Carrapatos são artrópodos hematófagos com ampla distribuição mundial, sendo capazes de parasitar quase todos os animais vertebrados terrestres. Estes parasitos causam grandes impactos em saúde pública e na produção animal por serem vetores de diversas doenças infecciosas e, também, pelos distúrbios diretos associados ao parasitismo. O Ornithodoros brasiliensis, conhecido popularmente como carrapato mouro, é uma espécie de carrapato somente encontrada no Brasil, e com distribuição restrita à região dos Campos de Cima da Serra no Rio Grande do Sul (RS). Desde os primeiros relatos, O. brasiliensis tem sido considerado um potencial risco à saúde pública no RS devido a ocorrência de parasitismo em humanos. Somado a isto, alguns relatos indicam que sua picada é capaz de induzir distúrbios locais e sistêmicos severos nas pessoas e animais parasitados, possivelmente enquadrando-se na definição de toxicose induzida pela picada de carrapatos. Depois de mais de 50 anos sem relatos sobre O. brasiliensis, recentemente, novos casos de parasitismo humano e animal foram observados na mesma região onde este carrapato foi originalmente descrito. O objetivo deste trabalho é caracterizar o parasitismo pelo carrapato O. brasiliensis, bem como a síndrome tóxica desencadeada nos hospedeiros parasitados. Com esta finalidade, neste trabalho realizamos: (i) um estudo epidemiológico sobre o parasitismo por O. brasiliensis, determinando sua prevalência em propriedades situadas nos municípios de São Francisco de Paula e Jaquirana (regiões de ocorrência natural), descrevendo características de seu habitat e determinando possíveis fatores de risco associados à sua ocorrência; (ii) a descrição de casos retrospectivos de parasitismo em humanos e animais domésticos; (iii) a descrição de um caso clínico de toxicose induzida por O. brasiliensis em cão naturalmente parasitado; (iv) a caracterização clínico-patológica do parasitismo e da toxicose experimentalmente induzida pela picada de O. brasiliensis em ratos; (v) a caracterização dos efeitos do conteúdo salivar sobre o processo de cicatrização in vivo e sobre células endoteliais. Entre as propriedades incluídas na amostragem, em 16,7% foram coletados exemplares de O. brasiliensis. O. brasiliensis foram encontrados perto das habitações humanas e enterrados no solo em pequenas áreas com grande densidade de parasitos, em uma espécie de “ninho”. A análise dos fatores de risco demonstrou que somente duas das 17 variáveis estudadas (falta de higiene e a presença de porão com chão de terra na propriedade) aumentam a probabilidade de ocorrência deste carrapato. Foram identificadas 28 pessoas e 11 cães previamente parasitados. A partir de relatos foi possível identificar os principais sintomas associados ao parasitismo: prurido, eritema, edema, dor, lesão de lenta cicatrização. Evidências indicam que, além do homem e do cão, Conepatus chinga (zorrilho) e Dasypus hybridus (tatu-mulita) podem servir de hospedeiros. A síndrome associada à picada do O. brasiliensis foi caracterizada em detalhes através do registro do progresso clínico de um cão naturalmente exposto ao carrapato, e em ratos experimentalmente infestados. Os achados decorrentes destas observações são compatíveis entre si e com as descrições dos casos retrospectivos em humanos, e evidenciam a severidade do quadro induzido pela picada de O. brasiliensis. Por fim, foi demonstrado que o extrato de glândula salivar de O. brasiliensis inibe a cicatrização in vivo, possivelmente por inibir a proliferação endotelial e por ser citotóxico. Esta atividade relaciona-se diretamente a uma das características marcantes da picada por O. brasiliensis: a indução de uma lesão de lenta cicatrização. O conjunto dos resultados aqui mostrados fornece novos subsídios para a compreensão das peculiaridades evolutivas, de habitat, biológicas e ecológicas do O. brasiliensis, bem como dos efeitos desencadeados pelo seu parasitismo. / Ticks are blood-sucking arthropods distributed worldwide and able to parasitize almost all terrestrial vertebrate animals. Ticks are considered a great hazard for both public health and animal breeding due to their ability to transmit infectious diseases and to induce direct toxic effects leading to clinical disturbances. Ornithodoros brasiliensis, popularly known as mouro tick, is a specie only found in highlands region of Rio Grande do Sul state, Southern Brazil. Since initial reports, O. brasiliensis is considered a potential public health hazard in Rio Grande do Sul state due to the possibility of human parasitism. In addition, few reports indicate that O. brasiliensis bite is able to induce severe local and systemic disturbances in humans and animals. This clinical profile possibly fits in the definition of tick toxicosis syndrome. After more than 50 years without reports about O. brasiliensis, new cases of human and animal parasitism were recently reported in the same region of its original description. This work aimed to describe the characteristics of O. brasiliensis parasitism, as well as the toxicosis observed in bitten hosts. Thus, we performed: (i) an epidemiological study about O. brasiliensis parasitism in farms located in the municipalities of São Francisco de Paula and Jaquirana (endemic region) to determine its prevalence and risk factors, and also, to describe the characteristics of its habitat; (ii) an analysis of retrospective cases of human and animal parasitism; (iii) the report of a clinical case of tick toxicosis in a dog naturally parasitized; (iv) a clinico-pathological characterization of the toxicosis induced in experimentally bitten rats; (v) the evaluation of salivary glands effects on skin wound healing. in vivo and upon endothelial cells. Specimens of O. brasiliensis were collected in 16.7% of sampled farms. Ticks were found near human houses and buried in sand. They were found in a very restricted area and in great density, in a kind of tick “nest”. Risk factor analysis showed that only two of the 17 studied variables (insufficient sanitary conditions and presence of house basement with sand floor) were significantly associated with an increased probability to find O. brasiliensis. Twenty-eight people and eleven bitten dogs were identified by analysis of retrospective cases. This analysis also showed the most common symptoms associated to parasitism (pruritus, erythema, edema, pain and slow healing lesion). Our results indicate that in addition to human and dogs, Conepatus chinga (hog-nosed skunk) and Dasypus hybridus (long nosed armadillo) may serve as potential hosts. The toxicosis syndrome induced by tick bite was characterized registering the clinical progress of a naturally bitten dog, and in experimentally infested rats. The clinical findings both in the dog and in rats were similar, and also were compatible with the symptoms reported by bitten humans. The set of all clinical findings evinced the clinical severity of this syndrome. Finally, it was shown that salivary gland extract inhibit in vivo wound healing, possibly due to inhibition of endothelial cell proliferation and cytotoxicity. This biological activity may explain one of the most common symptoms of O. brasiliensis bite: the induction of a slow healing lesion. The set of all results showed here gives new evidences for the understanding of habitat distribution, biology, ecology and peculiarities of O. brasiliensis, as well as for the clinical signs induced by its parasitism.

