Preventive effect of permethrin-impregnated clothing to Ixodes ricinus ticks and associated Borrelia burgdorferi s.l. in Germany

The protective effectiveness of a factory-based, permethrin-impregnated military battle dress uniform (BDU) trouser using a new high-residual, polymer-coating technique has been evaluated by field testing against Ixodes ricinus, a vector of Borrelia burgdorferi s.l. During 36 h exposure of test subjects walking in known tick-infested habitats in the Kühkopf mountain area, Koblenz, Germany, between June and October 2006, 6 I. ricinus were found crawling with a visible excitatory effect on those legs covered with fabric impregnated with 1200 mg permethrin/m², whereas 132 ticks were collected from the negative control legs. B. burgdorferi s.l. infection was detected in 33% (2/6) adult male, 56% (9/16) adult female, 11% (6/56) nymphal, and 0% (0/46) larval I. ricinus sampled from the negative control legs. The few ticks collected from the impregnated fabric tested all negative for B. burgdorferi s.l. The mean tick infestation rate on the negative control legs of test subjects was 3.6±2.7 (mean±SD; range 0–12) per hour exposure. Permethrin-impregnated clothing conveyed a high mean protection rate of 95.5% against questing I. ricinus ticks, making it an excellent tool for the prevention of associated tick-borne diseases.     

Infection rate ofIxodes ricinus ticks withBorrelia afzelii,Borrelia garinii,andBorrelia burgdorferi sensu stricto in Slovenia

In spring 1993,Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection withBorrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium.Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19 %): the isolation rate from adult female ticks was 35 % (23/66 ticks cultured), from adult male ticks 22 % (20/91), and from nymphal ticks 13 % (26/206). Determination of the species of 60 isolates revealed that 32 wereBorrelia afzelii (53 %), 20 wereBorrelia garinii (33 %), and 8 wereBorrelia burgdorferi sensu stricto (13 %). In the Ljubljana regionBorrelia afzelii andBorrelia garinii predominated (43 % and 40 %, respectively), whereasBorrelia burgdorferi sensu stricto constituted only 17 % of isolates. In three other regions of the countryBorrelia afzeliiwas isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species ofBorrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m².     

Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus ticks removed from humans

A total of 131 Ixodes ricinus (51 females, 1 male and 79 nymphs) removed from persons living in Southern Germany were investigated by immunofluorescence assay for the presence of Borrelia burgdorferi with a polyvalent rabbit immune serum and monoclonal antibodies specific for outer surface proteins (Osp) A or C. Borreliae were detectable in 48 (36.6%) of the ticks. Infection rates of these adults and nymphs were significantly higher than infection rates of unfed ticks from Southern Germany. Borreliae in 31.3% (n = 15) of the infected ticks expressed solely OspA, solely OspC in 12.5% (n = 6), and both OspA and OspC in 39.6% (n = 19) of ticks, while in 16.7% (n = 8) of ticks neither were expressed. Presentation of OspC by B. burgdorferi in I. ricinus was correlated with tick weight: in females, OspC was detectable only in ticks with a minimum weight of about 3.5 mg, and in nymphs weighing at least 1 mg. These results indicate that in I. ricinus removed from humans OspC is up-regulated during the blood meal of the tick, but in most ticks OspA is still detectable and might even be present in the absence of OspC expression in the midgut and salivary glands of nearly fully engorged nymphal ticks. Furthermore, we found strong evidence that borreliae expressing solely OspA while in the salivary glands can cause Lyme borreliosis. Our findings indicate that during tick feeding, humans are exposed to borreliae that may express either OspA or OspC or both, or lack both OspA and C. These findings suggests that, at the minimum, both OspA and C should be considered as vaccine candidates for prophylaxis of Lyme borreliosis in Europe. Received: 28 July 1998     

