Development and validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato.

Reliable detection of bacteria belonging to the Borrelia burgdorferi sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of B. burgdorferi s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of B. burgdorferi s. l. Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of B. afzelii, B. burgdorferi sensu stricto or B. bavariensis. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays. The developed qPCR assays allow exclusive detection of B. burgdorferi s. l. with the exception of the M16 marker which also detect relapsing fever Borreliae. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the B. burgdorferi genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays. Within the defined limits, this multi-target qPCR method allows a versatile detection of B. burgdorferi s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions of limits of detection, thereby limiting result ambiguities.

Microbiome and mitogenomics of the chigger mite Pentidionis agamae: potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia.

BACKGROUND: Trombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria (Orientia spp.) responsible for scrub typhus, a zoonotic febrile illness. Orientia tsutsugamushi causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by Leptotrombidium spp. chiggers. However, in Dubai, Candidatus Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to Orientia, chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear. RESULTS: Ten chigger species were collected from rodents in southwestern Saudi Arabia. Chiggers were pooled according to species and screened for Orientia DNA by PCR. Two species (Microtrombicula muhaylensis and Pentidionis agamae) produced positive results for the htrA gene, although Ca. Orientia chuto DNA was confirmed by Sanger sequencing only in P. agamae. Metagenomic sequencing of three pools of P. agamae provided evidence for two other bacterial associates: a spirochaete and a Wolbachia symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated Borrelia spp. that are widely-distributed globally with no known vector. For the Wolbachia symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I (COI) barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three P. agamae pools and almost 50 SNPs were identified, despite a common geographic origin. CONCLUSIONS: P. agamae was identified as a potential vector of Ca. Orientia chuto on the Arabian Peninsula. The detection of an unusual Borrelia sp. and a divergent Wolbachia symbiont in P. agamae indicated links with chigger microbiomes in other parts of the world, while COI barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites.

Evidence for Bartonella quintana in Lice Collected from the Clothes of Ethiopian Homeless Individuals.

Human lice, Pediculus humanus, can transmit various pathogens, including Bartonella quintana, Borrelia recurrentis, and Rickettsia prowazekii. Xenosurveillance is an epidemiological approach to assessing human infection risks performed by screening vectors of infectious disease agents. In the proof-of-principle study reported herein, the DNA of 23 human lice was collected from the clothes of 30 homeless Ethiopian individuals. These samples were assessed using 16S rRNA gene-specific pan-eubacterial PCR for screening, followed by Bartonella genus 16S-23S internal transcribed spacer (ITS) sequence-specific PCR, Bartonella genus gltA gene-specific PCR, and 16S rRNA gene PCR with specificity for relapsing-fever-associated Borrelia spp. with subsequent sequencing of the amplicons. In one sample, the pan-eubacterial 16S rRNA gene-specific screening PCR, the Bartonella genus 16S-23S ITS sequence-specific PCR, and the Bartonella genus gltA gene-specific PCR allowed for the sequencing of B. quintana-specific amplicons. In two additional samples, Bartonella genus gltA gene-specific PCR also provided sequences showing 100% sequence identity with B. quintana. In total, 3/23 (13.0%) of the assessed lice were found to be positive for B. quintana. Correlating clinical data were not available; however, the assessment confirmed the presence of B. quintana in the local louse population and thus an associated infection pressure. Larger-sized cross-sectional studies seem advisable to more reliably quantify the infection risk of lice-infested local individuals. The need for prevention by providing opportunities to maintain standard hygiene for Ethiopian homeless individuals is stressed by the reported findings, especially in light of the ongoing migration of refugees.

Genome diversity of Borrelia garinii in marine transmission cycles does not match host associations but reflects the strains evolutionary history.

