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.

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.

Multistrain Infections with Lyme Borreliosis Pathogens in the Tick Vector.

Mixed or multiple-strain infections are common in vector-borne diseases and have important implications for the epidemiology of these pathogens. Previous studies have mainly focused on interactions between pathogen strains in the vertebrate host, but little is known about what happens in the arthropod vector. Borrelia afzelii and Borrelia garinii are two species of spirochete bacteria that cause Lyme borreliosis in Europe and that share a tick vector, Ixodes ricinus Each of these two tick-borne pathogens consists of multiple strains that are often differentiated using the highly polymorphic ospC gene. For each Borrelia species, we studied the frequencies and abundances of the ospC strains in a wild population of I. ricinus ticks that had been sampled from the same field site over a period of 3 years. We used quantitative PCR (qPCR) and 454 sequencing to estimate the spirochete load and the strain diversity within each tick. For B. afzelii, there was a negative relationship between the two most common ospC strains, suggesting the presence of competitive interactions in the vertebrate host and possibly the tick vector. The flat relationship between total spirochete abundance and strain richness in the nymphal tick indicates that the mean abundance per strain decreases as the number of strains in the tick increases. Strains with the highest spirochete load in the nymphal tick were the most common strains in the tick population. The spirochete abundance in the nymphal tick appears to be an important life history trait that explains why some strains are more common than others in nature. IMPORTANCE: Lyme borreliosis is the most common vector-borne disease in the Northern Hemisphere and is caused by spirochete bacteria that belong to the Borrelia burgdorferi sensu lato species complex. These tick-borne pathogens are transmitted among vertebrate hosts by hard ticks of the genus Ixodes Each Borrelia species can be further subdivided into genetically distinct strains. Multiple-strain infections are common in both the vertebrate host and the tick vector and can result in competitive interactions. To date, few studies on multiple-strain vector-borne pathogens have investigated patterns of cooccurrence and abundance in the arthropod vector. We demonstrate that the abundance of a given strain in the tick vector is negatively affected by the presence of coinfecting strains. In addition, our study suggests that the spirochete abundance in the tick is an important life history trait that can explain why some strains are more common in nature than others.

Acceptability of tick control interventions to prevent Lyme disease in Switzerland and Canada: a mixed-method study.

BACKGROUND: Lyme disease control strategies may include tick control interventions in high risk areas. Public authorities may be interested to assess how these types of interventions are perceived by the public which may then impact their acceptability. The aims of this paper are to compare socio-cognitive factors associated with high acceptability of tick control interventions and to describe perceived issues that may explain their low acceptability in populations living in two different regions, one being an endemic region for LD since the last 30 years, the Neuchâtel canton, in Switzerland, and another where the disease is emerging, the Montérégie region, in Canada. METHODS: A mixed methods' design was chosen. Quantitative data were collected using web-surveys conducted in both regions (n = 814). Multivariable logistic regressions were used to compare socio-cognitive factors associated with high acceptability of selected interventions. Qualitative data were collected using focus group's discussions to describe perceived issues relative to these interventions. RESULTS: Levels of acceptability in the studied populations were the lowest for the use of acaricides and landscaping and were under 50 % in both regions for six out of eight interventions, but were higher overall in Montérégie. High perceived efficacy of the intervention was strongly associated with high acceptability of tick control interventions. A high perceived risk about LD was also associated with a high acceptability of intervention under some models. High level of knowledge about LD was negatively associated with high acceptability of the use of acaricides in Neuchâtel. Perceived issues explaining low acceptability included environmental impacts, high costs to the public system, danger of individual disempowerment and perceptions that tick control interventions were disproportionate options for the level of LD risk. CONCLUSION: This study suggests that the perceived efficacy and LD risk perception may be key factors to target to increase the acceptability of tick control interventions. Community-level issues seem to be important considerations driving low acceptability of public health interventions. Results of this study highlight the importance for decision-makers to account for socio-cognitive factors and perceived issues that may affect the acceptability of public health interventions in order to maximize the efficacy of actions to prevent and control LD.

Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp.

