afzelii and important feeding hosts for larval ticks. In the study reported here, we examined how variation in rodent abundance is associated with B. afzelii infection prevalence in ticks, the density of nymphs (DON) and the density of infected nymphs (DIN) in the following year. We further analysed the relationships between the abundance of infected rodents and nymphal infection prevalence (NIP) and DIN. Methods: We conducted a study that combined experimental and observational approaches on 15 islands (10 small islands and 5 large islands) in Finland. On all of the islands, ticks and rodents were monitored and sampled during the summer of 2019, with the monitoring of tick abundance and sampling continuing into the spring of 2020. On five of the 10 small islands, captured rodents were removed from the island ("removal" islands), and on the other five small islands, captured rodents were released back to the trapping site after marking and sampling ("control" islands). On the five large islands, captured rodents were released back to the trapping site after marking and sampling. The presence of B. afzelii from nymph and rodent samples was examined. Results: The results of the experimental study showed that neither treatment (removal), rodent abundance index nor abundance index of infected rodents in 2019 was associated with DON, NIP or DIN in 2020. Based on data from the observational study, the NIP in 2020 decreased with increasing rodent abundance index and abundance index of infected rodents in 2019. However, the DIN in 2020 was not associated with the rodent abundance index or the abundance index of infected rodents in 2019. In addition, in the observational study, DON in 2020 increased with increasing rodent abundance index. Conclusions: Our results suggest that low rodent abundance during the tick activity period is not sufficient for reducing the disease hazard and, hence, rodent removal may not be a feasible control measure in natural ecosystems. [ABSTRACT FROM AUTHOR]

Abstract Background Borrelia are important disease-causing tick- and louse-borne spirochaetes than can infect a wide variety of vertebrates, including humans and reptiles. Reptile-associated (REP) Borrelia, once considered a peculiarity, are now recognised as a distinct and important evolutionary lineage, and are increasingly being discovered worldwide in association with novel hosts. Numerous novel Borrelia spp. associated with monitor lizards (Varanus spp.) have been recently identified throughout the Indo-Pacific region; however, there is a lack of genomic data on these Borrelia. Methods We used metagenomic techniques to sequence almost complete genomes of novel Borrelia spp. from Varanus varius and Varanus giganteus from Australia, and used long- and short-read technologies to sequence the complete genomes of two strains of a novel Borrelia sp. previously isolated from ticks infesting Varanus salvator from Indonesia. We investigated intra- and interspecies genomic diversity, including plasmid diversity and relatedness, among Varanus-associated Borrelia and other available REP Borrelia and, based on 712 whole genome orthologues, produced the most complete phylogenetic analysis, to the best of our knowledge, of REP Borrelia to date. Results The genomic architecture of Varanus-associated Borrelia spp. is similar to that of Borrelia spp. that cause relapsing fever (RF), and includes a highly conserved megaplasmid and numerous smaller linear and circular plasmids that lack structural consistency between species. Analysis of PF32 and PF57/62 plasmid partitioning genes indicated that REP Borrelia plasmids fall into at least six distinct plasmid families, some of which are related to previously defined Borrelia plasmid families, whereas the others appear to be unique. REP Borrelia contain immunogenic variable major proteins that are homologous to those found in Borrelia spp. that cause RF, although they are limited in copy number and variability and have low sequence identities to RF variable major proteins. Phylogenetic analyses based on single marker genes and 712 single copy orthologs also definitively demonstrated the monophyly of REP Borrelia as a unique lineage. Conclusions In this work we present four new genomes from three novel Borrelia, and thus double the number of REP Borrelia genomes publicly available. The genomic characterisation of these Borrelia clearly demonstrates their distinctiveness as species, and we propose the names Borrelia salvatorii, ‘Candidatus Borrelia undatumii’, and ‘Candidatus Borrelia rubricentralis’ for them. Graphical Abstract

Background: Changing geographical and seasonal activity patterns of ticks may increase the risk of tick infestation and tick-borne pathogen (TBP) transmission for both humans and animals. Methods: To estimate TBP exposure of dogs and cats, 3000 female I. ricinus from these hosts were investigated for Anaplasma phagocytophilum and Borrelia species. Results: qPCR inhibition, which was observed for ticks of all engorgement stages but not questing ticks, was eliminated at a template volume of 2 µl. In ticks from dogs, A. phagocytophilum and Borrelia spp. prevalence amounted to 19.0% (285/1500) and 28.5% (427/1500), respectively, while ticks from cats showed significantly higher values of 30.9% (464/1500) and 55.1% (827/1500). Accordingly, the coinfection rate with both A. phagocytophilum and Borrelia spp. was significantly higher in ticks from cats (17.5%, 262/1500) than dogs (6.9%, 104/1500). Borrelia prevalence significantly decreased with increasing engorgement duration in ticks from both host species, whereas A. phagocytophilum prevalence decreased only in ticks from dogs. While A. phagocytophilum copy numbers in positive ticks did not change significantly over the time of engorgement, those of Borrelia decreased initially in dog ticks. In ticks from cats, copy numbers of neither A. phagocytophilum nor Borrelia spp. were affected by engorgement. Borrelia species differentiation was successful in 29.1% (365/1254) of qPCR-positive ticks. The most frequently detected species in ticks from dogs were B. afzelii (39.3% of successfully differentiated infections; 70/178), B. miyamotoi (16.3%; 29/178), and B. valaisiana (15.7%; 28/178), while B. afzelii (40.1%; 91/227), B. spielmanii (21.6%; 49/227), and B. miyamotoi (14.1%; 32/227) occurred most frequently in ticks from cats. Conclusions: The differences in pathogen prevalence and Borrelia species distribution between ticks collected from dogs and cats may result from differences in habitat overlap with TBP reservoir hosts. The declining prevalence of A. phagocytophilum with increasing engorgement duration, without a decrease in copy numbers, could indicate transmission to dogs over the time of attachment. The fact that this was not observed in ticks from cats may indicate less efficient transmission. In conclusion, the high prevalence of A. phagocytophilum and Borrelia spp. in ticks collected from dogs and cats underlines the need for effective acaricide tick control to protect both animals and humans from associated health risks. [ABSTRACT FROM AUTHOR]

Background: The genus Borrelia comprises pathogenic species of bacteria that pose a significant risk to public health. Borrelia spp. are emerging or reemerging infectious agents worldwide with complex transmission cycles, and many species use rodents as vertebrate reservoir hosts. Spirochetes morphologically compatible with Borrelia have been recurrently observed in opossums; however, there is currently a lack of genetic evidence confirming infection or supporting that these marsupials are hosts of Borrelia spirochetes. Methods: During 2017, 53 serum samples of Didelphis marsupialis from the municipality of Colosó (department of Sucre, Colombia) were collected and allocated in a serum bank. DNA extracted from the serum samples was submitted to a Borrelia genus-specific real-time PCR targeting the 16S rRNA gene. Positive samples were subsequently derived from semi-nested PCR protocols to obtain large fragments of the 16S rRNA and flaB genes. Obtained amplicons were subjected to Sanger sequencing. One positive sample was randomly selected for next-generation sequencing (NGS). Obtained reads were mapped to genomes of Borrelia spp. and sequences of two genes used in a multilocus sequence typing scheme retrieved for taxonomic assignment and phylogenetic analyses. Results: Overall, 18.8% (10/53) of the samples were positive by qPCR. Of them, 80% (8/10) and 60% (6/10) were positive for the 16S rRNA and flaB genes after semi-nested PCRs, respectively. Bioinformatic analysis of one sample sequenced with NGS yielded 22 reads of genus Borrelia with different sizes. Two housekeeping genes, rplB and pyrG, were recovered. Nucleotide pairwise comparisons and phylogenetic analyses of 16S rRNA, flaB, rplB and pyrG genes showed that the Borrelia sp. found in opossums from Colosó corresponded to Borrelia puertoricensis. Conclusions: We describe the first molecular evidence to our knowledge of B. puertoricensis in Colombia, specifically in opossums, and the first detection of this spirochete in a vertebrate host since its isolation from Ornithodoros puertoricensis in Panama. This detection is also relevant because of the epidemiological importance of opossums as reservoirs of zoonotic diseases to humans. [ABSTRACT FROM AUTHOR]

Background: Borrelia persica causes tick-borne relapsing fever in Israel, the eastern Mediterranean basin, and Asia. Relapsing fever is associated with severe illness and potentially death in humans and animals. Since B. persica infection has rarely been described in wild animals, the aim of this study was to evaluate the prevalence of infection with B. persica in wild carnivores in Israel. Methods: Spleen and blood clot samples from wild carnivores, which underwent necropsy, were tested for the presence of Borrelia DNA by real-time polymerase chain reaction (PCR). PCR products were sequenced, and the spirochete loads were quantified using a specific quantitative PCR (qPCR). Results: A total of 140 samples from 74 wild carnivores were analyzed for the presence of Borrelia DNA. Six out of the 74 (8.1%) animals were found positive for B. persica by PCR and sequencing of the flagellin B gene, of which 4/74 (5.4%) were also positive by PCR for the glycerophosphodiester phosphodiesterase (glpQ) gene. Positive samples were obtained from three European badgers, and one striped hyena, golden jackal, and red fox each. All B. persica-positive animals were young males (P < 0.0001). Quantifiable results were obtained from 3/5 spleen and 4/5 blood samples. The spirochete loads in the blood were significantly higher than those found in the spleen (P = 0.034). Conclusions: The prevalence of B. persica infection found in wild carnivores brought for necropsy was unexpectedly high, suggesting that this infection is widespread in some wild animal species in Israel. This is the first report of B. persica infection in the European badger and striped hyena. These carnivores have a wide geographical range of activity, and the results of this survey raise the possibility that they may serve as reservoir hosts for B. persica. [ABSTRACT FROM AUTHOR]

Background: Borrelia are important disease-causing tick- and louse-borne spirochaetes than can infect a wide variety of vertebrates, including humans and reptiles. Reptile-associated (REP) Borrelia, once considered a peculiarity, are now recognised as a distinct and important evolutionary lineage, and are increasingly being discovered worldwide in association with novel hosts. Numerous novel Borrelia spp. associated with monitor lizards (Varanus spp.) have been recently identified throughout the Indo-Pacific region; however, there is a lack of genomic data on these Borrelia. Methods: We used metagenomic techniques to sequence almost complete genomes of novel Borrelia spp. from Varanus varius and Varanus giganteus from Australia, and used long- and short-read technologies to sequence the complete genomes of two strains of a novel Borrelia sp. previously isolated from ticks infesting Varanus salvator from Indonesia. We investigated intra- and interspecies genomic diversity, including plasmid diversity and relatedness, among Varanus-associated Borrelia and other available REP Borrelia and, based on 712 whole genome orthologues, produced the most complete phylogenetic analysis, to the best of our knowledge, of REP Borrelia to date. Results: The genomic architecture of Varanus-associated Borrelia spp. is similar to that of Borrelia spp. that cause relapsing fever (RF), and includes a highly conserved megaplasmid and numerous smaller linear and circular plasmids that lack structural consistency between species. Analysis of PF32 and PF57/62 plasmid partitioning genes indicated that REP Borrelia plasmids fall into at least six distinct plasmid families, some of which are related to previously defined Borrelia plasmid families, whereas the others appear to be unique. REP Borrelia contain immunogenic variable major proteins that are homologous to those found in Borrelia spp. that cause RF, although they are limited in copy number and variability and have low sequence identities to RF variable major proteins. Phylogenetic analyses based on single marker genes and 712 single copy orthologs also definitively demonstrated the monophyly of REP Borrelia as a unique lineage. Conclusions: In this work we present four new genomes from three novel Borrelia, and thus double the number of REP Borrelia genomes publicly available. The genomic characterisation of these Borrelia clearly demonstrates their distinctiveness as species, and we propose the names Borrelia salvatorii, 'Candidatus Borrelia undatumii', and 'Candidatus Borrelia rubricentralis' for them. [ABSTRACT FROM AUTHOR]

