Cross-alteration of murine skin and tick microbiome concomitant with pathogen transmission after Ixodes ricinus bite.

BACKGROUND: Ticks are major vectors of diseases affecting humans such as Lyme disease or domestic animals such as anaplasmosis. Cross-alteration of the vertebrate host skin microbiome and the tick microbiome may be essential during the process of tick feeding and for the mechanism of pathogen transmission. However, it has been poorly investigated. METHODS: We used mice bitten by field-collected ticks (nymphs and adult ticks) in different experimental conditions to investigate, by 16S rRNA gene metabarcoding, the impact of blood feeding on both the mouse skin microbiome and the tick microbiome. We also investigated by PCR and 16S rRNA gene metabarcoding, the diversity of microorganisms transmitted to the host during the process of tick bite at the skin interface and the dissemination of the pathogen in host tissues (blood, heart, and spleen). RESULTS: Most of the commensal bacteria present in the skin of control mice were replaced during the blood-feeding process by bacteria originating from the ticks. The microbiome of the ticks was also impacted by the blood feeding. Several pathogens including tick-borne pathogens (Borrelia/Borreliella, Anaplasma, Neoehrlichia, Rickettsia) and opportunistic bacteria (Williamsia) were transmitted to the skin microbiome and some of them disseminated to the blood or spleen of the mice. In the different experiments of this study, skin microbiome alteration and Borrelia/Borreliella transmission were different depending on the tick stages (nymphs or adult female ticks). CONCLUSIONS: Host skin microbiome at the bite site was deeply impacted by the tick bite, to an extent which suggests a role in the tick feeding, in the pathogen transmission, and a potentially important impact on the skin physiopathology. The diversified taxonomic profiles of the tick microbiome were also modified by the blood feeding. Video Abstract.

Beech tree masting explains the inter-annual variation in the fall and spring peaks of Ixodes ricinus ticks with different time lags.

BACKGROUND: The tick Ixodes ricinus is an important vector of tick-borne diseases including Lyme borreliosis. In continental Europe, the nymphal stage of I. ricinus often has a bimodal phenology with a large spring peak and a smaller fall peak. There is consensus about the origin of the spring nymphal peak, but there are two alternative hypotheses for the fall nymphal peak. In the direct development hypothesis, larvae quest as nymphs in the fall of the same year that they obtained their larval blood meal. In the developmental diapause hypothesis, larvae overwinter in the engorged state and quest as nymphs one year after they obtained their larval blood meal. These two hypotheses make different predictions about the time lags that separate the larval blood meal and the density of questing nymphs (DON) in the spring and fall. METHODS: Inter-annual variation in seed production (masting) by deciduous trees is a time-lagged index for the density of vertebrate hosts (e.g., rodents) which provide blood meals for larval ticks. We used a long-term data set on the masting of the European beech tree and a 15-year study on the DON at 4 different elevation sites in western Switzerland to differentiate between the two alternative hypotheses for the origin of the fall nymphal peak. RESULTS: Questing I. ricinus nymphs had a bimodal phenology at the three lower elevation sites, but a unimodal phenology at the top elevation site. At the lower elevation sites, the DON in the fall was strongly correlated with the DON in the spring of the following year. The inter-annual variation in the densities of I. ricinus nymphs in the fall and spring was best explained by a 1-year versus a 2-year time lag with the beech tree masting index. Fall nymphs had higher fat content than spring nymphs indicating that they were younger. All these observations are consistent with the direct development hypothesis for the fall peak of I. ricinus nymphs at our study site. Our study provides new insight into the complex bimodal phenology of this important disease vector. CONCLUSIONS: Public health officials in Europe should be aware that following a strong mast year, the DON will increase 1 year later in the fall and 2 years later in the spring. Studies of I. ricinus populations with a bimodal phenology should consider that the spring and fall peak in the same calendar year represent different generations of ticks.

Masting by beech trees predicts the risk of Lyme disease.

