Search

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.

The incidence of tick-borne diseases caused by Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. has been rising in Europe in recent decades. Early pre-assessment of acarological hazard still represents a complex challenge. The aim of this study was to model Ixodes ricinus questing nymph density and its infection rate with B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in five European countries (Italy, Germany, Czech Republic, Slovakia, Hungary) in various land cover types differing in use and anthropisation (agricultural, urban and natural) with climatic and environmental factors (Normalized Difference Vegetation Index (NDVI), Normalized Difference Water Index (NDWI), Land Surface Temperature (LST) and precipitation). We show that the relative abundance of questing nymphs was significantly associated with climatic conditions, such as higher values of NDVI recorded in the sampling period, while no differences were observed among land use categories. However, the density of infected nymphs (DIN) also depended on the pathogen considered and land use. These results contribute to a better understanding of the variation in acarological hazard for Ixodes ricinus transmitted pathogens in Central Europe and provide the basis for more focused ecological studies aimed at assessing the effect of land use in different sites on tick–host pathogens interaction.

MultiLocus sequence typing (MLST) is considered a powerful method to unveil relationships within bacterial populations and it constitutes an economical and fast alternative to whole genome sequencing. We used this method to understand whether there are differences in human pathogenicity within and between different Borrelia burgdorferi sensu lato species. Therefore, 136 strains from human patients or ticks from Europe were included in MLST analyses. The scheme employed used eight chromosomally located housekeeping genes (i.e. clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA). We investigated Borrelia afzelii, one of the predominant species in Europe, and B. burgdorferi sensu stricto (s.s.), because it allowed comparative analysis to strains from the USA. We typed 113 patient isolates as well as 23 tick isolates. For further comparative purposes an additional 746 strains from Europe and the USA were included from the MLST website http://borrelia.mlst.net. We observed an overlap of the B. burgdorferi s.s. populations from Europe and the USA isolated from human patients while there was no overlap of the populations found in tick vectors. Further results indicate that B. afzelii was significantly less associated with disseminated infection than B. burgdorferi s.s. and that B. burgdorferi s.s. from Europe caused neuroborreliosis to a significantly greater extent than B. afzelii or B. burgdorferi s.s. in the USA. Our data suggest that there may be an evolutionary basis of differential interspecies pathogenicity in Borrelia. This was not evident within Borrelia species: we found the same sequence types in patients with disseminated or localized symptoms when the number of strains was sufficiently high. We hypothesize that the finding that B. burgdorferi s.s. in Europe is much more associated with neuroborreliosis than in the USA maybe linked to factor(s) related to the human host, the tick vector or the bacterium itself (e.g. plasmid content and structure).

For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection.Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10(4) to 10(-1)) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment.The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10(2) genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2% for B. miyamotoi and 11.8% for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples.The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

Urban, natural, and pasture areas were investigated for prevalences and 16S rRNA gene variants of Anaplasma phagocytophilum in questing Ixodes ricinus ticks. The prevalences differed significantly between habitat types, and year-to-year variations in prevalence and habitat-dependent occurrence of 16S rRNA gene variants were detected.

Borrelia persica, a bacterium transmitted by the soft tick Ornithodoros tholozani, causes tick-borne relapsing fever in humans in the Middle East, Central Asia and the Indian peninsula. Immunocompetent C3H/HeOuJ mice were infected intradermally with B. persica at varying doses: 1 x 106, 1 x 104, 1 x 102 and 4 x 100 spirochetes/mouse. Subsequently, blood samples were collected and screened for the presence of B. persica DNA. Spirochetes were detected in all mice infected with 1 x 106, 1 x 104 and 1 x 102 borrelia by real-time PCR targeting the flaB gene of the bacterium. Spirochetemia developed with a one- to two-day delay when 1 x 104 and 1 x 102 borrelia were inoculated. Mice injected with only four organisms were negative in all tests. No clinical signs were observed when infected mice were compared to negative control animals. Organs (heart, spleen, urinary bladder, tarsal joint, skin and brain) were tested for B. persica-specific DNA and cultured for the detection of viable spirochetes. Compiled data show that the target organs of B. persica infections are the brain and the skin. A newly developed serological two-tiered test system (ELISA and western blot) for the detection of murine IgM, IgG and IgA antibody titers against B. persica showed a vigorous antibody response of the mice during infection. In conclusion, the infection model described here for B. persica is a platform for in vivo studies to decipher the so far unexplored survival strategies of this Borrelia species., Author Summary The spirochete Borrelia persica is a tick-borne bacterium that is transmitted by the vector Ornithodoros tholozani to its vertebrate host in the Middle East, Central Asia and the Indian peninsula. Current migration of vast numbers of individuals from this area increases the likelihood that B. persica infections will be introduced into new geographic regions. After infection and distribution by the bloodstream, relapsing fever episodes occur in humans. Since no reliable in vivo tools have existed so far to study this organism, a murine model was established in this study to characterize the infection kinetics in immunocompetent mice. Aspects of the potential infectivity of the laboratory strain and of potential clinical signs, spirochetemia and antibody response as well as organ tropism and histopathological reactions were studied. With the successful infection model presented here, further studies are conceivable in order to gain advanced insights into the pathogenesis of B. persica infection and to characterize in detail the host immune response mounted against the bacterium. We propose that this model could also be used for the development of new rapid diagnostic approaches to initiate or monitor treatment regimes in order to clear or prevent the infection with B. persica.

The hard tick Ixodes ricinus is the most common tick in Central Europe and plays an important role as a vector of several pathogens. In the complex life cycles of these pathogens, the role of wild animals as natural reservoirs has been discussed. The aims of this study were to investigate the role of roe deer (Capreolus capreolus) as a potential reservoir host for Babesia spp., Anaplasma phagocytophilum, and Rickettsia spp. Therefore, we explored the differences in the infection rates of roe deer and engorged and questing ticks with these pathogens from a single forest site with special attention to coinfection. Blood, spleen, and skin samples of a total of 95 roe deer individuals were screened by molecular methods for these pathogens from September 2010 to January 2012 in the ‘Angelberger Forst’ (Bavaria, Germany). Moreover, 331 engorged ticks from 44 roe deer individuals and 199 host-seeking ticks from the same area were screened. Altogether, the following prevalence rates and a high diversity of species were detected for the respective pathogens in individual animals and ticks: (i) Babesia spp.: roe deer, 89.5%; engorged ticks, 7.3%; questing ticks: adults, 2.5%, nymphs, 3.3%. Sequencing revealed B. venatorum, B. capreoli, and B. microti. (ii) A. phagocytophilum: roe deer 98.9%; engorged ticks, 86.1%; questing ticks: adults, 8.9%, nymphs, 0.8%. (iii) Rickettsia spp.: roe deer, 0%; engorged ticks, 16.6%; questing ticks: adults, 13.9%, nymphs, 17.5%. Sequencing revealed R. helvetica. Furthermore, several coinfections were detected in both roe deer and ticks. The high prevalence rates of B. capreoli and A. phagocytophilum in roe deer support their role as reservoir hosts for these pathogens, but no evidence for a role of roe deer in the life cycle of R. helvetica could be provided.