Abstract Background Ticks and tick-borne pathogens significantly impact both human and animal health and therefore are of major concern to the scientific community. Knowledge of tick-borne pathogens is crucial for prescription of mitigation measures. In Africa, much research on ticks has focused on domestic animals. Little is known about ticks and their pathogens in wild habitats and wild animals like the endangered chimpanzee, our closest relative. Methods In this study, we collected ticks in the forested habitat of a community of 100 chimpanzees living in Kibale National Park, Western Uganda, and assessed how their presence and abundance are influenced by environmental factors. We used non-invasive methods of flagging the vegetation and visual search of ticks both on human team members and in chimpanzee nests. We identified adult and nymph ticks through morphological features. Molecular techniques were used to detect and identify tick-borne piroplasmids and bacterial pathogens. Results A total of 470 ticks were collected, which led to the identification of seven tick species: Haemaphysalis parmata (68.77%), Amblyomma tholloni (20.70%), Ixodes rasus sensu lato (7.37%), Rhipicephalus dux (1.40%), Haemaphysalis punctaleachi (0.70%), Ixodes muniensis (0.70%) and Amblyomma paulopunctatum (0.35%). The presence of ticks, irrespective of species, was influenced by temperature and type of vegetation but not by relative humidity. Molecular detection revealed the presence of at least six genera of tick-borne pathogens (Babesia, Theileria, Borrelia, Cryptoplasma, Ehrlichia and Rickettsia). The Afrotopical tick Amblyomma tholloni found in one chimpanzee nest was infected by Rickettsia sp. Conclusions In conclusion, this study presented ticks and tick-borne pathogens in a Ugandan wildlife habitat whose potential effects on animal health remain to be elucidated. Graphical Abstract

Abstract Relapsing fever group Borrelia (RFGB) are motile spirochetes transmitted to mammalian or avian hosts through the bite of hematophagous arthropods, such as soft ticks (Argasidae), hard ticks (Ixodidae) and the human clothing lice. RFGB can infect pets such as dogs and cats, as well as birds, cattle and humans. Borrelia recurrentis, B. anserina and B. theileri are considered to have worldwide distribution, affecting humans, domestic birds and ruminants, respectively. Borrelia spp. associated with soft ticks are transmitted mainly by Ornithodoros ticks and thrive in endemic foci in tropical and subtropical latitudes. Nowadays, human cases of soft tick-borne relapsing fever remain neglected diseases in several countries, and the impact these spirochetes have on the health of wild and domestic animals is largely understudied. Human infection with RFGB is difficult to diagnose, given the lack of distinguishing clinical features (undifferentiated febrile illness). Clinically, soft tick or louse-borne relapsing fever is often confused with other etiologies, such as malaria, typhoid or dengue. In Latin America, during the first half of the twentieth century historical documents elaborated by enlightened physicians were seminal, and resulted in the identification of RFGB and their associated vectors in countries such as Mexico, Panama, Colombia, Venezuela, Peru and Argentina. Almost 80 years later, research on relapsing fever spirochetes is emerging once again in Latin America, with molecular characterizations and isolations of novel RFGB members in Panama, Bolivia, Brazil and Chile. In this review we summarize historical aspects of RFGB in Latin America and provide an update on the current scenario regarding these pathogens in the region. To accomplish this, we conducted an exhaustive search of all the published literature for the region, including old medical theses deposited in libraries of medical academies. RFGB were once common pathogens in Latin America, and although unnoticed for many years, they are currently the focus of interest among the scientific community. A One Health perspective should be adopted to tackle the diseases caused by RFGB, since these spirochetes have never disappeared and the maladies they cause may be confused with etiologies with similar symptoms that prevail in the region. Graphical Abstract

Abstract Background The main objective of this study was to determine the prevalence of nine vector-borne pathogens or pathogen genera in roe deer (Capreolus capreolus) in the Netherlands, and to identify which host variables predict vector-borne pathogen presence in roe deer. The host variables examined were the four host factors ‘age category’, ‘sex’, ‘nutritional condition’ and ‘health status’, as well as ‘roe deer density’. Methods From December 2009 to September 2010, blood samples of 461 roe deer were collected and analysed by polymerase chain reaction (PCR) for the presence of genetic material from Anaplasma phagocytophilum, Bartonella spp., Babesia spp., Borrelia burgdorferi sensu lato (s.l.), Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia spp., and epizootic haemorrhagic disease virus (EHDV), and by commercial enzyme-linked immunosorbent assay (ELISA) for antibodies against bluetongue virus (BTV). The possible associations of host factors and density with pathogen prevalence and co-infection, and in the case of A. phagocytophilum with bacterial load, were assessed using generalized linear modelling. Results and conclusion Analysis revealed the following prevalence in roe deer: A. phagocytophilum 77.9%, Bartonella spp. 77.7%, Babesia spp. 17.4%, Rickettsia spp. 3.3%, B. burgdorferi sensu lato 0.2%. Various co-infections were found, of which A. phagocytophilum and Bartonella spp. (49.7% of infected roe deer) and A. phagocytophilum, Bartonella spp. and Babesia spp. (12.2% of infected roe deer) were the most common. Anaplasma phagocytophilum, Babesia spp., and co-infection prevalence were significantly higher in calves than in adult roe deer, whereas the prevalence of Bartonella spp. was lower in roe deer in good nutritional condition than in deer in poor nutritional condition. Local roe deer density was not associated with pathogen presence. The high prevalence of A. phagocytophilum, Bartonella spp., and Babesia spp. is evidence for the role of roe deer as reservoirs for these pathogens. Additionally, the results suggest a supportive role of roe deer in the life-cycle of Rickettsia spp. in the Netherlands. Graphical Abstract

Background: The composition of the microbial flora associated with ixodid ticks has been studied in several species, revealing the importance of geographical origin, developmental stage(s) and feeding status of the tick, as well as substantial differences between tissues and organs. Studying the microbiome in the correct context and scale is therefore necessary for understanding the interactions between tick-borne pathogens and other microorganisms as well as other aspects of tick biology. Methods: In the present study the microbial flora of whole Ixodes ricinus, I. persulcatus and I. trianguliceps ticks were analyzed with 16S rRNA amplicon sequencing. Additionally, tick organs (midguts, Malpighian tubules, ovaries, salivary glands) from flat and engorged I. ricinus female ticks were examined with the same methodology. Results: The most abundant bacteria belonged to the group of Proteobacteria (Cand. Midichloria mitochondrii and Cand. Lariskella). 16S amplicon sequencing of dissected tick organs provided more information on the diversity of I. ricinus-associated microbial flora, especially when organs were collected from engorged ticks. Bacterial genera significantly associated with tick feeding status as well as genera associated with the presence of tick-borne pathogens were identified. Conclusions: These results contribute to the knowledge of microbial flora associated with ixodid ticks in their northernmost distribution limit in Europe and opens new perspectives for other investigations on the function of these bacteria, including those using other approaches like in vitro cultivation and in vitro models. [ABSTRACT FROM AUTHOR]

Background: Ticks and tick-borne pathogens significantly impact both human and animal health and therefore are of major concern to the scientific community. Knowledge of tick-borne pathogens is crucial for prescription of mitigation measures. In Africa, much research on ticks has focused on domestic animals. Little is known about ticks and their pathogens in wild habitats and wild animals like the endangered chimpanzee, our closest relative. Methods: In this study, we collected ticks in the forested habitat of a community of 100 chimpanzees living in Kibale National Park, Western Uganda, and assessed how their presence and abundance are influenced by environmental factors. We used non-invasive methods of flagging the vegetation and visual search of ticks both on human team members and in chimpanzee nests. We identified adult and nymph ticks through morphological features. Molecular techniques were used to detect and identify tick-borne piroplasmids and bacterial pathogens. Results: A total of 470 ticks were collected, which led to the identification of seven tick species: Haemaphysalis parmata (68.77%), Amblyomma tholloni (20.70%), Ixodes rasus sensu lato (7.37%), Rhipicephalus dux (1.40%), Haemaphysalis punctaleachi (0.70%), Ixodes muniensis (0.70%) and Amblyomma paulopunctatum (0.35%). The presence of ticks, irrespective of species, was influenced by temperature and type of vegetation but not by relative humidity. Molecular detection revealed the presence of at least six genera of tick-borne pathogens (Babesia, Theileria, Borrelia, Cryptoplasma, Ehrlichia and Rickettsia). The Afrotopical tick Amblyomma tholloni found in one chimpanzee nest was infected by Rickettsia sp. Conclusions: In conclusion, this study presented ticks and tick-borne pathogens in a Ugandan wildlife habitat whose potential effects on animal health remain to be elucidated. [ABSTRACT FROM AUTHOR]

