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

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

Co-infection of Borrelia garinii and B. afzelii in a population of wild rodents from woodland

The maintenance of Borrelia burgdorferi s.l. in the environment is dependent on the zoonotic cycle involving tick vectors and certain reservoir hosts. It is well known, that the same species of wild rodents, as well as the vector Ixodes ricinus, are often co-infected with at least two genomospecies of B. burgdorferi s.l.: B. afzelii and B. garinii. The ticks collected from two rodent species: Clethrionomys glareolus and Apodemus flavicollis were examined for the presence of B. burgdorferi s.l., as well as for B. garinii and B. afzelii. In this study, an immunofluorescent antibody assay (IFA) and polymerase chain reaction (PCR) protocols were used. The high level of infestation in rodents (90% for C. glareolus and nearly 100% for A. flavicollis) shows that wild rodents are important hosts of the immature stages of I. ricinus. A high percent of Borrelia positive ticks collected from bank voles and yellow necked mice; above 7% determined by IFA and 2% determined by PCR, clearly revealed that these species of animals are competent zoonotic reservoirs of B. burgdorferi s.l.     

Clethrionomys glareolus, but not Apodemus flavicollis, acquires resistance to lxodes ricinus L, the main European vector of Borrelia burgdorferi

The European rodents Clethrionomys glareolus (bank vole) and Apodemus flavicollis (yellow-necked-mouse) are important hosts of the tick species Ixodes ricinus, the main European vector of Borrelia burgdorferi. We have addressed the question whether or not these tick hosts develop resistance to I. ricinus larvae. C. glareolus and A. flavicollis were exposed to 40 I. ricinus larvae for five consecutive times at two week intervals. Resistance was tested by the following parameters: percentage of ticks fully engorged, time of attachment, engorgement index, percentage of recovered ticks and proportion of larvae moulting to nymphs. Repeated infestation ofC. glareolus resulted in progressive and significant reductions in the percentage of fully engorged ticks, the time of attachment of partially engorged ticks, the scutal index of partially engorged ticks and the moulting success. In contrast, repeatedly infested A. flavicollis did not acquire resistance to larval I. ricinus. Effects of resistance in C glareolus could be partially disrupted by treatment with the immunosuppressive agent, cyclosporin A (CsA), indicating that T helper cells participate in the immune responses to tick bites. The data suggest that acquired immunity to I. ricinus larvae in C. glareolus is a density-dependent factor regulating natural tick burdens and that it may have an impact on the transmission cycle of B. burgdorferi in Central Europe.     

Absence of Lyme disease spirochetes in larval Ixodes ricinus ticks

To determine which kind of spirochete infects larval Ixodes ricinus, we examined questing larvae and larvae derived from engorged females for the presence of particular spirochetal DNA that permitted species differentiation. Borrelia miyamotoi was the sole spirochete detected in larval ticks sampled while questing on vegetation. Questing nymphal and adult ticks were infected mainly by Borrelia afzelii, whereas larval ticks resulting from engorged females of the same population were solely infected by B. miyamotoi. Since larvae acquire Lyme disease spirochetes within a few hours of attachment to an infected rodent, questing larvae in nature may have acquired Lyme disease spirochetes from an interrupted host contact. Even if transovarial transmission of Lyme disease spirochetes may occasionally occur, it seems to be an exceedingly rare event. No undisputable proof exists for vertical transmission of Lyme disease spirochetes, whereas B. miyamotoi appears to be readily passed between generations of vector ticks.     

Density of questing Ixodes ricinus nymphs and adults infected by Borrelia burgdorferi sensu lato in Switzerland: spatio-temporal pattern at a regional scale

