Uninfected larvae of Ixodes dammini, lxodes cookei, and Amblyomma americanum were fed on hamsters that had been injected intraperitoneally with a 0.5.ml sample of Borrelia burgdorferl (2.5 X 107 spirochetes per ml) 21 days earlier. A total of 108 of these larvae comprised of 36 1. dammini, 36 i. cookei, and 36 A. americanum were aseptically dissected and examined by darkfield and immunofluorescent microscopy for the presence of B. burgdorferl within 48 hours of feeding on the B. burgdorferi infected hamsters. The removal and examination of the midgut diverticula revealed that 32/36 (88.9%) of the l. dammini larvae contained B. burgdorferl. Only 5/36 (13.9%) of the l. cookei larvae and 7/36 of the A. americanum larvae harbored spirochetes in their midgut diverticula.A portion of the nymphs that molted from the above larvae were also dissected and examined by darkfleld and indirect immunofluorescent techniques. Borrelia burgdorferi were observed in the midgut diverticula of 94/107 (87.8%) of the l. dammini nymphs. None of the 30 (0%) l. cookei nymphs examined were found positive for spirochetes and only 1/60 (1.7%) of the A. americanum nymphs was found positive for B. burgdorrerl.A total of 83 lL dammini, 53 A. americanum, and 161. cookei nymphs reared from larvae that fed to repletion on hamsters infected with B. burgdorrerl were allowed to feed on uninfected hamsters to assess transmission of B. burgdorrerl. Transmission was demonstrated only by the l. dammlnl nymphs. The findings of this study suggest that it is extremely unlikely that l. cookei can serve as a vector for B. burgdorrerl, but do not rule out completely the possibility that A. americanum may be able to maintain B, burgdorrerl infections transstadially and, under certain conditions, transmit the organisms to vertebrate hosts. / Department of Physiology and Health Science

Uninfected nymphs of Ixodes scapularis and Amblyomma americanum were fed on hamsters that had been injected intraperitoneally with a 0.5 ml sample of Borrelia burgdorferi (2.5 X 10' spirochetes per ml) 30 days earlier. All nymphs fed to repletion and were then housed during the molting process. In order to assess their ability to transmit the spirochetes, the resulting l. scapularis and A. americanum adults were allowed to feed on uninfected rabbits.Dissection of the adult l. scapularis ticks revealed 9/12 (75%) to harbor motile spirochetes, identified as B. burgdorferi by darkfield microscopy, isolation in BSK II medium, and indirect immunofluorescent antibody staining with the monoclonal antibody H5332. Transmission was successful to one of two New Zealand White rabbits by these infected ticks.Dissection of the adult A. americanum ticks revealed 0/150 (0%) to harbor spirochetes. Transmission to each of three rabbits was unsuccessful. However, 5/90 (5.6%) cultures of midgut material from these same ticks, harbored non-motile spirochete-like bodies. The identity of these "spirochetes" is unknown. / Department of Biology

Thesis (M.S.)--University of Tennessee, Knoxville, 2009. / Title from title page screen (viewed on Mar. 18, 2010). Thesis advisor: Graham Hickling. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Rhode Island, 2004. / Typescript. Includes bibliographical references (leaves 77-88).

This study is focused on characterization of fibrinogen-related proteins (FREPs) from the tick Ixodes ricinus using molecular methods - PCR, cloning, qRT-PCR, RNA interference via dsRNA synthesis and injection, and also pathogen (Borrelia sp.) transmission on animal model.