Background: The genus Borrelia is composed of two well-defined monophyletic groups, the Borrelia burgdorferi sensu lato complex (Bb) and the relapsing fever (RF) group borreliae. Recently, a third group, associated with reptiles and echidnas, has been described. In general, RF group borreliae use rodents as reservoir hosts; although neotropical bats may also be involved as important hosts, with scarce knowledge regarding this association. The objective of this study was to detect the presence of Borrelia spp. DNA in bats from the department of Córdoba in northwest Colombia. Methods: During September 2020 and June 2021, 205 bats were captured in six municipalities of Córdoba department, Colombia. Specimens were identified using taxonomic keys and DNA was extracted from spleen samples. A Borrelia-specific real-time PCR was performed for the 16S rRNA gene. Fragments of the 16S rRNA and flaB genes were amplified in the positive samples by conventional PCR. The detected amplicons were sequenced by the Sanger method. Phylogenetic reconstruction was performed in IQ-TREE with maximum likelihood based on the substitution model TPM3+F+I+G4 with bootstrap values deduced from 1000 replicates. Results: Overall, 10.2% (21/205) of the samples were found positive by qPCR; of these, 81% (17/21) and 66.6% (14/21) amplified 16S rRNA and flaB genes, respectively. qPCR-positive samples were then subjected to conventional nested and semi-nested PCR to amplify 16S rRNA and flaB gene fragments. Nine positive samples for both genes were sequenced, and seven and six sequences were of good quality for the 16S rRNA and flaB genes, respectively. The DNA of Borrelia spp. was detected in the insectivorous and fruit bats Artibeus lituratus, Carollia perspicillata, Glossophaga soricina, Phyllostomus discolor, and Uroderma sp. The 16S rRNA gene sequences showed 97.66–98.47% identity with "Borrelia sp. clone Omi3," "Borrelia sp. RT1S," and Borrelia sp. 2374; the closest identities for the flaB gene were 94.02–98.04% with "Borrelia sp. Macaregua." For the 16S rRNA gene, the phylogenetic analysis showed a grouping with "Candidatus Borrelia ivorensis" and "Ca. Borrelia africana," and for the flaB gene showed a grouping with Borrelia sp. Macaregua and Borrelia sp. Potiretama. The pathogenic role of the Borrelia detected in this study is unknown. Conclusions: We describe the first molecular evidence of Borrelia spp. in the department of Córdoba, Colombia, highlighting that several bat species harbor Borrelia spirochetes. [ABSTRACT FROM AUTHOR]

Abstract Complement has been considered as an important factor impacting the host–pathogen association of spirochetes belonging to the Borrelia burgdorferi sensu lato complex, and may play a role in the spirochete’s ecology. Birds are known to be important hosts for ticks and in the maintenance of borreliae. Recent field surveys and laboratory transmission studies indicated that certain avian species act as reservoir hosts for different Borrelia species. Nevertheless, our current understanding of the molecular mechanisms determining host tropism of Borrelia is still in its fledgling stage. Concerning the role of complement in avian-host tropism, only a few bird species and Borrelia species have been analysed so far. Here, we performed in vitro serum bactericidal assays with serum samples collected from four bird species including the European robin Erithacus rubecula, the great tit Parus major, the Eurasian blackbird Turdus merula, and the racing pigeon Columba livia, as well as four Borrelia species (B. afzelii, B. garinii, B. valaisiana, and B. burgdorferi sensu stricto). From July to September 2019, juvenile wild birds were caught using mist nets in Portugal. Racing pigeons were sampled in a loft in October 2019. Independent of the bird species analysed, all Borrelia species displayed an intermediate serum-resistant or serum-resistant phenotype except for B. afzelii challenged with serum from blackbirds. This genospecies was efficiently killed by avian complement, suggesting that blackbirds served as dead-end hosts for B. afzelii. In summary, these findings suggest that complement contributes in the avian–spirochete–tick infection cycle and in Borrelia-host tropism.

BackgroundBorrelia burgdorferi (sensu lato), the causative agent of Lyme borreliosis is a bacterium transmitted by hard ticks, Ixodes spp. Bacteria are injected into the host skin during the tick blood meal with tick saliva. There, Borrelia and saliva interact together with skin cells such as keratinocytes, fibroblasts, mast cells and other specific immune cells before disseminating to target organs.MethodsTo study the role of mast cells in the transmission of Lyme borreliosis, we isolated mouse primary mast cells from bone marrow and incubated them in the presence of Borrelia burgdorferi (sensu stricto) and tick salivary gland extract. We further analyzed their potential role in vivo, in a mouse model of deficient in mast cells (Kit wsh-/- mice).ResultsTo our knowledge, we report here for the first time the bacteria ability to induce the inflammatory response of mouse primary mast cells. We show that OspC, a major surface lipoprotein involved in the early transmission of Borrelia, induces the degranulation of primary mast cells but has a limited effect on the overall inflammatory response of these cells. In contrast, whole bacteria have an opposite effect. We also show that mast cell activation is significantly inhibited by tick salivary gland extract. Finally, we demonstrate that mast cells are likely not the only host cells involved in the early transmission and dissemination of Borrelia since the use of mast cell deficient Kit wsh-/- mice shows a limited impact on these two processes in the context of this mouse genetic background.ConclusionsThe absence of mast cells did not change the replication rate of Borrelia in the skin. However, in the absence of mast cells, Borrelia dissemination to the joints was faster. Mast cells do not control skin bacterial proliferation during primary infection and the establishment of the primary infection, as shown in the C57BL/6 mouse model studied. Nevertheless, the Borrelia induced cytotokine modulation on mast cells might be involved in long term and/or repeated infections and protect from Lyme borreliosis due to the development of a hypersensitivity to tick saliva.

Abstract Background Lyme borreliosis (LB) is the most common vector-borne disease in Europe. Monitoring changes in the prevalence of different Borrelia species in ticks may be an important indicator of risk assessment and of differences in pathogenicity in humans. The objective of our study was to assess the prevalence, co-infection and distribution of Borrelia and Babesia species in ticks removed from humans in a large sample collected during a study period of 4 years. Methods The ticks were collected throughout Poland from March to November over 4-year period from 2016 to 2019. All ticks (n = 1953) were morphologically identified in terms of species and developmental stage. Molecular screening for Borrelia and Babesia by amplification of the flagellin gene (flaB) or 18S rRNA marker was performed. Pathogen identity was confirmed by Sanger sequencing or PCR–restriction fragment length polymorphism analysis. Results The ticks removed from humans in Poland during this study belonged to two species: Ixodes ricinus (97%) and Dermacentor reticulatus (3%). High Borrelia prevalence (25.3%), including B. miyamotoi (8.4%), was confirmed in Ixodes ricinus ticks removed from humans, as was the change in frequency of occurrence of Borrelia species during the 4-year study. Despite Babesia prevalence being relatively low (1.3%), the majority of tested isolates are considered to be pathogenic to humans. Babesia infection was observed more frequently among Borrelia-positive ticks (2.7%) than among ticks uninfected with Borrelia (0.8%). The most frequent dual co-infections were between Borrelia afzelii and Babesia microti. The presence of Borrelia was also confirmed in D. reticulatus (12.7%); however the role of these ticks in spirochete transmission to susceptible hosts is still unclear. Conclusions Although the overall risk of developing LB after a tick bite is low in Europe, knowledge of the prevalence and distribution of Borrelia and Babesia species in ticks might be an important indicator of the risk of both these tick-borne diseases. Graphical abstract

Relapsing fever group Borrelia (RFGB) are motile spirochetes transmitted to mammalian or avian hosts through the bite of hematophagous arthropods, such as soft ticks (Argasidae), hard ticks (Ixodidae) and the human clothing lice. RFGB can infect pets such as dogs and cats, as well as birds, cattle and humans. Borrelia recurrentis, B. anserina and B. theileri are considered to have worldwide distribution, affecting humans, domestic birds and ruminants, respectively. Borrelia spp. associated with soft ticks are transmitted mainly by Ornithodoros ticks and thrive in endemic foci in tropical and subtropical latitudes. Nowadays, human cases of soft tick-borne relapsing fever remain neglected diseases in several countries, and the impact these spirochetes have on the health of wild and domestic animals is largely understudied. Human infection with RFGB is difficult to diagnose, given the lack of distinguishing clinical features (undifferentiated febrile illness). Clinically, soft tick or louse-borne relapsing fever is often confused with other etiologies, such as malaria, typhoid or dengue. In Latin America, during the first half of the twentieth century historical documents elaborated by enlightened physicians were seminal, and resulted in the identification of RFGB and their associated vectors in countries such as Mexico, Panama, Colombia, Venezuela, Peru and Argentina. Almost 80 years later, research on relapsing fever spirochetes is emerging once again in Latin America, with molecular characterizations and isolations of novel RFGB members in Panama, Bolivia, Brazil and Chile. In this review we summarize historical aspects of RFGB in Latin America and provide an update on the current scenario regarding these pathogens in the region. To accomplish this, we conducted an exhaustive search of all the published literature for the region, including old medical theses deposited in libraries of medical academies. RFGB were once common pathogens in Latin America, and although unnoticed for many years, they are currently the focus of interest among the scientific community. A One Health perspective should be adopted to tackle the diseases caused by RFGB, since these spirochetes have never disappeared and the maladies they cause may be confused with etiologies with similar symptoms that prevail in the region. [ABSTRACT FROM AUTHOR]

Abstract Background The universal nature of the human–companion animal relationship and their shared ticks and tick-borne pathogens offers an opportunity for improving public and veterinary health surveillance. With this in mind, we describe the spatiotemporal trends for blacklegged tick (Ixodes scapularis) submissions from humans and companion animals in Ontario, along with pathogen prevalence. Methods We tested tick samples submitted through passive surveillance (2011–2017) from humans and companion animals for Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum and Babesia microti. We describe pathogen prevalence in ticks from humans and from companion animals and constructed univariable Poisson and negative binomial regression models to explore the spatiotemporal relationship between the rates of tick submissions by host type. Results During the study, there were 17,230 blacklegged tick samples submitted from humans and 4375 from companion animals. Tick submission rates from companion animals were higher than expected in several public health units (PHUs) lacking established tick populations, potentially indicating newly emerging populations. Pathogen prevalence in ticks was higher in PHUs where established blacklegged tick populations exist. Borrelia burgdorferi prevalence was higher in ticks collected from humans (maximum likelihood estimate, MLE = 17.5%; 95% confidence interval, CI 16.97–18.09%) than from companion animals (9.9%, 95% CI 9.15–10.78%). There was no difference in pathogen prevalence in ticks by host type for the remaining pathogens, which were found in less than 1% of tested ticks. The most common co-infection B. burgdorferi + B. miyamotoi occurred in 0.11% of blacklegged ticks from humans and animals combined. Borrelia burgdorferi prevalence was higher in unengorged (21.9%, 95% CI 21.12–22.65%) than engorged ticks (10.0%, 95% CI 9.45–10.56%). There were no consistent and significant spatiotemporal relationships detected via regression models between the annual rates of submission of each host type. Conclusions While B. burgdorferi has been present in blacklegged ticks in Ontario for several decades, other tick-borne pathogens are also present at low prevalence. Blacklegged tick and pathogen surveillance data can be used to monitor risk in human and companion animal populations, and efforts are under consideration to unite surveillance efforts for the different target populations. Graphic Abstract

