Identification of host bloodmeal source and Borrelia burgdorferi sensu lato in field-collected Ixodes ricinus ticks in Chaumont (Switzerland).

To evaluate the importance of vertebrate species as tick hosts and as reservoir hosts in two endemic areas for Lyme borreliosis in Switzerland, we applied molecular methods for the analysis of bloodmeal source and Borrelia infection in questing Ixodes ricinus L. ticks. In total, 1326 questing ticks were simultaneously analyzed for Borrelia and for blood meal remnants by using reverse line blot. An overall infection prevalence of 19.0% was recorded for Borrelia sp., with similar rates in both sites. Using a newly developed method for the analysis ofbloodmeal targeting the 12S rDNA mitochondrial gene, identification of host DNA from field-collected ticks was possible in 43.6% of cases. Success of host identification at the genus and species level reached 72%. In one site, host identification success reached its maximum in spring (93% in May), decreasing in summer (20% in July) and rising in autumn (73% in October). In the other site, identification rate in ticks remained low from April to July and increased in autumn reaching 68% in October and November. The most prevalent identified host DNA was artiodactyls in both sites. Red squirrel DNA was significantly more frequently detected in ticks collected in one site, whereas insectivore DNA was more frequent in ticks in the other site. DNA from more than one vertebrate host was detected in 19.5% of nymphs and 18.9% of adults. Host DNA was identified in 48.4% of the Borrelia infected ticks. Although DNA from all Borrelia species was found in at least some ticks with DNA from mammals and some ticks with DNA from birds, our results confirm a general association of B. afzelii and B. burgdorferi sensu stricto with rodents, and B. valaisiana and B. garinii with birds.

Molecular identification of bloodmeal source in Ixodes ricinus ticks using 12S rDNA as a genetic marker.

We developed an efficient molecular method for the identification of the bloodmeal sources in the tick Ixodes ricinus (L.), the European vector of the agents of Lyme borreliosis and tick-borne encephalitis. An approximately 145-bp orthologous fragment of the vertebrate mitochondrial 12S rDNA was used as a molecular marker to discriminate host vertebrate species. The method consists of a single run polymerase chain reaction amplification of the 12S rDNA molecular marker by using nondegenerate primers followed by a reverse line blot hybridization assay by using specific oligonucleotide probes. The palette of probes allowed us to distinguish major groups of host vertebrates (e.g., mammals, small rodents, artiodactyls, birds, lizards) and to identify the bloodmeal sources at the genus or species level. External primers were designed and used to sequence the 12S rDNA molecular marker of a broad range of known or potential host vertebrate species (n = 60), including mammal (n = 28), bird (n = 31), and reptile (n = 1) species. The use of this technique coupled with known methods for identification of tick-borne pathogens (e.g., Borrelia burgdorferi sensu lato) allowed us to determine the source of infective bloodmeal and to identify reservoir species. The present method was successfully used to identify the source of bloodmeals in all feeding I. ricinus ticks and in half of questing field-collected I. ricinus ticks. Moreover, the bloodmeal source was identified in 65% of ticks infected with B. burgdorferi sensu lato. Further development of this technique may be envisaged for the detection of other vector-borne pathogens and their reservoir hosts.

Phenology of Ixodes ricinus and infection with Borrelia burgdorferi sensu lato along a north- and south-facing altitudinal gradient on Chaumont Mountain, Switzerland.

Questing Ixodes ricinus L. ticks were collected monthly from 2003 to 2005 on the north- and south-facing slopes of Chaumont Mountain in Neuchâtel, Switzerland, at altitudes varying from 620 to 1,070 m. On the south-facing slope, questing tick density was higher than on the north-facing slope, and it decreased with altitude. Density tended to increase with altitude on the north-facing slope. Saturation deficit values higher than 10 mmHg and lasting for >2 mo were often recorded on the south-facing slope, explaining seasonal patterns of questing tick activity. The overall prevalence of Borrelia burgdorferi sensu lato was 22.4%, and prevalence differed according to exposure and among years. No difference was noticed between nymphs and adults. Four Borrelia species were identified. Mixed infections were detected in 52 ticks, B. garinii and B. valaisiana (n = 21) and B. afzelii and B. burgdorferi s.s. (n = 20) were the most frequent associations observed. The density of infected ticks varied from 3.6 to 78.7 infected nymphs per 100 m2 and from 0.6 to 16.9 infected adults per 100 m2, both slopes combined. The study on the south-facing slope was a follow-up of a previous study carried out at the same location during 1999-2001. Comparison of climatic data between the two periods showed a marked increase in saturation deficit. Substantial differences in density and phenology of ticks also were observed. At high elevations, ticks were significantly more abundant during the current study. This can be explained by rising temperatures recorded during summer at altitude, reaching values similar to those registered in the first study beneath. At the lowest altitude, adults were significantly less abundant, probably due to long-lasting high saturation deficits that impaired nymphal survival. The density of Borrelia-infected ticks was higher than in the previous study.