Carrapatos são artrópodos hematófagos com ampla distribuição mundial, sendo capazes de parasitar quase todos os animais vertebrados terrestres. Estes parasitos causam grandes impactos em saúde pública e na produção animal por serem vetores de diversas doenças infecciosas e, também, pelos distúrbios diretos associados ao parasitismo. O Ornithodoros brasiliensis, conhecido popularmente como carrapato mouro, é uma espécie de carrapato somente encontrada no Brasil, e com distribuição restrita à região dos Campos de Cima da Serra no Rio Grande do Sul (RS). Desde os primeiros relatos, O. brasiliensis tem sido considerado um potencial risco à saúde pública no RS devido a ocorrência de parasitismo em humanos. Somado a isto, alguns relatos indicam que sua picada é capaz de induzir distúrbios locais e sistêmicos severos nas pessoas e animais parasitados, possivelmente enquadrando-se na definição de toxicose induzida pela picada de carrapatos. Depois de mais de 50 anos sem relatos sobre O. brasiliensis, recentemente, novos casos de parasitismo humano e animal foram observados na mesma região onde este carrapato foi originalmente descrito. O objetivo deste trabalho é caracterizar o parasitismo pelo carrapato O. brasiliensis, bem como a síndrome tóxica desencadeada nos hospedeiros parasitados. Com esta finalidade, neste trabalho realizamos: (i) um estudo epidemiológico sobre o parasitismo por O. brasiliensis, determinando sua prevalência em propriedades situadas nos municípios de São Francisco de Paula e Jaquirana (regiões de ocorrência natural), descrevendo características de seu habitat e determinando possíveis fatores de risco associados à sua ocorrência; (ii) a descrição de casos retrospectivos de parasitismo em humanos e animais domésticos; (iii) a descrição de um caso clínico de toxicose induzida por O. brasiliensis em cão naturalmente parasitado; (iv) a caracterização clínico-patológica do parasitismo e da toxicose experimentalmente induzida pela picada de O. brasiliensis em ratos; (v) a caracterização dos efeitos do conteúdo salivar sobre o processo de cicatrização in vivo e sobre células endoteliais. Entre as propriedades incluídas na amostragem, em 16,7% foram coletados exemplares de O. brasiliensis. O. brasiliensis foram encontrados perto das habitações humanas e enterrados no solo em pequenas áreas com grande densidade de parasitos, em uma espécie de “ninho”. A análise dos fatores de risco demonstrou que somente duas das 17 variáveis estudadas (falta de higiene e a presença de porão com chão de terra na propriedade) aumentam a probabilidade de ocorrência deste carrapato. Foram identificadas 28 pessoas e 11 cães previamente parasitados. A partir de relatos foi possível identificar os principais sintomas associados ao parasitismo: prurido, eritema, edema, dor, lesão de lenta cicatrização. Evidências indicam que, além do homem e do cão, Conepatus chinga (zorrilho) e Dasypus hybridus (tatu-mulita) podem servir de hospedeiros. A síndrome associada à picada do O. brasiliensis foi caracterizada em detalhes através do registro do progresso clínico de um cão naturalmente exposto ao carrapato, e em ratos experimentalmente infestados. Os achados decorrentes destas observações são compatíveis entre si e com as descrições dos casos retrospectivos em humanos, e evidenciam a severidade do quadro induzido pela picada de O. brasiliensis. Por fim, foi demonstrado que o extrato de glândula salivar de O. brasiliensis inibe a cicatrização in vivo, possivelmente por inibir a proliferação endotelial e por ser citotóxico. Esta atividade relaciona-se diretamente a uma das características marcantes da picada por O. brasiliensis: a indução de uma lesão de lenta cicatrização. O conjunto dos resultados aqui mostrados fornece novos subsídios para a compreensão das peculiaridades evolutivas, de habitat, biológicas e ecológicas do O. brasiliensis, bem como dos efeitos desencadeados pelo seu parasitismo. / Ticks are blood-sucking arthropods distributed worldwide and able to parasitize almost all terrestrial vertebrate animals. Ticks are considered a great hazard for both public health and animal breeding due to their ability to transmit infectious diseases and to induce direct toxic effects leading to clinical disturbances. Ornithodoros brasiliensis, popularly known as mouro tick, is a specie only found in highlands region of Rio Grande do Sul state, Southern Brazil. Since initial reports, O. brasiliensis is considered a potential public health hazard in Rio Grande do Sul state due to the possibility of human parasitism. In addition, few reports indicate that O. brasiliensis bite is able to induce severe local and systemic disturbances in humans and animals. This clinical profile possibly fits in the definition of tick toxicosis syndrome. After more than 50 years without reports about O. brasiliensis, new cases of human and animal parasitism were recently reported in the same region of its original description. This work aimed to describe the characteristics of O. brasiliensis parasitism, as well as the toxicosis observed in bitten hosts. Thus, we performed: (i) an epidemiological study about O. brasiliensis parasitism in farms located in the municipalities of São Francisco de Paula and Jaquirana (endemic region) to determine its prevalence and risk factors, and also, to describe the characteristics of its habitat; (ii) an analysis of retrospective cases of human and animal parasitism; (iii) the report of a clinical case of tick toxicosis in a dog naturally parasitized; (iv) a clinico-pathological characterization of the toxicosis induced in experimentally bitten rats; (v) the evaluation of salivary glands effects on skin wound healing. in vivo and upon endothelial cells. Specimens of O. brasiliensis were collected in 16.7% of sampled farms. Ticks were found near human houses and buried in sand. They were found in a very restricted area and in great density, in a kind of tick “nest”. Risk factor analysis showed that only two of the 17 studied variables (insufficient sanitary conditions and presence of house basement with sand floor) were significantly associated with an increased probability to find O. brasiliensis. Twenty-eight people and eleven bitten dogs were identified by analysis of retrospective cases. This analysis also showed the most common symptoms associated to parasitism (pruritus, erythema, edema, pain and slow healing lesion). Our results indicate that in addition to human and dogs, Conepatus chinga (hog-nosed skunk) and Dasypus hybridus (long nosed armadillo) may serve as potential hosts. The toxicosis syndrome induced by tick bite was characterized registering the clinical progress of a naturally bitten dog, and in experimentally infested rats. The clinical findings both in the dog and in rats were similar, and also were compatible with the symptoms reported by bitten humans. The set of all clinical findings evinced the clinical severity of this syndrome. Finally, it was shown that salivary gland extract inhibit in vivo wound healing, possibly due to inhibition of endothelial cell proliferation and cytotoxicity. This biological activity may explain one of the most common symptoms of O. brasiliensis bite: the induction of a slow healing lesion. The set of all results showed here gives new evidences for the understanding of habitat distribution, biology, ecology and peculiarities of O. brasiliensis, as well as for the clinical signs induced by its parasitism.

