Infections and coinfections of questing Ixodes ricinus ticks by emerging zoonotic pathogens in Western Switzerland

In Europe, Ixodes ricinus is the vector of many pathogens of medical and veterinary relevance, among them Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which have been the subject of numerous investigations. Less is known about the occurrence of emerging tick-borne pathogens like Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and Anaplasma phagocytophilum in questing ticks. In this study, questing nymph and adult I. ricinus ticks were collected at 11 sites located in Western Switzerland. A total of 1,476 ticks were analyzed individually for the simultaneous presence of B. burgdorferi sensu lato, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and A. phagocytophilum. B. burgdorferi sensu lato, Rickettsia spp., and "Candidatus Neoehrlichia mikurensis" were detected in ticks at all sites with global prevalences of 22.5%, 10.2%, and 6.4%, respectively. Babesia- and A. phagocytophilum-infected ticks showed a more restricted geographic distribution, and their prevalences were lower (1.9% and 1.5%, respectively). Species rarely reported in Switzerland, like Borrelia spielmanii, Borrelia lusitaniae, and Rickettsia monacensis, were identified. Infections with more than one pathogenic species, involving mostly Borrelia spp. and Rickettsia helvetica, were detected in 19.6% of infected ticks. Globally, 34.2% of ticks were infected with at least one pathogen. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of coinfections underline the need to take them seriously into consideration when evaluating the risks of infection following a tick bite.     

Problems of isolating Borrelia burgdorferi from ticks collected in United Kingdom foci of Lyme disease

Abstract. Many isolates of Borrelia burgdorferi have been obtained from ticks and vertebrate tissues collected in North America and continental Europe but only one established culture of United Kingdom Borrelia burgdorferi has been recorded. In this paper we report the isolation of B.burgdorferi from one of 108 tick pools representing 733 ticks and eighty-four tissue samples from twenty-six rodents collected in the U.K., and the subsequent failure to establish the isolate (from ticks collected in Fordingbridge) in culture. In contrast, using identical techniques and culture medium, B.burgdorferi was isolated from one of seven tick pools collected in Switzerland, and from a single pool of ticks collected in Slovakia, and both isolates were successfully passaged. Analysis of questing I.ricinus collected from Fordingbridge by direct immunofluorescence showed 6/32 (19%) of adults and 8/108 (7%) of nymphs were positive for B. burgdorferi , although only one nymph contained ≥ 1000 spirochaetes. To examine further the problem of isolating U.K. B.burgdorferi , twelve Ixodes ricinus tick samples from Fordingbridge, a recognized focus of Lyme disease, were subjected to isolation and culturing techniques, and the procedures monitored by use of the polymerase chain reaction (PCR). Whereas 11/12 samples were PCR positive after 2 weeks in culture, only one was PCR positive after 4 weeks. Motile spirochaetes were not visible by dark-field microscopy in any of the cultures. The results indicate that the standard BSK II medium routinely used to isolate and culture B. burgdorferi does not readily support the replication of the Borrelia species endemic to the U.K.     

Tick species parasitizing people in an area endemic for tick-borne diseases in north-western Italy

In 1995 and 1996, 318 ticks were recovered from 240 people in Liguria (Province of Savona, Italy). Most of the ticks (284; 89.3%) were sheep ticks, Ixodes ricinus. Rhipicephalus sanguineus (31; 9.8%) and Dermacentor marginatus (3; 0.9%) were also recorded. All three life stages of I. ricinus were found on humans while only nymphs and adults of R. sanguineus and adults of D. marginatus were collected. Human tickbites were most frequent in the municipalities of the Province where roe deer density was highest. The number of tickbites in this area was about 500 per 100,000 residents over the period of observation. Most cases of tickbite were observed in children (11.2%), students (25.6%), workers (22.4%) and retired people (24%). Although ticks were recovered from people throughout the year, the highest frequencies of I. ricinus bites were in May, June and July. In the study area natural hosts were also studied. Six tick species were identified (I. ricinus, I. hexagonus, D. marginatus, R. bursa, R. sanguineus, R. turanicus). I. ricinus and R. sanguineus were the most abundant species. I. ricinus was recorded more frequently from ruminants particularly roe deer, while R. sanguineus was found to be associated with the dog.     