BACKGROUND: The incidence of Lyme borreliosis and other tick-borne diseases is increasing in Europe and North America. There is currently much interest in identifying the ecological factors that determine the density of infected ticks as this variable determines the risk of Lyme borreliosis to vertebrate hosts, including humans. Lyme borreliosis is caused by the bacterium Borrelia burgdorferi sensu lato (s.l.) and in western Europe, the hard tick Ixodes ricinus is the most important vector. METHODS: Over a 15-year period (2004-2018), we monitored the monthly abundance of I. ricinus ticks (nymphs and adults) and their B. burgdorferi s.l. infection status at four different elevations on a mountain in western Switzerland. We collected climate variables in the field and from nearby weather stations. We obtained data on beech tree seed production (masting) from the literature, as the abundance of Ixodes nymphs can increase dramatically 2 years after a masting event. We used generalized linear mixed effects models and AIC-based model selection to identify the ecological factors that influence inter-annual variation in the nymphal infection prevalence (NIP) and the density of infected nymphs (DIN). RESULTS: We found that the NIP decreased by 78% over the study period. Inter-annual variation in the NIP was explained by the mean precipitation in the present year, and the duration that the DNA extraction was stored in the freezer prior to pathogen detection. The DIN decreased over the study period at all four elevation sites, and the decrease was significant at the top elevation. Inter-annual variation in the DIN was best explained by elevation site, year, beech tree masting index 2 years prior and the mean relative humidity in the present year. This is the first study in Europe to demonstrate that seed production by deciduous trees influences the density of nymphs infected with B. burgdorferi s.l. and hence the risk of Lyme borreliosis. CONCLUSIONS: Public health officials in Europe should be aware that masting by deciduous trees is an important predictor of the risk of Lyme borreliosis.

Competition between strains of Borrelia afzelii in the host tissues and consequences for transmission to ticks.

Pathogen species often consist of genetically distinct strains, which can establish mixed infections or coinfections in the host. In coinfections, interactions between pathogen strains can have important consequences for their transmission success. We used the tick-borne bacterium Borrelia afzelii, which is the most common cause of Lyme disease in Europe, as a model multi-strain pathogen to investigate the relationship between coinfection, competition between strains, and strain-specific transmission success. Mus musculus mice were infected with one or two strains of B. afzelii, strain transmission success was measured by feeding ticks on mice, and the distribution of each strain in six different mouse organs and the ticks was measured using qPCR. Coinfection and competition reduced the tissue infection prevalence of both strains and changed their bacterial abundance in some tissues. Coinfection and competition also reduced the transmission success of the B. afzelii strains from the infected hosts to feeding ticks. The ability of the B. afzelii strains to establish infection in the host tissues was strongly correlated with their transmission success to the tick vector. Our study demonstrates that coinfection and competition between pathogen strains inside the host tissues can have major consequences for their transmission success.

Climate and tree seed production predict the abundance of the European Lyme disease vector over a 15-year period.

BACKGROUND: To predict the risk of tick-borne disease, it is critical to understand the ecological factors that determine the abundance of ticks. In Europe, the sheep tick (Ixodes ricinus) transmits a number of important diseases including Lyme borreliosis. The aim of this long-term study was to determine the abiotic and biotic factors driving the annual abundance of I. ricinus at a location in Switzerland where Lyme borreliosis is endemic. METHODS: Over a 15-year period (2004 to 2018), we monitored the abundance of I. ricinus ticks on a monthly basis at three different elevations on Chaumont Mountain in Neuchâtel, Switzerland. We collected climate variables in the field and from nearby weather stations. We obtained data on beech tree seed production from the literature, as the abundance of Ixodes nymphs can increase dramatically two years after a masting event. We used AIC-based model selection to determine which ecological variables drive annual variation in tick density. RESULTS: We found that elevation site, year, seed production by beech trees two years prior, and mean annual relative humidity together explained 73.2% of the variation in our annual estimates of nymph density. According to the parameter estimates of our models, (i) the annual density of nymphs almost doubled over the 15-year study period, (ii) changing the beech tree seed production index from very poor mast (1) to full mast (5) increased the abundance of nymphs by 86.2% two years later, and (iii) increasing the field-collected mean annual relative humidity from 50.0 to 75.0% decreased the abundance of nymphs by 46.4% in the same year. Climate variables collected in the field were better predictors of tick abundance than those from nearby weather stations indicating the importance of the microhabitat. CONCLUSIONS: From a public health perspective, the increase in nymph abundance is likely to have increased the risk of tick-borne disease in this region of Switzerland. Public health officials in Europe should be aware that seed production by deciduous trees is a critical driver of the abundance of I. ricinus, and hence the risk of tick-borne disease.

