Made available in DSpace on 2016-04-28T20:15:23Z (GMT). No. of bitstreams: 1 2003-Katia Roberta Fernandes.pdf: 342140 bytes, checksum: e0a9b9e260d5d0a6cd41c984d6cfada1 (MD5) Previous issue date: 2003-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / There were made sensibility-tests in vitro of acaricides in engorged female Boophilus microplus, ticks coming from 12 cattle farms localized in different municipes in Rio de Janeiro-state, from March to September 2002. The following active principles, with the respective concentrations, were tested: Amitraz 250 ppm, cipermetrine high CIS 250 ppm, cipermetrine high CIS + DDVP 500 ppm and clorpyrifos + DDVP 500 ppm. Two groups of 10 engorged female ticks were used for each principle. Two groups with 10 females were used as controls, being treated with destilled water. The found results of the medium efficacy and the range (on amplitude) were: amitraz 45.40% (10.16 87.54); cipermetrine high CIS 37.61% (13.62 61.71); cipermetrine high CIS + DDVP 42.27% (23.87 76.87) and clorpyrifos + DDVP 88.95% (62.76 99.73). The active principles amitraz, cipermetrine high CIS and cipermetrine high CIS + DDVP were less efficient than the product containing clorpyrifos + DDVP, who proved out to being 11 of the studied cattlefarms. These differences in efficient treatment and control of tick B. microplus point out to the necessity of tests as well as for permanent monitoring control in order to find out the best acaricides for each farm. / Realizaram-se testes de sensibilidade in vitro a acaricidas em amostras de f?meas ingurgitadas de Boophilus microplus procedentes de 12 propriedades pecu?rias com atividade leiteria e de corte, localizadas em diferentes munic?pios do Estado do Rio de Janeiro, no per?odo de mar?o a junho de 2002. Para a an?lise da susceptibilidade das amostras de carrapatos, foram utilizados os princ?pios ativos nas seguintes concentra??es: amitraz 250 ppm, cipermetrina high CIS 250 ppm, cipermetrina high CIS + DDVP 500 ppm e clorpirif?s + DDVP 500 ppm; empregando-se dois grupos de 10 f?meas ingurgitadas para cada princ?pio ativo e um grupo controle (dois grupos de 10 f?meas ingurgitadas) submetido ? imers?o em ?gua destilada. Os resultados obtidos da m?dia de efic?cia e a amplitude foram, respectivamente: amitraz - 45,40% (10,16 - 87,54); cipermetrina high CIS - 37,61% (13,62 61,71); cipermetrina high CIS + DDVP- 42,27% (23,87 76,87) e clorpirif?s + DDVP - 88,95% (62,76 - 99,73). Os princ?pios ativos amitraz, cipermetrina high CIS e cipermetrina high CIS + DDVP foram os menos eficazes, e o produto a base de clorpirif?s + DDVP mostrou-se o mais eficiente em 11 das propriedades estudadas. A grande variabilidade na efic?cia dos princ?pios ativos avaliados no controle do carrapato B. microplus, demonstra a import?ncia de um monitoramento permanente para a indica??o dos acaricidas mais apropriados para cada propriedade.

