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Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.

The western European hedgehog (Erinaceus europaeus) and the northern white-breasted hedgehog (E. roumanicus) are natural hosts of the tick Ixodes hexagonus, the vector of tick-borne pathogens such as the Borreliella bacteria responsible for Lyme disease. The aim of this study was to identify these pathogens in ticks collected from hedgehogs in northwestern Poland and to assess their genetic diversity by molecular analysis of the detected pathogens based on the flaB gene and the mag-trnI intergenic spacer. Among 101 hedgehogs examined, 737 ticks were found on 56 (55.45%) individuals, including 501 females of I. hexagonus. Borreliella spirochete infection was confirmed in 9 females of I. hexagonus (1.8%) obtained from 4 (3.96%) hedgehogs, detecting Borreliella (Bl.) afzelii (8/89%) and Bl. spielmanii (1/11%). Phylogenetic analysis based on the flaB gene and the mag-trnI intergenic spacer showed a lack of diversity in Bl. afzelii detected in I. hexagonus ticks collected from hedgehogs as well as little diversity against reference strains detected in small mammals and ticks collected from them. The results confirm that hedgehogs play an important role in the circulation of the detected spirochete species, at least as hosts of I. hexagonus ticks infected with them, indicating their potential to spread Borreliella spirochetes.

ABSTRACT Lyme disease and anaplasmosis are tick-borne bacterial diseases caused by Borreliella and Anaplasma species, respectively. A comprehensive analysis of the exposure of eastern coyotes (Canis latrans) in the northeastern United States to tick-borne pathogens has not been conducted. In this report, we assess the serological status of 128 eastern coyotes harvested in Pennsylvania in 2015 and 2017 for antibodies to Borreliella burgdorferi and Anaplasma phagocytophilum. Immunoblot and dot blot approaches were employed to test each plasma sample by using cell lysates and recombinant proteins as detection antigens. The results demonstrate high seropositivity incidences of 64.8% and 72.7% for B. burgdorferi and A. phagocytophilum, respectively. Antibodies to both pathogens were detected in 51.5% of the plasma samples, indicating high potential for coinfection. Antibodies to the B. burgdorferi proteins DbpB, VlsE, DbpA, BBA36, and OspF (BBO39) were detected in 67.2, 63.3, 56.2, 51.6, and 48.4% of the plasma samples, respectively. Antibodies to the A. phagocytophilum P44 and P130 proteins were detected in 72.7 and 60.9% of the plasma samples, respectively. IMPORTANCE The incidence of Lyme disease (Borreliella burgdorferi) and anaplasmosis (Anaplasma phagocytophilum) are increasing in North America and Europe. The causative agents of these debilitating tick-transmitted infections are maintained in nature in an enzootic cycle involving Ixodes ticks and diverse mammals and birds. It has been postulated that predators directly or indirectly influence the dynamics of the enzootic cycle and disease incidence. Here, we demonstrate high seropositivity of eastern coyotes for B. burgdorferi and A. phagocytophilum. As coyotes become established in urban and suburban environments, interactions with humans, companion animals, and urban/suburban wildlife will increase. Knowledge of the pathogens that these highly adaptable predators are exposed to or carry, and their potential to influence or participate in enzootic cycles, is central to efforts to reduce the risk of tick-borne diseases in humans and companion animals.

•Chv chimeritopes contain 18 linear epitopes from different OspC variants.•Epitope ordering in OspC chimeritopes influences antibody responses.•Inter-epitope linkers were not required for optimal antibody responses.•Chv chimeritopes elicit broadly cross-reactive antibody.•Immunization induced antibody has potent bactericidal activity. Experimental Outer surface protein (Osp) C based subunit chimeritope vaccinogens for Lyme disease (LD) were assessed for immunogenicity, structure, ability to elicit antibody (Ab) responses to divergent OspC proteins, and bactericidal activity. Chimeritopes are chimeric epitope based proteins that consist of linear epitopes derived from multiple proteins or multiple variants of a protein. An inherent advantage to chimeritope vaccinogens is that they can be constructed to trigger broadly protective Ab responses. Three OspC chimeritope proteins were comparatively assessed: Chv1, Chv2 and Chv3. The Chv proteins possess the same set of 18 linear epitopes derived from 9 OspC type proteins but differ in the physical ordering of epitopes or by the presence or absence of linkers. All Chv proteins were immunogenic in mice and rats eliciting high titer Ab. Immunoblot and enzyme linked immunosorbent assays demonstrated that the Chv proteins elicit IgG that recognizes a diverse array of OspC type proteins. The panel included OspC proteins produced by N. American and European strains of the LD spirochetes. Rat anti-Chv antisera uniformly labeled intact, non-permeabilized Borreliella burgdorferi demonstrating that vaccinal Ab can bind to targets that are naturally presented on the spirochete cell surface. Vaccinal Ab also displayed potent complement dependent-Ab mediated killing activity. This study highlights the ability of OspC chimeritopes to serve as vaccinogens that trigger potentially broadly protective Ab responses. In addition to the current use of an OspC chimeritope in a canine LD vaccine, chimeritopes can serve as key components of human LD subunit vaccines.