Carrapatos são artrópodos hematófagos com ampla distribuição mundial, sendo capazes de parasitar quase todos os animais vertebrados terrestres. Estes parasitos causam grandes impactos em saúde pública e na produção animal por serem vetores de diversas doenças infecciosas e, também, pelos distúrbios diretos associados ao parasitismo. O Ornithodoros brasiliensis, conhecido popularmente como carrapato mouro, é uma espécie de carrapato somente encontrada no Brasil, e com distribuição restrita à região dos Campos de Cima da Serra no Rio Grande do Sul (RS). Desde os primeiros relatos, O. brasiliensis tem sido considerado um potencial risco à saúde pública no RS devido a ocorrência de parasitismo em humanos. Somado a isto, alguns relatos indicam que sua picada é capaz de induzir distúrbios locais e sistêmicos severos nas pessoas e animais parasitados, possivelmente enquadrando-se na definição de toxicose induzida pela picada de carrapatos. Depois de mais de 50 anos sem relatos sobre O. brasiliensis, recentemente, novos casos de parasitismo humano e animal foram observados na mesma região onde este carrapato foi originalmente descrito. O objetivo deste trabalho é caracterizar o parasitismo pelo carrapato O. brasiliensis, bem como a síndrome tóxica desencadeada nos hospedeiros parasitados. Com esta finalidade, neste trabalho realizamos: (i) um estudo epidemiológico sobre o parasitismo por O. brasiliensis, determinando sua prevalência em propriedades situadas nos municípios de São Francisco de Paula e Jaquirana (regiões de ocorrência natural), descrevendo características de seu habitat e determinando possíveis fatores de risco associados à sua ocorrência; (ii) a descrição de casos retrospectivos de parasitismo em humanos e animais domésticos; (iii) a descrição de um caso clínico de toxicose induzida por O. brasiliensis em cão naturalmente parasitado; (iv) a caracterização clínico-patológica do parasitismo e da toxicose experimentalmente induzida pela picada de O. brasiliensis em ratos; (v) a caracterização dos efeitos do conteúdo salivar sobre o processo de cicatrização in vivo e sobre células endoteliais. Entre as propriedades incluídas na amostragem, em 16,7% foram coletados exemplares de O. brasiliensis. O. brasiliensis foram encontrados perto das habitações humanas e enterrados no solo em pequenas áreas com grande densidade de parasitos, em uma espécie de “ninho”. A análise dos fatores de risco demonstrou que somente duas das 17 variáveis estudadas (falta de higiene e a presença de porão com chão de terra na propriedade) aumentam a probabilidade de ocorrência deste carrapato. Foram identificadas 28 pessoas e 11 cães previamente parasitados. A partir de relatos foi possível identificar os principais sintomas associados ao parasitismo: prurido, eritema, edema, dor, lesão de lenta cicatrização. Evidências indicam que, além do homem e do cão, Conepatus chinga (zorrilho) e Dasypus hybridus (tatu-mulita) podem servir de hospedeiros. A síndrome associada à picada do O. brasiliensis foi caracterizada em detalhes através do registro do progresso clínico de um cão naturalmente exposto ao carrapato, e em ratos experimentalmente infestados. Os achados decorrentes destas observações são compatíveis entre si e com as descrições dos casos retrospectivos em humanos, e evidenciam a severidade do quadro induzido pela picada de O. brasiliensis. Por fim, foi demonstrado que o extrato de glândula salivar de O. brasiliensis inibe a cicatrização in vivo, possivelmente por inibir a proliferação endotelial e por ser citotóxico. Esta atividade relaciona-se diretamente a uma das características marcantes da picada por O. brasiliensis: a indução de uma lesão de lenta cicatrização. O conjunto dos resultados aqui mostrados fornece novos subsídios para a compreensão das peculiaridades evolutivas, de habitat, biológicas e ecológicas do O. brasiliensis, bem como dos efeitos desencadeados pelo seu parasitismo. / Ticks are blood-sucking arthropods distributed worldwide and able to parasitize almost all terrestrial vertebrate animals. Ticks are considered a great hazard for both public health and animal breeding due to their ability to transmit infectious diseases and to induce direct toxic effects leading to clinical disturbances. Ornithodoros brasiliensis, popularly known as mouro tick, is a specie only found in highlands region of Rio Grande do Sul state, Southern Brazil. Since initial reports, O. brasiliensis is considered a potential public health hazard in Rio Grande do Sul state due to the possibility of human parasitism. In addition, few reports indicate that O. brasiliensis bite is able to induce severe local and systemic disturbances in humans and animals. This clinical profile possibly fits in the definition of tick toxicosis syndrome. After more than 50 years without reports about O. brasiliensis, new cases of human and animal parasitism were recently reported in the same region of its original description. This work aimed to describe the characteristics of O. brasiliensis parasitism, as well as the toxicosis observed in bitten hosts. Thus, we performed: (i) an epidemiological study about O. brasiliensis parasitism in farms located in the municipalities of São Francisco de Paula and Jaquirana (endemic region) to determine its prevalence and risk factors, and also, to describe the characteristics of its habitat; (ii) an analysis of retrospective cases of human and animal parasitism; (iii) the report of a clinical case of tick toxicosis in a dog naturally parasitized; (iv) a clinico-pathological characterization of the toxicosis induced in experimentally bitten rats; (v) the evaluation of salivary glands effects on skin wound healing. in vivo and upon endothelial cells. Specimens of O. brasiliensis were collected in 16.7% of sampled farms. Ticks were