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.

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.

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.

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.

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.

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.

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.

Survival of Ixodes ricinus (Acari: Ixodidae) under challenging conditions of temperature and humidity is influenced by Borrelia burgdorferi sensu lato infection.

ABSTRACT To determine whether Borrelia burgdorferi sensu lato (s.l.) influences tick survival under thermohygrometric stress, Ixodesricinus (L.) (Acari: Ixodidae) questing ticks were tested under various relative humidities (13, 32, 51.5, 61, and 89% RH) at two different temperatures (12.5 and 25 degrees C) and investigated for Borrelia infection. Survival rate of females was highest (77.6%), followed by males (51.6%), and nymphs (43.2%). The thermohygrometric factor that most importantly determined survival was saturation deficit (SD). As SD increased, tick survival rate decreased in all stages. Among the 1,500 ticks tested for B. burgdorferi s.l., 34.8% (n = 522) were infected. Adult infection rate (39.6%) was higher than that of nymphs (25.5%). Infection load in real-time polymerase chain reaction ranged from 1 to 1.2 million spirochetes per tick. B. afzelii (39.7%), B. burgdorferi sensu stricto (12.1%), B. garinii (37.9%), B. myamotoi (3.6%), and B. valaisiana (23.8%) were recorded. B. garinii infected significantly less nymphs than adults whereas B. afzelii displayed the opposite trend. Survival rate of nymphal and adult I. ricinus was significantly enhanced by infection by B. burgdorferi s.l. (Chi(2): nymph, P = 0.008; adult, P = 0.021). In adults, a negative effect of infection on tick survival was observed when spirochete load overcame a threshold estimated at 160,000 spirochetes per tick but not in nymphs. Moreover, ticks infected by B. afzelii survived better than other ticks (infected by other genospecies or not). The results here indicate that infection by B. burgdorferi s.l., and more specifically infection by B. afzelii, confers survival advantages to I. ricinus under challenging thermohygrometric conditions.

Borrelia hispanica relapsing fever, Morocco.

We found that 20.5% of patients with an unexplained fever in northwestern Morocco had tick-borne relapsing fever. Molecular detection specific for the 16S rRNA gene identified Borrelia hispanica. The noncoding intergenic spacer sequence domain showed high sensitivity and good resolution for this species.

Tick bites in a Lyme borreliosis highly endemic area in Switzerland.

The duration of tick feeding is an important indicator to evaluate the risk of Borrelia burgdorferi sensu lato transmission, which increases considerably with the blood meal duration. This blood meal duration may be estimated from scutal index, the ratio between body length (idiosoma) and scutum width. For the estimation of blood meal duration in Ixodes ricinus, nymphal and adult female ticks were detached at predetermined intervals (24, 48, 72, and 96h) from laboratory mice and rabbits and their scutal index calculated. From this, non-linear regression equations were developed to determine the duration of attachment for nymphal and adult female I. ricinus ticks. As part of an epidemiological study addressing the risk of subclinical (seroconversion) and clinical infections after a tick bite in the Neuchâtel area (Switzerland) over 3 years (2003-2005), duration of tick attachment and anatomical site of bites collected on participants as well as seasonal distribution of tick bites were studied. Tick attachment duration was estimated in all ticks collected during this study (n=261). Nymphs were attached for a mean (+/- standard error, SE) of 31.6h (+/-2.6) and females for a mean (+/-SE) of 29.6h (+/-3.2). Most nymphs were removed after 24h of blood meal whereas most females were removed before 24h. Legs were the major anatomical sites of bites for women (40.7%), men (44.4%), and almost all age classes. Only children <10 years old were bitten more frequently on the head (41.2%) and on the neck (38.5%) than participants >10 years. The majority of tick bites were recorded from May to July during the 3 years. Attachment sites can influence the discovery of ticks, hence the duration of the tick bite. A detailed body examination after each outing in forest and an early withdrawal of an attached tick is an effective way to prevent Lyme borreliosis.

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.

Ixodes ricinus density and infection prevalence of Borrelia burgdorferi sensu lato along a North-facing altitudinal gradient in the Rhône Valley (Switzerland).