INTRODUCTION: Ticks transmit a great variety of pathogenic microorganisms to humans and animals. The detection of tick-borne pathogens (TBP) is mainly by molecular techniques based on polymerase chain reactions (PCR). OBJECTIVE: To design and evaluate a multiplex PCR for the molecular screening of zoonotic TBP for exploratory studies. MATERIAL AND METHODS: Control DNA from reference strains, DNA from experimentally-infected biological specimens, and from Rhipicephalus sanguineus ticks collected from domestic and homeless dogs were used. A multiplex PCR assay to detect the presence of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. was designed and optimized using primers previously reported for B. burgdorferi sensu lato and Anaplasma spp., while for Babesia spp. they were designed in silico. The multiplex PCR was evaluated on the DNA from biological samples. RESULTS: A new set of specific primers for Babesia spp. was designed. Adjustment of the master mix reactive concentrations and amplification conditions for the multiplex PCR allowed the successful amplification of the specific amplicons for each microbial group from the control DNA and experimentally-infected biological specimens. The efficiency of the multiplex PCR amplifying three DNA targets was confirmed. Individual and co-infection of Anaplasma spp. and Babesia spp. were detected in the R. sanguineus ticks from dogs. CONCLUSIONS: A multiplex PCR assay for the screening of three TBP is available. By using it, B. burgdorferi sensu lato, Anaplasma spp. and Babesia spp. can be detected accurately in one PCR reaction.

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.

Adaptation and Evaluation of a Multi-Criteria Decision Analysis Model for Lyme Disease Prevention.

Designing preventive programs relevant to vector-borne diseases such as Lyme disease (LD) can be complex given the need to include multiple issues and perspectives into prioritizing public health actions. A multi-criteria decision aid (MCDA) model was previously used to rank interventions for LD prevention in Quebec, Canada, where the disease is emerging. The aim of the current study was to adapt and evaluate the decision model constructed in Quebec under a different epidemiological context, in Switzerland, where LD has been endemic for the last thirty years. The model adaptation was undertaken with a group of Swiss stakeholders using a participatory approach. The PROMETHEE method was used for multi-criteria analysis. Key elements and results of the MCDA model are described and contrasted with the Quebec model. All criteria and most interventions of the MCDA model developed for LD prevention in Quebec were directly transferable to the Swiss context. Four new decision criteria were added, and the list of proposed interventions was modified. Based on the overall group ranking, interventions targeting human populations were prioritized in the Swiss model, with the top ranked action being the implementation of a large communication campaign. The addition of criteria did not significantly alter the intervention rankings, but increased the capacity of the model to discriminate between highest and lowest ranked interventions. The current study suggests that beyond the specificity of the MCDA models developed for Quebec and Switzerland, their general structure captures the fundamental and common issues that characterize the complexity of vector-borne disease prevention. These results should encourage public health organizations to adapt, use and share MCDA models as an effective and functional approach to enable the integration of multiple perspectives and considerations in the prevention and control of complex public health issues such as Lyme disease or other vector-borne and zoonotic diseases.

Factors associated with preventive behaviors regarding Lyme disease in Canada and Switzerland: a comparative study.

BACKGROUND: Lyme disease (LD) is a vector-borne disease that is endemic in many temperate countries, including Switzerland, and is currently emerging in Canada. This study compares the importance of knowledge, exposure and risk perception for the adoption of individual preventive measures, within and between two different populations, one that has been living in a LD endemic region for several decades, the Neuchâtel canton in Switzerland, and another where the disease is currently emerging, the Montérégie region in the province of Québec, Canada. METHODS: A web-based survey was carried out in both study regions (814 respondents) in 2012. Comparative analysis of the levels of adoption of individual preventive measures was performed and multivariable logistic regression analyses were used to test and compare how knowledge, exposure and risk perception were associated with the adoption of selected measures in both regions and globally. RESULTS: In Montérégie, the proportion of reported adoption of five of the most commonly recommended preventive measures varied from 6% for 'applying acaricides on one's property' to 49% for 'wearing protective clothing', and in Neuchâtel, proportions ranged from 6% (acaricides) to 77% for 'checking for ticks (tick check)'. Differences were found within gender, age groups and exposure status in both regions. The perceived efficacy of a given measure was the strongest factor associated with the adoption of three specific preventive behaviors for both regions: tick check, protective clothing and tick repellent. Risk perception and a high level of knowledge about LD were also significantly associated with some of these specific behaviors, but varied by region. CONCLUSIONS: These results strongly suggest that social and contextual factors such as the epidemiological status of a region are important considerations to take into account when designing effective prevention campaigns for Lyme disease. It furthermore underlines the importance for public health authorities to better understand and monitor these factors in targeted populations in order to be able to implement preventive programs that are well adapted to a population and the epidemiological contexts therein.