Combined infection of<Emphasis Type="Italic">Ixodes ricinus</Emphasis> with three<Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato genotypes

Ixodes ricinus ticks were collected by random collections from western and central Slovakia during the years 1996-98. The midgut content of 240 ticks was examined by dark-field microscopy and submitted for cultivation for the presence of borrelias. Spirochetes were found in 21 unfed and host-seeking adults and nymphs (8.8%). By the analysis of restriction fragment length polymorphism (RFLP) one sample from unfed I. ricinus male from western Slovakia was identified as a triple infection of Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii. The simultaneous presence of different B. burgdorferi genospecies in one midgut sample (triple infection in the tick) could be observed only after the multipart amplification of denaturated DNA and subsequent pooling of the products for further analysis.     

Risk factors in habitats of the tick Ixodes ricinus influencing human exposure to Ehrlichia phagocytophila bacteria

Ixodes ricinus L. (Acari: Ixodida) were sampled during 1996-99 in southern Scotland, on vegetation using cloth drags, on humans by removal from clothing and on roe deer (Capreolus capreolus L.) by searching legs of culled deer. Developmental microclimate was recorded by automatic recorders and questing microclimate by portable instruments during tick collections. Ticks and deer were examined for infection with Ehrlichia phagocytophila bacteria (Rickettsiales) using microscopy and polymerase chain reaction. This pathogen causes tick-borne fever of sheep in Europe and human granulocytic ehrlichiosis in North America, but in Europe human clinical ehrlichiosis due to E. phagocytophila has not been recorded despite serological evidence of exposure. Among three types of habitat, coniferous woodland was most infested with questing ticks (560 ticks/km of drag; mean numbers collected on long trousers: 24.3 larvae, 13.5 nymphs and 0.8 adult ticks/km walked), deciduous woodland had slightly lower infestation (426 ticks/km drag) and upland sheep pasture had much lower infestation (220 ticks/km drag). Of the three main vegetation types, bracken was least infested (360 ticks/km drag), ericas most (430 ticks/km drag) and grassland had intermediate infestation density (413 ticks/km drag). Questing and developmental microclimates were poor predictors of exposure within these habitats, except lower infestation of pastures was attributed to greater illumination there. Collectors who walked a total of 300 km through all habitats (taking 360 h in all seasons), wearing cotton trousers hanging outside rubber boots, were bitten by only four nymphs and 11 larvae of I. ricinus (but no adult ticks). There was a negative correlation between densities of deer and ticks collected, although presence of deer remains a major indicator of exposure. The proportion of infected ticks was fairly uniform at four sites studied. Overall prevalence of E. phagocytophila in I. ricinus was 3.3% in nymphs (40/1203) but only approximately 1.5% in adults of both sexes (although males do not bite). It was estimated that nymphs of I. ricinus gave 4.4% probability of one infected bite/person/year (for occupational exposure during this research) due to presence in all seasons and habitats, their human biting rate of 0.011 nymphs/h or 0.013 nymphs/km and widespread infection with E. phagocytophila. The frequency distribution of intensity of infection in ticks was approximately normal (mean 98 morulae/nymph infected), thus there is a high risk of receiving a high dose from any one infected tick bite.     

Influence of environmental factors on the occurrence of Ixodes ricinus ticks in the urban locality of Brno - Pisárky, Czech Republic.