Competition Between Strains of Borrelia afzelii in Immature Ixodes ricinus Ticks Is Not Affected by Season.

Vector-borne pathogens often consist of genetically distinct strains that can establish co-infections in the vertebrate host and the arthropod vector. Co-infections (or mixed infections) can result in competitive interactions between strains with important consequences for strain abundance and transmission. Here we used the spirochete bacterium, Borrelia afzelii, as a model system to investigate the interactions between strains inside its tick vector, Ixodes ricinus. Larvae were fed on mice infected with either one or two strains of B. afzelii. Engorged larvae were allowed to molt into nymphs that were subsequently exposed to three seasonal treatments (artificial summer, artificial winter, and natural winter), which differed in temperature and light conditions. We used strain-specific qPCRs to quantify the presence and abundance of each strain in the immature ticks. Co-infection in the mice reduced host-to-tick transmission to larval ticks and this effect was maintained in the resultant nymphs at 1 and 4 months after the larva-to-nymph molt. Competition between strains in co-infected ticks reduced the abundance of both strains. This inter-strain competition occurred in the three life stages that we investigated: engorged larvae, recently molted nymphs, and overwintered nymphs. The abundance of B. afzelii in the nymphs declined by 40.5% over a period of 3 months, but this phenomenon was not influenced by the seasonal treatment. Future studies should investigate whether inter-strain competition in the tick influences the subsequent strain-specific transmission success from the tick to the vertebrate host.

Competition between strains of Borrelia afzelii inside the rodent host and the tick vector.

Multiple-strain pathogens often establish mixed infections inside the host that result in competition between strains. In vector-borne pathogens, the competitive ability of strains must be measured in both the vertebrate host and the arthropod vector to understand the outcome of competition. Such studies could reveal the existence of trade-offs in competitive ability between different host types. We used the tick-borne bacterium Borrelia afzelii to test for competition between strains in the rodent host and the tick vector, and to test for a trade-off in competitive ability between these two host types. Mice were infected via tick bite with either one or two strains, and these mice were subsequently used to create ticks with single or mixed infections. Competition in the rodent host reduced strain-specific host-to-tick transmission and competition in the tick vector reduced the abundance of both strains. The strain that was competitively superior in host-to-tick transmission was competitively inferior with respect to bacterial abundance in the tick. This study suggests that in multiple-strain vector-borne pathogens there are trade-offs in competitive ability between the vertebrate host and the arthropod vector. Such trade-offs could play an important role in the coexistence of pathogen strains.

Whole body transcriptomes and new insights into the biology of the tick Ixodes ricinus.