As metaloproteases (MPs) são proteínas que participam em diversos processos fisiológicos e patológicos. Neste trabalho, relatamos a identificação de MPs em três carrapatos de importância econômica: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) e Rhipicephalus microplus (BrRm-MPs). O perfil transcricional em vários tecidos e fases de vida mostra que MPs são transcritas em glândula salivar durante a alimentação de fêmeas e não são transcritos no macho (com exceção de uma MP, BrRm-MP4) e ovos, o que sugere que esta família da proteínas são componentes funcionais necessários para interferir nas defesas do hospedeiro, apoiando o processo de hematofagia. A presença de um sítio de ligação de zinco, uma dobra“Met-turn” e um domínio rico em cisteína na região C-terminal indica que todas os transcritos obtidos codificam para a família de MPs reproplisina (metzincina). Uma das MPs de R. microplus (BrRm-MP4) foi selecionada para uma investigação mais aprofundada quanto ao potencial como um antígeno vacinal, devido ao seu padrão de transcrição ubíquos e devido àprevisão in silico de epítopos imunogênicos, em comparação com as demais MPs identificadas. BrRm-MP4 recombinante (rBrRm-MP4) foi expressa em Escherichia coli e testadas como um antígeno contra infestação de R. microplus. Imunoblot mostrou que o soro de bovinos imunizados com rBrRm-MP4 reconhece BrRm-MP4 da glândula salivar, ovário e larvas de R. microplus. Comparando com o controle, a vacinação com rBrRm-MP4 proporcionou uma redução de 43% no número de fêmeas adultas, redução de 14,80% na postura de ovos e redução de 17,53 % na capacidade de eclosão da larva, realizando um proteção total de 60% estatisticamente significativa. Os resultados indicam que rBrRm-MP4 é um potencial candidato a ser incluído como imunógeno em uma vacina anti-carrapato. / Metalloproteases (MPs) are proteins participating in several physiological and pathological processes. Here, we report the identification of MPs in three economically important ticks: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) and Rhipicephalus microplus (BrRm-MPs). Transcriptional profile in various tissues and life stages revealed the presence of all MPs transcripts in salivary glands during feeding stages and absence in males (except one MP, BrRm-MP4) and eggs, suggesting this family proteins are functional components required to interfere with host defenses, supporting tick hematophagy. The presence of a zinc binding site, a “Met-turn” and C-terminal cysteine rich domain indicates all obtained transcripts encode for proteins belonging to the reproplysin (metzincin) family of MPs. A R. microplus MP (BrRm-MP4) was selected for further investigation concerning its potential as a vaccinal antigen due to its ubiquitous transcription pattern and in silico prediction of immunogenic epitopes, comparing to other obtained MPs. Recombinant BrRm-MP4 (rBrRm-MP4) was expressed in Escherichia coli and tested as an immune-protective antigen against R. microplus infestation. Immunoblot showed specific antibodies against rBrRm-MP4 in immunized calves sera. Sera from immunized calves recognized BrRm-MP4 in R. microplus salivary gland, ovary and larvae. Comparing to ticks reared non-vaccinated calves, rBrRm-MP4 provided 43% reduction in adult female number, 14.80% reduction in egg laying and 17.53% reduction in larva hatching capacity, performing an overall 60% statistically significant protection. We assume rBrRm-MP4 is a potential candidate to be included as immmunogen for further development in an anti-tick vaccine.

ML (MD-2-related lipid-recognition) domain containing proteins are recognized as immune-related molecules. They do not belong among well-studied proteins in ticks although their occurence is quite often. Generally, ML proteins are involved in innate immunity processes, lipid binding and transport. Usually, expression of tick ML domain containing proteins is induced by blood feeding. Two members of the ML protein family, ML-domain containing protein and Der-p2 allergen-like protein were isolated from Ixodes ricinus and characterized for the first time.

Lyme disease is the most common vector-borne infection in the Western world with an annual incidence usually in excess of 100 cases per 100 000 people in temperate areas of the United States and Europe. Same as other infectious diseases, Lyme borreliosis wreaks havoc on the host they have invaded. B. burgdorferi, the causative agent of this disease, circulates among wildlife vertebrate hosts and Ixodes tick vectors but may sometimes infect humans. Its natural enzootic cycle usually occurs as follows: The larval/nymphal stage tick feeds on an infected host. During this engorgement, the spirochetes reach the tick gut and stay confined to it. After the tick molts into the next developmental stage, it finds a second host. The new bloodmeal triggers the spirochetes to multiply within the gut and traverse the gut endothelium in a highly organized manner. They finally disseminate through the hemocoel up to the tick salivary glands and into the new host. We studied whether B. burgdorferi is capable of reaching the tick salivary glands during the first infective feeding period in uninfected ticks.