Time of repletion of subadultIxodes ricinus ticks feeding on diverse hosts

For a comparison of the times of day at which the subadult stages ofIxodes ricinus detach from nocturnal vs diurnal hosts, these ticks were placed on a variety of indigenous and experimental animals. The time of detachment appears to depend more on properties of the host than on the periodicity of tick behavior. Ticks on rodents, regardless of host periodicity, tended to detach late in the afternoon; those on a hedgehog detached around midnight, and those on lizards and birds, during the morning. Ticks on carnivores (dog, cat) detached throughout the daylight hours. The ability of these parasites to survive to the next developmental stage and, ultimately, to come into contact with another suitable host may be influenced by the identity of the host and, hence, by the circumstances of detachment. SubadultI. ricinus probably concentrate in the host's nest when feeding on mammals but are scattered over the ground when feeding on lizard or avian hosts an aspect of engorgement behavior that may profoundly affect the capacity of this tick as a vector of agents of Lyme disease and other infections.     

The subgenusPersicargas (Ixodoidea: Argasidae:Argas). 33. Effect of gamma radiation on first nymphal instarA. (P.) arboreus and adult fertility

Gamma radiation doses higher than 1,000 rads are lethal to first nymphal instarArgas (Persicargas) arboreus. Only 50% of first nymphal instars receiving 1,000 rads reach adulthood but those receiving 100–500 rads survive normally. Males resulting from irradiated first nymphal instars are almost normally fertile. Female germinal cells tolerate higher radiation doses received in the first nymphal instar stage than in the adult stage. Females resulting from first nymphal instars receiving doses higher than 100 rads are less fertile (egg number and percent hatch) than normal and produce F₁ larvae of lower than normal viability. Progenies of females irradiated as nymphs apparently inherit lethal genes, which may be useful, if irradiated at critical stages, in tick control.From Research Project MR041.09.01-0152, Naval Medical Research and Development Command, National Naval Medical Center, Bethesda, Maryland. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Department of the Navy or the naval service at large. This study was assisted by Agreement 03-036-N between the National Institute of Allergy and Infectious Diseases (N.I.H.) and NAMRU-3. (Request reprints from Medical Zoology Department, NAMRU-3, FPO New York 09527)     

Absence of Borrelia spp., Candidatus Neoehrlichia mikurensis, and Anaplasma phagocytophilum in questing adult Dermacentor reticulatus ticks

To determine whether Dermacentor reticulatus ticks are infected by Borrelia spp., Candidatus Neoehrlichia mikurensis, or Anaplasma phagocytophilum, we collected questing adults in the outskirts of Berlin, Germany, examined them for the presence of DNA of these pathogens, and compared the infection rates to those of sympatric Ixodes ricinus ticks. Questing D. reticulatus adults appeared not to harbor the bacterial pathogens that are prevalent in I. ricinus ticks. Based on our sample size, the estimated prevalence of each of these pathogens in D. reticulatus ticks would be well below three tenth of a percent (<0.3 %). For pathogens which so rarely infect D. reticulatus ticks, this tick likely plays no epidemiologic vector role for either their enzootic transmission cycle in nature or their transmission to people.     

Development ofTheileria mutans (Theiler, 1906) in the gut and the haemolymph of the tickAmblyomma variegatum (Fabricius, 1794)

The development ofTheileria mutans in the gut and haemolymph of its tick vector,Amblyomma variegatum, was studied by Giemsa-stained smears after the nymphal tick had completed engorgement on parasitaemic calves. Sexual stages occurred in the gut between five and seven days after repletion. The zygotes were observed in the gut from day 29 and the kinetes from day 30, i.e. three and four days after the nymphs moulted to adults. From day 34 kinetes were observed in the haemolymph. The developmental stages ofT. mutans in the tick were compared with those of otherTheileria spp. of cattle.     

