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The analysis of different multi-host systems suggests that even hosts that are not capable of transmitting Borrelia burgdorferi sensu lato (s.l.) to the tick vector, Ixodes ricinus, or that are secondary reservoirs for these agents contribute to the intensity of transmission and to the overall risk of Lyme borreliosis, through the process of vector augmentation and pathogen amplification. On the other hand, above certain threshold densities, or in the presence of competition with primary reservoir hosts or low attachment rate of ticks to reservoir hosts, incompetent or less competent hosts may reduce transmission through dilution. The transmission of B. burgdorferi s.l. is affected by molecular processes at the tick-host interface including mechanisms for the protection of spirochaetes against the host's immune response. Molecular biology also increasingly provides important identification tools for the study of tick-borne disease agents. Ixodes ricinus and B. burgdorferi s.l. are expanding their geographical range to northern latitudes and to higher altitudes through the effects of climate change on host populations and on tick development, survival and seasonal activity. The integration of quantitative ecology with molecular methodology is central to a better understanding of the factors that determine the main components of Lyme borreliosis eco-epidemiology and should result in more accurate predictions of the effects of climate change on the circulation of pathogens in nature., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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.

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE) and causes neurological disease in humans in Eurasia. TBEV is transmitted by ticks of the genus Ixodes. Currently 10,000-12,000 clinical cases are reported annually in approximately 30 TBE endemic countries. Since 1990 the epidemiology of TBE is characterized by a global increase of clinical cases and an expansion of risk areas. Similar trends are also observed in Switzerland but few studies confirmed the emergence of new TBE foci by detecting viral RNA in field-collected ticks. In this study, free-living Ixodes ricinus (L.) ticks from one nonendemic and three new TBE endemic regions located in the Western part of Switzerland were screened during four consecutive years (2007-2010) for the presence of TBEV. A total of 9,868 I. ricinus ticks (6,665 nymphs and 3,203 adults) were examined in pools for TBEV by real-time reverse transcription polymerase chain reaction. Our results confirmed the presence of viral RNA in 0.1% (6/6120) of questing ticks collected in one new endemic region. Among TBE endemic sites, the minimal infection rate per 100 ticks tested ranged from 0.21 (1/477) to 0.95 (1/105). Four positive samples were sequenced and phylogenetic analysis of the NS5 gene showed that all TBEV nucleotide sequences belonged to the European subtype and were split into two distinct lineages originating probably independently from two distinct foci located North-East and East of the study region.

The focal distribution of tick-borne encephalitis virus (TBEV; Flaviviridae, Flavivirus) appears to depend mainly on cofeeding transmission between infected Ixodes ricinus L. nymphs and uninfected larvae. To better understand the role of cofeeding ticks in the transmission of TBEV, we investigated tick infestation of rodents and the influence of microclimate on the seasonality of questing I. ricinus ticks. A 3-yr study was carried out at four sites, including two confirmed TBEV foci. Free-living ticks and rodents were collected monthly, and microclimatic data were recorded. A decrease in questing nymph density was observed in 2007, associated with low relative humidity and high temperatures in spring. One site, Thun, did not show this decrease, probably because of microclimatic conditions in spring that favored the questing nymph population. During the same year, the proportion of rodents carrying cofeeding ticks was lower at sites where the questing nymph density decreased, although the proportion of infested hosts was similar among years. TBEV was detected in 0.1% of questing ticks, and in 8.6 and 50.0% of larval ticks feeding on two rodents. TBEV was detected at all but one site, where the proportion of hosts with cofeeding ticks was the lowest. The proportion of hosts with cofeeding ticks seemed to be one of the factors that distinguished a TBEV focus from a non-TBEV focus. The enzootic cycle of TBEV might be disrupted when dry and hot springs occur during consecutive years.

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.

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.

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.

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.

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.