BACKGROUND: Ixodes ricinus is the most important vector of tick-borne diseases in Europe. A better knowledge of its genome and transcriptome is important for developing control strategies. Previous transcriptomic studies of I. ricinus have focused on gene expression during the blood meal in specific tissues. To obtain a broader picture of changes in gene expression during the blood meal, our study analysed the transcriptome at the level of the whole body for both nymphal and adult ticks. Ixodes ricinus ticks from a highly inbred colony at the University of Neuchâtel were used. We also analysed previously published RNAseq studies to compare the genetic variation between three wild strains and three laboratory strains, including the strain from Neuchâtel. RESULTS: RNA was extracted from whole tick bodies and the cDNA was sequenced, producing 162,872,698 paired-end reads. Our reference transcriptome contained 179,316 contigs, of which 31% were annotated using Trinotate. Gene expression was compared between ticks that differed by feeding status (unfed vs partially fed). We found that blood-feeding in nymphs and female adult ticks increased the expression of cuticle-associated genes. Using a set of 3866 single nucleotide polymorphisms to calculate the heterozygosity, we found that the wild tick populations of I. ricinus had much higher levels of heterozygosity than the three laboratory populations. CONCLUSION: Using high throughput strand-oriented sequencing for whole ticks in different stages and feeding conditions, we obtained a de novo assembly that significantly increased the genomic resources available for I. ricinus. Our study illustrates the importance of analysing the transcriptome at the level of the whole body to gain additional insights into how gene expression changes over the life-cycle of an organism. Our comparison of several RNAseq datasets shows the power of transcriptomic data to accurately characterize genetic polymorphism and for comparing different populations or sources of sequencing material.

Influence of climatic factors on Ixodes ricinus nymph abundance and phenology over a long-term monthly observation in Switzerland (2000-2014).

BACKGROUND: One of the major public health challenges in the field of communicable diseases consists of being able to predict where and when a population is at risk of being infected by a pathogen. In the case of vector-borne diseases, such predictions often require strong ecological knowledge of the vector life-cycle and the environmental conditions promoting or preventing its establishment and maintenance. In this study, we analyse how climatic factors influence the abundance and phenology of the Lyme borreliosis vector Ixodes ricinus in a Swiss temperate forest, based on a long-term monthly observation over a period of 15 years (2000 and 2014). RESULTS: Our results show that questing nymph density significantly decreased during the study period in the sampling area. Although the analyses of climatic variables point out the relative importance of air temperature, relative humidity and saturation deficit on nymph questing activity, the global trends followed by these variables over the study period failed to fully explain the observed decline. However, nymph phenology was additionally explained by the presence of climatic thresholds that limit the questing behaviours of ticks. Most notably, we found that the presumed upper threshold of air saturation deficit, which strongly limits the increase of questing nymph density and is typically reached in the middle of spring, was reached significantly earlier and earlier over years. CONCLUSIONS: In addition to phenology per se, the use of climatic thresholds may help to predict the presence and abundance of questing ticks in Lyme borreliosis endemic areas. Tick sensitivity to temperature or saturation deficit thresholds also suggests that extreme climatic events more than global trends may affect tick population dynamics. These two points may be of high importance in epidemiological short-term as well as long-term predictions. However, the highly unexplained variability in nymph density underlines the need for further studies that include other factors such as tick host abundance or tick microhabitats, two potentially influent factors that were not assessed in the present study.

Prevalence of tick-borne pathogens in questing Ixodes ricinus ticks in urban and suburban areas of Switzerland.

BACKGROUND: Throughout Europe, Ixodes ricinus transmits numerous pathogens. Its widespread distribution is not limited to rural but also includes urbanized areas. To date, comprehensive data on pathogen carrier rates of I. ricinus ticks in urban areas of Switzerland is lacking. RESULTS: Ixodes ricinus ticks sampled at 18 (sub-) urban collection sites throughout Switzerland showed carrier rates of 0% for tick-borne encephalitis virus, 18.0% for Borrelia burgdorferi (sensu lato), 2.5% for Borrelia miyamotoi, 13.5% for Rickettsia spp., 1.4% for Anaplasma phagocytophilum, 6.2% for "Candidatus Neoehrlichia mikurensis", and 0.8% for Babesia venatorum (Babesia sp., EU1). Site-specific prevalence at collection sites with n > 45 ticks (n = 9) significantly differed for B. burgdorferi (s.l.), Rickettsia spp., and "Ca. N. mikurensis", but were not related to the habitat type. Three hundred fifty eight out of 1078 I. ricinus ticks (33.2%) tested positive for at least one pathogen. Thereof, about 20% (71/358) were carrying two or three different potentially disease-causing agents. Using next generation sequencing, we could detect true pathogens, tick symbionts and organisms of environmental or human origin in ten selected samples. CONCLUSIONS: Our data document the presence of pathogens in the (sub-) urban I. ricinus tick population in Switzerland, with carrier rates as high as those in rural regions. Carriage of multiple pathogens was repeatedly observed, demonstrating the risk of acquiring multiple infections as a consequence of a tick bite.

