SALP16, a gene induced in Ixodes scapularis salivary glands during tick feeding

Guinea pigs infested with Ixodes scapularis acquire antibody-mediated resistance to tick bites, a phenomenon known as tick-immunity. An I. scapularis salivary gland cDNA expression library was therefore probed with sera from tick-immune guinea pigs to identify antigens that elicit humoral responses in the host. Sera from sensitized guinea pigs strongly recognized 3 of 4,500 library clones in an initial screening. The open reading frames of all 3 clones encoded a putative 16.4-kD acidic protein, designated Salp16, with an N-terminal signal sequence and signal peptidase cleavage sites specific for secretory proteins. The salp16 mRNA and Salp16 protein were detected in the salivary glands of engorged, but not unfed, nymphal and adult ticks, and Salp16 was also found in the saliva of engorged ticks. Immunization with recombinant Salp16 induced high antibody titers in guinea pigs, but did not elicit tick-immunity. Salp16 is the first feeding inducible gene that has been cloned from L. scapularis. Molecular characterization of I. scapularis salivary antigens that are induced upon tick feeding should help to facilitate our understanding of tick-host interactions.     

Ixodes ricinus tick salivary gland extract inhibits IL-10 secretion and CD69 expression by mitogen-stimulated murine splenocytes and induces hyporesponsiveness in B lymphocytes

Tick saliva contains immunosuppressive factors allowing this blood-feeding ectoparasite to remain on hosts and enhancing pathogen transmission. In this study, we examined the modulation of mitogen-induced activation of naive murine splenocytes by the saliva and salivary gland extract (SGE) of I. ricinus ticks. We found that saliva-specific factors reduced IL-10 production by both concanavalin A (ConA) and lipopolysaccharide (LPS) stimulated splenocytes. The LPS-induced IL-10 production is 10 times more sensitive to SGE than the ConA-induced IL-10 production. Flow cytometric analysis determined that SGE particularly inhibited B (B220+) cell IL-10 production in mitogen-stimulated splenocyte preparations. Moreover, SGE reduced the early activation marker CD69 expression on ConA-activated T cells and also on B cells in presence of ConA or LPS. Annexin V and Via-probe staining demonstrated that SGE did not increase cell death in activated splenocytes and slightly decreased apoptosis in B lymphocytes. By employing assays with isolated B cells, we further showed that SGE had a direct effect on B cells and inhibited LPS-induced B cell proliferation. Taken together, our results indicate that salivary immunomodulators induce hyporesponsiveness to mitogen in both T and B cells, and that a direct B-cell inhibitory activity is present in tick saliva.     

Anaplasma phagocytophilum groEL gene heterogeneity in Ixodes ricinus larvae feeding on roe deer in Northeastern Italy

Anaplasma phagocytophilum is an emerging tick-borne pathogen with both veterinary and human health implications. The role of wildlife hosts for this pathogen are not well defined, even thought roe deer (Capreolus capreolus) has been suggested to contribute to the occurrence of this tick-borne diseases in Europe. Therefore the aim of the present study was to investigate the potential role of this ungulate species as a reservoir of human pathogenic strains of A. phagocytophilum in a tick-borne diseases endemic area in Northeastern Italy. Ixodes ricinus feeding on roe deer were collected and analyzed for the presence for A. phagocytophilum by a molecular approach targeting 16S rRNA and groEL genes. The mean prevalence of A. phagocytophilum recorded was 5.11%, highlighting the ability of roe deer to infect the I. ricinus larval stage. The results of further genetic characterization of the strains of A. phagocytophilum herein isolated, based on phylogenetic information contained in groEL gene sequences, showed substantial heterogeneity among sequences analyzed. Nevertheless, these findings suggest that the roe deer population of the Trentino region of Italy harbors strains of A. phagocytophilum of unknown pathogenicity for humans.     

