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The rickettsial pathogen Ehrlichia ruminantium, transmitted by ticks of the genus Amblyomma, causes heartwater, an economically important, often fatal disease of domestic and wild ruminants in sub-Saharan Africa and in the Caribbean. The studies described in this thesis have contributed to understanding several aspects of heartwater. First, a real-time PCR method was developed in order to study the kinetics of infection with E. ruminantium in the mammalian host. The assay was validated for specificity and sensitivity and was used to estimate numbers of the organisms in the blood of infected sheep. However, organisms were only detected during the clinical phase of infection, indicating that the way in which it was applied did not provide sufficient sensitivity to follow the early stages of infection. This PCR assay was then used, together with transcription and proteomic analyses, to investigate differential gene expression of E. ruminantium in the arthropod and mammalian hosts, in order to identify genes that may allow the organisms to successfully adapt to different environments. These studies used in vitro tick and mammalian cell culture systems, as well as tissues from infected A. variegatum ticks, and initially focused on the map1 multigene family. Although transcripts for most of the map1 paralogs were detected in organisms grown in vitro, in both mammalian and tick cells, only transcripts from map1 and map1-1 were detected in infected ticks. Moreover, map1-1 transcripts were more abundant in midguts than in salivary glands whereas map1 transcripts were most abundant in salivary glands and were expressed at higher levels following several days of tick feeding on a mammalian host. Because of the quantities of material required, proteomic analysis was only possible using in vitro-cultured organisms. Comparison of proteins encoded by the map1 cluster in E. ruminantium grown in tick or bovine endothelial cell cultures, using 2D gels and MALDI-TOF analysis, revealed that different proteins predominated in the corresponding spots in 2D gels from the different cultures; products of the map1-1 gene were abundant in tick cells, while products of map1 were abundant in endothelial cells. The detection of higher levels of map1 transcripts in salivary glands than in midguts of infected ticks, together with the presence of abundant MAP1 protein in organisms grown in mammalian but not in tick cell lines, suggest that expression of this protein may be associated with infectivity for mammalian cells. In contrast, map1-1 transcripts were abundant both in midguts of infected ticks and in tick cell lines, and the protein was expressed at high levels in infected tick cell cultures. Since both of these stages have low infectivity for sheep, these results suggest that the MAP1-1 protein may play an important role within the vector, possibly associated with colonisation and replication of E. ruminantium in the tick midgut. Collectively these findings suggest that this multigene family is involved in functions of biological relevance in different stages of the life cycle of E. ruminantium. Lastly the suppression subtractive hybridisation (SSH) technique was applied to RNA extracted from E. ruminantium-infected endothelial and tick cell cultures in an attempt to sample a large portion of the E. ruminantium genome for differentially expressed genes; although not resulting in identification of any differentially transcribed genes in the present study, this method was shown to work in principle.

Ticks are obligate haematophagous arthropods that represent a major economic drain upon the world's livestock as well being a significant medical and veterinary risk through the transmission of tick-borne pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. The tick-host relationship is a function of both ecological and physiological factors. Successful feeding requires the effective acquisition and digestion of a bloodmeal by the tick. Acquisition relies upon the ability of the tick to counteract host immune responses induced by the extended feeding periods of ixodid ticks (up to 2 weeks). The host response to tick infestation and the consequent countermeasures employed by the tick, constitute the tick-host interface. The immune response of hosts to Ixodes ricinus infestations was examined through antigenic profiling. The antigens exposed to the host were shown to vary throughout the feeding period and differed between the different development stages of I. ricinus. It was also shown that different host species infested with I. ricinus recognised different antigens. This was true of both natural and non-natural hosts, and even closely related species. Anti-complement activity was investigated in the salivary glands of Ixodes ticks. This activity was shown to inhibit some host species but not others. The pattern of inhibitory activity varied between the tick species tested in a way that was consistent with known tick host-preferences. The mechanisms of anti-complement activity in I. ricinus salivary glands were explored. The alternative but not the classical pathway of complement was inhibited. Activity was present in unfed ticks and throughout the feeding period. Three targets of the complement system were identified as being modulated by the tick. Digestion of the bloodmeal was explored and a haemolytic activity was associated with the salivary glands of I. ricinus ticks. The activity was demonstrated to be Mg²⁺- dependent. In addition, a subtractive cDNA library enriched for saliva-associated transcripts was successfully produced. Random sampling identified putative differentially expressed genes. The results of this thesis illustrate the complexity of tick-host interactions at the molecular level. It is apparent that the research described poses many more questions than answers.