Laboratory-infected Ehrlichia chaffeensis female adult Amblyomma americanum salivary glands reveal differential gene expression

Ticks are efficient ectoparasites that are able to steal blood, a rich source of nutrients, from their vertebrate hosts. The nymphal developmental stage of ticks plays an important role for pathogen transmission to human and other animal hosts. In this article, we describe a bloodmeal-based sex differentiation tool to generate adult female ticks infected with Ehrlichia chaffeensis to investigate vector-pathogen interactions (functional genomics and gene expression studies). We demonstrate that there is a correlation between the uptake of blood during nymph attachment and the molting into male or female adult ticks. The data obtained from the bloodmeal experiments suggest that nymphs that molt into females presumably imbibe more blood than those that become male during the nymphal stage. The natural low E. chaffeensis infection rate in female adult Amblyomma americanum (L.) is a major limiting factor to investigate Ehrlichia-Amblyomma interactions. To generate Ehrlichia-infected female adult ticks, we inoculated obligate E. chaffeensis (Arkansas strain) infected DH82 cells into heavier engorged nymphs (> 12 mg) and allowed them to molt. Freshly molted adults were used to test the E. chaffeensis infection rate. E. chaffeensis genomic DNA was extracted from individual unfed and partially blood fed tick midgut and salivary gland tissues. The tissue samples were tested for the presence of E. chaffeensis using the nested polymerase chain reaction process. Polymerase chain reaction-amplified fragments were detected in unfed and partially fed tissues, demonstrating successful E. chaffeensis infection of tick tissues. This method was used to successfully show differential expression of selected tick genes in E. chaffeensis-infected midguts and salivary glands.     

Repetitive detection by immunoblotting of an integumental 25-kDa antigen inIxodes ricinus and a corresponding 20-kDa antigen inRhipicephalus appendiculatus with sera of pluriinfested mice and rabbits

Mice were pluriinfested with nymphs and rabbits, with adultIxodes ricinus. As determined by immunoblotting, >50% of sera from these animals reacted against a tick antigen with a molecular weight of 25 kDa, which was detected in total extracts of partially fedI. ricinus females and in tick integumental extract. It was also found in engorged nymphs but was absent from larvae. Sera ofI. ricinus-infested rabbits and mice or of rabbits infested withRhipicephalus appendiculatus adults reacted with a 20-kDa antigen in total extracts of partially fedR. appendiculatus females and the integument of this species.     

Coinfection of Ixodes ricinus (Acari: Ixodidae) in northern Poland with the agents of Lyme borreliosis (LB) and human granulocytic ehrlichiosis (HGE)

Adult Ixodes ricinus ticks were collected from Pomerania province, northern Poland, to determine the presence of infection with agents of human granulocytic ehrlichiosis (HGE) and Lyme borreliosis by using the polymerase chain reaction (PCR). Of the 424 ticks 19.2% and 11.6% contained ehrlichiae and spirochetes, respectively. Frequency of single infection with the HGE agent was 63/424 while frequency of single infection with Borrelia burgdorferi sensu lato was 28/424. As many as 21/424 ticks (5%) contained both pathogens. This finding supports suggestions that both HGE agent and B. burgdorferi s. 1. perpetuate in the same foci and frequently co-infect the same tick vector thereby increasing the risk of humans acquiring mixed infection.     

Prevalence of tick‐borne encephalitis virus in Ixodes ricinus ticks from three islands in north‐western Norway

Tick‐borne encephalitis (TBE) is the most important viral tick‐borne disease in Europe and can cause severe disease in humans. In Norway, human cases have been reported only from the southern coast. The aim of this study was to investigate the prevalence of tick‐borne encephalitis virus (TBEV) in questing Ixodes ricinus ticks from the north‐western part of Norway. A total of 4509 ticks were collected by flagging in May and June 2014. A subpopulation of 2220 nymphs and 162 adult ticks were analysed by real‐time PCR and positive samples were confirmed by pyrosequencing. The estimated prevalence of TBEV was 3.08% among adult ticks from Sekken in Møre og Romsdal County and 0.41% among nymphs from both Hitra and Frøya in Sør‐Trøndelag County. This study indicates that TBEV might be more widespread than the distribution of reported human cases suggests.     

