Host identification in unfed ticks from stable isotope compositions (δ13 C and δ15 N).

Determination of the ratios of natural stable isotopes (13 C/12 C and 15 N/14 N) in unfed Ixodes ricinus nymphs and adults, which, in their previous stage, fed on captive wild rodents (Apodemus sylvaticus and Myodes glareolus), wild birds (Parus major and Cyanistes caeruleus) or domestic ruminants (Ovis aries and Bos taurus), demonstrated that it is possible to identify each host category with confidence. First, the tick-blood spacing, which is the difference between values obtained from ticks and the blood of hosts that they had fed on in the previous stage, was consistent (152 spacings investigated from 15 host individuals in total). Second, potential confounding factors (tick age and sex) did not affect the discriminatory power of the isotope patterns, nor did different rearing conditions (room temperature vs. 4 °C) or the duration of development (maximum of 430 days). The findings that the tick-blood isotope spacings, across a diverse range of hosts, were similar and predictable, and that confounders had little or no effect on this, strongly support the usage of the isotope approach. Because each of the host categories has a different role in the population dynamics of I. ricinus and in tick-borne pathogen ecology, the method described here has great potential for the clarification of tick and tick-borne pathogen ecology in the field.

Birds as reservoirs for Borrelia burgdorferi s.l. in Western Europe: circulation of B. turdi and other genospecies in bird-tick cycles in Portugal.

Birds are important in the ecology of Borrelia burgdorferi sensu lato (s.l.) because they are important hosts for vector tick immature stages and are known reservoirs for some Borrelia genospecies. The aim of our study was to assess the role of common passerine bird species as reservoirs for B. burgdorferi s.l. in Western Europe. We surveyed birds in enzootic areas in Portugal, where no information is available for birds as reservoirs for this aetiologic agent and where B. lusitaniae, for which few reservoirs have been identified, is the dominant genospecies. Twenty-three birds (2.9%), including Turdus merula, T. philomelos, Parus major and Fringilla coelebs harboured infected ticks, but only Turdus sp. harboured infected tick larvae. In one study area, although B. lusitaniae was dominant in questing Ixodes ricinus, no ticks feeding on birds were infected with this genospecies, and B. valaisiana was the dominant genospecies in I. ricinus larvae feeding on birds. In the other area ticks collected from birds were mainly I. frontalis which were infected with B. turdi. Two skin biopsies (4.2%) from two T. merula were positive, one for B. valaisiana and the other for B. turdi. This is the first report for B. turdi in Western Europe.

Do ticks and Borrelia burgdorferi s.l. constitute a burden to birds?

Ticks consume resources from their hosts shaping their life-history traits and are vectors of many zoonotic pathogens. Several studies have focused on the health effects of blood-sucking ectoparasites on avian hosts, but there is limited information on the effects of ticks on adult and sub-adult birds, which may actively avoid ticks and are likely to present low infestation intensities. We evaluated the effects of the presence of feeding ticks and intensity of infestation on health variables of avian hosts. We also evaluated whether these variables were affected by tick infection by Borrelia burgdorferi sensu lato (s.l.) and by the presence of Borrelia infection on the birds' skin. Presence of parasite association among ticks, haemosporidea and Borrelia within the bird-host was also tested. We found that infestation by ticks significantly increased heterophyl/lymphocyte ratio in Turdus merula suggesting increased stress. This was especially evident at high infestation intensities when a significant decrease in body mass and body condition (body mass corrected for size) was also observed. Erithacus rubecula infested with more than 10 larvae tended to have lower haematocrit and blood haemoglobin. Plasma globulin concentration in T. merula tended to be affected by the presence of attached ticks and their infection with Borrelia, but this depended on the age of the bird. No association was detected among ticks, haemosporidea and Borrelia infection. We showed that ticks have detrimental effects on their avian hosts even under natural infestation conditions and that confirmed Borrelia reservoir hosts may also present symptoms of infection, though these may be subtle.

Do the level of energy reserves, hydration status and Borrelia infection influence walking by Ixodes ricinus (Acari: Ixodidae) ticks?