Borrelia burgdorferi sensu lato is a species complex of spirochetal bacteria that occupy different ecological niches which is reflected in their reservoir host- and vector-associations. Borrelia genomes possess numerous linear and circular plasmids. Proteins encoded by plasmid genes play a major role in host- and vector-interaction and are important for Borrelia niche adaptation. However, the plasmid composition and therewith the gene repertoire may vary even in strains of a single species. Borrelia garinii, one of the six human pathogenic species, is common in Europe (vector Ixodes ricinus), Asia (vector Ixodes persulcatus) and in marine birds (vector Ixodes uriae). For the latter, only a single culture isolate (Far04) and its genome were previously available. The genome was rather small containing only one circular and six linear plasmids with a notable absence of cp32 plasmids. To further investigate B. garinii from marine transmission cycles and to explore i) whether the small number of plasmids found in isolate Far04 is a common feature in B. garinii from marine birds and presents an adaptation to this particular niche and ii) whether there may be a correlation between genome type and host species, we initiated in vitro cultures from live I. uriae collected in 2017 and 2018 from marine avian hosts and their nests. Hosts included common guillemots, Atlantic Puffin, razorbill, and kittiwake. We obtained 17 novel isolates of which 10 were sequenced using Illumina technology, one also with Pacific Bioscience technology. The 10 genomes segregated into five different genome types defined by plasmid types (based on PFam32 loci). We show that the genomes of seabird associated B. garinii contain fewer plasmids (6-9) than B. garinii from terrestrial avian species (generally ≥10), potentially suggesting niche adaptation. However, genome type did not match an association with the diverse avian seabird hosts investigated but matched the clonal complex they originated from, perhaps reflecting the isolates evolutionary history. Questions that should be addressed in future studies are (i) how is plasmid diversity related to host- and/or vector adaptation; (ii) do the different seabird species differ in reservoir host competence, and (iii) can the genome types found in seabirds use terrestrial birds as reservoir hosts.

German Ixodes inopinatus samples may not actually represent this tick species.

Ticks are important vectors of human and animal pathogens, but many questions remain unanswered regarding their taxonomy. Molecular sequencing methods have allowed research to start understanding the evolutionary history of even closely related tick species. Ixodes inopinatus is considered a sister species and highly similar to Ixodes ricinus, an important vector of many tick-borne pathogens in Europe, but identification between these species remains ambiguous with disagreement on the geographic extent of I. inopinatus. In 2018-2019, 1583 ticks were collected from breeding great tits (Parus major) in southern Germany, of which 45 were later morphologically identified as I. inopinatus. We aimed to confirm morphological identification using molecular tools. Utilizing two genetic markers (16S rRNA, TROSPA) and whole genome sequencing of specific ticks (n = 8), we were able to determine that German samples, morphologically identified as I. inopinatus, genetically represent I. ricinus regardless of previous morphological identification, and most likely are not I. ricinus/I. inopinatus hybrids. Further, our results showed that the entire mitochondrial genome, let alone singular mitochondrial genes (i.e., 16S), is unable to distinguish between I. ricinus and I. inopinatus. Our results suggest that I. inopinatus is geographically isolated as a species (northern Africa and potentially southern Spain and Portugal) and brings into question whether I. inopinatus exists in central Europe. Our results highlight the probable existence of I. inopinatus and the power of utilizing genomic data in answering questions regarding tick taxonomy.

A high fidelity approach to assembling the complex Borrelia genome.

BACKGROUND: Bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex can cause Lyme borreliosis. Different B. burgdorferi s.l. genospecies vary in their host and vector associations and human pathogenicity but the genetic basis for these adaptations is unresolved and requires completed and reliable genomes for comparative analyses. The de novo assembly of a complete Borrelia genome is challenging due to the high levels of complexity, represented by a high number of circular and linear plasmids that are dynamic, showing mosaic structure and sequence homology. Previous work demonstrated that even advanced approaches, such as a combination of short-read and long-read data, might lead to incomplete plasmid reconstruction. Here, using recently developed high-fidelity (HiFi) PacBio sequencing, we explored strategies to obtain gap-free, complete and high quality Borrelia genome assemblies. Optimizing genome assembly, quality control and refinement steps, we critically appraised existing techniques to create a workflow that lead to improved genome reconstruction. RESULTS: Despite the latest available technologies, stand-alone sequencing and assembly methods are insufficient for the generation of complete and high quality Borrelia genome assemblies. We developed a workflow pipeline for the de novo genome assembly for Borrelia using several types of sequence data and incorporating multiple assemblers to recover the complete genome including both circular and linear plasmid sequences. CONCLUSION: Our study demonstrates that, with HiFi data and an ensemble reconstruction pipeline with refinement steps, chromosomal and plasmid sequences can be fully resolved, even for complex genomes such as Borrelia. The presented pipeline may be of interest for the assembly of further complex microbial genomes.

Out of Asia? Expansion of Eurasian Lyme borreliosis causing genospecies display unique evolutionary trajectories.