Ixodes ricinus is the most important human-biting tick in Europe and the principal vector of Lyme borreliosis. In addition, this hard tick species transmits a large number of microbial pathogens that are of importance to animal and human health. Little is known about the diversity and genetic population structure of I. ricinus across Europe. Genetic diversity of these tick populations may have implications on disease transmission. I. ricinus primers were designed for a number of mitochondrial genes and a Multilocus Sequence Typing-like Scheme (MLST) was devised. This was termed mitochondrial MLST (mtMLST). MLST has so far mainly been used for typing microbes, and the development of a MLST scheme for an arthropod vector is novel. Understanding the geographic structure of I. ricinus populations, in combination with studies regarding the migration of tick-borne microbial infections, e.g. Lyme borreliosis, is likely to illuminate important processes in the evolution and spread of tick-borne diseases.

Resumo: O presente estudo apresenta a descrição da morfologia externa de larvas e a redescrição de ninfas do carrapato Amblyomma brasiliense, utilizando microscopia de luz, lupa estereoscópica e microscopia eletrônica de varredura. As larvas apresentaram a base do capítulo retangular, palpos curtos e idiossoma arredondado, coxa I com dois espinhos, sendo o externo mais longo que o interno e coxas II e III com um espinho, enquanto as ninfas apresentaram a base do capítulo também retangular, com córnua pontiaguda, idiossoma oval, coxa I com dois espinhos evidentes, sendo o externo mais longo que o interno, coxas II e III com um espinho curto em cada uma, e coxa IV, com um espinho muito pequeno e a presença de tubérculos quitinosos na superfície interna da borda posterior dos festões. Além disso, a morfologia do ovário e o processo de dinâmica da vitelogênese dos ovócitos de fêmeas ingurgiradas desta espécie de carrapato também são apresentados. O ovário, classificado como panoístico por não apresentar células foliculares e nutridoras, consiste em estrutura tubular única e contínua com ovócitos de vários tamanhos e diferentes estádios de desenvolvimento, presos a parede epitelial por meio do pedicelo. Tais ovócitos são aqui classificados em cinco estádios de acordo com a aparência do citoplasma, observação da vesícula germinal, aspecto dos grãos de vitelo e deposição do córion. Pela primeira vez foi observado em carrapatos ovócitos com o córion esculturado. / Abstract: The present study shows the external morphological description of larvae and the redescription of nymphs of Amblyomma brasiliense ticks under light microscope, stereoscopic microscope and scanning electron microscope. Larvae present basis capituli rectangular, short palpi, idiosoma rounded, coxa I with two spurs being the external longer than the internal one and coxae II and III each with one short spur. Nymphs present basis capituli rectangular with a sharp pointed cornua, oval idiosoma with scutum reaching coxa III and coxae I with two evident spurs being the external longer than the internal one; coxae IIIII each with one short spur and coxae IV with a very short spur and chitinous tubercles on internal surface of posterior border of festoons. Furthermore, the ovary morphology and the dynamics of the vitellogenesis process in oocytes of Amblyomma brasiliense engorged females. The ovary is classified as the panoistic type; therefore, it lacks nurse and follicular cells. This organ consists of a single tubular structure, continuous, with oocytes of various sizes and at different developmental stages remain attached to the ovary through a cellular pedicel. The oocytes were classified into five stages described according to cytoplasm appearance, presence of the germ vesicle, yolk granules aspects, and chorion deposition. For the first time was related in ticks oocytes chorium sculptured. / Orientador: Maria Izabel Camargo Mathias / Coorientador: Gervásio Henrique Bechara / Banca: Gilson Pereira de Oliveira / Banca: Marcelo B. Labruna / Mestre