As metaloproteases (MPs) são proteínas que participam em diversos processos fisiológicos e patológicos. Neste trabalho, relatamos a identificação de MPs em três carrapatos de importância econômica: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) e Rhipicephalus microplus (BrRm-MPs). O perfil transcricional em vários tecidos e fases de vida mostra que MPs são transcritas em glândula salivar durante a alimentação de fêmeas e não são transcritos no macho (com exceção de uma MP, BrRm-MP4) e ovos, o que sugere que esta família da proteínas são componentes funcionais necessários para interferir nas defesas do hospedeiro, apoiando o processo de hematofagia. A presença de um sítio de ligação de zinco, uma dobra“Met-turn” e um domínio rico em cisteína na região C-terminal indica que todas os transcritos obtidos codificam para a família de MPs reproplisina (metzincina). Uma das MPs de R. microplus (BrRm-MP4) foi selecionada para uma investigação mais aprofundada quanto ao potencial como um antígeno vacinal, devido ao seu padrão de transcrição ubíquos e devido àprevisão in silico de epítopos imunogênicos, em comparação com as demais MPs identificadas. BrRm-MP4 recombinante (rBrRm-MP4) foi expressa em Escherichia coli e testadas como um antígeno contra infestação de R. microplus. Imunoblot mostrou que o soro de bovinos imunizados com rBrRm-MP4 reconhece BrRm-MP4 da glândula salivar, ovário e larvas de R. microplus. Comparando com o controle, a vacinação com rBrRm-MP4 proporcionou uma redução de 43% no número de fêmeas adultas, redução de 14,80% na postura de ovos e redução de 17,53 % na capacidade de eclosão da larva, realizando um proteção total de 60% estatisticamente significativa. Os resultados indicam que rBrRm-MP4 é um potencial candidato a ser incluído como imunógeno em uma vacina anti-carrapato. / Metalloproteases (MPs) are proteins participating in several physiological and pathological processes. Here, we report the identification of MPs in three economically important ticks: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) and Rhipicephalus microplus (BrRm-MPs). Transcriptional profile in various tissues and life stages revealed the presence of all MPs transcripts in salivary glands during feeding stages and absence in males (except one MP, BrRm-MP4) and eggs, suggesting this family proteins are functional components required to interfere with host defenses, supporting tick hematophagy. The presence of a zinc binding site, a “Met-turn” and C-terminal cysteine rich domain indicates all obtained transcripts encode for proteins belonging to the reproplysin (metzincin) family of MPs. A R. microplus MP (BrRm-MP4) was selected for further investigation concerning its potential as a vaccinal antigen due to its ubiquitous transcription pattern and in silico prediction of immunogenic epitopes, comparing to other obtained MPs. Recombinant BrRm-MP4 (rBrRm-MP4) was expressed in Escherichia coli and tested as an immune-protective antigen against R. microplus infestation. Immunoblot showed specific antibodies against rBrRm-MP4 in immunized calves sera. Sera from immunized calves recognized BrRm-MP4 in R. microplus salivary gland, ovary and larvae. Comparing to ticks reared non-vaccinated calves, rBrRm-MP4 provided 43% reduction in adult female number, 14.80% reduction in egg laying and 17.53% reduction in larva hatching capacity, performing an overall 60% statistically significant protection. We assume rBrRm-MP4 is a potential candidate to be included as immmunogen for further development in an anti-tick vaccine.

Light microscopy was used to characterize digestive cells in the gut of the hard tick Ixodes ricinus during feeding and different phases of digestion. Indirect immunofluorescent microscopy was used to localize three digestive peptidases - cathepsin B, cathepsin L, cathepsin D in the gut of the hard tick Ixodes ricinus.