Made available in DSpace on 2016-04-28T20:15:25Z (GMT). No. of bitstreams: 1 2007-Vanessa de Almeida Raia.pdf: 1677171 bytes, checksum: e3f8cffe7eb95e4096f3adf06552ae0c (MD5) Previous issue date: 2007-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / To fill some gaps on intrinsic mechanisms of the biology of the oviposition and embryogenesis of R. (B.) microplus and R. (R.) sanguineus, was evaluated the variability protein in eggs per day of posture and incubation. For this, engorged female laid eggs in controlled environment chamber (27 ? 1oC, 80 ? 5% UR, and darkness). As soon as the female initiated the oviposition, egg samples of 50 mg was collected daily, conditioned in eppendorf tube and preserved in freezer - 20?C, characterizing the period of posture. Samples of 50 mg was removed from a fresh egg mixture and put in perforated eppendorf tube, kept in environment chamber (27 ? 1oC, 80 ? 5% UR, and darkness). Daily, one tube was transferred to freezer -20?C until the first larva hatch. Thus a sequence of different stage of embryogenesis was obtained. The Bradford method was used to measure the protein concentration and subsequently, the electrophoresis profiles was performed in SDS-PAGE. The protein concentrations was correlated with the oviposition and embryogenesis days using the Pearson (r) correlation and for this, the data was transformed in logarithmic value [log (X+1)] after the normality to be discarded (test of Shapiro-Wilk). During oviposition the protein concentrations of the eggs of R. (B.) microplus and R. (R.) sanguineus remained constant until the last days when abrupt increase was observed. In both of species, variation in the concentration of protein was observed during all embryonic period. The number of detectable bands of proteins decreasing during oviposition and embryogenesis days, but in the last days a new band was found. It can inferred that the proteins variation in the eggs of R. (B.) microplus and R. (R.) sanguineus is correlated with the days oviposition and incubation. The ticks R. (B.) microplus and R. (R.) sanguineus have a different oviposition profile proteins model that can be use as phenetic feature. As well, a different way of degradation of protein for each species was characterized. / Objetivando preencher algumas lacunas sobre mecanismos intr?nsecos da biologia da oviposi??o e embriog?nese de R. (B.) microplus e R. (R.) sanguineus, foi avaliada a variabilidade prot?ica dos ovos por dia de postura e incuba??o. Para tal, f?meas ingurgitadas foram colocadas para efetuar postura em estufa biol?gica sob condi??es controladas (27 ? 1oC, 80 ? 5% UR, escotofase). Ap?s in?cio da postura, amostras di?rias de 50 mg de ovos foram coletadas, acondicionadas e preservadas a 20?C, caracterizando o per?odo de postura. A partir de um pool de ovos rec?m colocados, foram obtidas al?quotas de 50 mg que acondicionadas em tubos de eppendorf perfurados foram mantidas em estufa biol?gica nas mesmas condi??es controladas descritas acima. Desde a separa??o das al?quotas at? o surgimento da primeira larva, diariamente uma amostra foi transferida para freezer ? -20?C, obtendo-se assim ovos seq?encialmente em diferentes momentos da embriog?nese. Para dosagem das prote?nas utilizou-se o m?todo de Bradford, e os perfis eletrofor?ticos foram tra?ados atrav?s de eletroforese em gel de poliacrilamida. Os dados das concentra??es prot?icas foram correlacionados com os dias de postura e de embriog?nese. Para isso, utilizou-se o coeficiente de correla??o de Pearson (r), com os dados da concentra??o transformados logar?timicamente [log (X+1)]. Os dados foram transformados ap?s o descarte da normalidade (teste de Shapiro-Wilk). De modo geral as concentra??es das prote?nas nos ovos de R. (B.) microplus e R. (R.) sanguineus durante a postura mantiveram-se constantes at? os ?ltimos dias quando se observou aumento abrupto das concentra??es. Nas duas esp?cies, foram observadas varia??es nas concentra??es das prote?nas durante o per?odo embrion?rio. Ainda em ambas esp?cies, na an?lise das bandas prot?icas, o n?mero de bandas detect?veis diminuiu ao longo do per?odo de postura e embriog?nese, sendo observado nos ?ltimos dias surgimento de uma nova banda. Pode-se depreender que a varia??o na concentra??o das prote?nas dos ovos de R. (B.) microplus e R. (R.) sanguineus est? correlacionada com os dias de postura e incuba??o, atrav?s do aumento na concentra??o de prote?nas ? medida que o final da postura e eclos?o da larva se aproximam. Devido ?s diferen?as entre os perfis prot?icos de R. (B.) microplus e R.(R.) sanguineus ao longo dos dias de postura, conclui-se que as prote?na s disponibilizadas aos ovos durante o per?odo de postura s?o diferentes entre estas duas esp?cies e que os zimogramas podem ser utilizados como marcadores fen?ticos. Ainda pode-se concluir que, ao longo da embriog?nese, devido ao desaparecimento e surgimento de bandas prot?icas, as prote?nas dispon?veis para o embri?o de R. (B.) microplus e R.(R.) sanguineus s?o biotransformadas de modo que h? um perfil de degrada??o particular para cada esp?cie.