found near human houses and buried in sand. They were found in a very restricted area and in great density, in a kind of tick “nest”. Risk factor analysis showed that only two of the 17 studied variables (insufficient sanitary conditions and presence of house basement with sand floor) were significantly associated with an increased probability to find O. brasiliensis. Twenty-eight people and eleven bitten dogs were identified by analysis of retrospective cases. This analysis also showed the most common symptoms associated to parasitism (pruritus, erythema, edema, pain and slow healing lesion). Our results indicate that in addition to human and dogs, Conepatus chinga (hog-nosed skunk) and Dasypus hybridus (long nosed armadillo) may serve as potential hosts. The toxicosis syndrome induced by tick bite was characterized registering the clinical progress of a naturally bitten dog, and in experimentally infested rats. The clinical findings both in the dog and in rats were similar, and also were compatible with the symptoms reported by bitten humans. The set of all clinical findings evinced the clinical severity of this syndrome. Finally, it was shown that salivary gland extract inhibit in vivo wound healing, possibly due to inhibition of endothelial cell proliferation and cytotoxicity. This biological activity may explain one of the most common symptoms of O. brasiliensis bite: the induction of a slow healing lesion. The set of all results showed here gives new evidences for the understanding of habitat distribution, biology, ecology and peculiarities of O. brasiliensis, as well as for the clinical signs induced by its parasitism.

Mans (2002) hypothesized that the two domains of savignin interact with each other, giving a globular form in the absence of thrombin. Binding of the C-terminal domain of the inhibitor to the fibrinogen-binding site of thrombin leads to the dissociation of the domains. This would yield an extended conformation that would allow binding of the N-terminal residues of the N-terminal domain to thrombin’s active site. To test this hypothesis, both theoretical and experimental approaches were employed to determine the molecular dimensions of uncomplexed savignin. In the theoretical approach, the hydrodynamic radius (Rh) of the extended form of savignin was calculated from the crystal structure data of the thrombin-ornithodorin complex, and found to be 2.319 nm. With the same programme, based on the crystal structure data for bikunin, a protein in which both domains are closely associated, the Rh value for the compact form of savignin was estimated as 1.96 nm. Using the equation that relates Rh to molecular mass, a value of 1.84 nm was calculated for savignin (12 430 Da). In the experimental approach, the SEC of salivary gland extracts, using lysozyme (Rh = 1.99 nm) as standards and chrymotrypsinogen (Rh = 2.31nm) indicated that uncomplexed savignin exists in both the globular and extended conformations. However, the majority of inhibitory activity was associcated with the extended form. Heat stability assays as well as SDS-PAGE experiments indicated the possible existence of the compact form of savignin. Generation of adequate amounts of savignin will allow further structural studies, to determine the structure of savignin in the uncomplexed form and in complex with thrombin. Expression of full length savignin and the separate N- and C-domains will facilitate further kinetic analysis. In the recombinant production of savignin, various factors were investigated: cell-lines, transformation efficiency, induction times, purification strategy and protease cleavage of expressed fusion protein. Even though large quantities of expressed fusion protein were obtained, cleavage of the target protein and its separation from the fusion partner by enzymatic means presented a major hurdle. Expression of Nsav was not observed and is most likely as a result of misfolding of the recombinant form. Due to the probable non-specific cleavage of the fusion protein, switching the prokaryotic expression system to other expression systems, like yeast- or baculovirus-insect cell-expression systems, is warranted. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / unrestricted

In this study the structural characteristics and antibacterial activities of two peptides derived from the carboxy-terminal of a tick defensin were investigated. Two defensin isoforms (OsDef1 and OsDef2) were previously identified in the midgut of the tick, Ornithodoros savignyi. Both OsDef1 and OsDef2 were found to be active against Gram-positive bacteria but showed no antibacterial activity against Gram-negative bacteria. OsDef2 was found to be slightly more active than OsDef1 and was, therefore, used as the template for the design of smaller antimicrobial peptides. Two peptide analogues were synthesised using the carboxy-terminal sequence of OsDef2 and differed in that in the one peptide the cysteine residues were present (Os) and in the other the cysteine residues were omitted (Os-C). Structurally, Os contained more α-helical properties than Os-C, whereas Os-C was more β-sheeted when prepared in 25 mM SDS. Both Os and Os-C showed no antibacterial activity when tested in Luria-Bertani broth or Mueller-Hinton broth indicating that the activities of Os and Os-C were influenced by the presence of broth salts and proteins. When tested in sodium phosphate buffer, both Os and Os-C exhibited Gram-positive and Gram-negative antibacterial activity. Os was slightly more active than Os-C against 3 of the 4 tested strains, with minimum bactericidal concentrations (MBCs) ranging from 0.94 μg/ml to 3.75 μg/ml. Os retained bactericidal activity against both Staphylococcus aureus and Escherichia coli when tested in the presence of 100 mM NaCl or 30% human serum. Os-C retained activity against E. coli in the presence of NaCl but became inactive in 30% human serum against both bacterial strains. At the MBCs, Os exhibited faster killing kinetics than Os-C killing both Bacillus subtilis and E. coli within 5 min, whereas Os-C took up to 120 min and 60 min, respectively. SYTOX Green permeabilization assays showed that both Os and Os-C caused permeabilization of E. coli membranes after 30 min exposure. At high peptide concentrations, both Os and Os-C were shown to interact with plasmid DNA. Both Os and Os-C exhibited no cytotoxic effects against SC-1 and Caco-2 cell lines, even at peptide concentrations 32 times higher than the highest MBC. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Biochemistry / unrestricted