Ticks infected via co-feeding transmission can transmit Lyme borreliosis to vertebrate hosts.

Vector-borne pathogens establish systemic infections in host tissues to maximize transmission to arthropod vectors. Co-feeding transmission occurs when the pathogen is transferred between infected and naive vectors that feed in close spatiotemporal proximity on a host that has not yet developed a systemic infection. Borrelia afzelii is a tick-borne spirochete bacterium that causes Lyme borreliosis (LB) and is capable of co-feeding transmission. Whether ticks that acquire LB pathogens via co-feeding are actually infectious to vertebrate hosts has never been tested. We created nymphs that had been experimentally infected as larvae with B. afzelii via co-feeding or systemic transmission, and compared their performance over one complete LB life cycle. Co-feeding nymphs had a spirochete load that was 26 times lower than systemic nymphs but both nymphs were highly infectious to mice (i.e., probability of nymph-to-host transmission of B. afzelii was ~100%). The mode of transmission had no effect on the other infection phenotypes of the LB life cycle. Ticks that acquire B. afzelii via co-feeding transmission are highly infectious to rodents, and the resulting rodent infection is highly infectious to larval ticks. This is the first study to show that B. afzelii can use co-feeding transmission to complete its life cycle.

Fitness estimates from experimental infections predict the long-term strain structure of a vector-borne pathogen in the field.

The populations of many pathogen species consist of a collection of common and rare strains but the factors underlying this strain-specific variation in frequency are often unknown. Understanding frequency variation among strains is particularly challenging for vector-borne pathogens where the strain-specific fitness depends on the performance in both the vertebrate host and the arthropod vector. Two sympatric multiple-strain tick-borne pathogens, Borrelia afzelii and B. garinii, that use the same tick vector, Ixodes ricinus, but different vertebrate hosts were studied. 454-sequencing of the polymorphic ospC gene was used to characterize the community of Borrelia strains in a local population of I. ricinus ticks over a period of 11 years. Estimates of the reproduction number (R0), a measure of fitness, were obtained for six strains of B. afzelii from a previous laboratory study. There was substantial variation in prevalence among strains and some strains were consistently common whereas other strains were consistently rare. In B. afzelii, the strain-specific estimates of R0 in laboratory mice explained over 70% of the variation in the prevalences of the strains in our local population of ticks. Our study shows that laboratory estimates of fitness can predict the community structure of multiple-strain pathogens in the field.

Strain-specific antibodies reduce co-feeding transmission of the Lyme disease pathogen, Borrelia afzelii.

Vector-borne pathogens use a diversity of strategies to evade the vertebrate immune system. Co-feeding transmission is a potential immune evasion strategy because the vector-borne pathogen minimizes the time spent in the vertebrate host. We tested whether the Lyme disease pathogen, Borrelia afzelii, can use co-feeding transmission to escape the acquired immune response in the vertebrate host. We induced a strain-specific, protective antibody response by immunizing mice with one of two variants of OspC (A3 and A10), the highly variable outer surface protein C of Borrelia pathogens. Immunized mice were challenged via tick bite with B. afzelii strains A3 or A10 and infested with larval ticks at days 2 and 34 post-infection to measure co-feeding and systemic transmission respectively. Antibodies against a particular OspC variant significantly reduced co-feeding transmission of the targeted (homologous) strain but not the non-targeted (heterologous) strain. Cross-immunity between OspC antigens had no effect in co-feeding ticks but reduced the spirochaete load twofold in ticks infected via systemic transmission. In summary, OspC-specific antibodies reduced co-feeding transmission of a homologous but not a heterologous strain of B. afzelii. Co-feeding transmission allowed B. afzelii to evade the negative consequences of cross-immunity on the tick spirochaete load.