Lyme borreliosis, the most important vector-borne disease in the Northern hemisphere, causes health problem for populations in endemic areas. In the present study, the density of questing Ixodes ricinus ticks and their infection with Borrelia burgdorferi sensu lato (sl) was examined in 11 areas located on the Swiss Plateau and in an alpine valley. From 1999 to 2001, free-living I. ricinus ticks were collected on a monthly basis by flagging vegetation in these areas. Each tick was examined for the presence of B. burgdorferi sl using direct fluorescent antibody assay, and for isolation of the bacteria. Borreliae were characterized by PCR followed by RFLP. Density of questing ticks varied greatly between studied areas. Borreliae were observed in ticks collected in all investigated sites. However, the prevalence of infection differed significantly among areas. Infection prevalence varied from 9% to 40% in nymphs and from 22% to 47% in adults. Adult ticks were significantly more infected (129/366, 35%) than nymphs (109/552, 20%). There was no correlation between nymphal density and infection prevalence as well as between adult density and infection prevalence, but there was a correlation between density of ticks and density of infected ticks. During the spring peak of questing tick density, a range of 2-30.3 infected ticks per 100 m(2) was observed. B. burgdorferi sl isolates (n = 129) were obtained from ticks collected in 10/11 areas. Five Borrelia species were identified: B. garinii, B. burgdorferi sensu stricto, B. afzelii, B. valaisiana, B. lusitaniae, and six mixed infections were also obtained. Borrelia species were heterogeneously distributed in the different areas.     

Terrestrial distribution of the Lyme borreliosis agent Borrelia burgdorferi sensu lato in East Asia

Lyme borreliosis is the most prevalent tick-borne zoonosis and an important emerging infection in Europe, North America, and Far Eastern countries. The geographical distribution of Borrelia spp. and the relationship between Borrelia spp. and tick spp. in East Asian countries have been studied. In Northern Asian countries, Ixodes persulcatus carries Eurasian-type Borrelia garinii (20047 type), Borrelia afzelii, and Asian-type B. garinii (variant NT29), whereas Borrelia burgdorferi sensu stricto has not been isolated. In contrast, Asian-type B. garinii has not been found in the European vector tick Ixodes ricinus. These Borrelia spp. cause Lyme borreliosis in their respective countries. The specificity between Borrelia spp. and tick spp. has been confirmed from studies in the Moscow region, which is a sympatric region for the tick spp. I. persulcatus and I. ricinus. In Southeast Asian countries including the southernmost island of Okinawa, the Borrelia valaisiana-related genomic group is carried by Ixodes granulatus. In Japan, a similar borrelia strain Am501, is transmitted by Ixodes columnae. Ixodes ovatus transmits Borrelia japonica but not other species. On other hand, in central China and Nepal, the ticks harbor Borrelia sinica. It is believed that these molecular epidemiological surveys will provide useful information for the diagnoses and prevention of Lyme borreliosis in these countries.     

Hosts on which nymphal Ixodes ricinus most abundantly feed

To identify hosts that may serve as European reservoirs for the agent of Lyme disease, Borrelia burgdorferi, we determined whether nymphal Ixodes ricinus feed mainly on particular mice (Apodemus flavicollis or A. agrarius), voles (Clethrionomys glareolus) or on sand lizards (Lacerta agilis) and whether the abundance of these hosts corresponds to the seasonal activity of the subadult stages of the vector tick. In all sites, the mice appeared most heavily infested by larvae; at least seven parasitized each mouse, about three per vole and four per lizard. Many fewer nymphal I. ricinus parasitized A. flavicollis and C. glareolus than did larvae. Although more than 30 times as many larval than nymphal ticks parasitized the two most abundant hosts (C. glareolus and A. flavicollis), about 15 times as many fed on A. agrarius and twice as many on lizards. Nymphal and larval ticks fed on rodents at about the same time. Lizards were most abundantly parasitized by nymphs somewhat earlier than by larvae. Early in the season of transmission of Lyme disease, virtually all A. agrarius as well as lizards were potentially exposed to spirochetes borne by nymphal I. ricinus. We concluded that larval and nymphal I. ricinus differentially parasitize different hosts. Because so many of these nymphs feed on them, A. agrarius may more effectively serve as reservoirs for the agent of Lyme disease than do other putative reservoir hosts. The presence of lizards may inhibit transmission.     

Prevalence of Anaplasma phagocytophilum and coinfection with Borrelia burgdorferi sensu lato in the hard tick Ixodes ricinus in the city of Hanover (Germany)

The castor bean tick Ixodes ricinus has been found to be the main vector for Lyme borreliosis spirochetes and Anaplasma phagocytophilum in Central Europe. 1646 I. ricinus ticks from Hanover, a city located in Northern Germany, were examined for infection with A. phagocytophilum and coinfection with Borrelia burgdorferi sensu lato (sl) to obtain so far missing prevalence data for this region. The total A. phagocytophilum infection rate was 3.2% (52/1646 ticks), divided into 4.1% (32/777) adults and 2.3% (20/869) nymphs. Coinfections with B. burgdorferi sl were found in 0.9% of all tick stages. The detected genospecies were B. afzelii, B. garinii, B. burgdorferi sensu stricto (ss), and B. garinii, which was the most frequent species in coinfected ticks.     