The occurrence of Ixodes ricinus ticks was observed in the suburban locality of Brno-Pisárky (South Moravia, Czech Republic) from March to November, 1996 to 2002. A total of 2,813 ticks was collected. Statistical tests divided the activity of ticks into three periods during the year. The curve of seasonality had two peaks with a maximum in May and August, with a significantly larger number of specimens collected during this period compared to other months. The abundance of ticks during Spring and Autumn months was comparable. All developmental stadia of ticks were found in this locality. The number of larvae, males, and females was not significantly different, but the occurrence of nymphs was significantly greater. Except for the year 2000, there were no statistically significant differences in tick abundance. Tick activity was not dependent on humidity but did vary directly with temperature. This relation had a linear character and could be described by the equation y = 8.3 + 1.8x.     

Genomic comparison of Rickettsia helvetica and other Rickettsia species

We report the complete and annotated genome sequence of Rickettsia helvetica strain C9P9, which was first isolated in 1979 from Ixodes ricinus ticks in Switzerland and is considered a human pathogen.     

IgG responses to salivary gland extract of Ixodes ricinus ticks vary inversely with resistance in naturally exposed sheep

Enzyme-linked immunosorbent assay (ELISA) was used to investigate the antibody responses of control sheep, and sheep naturally exposed to Ixodes ricinus Linné (Acari: Ixodidae) ticks, to salivary gland extract (SGE) proteins of partially fed, adult I. ricinus. Comparisons between responses of control sheep and naturally infested sheep by Western blot analysis suggested that variations in IgG responses of I. ricinus-exposed sheep were mostly associated with specific responses to I. ricinus SGE antigens. Sheep IgG responses were positively related to the numbers of adult ticks feeding per sheep at the time samples were collected, were greater during the spring than the autumn periods of I. ricinus activity and were inversely related to sheep resistance to ticks measured by the weights of nymphal I. ricinus that engorged on the sheep. These findings suggest that sheep lose their resistance to ticks due to polarization of a Th1 type response to some tick antigens towards a Th2 type response when sheep are exposed to high, natural tick infestations, or to seasonal conditions of relative nutritional stress. Potential consequences for the epidemiology of tick-borne diseases are discussed.     

Longitudinal surveillance of the tick Ixodes ricinus for borreliae

Host-seeking Ixodes ricinus (L.) (Acari: Ixodidae) were monitored for borreliae (Borrelia burgdorferi s.l.) using dark-field microscopy in South Moravia (Czech Republic) each May from 1991 to 2001 (150 nymphs, 100 females and 100 males each year). This survey revealed a mean annual percentage of infected ticks of 16.8% (range, 11.7-24.2) in nymphs, 24.9% (range, 16.5-33.6) in females and 26.1% (range, 17.1-37.3) in males. Annual incidence of Lyme borreliosis in humans of the area in the same period (range, 8.7-41.7 per 100,000) correlated significantly with the frequency (number of ticks per flag per hour) of nymphs infected with >50 borreliae or all nymphal ticks, but not with the frequency of females, infected females or the infection rate (% of ticks infected) of either nymphal or female ticks. A prediction of the annual incidence of Lyme borreliosis, based on the frequency of heavily infected or all nymphal I. ricinus ticks, is feasible. The infection rate in I. ricinus correlated significantly with the North Atlantic Oscillation winter index of the last year (in nymphs) or of the year before last (in adults).     

Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus

The tick Ixodes ricinus is responsible for the transmission of a number of bacterial, protozoan and viral diseases to humans and animals in Europe and Northern Africa. Female I. ricinus from England, Switzerland and Italy have been found to harbour an intracellular alpha-proteobacterium, designated IricES1, within the cells of the ovary. IricES1 is the only prokaryote known to exist within the mitochondria of any animal or multicellular organism. To further examine the distribution, prevalence and mode of transmission of IricES1, we performed polymerase chain reaction screening of I. ricinus adults from 12 countries across its geographic distribution, including tick colonies that have been maintained in the laboratory for varying periods of time. IricES1 was detected in 100% of field-collected female ticks from all countries examined (n = 128), while 44% of males were found to be infected (n = 108). Those males that are infected appear to harbour fewer bacteria than females. Sequencing of fragments of the 16S rRNA and gyrB genes revealed very low nucleotide diversity among various populations of IricES1. Transmission of IricES1 from engorged adult females to eggs was found to be 100% (n = 31). In tick colonies that had been maintained in the laboratory for several years, a relatively low prevalence was found in females (32%; n = 25). To our knowledge, IricES1 is the most widespread and highly prevalent of any tick-associated symbiont.     