Cross-reactive acquired immunity influences transmission success of the Lyme disease pathogen, Borrelia afzelii.

Cross-reactive acquired immunity in the vertebrate host induces indirect competition between strains of a given pathogen species and is critical for understanding the ecology of mixed infections. In vector-borne diseases, cross-reactive antibodies can reduce pathogen transmission at the vector-to-host and the host-to-vector lifecycle transition. The highly polymorphic, immunodominant, outer surface protein C (OspC) of the tick-borne spirochete bacterium Borrelia afzelii induces a strong antibody response in the vertebrate host. To test how cross-immunity in the vertebrate host influences tick-to-host and host-to-tick transmission, mice were immunized with one of two strain-specific recombinant OspC proteins (A3, A10), challenged via tick bite with one of the two B. afzelii ospC strains (A3, A10), and infested with xenodiagnostic ticks. Immunization with a given rOspC antigen protected mice against homologous strains carrying the same major ospC group allele but provided little or no cross-protection against heterologous strains carrying a different major ospC group allele. There were cross-immunity effects on the tick spirochete load but not on the probability of host-to-tick transmission. The spirochete load in ticks that had fed on mice with cross-immune experience was reduced by a factor of two compared to ticks that had fed on naive control mice. In addition, strain-specific differences in mouse spirochete load, host-to-tick transmission, tick spirochete load, and the OspC-specific IgG response revealed the mechanisms that determine variation in transmission success between strains of B. afzelii. This study shows that cross-immunity in infected vertebrate hosts can reduce pathogen load in the arthropod vector with potential consequences for vector-to-host pathogen transmission.

Cross-Immunity and Community Structure of a Multiple-Strain Pathogen in the Tick Vector.

Many vector-borne pathogens consist of multiple strains that circulate in both the vertebrate host and the arthropod vector. Characterization of the community of pathogen strains in the arthropod vector is therefore important for understanding the epidemiology of mixed vector-borne infections. Borrelia afzelii and B. garinii are two species of tick-borne bacteria that cause Lyme disease in humans. These two sympatric pathogens use the same tick, Ixodes ricinus, but are adapted to different classes of vertebrate hosts. Both Borrelia species consist of multiple strains that are classified using the highly polymorphic ospC gene. Vertebrate cross-immunity against the OspC antigen is predicted to structure the community of multiple-strain Borrelia pathogens. Borrelia isolates were cultured from field-collected I. ricinus ticks over a period spanning 11 years. The Borrelia species of each isolate was identified using a reverse line blot (RLB) assay. Deep sequencing was used to characterize the ospC communities of 190 B. afzelii isolates and 193 B. garinii isolates. Infections with multiple ospC strains were common in ticks, but vertebrate cross-immunity did not influence the strain structure in the tick vector. The pattern of genetic variation at the ospC locus suggested that vertebrate cross-immunity exerts strong selection against intermediately divergent ospC alleles. Deep sequencing found that more than 50% of our isolates contained exotic ospC alleles derived from other Borrelia species. Two alternative explanations for these exotic ospC alleles are cryptic coinfections that were not detected by the RLB assay or horizontal transfer of the ospC gene between Borrelia species.

Seasonality of Ixodes ricinus ticks on vegetation and on rodents and Borrelia burgdorferi sensu lato genospecies diversity in two Lyme borreliosis-endemic areas in Switzerland.

