Les tiques sont des arthropodes ectoparasites obligatoires qui se nourrissent sur une grande variété de vertébrés sur une large partie du globe. Au cours de leur repas, les tiques sécrètent dans leur salive de nombreux facteurs leur permettant de contourner bon nombre des défenses de l’hôte. Bien que la littérature rapporte beaucoup d’informations au sujet des effets du repas de la tique sur l’hôte, la nature des facteurs actifs exprimés par les glandes salivaires de la tique est peu connue. Au cours d’anciens travaux au sein du laboratoire, le crible de deux banques d’ADN complémentaires - issues de la rétro-transcription des ARN messagers synthétisés par les glandes salivaires de la tique Ixodes ricinus – a permis l’identification de 27 protéines dont l'expression est spécifiquement induite ou régulée positivement pendant le repas sanguin de la tique I. ricinus. Parmi ces protéines, la protéine Seq24, induite au cours du repas sanguin, présente la capacité de moduler les immunités innée et acquise de l’hôte. En conséquence, la protéine Seq24 a été nommée Iris pour « Ixodes ricinus Immunosuppressor ». Au cours de la présente étude, notre but fût de caractériser le rôle d’Iris et de déterminer son importance dans le repas sanguin de la tique I. ricinus. La protéine Iris appartient à la famille des inhibiteurs de sérine protéases et présente une homologie significative avec l’inhibiteur d’élastase de leucocytes. Une analyse in silico a confirmé qu’Iris présentait la structure des serpines, et notamment le RCL (Reactive Center Loop), boucle responsable de l’activité anti-protéasique. Comme attendu (sur base de l’analyse in silico), Iris inhibe de manière spécifique l’activité de plusieurs sérine protéases, et en particulier l’élastase de leucocyte. Ces tests effectués, nous avons essayé de comprendre quel(s) pouvai(en)t être le(s) rôle(s) d’Iris dans l’accomplissement du repas sanguin de la tique, c’est à dire dans la lutte contre les différents systèmes de défenses de l’hôte. Tout d’abord, des tests ont démontré la capacité d’Iris à inhiber les mécanismes de l’hémostase. Des tests sur du plasma et du sang complet ont montré qu’Iris allonge le temps de fibrinolyse, la voie intrinsèque de la coagulation et l’adhésion plaquettaire. L’utilisation de mutants a également démontré que si les deux premières activités sont dépendantes du RCL, et donc d’un mode de fonctionnement anti-protéolytique, l’adhésion plaquettaire est indépendante de ce système. Ce résultat met en évidence l’existence d’autres sites actifs, isolés par analyse in silico, nommés Receptor Binding Domain (RBD). Un travail antérieur du laboratoire avait permis d’indiquer la capacité de la protéine recombinante Iris semi-purifiée à inhiber la production de TNF-a, d’IL-6, et d’IL-8 (cytokines pro-inflammatoires) ainsi que l’IFN-g par des PBMCs (Peripherical Blood Mononuclear Cells) humaines. Ces résultats ont été confirmés avec de la protéine purifiée. Des analyses complémentaires ont démontré qu’un mutant d’Iris - dépourvu d’activité anti-protéasique - conserve l’activité pro-inflammatoire. Là encore, ce mécanisme semble impliquer un ou plusieurs RBD. L’utilisation d’anticorps dirigés contre ces zones a permis de déterminer le domaine d’interaction (aa : 105-120) impliqué dans cette fonction. D’autre part, une analyse par FACS a permis de démontrer qu’Iris interagit uniquement avec les cellules d’origine monocytaire. Enfin, nous avons également analysé l’importance d’Iris au cours du repas sanguin de la tique par une approche vaccinale. Les résultats observés indiquent que 30 % des tiques nourries sur des lapins immunisés par la protéine rIris ne survivent pas au repas.