Ticks are blood feeding ectoparasites that ingest large volumes of vertebrate blood. They are the most important arthropods that are capable of transmitting pathogens which cause disease in humans and domestic animals. Ticks are exposed to various microorganisms during feeding as well as in their habitat. They therefore must have a very good immune system to recognize and destroy these microorganisms. In the present study a micro-broth dilution assay was used to determine whether antifungal activity was present in different tick tissue extracts with or without challenge. The midguts gave the highest inhibition of yeast growth, followed by the salivary glands and then the hemolymph. This was seen with unchallenged tick tissue extracts, as well as tissue extracts collected after yeast challenge (2 hours). Thus all of the tick tissue extracts that was analyzed in this study had antifungal activity. Proteomics was used to determine whether proteins were differentially expressed in the hemolymph plasma, after a fungal challenge. 2DE was used since proteins are not only separated by molecular mass, but also by their charge. The proteins that were separated on the 2D-gels ranged between 17.5-76 kDa and not all proteins present on the 1D-gels (14-97 kDa) could be seen on the 2D-gels. Ticks were challenged for 2 hours to define the proteins that play a role in the short term innate immune response during a fungal infection. Various proteins were differentially expressed in the hemolymph samples that were collected 2 hours after ticks were injected with saline, â-1,3-glucan or yeast (or 72 hours). Injury and fungal challenge play a role in producing proteins that might play a role in the fungal response of the tick. Five spots that were statistically significant in the hemolymph collected 2 hours after ticks were injected with yeast cells were analyzed with MS/MS. No matches were found with MASCOT database searching or with EST searching. This can be due to the limited information that is available on the soft ticks, as only hard tick ESTs heve been published. It was also attempted to identify hemolymph proteins that might play a role in the recognition of fungi. Hemolymph was incubated with live Candida albicans cells and eluted with buffer. Three protein bands (97, 88 and 26 kDa) were found to be present whether ticks were challenged or unchallenged. These proteins were subjected to MS/MS analysis and database searching was performed revealing no matches to other known proteins. The antifungal response was found to be present in the soft tick O. savignyi and might play a vital function in the innate immune response during a fungal infection. These proteins may serve as lead molecules that could be used in the development of novel antifungal drugs, as well as in vaccine development. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

Relapsing fever Borrelia are transmitted through saliva of argasid ticks. Given the 10 to 60 minute long bloodmeal, we characterized the rapidity of Borrelia turicatae transmission from their vector Ornithodoros turicata. Infection rates were compared in mice when cohorts of ticks fed to repletion on animals in which groups of O. turicata were removed 15 seconds after attachment. Infection was evaluated by examining the blood by dark field microscopy, quantitative PCR, and serological responses generated against B. turicatae. Scanning electron microscopy was also performed on cryofractured tick salivary glands to determine spirochete localization. Dissemination of B. turicatae into murine blood was evaluated by removing the bite site after ticks engorged. Our findings indicate that B. turicatae is localized in the lumen of salivary gland acini of O. turicata and transmission to and dissemination in the mammal occur shortly after tick attachment and do not require a complete bloodmeal.

Ornithodoros brasiliensis Aragão é um carrapato endêmico do Brasil, restrito às regiões serranas e frias do estado do Rio Grande do Sul. É uma espécie bastante agressiva aos humanos, causando febre, muita dor e intensa resposta inflamatória no local da picada. O ciclo biológico de O. brasiliensis (ovo a adulto, única geração) realizado nos anos 50, utilizando ratos brancos como hospedeiros, levou um total de 103 e 184 dias de amplitude, com máximo de 7 ínstares ninfais observados e com emergência de adultos a partir de N4, estando os machos em maior proporção. Os hospedeiros apresentaram febre e uma bactéria espiroqueta denominada Borrelia brasiliensis foi descrita de esfregaços sanguíneos desses animais. Porém as borrélias não puderam ser cultivadas por muito tempo, e acabaram desaparecendo. Houve um silêncio de mais de 50 anos sobre relatos de ocorrência de O. brasiliensis, deixando inclusive dúvidas se a espécie estava extinta de fato. No entanto, há cerca de quatro anos, alguns espécimes de carrapatos da mesma localidade foram coletados e identificados como O. brasiliensis. O presente trabalho teve por objetivos estudar a morfologia e a biologia de O. brasiliensis, em