A total of 874 Ixodes ricinus ticks were collected in Switzerland to investigate the genetic diversity of the Borrelia population. We integrated to the RT-PCR method the DNA sequence analysis of a 162-bp fragment of the recA gene. Five genospecies were detected: Borrelia afzelii, Borrelia burgdorferi s.s., Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. A heterogeneous distribution was observed within the B. burgdorferi s.l. genospecies. The most prevalent and diverse genospecies found in Switzerland was Borrelia afzelii, which might suggest a rapid evolution of this genospecies.

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.

In this study, we measured the phenology of Ixodes ricinus ticks and their infection with Borrelia burgdorferi sensu lato (sl) simultaneously along an altitudinal gradient to assess the impact of climate on the phenology of ticks and on their infection with B. burgdorferi sl. From 1999 to 2001, free-living I. ricinus ticks were collected monthly by flagging vegetation at three different altitudes (620, 740, and 900 m above sea level) on the slope of a mountain in Chaumont (Neuchâtel, Switzerland). I. ricinus ticks were examined for the presence of B. burgdorferi sl by using direct fluorescent antibody assay and isolation of spirochetes. Borrelia species were characterized by polymerase chain reaction followed by restriction fragment-length polymorphism. Tick density and tick phenology varied with altitude. Although the peak tick density decreased and the onset of ticks was delayed with altitude, the phenology was much more stable among years at the highest altitudes than at the lowest. The prevalence of B. burgdorferi infection in nymphs and adults decreased with altitude. The prevalence of infection differed significantly among years, and it was significantly higher in adults (30%) than in nymphs (21%). B. burgdorferi infection in adults was positively related with adult density, but this was not observed for nymphs. Five B. burgdorferi sl genospecies were successfully isolated: B. garinii, B. burgdorferi sensu stricto, B. afzelii, B. valaisiana, and B. lusitaniae. Mixed infections were obtained from five of 140 infected ticks. The greatest diversity in Borrelia species was observed at the lowest altitude where all five Borrelia species were present, whereas at the two highest altitudes, B. lusitaniae was not observed.

Lyme disease is caused by genetically divergent spirochetes, including 3 pathogenic genospecies: Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii. Serodiagnosis is complicated by this genetic diversity. A synthetic peptide (C(6)), based on the 26-mer invariable region (IR(6)) of the variable surface antigen of B. burgdorferi (VlsE), was used as ELISA antigen, to test serum samples collected from mice experimentally infected with the 3 genospecies and from European patients with Lyme disease. Regardless of the infecting strains, mice produced a strong antibody response to C(6), which indicates that IR(6) is antigenically conserved among the pathogenic genospecies. Twenty of 23 patients with culture-confirmed erythema migrans had a detectable antibody response to C(6). A sensitivity of 95.2% was achieved, with serum samples collected from patients with well-defined acrodermatitis chronica atrophicans. Fourteen of 20 patients with symptoms of late Lyme disease also had a positive anti-IR(6) ELISA. Thus, it is possible that C(6) may be used to serodiagnose Lyme disease universally.

Free-living adult Ixodes ricinus L, were collected in Amdoun, situated in the Kroumiry mountains in northwestern Tunisia (North Africa). Using direct fluorescence antibody assay, the infection rate of field-collected I. ricinus by Borrelia burgdorferi sensu lato was 30.5% (n = 72). No difference in infection rate was observed between male and female ticks. Spirochetes that had been isolated from I. ricinus from Ain Drahim (Kroumiry Mountains) in 1988 were identified as Borrelia lusitaniae (formerly genospecies PotiB2). This is the first identification of a genospecies of Borrelia burgdorferi sensu lato from the continent of Africa.