Abstract Background Dogs play an important role as reservoirs of many zoonotic vector-borne pathogens worldwide, yet reports of canine vector-borne diseases (CVBDs) in Egypt are scarce. Methods Serum samples were collected from pet dogs (n = 500) of the three most common breeds (German Shepherd, Rottweiler and Pit Bull) in five Governates of Cairo (n = 230), Giza (n = 110), Al-Qalyubia (n = 60), Al-Gharbia (n = 60) and Kafr El-Sheikh (n = 40) with a hot desert climate. The presence of antibodies to Anaplasma spp. (A. phagocytophilum, A. platys), Ehrlichia spp. (E. canis, E. chaffeensis, E. ewingii), Borrelia burgdorferi (s.l.) and Dirofilaria immitis were assessed using IDEXX SNAP® 4Dx® ELISA tests. For each pathogen, risk factors (i.e. geographical area, keeping condition, sex, age, breed, tick infestation, weekly sanitation of dog enclosures and application of ectoparasiticides) were evaluated by logistic regression approach. Results In total, 18.2% (n = 91, 95% CI 15.1–21.8) of dogs scored seropositive for at least one pathogen, the most frequent being Ehrlichia spp. (n = 56; 11.2%; 95% CI 8.7–14.3) followed by Anaplasma spp. (n = 33; 6.6%, 95% CI 4.7–9.1), Borrelia burgdorferi (s.l.) (n = 9; 1.8%, 95% CI 0.9–3.4) and D. immitis (n = 7; 1.4%, 95% CI 0.9–2.9). In the tested population, 15.4% (95% CI 12.5–18.8) of dogs were exposed to a single pathogen while 2.4 (95% CI 1.4–4.2) and 0.4% (95% CI 0.1–1.4) were simultaneously exposed to two or three pathogens, respectively. Major risk factors associated with VBDs were living outdoors (Anaplasma spp., P = 0.0001; Ehrlichia spp., P = 0.0001), female sex (Ehrlichia spp., P = 0.005), German Shepherd breed (Anaplasma spp., P = 0.04; Ehrlichia spp., P = 0.03), tick infestation (Anaplasma spp., P = 0.0001; Ehrlichia spp., P = 0.0001; B. burgdorferi (s.l.), P = 0.003; D. immitis, P = 0.02), irregular sanitation (Anaplasma spp., P = 0.0001; Ehrlichia spp., P = 0.0001; B. burgdorferi (s.l.), P = 0.002; D. immitis, P = 0.01) and not using ectoparasiticides (Anaplasma spp., P = 0.0001; Ehrlichia spp., P = 0.0001; B. burgdorferi (s.l.), P = 0.007). Conclusion To our knowledge, this is the first large-scale seroepidemiological study of CVBDs in Egypt. Considering that all of the detected pathogens are potentially zoonotic, effective ectoparasite control strategies, regular examination of pet dogs and successful chemoprophylaxis are advocated.

Background: Relapsing fever borreliosis is an infectious disease caused by bacteria of the genus Borrelia, inflicting recurrent episodes of fever and spirochetemia in humans. Borrelia persica, the causative agent of relapsing fever in Israel, is prevalent over a broad geographic area that extends from India to Egypt. It is transmitted by the soft tick Ornithodoros tholozani and causes disease in humans as well as domestic cats and dogs. The goal of this study was to survey domestic dogs and cats in Israel for infection with B. persica. Methods: Blood, sera and demographic and clinical data were collected from dogs and cats brought for veterinary care in central Israel. PCR followed by DNA sequencing was used to detect B. persica DNA in blood samples, and an enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies reactive with B. persica antigens in sera from the same animals. This is the first serological survey of B. persica in dogs and the first survey for antibodies reactive with a relapsing fever Borrelia sp. in cats globally. Results: Four of the 208 dogs (1.9%) and three of 103 cats (2.9%) sampled were positive by PCR for B. persica DNA, and 24 dogs (11.5%) and 18 cats (17.5%) were seropositive for B. persica antigen by ELISA. The ratio between PCR-positivity and seropositivity in both the dog and cat populations was 1:6. All four PCR-positive dogs and two of three PCR-positive cats were seronegative, suggesting a probable recent infection. Thrombocytopenia showed significant association with seropositivity in dogs (P = 0.003). In cats, anemia had a significant association with seropositivity (P = 0.0001), and thrombocytopenia was associated with the combined prevalence of seropositivity or PCR-positivity (P = 0.022). Conclusions: Borrelia persica infection is more prevalent and widespread in domestic canine and feline populations in Israel than previously thought. Dogs and cats may play a role as reservoirs and sentinels for human infection. Precautions should be taken to prevent transfusion-transmitted infection between blood donor and recipient animals. [ABSTRACT FROM AUTHOR]

Background: Ticks and tick-borne diseases constitute a real threat for the livestock industry, which is increasing in Angola. In addition, ticks are vectors of zoonoses of public health concern, and scarce information is available from this country. In an effort to contribute to the prevention of zoonotic infectious diseases affecting humans and animals, the molecular screening of certain tick-related microorganisms collected on cattle in Angola was performed under a 'One Health' scope. Methods: Ticks collected from cattle in Cubal (Benguela Province, Angola) in July 2017 were analysed in pools using specific PCR assays for bacteria (Rickettsia, Anaplasmataceae, Borrelia, Coxiella and Spiroplasma) and protozoa (Theileria and Babesia) detection. Results: A total of 124 tick specimens were grouped in 25 pools (two Amblyomma variegatum, three Hyalomma truncatum, 16 Rhipicephalus decoloratus, two Rhipicephalus duttoni, one Rhipicephalus evertsi mimeticus and one Rhipicephalus sp.). The amplified microorganisms were (pools): Rickettsia africae (two A. variegatum and one R. decoloratus), Rickettsia aeschlimannii (three H. truncatum), Ehrlichia spp. (six R. decoloratus), Coxiella spp. (all but H. truncatum), Francisella sp. (one H. truncatum), Spiroplasma sp. closely related to Spiroplasma ixodetis (three R. decoloratus), Babesia bigemina (two R. decoloratus) and Babesia spp. (two A. variegatum). The obtained nucleotide sequences from Ehrlichia spp., two Coxiella genotypes (from R. duttoni and Rhipicephalus sp.), Francisella sp. and Babesia spp. (from A. variegatum) reached low identities with known genetically characterized species. Conclusions: This study demonstrates the circulation in Angola of the pathogen R. aeschlimannii and potential novel tick-related microorganisms belonging to Ehrlichia, Coxiella, Francisella, Spiroplasma and Babesia spp. and corroborates the presence of R. africae and B. bigemina. Our results should be considered in developing protocols for the management of fever of unknown origin and for veterinary practices. Further studies are required to evaluate the risk of tick-borne diseases in Angola. [ABSTRACT FROM AUTHOR]

Background: The main objective of this study was to determine the prevalence of nine vector-borne pathogens or pathogen genera in roe deer (Capreolus capreolus) in the Netherlands, and to identify which host variables predict vector-borne pathogen presence in roe deer. The host variables examined were the four host factors 'age category', 'sex', 'nutritional condition' and 'health status', as well as 'roe deer density'. Methods: From December 2009 to September 2010, blood samples of 461 roe deer were collected and analysed by polymerase chain reaction (PCR) for the presence of genetic material from Anaplasma phagocytophilum, Bartonella spp., Babesia spp., Borrelia burgdorferi sensu lato (s.l.), Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia spp., and epizootic haemorrhagic disease virus (EHDV), and by commercial enzyme-linked immunosorbent assay (ELISA) for antibodies against bluetongue virus (BTV). The possible associations of host factors and density with pathogen prevalence and co-infection, and in the case of A. phagocytophilum with bacterial load, were assessed using generalized linear modelling. Results and conclusion: Analysis revealed the following prevalence in roe deer: A. phagocytophilum 77.9%, Bartonella spp. 77.7%, Babesia spp. 17.4%, Rickettsia spp. 3.3%, B. burgdorferi sensu lato 0.2%. Various co-infections were found, of which A. phagocytophilum and Bartonella spp. (49.7% of infected roe deer) and A. phagocytophilum, Bartonella spp. and Babesia spp. (12.2% of infected roe deer) were the most common. Anaplasma phagocytophilum, Babesia spp., and co-infection prevalence were significantly higher in calves than in adult roe deer, whereas the prevalence of Bartonella spp. was lower in roe deer in good nutritional condition than in deer in poor nutritional condition. Local roe deer density was not associated with pathogen presence. The high prevalence of A. phagocytophilum, Bartonella spp., and Babesia spp. is evidence for the role of roe deer as reservoirs for these pathogens. Additionally, the results suggest a supportive role of roe deer in the life-cycle of Rickettsia spp. in the Netherlands. [ABSTRACT FROM AUTHOR]

Background: Currently, various zoonotic diseases are classified as emerging or reemerging. Because equids have a direct relationship with various vectors, they are possibly more frequently exposed to zoonotic agents than are humans. The undeniable importance of diseases such as human granulocytic anaplasmosis, spotted fever, and leishmaniasis for both public and animal health, as well as the possibility of equids acting as sources, reservoirs, or even sentinels for these pathogens, justifies the detection of their frequency and factors associated with infection in equids from northeastern Brazil. Methods: Blood samples were collected from 569 equids (528 horses, 33 donkeys, and 8 mules), 516 from a rural area and 53 from an urban area. Pathogen detection was carried out as follows: Borrelia spp. and Rickettsia spp., serological analysis; Leishmania spp., serological analysis and polymerase chain reaction (PCR); Anaplasma phagocytophilum, PCR. Determination of associated factors was carried out through generalized linear models. Results: The frequencies of positivity for the pathogens observed in equids were as follows: Borrelia spp., 13.9% (79/569); Leishmania spp., 3.5% (20/569); Rickettsia spp. 33.4% (190/569). Regarding factors associated with infection, male sex was associated with protection against Borrelia spp.; donkeys and mules were associated with protection against Rickettsia spp., while a younger age was a risk factor. The infection of A. phagocytophilum was not detected in the sampled population. Co-infection was detected in 5.1% (29/569) of the animals. Conclusions: Most of the studied pathogenic agents are present in the prospected area, indicating a possible risk for both human and animal health. This demonstrates that equids can be considered important sentinels in the assessment of pathogens with zoonotic potential in the region. [ABSTRACT FROM AUTHOR]

Background: The universal nature of the human–companion animal relationship and their shared ticks and tick-borne pathogens offers an opportunity for improving public and veterinary health surveillance. With this in mind, we describe the spatiotemporal trends for blacklegged tick (Ixodes scapularis) submissions from humans and companion animals in Ontario, along with pathogen prevalence. Methods: We tested tick samples submitted through passive surveillance (2011–2017) from humans and companion animals for Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum and Babesia microti. We describe pathogen prevalence in ticks from humans and from companion animals and constructed univariable Poisson and negative binomial regression models to explore the spatiotemporal relationship between the rates of tick submissions by host type. Results: During the study, there were 17,230 blacklegged tick samples submitted from humans and 4375 from companion animals. Tick submission rates from companion animals were higher than expected in several public health units (PHUs) lacking established tick populations, potentially indicating newly emerging populations. Pathogen prevalence in ticks was higher in PHUs where established blacklegged tick populations exist. Borrelia burgdorferi prevalence was higher in ticks collected from humans (maximum likelihood estimate, MLE = 17.5%; 95% confidence interval, CI 16.97–18.09%) than from companion animals (9.9%, 95% CI 9.15–10.78%). There was no difference in pathogen prevalence in ticks by host type for the remaining pathogens, which were found in less than 1% of tested ticks. The most common co-infection B. burgdorferi + B. miyamotoi occurred in 0.11% of blacklegged ticks from humans and animals combined. Borrelia burgdorferi prevalence was higher in unengorged (21.9%, 95% CI 21.12–22.65%) than engorged ticks (10.0%, 95% CI 9.45–10.56%). There were no consistent and significant spatiotemporal relationships detected via regression models between the annual rates of submission of each host type. Conclusions: While B. burgdorferi has been present in blacklegged ticks in Ontario for several decades, other tick-borne pathogens are also present at low prevalence. Blacklegged tick and pathogen surveillance data can be used to monitor risk in human and companion animal populations, and efforts are under consideration to unite surveillance efforts for the different target populations. [ABSTRACT FROM AUTHOR]

