Borrelia burgdorferi sensu lato genospecies in questing Ixodes ricinus ticks in Austria

Unfed ticks of all instars (Ixodes ricinus, n=853; Haemaphysalis concinna, n=11) collected in all nine federal states of Austria were individually examined for the presence of Borrelia burgdorferi sensu lato (s.l.) using PCR. The mean overall infection rate was 14.4%. Infection rates were 24.5% in adult ticks, 16.1% in nymphs, and 1.6% in larvae. Four genospecies were detected, including B. valaisiana which was detected for the first time in Austria. The most common B. burgdorferi s.l. genospecies was B. garinii (66.9%), followed by B. valaisiana (13.7%), B. afzelii (11.3%), and B. burgdorferi sensu stricto (s.s.) (6.5%). Two specimens (1.6%) could not be identified to the genospecies level. Geographically, the highest infection rates were detected in the federal state of Vorarlberg (33.3%), B. garinii and B. afzelii being the most prevalent genospecies. B. valaisiana occurred most often in the federal state of Lower Austria, and B. burgdorferi s.s. was focally distributed in the Tyrol, in the surroundings of Imst.     

Distribution of Borrelia burgdorferi sensu lato in Ixodes ricinus (Acari: Ixodidae) ticks from the Basque Country, Spain

Borrelia burgdorferi was found widespread in ixodid ticks from the Basque Country (Spain) during a two-step study. In the first part, a total of 7,835 ixodids of eight different species was collected from vegetation, classified, and processed using polymerase chain reaction (PCR) for detection of B. burgdorferi ospA DNA. B. burgdorferi DNA wasdetectedin < or = 12.5% of adults and > or = 0.6% of Ixodes ricinus (L., 1758) nymphs (mean 1.5 and 0.05%, respectively), and in < or = 14.3% of adult Hemaphysalis punctata (Canestrini & Fanzago, 1877) analyzed (mean 1.2%). The second part of the study was undertaken 2 yr later to characterize B. burgdorferi distribution by focusing on the areas where L. ricinus was the predominant species. Ten areas were selected from which 1,535 nymphs and adults of I. ricinus were collected and processed by PCR and culture techniques. Infected ticks were found in all zones. B. burgdorferi DNA was detected in a mean of 9.3 and 1.5% of adults and nymphs, respectively. Nine isolates of B. burgdorferi were obtained, belonging to four different genospecies (B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. lusitaniae). The results indicate that some areas of Spain have a potential risk for Lyme disease agent exposure and that B. borgdorferi appears to have an increasing occurrence in ticks in the Basque Country.     

Borrelia burgdorferi sensu lato infection in larval Ixodes ricinus (Acari: Ixodidae) feeding on blackbirds in northwestern Italy

Birds belonging to 59 species (n = 1,206) were live captured in Piemonte, northwestern Italy, in 2001. Ixodes ricinus (L.) larvae were collected from 59 birds belonging to nine species, and nymphs were recovered on 79 birds belonging to 10 species. Eurasian blackbirds, Turdus merula L., had significantly higher levels of infestation by ticks than other passerine species. Larval I. ricinus of blackbirds peaked in summer, when prevalence was 39% (95% confidence interval 24.2-55.5) and mean number of ticks per host was 3.3 (1.6-7.2), whereas nymphs peaked in spring, when prevalence was 72.2% (54.8-85.8) and mean number of ticks per host was 6.9 (4.4-10.7). Immature I. ricinus were coincidentally aggregated on blackbirds, with 15 blackbirds feeding 67.4% of nymphs and 40.3% of larvae, and coinfestation by both stages was relatively high in summer: Kappa = 0.64 (0.40-0.88). Borrelia burgdorferi sensu lato was identified by polymerase chain reaction (PCR) in 58.3% (35.9-78.5) of larvae with engorgement ratio > or = 3 that were collected from blackbirds. Larvae that were collected from other passerine species gave negative PCR results. Sixteen of 21 PCR-positive samples belonged to B. garinii (76.2%), and five (23.8%) were Borrelia valaisiana. Results of this study suggest that blackbirds play an important role as hosts for immature I. ricinus and as reservoir of Borrelia garinii in northwestern Italy.     