Ixodes ricinus horizontal movement within a humidity gradient and the influence of infection by Borrelia burgdorferi sensu lato (s.l.) on tick walking were investigated. Nymphs were placed within an arena containing a humidity gradient ranging from 45 to 95% relative humidity (RH). After 1 h of acclimation at 70% RH ticks were released so that they could either stay, or walk towards either the wet or the dry end. Their position was recorded 2 h post-release. Fat content was quantified and Borrelia infection was detected using real-time PCR and PCR followed by Reverse Line Blotting. Among the 1500 ticks tested, 29·85% were infected. More low-fat nymphs walked inside the arena than high-fat individuals. When nymphs walked, more low-fat ticks walked towards wetter than drier air, whereas more high-fat individuals walked towards drier than wetter air. Among high-fat nymphs, a lower proportion of Borrelia-infected ticks walked inside the arena compared to uninfected individuals, as though spirochetes manipulated their arthropod vector to stay. However, Borrelia infection had no effect on walking direction towards the dry or the wet end. Hence, it appears that I. ricinus nymphs walk horizontally over short distances within a humidity gradient depending on both energy resources and Borrelia infection.

Prevalence and genotyping of tick-borne encephalitis virus in questing Ixodes ricinus ticks in a new endemic area in western Switzerland.

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.

Diversity and seasonal patterns of ticks parasitizing wild birds in western Portugal.

The diversity and abundance of questing ticks and ticks parasitizing birds was assessed during 1 year in two recreational forests in western Portugal, a suburban forest and an enclosed game area. The aim of this study was to assess the distribution and seasonality of tick species and to understand the role of bird species as hosts for ticks. Ixodes ricinus was the most abundant questing tick collected in the enclosed game area, whereas in the suburban forest, only three ticks were collected by blanket dragging. Tick species parasitizing birds included I. ricinus, I. frontalis, I. arboricola, I. acuminatus, Haemaphysalis punctata, Hyalomma marginatum and H. lusitanicum. This is the first record of I. arboricola in Portugal. Tick prevalence and intensity of infestation differed between study areas and was higher in birds from the game area where a large population of deer and wild boar may support tick populations. Ground and shrub dwelling bird species such as Turdus merula, Erithacus rubecula and Sylvia melanocephala were the most heavily parasitized by ticks, but the importance of different bird species as hosts of larvae and nymphs of I. ricinus and I. frontalis differed. Therefore, different bird species may contribute differently for tick population maintenance.

Microclimate and the zoonotic cycle of tick-borne encephalitis virus in Switzerland.

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.

[Diseases and pathogenic agents transmitted by ticks in Switzerland]. / Maladies et agents pathogènes transmis par les tiques en Suisse.

Among the 20 tick species described in Switzerland, Ixodes ricinus, the most frequent one, is implicated in the transmission of pathogenic agents. Lyme borreliosis and tick-borne encephalitis (TBE) are the major tick-borne diseases transmitted to human. Presently 5 Borrelia species, belonging to the group Borrelia burgdorferi, are recognized as human pathogens. The risks of infection depend on the stage of the vector, the multiple hosts, the pathogenic agent, as well as human behavior in nature. The detection of other pathogenic agents in ticks: Anaplasma, Babesia and Rickettsia predispose to infections or co-infections. Results of sero-epidemiologic studies suggest human infections. Active surveillance by physicians is necessary and clinical studies are required to evaluate the importance of these infections in Switzerland.

Ticks and tick-borne pathogens from wildlife in the Free State Province, South Africa.

Eight ixodid tick species, associated with 59 free-ranging mammals belonging to 10 species, were collected at five different localities in the Free State Province, South Africa. Four of the study areas were nature reserves (Willem Pretorius, Sandveld, Tussen-die-Riviere, and Soetdoring), and one site was a private farm located in Senekal district. The collection was performed from March 2006 until June 2006. Ticks (n=569) and tissues from animals (n=52) were analyzed by polymerase chain reaction, reverse line blot, and sequencing for various tick-borne pathogens belonging to the genera Babesia, Theileria, Anaplasma, and Ehrlichia. Rhipicephalus (Boophilus) microplus, the known vector of Babesia bovis responsible for Asiatic redwater in South Africa, was found for the first time in the Free State Province. Rhipicephalus warburtoni [corrected] also was collected in areas in the Free State where it has not been previously described. Anaplasma marginale was detected for the first time in a gemsbok (Oryx gazella gazella). Gene sequences recovered in this study were 98-100% homologous with GenBank sequences for Anaplasma bovis, Theileria separata, and Theileria sp. Malelane sable antelope.

Assessment of intraspecific mtDNA variability of European Ixodes ricinus sensu stricto (Acari: Ixodidae).