Endosymbiosis in arthropods involves intracellular bacteria that supply an array of benefits to the host. Endosymbionts likely enhance the health of ticks by provisioning amino acids such as cysteine and tyrosine, and cofactors such as biotin and folic acid that are not available in blood--the sole nutrient source of ticks. Endosymbionts of ticks are of special interest due to their close evolutionary relationship with tick-vectored pathogens that impact livestock and human health. For example, ticks typically contain Coxiella-like endosymbionts (CLEs) that are the closest relatives of the human pathogen Coxiella burnetii. In order to understand the evolutionary relationship between the mammalian pathogen Francisella tularensis, which is vectored by ticks, and the Francisella-like endosymbionts (FLEs) present in several ticks, we assembled the genomes of the FLEs in the hard tick Amblyomma maculatum and the soft tick Ornithodoros moubata using high-throughput sequencing. While this project was in progress, another group described the genome of an FLE in the soft tick Argus (Persicargas) arboreus. Utilizing the three genomes, we show that all FLEs evolved from a mammalian pathogen, a relationship that is converse to that of C. burnetii, which likely evolved from a tick-associated non-pathogenic ancestor. Additionally, our analyses indicate that FLEs are horizontally transferred between ticks, and due to their superior metabolic capabilities could replace ancestral endosymbionts with reduced genomes.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Ticks are important vectors of pathogenic agents that cause disease in humans, domestic animals, and wildlife. They are also hosts for a variety of bacterial endosymbionts. However, little is known about the microbial diversity of many tick species, particularly those species that parasitize small mammals in western Canada. In this thesis, I used a combined morphological and molecular approach to identify, to the species-level, ticks that parasitized small mammals from three localities in Saskatchewan and British Columbia. The genetic diversity and phylogenetic relationships of these tick species was also examined. Comparisons were also made of the composition and diversity of bacteria within individuals of each tick species. Questions relating to the biology, systematics, and vector ecology of the vole tick (Ixodes angustus), the rotund tick (Ixodes kingi), the sculptured tick (Ixodes sculptus) and the Rocky Mountain wood tick (Dermacentor andersoni) were also addressed. The results of my thesis work revealed that I. kingi and I. sculptus were the most encountered tick species on northern pocket gophers (Thomomys talpoides) and Richardson’s ground squirrels (Spermophilus richardsonii), respectively, in Saskatchewan, while I. angustus was the most abundant tick on red-backed voles (Clethrionomys gapperi) in Kootenay National Park (British Columbia). At least 40 genera of bacteria were detected in the four tick species; however, there were significant differences in the composition of the bacteria among tick species. Two novel species of Rickettsia and three putative new species of Rickettsiella were also discovered. The findings of this thesis make an important contribution to our understanding of the evolution and ecology of ticks and tick-borne bacteria.

A set of studies was designed in order to better understand the exposure of horses in Canada to Ixodes-borne diseases, namely equine granulocytic anaplasmosis (EGA, caused by Anaplasma phagocytophilum) and Lyme borreliosis (LB, caused by Borrelia burgdorferi). In the first study, equine serum samples submitted to veterinary diagnostic laboratories in SK, MB and ON were tested for antibodies against A. phagocytophilum and B. burgdorferi, using the point-of-care SNAP® 4Dx® ELISA. Horses seropositive to EGA were found in SK and MB and horses seropositive to LB were found in SK, MB and ON. Overall seroprevalence according to the SNAP® 4Dx® ELISA was 0.53% for EGA and 1.6% for LB. Samples that tested positive for antibodies against A. phagocytophilum (n=2) and B. burgdorferi (n=6) by SNAP® 4Dx® ELISA and 2 randomly selected subsets of samples that tested negative (n=92 each) were then re-tested using currently recommended serologic methods, and test results were compared. A lack of agreement was found between the SNAP® 4Dx® ELISA and indirect immunofluorescent assay (IFA) for EGA (McNemar test p = 0.000001). Agreement of the SNAP® 4Dx® ELISA and ELISA confirmed with Western Blot (WB) for LB was only fair (Kappa 0.23). Due to the lack of agreement between serologic tests for EGA and LB in the first study, another study to further evaluate the agreement among available serologic tests was conducted. A set of 50 convenience serum samples submitted to the veterinary diagnostic laboratory in SK was tested by SNAP® 4Dx® Plus ELISA for antibodies against A. phagocytophilum and B. burgdorferi. Samples were also tested by IFA for antibodies against A. phagocytophilum in two referral laboratories, and by IFA, ELISA confirmed with WB and Equine Lyme multiplex assay for antibodies against B. burgdorferi in three referral laboratories. Again, test results varied between the different tests. For EGA, all 3 pair-wise test comparisons lacked agreement. For LB, agreement between tests ranged from poor to fair. Differences in test methodology and antigens used, cut-off settings between the laboratories and false positive or false negative results are likely the cause for the different assessment of the same sample as seropositive or seronegative. In the third study, the goal was to describe potential risk factors for exposure of horses in Canada to EGA and LB. Management factors in horses that tested seropositive or seronegative for EGA or LB, respectively, in the previous studies were evaluated. Horse owners were surveyed with regard to their horses’ signalment, timing of pasture housing, and province of residence, travel history, tick infestation history, history of Lyme vaccination and history of previously diagnosed tick-borne disease. Response rate (11.5%) and the number of seropositive horses available for evaluation were low, which precluded statistical analysis. The majority of seropositive horses resided in SK, was pastured in the fall, did not have a recent travel history and had not had visible tick infestation. These observations supported exposure of horses to tick-borne diseases within Canada. Potential risk factors require further investigation. As information about tick infestation in horses is scarce in general, a passive surveillance study of horse ticks in SK was conducted in 2012 and 2013. A total of 833 ticks from over 86 horses were received. All ticks were Dermacentor species, i.e. D. albipictus, D. andersoni and D. variabilis. D. albipictus ticks were mostly received in February and March, D. andersoni mainly in April and June and D. variabilis mostly in May and June. Geographic distribution of the species in SK was similar to that previously reported based on active and passive surveillance. No Ixodes species were received.