Infection of Ixodes ricinus (Acari: Ixodidae) by Borrelia burgdorferi sensu lato in North Africa

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.     

Life stage-related differences in density of questing ticks and infection with Borrelia burgdorferi sensu lato within a single cohort of Ixodes pacificus (Acari: Ixodidae)

The primary aims of this study were to quantify the density of Ixodes pacificus Cooley and Kohls nymphs and adults of the same generational cohort collected within a single year in six oak or madrone leaf litter habitats and to compare the prevalence of infection with Borrelia burgdorferi sensu lato (s.l.) in adults originating from nymphal cohorts with a low (<1%) versus high (>10%) infection prevalence. Because adult densities were very low both in and adjacent to several sites, direct comparisons of infection prevalence between nymphs and adults were possible only for two sites. Mean density in these sites decreased from 11.95/100 m2 for nymphs to 0/100 m2 for adults in leaf litter, and infection prevalence with B. burgdorferi s.l. was four-fold higher in nymphs (7.4%) versus adults (1.6%) of the same generational cohort collected in ecotones bordering the leaf litter areas. Assuming a density of adults in leaf litter of 0.04/100 m2 (mean for all six examined sites) and an infection prevalence similar to that found in adults collected from litter ecotones, the risk of encountering infected ticks in leaf litter decreased >1,000-fold from the nymphal to adult stage. Regardless of site-specific infection prevalence in the nymphal stage (n = 2 sites; 0.7 versus 14%), the infection prevalence for the adults of the same generational cohort was similarly low (1.5-1.6%). Peak densities of adult I. pacificus were 0-0.1/100 m2 in leaf litter, 0-6.5/100 m2 in ecotonal grasslands, and 2.0-39.0/100 m2 in ecotonal chaparral. Despite more intensive sampling efforts in leaf litter, the vast majority of the 282 adults collected came from grass or chaparral ecotones (98.9%, n = 279) rather than leaf litter (1.1%, n = 3). The study yielded eight B. burgdorferi s.l.-infected adults; four of these carried B. burgdorferi sensu stricto Johnson, Schmidt, Hyde, Steigerwalt, and Brenner, and the remaining four were infected with currently undescribed B. burgdorferi s.l. spirochetes. This is the first study comparing both density of I. pacificus nymphs and adults and prevalence of infection with B. burdorferi s.l in these ticks within the same generational cohort and sampling area.     

Prevalence of Borrelia burgdorferi sensu lato in the selected Ixodes ricinus (Acari: Ixodidae) population in Weilburg forests,Hesse, Germany

We studied the presence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in woodland areas in the vicinity of Weilburg (Hesse, Germany). Ticks (n = 13347) were collected in five localities in 1996 and were fixed in 70% ethanol. Out of them, 4404 specimens were analysed individually (n = 406) and in 796 pools of 2-10 specimens (n = 3998) by the polymerase chain reaction (PCR). The calculated minimal infection rate of individual and pooled ticks was 0% in larvae (n = 280), 6.9 % in nymphs (n = 3600), 13.9 % in males (n = 280) and 20.9 % in females (n = 244). Prevalence of B. burgdorferi s. 1. in I. ricinus was determined with respect to the abundance and seasonal activity of the ticks.     

Borrelia burgdorferi in ticks (Acari: Ixodidae) from coastal Virginia

Ixodid ticks removed from hosts and from vegetation during March-November 1987 at sites in coastal Virginia and North Carolina were examined for Borrelia burgdorferi. B. burgdorferi was evident in nine (22%) Ixodes cookei Packard removed from rice rats (Oryzomys palustris), a white-footed mouse (Peromyscus leucopus), and raccoons (Procyon lotor); four (6%) Amblyomma americanum (L.) removed from raccoons; and two (3%) Dermacentor variabilis (Say) removed from a raccoon and a rice rat. B. burgdorferi was also detected in Ixodes dentatus Marx removed from a brown thrasher (Toxostoma rufum), a Carolina wren (Thryothoros ludovicianus), and a towhee (Piplio erythrophthalamus); and in Haemaphysalis leporispalustris (Packard) removed from a brown thrasher and a white-throated sparrow (Zonotrichia albicollis) netted at Kiptopeke Beach, Va. Two Ixodes dammini Spielman, Clifford, Piesman & Corwin were collected on Parramore Island; one specimen was examined for spirochetes, and it was infected with B. burgdorferi. No spirochetes were detected in host-seeking A. americanum and Amblyomma maculatum Koch removed from vegetation. The plasma of one P. leucopus and sera obtained from two P. lotor contained antibodies to B. burgdorferi. All infected ticks and the seroreactive hosts were collected from the Eastern Shore of Virginia.     