Borrelia miyamotoi, a bacterium that causes relapsing fever, is found in ixodid ticks throughout the northern hemisphere. The first cases of human infection with B. miyamotoi were identified in 2011. In the eastern USA, blacklegged ticks (Ixodes scapularis) become infected by feeding on an infected vertebrate host, or through transovarial transmission. We surveyed B. miyamotoi prevalence in ticks within forested habitats in Dutchess County, New York, and identified possible reservoir hosts. To assess spatial variation in infection, we collected questing nymphal ticks at > 150 sites. To assess temporal variation in infection, we collected questing nymphs for 8 years at a single study site. We collected questing larval ticks from nine plots to estimate the amount of transovarial transmission. To evaluate potential reservoir hosts, we captured 14 species of mammal and bird hosts naturally infested with larval blacklegged ticks and held these hosts in the laboratory until ticks fed to repletion and molted to nymphs. We determined infection for all ticks using quantitative polymerase chain reaction. The overall infection prevalence of questing nymphal ticks across all sites was ~ 1%, but prevalence at individual sites was as high as 9.1%. We detected no significant increase in infection through time. Only 0.4% of questing larval ticks were infected. Ticks having fed as larvae from short-tailed shrews, red squirrels, and opossums tended to have higher infection prevalence than did ticks having fed on other hosts. Further studies of the role of hosts in transmission are warranted. The locally high prevalence of B. miyamotoi in the New York/New England landscape suggests the importance of vigilance by health practitioners and the public. [ABSTRACT FROM AUTHOR]

Complement has been considered as an important factor impacting the host–pathogen association of spirochetes belonging to the Borrelia burgdorferi sensu lato complex, and may play a role in the spirochete's ecology. Birds are known to be important hosts for ticks and in the maintenance of borreliae. Recent field surveys and laboratory transmission studies indicated that certain avian species act as reservoir hosts for different Borrelia species. Nevertheless, our current understanding of the molecular mechanisms determining host tropism of Borrelia is still in its fledgling stage. Concerning the role of complement in avian-host tropism, only a few bird species and Borrelia species have been analysed so far. Here, we performed in vitro serum bactericidal assays with serum samples collected from four bird species including the European robin Erithacus rubecula, the great tit Parus major, the Eurasian blackbird Turdus merula, and the racing pigeon Columba livia, as well as four Borrelia species (B. afzelii, B. garinii, B. valaisiana, and B. burgdorferi sensu stricto). From July to September 2019, juvenile wild birds were caught using mist nets in Portugal. Racing pigeons were sampled in a loft in October 2019. Independent of the bird species analysed, all Borrelia species displayed an intermediate serum-resistant or serum-resistant phenotype except for B. afzelii challenged with serum from blackbirds. This genospecies was efficiently killed by avian complement, suggesting that blackbirds served as dead-end hosts for B. afzelii. In summary, these findings suggest that complement contributes in the avian–spirochete–tick infection cycle and in Borrelia-host tropism. [ABSTRACT FROM AUTHOR]

Abstract Background Public interest for tick-borne pathogens in cattle livestock is rising due to their veterinary and zoonotic importance. Consequently, correct identification of these potential pathogens is crucial to estimate the level of exposition, the risk and the detrimental impact on livestock and the human population. Results Conventional PCR with generic primers was used to identify groups of tick-borne pathogens in cattle breeds from northern Cameroon. The overall prevalence in 1260 blood samples was 89.1%, with 993 (78.8%) positive for Theileria/Babesia spp., 959 (76.1%) for Anaplasma/Ehrlichia spp., 225 (17.9%) for Borrelia spp., and 180 (14.3%) for Rickettsia spp. Sanger sequencing of a subset of positively-tested samples revealed the presence of Theileria mutans (92.2%, 130/141), T. velifera (16.3%, 23/141), Anaplasma centrale (10.9%, 15/137), A. marginale (30.7%, 42/137), A. platys (51.1%, 70/137), Anaplasma sp. ‘Hadesa’ (10.9%, 15/137), Ehrlichia ruminantium (0.7%, 1/137), E. canis (0.7%, 1/137), Borrelia theileri (91.3%, 42/46), Rickettsia africae (59.4%, 19/32) and R. felis (12.5%, 4/32). A high level of both intra- and inter-generic co-infections (76.0%) was observed. To the best of our knowledge, B. theileri, T. mutans, T. velifera, A. platys, Anaplasma sp. ‘Hadesa’, R. felis and E. canis are reported for the first time in cattle from Cameroon, and for R. felis it is the first discovery in the cattle host. Babesia spp. were not detected by sequencing. The highest number of still identifiable species co-infections was up to four pathogens per genus group. Multifactorial analyses revealed a significant association of infection with Borrelia theileri and anemia. Whereas animals of older age had a higher risk of infection, the Gudali cattle had a lower risk compared to the other local breeds. Conclusion Co-infections of tick-borne pathogens with an overall high prevalence were found in all five study sites, and were more likely to occur than single infections. Fulani, Namchi and Kapsiki were the most infected breed in general; however, with regions as significant risk factor. A better-adapted approach for tick-borne pathogen identification in co-infected samples is a requirement for epidemiological investigations and tailored control measures.

Abstract Hematophagous arthropods are responsible for the transmission of a variety of pathogens that cause disease in humans and animals. Ticks of the Ixodes ricinus complex are vectors for some of the most frequently occurring human tick-borne diseases, particularly Lyme borreliosis and tick-borne encephalitis virus (TBEV). The search for vaccines against these diseases is ongoing. Efforts during the last few decades have primarily focused on understanding the biology of the transmitted viruses, bacteria and protozoans, with the goal of identifying targets for intervention. Successful vaccines have been developed against TBEV and Lyme borreliosis, although the latter is no longer available for humans. More recently, the focus of intervention has shifted back to where it was initially being studied which is the vector. State of the art technologies are being used for the identification of potential vaccine candidates for anti-tick vaccines that could be used either in humans or animals. The study of the interrelationship between ticks and the pathogens they transmit, including mechanisms of acquisition, persistence and transmission have come to the fore, as this knowledge may lead to the identification of critical elements of the pathogens’ life-cycle that could be targeted by vaccines. Here, we review the status of our current knowledge on the triangular relationships between ticks, the pathogens they carry and the mammalian hosts, as well as methods that are being used to identify anti-tick vaccine candidates that can prevent the transmission of tick-borne pathogens.

Background: Borrelia miyamotoi is a newly described relapsing fever spirochete transmitted by ixodid tick species. Little is known about the prevalence of B. miyamotoi infections in humans and ticks in Inner Mongolia, China. Therefore, we investigated the prevalence of B. miyamotoi in Ixodes persulcatus ticks, and we aimed to isolateB. miyamotoi from I. persulcatus from four regions of Greater Khingan, Inner Mongolia, China. Methods: From May to June each year during the period 2016–2019, host-seeking adult I. persulcatus ticks were collected from vegetation. Genomic DNA was prepared from half of each tick body for PCR template, and the remaining half was used to cultivate B. miyamotoi in BSK-M medium. We employed quantitative real-time PCR (qPCR) to detect Borrelia DNA in the ticks and to calculate the prevalence of B. miyamotoi and infections with other borreliae. For characterization of the isolated B. miyamotoi, we performed draft genome sequencing and multilocus sequencing analysis (MLSA). Results: A total of 2656 adult I. persulcatus ticks were collected. The overall prevalence of relapsing fever (RF) borreliae in ticks was 5.0% (134/2656) and that of Lyme disease (LD) borreliae was 43.8% (1164/2656). Co-infection with RF and LD borreliae was observed in 63 ticks (2.4%). Ticks that were positive for RF borreliae by qPCR were subjected to glycerophosphodiester diester phosphodiesterase gene (glpQ) PCR amplification and sequencing, through which we identified the RF borrelia specimens as B. miyamotoi. Furthermore, the B. miyamotoi strain Hetao-1 was isolated from I. persulcatus, and a draft genome sequence was obtained from the isolate. Sequencing determined the strain Hetao-1 genome to be approximately 906.1 kbp in length (28.9% average GC content), and MLSA identified the strain as ST633, which has previously been reported in Japan and Mongolia. Conclusion: We detected B. miyamotoi from I. persulcatus ticks collected in Inner Mongolia, and successfully isolated a B. miyamotoi strain. To our knowledge, this is the first study to culture a B. miyamotoi isolate from China. The data on the prevalence of B. miyamotoi and other borreliae in I. persulcatus ticks will be fundamental for future epidemiological studies of B. miyamotoi disease in Inner Mongolia. [ABSTRACT FROM AUTHOR]

Background: Lyme borreliosis (LB) is the most common vector-borne disease in Europe. Monitoring changes in the prevalence of different Borrelia species in ticks may be an important indicator of risk assessment and of differences in pathogenicity in humans. The objective of our study was to assess the prevalence, co-infection and distribution of Borrelia and Babesia species in ticks removed from humans in a large sample collected during a study period of 4 years. Methods: The ticks were collected throughout Poland from March to November over 4-year period from 2016 to 2019. All ticks (n = 1953) were morphologically identified in terms of species and developmental stage. Molecular screening for Borrelia and Babesia by amplification of the flagellin gene (flaB) or 18S rRNA marker was performed. Pathogen identity was confirmed by Sanger sequencing or PCR–restriction fragment length polymorphism analysis. Results: The ticks removed from humans in Poland during this study belonged to two species: Ixodes ricinus (97%) and Dermacentor reticulatus (3%). High Borrelia prevalence (25.3%), including B. miyamotoi (8.4%), was confirmed in Ixodes ricinus ticks removed from humans, as was the change in frequency of occurrence of Borrelia species during the 4-year study. Despite Babesia prevalence being relatively low (1.3%), the majority of tested isolates are considered to be pathogenic to humans. Babesia infection was observed more frequently among Borrelia-positive ticks (2.7%) than among ticks uninfected with Borrelia (0.8%). The most frequent dual co-infections were between Borrelia afzelii and Babesia microti. The presence of Borrelia was also confirmed in D. reticulatus (12.7%); however the role of these ticks in spirochete transmission to susceptible hosts is still unclear. Conclusions: Although the overall risk of developing LB after a tick bite is low in Europe, knowledge of the prevalence and distribution of Borrelia and Babesia species in ticks might be an important indicator of the risk of both these tick-borne diseases. [ABSTRACT FROM AUTHOR]