Lyme borreliosis

Lyme borreliosis is a multi-organ infection caused by spirochetes of the Borrelia burgdorferi sensu lato group with its species B burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, which are transmitted by ticks of the species Ixodes. Laboratory testing of Lyme borreliosis includes culture, antibody detection using ELISA with whole extracts or recombinant chimeric borrelia proteins, immunoblot, and PCR with different levels of sensitivity and specificity for each test. Common skin manifestations of Lyme borreliosis include erythema migrans, lymphocytoma, and acrodermatitis chronica atrophicans. The last two conditions are usually caused by B garinii and B afzelii, respectively, which are seen more frequently in Europe than in America. Late extracutaneous manifestations of Lyme borreliosis are characterised by carditis, neuroborreliosis, and arthritis. We present evidence-based treatment recommendations for Lyme borreliosis and review the prevention of Lyme borreliosis, including the Lyme vaccines.     

Occurrence of Borrelia afzelii and Borrelia garinii in Ixodes ricinus ticks from southern Moravia, Czech Republic

During the years 1996-2000, a total of 2398 Ixodes ricinus ticks were collected in three areas of southern Moravia and eastern Bohemia, Czech Republic, and examined by dark-field microscopy for the presence of spirochetes. The prevalence of B. burgdorferi sensu lato in Ixodes ricinus ticks varied and depended upon the year. In 1996, the prevalence 6.8% was observed, during 1997 and 1998, it increased to 8.4% and 12.3%, respectively. The lowest prevalence was observed in 1999 (3.6%), and in 2000 it increased to 4.0%. The mean rate of infection was 6.5%, and the proportions of infected ticks were 12.2% in 263 male ticks, 8.3% in 289 female ticks, 6.0% in 1621 nymphs, and 1.3% in 225 larvae. From the total of 156 highly infected ticks (>100 spirochetes per sample) transferred into BSK-H medium for isolation attempts, 13 isolates were obtained. PCR-RFLP and electrophoresis (SDS-PAGE) were used for the identification and characterization of Borrelia strains. Ten tick isolates were identified as Borrelia afzelii, and the other three isolates were found to be Borrelia garinii. The results indicate the epidemiological importance of B. afzelii and B. garinii in central Europe, and emphasize the role of I. ricinus in the ecology of B. burgdorferi and in the epidemiology and epizootiology of Lyme disease.     

Geographic and temporal variations in population dynamics of Ixodes ricinus and associated Borrelia infections in The Netherlands

In a countrywide investigation of the ecological factors that contribute to Lyme borreliosis risk, a longitudinal study on population dynamics of the sheep tick Ixodes ricinus and their infections with Borrelia burgdorferi sensu lato (s.l.) was undertaken at 24 sites in The Netherlands from July 2006 to December 2007. Study sites were mature forests, dune vegetations, or new forests on land reclaimed from the sea. Ticks were sampled monthly and nymphal ticks were investigated for the presence of Borrelia spp. I. ricinus was the only tick species found. Ticks were found in all sites, but with significant spatial and temporal variations in density between sites. Peak densities were found in July and August, with lowest tick numbers collected in December and January. In some sites, questing activities of I. ricinus nymphs and adults were observed in the winter months. Mean monthly Borrelia infections in nymphs varied from 0% to 29.0% (range: 0%-60%), and several sites had significantly higher mean nymphal Borrelia infections than others. Four genospecies of Borrelia burgdorferi s.l. were found, with B. afzelii being dominant at most sites. Borrelia infection rates in nymphal ticks collected in July, September, and November 2006 were significantly higher (23.7%, p<0.01) than those in the corresponding months of 2007 (9.9%). The diversity in Borrelia genospecies between sites was significantly different (p<0.001). Habitat structure (tree cover) was an effective discriminant parameter in the determination of Borrelia infection risk, as measured by the proportion of nymphal ticks infected with B. burgdorferi s.l. Thickness of the litter layer and moss cover were positively related to nymphal and adult tick densities. The study shows that Borrelia-infected ticks are present in many forest and dune areas in The Netherlands and suggests that in such biotopes, which are used for a wide variety of recreational activities, the infection risk is high.     