Babesia spp. identified by PCR in ticks collected from domestic and wild ruminants in southern Switzerland

Concurrent infections with vector-borne pathogens affected a cattle herd in Switzerland, and one of the pathogens was identified as Babesia bigemina, which had never been observed in this country before. Therefore, a survey of the occurrence of ruminant Babesia spp. and their tick vectors in Switzerland was conducted. A total of 2,017 ticks were collected from sheep, goats, cattle, and wild ruminants (deer, roe deer, and chamois) in southern parts of Switzerland and identified morphologically. The vast majority of the ticks (99.2%) were Ixodes ricinus, but 14 ticks from sheep and goats were identified as Dermacentor marginatus and two ticks from wild ruminants were identified as Hemaphysalis punctata. PCR analyses of 700 ticks revealed the presence of Babesia divergens (n = 6), Babesia sp. genotype EU1 (n = 14), and B. major (n = 2), whose suggested occurrence was confirmed in this study by molecular analysis, and the presence of novel Babesia sp. genotype CH1 (n = 4), which is closely related to B. odocoilei and to Babesia sp. genotype RD61 reported from North America. The identification of B. divergens and B. major in ticks collected from wild ruminants cast doubt on the postulated strict host specificity of these bovine Babesia species. Furthermore, the zoonotic Babesia sp. genotype EU1 was detected in ticks collected from domestic animals but was obtained predominantly from ticks collected from wild ruminants. More than one tick containing DNA of different Babesia spp. were collected from two red deer. Hence, the role of these game animals as reservoir hosts of Babesia spp. seems to be important but requires further investigation.     

Molecular evidence of<Emphasis Type="Italic">Bartonella</Emphasis> DNA in ixodid ticks in Czechia

We report the development of a novel method for detection of Bartonella DNA in ixodid ticks. The assay is based on a specific amplification of a part of 16S rRNA gene and 16S-23S rRNA intergenic spacer region of Bartonella sp. by nested PCR and Southern blot hybridization with specific DNA probe; the method is highly sensitive and specific. The screening of 327 unfed ticks collected in different urban and suburban areas of Czechia in 2003-2005 revealed the presence of Bartonella DNA in four Ixodes ricinus individuals (1.2%), two males, one female and one nymph.     

A symbiont of the tick Ixodes ricinus invades and consumes mitochondria in a mode similar to that of the parasitic bacterium Bdellovibrio bacteriovorus

We have recently performed molecular characterisation of an intracellular alpha-proteobacterium, named IricES1, which resides in the ovarian tissue of female Ixodes ricinus ticks from Italy. A unique characteristic of this bacterium is its ability to invade the mitochondria of the cells in which it resides. Although some ultrastructural studies have been performed on close relatives of this bacterium from I. ricinus in England and Switzerland, a number of questions remain about its movement within ovarian tissues and mitochondria. We have performed the first detailed ultrastructural examination of IricES1 in engorged female adult I. ricinus. Among our findings was that the bacterium enters mitochondria in a similar way to that employed by the 'predatory' bacterium Bdellovibro bacteriovorus, that is, between the inner and outer membranes. It then appears to multiply, with the new 'colony' consuming the mitochondrial matrix. Despite having many of their mitochondria consumed, oocytes appear to develop normally, and the bacteria are likely to be vertically transferred to all eggs.     