Experimental infection of dogs with<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu stricto using<Emphasis Type="Italic"> Ixodes scapularis</Emphasis> ticks artificially infected by capillary feeding

Specific pathogen-free dogs were experimentally infected with Borrelia burgdorferi sensu stricto using nymphal or adult female Ixodes scapularis ticks artificially infected with spirochetes by capillary feeding. The ticks were capillary fed B. burgdorferi isolate 610, previously isolated from a dog with Lyme disease and grown in BSK medium. This isolate induced clinical signs in the dogs similar to those for dogs infested with ticks naturally infected with B. burgdorferi. Adult ticks were more efficient than nymphs in transmitting spirochetes to the dogs. One of five dogs infested with nymphal ticks capillary fed B. burgdorferi was skin biopsy culture and serologically positive, and demonstrated lameness. In contrast, all five dogs infested with adult female ticks that had been capillary fed with B. burgdorferi were culture and serologically positive, with one dog developing lameness. The immunoblot profiles of dogs challenged with female ticks infected by capillary feeding (8 weeks post challenge) were similar to immunoblots (4 weeks post challenge) from dogs challenged with naturally infected females collected in the field. These studies demonstrated that B. burgdorferi cultured in BSK medium can be capillary fed to either nymphal or adult female ticks under laboratory controlled conditions for the purpose of transmitting the spirochete to dogs during the tick's blood meal. This tick infection system would be useful for a controlled and defined challenge of vaccinated and non-vaccinated dogs for proper evaluation of vaccine efficacy, which is difficult to achieve using field-collected ticks. Furthermore, this system may also be useful for investigation of the pathogenesis of Lyme disease, evaluation of the pathogenicity of new isolates of B. burgdorferi, or evaluation of antibiotic therapy.     

Blood donor Borrelia burgdorferi sensu lato seroprevalence and history of tick bites at a northern limit of the vector distribution

In order to study the antibody seroprevalence of the causal agent of Lyme borreliosis, Borrelia burgdorferi sensu lato (s.l.), and the history of tick bites at a geographical distribution limit of Ixodes ricinus, we compared healthy blood donors in geographically extreme regions: the borreliosis‐endemic Vestfold County (59°N) and the region of northern Norway. Blood samples were screened using IgG/VlsE ELISA, and positive/borderline samples were confirmed using C6 ELISA and immunoblot assays. Also, donors completed a questionnaire consisting of several items including the places they have lived, and whether they owned any pets. The seroprevalence was 0.48% (5/1048) in northern Norway and 9.25% (48/519) in Vestfold County. Seven donors (of 1048) had experienced a single tick bite in the southern part of Nordland County (65°N) in northern Norway. This first study on B. burgdorferi s.l. antibody seroprevalence and tick bites on humans and pets in northern Norway showed that the seroprevalence of B. burgdorferi s.l. infection and the risk of tick bite in northern Norway are insignificant; the fact that only five positive IgG samples were detected underscores the very low background seroprevalence. These results suggest that so far I. ricinus has not expanded north of the previously established geographical distribution limit.     

Mitotic activity of the hemocytes in the tick Ixodes ricinus (Acari; Ixodidae)

 The blood cells, or hemocytes, of Ixodes ricinus have been shown to recognize, attack, and phagocytose microorganisms invading the body cavity, or hemocoel, of this tick. Regulated proliferation and differentiation of hemocytes, also referred to as immunocytes, is basic to an effective immune response to invading microorganisms. Therefore, this study dealt with hemopoiesis in I. ricinus, the vector tick of the Lyme disease spirochete Borrelia burgdorferi. Histological evidence for the presence of hemopoietic tissue, a preferential proliferation site of hemocytes, is presented. Mainly the mitotic activity of free-floating hemocytes was examined. By means of microscopical photometry and flow cytometry, all three types of hemocytes in engorging female I. ricinus were found in different stages of the cell cycle. In the engorging tick, up to 40% of the hemocytes counted were in the S phase or the G₂/M phase. From this study we conclude that the differentiated hemocyte types do not differentiate from stem cells in the adult tick. Moreover, microorganisms entering the hemocoel of engorging ticks are confronted with high numbers of hemocytes and, therefore, with an effective cellular immune response. Received: 20 October 1995 / Accepted: 30 January 1996     