Prevalence and genetic heterogeneity of Borrelia burgdorferi sensu lato in Ixodes ticks in Belgium

Borrelia burgdorferi sensu lato is a genetically diverse group of spirochetes that includes the agent of Lyme borreliosis in which genospecies tend to be associated with specific clinical features. The aim of the study was to determine the prevalence and genetic diversity of Borrelia burgdorferi sensu lato in 524 ticks collected in woods of a western province of Belgium. Presence of spirochetes in ticks was determined by phase contrast microscopy. The mean infection rate of ticks was 12.0%. Variability was observed in the prevalence of infection among the five sites examined, ranging from 2.8 to 21.6%. Identification to genospecies was determined by PCR and sequencing. The most common genomospecies were Borrelia afzelii (55%) and Borrelia garinii (21%). For the first time in Belgium, we detected Borrelia valaisiana and Borrelia spielmanii, representing 14% and 2%, respectively. Borrelia burgdorferisensu stricto counted only for 2%. Co-infections were present in 8% of ticks. We emphasize the need for clinical studies to assess the prevalence of specific genospecies-related clinical manifestations of Lyme borreliosis in Belgium.     

Diversity of Ixodes-borne Borrelia species--clinical,pathogenetic, and diagnostic implications and impact on vaccine development

Among Borrelia spirochetes carried by hard ticks belonging to the various Ixodes species, at least 10 species can be distinguished. Of these, Borrelia burgdorferi sensu stricto is involved in human Lyme borreliosis in North America and Europe, and Borrelia garinii and Borrelia afzelii in human disease in Europe and Asia. The pathogenetic significance of the other species is uncertain. Although some of the Borrelia species are restricted to certain tick species, Ixodes ricinus, the vector of Lyme borreliosis in Europe, can be infested by at least five different species, including all three pathogenic species. There is evidence that different Borrelia species are preferentially found in different hosts: In Europe, B. afzelii is frequently found in small mammals, whereas B. garinii and Borrelia valaisiana are often found in birds. This could very well be related to differential sensitivity of these species to complement-mediated bactericidal activity of different hosts. Borrelial complement regulator acquiring proteins, among them OspE or Erp proteins, bind to host factor H and related proteins, and this binding protects against activation of complement by the spirochetal surface. The binding is different for proteins originating from different species and is also depending on the host origin of factor H. In Europe, B. garinii is mainly found in neuroborreliosis, whereas in skin disease B. afzelii is more frequently found. The reason is unclear. The majority of human sera cross-react between proteins of different Borrelia species, but some sera react only with proteins from one of the species. This holds especially for reactivity with OspC. A vaccine against B. burgdorferi sensu stricto has been licensed, but was recently redrawn from the market because of commercial reasons. A vaccine protecting against all three pathogenic species is not yet available.     

Lyme borreliosis incidence in two French departments: correlation with infection of Ixodes ricinus ticks by Borrelia burgdorferi sensu lato

We conducted a prospective study to estimate the Lyme borreliosis incidence in two rural French departments, Meuse and Puy-de-D?me. Concurrently, we investigated the prevalence of ticks infected with Borrelia burgdorferi sensu lato (sl) and Anaplasma phagocytophilum. The incidence of Lyme borreliosis decreased from 156 to 109/100,000 inhabitants in Meuse and from 117 to 76/100,000 inhabitants in Puy-de-D?me in 2004 and 2005, respectively, corresponding to a decrease in the density of Ixodes ricinus nymphs infected with B. burgdorferi sl. During the same period, the density of adult ticks increased. Interestingly, B. valaisiana, a nonpathogenic species, infected adult ticks more often than nymphs. These results confirmed the correlation between the Lyme borreliosis incidence and the density of infected nymphs, a stage preferentially infected with B. afzelii. In contrast, we found a low rate of infection by A. phagocytophilum, ranging from 0% to 0.4% in Puy-de-D?me and from 0.8% to 1.4% in Meuse, suggesting a low risk for humans.     