Made available in DSpace on 2016-04-28T20:15:30Z (GMT). No. of bitstreams: 1 2008- Jania de Resende.pdf: 467062 bytes, checksum: cf6acc7c0e32afbfa397b5b1c07d0d87 (MD5) Previous issue date: 2008-02-22 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Embryonic cells of tick in vitro constitute an important one tool for culture and study of the biology of B. burgdorferi. Spirochetes Borrelia burgdorferi is the aetiologic agent of borreliose of Lyme in U.S.A. and Europe, where it is transmitted by tick of the Ixodes genus. The aim of this work was in vitro to cultivate B. burgdorferi (American Cepa G39/40) in primary culture of embryonic cells of Rhipicephalus (B.) microplus and the A. cajennense. From the primary culture of embryonic cells of R. (B.) microplus were performed subculture maintained with medium Leibovitz's (L-15) free of antibiotic, that later it were changed by medium Barbour-Stoener-Kelly (BSK). After the addition of the BSK inoculated B. burgdorferi of and also in Tube of Leighton (TL) with free BSK of cells (control) in a final concentration of 6x106 spirochetes/mL. The embryonic cells of the A. cajennense initially were cultivated in medium L-15 with antibiotic, which was substituted by the BSK. Later, 1,1x107 spirochetes/mL in medium BSK cultivated was inoculated, and also in TL controlled free of cells. All the culture was incubated at 34?C. The development of the culture was observed in microscope of inverted contrast of phase, as well as the countings of B. burgdorferi performed in chamber of neubauer. Cover glass of the TL had been stained with Giemsa. It was evidenced by the observation in microscope of inverted contrast of phase the survival, attach and multiplication of B. burgdorferi, in the embryonic cells of R. (B.) microplus and A. cajennense. It did not have differences in the finale counting of spirochetes cultivated in cells of R. (B.) microplus when compared with the free control of cells, but on with cells of the A. cajennense, the number amount approximately was 1,9x107 spirochetes/mL, and while in the tube it has controlled free of cells was 1x106 spirochetes /mL. The culture of cells of tick R. (B.) microplus and the A. cajennense have potential to be used as substratum for culture of B. burgdorferi, and study of its development. / C?lulas embrion?rias de carrapatos mantidas in vitro constituem uma importante ferramenta para cultivo e estudo da biologia de Borrelia burgdorferi. A espiroquetas B. burgdorferi ? o agente etiol?gico da borreliose de Lyme nos EUA e Europa, onde ? transmitida por carrapatos do g?nero Ixodes. O objetivo deste trabalho foi cultivar in vitro Borrelia burgdorferi (Cepa Americana G39/40) em cultura prim?ria de c?lulas embrion?rias dos carrapatos Rhipicephalus (Boophilus) microplus e Amblyomma cajennense. A partir da cultura prim?ria de c?lulas embrion?rias de R. (Boophilus) microplus foram realizados subcultivos mantidos com meio Leibovitz s L-15 livre de antibi?tico, que posteriomente foi trocado pelo meio Barbour-Stoener-Kelly (BSK). Ap?s a adi??o do BSK na cultura, inoculou-se B. burgdorferi e tamb?m em Tubo de Leighton (TL) com BSK livres de c?lulas (controle), numa concentra??o final de 6x106 espiroquetas/mL. As c?lulas embrion?rias de A. cajennense foram inicialmente cultivadas em meio L-15 com antibi?tico, o qual foi substitu?do pelo BSK. Posteriormente, inoculou-se 1,1x107 espiroquetas/mL cultivadas em meio BSK, e tamb?m em TL controle livre de c?lulas. Todos os cultivos foram incubados em estufa bacteriol?gica a 34?C. O desenvolvimento dos cultivos foram observados em microsc?pio de contraste de fase invertido, assim como as contagens de B. burgdorferi realizados em c?mara de neubauer. As lam?nulas dos TL foram coradas com Giemsa. Foi constatado pela observa??o em microsc?pio de contraste de fase invertido a sobreviv?ncia, ader?ncia e multiplica??o de B. burgdorferi, nas c?lulas embrion?rias de R. (Boophilus) microplus e A. cajennense. N?o houve diferen?as na contagem final de espiroquetas cultivadas em c?lulas de R. (Boophilus) microplus quando comparada ao controle livre de c?lulas, mas sobre c?lulas de A. cajennense o valor total de aproximadamente 1,9x107 espiroquetas/mL, e enquanto no tubo controle livre de c?lulas foi 1x106 espiroquetas/mL. O cultivo de c?lulas do carrapato R. (Boophilus) microplus e A. cajennense t?m potencial para ser utilizado como substrato para cultivo de B. burgdorferi e para estudo de seu desenvolvimento.