We compared Ixodes ricinus questing density, the infestation of rodents by immature stages, and the diversity of Borrelia burgdorferi sensu lato (sl) in questing ticks and ticks collected from rodents in two Lyme borreliosis (LB)-endemic areas in Switzerland (Portes-Rouges [PR] and Staatswald [SW]) from 2003 to 2005. There were variations in the seasonal pattern of questing tick densities among years. Questing nymphs were globally more abundant at PR than at SW, but the proportion of rodents infested by immature ticks was similar (59.4% and 61%, respectively). Questing tick activity lasted from February to November with a strong decline in June. The seasonal pattern of ticks infesting rodents was different. Ticks infested rodents without decline in summer, suggesting that the risk of being bitten by ticks remains high during the summer. Rodents from SW showed the highest infestation levels (10±21.6 for larvae and 0.54±1.65 for nymphs). The proportion of rodents infested simultaneously by larvae and nymphs (co-feeding ticks) was higher at SW (28%) than at PR (11%). Apodemus flavicollis was the species the most frequently infested by co-feeding ticks, and Myodes glareolus was the most infective rodent species as measured by xenodiagnosis. At PR, the prevalence of B. burgdorferi sl in questing ticks was higher (17.8% for nymphs and 32.4% for adults) than at SW (10.4% for nymphs and 24.8% for adults), with B. afzelii as the dominant species, but B. garinii, B. burgdorferi sensu stricto, and B. valaisiana were also detected. Rodents transmitted only B. afzelii (at PR and at SW) and B. bavariensis (at SW) to ticks, and no mixed infection by additional genospecies was observed in co-feeding ticks. This implies that co-feeding transmission does not contribute to genospecies diversity. However, persistent infections in rodents and co-feeding transmission contribute to the perpetuation of B. afzelii in nature.

Prospective study on the incidence of infection by Borrelia burgdorferi sensu lato after a tick bite in a highly endemic area of Switzerland.

The periurban forest of Neuchâtel (Switzerland) is a high-risk area for Lyme Borreliosis, due to a high density of infected Ixodes ricinus ticks. In this study, we evaluated the risk of subclinical (seroconversion) and clinical infection after a tick bite in Neuchâtel inhabitants from 2003 to 2005. Inhabitants have been invited, through media, to visit a physician after a tick bite. A questionnaire was filled out and two blood samples were taken at 8-week interval. EIA screening tests for IgM and IgG (IMX system, Abbott) were applied for paired sera. In case of a change in antibody titres between both samples, a homemade Western-blot using Borrelia afzelii, B. burgdorferi sensu stricto and B. garinii as antigens was performed. Participants were included into two groups. Group one included asymptomatic participants (n=255). Among them, nine (3.5%) seroconverted with seroconversion rates varying between 6.8% in 2003, 2.1% in 2004 and 2.3% in 2005. Participants who developed clinical symptoms of LB were included into group two (n=14). Erythema migrans (EM) was reported in 5.2% of participants (5.2%), varying between 7.5% in 2003, 5% in 2004 and 3.4% in 2005. Ticks obtained from 186 participants were examined for B. burgdorferi infection by PCR/Reverse Line Blotting, and by Real Time PCR and tick attachment duration was estimated. Among I. ricinus ticks collected from participants, 32.8% were infected by B. burgdorferi sensu lato. B. afzelii predominated among these ticks. Globally, 65.9% of nymphs remained attached for more than 24h whereas only 38.3% of female ticks remained attached for more than 24h. We observed that 6.6% and 2.4% of participants bitten by infected and uninfected ticks, respectively, developed EM.

Borrelia afzelii ospC genotype diversity in Ixodes ricinus questing ticks and ticks from rodents in two Lyme borreliosis endemic areas: contribution of co-feeding ticks.

