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.

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-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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Investigations on the mode and dynamics of transmission and infectivity of Borrelia burgdorferi sensu stricto and Borrelia afzelii in Ixodes ricinus ticks.

Borrelia burgdorferi sensu lato (sl), the agent of Lyme disease, is transmitted to the host during the blood meal of Ixodes ticks. In most unfed ticks, spirochetes are present in the midgut and migrate during blood feeding to the salivary glands, from which they are transmitted to the host via saliva. In the present study, the efficiency of Ixodes ricinus ticks to transmit B. afzelii and B. burgdorferi sensu stricto (ss) and their infectivity for mice were examined in relation to the duration of the blood meal. In addition, we investigated whether these two Borrelia species can penetrate intact skin. Three modes of infection of mice were studied: tick-bite infection, inoculation of tick homogenates, and transcutaneous infection by topical application of tick homogenates on mouse skin. Transmission of B. burgdorferi sl from I. ricinus nymphs to mouse increased with duration of tick attachment. B. afzelii-infected ticks start to transmit infection earlier (< or = 48 h) than B. burgdorferi ss-infected ticks. As previously shown for B. burgdorferi ss in Ixodes scapularis, B. burgdorferi ss and B. afzelii in unfed I. ricinus were noninfectious for mice when tick homogenates were inoculated. However, the inoculation of homogenates of ticks fed for 24 h readily produced infection in mice. Therefore, B. burgdorferi ss and B. afzelii spirochetes are potentially infectious in the tick before natural transmission can occur. None of the mice (n = 33) became infected by transcutaneous transmission when tick homogenates were applied on mouse skin, showing that B. burgdorferi ss and B. afzelii are unable to penetrate intact skin, in contrast to relapsing fever spirochetes. This study also shows that B. afzelii is transmitted by I. ricinus to the host earlier than B. burgdorferi ss and that I. ricinus seems to be a more efficient vector of B. afzelii than B. burgdorferi ss.

Influence of climate on the proportion of Ixodes ricinus nymphs and adults questing in a tick population.

We studied the relationship between climate and Ixodes ricinus L. tick behavior by following every day the proportion of ticks questing in a tick population placed in polyamide mesh-delimited arenas in the field. Simultaneously, the phenology of the questing density of nymphs and adults was studied by sampling ticks in a close location. At any time during the year, the proportion of questing adults was significantly higher (mean 24%) than the proportion of questing nymphs (mean 12%). The proportion of questing nymphs and adults decreased stepwise with time. The proportion of questing adults partially recovered after each decrease. In contrast, the proportion of questing nymphs was strongly reduced during a single short period in June and did not recover even partially. Decrease in the proportion of questing ticks was strongly related either to a peak in saturation deficit or to a drop in maximal relative humidity. No increase in the proportion of questing nymphs was observed in the arenas during autumn, although an autumn peak of nymphs was observed at the sampling location close to the arenas. This suggests that the autumn peak of nymphs observed in nature was due to newly emerged spring-fed larvae and not to reactivated spring active nymphs.