Made available in DSpace on 2016-04-28T20:15:31Z (GMT). No. of bitstreams: 1 2008 - Charles Passos Rangel.pdf: 970968 bytes, checksum: 8211b14a033b6089d8ed8bbdef93fe27 (MD5) Previous issue date: 2008-08-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Artificial feeding is an important tool, as an option to minimize the use of animals in scientific experiments, providing study of the transmission of bioagentes in the absence of vertebrate host. The objectives of this study were artificially food through capillary tubes females partially engorged of the tick Rhipicephalus (Boophilus) microplus previously fed on cattle and to check the influence of the technique in the biological aspects of non-parasitic phase this species. Females weighing between 40,6 and 69,7 milligrams were separated into four groups of homogeneous weight, with 10 females in each and set on trays of styrofoam with double-sided tape. For artificial feeding tubes of microhematocrit containing blood citrated cattle were placed on the apparatus mouthpiece of ticks. The groups were fed by six, 12, 24, 36 hours, being kept under temperature of 27 ? 1 ? C and humidity above 80%. After artificial feeding, the ticks were weighed to verify the ingestion of blood. To monitoring the biological aspects, the ticks were set in Petri dishes, and kept in the same controled temperature and humidity, describe above. The control group was formed by engorged females from cattle, being kept in the same conditions of temperature and humidity of the groups artificially fed. For statistical analysis was used variance analysis and Tukey's test with a significance level of 5%, for comparisons of means. The average weights acquired in milligrams were 12.33 ? 15.17; 33.41 ? 21.27; 67.53 ? 27.57; 79.47 ? 45.53 in groups 6, 12, 24, 36 hours, respectively. The two groups exposed for less time to capillaries, showed statistical difference compared with the gain average of weight of the group of 36 hours. From 24 hours of feeding was observed significant difference between the biological aspects of the groups artificially fed, except for the posture period. Although the females artificially fed through capillary tubes have not reached full engorgiment, the findings show that the technique did not presented deleterious effect on the biological aspects of this species. / Alimenta??o artificial ? uma ferramenta importante, por constituir op??o para minimizar o uso de animais na experimenta??o cient?fica propiciando estudo da transmiss?o de bioagentes na aus?ncia do hospedeiro vertebrado. Os objetivos deste estudo foram alimentar artificialmente, por meio de tubos capilares, f?meas parcialmente ingurgitadas do carrapato Rhipicephalus (Boophilus) microplus previamente alimentadas em bovinos e verificar a influ?ncia da t?cnica, nos aspectos biol?gicos da fase n?o-parasit?ria desta esp?cie. F?meas pesando entre 40,6 e 69,7 miligramas foram separadas em quatro grupos de peso homog?neo, com 10 f?meas cada e fixadas em bandejas de isopor com aux?lio de fita dupla face. Para alimenta??o artificial, tubos de microhemat?crito contendo sangue bovino citratado foram posicionados sobre o aparelho bucal dos carrapatos. Os grupos foram alimentados por 6, 12, 24, 36 horas, sendo mantidos em estufa, ? temperatura de 27 ? 1?C e umidade superior a 80%. Ap?s alimenta??o artificial, os carrapatos foram pesados para verifica??o da ingest?o de sangue. Para acompanhamento dos aspectos biol?gicos, os carrapatos foram fixados em placas de Petri, e incubados nas mesmas condi??es de temperatura e umidade descritas acima. O grupo controle foi formado a partir de f?meas ingurgitadas oriundas de bovinos infestados experimentalmente, mantidas nas mesmas condi??es de temperatura e umidade dos grupos alimentados artificialmente. Para an?lise estat?stica utilizou-se an?lise de vari?ncia e teste de Tukey com n?vel de signific?ncia 5%, para compara??es das m?dias. Os pesos m?dios em miligrama adquiridos foram 12,33?15,17; 33,41?21,27; 67,53?27,57; 79,47?45,53 nos grupos 6, 12, 24, 36 horas, respectivamente. Os dois grupos expostos por menos tempo aos capilares apresentaram diferen?a estat?stica em rela??o ao ganho m?dio de peso do grupo de 36 horas. A partir de 24 horas de alimenta??o foi observada diferen?a significativa entre os aspectos biol?gicos dos grupos alimentados artificialmente, exceto para o per?odo de postura. Embora as f?meas alimentadas artificialmente por meio de tubos capilares n?o tenham atingido ingurgitamento total, os resultados apresentados demonstram que a t?cnica n?o apresentou efeito delet?rio sobre os aspectos biol?gicos da esp?cie.