The Ixodes ricinus complex is composed of 14 species distributed worldwide. Some members of this complex are involved in the transmission of a number of diseases to animals and humans, in particular Lyme borreliosis, tick-borne encephalitis, ehrlichiosis and babesiosis. While the phylogenetic relationships between species of the I. ricinus complex have been investigated in the past, still little is known about the genetic structure within the species I. ricinus sensu stricto. We have investigated the intraspecific variability among 26 I. ricinus s.s. ticks collected in various European countries, including Switzerland, Italy, Austria, Denmark, Sweden, and Finland by using five mitochondrial gene fragments corresponding to the control region, 12S rDNA, cytb, COI, and COII. The five genes considered here showed a low genetic variability (1.6-5%). Our results based on both statistical parsimony (applied to the COI + COII + cytb + 12S + CR data set, for a total of 3423 bp) and maximum parsimony (applied to the COI + COII + cytb + 12S data set, for a total of 2980 bp) did not provide any evidence for a correlation between the identified haplotypes and their geographic origin. Thus, the European I. ricinus s.s. ticks do not seem to show any phylogeography structure.

Borrelia burgdorferi sensu lato, the agent of lyme borreliosis: life in the wilds.

In Europe, Borrelia burgdorferi sensu lato (sl) the agent of Lyme borreliosis circulates in endemic areas between Ixodes ricinus ticks and a large number of vertebrate hosts upon which ticks feed. Currently, at least 12 different Borrelia species belonging to the complex B. burgdorferi sl have been identified among which seven have been detected in I. ricinus: B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. valaisiana, B. spielmanii and B. bissettii. A few dozens of vertebrate hosts have been identified as reservoirs for these Borrelia species. Specific associations were rather early observed between hosts, ticks and borrelia species, like for example between rodents and B. afzelii and B. burgdorferi ss, and between birds and B. garinii and B. valaisiana. The complement present in the blood of the hosts is the active component in the Borrelia host specificity. Recent studies confirmed trends toward specific association between Borrelia species and particular host, but also suggested that loose associations may be more frequent in transmission cycles in nature than previously thought.

[Cattle farmers' perception of the nuisances caused by insects and ticks to cattle in the Canton Jura]. / Perception par les éleveurs des nuisances causées par les in-sectes et les tiques aux bovins dans le Canton du Jura.

During summer 2005, a survey of 172 farmers from the Canton Jura was carried out to determine their perception of the nuisances caused by insects and ticks to cattle. The presence of ticks was significantly more often spontaneously mentioned by farmers in the Clos-du-Doubs and in the district of Delémont (95% and 72%, respectively) than in Ajoie and in the Franches-Montagnes (29% and 19%, respectively, p < 0.0001). 20% of the farmers perceived the tick populations to be increasing. The following diseases were spontaneously listed: keratoconjunctivitis (59%), mastitis related to flies (31%), hypodermosis (15%), ehrlichiosis (12%) and babesiosis (10%). Suspicion of ehrlichiosis was significantly more often mentioned in the district of Delémont than in the rest of the canton (p < 0.001) which was associated with the presence of ticks observed by farmers (p < 0.001). Cases of clinical babesiosis, which is common in the Clos_du_Doubs, were mentioned by farmers outside this area: Delémont (3/57) and Ajoie (1/26). In this study babesiosis depends on the production area, on the presence of ticks observed by farmers and on the presence of water in the pasture (p < 0.05). This study shows that cattle farmers are well aware of the presence of the diseases linked to insects and ticks and that the perception of their distribution is in accordance with the epidemiological data. An overview of the epidemiology of tick- and insect-related diseases can therefore be established by surveying farmers' perception, but should then be confirmed by a biomedical study. Due to their accurate observations, farmers are key participants at the first level of surveillance systems of animal diseases.

[The biology of the Ixodes ricinus tick]. / Die Biologie der Ixodes ricinus Zecke.

More than 800 tick species have been reported world-wide however only about 30 tick species feed on humans, among them Ixodes ricinus, which is the most frequent tick species biting humans in Europe. It is the vector of Borrelia burgdorferi, which causes Lyme disease and of the tick-borne encephalitis virus. I. ricinus ticks pass through three developmental stages: larvae, nymphs and adults (females and males). More than 300 animal species have been reported as natural hosts for I. ricinus and 50 vertebrate species have been identified as reservoir hosts for B. burgdorferi. The density of this tick species may be very high, reaching in some places more than 300 ticks/100 m2. Attached ticks should be removed without delay with a blunt forceps.

The wild hidden face of Lyme borreliosis in Europe.

Lyme borreliosis is a zoonosis affecting humans in the Northern hemisphere. The pathogen, Borrelia burgdorferi sensu lato (sl), persists in endemic areas through a maintenance cycle involving ticks and wild animals. The description of different genospecies associated with Lyme borreliosis in Europe has generated the question concerning the maintenance of these pathogens in nature: how do closely related bacterial species like B. burgdorferi sl circulate between one main tick species, Ixodes ricinus, and several vertebrate host species? Recent studies have provided evidence that specific associations exist in some areas between Borrelia species and vertebrate hosts. The present paper based on this recent knowledge discusses various aspects of the ecology of the disease in Western Europe, in particular the maintenance and dispersal of the pathogens, and brings up some interesting questions.