Feeding ticks are generally spatially distributed in clusters on vertebrate hosts. To test the effect of clustering on transmission of a tick-borne pathogen, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner-infected Ixodes ricinus L. nymphs and uninfected I. ricinus larvae were allowed to feed together in retaining chambers on uninfected AKR/N mice. Engorged infective nymphs dropped off at days 5, 6, and 7, and the 1st infected larvae that fed in the chambers together with the infected nymphs dropped off at day 5. In contrast, ear biopsies and xenodiagnostic larvae placed on the head remained negative during that period. These results suggest that a cofeeding transmission occurred between B. burgdorferi-infected ticks and noninfected ones in the absence of a disseminated infection. Further investigations are being undertaken to determine whether the mechanism responsible for this cofeeding transmission is similar to that described previously with virus-infected ticks.

The relationship among Lyme borreliosis, Borrelia genospecies, rodent reservoirs, and Ixodes ricinus L. ticks was studied in two endemic areas in Switzerland. Ear punch biopsies and sampling of internal organs were used to isolate Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt and Brenner) from small mammals, Apodemus sylvaticus L., A. flavicollis Melchoir, Clethrionomys glareolus Schreber. Spirochetes were isolated from ear tissue and spleen of the rodents. Isolates were homogeneous and belonged to typing group II identified as B. afzelii (Canica, Nato, du Merle, Mazie, Baranton and Postic). Our data show that a specific association exists between B. afzelii and rodent reservoirs in European foci. Borreliae were also isolated from field-collected I. ricinus ticks from the same study areas. Proteinic and antigenic analysis indicated that more than one genospecies were present in the tick population. This suggests that other vertebrate hosts may serve as reservoirs of other Borrelia genospecies implicated with Lyme disease.

Free-living larvae from natural areas in five regions in Switzerland were collected to determine the infection rate of Ixodes ricinus L. larvae by Borrelia burgdorferi Johnson. Larvae were examined for the spirochetes using direct fluorescent antibody assay. Of 652 larvae examined, spirochetes were detected in 20 (3.1%). No differences in infection rate among sites were detected. The low prevalence of B. burgdorferi in larvae, compared with higher infection rate in nymphs (12.8%) and adults (14.5%), suggests that transovarial transmission is inefficient. These results suggest that I. ricinus can serve as a reservoir for B. burgdorferi in nature. Further study is needed on the relative importance of ticks, compared with mammals and birds, as reservoirs for B. burgdorferi.

Mice were infected with Borrelia burgdorferi by infection via Ixodes ricinus and experimental inoculation to determine whether transmission rates of spirochetes and antibody responses are influenced. Mice infected by the natural route were substantially more infective for ticks; two- to sixfold more tick larvae were positive for B. burgdorferi than those fed on experimentally inoculated mice. In natural infection, spirochetemia may be greater or spirochetes may be more accessible for transmission. Thus, this form of xenodiagnosis could be used to determine levels of spirochetes in the vertebrate host. Similar levels of antibody were present in all mice; however, those infected by the natural route lacked antibodies to outer surface proteins (Osp) A and B. The small antigen dose given through a tick bite may not have been sufficient to induce rapid OspA or OspB antibodies, thereby allowing the later development of higher levels of spirochetemia.

A past history of clinical Lyme borreliosis and the 6-month incidence of clinical and asymptomatic Lyme borreliosis was studied prospectively in a high-risk population. In the spring, blood samples were drawn from 950 Swiss orienteers, who also answered a questionnaire. IgG anti-Borrelia burgdorferi antibodies were detected by ELISA. Positive IgG antibodies were seen in 248 (26.1%), in contrast to 3.9%-6.0% in two groups of controls (n = 101). Of the orienteers, 1.9%-3.1% had a past history of definite or probable clinical Lyme borreliosis. Six months later a second blood sample was obtained from 755 participants, 558 (73.9%) of whom were seronegative initially; 45 (8.1%) had seroconverted from negative to positive. Only 1 (2.2%) developed clinical Lyme borreliosis. Among all participants, the 6-month incidence of clinical Lyme borreliosis was 0.8% (6/755) but was much higher (8.1%) for asymptomatic seroconversion (45/558). In conclusion, positive Lyme serology was common in Swiss orienteers, but clinical disease occurred infrequently.