In Indiana, Ixodes scapularis is an important tick in public health because it feeds on a variety of hosts including humans, and transmits Borrelia burgdorferi (Lyme disease), Anaplasma phagocytophilum (human granulocytic ehrlichiosis), and Babesia microti (babesiosis). Symbiotic, non-pathogenic Rickettsia found in Ixodes scapularis may play a role in excluding pathogenic species of Rickettsia from being transovarially transmitted. In order to investigate this idea further in Indiana, a total of 378 adult I. scapularis from 4 different counties (Jasper, Pulaski, Newton and Starke) were tested by polymerase chain reaction analysis (PCR) for the presence of Rickettsia sp. Four positive samples from the PCR (using Rocky Mountain spotted fever group specific primers to target the rOmpA gene; Rr190.70p and RH 90.602n) reactions were sequenced to verify identity. These four samples matched closest to the reference number AB002268 from GenBank which describes, I. scapularis endosymbiont DNA for rOmpA. A total of 62 engorged females were tested; 53 (85.5%) harbored the rickettsial symbiont. A total of 41 questing females were tested; 33 (80.5%) were positive. Of the 249 males tested, 14 (5.6%) were positive. A restriction digestion on some of the positive samples revealed that the 1 scapularis symbiont was different from R. montana and R. rickettsii. The second goal of this study was to identify the presence of B. microti. In I. scapularis ticks, this would be the first time this pathogen was identified in Indiana. To accomplish this goal 106, ticks were tested using the primers Babl and Bab4, which target the 18S rRNA gene specific for B. microti. Three tick samples were found to harbor B. microti as determined by sequencing. However, sequencing of amplification band in the negative control also yielded B. microti. Thus, the presence of B. microti in Indiana ticks could not be confirmed. A negative control was also sequenced and was identified as Babesia microti indicating that there was a contamination so it is not possible to conclude that B. microti was found in Indiana ticks. / Department of Physiology and Health Science

Lyme borreliosis (LB) is the most frequent vectorborne disease in the Northern Hemisphere. It is a complex bacterial zoonosis involving vertebrate hosts and hard ticks of the genus Ixodes. The causative agents, bacteria of the LB group of spirochaetes, form a species complex comprising 17 named species. As is the case for most microbial pathogens, epidemiological and ecological studies require appropriate genotyping. Although the use of single loci may provide rapid results, there are serious disadvantages, in particular when inferring evolutionary relationships or geographic population structure. A novel multilocus sequence analysis (MLSA) system of the LB group spirochaetes has been developed based on housekeeping genes to overcome these problems. Here, the system is optimized and tested using extracted spirochaetal DNA directly from ticks and then utilized to obtain insights into the migration and spread of individual species as well as to investigate the evolutionary origins of the species complex. Species belonging to the LB group of spirochetes display different patterns and levels of host specialisation which makes this an ideal system to study the impact of host associations on spread of zoonotic tickborne diseases. For example, Borrelia valaisiana and B. garinii are transmitted exclusively by birds while B. afzelii is transmitted by rodents. I demonstrate that the migration of the LB species is dependent on, and limited by, the migration of their respective hosts. I also show the presence of B. afzelii strains in England and, through the use of the MLSA scheme, demonstrate that the strains are highly structured. A close evolutionary relationship between B. afzelii and its rodent host species is shown. Furthermore, through phylogenetic analyses, some evidence of a coevolutionary relationship between the LB group species and their major group of vector species, the Ixodes persulcatus species complex, is presented and a Eurasian origin for the species group is suggested.