In Europe, the Lyme borreliosis (LB) agents like Borrelia burgdorferi sensu stricto (ss), B. afzelii, and B. garinii are maintained in nature by enzoonotic transmission cycles between vertebrate hosts and Ixodes ricinus ticks. The outer surface protein C is a highly antigenic protein expressed by spirochaetes during transmission from ticks to mammals as well as during dissemination in the vertebrate hosts. Previous studies based on analysis of ospC gene sequences have led to the classification of ospC genotypes into ospC groups. The aim of this study was to analyse and compare ospC group distribution among isolates of the rodent-associated genospecies, B. afzelii, at 3 levels (questing ticks, ticks feeding on rodents, and xenodiagnostic ticks). Isolates were obtained during a study carried out in 2 LB endemic areas located on the Swiss Plateau [Portes-Rouges (PR) and Staatswald (SW)], where rodents were differently infested by co-feeding ticks (Pérez et al., unpublished data). Overall, we identified 10 different ospC groups with different distributions among isolates from questing ticks, ticks that detached from rodents, and xenodiagnostic ticks at the 2 sites. We observed a higher ospC diversity among isolates from ticks that fed on rodents at SW, and mixed infections with 2 ospC groups were also more frequent among isolates from ticks that fed on rodents at SW (n=18) than at PR (n=1). At both sites, B. afzelii isolates obtained from larvae that were feeding on the rodents simultaneously with nymphs displayed a higher diversity of ospC groups (mean number of ospC groups: 2.25 for PR and 1.75 for SW) than isolates from larvae feeding without nymphs (mean number of ospC groups: 1.17 for PR and 1 for SW). We suggest that co-feeding transmission of Borrelia, previously described in laboratory models, contributes in nature in promoting and maintaining ospC diversity within local tick populations.

Prevalence of three zoonotic Babesia species in Ixodes ricinus (Linné, 1758) nymphs in a suburban forest in Switzerland.

The tick Ixodes ricinus (Linné, 1758) is known as the vector of various Babesia spp. pathogenic for humans. In Switzerland, three of them, Babesia divergens, Babesia venatorum (also known as Babesia EU1), and Babesia microti, have been reported in I. ricinus ticks from various areas. The aim here was to determine how frequently these species infect I. ricinus nymphs in a suburban forest and to determine their prevalence over 3 years along a pathway delimited in four different sections. Babesia spp. was detected and identified in 44/2568 (1.7%) I. ricinus nymphs using Reverse Line Blot. B. venatorum was infecting 1.1% (27/2568) of nymphs, B. divergens 0.2% (4/2568), and B. microti 0.7% (13/1908). Tick infection rates by these three Babesia species between years were not different except for B. microti, which was significantly less frequent in ticks in 2008 than in 2006 and 2007 according to a test using trusted intervals of percentages. B. microti was displaying the greater difference of prevalence among sampling sections, ranging from 1.6% in section 1 to 0% in section 4. The presence of these three Babesia species that are of medical relevance in a suburban forest where I. ricinus tick density is high requires attention from physicians, particularly for patients presenting unspecific symptoms and for patients who are immunocompromised, and who have history of contact with tick biotopes.

Diversity of Borrelia genospecies in Ixodes ricinus ticks in a Lyme borreliosis endemic area in Switzerland identified by using new probes for reverse line blotting.

In Europe, 7 Borrelia species belonging to the Borrelia burgdorferi sensu lato complex have been reported in Ixodes ricinus ticks. In addition, another Borrelia, related to the relapsing fever spirochaetes, has also been described. In the present study, we designed probes for reverse line blotting allowing detection and identification of all these Borrelia species after amplification of the variable spacer region between the 23S and 5S ribosomal genes. These new probes allowed us investigate the diversity of Borrelia in 915 I. ricinus collected on the south-facing slope of Chaumont (Switzerland). Among the 159 infected ticks, 7 Borrelia species were identified, and B. spielmanii and relapsing fever-like (RFL) spirochaetes were identified in this area for the first time. B. valaisiana and B. spielmanii were significantly less present in male than in female or nymphal ticks. Mixed infection with RFL spirochaetes and Lyme borreliosis spirochaetes were detected in 4 ticks. In addition, the set of probes could identify the recently described species, B. bavariensis.