Using whole genome sequencing, we demonstrated that the bacteria that are transmitted by ticks and other arthropod vectors that cause Lyme disease and relapsing fevers, while related, do not belong within the same genus classification. In addition, through characterization of their highly atypical genomic structure, we were able to develop a genetic typing system that will help with future studies of how they cause disease while also providing targets for medical interventions.

Background Ixodes ricinus ticks are commonly encountered in either natural or urban areas, contributing to Lyme disease agents Borreliella [( Borrelia burgdorferi ( sensu lato )] spp. and Borrelia miyamotoi enzootic cycles in cities. It is an actual problem whether urbanization affects pathogen circulation and therefore risk of infection. The aim of the study was to evaluate main tick-borne disease risk factors in natural, endemic areas of north-east (NE) Poland (Białowieża) and urban areas of central Poland (Warsaw), measuring tick abundance/density, prevalence of infection with spirochaetes and diversity of these pathogens in spring-early summer and late summer-autumn periods between 2012 and 2015. Methods Questing I. ricinus ticks were collected from three urban sites in Warsaw, central Poland and three natural sites in Białowieża, NE Poland. A total of 2993 ticks were analyzed for the presence of Borreliella spp. and/or Borrelia miyamotoi DNA by PCR. Tick abundance was analyzed by General Linear Models (GLM). Prevalence and distribution of spirochaetes was analyzed by Maximum Likelihood techniques based on log-linear analysis of contingency tables (HILOGLINEAR). Species typing and molecular phylogenetic analysis based on the sequenced flaB marker were carried out. Results Overall 4617 I. ricinus ticks were collected (2258 nymphs and 2359 adults). We report well established population of ticks in urban areas (10.1 ± 0.9 ticks/100 m 2 ), as in endemic natural areas with higher mean tick abundance (16.5 ± 1.5 ticks/100 m 2 ). Tick densities were the highest in spring-early summer in both types of areas. We observed no effect of the type of area on Borreliella spp. and B. miyamotoi presence in ticks, resulting in similar prevalence of spirochaetes in urban and natural areas [10.9% (95% CI: 9.7–12.2%) vs 12.4% (95% CI: 10.1–15.1%), respectively]. Prevalence of spirochaetes was significantly higher in the summer-autumn period than in the spring-early summer [15.0% (95% CI: 12.8–17.5%) vs 10.4% (95% CI: 9.2–11.6%), respectively]. We have detected six species of bacteria present in both types of areas, with different frequencies: dominance of B. afzelii (69.3%) in urban and B. garinii (48.1%) in natural areas. Although we observed higher tick densities in forests than in maintained parks, the prevalence of spirochaetes was significantly higher in the latter [9.8% (95% CI: 8.6–11.0%) vs 17.5% (95% CI: 14.4–20.5%)]. Conclusions Surprisingly, a similar risk of infection with Borreliella spp. and/or B. miyamotoi was discovered in highly- and low-transformed areas. We suggest that the awareness of presence of these disease agents in cities should be raised.

The genus Borrelia contains two groups of organisms: the causative agents of Lyme disease and their relatives and the causative agents of relapsing fever and their relatives. These two groups are morphologically indistinguishable and are difficult to distinguish biochemically. In this work, we have carried out detailed comparative genomic analyses on protein sequences from 38 Borrelia genomes to identify molecular markers in the forms of conserved signature inserts/deletions (CSIs) that are specifically found in the Borrelia homologues, and conserved signature proteins (CSPs) which are uniquely present in Borrelia species. Our analyses have identified 31 CSIs and 82 CSPs that are uniquely shared by all sequenced Borrelia species, providing molecular markers for this group of organisms. In addition, our work has identified 7 CSIs and 21 CSPs which are uniquely found in the Lyme disease Borrelia species and eight CSIs and four CSPs that are specific for members of the relapsing fever Borrelia group. Additionally, 38 other CSIs, in proteins which are uniquely found in Borrelia species, also distinguish these two groups of Borrelia. The identified CSIs and CSPs provide novel and highly specific molecular markers for identification and distinguishing between the Lyme disease Borrelia and the relapsing fever Borrelia species. We also report the results of average nucleotide identity (ANI) analysis on Borrelia genomes and phylogenetic analysis for these species based upon 16S rRNA sequences and concatenated sequences for 25 conserved proteins. These analyses also support the distinctness of the two Borrelia clades. On the basis of the identified molecular markers, the results from ANI and phylogenetic studies, and the distinct pathogenicity profiles and arthropod vectors used by different Borrelia spp. for their transmission, we are proposing a division of the genus Borrelia into two separate genera: an emended genus Borrelia, containing the causative agents of relapsing fever and a novel genus, Borreliella gen. nov., containing the causative agents of Lyme disease.