Efficiency of experimental infection of Ixodes ricinus ticks with Borrelia burgdorferi spirochetes

Feeding of an ixodid tick on a Borrelia-infected vertebrate is the natural route of tick infection. To obtain a cohort of nymphs with a high prevalence of infection, the immersion of Ixodes ricinus larvae in the suspension of B. burgdorferi sensu stricto spirochetes was used. Immersed larvae fed on C3H mice until full engorgement, molted to nymphs and 9 of 10 nymphs were shown to be infected by PCR with specific primers. Specific antibodies in the sera of the mice, on which either immersion-infected larvae or infected nymphs had engorged, were not detected. However, nymphs molted from immersed larvae were able to infect naïve mice, and all the resulting 10 adults were infected.     

Vector competence of Ixodes muris (Acari: Ixodidae) for Borrelia burgdorferi

The vector competence of Ixodes muris (Bishopp & Smith) was determined for Borrelia burgdorferi, the etiologic agent of Lyme disease. Larval I. muris were fed on ICR outbred mice infected with the B-31 laboratory strain of B. burgdorferi. Replete larvae, at 5 d after feeding, were assayed for infection by culture in Barbour-Stoner-Kelly (BSK-H) media. Infection frequency in I. muris replete larvae was 66%. Resultant nymphs were fed on naive ICR outbred mice to determine the ability of I. muris to transmit infection. Infection frequency in fed nymphs declined to 38% and only 1/5 mice was positive for B. burgdorferi on ear biopsy culture. We demonstrated that I. muris is capable of acquiring and transmitting B. burgdorferi but is a relatively poor vector compared with I. scapularis (Say).     

Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in urban recreational areas of Helsinki

Lyme borreliosis, an infection caused by the tick-borne spirochete Borrelia burgdorferi, is a major health problem for populations in areas of endemicity in the Northern Hemisphere. In the present study we assessed the density of ticks and the prevalence of B. burgdorferi sensu lato among ticks in popular urban recreational areas of Helsinki, Finland. Altogether 1,688 Ixodes ricinus ticks were collected from five areas located within 5 km of the downtown section of Helsinki, and 726 of them (303 nymphs, 189 females, and 234 males) were randomly chosen for laboratory analysis. The midguts of the ticks were divided into three pieces, one for dark-field microscopy, one for cultivation in BSK-II medium, and one for PCR analysis. Ticks were found in all the study areas; their densities varied from 1 to 36 per 100 m along which a cloth was dragged. The rate of tick infection with B. burgdorferi sensu lato varied from 19 to 55%, with the average being 32%. Borellia afzelii was the most predominant genospecies in all the areas, and no B. burgdorferi sensu stricto isolates were detected. Only two ticks were concurrently infected with both B. afzelii and Borrelia garinii. Dark-field microscopy gave more positive results for B. burgdorferi than did cultivation or PCR analysis. However, the agreement between all three methods was fairly good. We conclude that Lyme borreliosis can be contracted even in urban environments not populated with large mammals like deer or elk. The disease should be taken into account in the differential diagnosis of certain symptoms of patients from these areas, and the use of measures to improve the awareness of the general population and health care officials of the risk of contracting the disease is warranted.     