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.     

Elimination of Borrelia burgdorferi and Anaplasma phagocytophilum in rodent reservoirs and Ixodes scapularis ticks using a doxycycline hyclate-laden bait

A field trial was conducted in a Lyme disease-endemic area of New Jersey to determine the efficacy of a doxycyline hyclate rodent bait to prophylactically protect and cure small-mammal reservoirs and reduce infection rates in questing Ixodes scapularis ticks for Borrelia burgdorferi and Anaplasma phagocytophilum. The doxycycline-laden bait was formulated at a concentration of 500 mg/kg and delivered during the immature tick feeding season in rodent-targeted bait boxes. The percentage of infected small mammals recovered from treated areas after 2 years of treatment was reduced by 86.9% for B. burgdorferi and 74% for A. phagocytophilum. Infection rates in questing nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 94.3% and 92%, respectively. Results from this study indicate that doxycycline-impregnated bait is an effective means of reducing infection rates for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing I. scapularis ticks.     

Genetic diversity of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in north-west Poland

Borrelia burgdorferi sensu lato (s. l.), the etiological agent of Lyme diesease, is transmitted by the bite of Ixodes ricinus. During May and September 1999, field surveys on Lyme disease spirochetes were conducted in three locations of a region of north-west Poland, known as recreational districts visited by many people. The ticks Ixodes ricinus were collected in natural habitats by dragging a flanel cloth over the vegetation. Sex and developmental stage of each tick were determined. Based on a polymerase chain reaction test with primers that recognize a chromosomal gene of all strains, out of the total 1414 specimens collected, 126 (8.9%) were found to be infected. The species B. burgdorferi s. l. comprises at least three pathogenic genomospecies, B. burgdorferi sensu stricto (s. s.), Borrelia garinii, and Borerelia afzelii, witch could be distinguished in nested-PCR tests with species-specific primers. B. burgdorferi s. s. was most prevalent (96% of infected ticks), followed by B. garinii (1.3%), and B. afzelii. was not found. Of the infected ticks, over the 99% were infected with a single species, one specimens was infected with two species. For 4 ticks, the infecting species could not be identied. The difference in rates of prevalence was observed among the tree locations (17%--5.3%--3.2%).     

Capacity of European animals as reservoir hosts for the Lyme disease spirochete.

The abundance of spirochete-infected Ixodes ricinus ticks naturally derived from endemic rodents was compared to identify the reservoir hosts of the agent of Lyme disease at a series of enzootic sites in Central Europe. Black-striped mice appear to be the most important rodent host for the tick and infect more ticks with Borrelia burgdorferi than do other mice and voles tested. Although rodents infect about half of the ticks that feed on them, lizards infect none. These spirochete-incompetent hosts dilute the force of transmission because many nymphal ticks feed on them. Older male hosts are more heavily infested by subadult ticks than are younger or female hosts. The intensity of transmission of the agent of Lyme disease at particular Central European sites appears to correlate directly with the presence of black-striped mice and inversely with that of lizards.     

Molecular characterization of Borrelia burgdorferi isolated from Ixodes ricinus in northern Sweden.

Ixodes ricinus ticks, harbouring Borrelia burgdorferi, were found in an area in northern Sweden, not thought to be endemic for Lyme borreliosis. This investigation took place at Norrbysk?r, an island situated in the Bothnian Gulf, 63 degrees 33'N/19 degrees 52'E. One of 42 nymphal and 8/43 adult I. ricinus ticks collected carried spirochetes as seen by phase contrast microscopy. Pure bacterial cultures were obtained from 2 of the ticks. Western blot analysis using species-specific monoclonal antibodies showed that the isolated spirochetes were B. burgdorferi. The identity of the isolated spirochetes was confirmed by DNA amplification using B. burgdorferi OspA and flagellin gene specific oligonucleotides as well as partial DNA sequencing of the respective OspA and flagellin genes. The 2 isolated spirochaete populations were different as shown by their protein profiles in sodium dodecyl sulphate polyacrylamide gels. Moreover, the demonstration of Lyme borreliosis in a patient from the island of Norrbysk?r indicates the need for clinical consideration of this disease in northern Sweden.