Vector-borne pathogens exist in obligate transmission cycles between vector and reservoir host species. Host and vector shifts can lead to geographic expansion of infectious agents and the emergence of new diseases in susceptible individuals. Three bacterial genospecies (Borrelia afzelii, Borrelia bavariensis, and Borrelia garinii) predominantly utilize two distinct tick species as vectors in Asia (Ixodes persulcatus) and Europe (Ixodes ricinus). Through these vectors, the bacteria can infect various vertebrate groups (e.g., rodents, birds) including humans where they cause Lyme borreliosis, the most common vector-borne disease in the Northern hemisphere. Yet, how and in which order the three Borrelia genospecies colonized each continent remains unclear including the evolutionary consequences of this geographic expansion. Here, by reconstructing the evolutionary history of 142 Eurasian isolates, we found evidence that the ancestors of each of the three genospecies probably have an Asian origin. Even so, each genospecies studied displayed a unique substructuring and evolutionary response to the colonization of Europe. The pattern of allele sharing between continents is consistent with the dispersal rate of the respective vertebrate hosts, supporting the concept that adaptation of Borrelia genospecies to the host is important for pathogen dispersal. Our results highlight that Eurasian Lyme borreliosis agents are all capable of geographic expansion with host association influencing their dispersal; further displaying the importance of host and vector association to the geographic expansion of vector-borne pathogens and potentially conditioning their capacity as emergent pathogens.

Epidemiological and Serological Analysis of a SARS-CoV-2 Outbreak in a Nursing Home: Impact of SARS-CoV-2 Vaccination and Enhanced Neutralizing Immunity Following Breakthrough Infection.

Background: Despite a vaccination rate of 82.0% (n = 123/150), a SARS-CoV-2 (Alpha) outbreak with 64.7% (n = 97/150) confirmed infections occurred in a nursing home in Bavaria, Germany. Objective: the aim of this retrospective cohort study was to examine the effects of the Corminaty vaccine in a real-life outbreak situation and to obtain insights into the antibody response to both vaccination and breakthrough infection. Methods: the antibody status of 106 fully vaccinated individuals (54/106 breakthrough infections) and epidemiological data on all 150 residents and facility staff were evaluated. Results: SARS-CoV-2 infections (positive RT-qPCR) were detected in 56.9% (n = 70/123) of fully vaccinated, compared to 100% (n = 27/27) of incompletely or non-vaccinated individuals. The proportion of hospitalized and deceased was 4.1% (n = 5/123) among fully vaccinated and therewith lower compared to 18.5% (n = 5/27) hospitalized and 11.1% (n = 3/27) deceased among incompletely or non-vaccinated. Ct values were significantly lower in incompletely or non-vaccinated (p = 0.02). Neutralizing antibodies were detected in 99.1% (n = 105/106) of serum samples with significantly higher values (p < 0.001) being measured post-breakthrough infection. α-N-antibodies were detected in 37.7% of PCR positive but not in PCR negative individuals. Conclusion: Altogether, our data indicate that SARS-CoV-2 vaccination does provide protection against infection, severe disease progression and death with regards to the Alpha variant. Nonetheless, it also shows that infection and transmission are possible despite full vaccination. It further indicates that breakthrough infections can significantly enhance α-S- and neutralizing antibody responses, indicating a possible benefit from booster vaccinations.

Phylogenomic Diversity Elucidates Mechanistic Insights into Lyme Borreliae-Host Association.