Study of Lyme borreliosis in Ukraine

Cases of human Lyme borreliosis (LB) are registered in all administrative regions of Ukraine and in the Autonomous Republic of Crimea with an increasing tendency. The hard tick Ixodes ricinus was proved as the main vector of Borrelia burgdorferi s.l., the causative agents of LB. We identified active natural LB foci, in which almost 25% of the adult I. ricinus ticks contained borrelias and more than 30% of the human population were seropositive for Lyme borreliae. The role of Mus musculus, Microtus arvalis, Myodes glareolus, Apodemus agrarius, and A. sylvaticus in the transmission cycle of the LB agent(s) was revealed. Combined foci and cases of LB/TBE (tick-borne encephalitis) mixed infection were registered in 8 oblasts. A considerable spectrum of different clinical manifestations of LB has been found including erythema migrans in 64.9% of cases, neurologic symptoms in 26.1%, rheumatic affliction in 24.7%, and cardiovascular problems in 8.3%.     

Characterization of Borrelia burgdorferi isolated from different organs of Ixodes ricinus Ticks collected in nature

Borrelia burgdorferi was isolated from 22 out of 133 adult Ixodes ricinus ticks collected from vegetation at two sites in Switzerland. From 17 ticks, spirochetes could be isolated from more than one organ. When the different isolates obtained from one tick were compared by SDS-PAGE analysis, differences in the protein profiles were observed in 8 cases. The isolates were further compared by immunological methods using mono- and polyclonal antibodies. Differences were observed in the proteins of 31–35 kDA and 18–25 kDa. Genetic divergence among isolates was evaluated by use of a B. burgdorferi specific gene probe for ospA. Correlation could be observed between immunological differences in OspA defined by monoclonal antibody LA31 and genetic variation of ospA as judged by restriction fragment length polymorphism (RFLP). Our findings indicate that systemic infection in unfed I. ricinus adults, as reflected by isolation of B. burgdorferi from multiple organs of one tick, is more frequent (8/22, 36%) than previously described (5%). Moreover, the presence of different B. burgdorferi phenotypes/genotypes in one tick is described for the first time. The findings may have bearings (i) on the time of tick attachment required for spirochete transmission since borreliae are already present in the salivary glands of systemically infected ticks at the beginning of the blood meal and (ii) perhaps also on the diversity of B. burgdorferi phenotypes inoculated by these ticks.     

Serological and Molecular Evidence of Human Granulocytic Ehrlichiosis Focus in the Białowieża Primeval Forest (Puszcza Białowieska), Northeastern Poland

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne zoonosis. First described in the USA, it is being increasingly reported from several European countries. This study was undertaken to provide serological and molecular evidence of the occurrence of the HGE focus in the Białowieża Primeval Forest, located in northeastern Poland. To this end, the seroprevalence of HGE in this area, where Lyme borreliosis and tickborne encephalitis are highly endemic, was determined by means of an indirect immunofluorescence antibody assay. In addition, the frequency of granulocytic Ehrlichia spp. infection in Ixodes ricinus ticks from the same area was estimated using a polymerase chain reaction method with EHR 521 and EHR 747 primers, which amplified a fragment of 16S rDNA. The rate of seropositivity for HGE was 6.2% (8/130 subjects). Individuals seropositive for Lyme borreliosis were more likely to have anti-HGE antibodies than seronegative ones (P<0.05; OR=6.34, 95%CI=1.12–36.98). There was no association between self-reported frequency of tick bites or forestry employment and HGE seropositivity. Sixty of 376 (16%) Ixodes ricinus ticks tested were positive for the Ehrlichia phagocytophila genogroup by polymerase chain reaction. Ehrlichial DNA was present in 59 of 302 (19.5%) adult ticks and in 1 of 74 nymphs (1.4%). There was a significantly higher infection rate among female ticks (32.9%; 49/149) than among male ticks (6.5%; 10/153) (P<0.05). Dual infection with Ehrlichia spp. and Borrelia burgdorferi sensu lato was detected in 10 samples that were positive for ehrlichiae. The results obtained confirm the perpetuation of the HGE agent in the primeval forest ecosystem of northeastern Poland. Electronic Publication     