Background: Dogs play an important role as reservoirs of many zoonotic vector-borne pathogens worldwide, yet reports of canine vector-borne diseases (CVBDs) in Egypt are scarce. Methods: Serum samples were collected from pet dogs (n = 500) of the three most common breeds (German Shepherd, Rottweiler and Pit Bull) in five Governates of Cairo (n = 230), Giza (n = 110), Al-Qalyubia (n = 60), Al-Gharbia (n = 60) and Kafr El-Sheikh (n = 40) with a hot desert climate. The presence of antibodies to Anaplasma spp. (A. phagocytophilum, A. platys), Ehrlichia spp. (E. canis, E. chaffeensis, E. ewingii), Borrelia burgdorferi (s.l.) and Dirofilaria immitis were assessed using IDEXX SNAP® 4Dx® ELISA tests. For each pathogen, risk factors (i.e. geographical area, keeping condition, sex, age, breed, tick infestation, weekly sanitation of dog enclosures and application of ectoparasiticides) were evaluated by logistic regression approach. Results: In total, 18.2% (n = 91, 95% CI 15.1–21.8) of dogs scored seropositive for at least one pathogen, the most frequent being Ehrlichia spp. (n = 56; 11.2%; 95% CI 8.7–14.3) followed by Anaplasma spp. (n = 33; 6.6%, 95% CI 4.7–9.1), Borrelia burgdorferi (s.l.) (n = 9; 1.8%, 95% CI 0.9–3.4) and D. immitis (n = 7; 1.4%, 95% CI 0.9–2.9). In the tested population, 15.4% (95% CI 12.5–18.8) of dogs were exposed to a single pathogen while 2.4 (95% CI 1.4–4.2) and 0.4% (95% CI 0.1–1.4) were simultaneously exposed to two or three pathogens, respectively. Major risk factors associated with VBDs were living outdoors (Anaplasma spp., P = 0.0001; Ehrlichia spp., P = 0.0001), female sex (Ehrlichia spp., P = 0.005), German Shepherd breed (Anaplasma spp., P = 0.04; Ehrlichia spp., P = 0.03), tick infestation (Anaplasma spp., P = 0.0001; Ehrlichia spp., P = 0.0001; B. burgdorferi (s.l.), P = 0.003; D. immitis, P = 0.02), irregular sanitation (Anaplasma spp., P = 0.0001; Ehrlichia spp., P = 0.0001; B. burgdorferi (s.l.), P = 0.002; D. immitis, P = 0.01) and not using ectoparasiticides (Anaplasma spp., P = 0.0001; Ehrlichia spp., P = 0.0001; B. burgdorferi (s.l.), P = 0.007). Conclusion: To our knowledge, this is the first large-scale seroepidemiological study of CVBDs in Egypt. Considering that all of the detected pathogens are potentially zoonotic, effective ectoparasite control strategies, regular examination of pet dogs and successful chemoprophylaxis are advocated. [ABSTRACT FROM AUTHOR]

Abstract Background Ticks participate as arthropod vectors in the transmission of pathogenic microorganisms to humans. Several tick-borne infections have reemerged, along with newly described agents of unexplored pathogenicity. In an attempt to expand current information on tick-associated bacteria and protozoans, we performed a cross-sectional screening of ticks, using next-generation sequencing. Ticks seeking hosts and infesting domestic animals were collected in four provinces across the Aegean, Mediterranean and Central Anatolia regions of Turkey and analyzed by commonly used procedures and platforms. Results Two hundred and eighty ticks comprising 10 species were evaluated in 40 pools. Contigs from tick-associated microorganisms were detected in 22 (55%) questing and 4 feeding (10%) tick pools, with multiple microorganisms identified in 12 pools. Rickettsia 16S ribosomal RNA gene, gltA, sca1 and ompA sequences were present in 7 pools (17.5%), comprising feeding Haemaphysalis parva and questing/hunting Rhipicephalus bursa, Rhipicephalus sanguineus (sensu lato) and Hyalomma marginatum specimens. A near-complete genome and conjugative plasmid of a Rickettsia hoogstraalii strain could be characterized in questing Ha. parva. Coxiella-like endosymbionts were identified in pools of questing (12/40) as well as feeding (4/40) ticks of the genera Rhipicephalus, Haemaphysalis and Hyalomma. Francisella-like endosymbionts were also detected in 22.5% (9/40) of the pools that comprise hunting Hyalomma ticks in 8 pools. Coxiella-like and Francisella-like endosymbionts formed phylogenetically distinct clusters associated with their tick hosts. Borrelia turcica was characterized in 5% (2/40) of the pools, comprising hunting Hyalomma aegyptium ticks. Co-infection of Coxiella-like endosymbiont and Babesia was noted in a questing R. sanguineus (s.l.) specimen. Furthermore, protozoan 18S rRNA gene sequences were detected in 4 pools of questing/hunting ticks (10%) and identified as Babesia ovis, Hemolivia mauritanica, Babesia and Theileria spp. Conclusions Our metagenomic approach enabled identification of diverse pathogenic and non-pathogenic microorganisms in questing and feeding ticks in Anatolia.

Abstract Background Lyme borreliosis in humans results in a range of clinical manifestations, thought to be partly due to differences in the pathogenicity of the infecting strain. This study compared European human clinical strains of Borreliella afzelii (previously named Borrelia afzelii) using multilocus sequence typing (MLST) to determine their spatial distribution across Europe and to establish whether there are associations between B. afzelii genotypes and specific clinical manifestations of Lyme borreliosis. For this purpose, typing was performed on 63 strains, and data on a further 245 strains were accessed from the literature. Results All 308 strains were categorized into 149 sequence types (STs), 27 of which are described here for the first time. Phylogenetic and goeBURST analyses showed short evolutionary distances between strains. Although the main STs differed among the countries with the largest number of strains of interest (Germany, the Netherlands, France and Slovenia), the B. afzelii clinical strains were less genetically structured than those previously observed in the European tick population. Two STs were found significantly more frequently in strains associated with clinical manifestations involving erythema migrans, whereas another ST was found significantly more frequently in strains associated with disseminated manifestations, especially neuroborreliosis. Conclusions The MLST profiles showed low genetic differentiation between B. afzelii strains isolated from patients with Lyme borreliosis in Europe. Also, clinical data analysis suggests the existence of lineages with differential dissemination properties in humans.

Abstract Background Lyme borreliosis caused by spirochetes of the Borrelia burgdorferi (sensu lato) complex is still the most common tick-borne disease in Europe, posing a considerable threat to public health. The predominant vector in Europe is the widespread hard tick Ixodes ricinus, which also transmits the relapsing fever spirochete B. miyamotoi as well as pathogenic Rickettsiales (Anaplasma phagocytophilum, Rickettsia spp.). To assess the public health risk, a long-term monitoring of tick infection rates with the named pathogens is indispensable. Methods The present study is the first German 10-year follow-up monitoring of tick infections with Borrelia spp. and co-infections with Rickettsiales. Furthermore, a specific Reverse Line Blot (RLB) protocol for detection of B. miyamotoi and simultaneous differentiation of B. burgdorferi (s.l.) geno-species was established. Results Overall, 24.0% (505/2100) of ticks collected in the city of Hanover were infected with Borrelia. In detail, 35.4% (203/573) of adult ticks [38.5% females (111/288) and 32.3% males (92/285)] and 19.8% nymphs (302/1527) were infected, representing consistent infection rates over the 10-year monitoring period. Geno-species differentiation using RLB determined B. miyamotoi in 8.9% (45/505) of positive ticks. Furthermore, a significant decrease in B. afzelii and B. spielmanii infection rates from 2010 to 2015 was observed. Co-infections with Rickettsia spp. and A. phagocytophilum increased between 2010 and 2015 (7.3 vs 10.9% and 0.3 vs 1.1%, respectively). Conclusions Long-term monitoring is an essential part of public health risk assessment to capture data on pathogen occurrence over time. Such data will reveal shifts in pathogen geno-species distribution and help to answer the question whether or not climate change influences tick-borne pathogens.

Abstract Background Ticks are important carriers of many different zoonotic pathogens. To date, there are many studies about ticks and tick-borne pathogens (TBP), but only a few were carried out in Bulgaria. The present study intends to detect the prevalence of tick-borne bacteria and parasites occurring at the Black Sea in Bulgaria to evaluate the zoonotic potential of the tick-borne pathogens transmitted by ticks in this area. Methods In total, cDNA from 1541 ticks (Dermacentor spp., Haemaphysalis spp., Hyalomma spp., Ixodes spp. and Rhipicephalus spp.) collected in Bulgaria by flagging method or from hosts was tested in pools of ten individuals each for Anaplasma phagocytophilum, Babesia spp., Borrelia burgdorferi (s.l.), Rickettsia spp. and “Candidatus Neoehrlichia mikurensis” via conventional and quantitative real-time PCR. Subsequently, samples from positive pools were tested individually and a randomized selection of positive PCR samples was purified, sequenced, and analyzed. Results Altogether, 23.2% of ticks were infected with at least one of the tested pathogens. The highest infection levels were noted in nymphs (32.3%) and females (27.5%). Very high prevalence was detected for Rickettsia spp. (48.3%), followed by A. phagocytophilum (6.2%), Borrelia burgdorferi (s.l.) (1.7%), Babesia spp. (0.4%) and “Ca. Neoehrlichia mikurensis” (0.1%). Co-infections were found in 2.5% of the tested ticks (mainly Ixodes spp.). Sequencing revealed the presence of Rickettsia monacensis, R. helvetica, and R. aeschlimannii, Babesia microti and B. caballi, and Theileria buffeli and Borrelia afzelli. Conclusion This study shows very high prevalence of zoonotic Rickettsia spp. in ticks from Bulgaria and moderate to low prevalence for all other pathogens tested. One should take into account that tick bites from this area could lead to Rickettsia infection in humans and mammals.