Host association-the selective adaptation of pathogens to specific host species-evolves through constant interactions between host and pathogens, leaving a lot yet to be discovered on immunological mechanisms and genomic determinants. The causative agents of Lyme disease (LD) are spirochete bacteria composed of multiple species of the Borrelia burgdorferi sensu lato complex, including B. burgdorferi (Bb), the main LD pathogen in North America-a useful model for the study of mechanisms underlying host-pathogen association. Host adaptation requires pathogens' ability to evade host immune responses, such as complement, the first-line innate immune defense mechanism. We tested the hypothesis that different host-adapted phenotypes among Bb strains are linked to polymorphic loci that confer complement evasion traits in a host-specific manner. We first examined the survivability of 20 Bb strains in sera in vitro and/or bloodstream and tissues in vivo from rodent and avian LD models. Three groups of complement-dependent host-association phenotypes emerged. We analyzed complement-evasion genes, identified a priori among all strains and sequenced and compared genomes for individual strains representing each phenotype. The evolutionary history of ospC loci is correlated with host-specific complement-evasion phenotypes, while comparative genomics suggests that several gene families and loci are potentially involved in host association. This multidisciplinary work provides novel insights into the functional evolution of host-adapted phenotypes, building a foundation for further investigation of the immunological and genomic determinants of host association. IMPORTANCE Host association is the phenotype that is commonly found in many pathogens that preferential survive in particular hosts. The Lyme disease (LD)-causing agent, B. burgdorferi (Bb), is an ideal model to study host association, as Bb is mainly maintained in nature through rodent and avian hosts. A widespread yet untested concept posits that host association in Bb strains is linked to Bb functional genetic variation conferring evasion to complement, an innate defense mechanism in vertebrate sera. Here, we tested this concept by grouping 20 Bb strains into three complement-dependent host-association phenotypes based on their survivability in sera and/or bloodstream and distal tissues in rodent and avian LD models. Phylogenomic analysis of these strains further correlated several gene families and loci, including ospC, with host-specific complement-evasion phenotypes. Such multifaceted studies thus pave the road to further identify the determinants of host association, providing mechanistic insights into host-pathogen interaction.

Borrelia Ecology and Evolution: Ticks and Hosts and the Environment.

The genus Borrelia encompasses bacterial pathogens that can cause Lyme borreliosis (LB) and relapsing fever (RF) [...].

Evidence of taxonomic bias in public databases: The example of the genus Borrelia.

The taxon names used in public databases are of critical importance in all areas of biology because they are needed for linking organisms to sequence data and other information. Since most users of taxonomic classifications may be unprepared for dealing with synonyms, the names that are preferred in such databases are of high impact. Using the genus Borrelia as an example, we here show how simplistic approaches for determining the preferred synonym may lead to biases regarding the preferences for taxonomic opinions. We highlight that in this and other cases where genera were split, for reverting to the previous "merged" genus it is neither possible nor necessary to generate validly published and legitimate names that are newer than those that were proposed as new combinations when the genus was split. The policy to always prefer the latest validly published name in a public database may thus render this database oblivious to reversals in taxonomic opinion. We emphasize that users of public databases should be aware of such potential shortcomings, and that curators of databases which provide nomenclatural information should be open-minded about taxonomic views expressed in the literature.

Molecular Characterization of a Novel Relapsing Fever Borrelia Species from the Desert Cottontail (Sylvilagus audubonii) in New Mexico, USA.

The Borrelia genus comprises vector-borne, spirochete bacteria infecting vertebrates worldwide. We characterized a novel relapsing fever Borrelia species from a desert cottontail (Syvilagus audubonii) from New Mexico, US, using an established multilocus sequence analysis approach. Phylogenetic analysis of the flagellin gene (flaB) and four other protein-coding loci (clpX, pepX, recG, rplB) grouped the novel Borrelia species with hard tick relapsing fever borreliae Borrelia lonestari, Borrelia theileri, and Borrelia miyamotoi. The identity of the vectors and other vertebrate hosts, geographic distribution, and zoonotic potential of this novel Borrelia species deserve further investigation.

The evolving story of Borrelia burgdorferi sensu lato transmission in Europe.

Beside mosquitoes, ticks are well-known vectors of different human pathogens. In the Northern Hemisphere, Lyme borreliosis (Eurasia, LB) or Lyme disease (North America, LD) is the most commonly occurring vector-borne infectious disease caused by bacteria of the genus Borrelia which are transmitted by hard ticks of the genus Ixodes. The reported incidence of LB in Europe is about 22.6 cases per 100,000 inhabitants annually with a broad range depending on the geographical area analyzed. However, the epidemiological data are largely incomplete, because LB is not notifiable in all European countries. Furthermore, not only differ reporting procedures between countries, there is also variation in case definitions and diagnostic procedures. Lyme borreliosis is caused by several species of the Borrelia (B.) burgdorferi sensu lato (s.l.) complex which are maintained in complex networks including ixodid ticks and different reservoir hosts. Vector and host influence each other and are affected by multiple factors including climate that have a major impact on their habitats and ecology. To classify factors that influence the risk of transmission of B. burgdorferi s.l. to their different vertebrate hosts as well as to humans, we briefly summarize the current knowledge about the pathogens including their astonishing ability to overcome various host immune responses, regarding the main vector in Europe Ixodes ricinus, and the disease caused by borreliae. The research shows, that a higher standardization of case definition, diagnostic procedures, and standardized, long-term surveillance systems across Europe is necessary to improve clinical and epidemiological data.