condições de laboratório, bem como investigar a presença de bactérias do gênero Rickettsia em espécimes coletados. Ninfas e adultos foram alimentados em cobaias de laboratório e as fêmeas acasaladas realizaram posturas que resultaram nas gerações sucessivas. A larva e os adultos foram descritos e redescritos, e comparados com as espécies próximas, O. rostratus e O. turicata. Análises moleculares de uma porção do gene 16S rDNA mitocondrial foram comparadas com as sequências depositadas no GenBank e apresentaram valores de máximo bootstrap de 100% para ambas, O. rostratus e O. turicata. O ciclo biológico completo (ovo a ovo) de O. brasiliensis foi obtido entre 215,4 e 195 dias, em média, para duas gerações consecutivas, respectivamente. Os períodos médios de pré-oviposição, oviposição e incubação dos ovos (em dias), para a geração F1 foram 76,7 ± 27,01 (31-117); 15,55 ± 5,36 (10-24); 10,6 ± 4,5 (3-19) e para geração F2 foram 54,1 ± 8,7 (40-65), 15,8 ± 8,9 (7-36); 15,4. Os períodos médios de pré-ecdise larval foram de 12,5 ± 1,8 (10-15), considerando as duas gerações. A larva não se alimenta, enquanto que há um repasto sanguíneo para cada instar ninfal, antes de cada muda. Um total de 5 instares ninfais foi observado, sendo que os períodos médios de préecdise para os instares N1, N2, N3, N4 e N5, respectivamente para as duas gerações foram de 33,3 ± 4 (32-45), 38,6 ± 2,5 (36-47), 31,1 ± 3,4 (28-42), 34,8 ± 3,3 (31-43), 37,2 ± 2,4 (35-41) e 32,5 ± 3,3 (29-40), 37,5 ± 2,5 (34-44), 34,6 ± 3,2 (31- 37), 36,5 ± 2,5 (34-42), 32,5 ± 0,7 (32-33). Os períodos médios de alimentação para os ínstares ninfais, foram semelhantes nas duas gerações, sendo de 28,62 ± 3,67 (25,2 35,1) minutos. Adultos alimentam-se repetidas vezes, e as posturas sempre são precedidas de repasto sanguíneo, embora somente dois ciclos gonotróficos foram observados. O primeiro ciclo gonotrófico apresentou maior número de ovos depositados, com 139 ± 13,8 (53 197) em relação ao segundo 73,8 ± 10,6 (53 123). A investigação da presença de riquétsia foi avaliada em 107 espécimes recentemente coletados, evidenciando um total de 12 exemplares positivos. O produto amplificado pela PCR foi sequenciado e as análises moleculares apresentaram homologia de 98% com sequências disponibilizadas no Genbank, correspondentes a uma espécie asiática denominada RDa420 (AF497584) e outra brasileira, Rickettsia bellii (DQ865204) . / Ornithodoros brasiliensis Aragão is an endemic tick to Brazil, restricted to the highlands and cold regions of the state of Rio Grande do Sul. This species is very aggressive to humans, causing fever, great pain and intense inflammatory response at the site of the bite. The life cycle of O. brasiliensis (egg to adult, single generation) previously performed (studied during 50\') using white mice as host, spent a total of 103 and 184 days of amplitude, with maximum 7 nymphal instars observed, with adult emerging from N4, and males in greater proportion. The hosts had fever and spirochete bacteria called Borrelia brasiliensis was described from blood of the animals used. Nevertheless borrelias could not be cultivated for a long time, and they eventually disappeared. A silence of more than 50 years on reports of occurrence of O. brasiliensis, left doubts about whether the species was extinct. However, about four years ago, some specimens of ticks were collected in the same locality and identified as O. brasiliensis. This work aimed to study the morphology and biology of O. brasiliensis, under laboratory conditions, and investigate the presence of bacteria of the genus Rickettsia in the collected ticks. Nymphs and adults were fed on laboratory guinea pigs, and mated females laid eggs that resulted in the successive generations. The larvae and adults have been described and redescribed, respectively, and compared with the closely related species, O. rostratus and O. turicata. Molecular analyzes of a portion of the mitochondrial 16S rDNA gene was compared with the sequences deposited in GenBank, showing maximum bootstrap values out of 100% for both O. rostratus and O. turicata. In this study, the complete life cycle (egg to egg) of O. brasiliensis was obtained between 215,4 and 195 days, on average, for the two generations studied, respectively. The average periods of preoviposition, oviposition and incubation of the eggs to the F1 generation were 76,7 ± 27,01 (31-117), 15,55 ± 5,36 (10-24), and to F2 were 54,1 ± 8,7 (40-65), 15,8 ± 8,9 (7-36). The averaging periods to the preecdysis of the larvae were 12,5 ± 1,8 (10-15), considering the two generations. The larva does not feed, while for the nymphs there is a blood meal for each instar, before each molt. A total of five nymphal instars was observed, and the average periods of pre-molt instars for N1, N2, N3, N4 and N5, for two generations, respectively, were 33,3 ± 4 (32-45), 38,6 ± 2,5 (36-47), 31,1 ± 3,4 (28-42), 34,8 ± 3,3 (31-43), 37,2 ± 2,4 (35-41) and 32,5 ± 3,3 (29-40), 37,5 ± 2,5 (34- 44), 34,6 ± 3,2 (31-37), 36,5 ± 2,5 (34-42), 32,5 ± 0,7 (32-33). The average feeding time of nymphal instars, were similar in the two generations, being 28,62 ± 3,67 (25,2 35,1) minutes. Adults fed repeatedly, and the postures were always preceded by blood meal, although only two gonotrophic cycles have been observed. The first gonotrophic cycle presented higher average number of eggs deposited 139 ± 13,8 (53-197) that the second 73,8 ± 10,6 (53 123). The investigation to the presence of rickettsia was performed in 109 recently collected ticks, showing a total of 12 positive specimens. The amplified PCR product was sequenced and molecular analyzes showed 98% homology with sequences available in Genbank, corresponding to an Asian species named RDa420 (AF497584) e another Brazilian strain named Rickettsia bellii (DQ865204).