The Australian paralysis tick, Ixodes holocyclus causes a potentially fatal paralysis in domestic animals, livestock and humans with companion animals (mainly dogs) most commonly affected. Current treatment regimes include administration of a commercial tick anti-serum (TAS), prepared as hyperimmune serum in dogs, to neutralise the effects of the toxin. However, each new batch must be standardised using an expensive and highly subjective bioassay performed in neonatal mice. There is currently an urgent need for a more cost effective and rapid in vitro assay that can be more objectively and accurately quantified. Further understanding of the composition of the toxin molecule is also required to develop toxin-specific reagents necessary for these assays. One of the main objectives of this study was to develop a suitable immunoassay to replace the existing mouse bioassay for assessing batches of tick anti-sera for use in tick paralysis therapy in dogs. Initially an enzyme-linked immunosorbent assay (ELISA) was established to detect and quantify antibody specific for I. holocyclus toxin in dog sera. Using a partially purified antigen extracted from I. holocyclus salivary glands, good discrimination was achieved between reactive (hyperimmune) and non-reactive (naïve) sera. The hyperimmune dog sera reacted very strongly with the antigen compared to negligible reactions of serum from dogs not exposed to I. holocyclus. The reactions of hyperimmune sera were also significantly weaker to a non-toxin antigen control extracted from the salivary glands of the non-toxic tick Rhipicephalus microplus, indicating the assay was detecting toxin-specific responses. Furthermore, each of the hyperimmune sera that reacted strongly and specifically with the I. holocyclus antigen in the ELISA also strongly neutralised toxin in the mouse bioassay. Together these findings support the suitability of this ELISA for assessing the potency of batches of commercial dog hyperimmune sera for use as therapy for tick paralysis in dogs. Sera from dogs that were experimentally infested with ticks and sera from patient dogs, presenting at veterinary clinics with signs of tick paralysis, were also screened for antibodies to I. holocyclus antigen using the ELISA. Twenty-eight out of 29 sera from animals with single or multiple exposures to ticks failed to recognise the I. holocyclus antigen indicating the ELISA is not suitable as a diagnostic test to detect toxin-specific antibodies in animals with limited exposure to I. holocyclus infestation. A panel of toxin-specific monoclonal antibodies (mAbs) was produced as research tools to analyse and purify tick toxin components. Rats were successfully immunised against tick toxin using a combination of inoculation of partially purified salivary gland antigen and exposure to tick infestation. The latter approach preserved the native confirmation of the toxin using a natural route of immunisation and rats were chosen due to their high tolerance of multiple tick infestations over several days. While fusion of rat spleen cells with mouse myeloma cells has been reported several times in the literature, the resulting hybridomas are unstable with fastidious culture requirements. Optimisation of the culture conditions revealed that most rat-mouse hybridoma lines grew best in serum-free medium supplemented with 5% foetal bovine serum. Of 600 hybridomas produced, only 12 were shown to be specific for the Ixodes antigen, as determined by ELISA. A selection of these hybridomas representing various patterns of affinity and/or antigen specificity were further analysed for toxin-neutralising ability in a mouse bioassay. Notably, the most potent toxin-neutralising mAb in mice, showed a specific but relatively moderate reaction to Ixodes antigen in the ELISA. The most potent toxin-neutralising mAbs inactivated toxin as strongly as the commercial TAS used for immunotherapy in dogs with tick paralysis. This suggests that mAbs may present an alternative source of immunotherapy, providing a potentially endless supply of a highly consistent reagent and negating the need to use live animals for both the production of tick antiserum and the continual testing of reagent batches. The toxin-neutralising mAbs were also used to analyse I. holocyclus toxin in polyacrylamide gel electrophoresis (PAGE) and Western blot to identify specific toxin proteins. The most potent neutralising mAbs consistently recognised high MW proteins (100-200 kDa) in a smeared pattern. Although this was contrary to previous reports of low molecular weight components (3-5 kDa) in holocyclotoxin, this study was the first to use mAbs prepared to native toxin. The large molecular weight structures likely represent presucursors to, or complexes of the smaller peptides, previously identified. When the Toxin-neutralising mAbs were assessed as ligands to affinity purify toxin components from crude Ixodes SG extracts, toxin components of 110 and 32 kDa were consistently identified. These purified proteins represent good candidates for N-terminal sequencing to further identify the toxin components in I.holocyclus salivary glands.

<p>This thesis examines seasonal, diel and vertical distribution patterns of activity of host-seeking <i>Ixodes ricinus</i> (L.) ticks at three localities in south-central Sweden. In addition, by examining the prevalence of infection in ticks with Lyme borreliosis (LB) spirochetes, <i>Borrelia burgdorferi</i> s.l, information for estimating relative LB risk in humans and the effect of control measures directed against this tick vector is provided.</p><p>The seasonal activity pattern of <i>I. ricinus</i> was, in general, bimodal with peaks of activity in May-June and August-September. Tick densities were generally high at Torö and low at Kungshamn-Morga. The greatest variation in tick density occurred at the sample site level, which indicated a patchy distribution of ticks. The diel activity of adult <i>I. ricinus</i> sampled at Bogesund showed a distinct nocturnal activity peak while nymphal ticks exhibited no particular diel variation. At the meadow site, there was a strong negative association between activity of each tick stage and ambient air temperature, and larval ticks also showed a nocturnal activity peak. <i>I. ricinus</i> of all stages were present in the vegetation up to at least 140 cm above ground level. At Torö, host-seeking larvae were found at significantly lower levels (below 20 cm) in the vegetation compared to nymphs and adults (50-59 and 60-79 cm, respectively). Vegetation structure is likely to be the main factor governing tick vertical distribution at this locality. The northern limit of the geographical distribution of <i>I. ricinus</i> in Sweden corresponds with the southern boundary of the taiga zone, as well as with several other climatic or vegetational isoclines primarily associated with the vegetation period.</p><p>The prevalence rates of <i>Borrelia</i> spirochetes, recorded by phase-contrast microscopy in host-seeking <i>I. ricinus</i>, were 0% in larvae, 5.8-13.1% in nymphs and 14.5-28.6% in adult ticks. The human LB risk, estimated by the number of <i>Borrelia</i>-infected nymphs per hectare, was greater at Torö than at Kungshamn-Morga and greater in woodland than in open areas. The risk also possessed a bimodal seasonal pattern similar to that of subadult host-seeking activity. Controlling the number of infected nymphs through de-ticking of reservoir hosts seems not to be an effective control measure in Sweden due to the ubiquitous availability of alternative reservoir hosts.</p>