Neste estudo avaliamos a ocorrência de fagocitose por parte de alguns tipos celulares presentes na hemolinfa do carrapato bovino B. microplus e a produção de espécies reativas de oxigênio durante a resposta imune. As técnicas empregadas para avaliação da produção de espécies reativas de oxigênio foram luminescência amplificada por luminol, oxidação de fenol vermelho, microscopia de fluorescência e fluorimetria com o corante dihidrorrodamina 123 (DHR). Observamos um aumento da luminescência amplificada por luminol quando hemócitos foram incubados na presença de bactérias Micrococcus luteus ou zimosam ou PMA. Esta luminescência foi inibida por superóxido dismutase (SOD) e por catalase (CAT), enzimas antioxidantes que removem superóxido e peróxido de hidrogênio, respectivamente. LPS não elicitou aumento da luminescência dos hemócitos em relação ao controle. Através da oxidação de fenol vermelho em reação inibida por CAT, verificamos aumento nos níveis de H2O2 produzido pelos hemócitos quando estimulados com PMA e Micrococcus luteus, enquanto não houve aumento quando o estímulo foi LPS, corroborando os resultados da luminescência. Usando microscopia de fluorescência para avaliar a produção de ERO pelos hemócitos, encontramos que cerca de 25% dos hemócitos fluorescem com maior intensidade quando estimulados com zimosam, sendo esta fluorescência inibida por CAT. Através de fluorimetria usando DHR observamos um aumento na intensidade de fluorescência dos hemócitos estimulados com PMA em reação inibida por cisteína, substância redutora que remove peróxido de hidrogênio e peroxinitrito. Nosso conjunto de resultados permitem concluir que os hemócitos do carrapato bovino produzem espécies reativas de oxigênio durante a resposta imune, semelhantemente ao que ocorre em vertebrados e em invertebrados como moluscos, crustáceos e insetos. Este é o primeiro trabalho mostrando produção de ERO pelos hemócitos de aracnídeos. / The phagocytic activity and the reactive oxygen species (ROS) production during immune response of some hemocytes of the cattle tick Boophilus microplus were evaluated in this study. The ROS production was evaluated by luminol amplified luminescence, phenol red oxidation, dyhydrorhodamine (DHR) fluorescence microscopy and fluorimetry. The luminol-amplified luminescence increased when hemocytes were incubated with bacteria (Micrococcus luteus) or zymosam or phorbol 12-miristate 13 acetate (PMA). The superoxide dismutase (SOD) and catalase (CAT), antioxidant enzymes that removes superoxide and hydrogen peroxide, respectively, inhibited this luminescence. Lipopolysaccharide (LPS) did not elicit luminescence of hemocytes in relation to controls. The catalase-inhibittable phenol red oxidation assay also showed an increase in the level of hydrogen peroxide produced by hemocytes stimulated with PMA or Micrococcus luteus. LPS did not stimulate the hemocytes, similarly to the observed by luminescence assay's. We also evaluated ROS production by fluorescence microscopy and we found approximately 25% more fluorescent hemocytes when zymosam was used. This fluorescence was inhibited by catalase. In DHR fluorimetry assay we observed an increase in the intensity of fluorescence in PMA stimulated hemocytes. This fluorescence was inhibited by cystein, a reducing agent that removes hydrogen peroxide and peroxinitrite. We conclude that hemocytes of the tick, like other invertebrate such as mollusks, crustaceans and insects and vertebrate, produce reactive oxygen species during the immune response. This is the first report of reactive oxygen species production by arachnid hemocytes.