Questing Ixodes ricinus ticks were sampled monthly along a north-facing altitudinal gradient in the canton of Valais, Switzerland, from March 2004 to February 2005. Tick density and infection with Borrelia burgdorferi sensu lato were monitored. Ticks were collected by flagging vegetation at three different altitudes (750 m, 880 m, and 1020 m above sea level). Ticks were examined for Borrelia by polymerase chain reaction (PCR) followed by reverse line blot. At the three altitudes, questing tick activity was not observed under 10 degrees C and was reduced when saturation deficit was higher than 5 mm Hg, most questing tick activity was occurred between 2 mm Hg and 7 mm Hg. Tick density and peak tick density were highest at 1020 m. High saturation deficits at the lowest altitudes appear to impair the tick population. The prevalence of B. burgdorferi infection in nymphs and adults decreased with altitude. The prevalence of infection was higher in adult ticks (47%) than in nymphs (29%). Four B. burgdorferi sensu lato genospecies were detected: B. afzelii (40%), B. garinii (22%), B. valaisiana (12%) and B. burgdorferi sensu stricto (6%). Mixed infections were detected in 13% of infected ticks.

A comparison of two DNA extraction approaches in the detection of Borrelia burgdorferi sensu lato from live Ixodes ricinus ticks by PCR and reverse line blotting.

We tested two approaches to extract Borrelia DNA from live Ixodes ricinus ticks before polymerase chain reaction (PCR) and reverse line blotting (RLB): DNA extraction of one half of the tick after incubation in BSK medium and DNA extraction of the other half of the tick directly, using ammonium hydroxide. Among 2079 ticks, 31.2% (n=649) were found to be Borrelia-infected by PCR-RLB test using at least one of the DNA extraction methods. Five hundred four ticks (24.2%) were found infected after incubation in BSK and 481 (23.1%) after direct DNA extraction from the tick. The difference was not significant. However, these prevalences were significantly lower when only one method was applied (23.1% and 24.2%) compared to the prevalence obtained by the use of both methods (31.2%). In 313 infected ticks discordant results were obtained, i.e., one half of the tick was found to be infected whereas the other half was uninfected. Among these ticks, B. garinii and B. burgdorferi sensu stricto (ss) were significantly more frequently identified in the half tick incubated in BSK. No significant differences were observed for B. burgdorferi ss, B. valaisiana, and for undetermined Borrelia species.

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.

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.

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.

Detection and identification of Borrelia burgdorferi sensu lato genospecies in ticks from three different regions in Slovakia.

Lyme borreliosis is one of the most common tick-borne diseases that occur in Slovakia. In this study, Borrelia burgdorferi sensu lato was detected and cultivated from questing ticks collected in three areas of Slovakia. Two methods, restriction fragment length polymorphism and reverse line blot, were used for identification of isolates and determination of the prevalence of B. burgdorferi s.l. in the ticks. The prevalence of B. burgdorferi s.l. in I. ricinus detected by reverse line blot was 31.9%. Four genospecies, namely B. garinii, B. valaisiana, B. afzelii and B. burgdorferi sensu stricto were found. B. garinii was the most prevalent genospecies.

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.

Reservoir role of lizard Psammodromus algirus in transmission cycle of Borrelia burgdorferi sensu lato (Spirochaetaceae) in Tunisia.

To investigate the reservoir role of the lizard Psammodromus algirus for the Lyme disease spirochete, 199 lizards were trapped from April to October 2003 in El Jouza, northwestern Tunisia. In this site, the infection rate of free-living Ixodes ricinus (L.) by Borrelia was evaluated by immunofluorescence as 34.6% for adult ticks and 12.5% for nymphs. Eighty percent of P. algirus (117/146) captured during this study were infested by I. ricinus, the predominant tick species collected from lizards. The intensity of tick infestation of this host by larvae and nymphs ranged from 0.14 to 7.07 and from 1.5 to 6.58, respectively. These immature stages of I. ricinus were found on lizards in spring and the beginning of summer, with a peak of intensity during June (10.16 immature ticks by lizard). Tissue cultures from lizards and xenodiagnosis with larval I. ricinus were used to assess the infection and the ability, respectively, of infected lizards to transmit Borrelia to naive ticks. Seventeen percent of xenodiagnostic ticks (40/229) acquired B. lusitaniae while feeding on P. algirus. Therefore, we demonstrated the ability of the lizards to sustain Borrelia infection and to infect attached ticks, and we proved that P. algirus is a reservoir host competent to transmit B. lusitaniae.