Isolation of Borrelia burgdorferi sensu lato in ticks Ixodes ricinus by polymerase chain reaction (PCR)

Attempts were made to identify the causative orgamsm of Lyme disease in Szczecin from tick Ixodes ricinus as a vector. Ticks were collected in 1997 year in forest areas of Szczecin, from localites associated with numerous attendance of people. The method used in this study was the polymerase chain reaction (PCR) on the flagellin structural gene fla of Borrelia burgdorferi sensu stricto. The flagellin PCR primer set reaction was conservative for B. burgdorferi sensu stricto, B. afzelii and B. garinii. The overall prevalence of B. burgdorferi sensu lato, in tick population studied was 8.8%. The female, nymphs and larves of Ixodes ricinus were infected almost just the some--about 10%, when the male 2.5% only.     

Salivary gland extract from Ixodes ricinus ticks inhibits production of interferon-γ by the upregulation of interleukin-10

Tick saliva has been shown to modulate host immunity by a so far unknown mechanism. We have demonstrated an inverted effect of salivary gland extract (SGE), derived from partially fed Ixodes ricinus females, on the production of two cytokines, interferon (IFN)-gamma and interleukin (IL)-10, in vitro. While SGE markedly suppressed the elaboration of IFN-gamma by mouse splenocytes stimulated with lipopolysaccharide (LPS), the production of IL-10 was increased in comparison with SGE-untreated cultures. The suppressive effect of SGE could be abolished by the addition of an IL-10 neutralizing monoclonal antibody to splenocyte cultures. Similar results were obtained when live Borrelia afzelii spirochetes, which are transmitted in Europe by I. ricinus ticks, were used for the cytokine induction. These results suggest that tick saliva can upregulate the IL-10 production at the tick feeding site, which consecutively inhibits the elaboration of pro-inflammatory cytokines, for example IFN-gamma. This immunosuppression may facilitate the establishment of tick-transmitted pathogens in the host.     

Antigenic profile of Ixodes ricinus: effect of developmental stage, feeding time and the response of different host species

Antigens recognized by host species in response to ectoparasite infestation have been widely reported. Although differences in the immune responses of different host species have been described, only a very few of these studies compare the range of antigens recognized by different host species in response to infestation. We used Western blot analysis to investigate antigenic responses of different host species that were repeatedly infested with Ixodes ricinus ticks. Antigenic profiles of larval and nymphal whole tick homogenates were compared with the respective salivary gland extract (SGE) samples using sera from rabbits repeatedly infested with either adults, nymphs or larvae. SGE samples were also analysed using sera from hamsters infested with adults, nymphs or larvae. Sera from BALB/C mice, Apodemus flavicollis (yellow-necked mouse) or Clethrionomys glareolus (bank vole) repeatedly infested with larvae were used to compare the antigenic profiles of SGE and larval homogenate samples. We also investigated different sources of tick antigens, using rabbit sera, by comparing midgut extracts from female adult ticks and SGE from unfed ticks and from ticks throughout the 6-day feeding period with whole tick homogenates of female and male adults, nymphs and larvae. The pattern of antigenic tick-molecules recognized by infested host species varies with the period of feeding, developmental stage and the particular host species parasitized.     

Progressive sensitization of circulating basophils against Ixodes ricinus L. antigens during repeated infestations of rabbits

The sensitivity of rabbit basophils to antigens from Ixodes ricinus females has been studied by a degranulation test. Observations of basophil numbers and degranulation were made on the 6th day of each of four sequential infestations. Maximal degranulation of cells was observed after challenge of cells with antigen at a concentration of 10⁶ and 10⁷ pg/ml. At these concentrations, during a 1st infestation, 21.8 and 23.6% of cells degranulated. During a 2nd infestation, these percentages increased (34.8 and 33.8%) and reached 59.8 and 63.8% by the 4th infestation. A plasma factor which partially blocks basophil degranulation, is described. This was already present during the 1st infestation, since in its presence the percentage of degranulation was reduced by 2.8 and 4.0% respectively on challenge with 10⁶ and 10⁷ pg antigen/ml. Inhibition was maximal at the 4th infestation (difference: 16.5 and 20.5%). Basophil sensitization and inhibition of the degranulation are thus both progressive phenomena. After 10–15 infestations on four other rabbits, 75. 0 and 79. 8% degranulation was obtained. The inhibition of degranulation by plasma was also greater (difference: 25. 5 and 27. 4%). IgG specific anti- I. ricinus antibodies were identified by indirect immunofluorescence. In two animals, they were detected at the 6th day of the 1st infestation. Subsequently, they were generally present for all the animals.     