La peste porcine africaine (PPA) est une maladie hémorragique contagieuse dévastatrice pour l’élevage porcin pour laquelle aucun traitement ni vaccin ne sont disponibles. Cette infection est due à un virus à ADN, unique membre de la famille des Asfarviridae, qui se transmet directement entre suidés ou via un vecteur, la tique du genre Ornithodoros. Le rôle de ces tiques dans le cycle épidémiologique de la PPA consiste principalement à maintenir le virus dans les populations de suidés sauvages en Afrique. Elles ont aussi été identifiées à l’origine de certains cas de résurgence de la maladie dans des bâtiments d’élevage porcin, notamment dans la péninsule ibérique, lors des années 1970-80. La PPA, éradiquée fin des années 1980 en Europe (à l’exception de la Sardaigne), a été de nouveau introduite en 2007 d’abord en Géorgie pour progresser jusqu’à atteindre l’Est de l’Union Européenne. La question de la compétence vectorielle des tiques Ornithodores pour le virus de la PPA et des déterminants qui influencent cette compétence est posée dans l’évaluation des risques d’endémisation et/ou de dispersion de la maladie en Europe et ailleurs. Le premier chapitre de cette thèse vise à caractériser la compétence vectorielle des tiques Ornithodores pour le virus de la PPA et faire ressortir les patrons généraux qui la qualifient. Pour cela, a été réalisée une revue systématique des études ayant testé la compétence vectorielle d’une ou plusieurs espèces de tiques pour une ou plusieurs souches virales de PPA durant ces 50 dernières années. Au final, il en ressort une forte variabilité de résultats selon les couples tique-virus. En outre, il semble difficile de comparer ces résultats et d’établir des « profils types » du fait de l’évaluation partielle de la compétence vectorielle pour de nombreux couples tique-virus et de la diversité des méthodologies utilisées pour tester et mesurer la compétence. Pour autant chaque modalité d’étude révèle une partie des mécanismes et des adaptations auxquels sont soumis les couples tique–virus et suggère l’effet de certains déterminants dont deux sont traités dans les deux autres chapitres de la thèse. Le second chapitre de la thèse traite de l’adaptation tique-virus, par l’étude expérimentale de l’infection des tiques O. erraticus, O. porcinus et O. moubata à l’aide de deux souches de génotype II du virus de la PPA, et par un essai de transmission du virus des tiques aux porcs. Des souches du génotype II ont été choisies car ce génotype circule actuellement en Europe et risque d’infecter les tiques européennes O. erraticus s’il se propage jusqu’en péninsule ibérique. Alors qu’O. erraticus est capable de s’infecter et de transmettre différentes souches virales du génotype I, sa compétence à transmettre la souche Georgia2007/1 (génotype II) n’a pour l’instant pas été démontrée. Toutefois, nos études suggèrent que les résultats de compétence dépendent aussi des conditions d’expérimentation telles que la nature des colonies de tiques utilisées ou encore le titre viral utilisé pour infecter les tiques. Le dernier chapitre de la thèse porte sur l’effet de la salive de tique sur l’étape de transmission du virus de la PPA par la tique au porc. Durant le repas de sang, la salive est un élément essentiel qui va permettre l’accroche et le gorgement durable de la tique sur son hôte, avec des propriétés immuno-modulatrices importantes agissant directement sur l’hôte. Ainsi a été réalisée une étude expérimentale in vivo faisant intervenir la tique O. porcinus et le virus Ambat02 (génotype II) et testant l’effet local et systémique chez le porc d’un extrait de glandes salivaires de tique co-inoculé ou non avec le virus de la PPA, versus la piqûre naturelle de tiques non infectées. Les résultats de cette expérience nous montrent que la salive de tique est capable de moduler au niveau local le recrutement de cellules immunitaires dans la peau du porc et potentiellement influencer l’infection locale chez le porc. / African swine fever (ASF) is a contagious hemorrhagic disease with disastrous financial consequences for pig industry, as no vaccine or treatment exists. This infection is caused by a DNA virus, only member of the Asfarviridae family that can be directly transmitted between swine or by a non-compulsory vector, the Ornithodoros tick. Ornithodoros ticks play a role in the persistence of the disease within wild and domestic suids in Africa. They were also involved in resurgences of outbreaks in some pig farms in the Iberian Peninsula in 1970-1980. ASF, eradicated in Europe at the end of the 1980’s except in Sardinia, was reintroduced in Georgia in 2007 then spread towards the Eastern European Union. The question of the tick vector competence for ASF virus (ASFV) and its related determinants is of importance in the risk assessment of endemisation/spread of the disease in Europe or elsewhere.The first chapter of this thesis aims to characterize Ornithodoros tick vector competence for ASFV and to highlight a common pattern to qualify it. For this purpose, a systematic review of the studies carried out on the vector competence of one or more tick species for one or more ASFV, was performed on the last 50 years publications. A high variability of the results obtained for different couples “tick-virus” was highlighted. As most of the papers describe partial evaluation of the vector competence and because of the high number of methods used to perform these assessments, it was definitively very difficult to compare these results, and to propose common patterns. However, each of these studies revealed a part of the mechanisms that participated to the adaptation in the couple “tick-virus”, and suggested the importance of different determinants, out of them, two were experimentally assessed as described in the two other chapters.The second chapter of this thesis describes the adaptation “tick-virus” through the experimental infection of three different ticks, O. erraticus, O. porcinus and O. moubata by two ASFV strains belonging to the genotype II. O erraticus’s competence is known for ASFV strains belonging to the genotype I but has never demonstrated the ASFV Georgia 2007/1 strain (genotype II) and currently circulating in Europe. However, the experiments we performed, suggest that many experimental conditions could influence the results obtained on vector competence as the tick colony or the virus dose used for the tick infection.The third chapter describes the effect of the tick saliva on the ASFV transmission from the tick to the pig. Tick saliva contains important immunomodulatory molecules that interfere with the pig immune system permitting complete engorgement of the tick on its host. The host-vector and pathogen interactions were studied through an in vivo experimentation involving pig, O porcinus tick and ASFV Ambat02 strain (genotype II). The local and systemic effects on the pig immune responses were assessed with the ASFV alone or combined with tick gland extract, versus a healthy tick bite. Data analysis highlighted the tick saliva role on skin immune cell recruitment and its potential effect on local infection.

