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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 FROM AUTHOR]

In Europe, Ixodes ricinus tick is the vector of Lyme disease spirochetes and their relatives (Borreliella genus) and Borrelia miyamotoi. However, a newly described tick I. inopinatus with similar biological features and separated from I. ricinus may act as a vector for different Borrelia species. To date, eleven Borreliella species were detected in the natural populations of I. ricinus. Recently, two North American species have been detected in ticks parasitizing bats and red foxes in Europe, i.e., B. lanei and B. californiensis pointing to the necessity for searching for them in natural tick populations. In this study, using the coxI molecular marker only I. ricinus was identified in field-collected ticks with the exception of individual specimens of Haemaphysalis concinna. Using the flaB gene and mag-trnI intergenic spacer as molecular markers 14 Borreliaceae species have been detected with various frequencies in different parts of northern Poland. Among infected ticks, the most frequent were Borreliella (Bl.) afzelii (29.4%) and Bl. garinii (20.0%), followed by Bl. spielmanii, Bl. valaisiana, Bl. lanei, Bl. californiensis, B. miyamotoi, Bl. burgdorferi, Bl. carolinensis, Bl. americana, B. turcica, Bl. lusitaniae, Bl. bissettiae and Bl. finlandensis. Three of the above-mentioned species, i.e., Bl. lanei, Bl. californiensis and B. turcica were detected in this study for the first time in the natural ixodid tick population in Europe. The existence of the newly detected spirochetes increases their total diversity in Europe and points to the necessity of careful identification and establishment of the actual distribution of all Borreliaceae species transmitted by I. ricinus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To characterize Lyme arthritis, with a focus on management, and outcome. Observational retrospective multicentre study in Western France, of all consecutive cases of Lyme arthritis, documented by Borrelia burgdorferi IgG on ELISA serological testing, confirmed by Western blot, with or without positive Borrelia PCR in synovial fluid, with no alternative diagnosis. We enrolled 52 patients (29 males), with a mean age of 43 ± 19.4 years. Most patients had monoarthritis (n = 43, 82.7%), involving the knee (n = 51, 98.1%), with a median delay between symptoms onset and Lyme arthritis diagnosis of 5 months (interquartile range, 1.5–8). Synovial fluid analysis yielded median white cell count of 16,000/mm3 (9230–40,500), and positive PCR in 16 cases (39%), for B. burgdorferi sensu stricto (n = 5), B. garinii (n = 5), B. afzelii (n = 3), and undetermined (n = 3). All patients received antibiotics, for a median duration of 28 days (21–30), with doxycycline (n = 44, 84.6%), ceftriaxone (n = 6, 11.5%), or amoxicillin (n = 2). Twelve patients (23.1%) also received intra-articular injection of glucocorticoids as first-line treatment. Of 47 patients with follow-up, 35 (74.5%) had complete resolution of Lyme arthritis. Lyme arthritis in Western Europe may be due to B. burgdorferi ss, B. afzelii, or B. garinii. Clinical presentation is similar to Lyme arthritis in North America (i.e. chronic knee monoarthritis), with low sensitivity of synovial fluid PCR (39%). [ABSTRACT FROM AUTHOR]

In the tick-borne pathogens, Borreliella burgdorferi and Borrelia hermsii , c-di-GMP is produced by a single diguanylate cyclase (Rrp1). In these pathogens, the Plz proteins (PlzA, B and C) are the only c-di-GMP receptors identified to date and PlzA is the sole c-di-GMP receptor found in all Borreliella isolates. Bioinformatic analyses suggest that PlzA has a unique PilZN3-PilZ architecture with the relatively uncommon xPilZ domain. Here, we present the crystal structure of PlzA in complex with c-di-GMP (1.6 Å resolution). This is the first structure of a xPilz domain in complex with c-di-GMP to be determined. PlzA has a two-domain structure, where each domain comprises topologically equivalent PilZ domains with minimal sequence identity but remarkable structural similarity. The c-di-GMP binding site is formed by the linker connecting the two domains. While the structure of apo PlzA could not be determined, previous fluorescence resonance energy transfer data suggest that apo and holo forms of the protein are structurally distinct. The information obtained from this study will facilitate ongoing efforts to identify the molecular mechanisms of PlzA-mediated regulation in ticks and mammals. [ABSTRACT FROM AUTHOR]