Background: Because ixodid ticks are vectors of zoonotic pathogens, including Borrelia, information of their abundance, seasonal variation in questing behaviour and pathogen prevalence is important for human health. As ticks are invading new areas northwards, information from these new areas are needed. Taiga tick (Ixodes persulcatus) populations have been recently found at Bothnian Bay, Finland. We assessed seasonal variation in questing abundance of ticks and their pathogen prevalence in coastal deciduous forests near the city of Oulu (latitudes 64–65°) in 2019. Methods: We sampled ticks from May until September by cloth dragging 100 meters once a month at eight study sites. We calculated a density index (individuals/100 m2) to assess seasonal variation. Samples were screened for Borrelia burgdorferi (sensu lato) (including B. afzelii, B. garinii, B. burgdorferi (sensu stricto) and B. valaisana), Borrelia miyamotoi, Anaplasma phagocytophilum, Rickettsia spp., Neoehrlichia mikurensis, Francisella tularensis and Bartonella spp., Babesia spp. and for the tick-borne encephalitis virus. Results: All except one nymph were identified as I. persulcatus. The number of questing adults showed a strong peak in May (median: 6.5 adults/100 m2), which is among the highest values reported in northern Europe, and potentially indicates a large population size. After May, the number of questing adults declined steadily with few adults still sampled in August. Nymphs were present from May until September. We found a striking prevalence of Borrelia spp. in adults (62%) and nymphs (40%), with B. garinii (51%) and B. afzelii (63%) being the most common species. In addition, we found that 26% of infected adults were coinfected with at least two Borrelia genospecies, mainly B. garinii and B. afzelii, which are associated with different host species. Conclusions: The coastal forest environments at Bothnian Bay seem to provide favourable environments for I. persulcatus and the spread of Borrelia. High tick abundance, a low diversity of the host community and similar host use among larvae and nymphs likely explain the high Borrelia prevalence and coinfection rate. Research on the infestation of the hosts that quantifies the temporal dynamics of immature life stages would reveal important aspects of pathogen circulation in these tick populations. [ABSTRACT FROM AUTHOR]

Background: Borrelia miyamotoi is a relapsing fever Borrelia species transmitted by ticks of the Ixodes ricinus complex. Human disease caused by B. miyamotoi was first described in Russia and later in the USA and Japan. Additionally, five cases of meningoencephalitis in immunocompromised patients and one case in an apparently immunocompetent patient were described. Methods: We investigated the presence of B. miyamotoi in I. ricinus nymphs and in patients suspected of human granulocytic anaplasmosis, in Alsace (France), an endemic area for I. ricinus ticks and Lyme borreliosis, using direct (PCR) and indirect diagnosis (glycerophosphoryldiester-phosphodiesterase (GlpQ) serology). Results: Borrelia miyamotoi was found in 2.2% of 4354 ticks collected between 2013 and 2016. None of the 575 blood samples, collected from the patients suspected of HGA, was found positive for B. miyamotoi by PCR. Acute and late sera from 138 of these 575 patients were available. These paired sera were tested for IgM and IgG antibodies against the B. miyamotoi GlpQ antigen. A total of 14 out of 138 patients had at least one positive parameter (i.e. anti-GlpQ IgG and/or IgM). One patient seroconverted for IgG, and three had isolated IgM in the acute serum. These three patients were treated with doxycycline which could have prevented seroconversion. After reviewing clinical data and other biological tests performed, co-exposure among different microorganisms vectored by ticks or serological cross-reactivity could not be ruled out in these different cases. One patient had persistent IgG, which strongly suggests previous exposure to B. miyamotoi. Conclusions: Humans can be exposed to B. miyamotoi through tick bites in Alsace. We present serological data for possible B. miyamotoi exposure or infection of patients with fever after tick bite. Future studies should determine the incidence, clinical course and burden of this emerging tick-borne disease in other parts of Western Europe. [ABSTRACT FROM AUTHOR]

Background: Ixodes ricinus constitutes the main European vector tick for the Lyme borreliosis pathogen Borrelia burgdorferi (sensu lato), the relapsing fever borrelia Borrelia miyamotoi, as well as Anaplasma phagocytophilum and several Rickettsia species. Under laboratory conditions, a transovarial transmission to the next tick generation is described for Rickettsia spp. and Borrelia spp., especially regarding B. miyamotoi, whereas the efficiency of transovarial transfer under field conditions is largely unstudied. Methods: In order to better estimate the potential infection risk by tick larvae for humans and animals, 1500 I. ricinus larvae from 50 collected "nests" (larvae adhering to the flag in a clumped manner) were individually examined for Borrelia, Rickettsia and A. phagocytophilum DNA using quantitative real-time PCR (qPCR). Results: Thirty-nine of 50 nests each (78.0%, 95% CI: 64.0–88.5%) were positive for Borrelia spp. and Rickettsia spp. DNA, and in three nests (6.0%, 95% CI: 1.3–16.5%) A. phagocytophilum DNA was detected. Overall, DNA from at least one pathogen could be detected in 90.0% (45/50, 95% CI: 78.2–96.7%) of the nests. Of the 1500 larvae, 137 were positive for Borrelia spp. DNA (9.1%, 95% CI: 7.7–10.7%), 341 for Rickettsia spp. DNA (22.7%, 95% CI: 20.6–24.9%) and three for A. phagocytophilum DNA (0.2%, 95% CI: 0–0.6%). Quantity of Borrelia spp. and Anaplasma spp. DNA in positive larvae was low, with 2.7 × 100Borrelia 5S-23S gene copies and 2.4 × 101A. phagocytophilum msp2/p44 gene copies detected on average, while Rickettsia-positive samples contained on average 5.4 × 102gltA gene copies. Coinfections were found in 66.0% (33/50, 95% CI: 51.2–78.8%) of the nests and 8.6% (38/443, 95% CI: 6.1–11.6%) of positive larvae. In fact, larvae had a significantly higher probability of being infected with Borrelia spp. or Rickettsia spp. when both pathogens were present in the nest. Conclusions: This study provides evidence for transovarial transmission of Rickettsia spp. and Borrelia spp. in I. ricinus under field conditions, possibly facilitating pathogen persistence in the ecosystem and reducing the dependence on the presence of suitable reservoir hosts. Further studies are needed to prove transovarial transmission and to explain the surprisingly high proportion of nests containing Rickettsia and/or Borrelia DNA-positive larvae compared to infection rates in adult ticks commonly reported in other studies. [ABSTRACT FROM AUTHOR]

Abstract Background Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp. Results We collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98–99%), while the remaining OTUs belonged to Proteobacteria (51–81%), Actinobacteria (6–30%) and Firmicutes (2–7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa. Conclusions Two distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species.

Abstract Background Throughout Europe, Ixodes ricinus transmits numerous pathogens. Its widespread distribution is not limited to rural but also includes urbanized areas. To date, comprehensive data on pathogen carrier rates of I. ricinus ticks in urban areas of Switzerland is lacking. Results Ixodes ricinus ticks sampled at 18 (sub-) urban collection sites throughout Switzerland showed carrier rates of 0% for tick-borne encephalitis virus, 18.0% for Borrelia burgdorferi (sensu lato), 2.5% for Borrelia miyamotoi, 13.5% for Rickettsia spp., 1.4% for Anaplasma phagocytophilum, 6.2% for "Candidatus Neoehrlichia mikurensis", and 0.8% for Babesia venatorum (Babesia sp., EU1). Site-specific prevalence at collection sites with n > 45 ticks (n = 9) significantly differed for B. burgdorferi (s.l.), Rickettsia spp., and "Ca. N. mikurensis", but were not related to the habitat type. Three hundred fifty eight out of 1078 I. ricinus ticks (33.2%) tested positive for at least one pathogen. Thereof, about 20% (71/358) were carrying two or three different potentially disease-causing agents. Using next generation sequencing, we could detect true pathogens, tick symbionts and organisms of environmental or human origin in ten selected samples. Conclusions Our data document the presence of pathogens in the (sub-) urban I. ricinus tick population in Switzerland, with carrier rates as high as those in rural regions. Carriage of multiple pathogens was repeatedly observed, demonstrating the risk of acquiring multiple infections as a consequence of a tick bite.

Abstract Background Borrelia burgdorferi (sensu lato), the causative agent of Lyme borreliosis is a bacterium transmitted by hard ticks, Ixodes spp. Bacteria are injected into the host skin during the tick blood meal with tick saliva. There, Borrelia and saliva interact together with skin cells such as keratinocytes, fibroblasts, mast cells and other specific immune cells before disseminating to target organs. Methods To study the role of mast cells in the transmission of Lyme borreliosis, we isolated mouse primary mast cells from bone marrow and incubated them in the presence of Borrelia burgdorferi (sensu stricto) and tick salivary gland extract. We further analyzed their potential role in vivo, in a mouse model of deficient in mast cells (Kit wsh−/− mice). Results To our knowledge, we report here for the first time the bacteria ability to induce the inflammatory response of mouse primary mast cells. We show that OspC, a major surface lipoprotein involved in the early transmission of Borrelia, induces the degranulation of primary mast cells but has a limited effect on the overall inflammatory response of these cells. In contrast, whole bacteria have an opposite effect. We also show that mast cell activation is significantly inhibited by tick salivary gland extract. Finally, we demonstrate that mast cells are likely not the only host cells involved in the early transmission and dissemination of Borrelia since the use of mast cell deficient Kit wsh−/− mice shows a limited impact on these two processes in the context of this mouse genetic background. Conclusions The absence of mast cells did not change the replication rate of Borrelia in the skin. However, in the absence of mast cells, Borrelia dissemination to the joints was faster. Mast cells do not control skin bacterial proliferation during primary infection and the establishment of the primary infection, as shown in the C57BL/6 mouse model studied. Nevertheless, the Borrelia induced cytotokine modulation on mast cells might be involved in long term and/or repeated infections and protect from Lyme borreliosis due to the development of a hypersensitivity to tick saliva.

Background: Ixodes ricinus is the most common tick species in Europe and the main vector for Borrelia burgdorferi (sensu lato) and tick-borne encephalitis virus (TBEV). It is involved also in the transmission of Borrelia miyamotoi, a relapsing fever spirochete that causes health disorders in humans. Little is known regarding the circulation of Borrelia species and the natural foci of TBEV in north-eastern Germany. The goal of this study was to investigate the infection rates of Borrelia spp. and of TBEV in I. ricinus ticks from north-eastern Germany. Methods: Ticks were collected by flagging from 14 forest sites in Mecklenburg-Western Pomerania between April and October 2018. RNA and DNA extraction was performed from individual adult ticks and from pools of 2–10 nymphs. Real time reverse transcription PCR (RT-qPCR) targeted the 3′ non-coding region of TBEV, while DNA of Borrelia spp. was tested by nested PCR for the amplification of 16S-23S intergenic spacer. Multilocus sequence typing (MLST) was performed on B. miyamotoi isolates. Results: In total, 2407 ticks were collected (239 females, 232 males and 1936 nymphs). Female and male I. ricinus ticks had identical infection rates (both 12.1%) for Borrelia spp., while nymphal pools showed a minimum infection rate (MIR) of 3.3%. Sequencing revealed four Borrelia species: B. afzelii, B. garinii, B. valaisiana and B. miyamotoi. Borrelia afzelii had the highest prevalence in adult ticks (5.5%) and nymphs (MIR of 1.8%). Borrelia miyamotoi was identified in 3.0% of adults and registered the MIR of 0.8% in nymphs. Borrelia valaisiana was confirmed in 2.5% adult ticks and nymphs had the MIR of 0.7%, while B. garinii was present in 1.1% of adults and showed a MIR of 0.1% in nymphs. The MLST of B. miyamotoi isolates showed that they belong to sequence type 635. No tick sample was positive after RT-qPCR for TBEV RNA. Conclusions: The prevalence of B. miyamotoi in I. ricinus ticks registered similar levels to other reports from Europe suggesting that this agent might be well established in the local tick population. The detection of B. burgdorferi (s.l.) indicates a constant circulation in tick populations from this region. [ABSTRACT FROM AUTHOR]

Abstract Background In West Africa, tick-borne relapsing fever is a neglected arthropod-borne infection caused by Borrelia crocidurae transmitted by the argasid tick Ornithodoros sonrai. From an epidemiological point of view, it is of interest to know whether some genotypes of the vector are specialized in carrying certain genotypes of the pathogen. Findings Thirty-five O. sonrai ticks collected in Mali, Senegal, Mauritania and Morocco confirmed to be B. crocidurae-infected, were genotyped by 16S rRNA gene sequencing. B. crocidurae was genotyped by Multispacer Sequence Typing. The 35 O. sonrai ticks grouped into 12 genotypes with strong geographical structuration. MST resolved the 35 B. crocidurae isolates into 29 genotypes with pairwise divergence of 0.09 - 1.56 % without strict geographical structuration as genotype ST22 was found in Mali, Senegal and Mauritania. There was no evidence of tick-borrelia specialization as one O. sonrai genotype carried several B. crocidurae genotypes and one B. crocidurae genotype was found in different O. sonrai genotypes. Conclusions This report illustrates a non-specialized circulation of B. crocidurae borreliae within O. sonrai ticks in West Africa.