Ornithodoros brasiliensis Aragão é um carrapato endêmico do Brasil, restrito às regiões serranas e frias do estado do Rio Grande do Sul. É uma espécie bastante agressiva aos humanos, causando febre, muita dor e intensa resposta inflamatória no local da picada. O ciclo biológico de O. brasiliensis (ovo a adulto, única geração) realizado nos anos 50, utilizando ratos brancos como hospedeiros, levou um total de 103 e 184 dias de amplitude, com máximo de 7 ínstares ninfais observados e com emergência de adultos a partir de N4, estando os machos em maior proporção. Os hospedeiros apresentaram febre e uma bactéria espiroqueta denominada Borrelia brasiliensis foi descrita de esfregaços sanguíneos desses animais. Porém as borrélias não puderam ser cultivadas por muito tempo, e acabaram desaparecendo. Houve um silêncio de mais de 50 anos sobre relatos de ocorrência de O. brasiliensis, deixando inclusive dúvidas se a espécie estava extinta de fato. No entanto, há cerca de quatro anos, alguns espécimes de carrapatos da mesma localidade foram coletados e identificados como O. brasiliensis. O presente trabalho teve por objetivos estudar a morfologia e a biologia de O. brasiliensis, em condições de laboratório, bem como investigar a presença de bactérias do gênero Rickettsia em espécimes coletados. Ninfas e adultos foram alimentados em cobaias de laboratório e as fêmeas acasaladas realizaram posturas que resultaram nas gerações sucessivas. A larva e os adultos foram descritos e redescritos, e comparados com as espécies próximas, O. rostratus e O. turicata. Análises moleculares de uma porção do gene 16S rDNA mitocondrial foram comparadas com as sequências depositadas no GenBank e apresentaram valores de máximo bootstrap de 100% para ambas, O. rostratus e O. turicata. O ciclo biológico completo (ovo a ovo) de O. brasiliensis foi obtido entre 215,4 e 195 dias, em média, para duas gerações consecutivas, respectivamente. Os períodos médios de pré-oviposição, oviposição e incubação dos ovos (em dias), para a geração F1 foram 76,7 ± 27,01 (31-117); 15,55 ± 5,36 (10-24); 10,6 ± 4,5 (3-19) e para geração F2 foram 54,1 ± 8,7 (40-65), 15,8 ± 8,9 (7-36); 15,4. Os períodos médios de pré-ecdise larval foram de 12,5 ± 1,8 (10-15), considerando as duas gerações. A larva não se alimenta, enquanto que há um repasto sanguíneo para cada instar ninfal, antes de cada muda. Um total de 5 instares ninfais foi observado, sendo que os períodos médios de préecdise para os instares N1, N2, N3, N4 e N5, respectivamente para as duas gerações foram de 33,3 ± 4 (32-45), 38,6 ± 2,5 (36-47), 31,1 ± 3,4 (28-42), 34,8 ± 3,3 (31-43), 37,2 ± 2,4 (35-41) e 32,5 ± 3,3 (29-40), 37,5 ± 2,5 (34-44), 34,6 ± 3,2 (31- 37), 36,5 ± 2,5 (34-42), 32,5 ± 0,7 (32-33). Os períodos médios de alimentação para os ínstares ninfais, foram semelhantes nas duas gerações, sendo de 28,62 ± 3,67 (25,2 35,1) minutos. Adultos alimentam-se repetidas vezes, e as posturas sempre são precedidas de repasto sanguíneo, embora somente dois ciclos gonotróficos foram observados. O primeiro ciclo gonotrófico apresentou maior número de ovos depositados, com 139 ± 13,8 (53 197) em relação ao segundo 73,8 ± 10,6 (53 123). A investigação da presença de riquétsia foi avaliada em 107 espécimes recentemente coletados, evidenciando um total de 12 exemplares positivos. O produto amplificado pela PCR foi sequenciado e as análises moleculares apresentaram homologia de 98% com sequências disponibilizadas no Genbank, correspondentes a uma espécie asiática denominada RDa420 (AF497584) e outra brasileira, Rickettsia bellii (DQ865204) . / Ornithodoros brasiliensis Aragão is an endemic tick to Brazil, restricted to the highlands and cold regions of the state of Rio Grande do Sul. This species is very aggressive to humans, causing fever, great pain and intense inflammatory response at the site of the bite. The life cycle of O. brasiliensis (egg to adult, single generation) previously performed (studied during 50\') using white mice as host, spent a total of 103 and 184 days of amplitude, with maximum 7 nymphal instars observed, with adult emerging from N4, and males in greater proportion. The hosts had fever and spirochete bacteria called Borrelia brasiliensis was described from blood of the animals used. Nevertheless borrelias could not be cultivated for a long time, and they eventually disappeared. A silence of more than 50 years on reports of occurrence of O. brasiliensis, left doubts about whether the species was extinct. However, about four years ago, some specimens of ticks were collected in the same locality and identified as O. brasiliensis. This work aimed to study the morphology and biology of O. brasiliensis, under laboratory conditions, and investigate the presence of bacteria of the genus Rickettsia in the collected ticks. Nymphs and adults were fed on laboratory guinea pigs, and mated females laid eggs that resulted in the successive generations. The larvae and adults have been described and redescribed, respectively, and compared with the closely related species, O. rostratus and O. turicata. Molecular analyzes of a portion of the mitochondrial 16S rDNA gene was compared with the sequences deposited in GenBank, showing maximum bootstrap values out of 100% for both O. rostratus and O. turicata. In this study, the complete life cycle (egg to egg) of O. brasiliensis was obtained between 215,4 and 195 days, on average, for the two generations studied, respectively. The average periods of preoviposition, oviposition and incubation of the eggs to the F1 generation were 76,7 ± 27,01 (31-117), 15,55 ± 5,36 (10-24), and to F2 were 54,1 ± 8,7 (40-65), 15,8 ± 8,9 (7-36). The averaging periods to the preecdysis of the larvae were 12,5 ± 1,8 (10-15), considering the two generations. The larva does not feed, while for the nymphs there is a blood meal for each instar, before each molt. A total of five nymphal instars was observed, and the average periods of pre-molt instars for N1, N2, N3, N4 and N5, for two generations, respectively, were 33,3 ± 4 (32-45), 38,6 ± 2,5 (36-47), 31,1 ± 3,4 (28-42), 34,8 ± 3,3 (31-43), 37,2 ± 2,4 (35-41) and 32,5 ± 3,3 (29-40), 37,5 ± 2,5 (34- 44), 34,6 ± 3,2 (31-37), 36,5 ± 2,5 (34-42), 32,5 ± 0,7 (32-33). The average feeding time of nymphal instars, were similar in the two generations, being 28,62 ± 3,67 (25,2 35,1) minutes. Adults fed repeatedly, and the postures were always preceded by blood meal, although only two gonotrophic cycles have been observed. The first gonotrophic cycle presented higher average number of eggs deposited 139 ± 13,8 (53-197) that the second 73,8 ± 10,6 (53 123). The investigation to the presence of rickettsia was performed in 109 recently collected ticks, showing a total of 12 positive specimens. The amplified PCR product was sequenced and molecular analyzes showed 98% homology with sequences available in Genbank, corresponding to an Asian species named RDa420 (AF497584) e another Brazilian strain named Rickettsia bellii (DQ865204).