Genetic variation in transmission success of the Lyme borreliosis pathogen Borrelia afzelii.

The vector-to-host and host-to-vector transmission steps are the two critical events that define the life cycle of any vector-borne pathogen. We expect negative genetic correlations between these two transmission phenotypes, if parasite genotypes specialized at invading the vector are less effective at infecting the vertebrate host and vice versa. We used the tick-borne bacterium Borrelia afzelii, a causative agent of Lyme borreliosis in Europe, to test whether genetic trade-offs exist between tick-to-host, systemic (host-to-tick), and a third mode of co-feeding (tick-to-tick) transmission. We worked with six strains of B. afzelii that were differentiated according to their ospC gene. We compared the three components of transmission among the B. afzelii strains using laboratory rodents as the vertebrate host and a laboratory colony of Ixodes ricinus as the tick vector. We used next generation matrix models to combine these transmission components into a single estimate of the reproductive number (R0) for each B. afzelii strain. We also tested whether these strain-specific estimates of R0 were correlated with the strain-specific frequencies in the field. We found significant genetic variation in the three transmission components among the B. afzelii strains. This is the first study to document genetic variation in co-feeding transmission for any tick-borne pathogen. We found no evidence of trade-offs as the three pairwise correlations of the transmission rates were all positive. The R0 values from our laboratory study explained 45% of the variation in the frequencies of the B. afzelii ospC strains in the field. Our study suggests that laboratory estimates of pathogen fitness can predict the distribution of pathogen strains in nature.

Search for blood or water is influenced by Borrelia burgdorferi in Ixodes ricinus.

An increasing number of studies suggest that vector-borne parasites are able to alter phenotypic traits in their arthropod vectors so that microorganism transmission is enhanced. This review documents this phenomenon, which occurs between Borrelia burgdorferi bacteria, the causative agents of Lyme borreliosis, and their tick vectors belonging to the Ixodes ricinus complex. It also reviews the influence of other tick-borne pathogens on these ticks. Ticks belonging to the Ixodes ricinus complex benefit from Borrelia infection by an increased lifespan (more fat and more resistance to desiccation) and by an increased questing period (less need to move to the litter zone to rehydrate), which enhances tick chances to find a host and to subsequently transmit the pathogens.

From Lyme disease emergence to endemicity: a cross sectional comparative study of risk perceptions in different populations.

BACKGROUND: Lyme disease (LD) is a tick-borne emerging disease in Canada that has been endemic in many temperate countries for decades. Currently, one of the main approaches for LD prevention is the promotion of individual-level preventive behaviors against ticks. Health behaviors are influenced by individual and social factors, one important of which is risk perception. This study aims to describe and compare risk perception of LD, within and between general populations and experts living in two different regions: the Neuchâtel canton in Switzerland, where LD is endemic, and the Montérégie region in Québec (Canada), where LD is emerging. METHOD: A web-based survey was conducted in both study regions (814 respondents) in 2012, and a questionnaire was administered to 16 experts. Comparative analyses of knowledge, risk exposure and different components of LD risk perception were performed. Multivariate analyses were used to calculate a global risk perception score and to identify determinants of risk perception in both regions. RESULTS: In Montérégie, only 15% of the survey respondents had a good level of knowledge of LD compared to Neuchâtel where 51% of survey respondents had good levels of knowledge. In Montérégie, 24% of respondents perceived themselves as being at high or very high risk of contracting LD vs 54% in Neuchâtel; however, a higher percentage of respondents from this region believed that personal protection was simple to carry out (73% vs 58% in Montérégie). Based on the population surveys, almost all of the identified determinants of risk perception were different between both populations except for gender. A good level of knowledge, living in the risk zone and knowing someone who has had LD increased risk perception, while a high level of education and being 18-34 years of age decreased this perception. The majority of the studied components of risk perception were different between populations and their regional experts. CONCLUSION: This study suggests that risk perception of LD differs between populations and regional experts living in different epidemiological situations. Monitoring of knowledge and risk perception in local populations may help to better target LD communication efforts in accordance with population specific attributes thereby enhancing prevention efficacy.