Immunization against ticks: use of salivary gland antigens and infestations with Rhipicephalus appendiculatus (Acari: Ixodidae) in rabbits

Comparative immunogenicity of salivary gland antigens (SGA) derived from adult Rhipicephalus appendiculatus (Neumann) and experimental infestations with the three stages of the tick was investigated. The best immunization schedule judged by reduction of engorgement weights and hatchability resulted from nymphal and adult tick infestations. Inoculation of rabbits with SGA in Freund's incomplete adjuvant (FIA) induced immune responses comparable to those associated with larval infestations, but less than those produced by nymphal or adult tick infestations. High antibody titers directed against SGA were detected only in the vaccinated and adult tick-infested rabbits. The results of this experiment suggest that SGA prepared from partially fed ticks can be used in the experimental induction of immunity to ticks and that the antigen has potential as a vaccine against R. appendiculatus ticks.     

Winter activity of Ixodes ricinus in a Berlin forest

Nymphs and adults of the hard tick Ixodes ricinus typically quest in Central Europe from March/April to October/early November. The recent trend towards milder winters raises the question whether I. ricinus will gradually extend its seasonal activity and use mild winter periods for questing. The present study followed the host-seeking activity of I. ricinus from early September 2006 to early March 2007 in a Berlin forest. Field-collected larval and nymphal ticks were allowed to take a full blood meal in the laboratory and to moult. The resultant unfed nymphs (n=125) and adults (n=149) were released onto three field plots (1×1 m²) in early September 2006. A second group of field-collected unfed I. ricinus nymphs (n=79) and adults (n=47) were released onto a field plot in early September 2006. Each plot was covered by an approximately 10 cm thick layer of leaf litter (mostly of oak and beech) to provide a humid microclimate refuge and shelter for non-active ticks. Wooden rods (60 cm high) arranged in a 6×8 grid on these field plots were climbed by questing nymphs and adults. Active ticks were observed on each of 33 observation dates. The winter activity revealed by this experiment was confirmed by flagging for ticks on two occasions in January and February 2007 with remarkably high numbers of collected ticks. This appears to be the first time that extended or even continuous winter activity of I. ricinus nymphs and adults has been demonstrated in Central Europe, most probably caused by the extremely mild winter of 2006/2007. The fact that I. ricinus now can be active during the whole winter, a time of the year when these ticks historically have been dormant when the weather is normal, is relevant to the public because people who enter forest areas should be well aware of the risk of getting tick bites and tick-borne infections in mild winter periods.     

Identification of galactose‐α‐1,3‐galactose in the gastrointestinal tract of the tick Ixodes ricinus; possible relationship with red meat allergy

Patients with IgE antibodies against the carbohydrate epitope galactose‐α‐1,3‐galactose (α‐Gal) have reported severe allergic reactions after consumption of red meat. Investigations have revealed associations between IgE to α‐Gal and tick bites. We provide the first direct evidence that α‐Gal is present within ticks thus potentially explaining the relationship between tick exposure and sensitization to α‐Gal, with development of red meat allergy as a secondary phenomena. Serum from Swedish patients with delayed severe reactions to red meat was included in the study. A dose‐dependent inhibition of IgE responses to α‐Gal by the tick Ixodes ricinus is demonstrated. Furthermore, using cryostat‐cut sections of I. ricinus, we show that both a monoclonal and a polyclonal antibody against α‐Gal stains the gastrointestinal tract of the tick. The same pattern is seen when staining with patient sera IgE positive to α‐Gal. These results confirm that the α‐Gal epitope is present in I. ricinus and imply host exposure to α‐Gal during a tick bite. This provides further evidence that tick bites are associated with IgE responses to α‐Gal and red meat allergy.