O carrapato Amblyomma cajennense é o principal vetor da bactéria Rickettsia rickettsii, agente etiológico da febre maculosa brasileira (FMB). Os estágios imaturos destes artrópodes apresentam uma baixa especificidade para os hospedeiros, o que aumentam as chances de parasitismo em humanos. Nos anos de 2011 e 2012, a vigilância epidemiológica da FMB registrou 140 casos confirmados e letalidade de 50 por cento , a maior incidência desde a regulamentação da notificação compulsória no Estado de São Paulo, em 2001. Além disso, estudos indicam uma tendência de aumento de expansão geográfica e de número de casos da doença. A fim de aplicar medidas de controle para a FMB, a determinação de quais são os animais hospedeiros para as fases imaturas do carrapato é importante para identificar as fontes de infecção de bactérias. Entre a literatura científica não havia estudos sobre esse escopo para carrapatos da América do Sul. Neste estudo, uma abordagem para a detecção de hábito alimentar de A. cajennense foi padronizada. Resumidamente, as amostras de sangue foram coletadas a partir das seguintes espécies animais: frango, capivara, codorna, cavalo, cobaia, coelho, cachorro e um camundongo silvestre. Em seguida, o DNA foi extraído a partir destas amostras e, depois, testado para a amplificação por PCR utilizando-se três pares de diferentes oligonucleotídeos iniciadores para mamíferos, três para aves e cinco para os dois grupos de animais, além de oligonucleotídeos iniciadores específicos desenhados para roedores cricetídeos. Os genes alvos 12S rDNA, cyt b e COI resultou em positivo para a detecção de fragmentos de DNA. Por PCR foi testado posteriormente em laboratório repastos de carrapatos. Carrapatos adultos de A. cajennense que foram alimentados em coelhos quando larvas e ninfas tiveram o intestino extraído e processado para o isolamento de DNA que foi submetido à amplificação por PCR. Foi possível identificar a espécie hospedeira em 66,7 por cento dos carrapatos testados. O sequenciamento de DNA e a comparação das sequências consenso com todas as sequências do banco de dados (GenBank) permitiu a identificação em nível de espécie (coelho), com base em 98 por cento de similaridade. / The tick Amblyomma cajennense is the main vector of the bacterium Rickettsia rickettsii, the etiological agent of brazilian spotted fever (BSF). The subadult stages of this arthropod present a low specificity for hosts, which increases the chances of parasitism in humans. In the years of 2011 and 2012, the BSF epidemiological surveillance recorded 140 confirmed cases and 50 per cent case-letality rate, the highest incidence since the regulation of the compulsory notification in the State of São Paulo, in 2001. Furthermore, studies indicate an increase trend for geographical expansion and number of cases of the disease. In order to apply control measures for BSF, the determination of which is the vertebrate hosts for the immature stages of the tick is important to identify the sources of infection of bacteria. Among the scientific literature there was no studies on this scope for ticks of South America. In this study, it was standardized a approach for detection of feeding habits of A. cajennense. Briefly, blood samples were collected from the following animal species: chicken, capybara, quail, horse, guinea pig, rabbit, dog and a wild mouse. Then, DNA was extracted from these samples and afterwards tested for PCR amplification using three different pairs of primers for mammals, three for birds, and five for both groups of animals in addition to a specific designed primers for cricetidae rodents. The target gene 12S rDNA, cyt b and COI resulted in positive for detection of DNA fragments. PCR was tested thereafter on laboratory fed ticks. Adult A. cajennense ticks that were fed on rabbits as larvae and nymphs had the midguts extracted and processed for DNA isolation and underwent PCR amplification. It was possible to identify the host species on 66,7 per cent of tested ticks. The DNA sequencing and comparison of the consensus sequences of all the database sequences (GenBank) allowed the identification at the species level (rabbit), based on 98 per cent similarity.