Utilizing Two Borrelia bavariensis Isolates Naturally Lacking the PFam54 Gene Array To Elucidate the Roles of PFam54-Encoded Proteins.

Lyme borreliosis is the most common vector-borne disease in the Northern Hemisphere, caused by spirochetes belonging to the Borrelia burgdorferi sensu lato species complex, which are transmitted by ixodid ticks. B. burgdorferi sensu lato species produce a family of proteins on the linear plasmid 54 (PFam54), some of which confer the functions of cell adhesion and inactivation of complement, the first line of host defense. However, the impact of PFam54 in promoting B. burgdorferi sensu lato pathogenesis remains unclear because of the hurdles to simultaneously knock out all PFam54 proteins in a spirochete. Here, we describe two Borrelia bavariensis strains, PBN and PNi, isolated from patients naturally lacking PFam54 but maintaining the rest of the genome with greater than 95% identity to the reference B. bavariensis strain, PBi. We found that PBN and PNi less efficiently survive in human serum than PBi. Such defects were restored by introducing two B. bavariensis PFam54 recombinant proteins, BGA66 and BGA71, confirming the role of these proteins in providing complement evasion of B. bavariensis. Further, we found that all three strains remain detectable in various murine tissues 21 days post-subcutaneous infection, supporting the nonessential role of B. bavariensis PFam54 in promoting spirochete persistence. This study identified and utilized isolates deficient in PFam54 to associate the defects with the absence of these proteins, building the foundation to further study the role of each PFam54 protein in contributing to B. burgdorferi sensu lato pathogenesis. IMPORTANCE To establish infections, Lyme borreliae utilize various means to overcome the host's immune system. Proteins encoded by the PFam54 gene array play a role in spirochete survival in vitro and in vivo. Moreover, this gene array has been described in all currently available Lyme borreliae genomes. By investigating the first two Borrelia bavariensis isolates naturally lacking the entire PFam54 gene array, we showed that both patient isolates display an increased susceptibility to human serum, which can be rescued in the presence of two PFam54 recombinant proteins. However, both isolates remain infectious to mice after intradermal inoculation, suggesting the nonessential role of PFam54 during the long-term, but may differ slightly in the colonization of specific tissues. Furthermore, these isolates show high genomic similarity to type strain PBi (>95%) and could be used in future studies investigating the role of each PFam54 protein in Lyme borreliosis pathogenesis.

Mobile Colistin Resistance Genetic Determinants of Non-Typhoid Salmonella enterica Isolates from Russia.

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently poses a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carrying mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n = 586). mcr-1 genes were detected using a previously developed qPCR assay, and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates, including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans, carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene-carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological backgrounds.

Borrelia burgdorferi Sensu Lato in Questing and Engorged Ticks from Different Habitat Types in Southern Germany.

Borrelia burgdorferi sensu lato (s.l.) causes the most common tick-borne infection in Europe, with Germany being amongst the countries with the highest incidences in humans. This study aimed at (1) comparing infection rates of B. burgdorferi s.l. in questing Ixodes ricinus ticks from different habitat types in Southern Germany, (2) analysing genospecies distribution by habitat type, and (3) testing tissue and ticks from hosts for B. burgdorferi s.l. Questing ticks from urban, pasture, and natural habitats together with feeding ticks from cattle (pasture) and ticks and tissue samples from wild boars and roe deer (natural site) were tested by PCR and RFLP for species differentiation. B. burgdorferi s.l. was found in 29.8% questing adults and 15% nymphs. Prevalence was lower at the urban sites with occurrence of roe deer than where these were absent. Borrelia burgdorferi s.l. DNA was found in 4.8% ticks from roe deer, 6.3% from wild boar, and 7.8% from cattle. Six genospecies were identified in unfed ticks: Borrelia afzelii (48.6%), Borrelia burgdorferi sensu stricto (16%), Borrelia garinii (13.2%), Borrelia valaisiana (7.5%), Borrelia spielmanii (6.2%), and Borrelia bavariensis (0.9%). This study shows high infection levels and a great diversity of Borrelia in questing ticks. The presence of roe deer seems to reduce B. burgdorferi s.l. infection rates in tick populations.

Host association of Borrelia burgdorferi sensu lato: A review.