Absence of Lyme disease spirochetes in larval Ixodes ricinus ticks

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

First detection of Rickettsia helvetica in Ixodes ricinus ticks in Austria

A total of 853 Ixodes ricinus ticks collected from the nine federal states of Austria were examined by molecular methods for possible infections with Rickettsia spp. It was shown that roughly one-third of the ticks were infected with Rickettsia spp. Moreover, Rickettsia helvetica was detected in Austria for the first time.     

Ixodes ricinus spirochete infection as the cause of postinfectious arthritis in Sweden

Antibodies to Swedish Ixodes ricinus spirochete were determined by an enzyme-linked immunosorbent assay (ELISA) in sera from 298 patients with postinfectious arthritis. Sera from healthy individuals, patients with acute infectious meningitis of proven etiology and patients with multiple sclerosis served as controls. With the upper limit of normal values set at the 95 percentile of controls, 18 of 298 (6%) arthritis patients had positive serum antibody titres. Titres above the 100 percentile of controls were found in 5 of 298 (2%) arthritis patients. Two of the arthritis patients had extremely high titres--higher than any earlier found in Swedish patients with spirochetal meningitis. The clinical manifestations and laboratory findings in the 5 patients with high spirochetal antibody titres are described. It is concluded that a spirochetal etiology should be considered in patients with reactive or postinfectious arthritis of unknown origin.     

First isolation of Borrelia lusitaniae DNA from Ixodes ricinus ticks in Poland

European genospecies of B. burgdorferi sensu lato were identified by examining unfed I. ricinus ticks collected from 10 locations in northwest Poland. Research was conducted using 3 methods: PCR amplification of the fla gene with the FLA1 and FLA2 primer set conserved in all European species of B. burgdorferi sensu lato, PCR-RFLP, and sequencing. There were 5 restriction patterns obtained in this study: 4 characteristic for genospecies B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. afzelii and 1 untypical restriction pattern type. PCR products for all restriction patterns were sequenced. Polish sequences of the fla gene for B. burgdorferi s.s., B. garinii, B. valaisiana, and B. afzelii were identical with sequences from GeneBank at 99.79%, 99.58%, 100% and 100% respectively. The fifth sequence demonstrated 99.79% identity with sequences of a B. lusitaniae PotiB2 isolate from Portugal, and also clusters with this strain. B. lusitaniae DNA in I. ricinus was detected in 3 out of 10 localities and constituted 5.9% of infected individuals with I. ricinus.     

Differentiation of medically important Euro-Asian tick species Ixodes ricinus, Ixodes persulcatus, Ixodes hexagonus, and Dermacentor reticulatus by polymerase chain reaction

Understanding epidemiology of the tick-borne pathogens requires the accurate identification of the vector ticks. Morphological analysis of ticks is difficult and often leads to misidentification. Molecular techniques offer an alternative approach of tick identification. To date, no practical and reliable molecular assays for discrimination of Euro-Asian ticks are available. Our aim was to develop such an assay for discrimination between four Euro-Asian tick species of high medical importance such as Ixodes ricinus, Ixodes persulcatus, Ixodes hexagonus, and Dermacentor reticulatus. As a basis, we have chosen conventional species-specific polymerase chain reaction (PCR), a technique providing a good combination of simplicity and reliability. The DNA information available on ticks was searched for orthologous loci containing stretches of sequence dissimilarity sufficient for designing species-specific primers. ITS2 locus (second internal transcribed region of the rRNA gene cluster) was found to be the most favorable for primer design. Finally, for each of the three Ixodes species a PCR was developed amplifying only for the targeted species. One PCR amplified the entire ITS2 locus of the four species and allowed discrimination of D. reticulatus from the Ixodes species on the basis of the size difference of the respective PCR products. This PCR system was successfully tested for discrimination of the ticks at different maturation stages (larva, nymph, and adult) in engorged and unfed conditions, and therefore it may be useful for large-scale epidemiological studies. Differentiation between the closely related I. ricinus and I. persulcatus, the two species most often occurring in the tick-borne diseases in Eurasia, is of special importance.