Prevalence of Borrelia burgdorferi sensu lato in ticks collected from migratory birds in Switzerland.

The prevalence of ticks infected by Borrelia burgdorferi sensu lato on birds during their migrations was studied in Switzerland. A total of 1,270 birds captured at two sites were examined for tick infestation. Ixodes ricinus was the dominant tick species. Prevalences of tick infestation were 6% and 18.2% for birds migrating northward and southward, respectively. Borrelia valaisiana was the species detected most frequently in ticks, followed by Borrelia garinii and Borrelia lusitaniae. Among birds infested by infected ticks, 23% (6/26) were infested by B. lusitaniae-infected larvae. Migratory birds appear to be reservoir hosts for B. lusitaniae.

Non-Mendelian transmission of alleles at microsatellite loci: an example in Ixodes ricinus, the vector of Lyme disease.

Microsatellite loci are usually considered to be neutral co-dominant and Mendelian markers. We undertook to study the inheritance of five microsatellite loci in the European Lyme disease vector, the tick Ixodes ricinus. Only two loci appeared fully Mendelian while the three others displayed non-Mendelian patterns that highly frequent null alleles could not fully explain. At one locus, IR27, some phenomenon seems to hinder the PCR amplification of one allele, depending on its origin (maternal imprinting) and/or its size (short allele dominance). DNA methylation, which appeared to be a possible explanation of this amplification bias, was rejected by a specific test comparing the amplification efficiency that did not differ between unmethylated and experimentally methylated DNA. The role of allele size in heterozygous individuals was then revealed from the data available on field collected ticks and consistent with the results of a theoretical approach. These observations highlight the need for prudence while inferring reproductive systems (selfing rates), parentage or even allelic frequencies from microsatellite markers, in particular for parasitic organisms for which molecular approaches often represent the only way for population biology inferences.

PCR-reverse line blot typing method underscores the genomic heterogeneity of Borrelia valaisiana species and suggests its potential involvement in Lyme disease.

Detection of the Borrelia burgdorferi sensu lato complex in biological samples is currently done by conventional immunological and molecular biological methods. To improve on the accuracy of these methods and to simplify the procedure for testing large numbers of samples, a solid-phase sandwich hybridization system readily applicable to the detection of PCR products has been designed. This colorimetric detection system relies on the use of polybiotinylated detection probes and of specific capture oligonucleotides covalently linked at allocated positions on nylon membrane strips. From a phylogenetic analysis on a great number of ospA gene sequences, we have designed and synthesized a set of PCR primers specific to the five Borrelia burgdorferi sensu lato genospecies present in Europe and a subset of probes (capture and detection probes) specific to these five genospecies (B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. lusitaniae). This combined PCR hybridization system was evaluated with a large number of various B. burgdorferi isolates and clinical specimens. These analyses clearly showed that the system could be used as a typing method to distinguish five genospecies belonging to the B. burgdorferi sensu lato complex. In addition, the study showed that B. valaisiana strains might be more heterologous than suspected up to now and clustered into three genomic groups.

Birds and Borrelia.

After several years of controversy, the contribution of birds in the ecology of Borrelia burgdorferi sensu lato (sl) has become more and more obvious on the three continents where the pathogens are distributed. Evidence of the reservoir competence of particular bird species has been obtained using tick xenodiagnosis. B. burgdorferi sl circulates not only in terrestrial environment involving Ixodes ricinus and undergrowth-frequenting birds but also in marine environment involving I. uriae and seabirds. Migrating birds contribute to the spread of B. burgdorferi sl and of infected tick vectors along migration routes.