Background With approximately one-third of their genomes consisting of linear and circular plasmids, the Lyme disease agent cluster of species has the most complex genomes among known bacteria. We report here a comparative analysis of plasmids in eleven Borreliella (also known as Borrelia burgdorferi sensu lato) species. Results We sequenced the complete genomes of two B. afzelii, two B. garinii, and individual B. spielmanii , B. bissettiae, B. valaisiana and B. finlandensis isolates. These individual isolates carry between seven and sixteen plasmids, and together harbor 99 plasmids. We report here a comparative analysis of these plasmids, along with 70 additional Borreliella plasmids available in the public sequence databases. We identify only one new putative plasmid compatibility type (the 30th) among these 169 plasmid sequences, suggesting that all or nearly all such types have now been discovered. We find that the linear plasmids in the non- B. burgdorferi species have undergone the same kinds of apparently random, chaotic rearrangements mediated by non-homologous recombination that we previously discovered in B. burgdorferi . These rearrangements occurred independently in the different species lineages, and they, along with an expanded chromosomal phylogeny reported here, allow the identification of several whole plasmid transfer events among these species. Phylogenetic analyses of the plasmid partition genes show that a majority of the plasmid compatibility types arose early, most likely before separation of the Lyme agent Borreliella and relapsing fever Borrelia clades, and this, with occasional cross species plasmid transfers, has resulted in few if any species-specific or geographic region-specific Borreliella plasmid types. Conclusions The primordial origin and persistent maintenance of the Borreliella plasmid types support their functional indispensability as well as evolutionary roles in facilitating genome diversity. The improved resolution of Borreliella plasmid phylogeny based on conserved partition-gene clusters will lead to better determination of gene orthology which is essential for prediction of biological function, and it will provide a basis for inferring detailed evolutionary mechanisms of Borreliella genomic variability including homologous gene and plasmid exchanges as well as non-homologous rearrangements.

Borreliella (syn. Borrelia) burgdorferi is a spirochete bacterium that causes tick-borne Lyme disease. Along its lifecycle B. burgdorferi develops several pleomorphic forms with unclear biological and medical relevance. Surprisingly, these morphotypes have never been compared at the global transcriptome level. To fill this void, we grew B. burgdorferi spirochete, round body, bleb, and biofilm-dominated cultures and recovered their transcriptomes by RNAseq profiling. We found that round bodies share similar expression profiles with spirochetes, despite their morphological differences. This sharply contrasts to blebs and biofilms that showed unique transcriptomes, profoundly distinct from spirochetes and round bodies. To better characterize differentially expressed genes in non-spirochete morphotypes, we performed functional, positional, and evolutionary enrichment analyses. Our results suggest that spirochete to round body transition relies on the delicate regulation of a relatively small number of highly conserved genes, which are located on the main chromosome and involved in translation. In contrast, spirochete to bleb or biofilm transition includes substantial reshaping of transcription profiles towards plasmids-residing and evolutionary young genes, which originated in the ancestor of Borreliaceae. Despite their abundance the function of these Borreliaceae-specific genes is largely unknown. However, many known Lyme disease virulence genes implicated in immune evasion and tissue adhesion originated in this evolutionary period. Taken together, these regularities point to the possibility that bleb and biofilm morphotypes might be important in the dissemination and persistence of B. burgdorferi inside the mammalian host. On the other hand, they prioritize the large pool of unstudied Borreliaceae-specific genes for functional characterization because this subset likely contains undiscovered Lyme disease pathogenesis genes.

•OspC of the Lyme disease spirochetes triggers highly specific IgG responses in a diverse mammals.•IgG responses to OspC are not elicited by conserved regions of the protein.•The C7/C10 motif does not play a central role in triggering IgG responses.•The C7/C10 motif is not required for induction of bactericidal antibody. As Ixodes ticks spread to new regions, the incidence of Lyme disease (LD) in companion animals and humans will increase. Preventive strategies for LD in canines center on vaccination and tick control (acaricides). Both subunit and bacterin based LD veterinary vaccines are available. Outer surface protein C (OspC), a potent immunogen and dominant early antigen, has been demonstrated to elicit protective antibody (Ab) responses. However, a single OspC protein elicits a relatively narrow range of protection. There are conflicting reports as to whether the immunodominant epitopes of OspC reside within variable or conserved domains. A detailed understanding of the antigenic determinants of OspC is essential for understanding immune responses to this essential virulence factor and vaccinogen. Here, we investigate the contribution of the conserved C-terminal C10 motif in OspC triggered Ab responses. Using a panel of diverse recombinant full length OspC proteins and their corresponding C10 deletion variants (OspCΔC10), we demonstrate that the C10 motif does not significantly contribute to immunization or infection induced Ab responses in rabbits, rats, canines, horses and non-human primates. Furthermore, the C10 motif is not required to trigger potent bactericidal Ab responses. This study provides insight into the antigenic structure of OspC. The results enhance our understanding of immune responses that develop during infection or upon vaccination and have implications for interpretation of LD diagnostic assays that employ OspC.