Borreliae in Ixodes ricinus ticks feeding on humans

A total of 298 Ixodes ricinus (L.) ticks (Acari: Ixodidae) feeding on humans in the Czech Republic were tested for borreliae (Borrelia burgdorferi sensu lato) by darkfield microscopy between 1997 and 2003. A majority (68%) of the supplied I. ricinus ticks were nymphs, 25% were females and 7% were larvae. Overall, 20% of 74 examined females and 9% of 203 examined nymphs (but none of 21 examined larvae) were infected with borreliae. The proportion of ticks with a high infection load (>100 spirochetes) was 4% in females and 2% in nymphal I. ricinus. During the year, the highest numbers and proportions of infected nymphal and female ticks were taken from humans in June. Detection of borreliae in the ticks feeding on humans might be helpful in the prophylaxis of Lyme borreliosis.     

Prevalence of Borrelia burgdorferi sensu lato in ticks collected from migratory birds in Switzerland

The prevalence of ticks infected by Borrelia burgdorferi sensu lato on birds during their migrations was studied in Switzerland. A total of 1,270 birds captured at two sites were examined for tick infestation. Ixodes ricinus was the dominant tick species. Prevalences of tick infestation were 6% and 18.2% for birds migrating northward and southward, respectively. Borrelia valaisiana was the species detected most frequently in ticks, followed by Borrelia garinii and Borrelia lusitaniae. Among birds infested by infected ticks, 23% (6/26) were infested by B. lusitaniae-infected larvae. Migratory birds appear to be reservoir hosts for B. lusitaniae.     

The α-Gpdh polymorphism in the tick Ixodes ricinus: similar diurnal trends in genotypic composition in Irish and Swedish population samples

Analysis of the alpha-Gpdh locus, coding for the enzyme alpha-glycerophosphate dehydrogenase (glycerol-3-phosphate dehydrogenase), was conducted using starch-gel electrophoresis on samples from one Irish and one Swedish population of the tick Ixodes ricinus. Morning ('AM') and late afternoon ('PM') samples of unfed adults were collected in each case. The results show a higher frequency of homozygotes in females in the AM than in the PM sub-samples in both populations. No such diurnal differentiation was found in the case of male ticks. The results are discussed in relation to previous studies on the polymorphism in Ireland and in the context of the known metabolic role of alpha-glycerophosphate dehydrogenase in insect muscle. The possible selective influence of climatological factors is considered.     

Coexistence of emerging bacterial pathogens in Ixodes ricinus ticks in Serbia

The list of tick-borne pathogens is long, varied and includes viruses, bacteria, protozoa and nematodes. As all of these agents can exist in ticks, their co-infections have been previously reported. We studied co-infections of emerging bacterial pathogens (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Francisella tularensis) in Ixodes ricinus ticks in Serbia. Using PCR technique, we detected species-specific sequences, rrf-rrl rDNA intergenic spacer for B. burgdorferi s.l., p44/msp2 paralogs for A. phagocytophilum, and the 17 kDa lipoprotein gene, TUL4, for F. tularensis, respectively, in total DNA extracted from the ticks. Common infections with more than one pathogen were detected in 42 (28.8%) of 146 infected I. ricinus ticks. Co-infections with two pathogens were present in 39 (26.7%) of infected ticks. Simultaneous presence of A. phagocytophilum and different genospecies of B. burgdorferi s.l. complex was recorded in 16 ticks, co-infection with different B. burgdorferi s. l. genospecies was found in 15 ticks and eight ticks harbored mixed infections with F. tularensis and B. burgdorferi s.l. genospecies. Less common were triple pathogen species infections, detected in three ticks, one infected with A. phagocytophilum / B. burgdorferi s.s. / B. lusitaniae and two infected with F. tularensis / B. burgdorferi s.s. / B. lusitaniae. No mixed infections of A. phagocytophilum and F. tularensis were detected.     