Borrelia burgdorferi s.s. is a Gram‐negative spirochaete, the aetiological agent of Lyme disease, the most common vector‐borne disease in the Northern hemisphere. Reports on the presence of B. burgdorferi in central Mexico have been strongly criticized, since these were based only on unspecific serological methods. Furthermore, the worldwide genetic diversity of B. burgdorferi s.s. has not been evaluated. For this reason, the aim of the present study was to confirm the presence of B. burgdorferi in the central area of Mexico and to evaluate its relationship with regard to the global genetic diversity of B. burgdorferi s.s. To achieve this, fragments of the flagellin and the outer surface protein A genes were amplified from ear biopsies of the arboreal wild endemic mice Habromys schmidlyi. With these sequences, a concatenated Bayesian analysis was performed to confirm the identity of B. burgdorferi s.s. Afterwards, the global genetic diversity of this bacterial species was evaluated using our sequences and those available in GenBank. A prevalence of 10.4% (5/48) of H. schmidlyi infected with Borrelia sp. was detected, and the phylogenetic analyses confirmed the identity of B. burgdorferi s.s. Using both genes, the genetic diversity was low. However, genetic structuring analyses revealed that populations of western United States and those from Mexico formed slightly different genetic groups, separated from the populations of the rest of the world. Our study not only confirms the presence of this bacterium in central Mexico, but also shows the most southern record of this bacterium so far. It also highlights the importance of H. schmidlyi as a new potential host of this bacterial species. Our study also provides first genetic data on an incipient process of divergence in B. burgdorferi s.s. populations of eastern United States and central Mexico. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights • 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. Abstract 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. [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: 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 m2), as in endemic natural areas with higher mean tick abundance (16.5 ± 1.5 ticks/100 m2). 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 highlyand low-transformed areas. We suggest that the awareness of presence of these disease agents in cities should be raised. [ABSTRACT FROM AUTHOR]

In North America, the tick-borne pathogens Borreliella burgdorferi (Lyme disease; LD) and Anaplasma phagocytophilum (anaplasmosis) are a significant health threat to dogs. Little is known regarding the seroprevalence of maternal antibodies (Abs) to these pathogens in young dogs. The analysis of maternal antibody (Ab) profiles is important as it could bear on the interpretation of currently available diagnostic assays and the potential for vaccine interference in pups. In this pilot study, sera from 32 client-owned dogs (6–24 weeks of age; 3 serum samples per dog) from four veterinary hospitals in the United States were screened for IgG against B. burgdorferi and A. phagocytophilum using whole cell lysate immunoblots and recombinant protein-based ELISAs. As a control, the sera were also screened for Abs to canine parvovirus and canine distemper virus using a commercially available colorimetric assay. Maternally derived Abs against B. burgdorferi including the diagnostic antigen VlsE were detected in 2 of the 32 dogs, accounting for 12.5 % of dogs from LD endemic regions, and as expected, the Ab levels declined over time. Differentiating between maternal Ab and infection-induced Ab is of importance in interpreting serological tests for tick-borne diseases in young dogs and in making decisions regarding treatment and timing of vaccination. [ABSTRACT FROM AUTHOR]

• OspA and OspC antibody responses to canine Lyme disease (LD) vaccines were assessed. • Commercially available canine LD vaccines in North America were compared. • The OspC chimeritope Ch14 in the Subunit-AC vaccine elicited a robust IgG response. • The Subunit-AC vaccine elicited broadly cross-reactive OspC antibodies. • Bacterin vaccines elicited low OspC antibody titers that were not cross reactive. Lyme disease (LD), the most common tick-borne disease of canines and humans in N. America, is caused by the spirochete Borreliella burgdorferi. Subunit and bacterin vaccines are available for the prevention of LD in dogs. LD bacterin vaccines, which are comprised of cell lysates of two strains of B. burgdorferi , contain over 1000 different proteins and cellular constituents. In contrast, subunit vaccines are defined in composition and consist of either outer surface protein (Osp)A or OspA and an OspC chimeritope. In this study, we comparatively assessed antibody responses to OspA and OspC induced by vaccination with all canine bacterin and subunit LD vaccines that are commercially available in North America. Dogs were administered a two-dose series of the vaccine to which they were assigned (3 weeks apart): Subunit-AC, Subunit-A, Bacterin-1, and Bacterin-2. Antibody titers to OspA and OspC were determined by ELISA and the ability of each vaccine to elicit antibodies that recognize diverse OspC proteins (referred to as OspC types) assessed by immunoblot. While all of the vaccines elicited similar OspA antibody responses, only Subunit-AC triggered a robust and broadly cross-reactive antibody response to divergent OspC proteins. The data presented within provide new information regarding vaccination-induced antibody responses to key tick and mammalian phase antigens by both subunit and bacterin LD canine vaccine formulations. [ABSTRACT FROM AUTHOR]