Borrelia burgdorferi sensu lato (Bbsl) is a bacterial species complex that includes the etiological agents of the most frequently reported vector-borne disease in the Northern hemisphere, Lyme borreliosis. It currently comprises > 20 named and proposed genospecies that use vertebrate hosts and tick vectors for transmission in the Americas and Eurasia. Host (and vector) associations influence geographic distribution and speciation in Bbsl, which is of particular relevance to human health. To target gaps in knowledge for future efforts to understand broad patterns of the Bbsl-tick-host system and how they relate to human health, the present review aims to give a comprehensive summary of the literature on host association in Bbsl. Of 465 papers consulted (404 after exclusion criteria were applied), 96 sought to experimentally establish reservoir competence of 143 vertebrate host species for Bbsl. We recognize xenodiagnosis as the strongest method used, however it is infrequent (20% of studies) probably due to difficulties in maintaining tick vectors and/or wild host species in the lab. Some well-established associations were not experimentally confirmed according to our definition (ex: Borrelia garinii, Ixodes uriae and sea birds). We conclude that our current knowledge on host association in Bbsl is mostly derived from a subset of host, vector and bacterial species involved, providing an incomplete knowledge of the physiology, ecology and evolutionary history of these interactions. More studies are needed on all host, vector and bacterial species globally involved with a focus on non-rodent hosts and Asian Bbsl complex species, especially with experimental research that uses xenodiagnosis and genomics to analyze existing host associations in different ecosystems.

The Population Structure of Borrelia lusitaniae Is Reflected by a Population Division of Its Ixodes Vector.

Populations of vector-borne pathogens are shaped by the distribution and movement of vector and reservoir hosts. To study what impact host and vector association have on tick-borne pathogens, we investigated the population structure of Borrelia lusitaniae using multilocus sequence typing (MLST). Novel sequences were acquired from questing ticks collected in multiple North African and European locations and were supplemented by publicly available sequences at the Borrelia Pubmlst database (accessed on 11 February 2020). Population structure of B. lusitaniae was inferred using clustering and network analyses. Maximum likelihood phylogenies for two molecular tick markers (the mitochondrial 16S rRNA locus and a nuclear locus, Tick-receptor of outer surface protein A, trospA) were used to confirm the morphological species identification of collected ticks. Our results confirmed that B. lusitaniae does indeed form two distinguishable populations: one containing mostly European samples and the other mostly Portuguese and North African samples. Of interest, Portuguese samples clustered largely based on being from north (European) or south (North African) of the river Targus. As two different Ixodes species (i.e., I. ricinus and I. inopinatus) may vector Borrelia in these regions, reference samples were included for I. inopinatus but did not form monophyletic clades in either tree, suggesting some misidentification. Even so, the trospA phylogeny showed a monophyletic clade containing tick samples from Northern Africa and Portugal south of the river Tagus suggesting a population division in Ixodes on this locus. The pattern mirrored the clustering of B. lusitaniae samples, suggesting a potential co-evolution between tick and Borrelia populations that deserve further investigation.

Spatial variability in prevalence and genospecies distributions of Borrelia burgdorferi sensu lato from ixodid ticks collected in southern Germany.

Lyme borreliosis (LB) is the most common arthropod-borne disease in Europe and North America and is caused by members of the Borrelia burgdorferi sensu lato (Bbsl) species complex. These bacteria are transmitted by ixodid tick vectors and therefore human LB risk is influenced by the prevalence and distribution of Bbsl genospecies within tick vectors throughout the wild. These distributions can easily change over spatiotemporal scales and, to understand LB risk fully, up to date information on prevalence and distribution of Bbsl is required. The last survey of Bbsl in southern Germany, including parts of the Munich metropolitan area, was completed in 2006 and new data is needed. Ixodid ticks were collected in seven plots located in and around Munich, Germany, from March to July 2019 and were screened for Bbsl. Borrelia burgdorferi s. l. positive ticks (52 adults, 158 nymphs) were found in all plots and adults (0-61.5 % Bbsl positive/plot) and nymphs (17.4-59.5 % Bbsl positive/plot) did not differ significantly in their overall Bbsl prevalence. The number of Bbsl positive nymphs did vary significantly between plots but the number of positive adults did not. In total, six Bbsl genospecies were located with B. afzelii and B. garinii dominating. Additionally, the relapsing-fever species B. miyamotoi was found in two sampling plots. Our results highlight the variability in Bbsl prevalence and genospecies distribution over short geographic distances and aid in understanding LB risk in and around the Munich metropolitan area.