Prevalence and phylogenetic analysis of tick-borne encephalitis virus (TBEV) in field-collected ticks (Ixodes ricinus) in southern Switzerland.

BACKGROUND: Tick-borne encephalitis is the most common tick-borne viral infection in Europe with 3,000 human cases reported each year. In Western Europe, the castor bean tick, Ixodes ricinus, is the principal vector of the tick-borne encephalitis virus (TBEV). TBEV appears to be spreading geographically and was recently detected for the first time in Canton Valais in the southern part of Switzerland. The purpose of the present study was to survey the I. ricinus tick populations of Canton Valais for TBEV. METHODS: We collected a total of 19,331 I. ricinus ticks at 45 different sites in Canton Valais between 2010 and 2013. Ticks were processed in pools and tested for TBEV using reverse transcription quantitative PCR. The NS5 gene and the envelope gene of the TBEV isolates were partially sequenced for phylogenetic analysis. RESULTS: TBEV was detected in tick populations at six of the 45 sites. These six sites were all located in a 33 km transect along the Rhône River. TBEV was detected in two sites for three of the four years of the study showing the temporal persistence of the pathogen. Prevalence of TBEV in the six positive sites ranged from 0.16% to 11.11%. Phylogenetic analysis found that all TBEV isolates from Canton Valais belonged to the European subtype. Genetic analysis found two distinct lineages of TBEV suggesting that Canton Valais experienced two independent colonization events. CONCLUSIONS: TBEV appears to be well established at certain locations in Canton Valais.

Tick-borne pathogens in ticks collected from breeding and migratory birds in Switzerland.

From 2007 to 2010, 4558 migrating and breeding birds of 71 species were caught and examined for ticks in Switzerland. A total of 1205 specimens were collected; all were Ixodes ricinus ticks except one Ixodes frontalis female, which was found on a common chaffinch (Fringilla coelebs) for the first time in Switzerland. Each tick was analysed individually for the presence of Borrelia spp., Rickettsia spp., Anaplasma phagocytophilum and tick-borne encephalitis virus (TBEV). Altogether, 11.4% of birds (22 species) were infested by ticks and 39.8% of them (15 species) were carrying infected ticks. Bird species belonging to the genus Turdus were the most frequently infested with ticks and they were also carrying the most frequently infected ticks. Each tick-borne pathogen for which we tested was identified within the sample of bird-feeding ticks: Borrelia spp. (19.5%) and Rickettsia helvetica (10.5%) were predominantly detected whereas A. phagocytophilum (2%), Rickettsia monacensis (0.4%) and TBEV (0.2%) were only sporadically detected. Among Borrelia infections, B. garinii and B. valaisiana were largely predominant followed by B. afzelii, B. bavariensis, B. miyamotoi and B. burgdorferi ss. Interestingly, Candidatus Neoehrlichia mikurensis was identified in a few ticks (3.3%), mainly from chaffinches. Our study emphasizes the role of birds in the natural cycle of tick-borne pathogens that are of human medical and veterinary relevance in Europe. According to infection detected in larvae feeding on birds we implicate the common blackbird (Turdus merula) and the tree pipit (Anthus trivialis) as reservoir hosts for Borrelia spp., Rickettsia spp. and A. phagocytophilum.

Borrelia burgdorferi genospecies detection by RLB hybridization in Ixodes ricinus ticks from different sites of North-Eastern Poland.