• This is the first profiling of Borreliella burgdorferi specific maternal antibody in pups. • Sera from dam and pups had similar immunoreactivity patterns and were bactericidal. • Abs against diagnostic antigens (VlsE, OspF) persisted longer than Abs to other proteins. • Antibodies in the pups were derived from colostrum and not an active infection. • Vaccination triggered an immune memory response to diverse OspC types. Lyme disease (LD) is a tick-transmitted disease caused by Borreliella burgdorferi (Bb). Temporal studies of maternal antibody (Ab) profiles in Bb infected pregnant dogs and their pups have not been conducted. In this study, Ab profiles of a client-owned Bb C6 Ab positive Rottweiler and her nine pups were assessed. The dam presented with lameness 12 days prior to parturition and was C6 Ab positive with a Quant C6 Ab concentration of 237U/mL. Treatment with amoxicillin was initiated and 11 days later nine pups were delivered. Screening of the sera from the dam and pups against Bb cell lysates and a panel of antigens revealed similar immunoreactivity profiles. While antigen-specific IgG and IgM reactivity persisted in the dam for at least 7 months, a rapid decline in IgG specific for BBA36, BBK53, BB0238, BBA73 and outer surface protein (Osp) E in the pups occurred between days 29 and 52 post-parturition. In contrast, Ab specific for DbpA and the diagnostic antigens VlsE (C6) and OspF, remained elevated in the pups. Sera from the dam displayed potent complement-dependent bactericidal activity against Bb. Sera from the pups was also bactericidal but primarily through a complement-independent mechanism. Lastly, single dose vaccination of the dam at day 51 post-parturition with a LD subunit vaccine consisting of OspA and an OspC chimeritope triggered a broad anti-OspC Ab response indicative of an anamnestic response. Although this study focused on a single case, these findings add to our knowledge of maternal Ab profiles and will aid the interpretation of serological assays in pups delivered by a Bb C6 Ab positive dog. [ABSTRACT FROM AUTHOR]

Background: The evolutionary history of a species is frequently derived from molecular sequences, and the resulting phylogenetic trees do not include explicit functional information. Here, we aimed to assess the functional relationships among bacteria in the Spirochaetes phylum, based on the biological processes of 42,489 proteins in reference proteomes of 34 Spirochaetes species. We tested the hypothesis that the species in the genus Borrelia might be sufficiently different to warrant splitting them into two separate genera. Results: A detrended canonical analysis demonstrated that the presence/absence of biological processes among selected bacteria contained a strong phylogenetic signal, which did not separate species of Borrelia. We examined the ten biological processes in which most proteins were involved consistently. This analysis demonstrated that species in Borrelia were more similar to each other than to free-life species (Sediminispirochaeta, Spirochaeta, Sphaerochaeta) or to pathogenic species without vectors (Leptospira, Treponema, Brachyspira), which are highly divergent. A dendrogram based on the presence/absence of proteins in the reference proteomes demonstrated that distances between species of the same genus among free-life or pathogenic non-vector species were higher than the distances between the 19 species (27 strains) of Borrelia. A phyloproteomic network supported the close functional association between species of Borrelia. In the proteome of 27 strains of Borrelia, only a few proteins had evolved separately, in the relapsing fever and Lyme borreliosis groups. The most prominent Borrelia proteins and processes were a subset of those also found in free-living and non-vectored pathogenic species. In addition, the functional innovation (i.e., unique biological processes or proteins) of Borrelia was very low, compared to other genera of Spirochaetes. Conclusions: We found only marginal functional differences among Borrelia species. Phyloproteomic networks that included all pairwise combinations between species, proteins, and processes were more effective than other methods for evaluating the evolutionary relationships among taxa. With the limitations of data availability, our results did not support a split of the arthropod-transmitted spirochaetes into the proposed genera, Borrelia and Borreliella. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]