This thesis examines seasonal, diel and vertical distribution patterns of activity of host-seeking Ixodes ricinus (L.) ticks at three localities in south-central Sweden. In addition, by examining the prevalence of infection in ticks with Lyme borreliosis (LB) spirochetes, Borrelia burgdorferi s.l, information for estimating relative LB risk in humans and the effect of control measures directed against this tick vector is provided. The seasonal activity pattern of I. ricinus was, in general, bimodal with peaks of activity in May-June and August-September. Tick densities were generally high at Torö and low at Kungshamn-Morga. The greatest variation in tick density occurred at the sample site level, which indicated a patchy distribution of ticks. The diel activity of adult I. ricinus sampled at Bogesund showed a distinct nocturnal activity peak while nymphal ticks exhibited no particular diel variation. At the meadow site, there was a strong negative association between activity of each tick stage and ambient air temperature, and larval ticks also showed a nocturnal activity peak. I. ricinus of all stages were present in the vegetation up to at least 140 cm above ground level. At Torö, host-seeking larvae were found at significantly lower levels (below 20 cm) in the vegetation compared to nymphs and adults (50-59 and 60-79 cm, respectively). Vegetation structure is likely to be the main factor governing tick vertical distribution at this locality. The northern limit of the geographical distribution of I. ricinus in Sweden corresponds with the southern boundary of the taiga zone, as well as with several other climatic or vegetational isoclines primarily associated with the vegetation period. The prevalence rates of Borrelia spirochetes, recorded by phase-contrast microscopy in host-seeking I. ricinus, were 0% in larvae, 5.8-13.1% in nymphs and 14.5-28.6% in adult ticks. The human LB risk, estimated by the number of Borrelia-infected nymphs per hectare, was greater at Torö than at Kungshamn-Morga and greater in woodland than in open areas. The risk also possessed a bimodal seasonal pattern similar to that of subadult host-seeking activity. Controlling the number of infected nymphs through de-ticking of reservoir hosts seems not to be an effective control measure in Sweden due to the ubiquitous availability of alternative reservoir hosts.

Medicinsk entomologi

ABSTRACT The epidemiological study of intestinal helminths and ticks of Siberian weasel (Mustela sibirica) was conducted from June 2001 to May 2002 in Shan-Ping, southern Taiwan. Nine helminths were found, including seven nematodes, Filaroides sp. (prevalence 94.4%), Ancylostoma sp. (77.4%), Uncinaria sp. (35.5%), Trichuris sp. 1 (35.5%), Trichuris sp. 2 (19.3%), Capillaria sp. (6.5%) and Physaloptera sp. (3.2%); one trematode, Platynosomum sp. (74.1%) and one acanthocephalans, Macracanthorhynchus sp. (10%). There was a significant seasonal difference of intensity of the Platynosomum sp. Two species of ticks were observed, Ixodes ovatus (prevalence 55.56%) and Haemaphysalis sp. Seasonal differences were significant in the prevalence and intensity of the Ixodes ovatus infection. The intensities of ticks and helminths in different sampling area were similar. The diet composition was possibly the factor affecting the seasonal change of intensity of the Platynosomum sp. and host availability affecting the seasonal change of ticks, Ixodes ovatus.

The objectives of this study were to determine the distribution of Borrelia burgdorferi (Bb), the bacterium that causes Lyme disease, and its tick vector (Ixodes scapularis) in the Thousand Islands in Ontario, and to assess the effects of various factors, including host populations, temperature, and vegetation, on the distribution of Bb and ticks in this new endemic region. Data were collected via drag sampling and small mammal trapping at 12 sites and multimodel inference was used to evaluate variables. There was inter-island variation in the abundance of ticks and prevalence of Bb in ticks. Important predictors of tick numbers and infection prevalence included deer abundance, distance to the United States, temperature, species richness, and relative mouse abundance. These results will contribute to management strategies to reduce Lyme disease risk in the Thousand Islands and to our understanding of the effects of biodiversity on disease risk. / Canadian Cooperative Wildlife Health Centre; Ontario Ministry of Natural Resources; Parks Canada; Public Health Agency of Canada; Ontario Graduate Scholarship

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science