Subadult Ixodes ricinus (Acari: Ixodidae) on rodents in Berlin, West Germany

To identify hosts that may serve as reservoirs for the agent of Lyme disease in Central Europe, we determined whether Ixodes ricinus L. feed most frequently on certain rodents and whether the abundance of these hosts corresponds to the season of feeding activity of the tick in four sites in Berlin, Federal Republic of Germany. In addition, we correlated abundance of I. ricinus with that of particular rodent hosts. Two small rodents were more abundant than any others; a mouse, Apodemus flavicollis, predominated in a wooded site and a vole, Clethrionomys glareolus, in three brush- or grass-covered sites. The tick was most abundant in the mouse-infested site. Although A. flavicollis comprised only about a third of rodents collected, nearly 60% of all such rodent parasitizing I. ricinus fed on this mouse. These ticks were more abundant on mice than voles in each of the study sites and throughout the year, and more larvae fed on these rodents than did nymphs. Although larval as well as nymphal I. ricinus are most abundant during midsummer, they feed on rodents from April through October. Taken together, these observations suggest A. flavicollis as a potentially important reservoir host for I. ricinus-borne infections.     

Rates of Borrelia burgdorferi infection in Ixodes dammini (Acari: Ixodidae) in southwestern Wisconsin.

Ixodes dammini Spielman, Clifford, Piesman & Corwin collected from a study site in southwestern Wisconsin were examined for Borrelia burgdorferi infection. Adult spirochete infection rates varied seasonally (38.1%, spring 1990; 60.3%, fall 1990; 41.2%, spring 1991) and were similar to or higher than rates reported in various studies from the northeastern United States. Statistical comparison of seasonal infection rates indicated a significant difference between the fall 1990 adult population and the subsequent overwintered population of the same cohort (spring 1991). Additionally, a significant decrease in the intensity of spirochete infection was observed in the overwintered adult sample.     

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.     

Salivary gland extract from engorged Ixodes ricinus (Acari: Ixodidae) stimulates in vitro growth of Borrelia burgdorferi sensu lato

In vitro effect of salivary gland extract from fed Ixodes ricinus, the competent vector of Lyme borreliosis in Europe, on the growth of Borrelia burgdorferi sensu lato (B. garinii, B. afzelii and B. burgdorferi sensu stricto) was examined in BSK‐H medium. Motility rate, concentration of motile spirochetes and their morphology were estimated at intervals of 0, 2, 4, 6 and 8 days using darkfield microscopy. Salivary gland extract derived from I. ricinus stimulated markedly the growth of three genomic species of borreliae. The results confirm a substantial role of salivary glands in the mechanism of pathogen transmission to vertebrate host. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Aggregation in the tick Ixodes ricinus (Acari: Ixodidae): use and reuse of questing vantage points

Ongoing work in oak woods in Killarney National Park in southwestern Ireland is focusing on the factors influencing the fine-scale aggregated distribution of Ixodes ricinus L. (Acari: Ixodidae) on the ground. The extent of reuse of stems of vegetation as questing points by adult ticks was determined by paint-marking stems on which ticks were found, counting and removing these ticks, and subsequently reexamining the same stems for ticks on two further occasions. Overall, an estimated 2,967 stems in 123 separate rush plants (Juncus effusus L.) were examined. Statistical analysis of the data demonstrated a highly significant reoccupancy by ticks of stems previously and recently used. Furthermore, it is shown that the extent of stem reuse by ticks is significantly and positively correlated with the numbers of ticks originally observed on those stems. Although other factors may be involved in generating clumping of ticks, the results are compatible with the proposition that aggregation of I. ricinus on the ground is pheromone-mediated. The findings are discussed in relation to what is known about the powers of lateral movement of I. ricinus on the ground and the possible implications for the performance of tick traps.     

Isolation of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) from Ixodes scapularis and Dermacentor albipictus ticks (Acari: Ixodidae) in Oklahoma.

Borrelia burgdorferi was isolated from Ixodes scapularis Say and Dermacentor albipictus Packard that were removed as partially fed adults from white-tailed deer, Odocoileus virginianus Zimmermann, in Oklahoma. Isolation in media was accomplished only after homogenates of pooled field-collected ticks were inoculated into laboratory-reared Peromyscus leucopus and reisolated from the urinary bladder into BSK II media. Both isolates were confirmed by western blot analysis and reactivity with monoclonal antibody H5332. These are the first reported isolates of B. burgdorferi from Oklahoma from these two tick species and are the first isolates from ticks from the south-central United States that were infective for laboratory-reared P. leucopus.     