INTRODUCTION: RLB (Reverse Line Blot Hybridization) is a molecular biology technique that might be used for Borrelia burgdorferi sensu lato (sl) DNA detection with genospecies specification. Among B. burgdorferi sl genospecies at least 7 are regarded as pathogenic in Europe. OBJECTIVE: The aim of the study was to evaluate the frequency of different Borrelia genospecies DNA detection in Ixodes ricinus ticks in the endemic area of North-Eastern Poland by using RLB. MATERIALS AND METHOD: Ixodes ricinus ticks were collected in May - June, from 6 different sites in North-Eastern Poland (Jakubin, Kolno, Grajewo, Suwalki, Siemiatycze, Bialowieza) by flagging. Extracted DNA was amplified by polymerase chain reaction (PCR) targeting the intergenic spacer 5S 23S of B. burgdorferi sl. PCR products were hybridised to 15 different oligonucleotide probes for 9 different Borrelia genospecies (B. burgdorferi sl, B. burgdorferi ss, B. garinii, B. afzelii, B. valaisiana, B. lusitaniae, B. spielmanii, B. bissettii and B. relapsing fever-like spirochetes (B. myamotoi)) by RLB. RESULTS: Borrelia genospecies DNA was detected in 205 Ixodes ricinus ticks. Among 14 infected with Borrelia ticks, 4 were identified as B. garinii and 10 as B. afzelii. Higher numbers of infected ticks were noticed in the eastern part of the research area, where large forest complexes dominate. Nymphs appeared to be the most frequently infected tick stage, which has an epidemiological meaning in the incidence of Lyme borreliosis. CONCLUSIONS: The study demonstrated that RLB might be easily used in Borrelia DNA detection with genospecies-identification, and indicated the domination of B. afzelii and B. garinii in ticks from North-Eastern Poland.

Species co-occurrence patterns among Lyme borreliosis pathogens in the tick vector Ixodes ricinus.

Mixed infections have important consequences for the ecology and evolution of host-parasite interactions. In vector-borne diseases, interactions between pathogens occur in both the vertebrate host and the arthropod vector. Spirochete bacteria belonging to the Borrelia burgdorferi sensu lato genospecies complex are transmitted by Ixodes ticks and cause Lyme borreliosis in humans. In Europe, there is a high diversity of Borrelia pathogens, and the main tick vector, Ixodes ricinus, is often infected with multiple Borrelia genospecies. In the present study, we characterized the pairwise interactions between five B. burgdorferi sensu lato genospecies in a large data set of I. ricinus ticks collected from the same field site in Switzerland. We measured two types of pairwise interactions: (i) co-occurrence, whether double infections occurred more or less often than expected, and (ii) spirochete load additivity, whether the total spirochete load in double infections was greater or less than the sum of the single infections. Mixed infections of Borrelia genospecies specialized on different vertebrate reservoir hosts occurred less frequently than expected (negative co-occurrence) and had joint spirochete loads that were lower than the additive expectation (inhibition). In contrast, mixed infections of genospecies that share the same reservoir hosts were more common than expected (positive co-occurrence) and had joint spirochete loads that were similar to or greater than the additive expectation (facilitation). Our study suggests that the vertebrate host plays an important role in structuring the community of B. burgdorferi sensu lato genospecies inside the tick vector.

Blackbirds Turdus merula as competent reservoirs for Borrelia turdi and Borrelia valaisiana in Portugal: evidence from a xenodiagnostic experiment.

To confirm that thrushes, such as blackbirds Turdus merula, play a role as reservoir for some Borrelia genospecies, we performed a xenodiagnostic experiment with blackbirds captured in a mixed wood located in Western Portugal where Borrelia turdi, an uncommon genospecies in Europe, was the most prevalent genospecies associated with birds. Two out of five birds harboured B. turdi infected Ixodes frontalis at the time of capture. Four out of five birds transmitted spirochaetes to Ixodes ricinus xenodiagnostic ticks: two birds transmitted Borrelia valaisiana to 25.7% and 10.5% of ticks, and two transmitted B. turdi to 6.4% and 5.4% of ticks. Our results showed that blackbirds transmit B. valaisiana and B. turdi to I. ricinus feeding larvae, acting as reservoir hosts for these genospecies in nature.

Survival of Ixodes ricinus (Acari: Ixodidae) nymphs under cold conditions is negatively influenced by frequent temperature variations.