Immunization of mice by recombinant OspA preparations and protection against Borrelia burgdorferi infection induced by Ixodes ricinus tick bites.

The wide distribution of Borrelia burgdorferi, the spirochete causing Lyme borreliosis, represents a human health hazard in many areas of the world. Vaccination has been proposed as an effective prevention strategy. Vaccination experiments were conducted with preparations of recombinant outer surface protein A (OspA) derived from Borrelia burgdorferi strain ZS7. Mice received three doses (1 microgram each) of the antigens adsorbed to aluminum hydroxide. A strong immune response to the vaccine antigen was observed. Mice were challenged after immunization, using Ixodes ricinus nymphal ticks infected with Borrelia burgdorferi strain ZS7. Infection was investigated by ear biopsy culture, xenodiagnosis with uninfected larvae and serological response to Borrelia burgdorferi antigens. All unimmunized control animals were found to be infected, while all immunized animals were found to be protected against infection by Borrelia burgdorferi. In addition, most adult ticks derived from nymphs that fed on immunized mice were found to be free of spirochetes.

Distinct patterns of protective antibodies are generated against Borrelia burgdorferi in mice experimentally inoculated with high and low doses of antigen.

We have studied the development of clinical arthritis and the generation of protective antibodies in two normal, inbred strains of mice either infected by ticks or experimentally (subcutaneous) inoculated with increasing numbers of Borrelia burgdorferi organisms. AKR/N mice developed only mild and DBA/2 mice only marginal clinical arthritis irrespective of the route of infection or the numbers of spirochetes (10-10(8)) inoculated. In contrast, immunodeficient SCID mice developed severe chronic arthritis under similar conditions, but with a delayed onset at lower numbers of needle-inoculated spirochetes or after tick bite. AKR/N and DBA/2 mice inoculated with either 10(4) (and fewer) B. burgdorferi organisms or via experimentally infected ticks generated antibodies with specificities for a variety of B. burgdorferi antigens except those to the outer surface proteins A and B (OspA, OspB). In contrast, mice inoculated with more than 10(4) spirochetes (10(5)-10(8)) developed in addition antibodies to OspA and OspB. Most notably, all three types of immune sera taken from DBA/2 mice showed similar capacities to confer protection on SCID mice against subsequent challenge with viable B. burgdorferi organisms. The data not only demonstrate that the quality of humoral immune responses to B. burgdorferi in mice is determined by the antigenic load, they also indicate the existence of further protective antibodies with specificities distinct from OspA and OspB.

Relationship between Borrelia burgdorferi sensu lato species, red squirrels (Sciurus vulgaris) and Ixodes ricinus in enzootic areas in Switzerland.

The infection and reservoir status of red squirrels (Sciurus vulgaris) for Borrelia burgdorferi sensu lato were studied in Switzerland. B. burgdorferi sensu lato was isolated from 15 skin samples from 4/6 dead red squirrels, victims of road traffic. Isolates were identified using restriction fragment length polymorphism (RFLP): B. burgdorferi sensu stricto was present in 14 culture tubes containing skin samples and B. afzelii in two other tubes. A mixed infection was revealed in one case. A total of 227 ticks attached to squirrels were cultivated in BSKII medium and 90 isolates were obtained. Genotypic identification by RFLP showed that B. afzelii (59%) and B. burgdorferi sensu stricto (46%) dominated in ticks feeding on red squirrels. Data collected from one particular animal, highly infested with Ixodes ricinus and harbouring numerous Borrelia-infected Ixodes ricinus ticks, showed that transmission of B. burgdorferi sensu lato occurred from S. vulgaris to feeding ticks. More precisely, B. burgdorferi sensu stricto and B. afzelii were mainly transmitted from S. vulgaris to ticks. The present data emphasized the results obtained previously from small rodents and birds in Japan and in Switzerland, showing the occurrence of specific associations between host species and Borrelia genospecies.

Efficient transmission of Borrelia burgdorferi between cofeeding Ixodes ricinus ticks (Acari: Ixodidae).

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.

Infection of field-collected Ixodes ricinus (Acari: Ixodidae) larvae with Borrelia burgdorferi in Switzerland.

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.

Strain variation of Lyme disease spirochetes isolated from Ixodes ricinus ticks and rodents collected in two endemic areas in Switzerland.

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.