The Use of a 4% (w/w) Deltamethrin Collar (Scalibor® ProtectorBand) in the Extended Control of Ticks on Dogs

Deltamethrin (4%, w/w) formulated in a polymer matrix system (Scalibor ProtectorBand, Intervet International BV) was evaluated for the prevention of ticks on dogs. Controlled laboratory trials were conducted, wherein dogs wearing anti-tick collars were compared with control dogs and artificially infested by adult Ixodes ricinus and/or Rhipicephalus sanguineus ticks. The activity of the collars increased from 24 to 48 h post-treatment (pt) wherein tick numbers were reduced to 78.7% for I. ricinus and to 77% for R. sanguineus. The proportion of ticks controlled after the second infestation on D7 pt increased to 99% for I. ricinus and 99.5% for R. sanguineus. For I. ricinus adult ticks, an efficacy of 95.4% was reached over a period of 5.5 months and approximated 90% for R. sanguineus. Hence, the efficacy of the deltamethrin-impregnated dog collar was >90% for 6 months against both tick species. There was no significant difference between the efficacy of the collar against either R. sanguineus or I. ricinus. A subsequent field study wherein 82 dogs were wearing the collar and monitored for tick infestation indicated that the duration of efficacy was between 5 and 6 months.     

Autochthonous and migratory birds as a dispersion source for Ixodes ricinus in southern Italy

The present study was carried out in a protected wooded area, which is part of the Parco Regionale Gallipoli Cognato Piccole Dolomiti Lucane, one of the most important ecological reserves in southern Italy. From April 2010 to April 2011, 212 birds, comprising 22 species from 12 families, were captured and examined for ticks. A total of 75 (35.4?%) birds were found infested by ticks, with 451 ticks being collected. All ticks were identified as Ixodes ricinus, of which 241 (53.4?%) were larvae and 210 nymphs (46.6?%). The highest intensity of infestation was found in April 2010, when 117 ticks were retrieved on 25 birds. No ticks were found on birds (n?=?5) netted in December 2010. High infestation rates were recorded on blackbirds (Turdus merula) (90 %; 29 birds examined) and on mistle thrushes (Turdus viscivorus) (100?%; 2 birds examined). The highest intensity of infestation by larvae was found on wrens (5.6 larvae/bird) and by nymphs on mistle thrushes (11.5 nymphs/bird). Temperature and number of hours of light showed to influence the activity of larvae and nymphs. These data support the notion that birds may be responsible for the heterogeneous distribution of I. ricinus in Europe, thus playing a role in the epidemiology of certain tick-borne pathogens.     

Ethiological agents of rickettsiosis and anaplasmosis in ticks collected in Emilia-Romagna region (Italy) during 2008 and 2009

Ticks are the main vectors of rickettsiae of the spotted fever group, as well as of a variety of other Rickettsiales, including bacteria of the genus Anaplasma, that might cause diseases in humans and animals. Here we present the result of a survey for ticks and for tick-associated Rickettsiales in the Emilia Romagna region (Northern Italy). The study was focused on ticks collected from wild-hunted animals. Out of 392 ticks collected from these animals, 282 (72%) were identified as Ixodes ricinus, 110 (28%) as Dermacentor marginatus. The former was found on four vertebrate species, whereas the latter appeared more specific for wild boar. The presence of rickettsiae was demonstrated in 22.5% of I. ricinus (57/253) and in 29% of D. marginatus (32/110). Five ticks of the species I. ricinus were also positive for Anaplasma phagocytophilum (2%). In addition, we collected ticks by dragging in a natural park of the same region. All of the ticks captured by dragging were identified as I. ricinus. Thirty-six out of 200 analyzed ticks proved positive for Rickettsia monacensis and R. helvetica (16.5 and 1.5%, respectively). Our results highlight that that ticks present in wild areas, widely exploited for recreation and hunting in Emilia-Romagna, represent a risk for the transmission of spotted fevers and anaplasmosis to humans.