In this study, we tested the survival of Ixodes ricinus under cold conditions in the laboratory. We investigated how the frequency of temperature variations (from -5 °C or -10 °C to 13 °C), and infection with Borrelia burgdorferi sensu lato (s.l.) influenced survival of questing nymphs collected in spring and autumn 2011. In experiment 1, survival of 1760 nymphs was tested at -10 °C over a short period of time to simulate very cold winter conditions. In experiment 2, survival of 1600 nymphs was tested under cold condition (-5 °C) over a long period of time to simulate common winter conditions. Ticks used for survival tests at -5 °C were screened for Borrelia by quantitative PCR, and genospecies identification was achieved by reverse line blotting. Tick age and frequency of temperature variations had a highly significant effect on I. ricinus survival while Borrelia infection was marginally significant. Hence, survival rate was higher in younger (autumn) than older (spring) nymphs and in nymphs exposed to low rather than high-frequency temperature variations. Borrelia-infected ticks tended to survive better than their uninfected counterparts. These findings suggest that in nature (i) frequent temperature changes in winter threaten tick survival more importantly than very low temperatures, (ii) older (spring) ticks are less resistant to cold than younger (autumn) individuals, and (iii) Borrelia infection plays a marginal role in I. ricinus survival during winter conditions.

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.

Serological evidence of tick-borne encephalitis virus infection in rodents captured at four sites in Switzerland.

In a previous study, the presence of tick-borne encephalitis virus (TBEV) in questing Ixodes ricinus L. ticks and in field derived ticks that engorged on small mammals (n = 9,986) was investigated at four sites located in a TBE area in Switzerland. Two of these sites were already recognized as TBE foci (Thun and Belp) and the screening of ticks revealed the presence of TBEV in ticks at a third site, Kiesen, but not at the fourth one, Trimstein. The aim here was to test another approach to detect TBE endemic areas. Sera from 333 small mammals (Apodemus flavicollis, A. sylvaticus, Myodes glareolus) captured in 2006 and 2007 at the four sites were examined for the presence of antibodies against TBEV using immunofluorescence and avidity tests. Overall the prevalence of antibodies against TBEV in rodents reached 3.6% (12/333). At two sites known as TBE foci, Thun and Belp, anti-TBEV antibodies were detected in 9.9% (9/91) and 1.6% (1/63) of rodent sera, respectively. At the third site, Kiesen, recently identified as a TBE focus by the detection of TBEV in ticks, anti-TBEV antibodies were detected in 1.8% (2/113) of rodent sera. Finally, at Trimstein, none of the examined rodent sera had antibodies against TBEV (0/66). This study shows another approach to detect TBE foci by testing antibodies in small mammal sera that is less time-consuming and less expensive than molecular tools.

Infections and coinfections of questing Ixodes ricinus ticks by emerging zoonotic pathogens in Western Switzerland.

In Europe, Ixodes ricinus is the vector of many pathogens of medical and veterinary relevance, among them Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which have been the subject of numerous investigations. Less is known about the occurrence of emerging tick-borne pathogens like Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and Anaplasma phagocytophilum in questing ticks. In this study, questing nymph and adult I. ricinus ticks were collected at 11 sites located in Western Switzerland. A total of 1,476 ticks were analyzed individually for the simultaneous presence of B. burgdorferi sensu lato, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and A. phagocytophilum. B. burgdorferi sensu lato, Rickettsia spp., and "Candidatus Neoehrlichia mikurensis" were detected in ticks at all sites with global prevalences of 22.5%, 10.2%, and 6.4%, respectively. Babesia- and A. phagocytophilum-infected ticks showed a more restricted geographic distribution, and their prevalences were lower (1.9% and 1.5%, respectively). Species rarely reported in Switzerland, like Borrelia spielmanii, Borrelia lusitaniae, and Rickettsia monacensis, were identified. Infections with more than one pathogenic species, involving mostly Borrelia spp. and Rickettsia helvetica, were detected in 19.6% of infected ticks. Globally, 34.2% of ticks were infected with at least one pathogen. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of coinfections underline the need to take them seriously into consideration when evaluating the risks of infection following a tick bite.