Ixodes scapularis ticks (Acari: Ixodidae) from Louisiana are competent to transmit Borrelia burgdorferi, the agent of Lyme borreliosis

The principal vector of Borrelia burgdorferi, the Lyme borreliosis spirochete, in the Northeast and Midwestern regions of the United States is the blacklegged tick Ixodes scapularis. Because of a favorable environment, I. scapularis is also plentiful in the South; however, a correlation with Lyme borreliosis cases does not exist in this region of the United States. Concern existed that something intrinsic to ticks found in Louisiana could mitigate their ability to transmit B. burgdorferi. Therefore, we set out to assess the ability of I. scapularis ticks from Louisiana to become infected with and transmit B. burgdorferi using mice as hosts. In the laboratory, mating adult female ticks collected in southeastern Louisiana were fed on the ears of rabbits. After oviposition and egg hatching, the resulting larvae were fed on mice that had been needle-inoculated with two different strains of B. burgdorferi sensu stricto, B31 and JD1. Larvae were found to be positive for spirochetes. Additional fed larvae were allowed to molt into the nymphal stage. Flat nymphs remained infected with B. burgdorferi. Infected nymphs were allowed to feed on na?ve mice, all of which became infected as shown by culture of ear biopsy specimens. Na?ve larvae were then fed on these same mice to assess transmissibility. The resulting engorged larvae harbored spirochetes. We have demonstrated that the I. scapularis ticks found in Louisiana are fully competent to carry and transmit B. burgdorferi infection.     

Borrelia lusitaniae and spotted fever group rickettsiae in Ixodes ricinus (Acari: Ixodidae) in Tuscany, central Italy

Prevalence of infection by Borrelia burgdorferi s.l. and spotted fever group (SFG) rickettsiae was estimated in host-seeking ticks in an area in Tuscany, central Italy, where Lyme borreliosis was reported in a forestry worker. B. burgdorferi s.l. was identified by polymerase chain reaction in 16.7% (95% CI = 10.3, 24.8) of Ixodes ricinus (L.) nymphs and 39.6% (95% CI = 26.5, 54.0) of adults. Borrelia lusitaniae accounted for 82.9% of positive samples, followed by Borrelia garinii (9.8%), Borrelia afzelii (2.4%), and Borrelia burgdorferi s.s. (2.4%). One Rhipicephalus spp. adult was infected with B. garinii (prevalence = 8.3%; 95% CI = 0.21, 38.5). Prevalence of infection by SFG rickettsiae was 38.5% (95% CI = 26.7, 51.4) in I. ricinus nymphs, 34.6% (95% CI = 22.0, 49.1) in I. ricinus adults, and 50% (95% CI = 21.1, 78.9) in Rhipicephalus spp. adults. Phylogenetic analysis showed the similarity of B. lusitaniae strains that were identified in this study and of a strain that was previously isolated from a human patient in Portugal. Results of this study confirm the dominance of B. lusitaniae in areas in the Mediterranean basin and the infection by SFG rickettsiae in I. ricinus.     

Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi

We describe a procedure for the introduction of Borrelia burgdorferi, the spirochetal agent of Lyme disease, into larvae of the tick vector Ixodes scapularis. Internalized spirochetes were observed in larvae examined after 15 or 45 min immersion at 32 degrees C in liquid culture suspensions of low passage B. burgdorferi strain B31. Larval ticks immersed in low passage strain B31 were able to feed to repletion on white-footed mice. Midguts of larvae contained many spirochetes 1 wk postengorgement, while larvae incubated with high passage strain B31 were free of detectable spirochetes at the same interval. Larvae incubated with low passage strain B31 were competent to transmit the pathogen to mice, as shown by serology, reisolation of B. burgdorferi from mice, and xenodiagnosis. Ticks maintained the infection transstadially to the nymphal stage and transmitted the infection to naive mice, replicating an essential aspect of natural infection. This method requires no special equipment and allows artificial infection of large numbers of ticks at the larval stage. It will facilitate studies of the contribution of specific B. burgdorferi genetic loci to tick colonization.