Abstract Background With the exception of Bartonella spp. or Cytauxzoon felis, feline vector-borne pathogens (FVBP) have been less frequently studied in North America and are generally under-appreciated as a clinical entity in cats, as compared to dogs or people. This study investigated selected FVBP seroreactivity and PCR prevalence in cats using archived samples. Methods Feline blood samples submitted to the Vector Borne Diseases Diagnostic Laboratory (VBDDL) at North Carolina State University College of Veterinary Medicine (NCSU-CVM) between 2008 and 2013 were tested using serological assays and PCR. An experimental SNAP® Multi-Analyte Assay (SNAP® M-A) (IDEXX Laboratories, Inc. Westbrook, Maine, USA) was used to screen all sera for antibodies to Anaplasma and Ehrlichia genus peptides and A.phagocytophilum, A.platys, B.burgdorferi, E.canis, E.chaffeensis, and E.ewingii species-specific peptides. PCR assays were used to amplify Anaplasma or Ehrlichia DNA from extracted ethylenediaminetetraacetic acid (EDTA)-anti-coagulated blood samples. Amplicons were sequenced to identify species. Results Overall, 7.8 % (56/715) of cats were FVBP seroreactive and 3.2 % (13/406) contained Anaplasma or Ehrlichia DNA. Serologically, B.burgdorferi (5.5 %) was the most prevalent FVBP followed by A.phagocytophilum (1.8 %). Ehrlichia spp. antibodies were found in 0.14 % (12/715) of cats with species-specific seroreactivity to E.canis (n = 5), E.ewingii (n = 2) and E.chaffeensis (n = 1). Of seropositive cats, 16 % (9/56) were exposed to more than one FVBP, all of which were exposed to B.burgdorferi and either A.phagocytophilum (n = 7) or E.ewingii (n = 2). Based upon PCR and DNA sequencing, 4, 3, 3, 2, and 1 cat were infected with A.phagocytophilum, A.platys, E. ewingii, E. chaffeensis and E.canis, respectively. Conclusions Cats are exposed to and can be infected with vector-borne pathogens that commonly infect dogs and humans. To our knowledge, this study provides the first evidence for E.chaffeensis and E.ewingii infection in naturally-exposed cats in North America. Results from this study support the need for regional, serological and molecular FVBP prevalence studies, the need to further optimize serodiagnostic and PCR testing for cats, and the need for prospective studies to better characterize clinicopathological disease manifestations in cats infected with FVBP.

Abstract Background European hedgehogs (Erinaceus europaeus) are hosts for Ixodes hexagonus and I. ricinus ticks, which are vectors for zoonotic microorganisms. In addition, hedgehogs may carry several enteric zoonoses as well. It is unclear to what extent a presence of pathogens in hedgehogs poses a risk to public health, as information on the presence of zoonotic agents in hedgehogs in urban areas is relatively scarce. Methods Engorged ticks and hedgehog faeces were collected from rehabilitating hedgehogs. Ticks were screened individually for presence of Borrelia burgdorferi sensu lato, B. miyamotoi, Anaplasma phagocytophilum, and Candidatus Neoehrlichia mikurensis using PCR-based assays. Faecal samples were screened for presence of Campylobacter, Salmonella, Giardia, Cryptosporidium, and extended-spectrum cephalosporin-resistant-Escherichia coli (ESC)-resistant E. coli, using both culture-based and PCR-based methods. Results Anaplasma phagocytophilum and Borrelia genospecies B. afzelii, B. spielmanii, B. garinii, and B. burgdorferi sensu stricto were detected in both I. hexagonus and I. ricinus ticks. Despite their widespread distribution in the Netherlands, B. miyamotoi and Candidatus N. mikurensis were not detected in collected ticks. Analysis of hedgehog faecal samples revealed the presence of Salmonella enterica subspecies enterica and Campylobacter jejuni. In addition, ESC-resistant E. coli were observed in high prevalence in faecal samples, but no Shiga-toxin producing-E.coli were detected. Finally, potentially zoonotic protozoan parasites were observed in hedgehog faecal samples as well, including Giardia duodenalis assemblage A, Cryptosporidium parvum subtypes IIaA17G1R1 and IIcA5G3, and C. hominis subtype IbA10G2. Conclusions European hedgehogs in (sub)urban areas harbor a number of zoonotic agents, and therefore may contribute to the spread and transmission of zoonotic diseases. The relatively high prevalence of B. burgdorferi s.l. and A. phagocytophilum in engorged ticks, suggests that hedgehogs contribute to their enzootic cycles in (sub)urban areas. To what extent can hedgehogs maintain the enteric zoonotic agents in natural cycles, and the role of (spill-back from) humans remains to be investigated.

Background: “Candidatus Neoehrlichia mikurensis” is a gram-negative bacterium belonging to the family Anaplasmataceae that, in Europe, is transmitted by Ixodes ricinus ticks. “Candidatus N. mikurensis” can cause a severe systemic inflammatory syndrome, neoehrlichiosis, mostly in persons with other underlying diseases. To date, “Ca. N. mikurensis” has been found in ticks in different countries in Asia and Europe, but never as far north as at the Arctic Circle. Methods: A total of 1104 I. ricinus ticks collected from vegetation and from animals in northern Norway (64–68°N) were analysed for the prevalence of “Ca. N. mikurensis”. Of them, 495 ticks were collected from vegetation by flagging and 609 ticks were collected from dogs and cats. Total nucleic acid extracted from the ticks were converted to cDNA and analyzed with real-time PCR targeting the 16S rRNA gene of “Ca. N. mikurensis”. Positive samples were further analysed by nested PCR and sequencing. Results: “Candidatus N. mikurensis” was detected in 11.2% of all collected I. ricinus ticks in northern Norway. The prevalence differed between ticks collected from vegetation (18.2%; 90/495) compared to ticks collected from dogs and cats (5.6%; 34/609). The ticks from dogs and cats were collected in Brønnøy area and seven additional districts further north. The prevalence of “Ca. N. mikurensis” in these ticks differed between geographical localities, with the highest prevalence in the Brønnøy area. Conclusions: The detection of “Ca. N. mikurensis” in I. ricinus ticks from the Arctic Circle in northern Norway indicates potential risk for tick-bitten humans at this latitude to be infected with “Ca. N. mikurensis”. [ABSTRACT FROM AUTHOR]

Background: Lyme borreliosis in humans results in a range of clinical manifestations, thought to be partly due to differences in the pathogenicity of the infecting strain. This study compared European human clinical strains of Borreliella afzelii (previously named Borrelia afzelii) using multilocus sequence typing (MLST) to determine their spatial distribution across Europe and to establish whether there are associations between B. afzelii genotypes and specific clinical manifestations of Lyme borreliosis. For this purpose, typing was performed on 63 strains, and data on a further 245 strains were accessed from the literature. Results: All 308 strains were categorized into 149 sequence types (STs), 27 of which are described here for the first time. Phylogenetic and goeBURST analyses showed short evolutionary distances between strains. Although the main STs differed among the countries with the largest number of strains of interest (Germany, the Netherlands, France and Slovenia), the B. afzelii clinical strains were less genetically structured than those previously observed in the European tick population. Two STs were found significantly more frequently in strains associated with clinical manifestations involving erythema migrans, whereas another ST was found significantly more frequently in strains associated with disseminated manifestations, especially neuroborreliosis. Conclusions: The MLST profiles showed low genetic differentiation between B. afzelii strains isolated from patients with Lyme borreliosis in Europe. Also, clinical data analysis suggests the existence of lineages with differential dissemination properties in humans. [ABSTRACT FROM AUTHOR]

Background: Lyme borreliosis caused by spirochetes of the Borrelia burgdorferi ( sensu lato ) complex is still the most common tick-borne disease in Europe, posing a considerable threat to public health. The predominant vector in Europe is the widespread hard tick Ixodes ricinus , which also transmits the relapsing fever spirochete B. miyamotoi as well as pathogenic Rickettsiales ( Anaplasma phagocytophilum , Rickettsia spp.). To assess the public health risk, a long-term monitoring of tick infection rates with the named pathogens is indispensable. Methods: The present study is the first German 10-year follow-up monitoring of tick infections with Borrelia spp. and co-infections with Rickettsiales. Furthermore, a specific Reverse Line Blot (RLB) protocol for detection of B. miyamotoi and simultaneous differentiation of B. burgdorferi ( s.l. ) geno-species was established. Results: Overall, 24.0% (505/2100) of ticks collected in the city of Hanover were infected with Borrelia . In detail, 35. 4% (203/573) of adult ticks [38.5% females (111/288) and 32.3% males (92/285)] and 19.8% nymphs (302/1527) were infected, representing consistent infection rates over the 10-year monitoring period. Geno-species differentiation using RLB determined B. miyamotoi in 8.9% (45/505) of positive ticks. Furthermore, a significant decrease in B. afzelii and B. spielmanii infection rates from 2010 to 2015 was observed. Co-infections with Rickettsia spp. and A. phagocytophilum increased between 2010 and 2015 (7.3 vs 10.9% and 0.3 vs 1.1%, respectively). Conclusions: Long-term monitoring is an essential part of public health risk assessment to capture data on pathogen occurrence over time. Such data will reveal shifts in pathogen geno-species distribution and help to answer the question whether or not climate change influences tick-borne pathogens. [ABSTRACT FROM AUTHOR]