Leishmaniasis is a parasitic disease that affects humans and animals in more than 98 countries across the globe placing more than 1 billion people at risk for the disease and killing more than 20,000 people per year. In the United States the disease is enzootic within the hunting dog population and vertical transmission has been identified as the primary route of transmission in this population. In Brazil the disease is endemic in the human population and enzootic in the dog population with vector and vertical transmission having been reported. In many diseases reports have found there is increased disease severity when an individual is co-infected with another organism. Case reports have suggested this may also occur with tick borne diseases and leishmaniosis in dogs but there is limited longitudinal data to support this relationship. Even less is known and understood regarding the risk factors and basic reproduction number, number of secondary cases one infected individual can cause in a susceptible population, of leishmaniosis in regards to vertical transmission. The goal of the work presented in this thesis is to address host factors related to the transmission of L. infantum and the way in which co-infections affect the progression of the disease both in the U.S. and in Brazil. Understanding the risk factors associated with the transmission of the parasite Leishmania infantum, the causative agent of the disease, are necessary to controlling and potentially elimination the disease. Utilizing a large prospective cohort and both active and passive surveillance it was identified that leishmaniosis can be maintained in a population via vertical transmission at prevalence rates similar to other endemic countries, 20%. With this knowledge an additional study examining a longitudinal cohort and assessing the impact of tick borne disease co-infections upon disease transmission was performed. It was identified that dogs exposed to three or more tick borne diseases were 11x more likely to progress to clinical disease (Adjusted RR: 11.64 95% CI: 1.22-110.99 p-value: 0.03) than dogs with no tick borne disease exposures. Furthermore, dogs with Leishmania and tick borne disease were 5x more likely to die within the study (RR: 4.85 95% CI: 1.65-14.24 p-value: 0.0051). When examining this relationship in a cross-sectional study in Brazil it was found that dogs with multiple tick borne disease exposures had 1.68x greater risk of being positive for Leishmania (Adjusted RR: 1.68 95% CI: 1.09-2.61 p-value: 0.019). Using a retrospective cohort of dogs and information regarding their dam’s diagnostic status near the time of pregnancy risk factors associated with vertical transmission and the basic reproduction number were calculated. It was found that dogs who were born to dams that were ever diagnostically positive for exposure and/or infection with L. infantum were 13.84x more likely become positive for L. infantum within their lifetime (RR: 13.84 95% CI: 3.54-54.20 p-value < 0.0001). The basic reproduction number for vertically transmitted L. infantum within this cohort was 4.16. The results of these studies suggest that leishmaniosis can be maintained in a population through vertical transmission. Furthermore, the studies show the risk factors associated with vertical transmission relate to the mother’s diagnostic status at time of pregnancy. The results of the co-infection studies highlight the importance of tick prevention in order to reduce disease progression. With increased disease severity associated with increased transmission to potential vectors these studies underline the need for immunotherapies and prevention measures to reduce disease progression in order to reduce transmission. Furthermore, these studies highlight the need for public health control and prevention programs to address vertical transmission if elimination of the disease is to ever be successful.

本研究中，我們計算OGA演化投資策略在五類時間數列模型上之表現，這五類模型分別是線性模型、雙線性模型、自迴歸條件異質變異數模型、門檻模型以及混沌模型。我們選擇獲勝機率、累積報酬率、夏普比例以及幸運係數做為評斷表現之準則，並分別推導出其漸近統計檢定。有別於一般計算智慧在財務工程上之應用，利用蒙地卡羅模擬法，研究中將對各表現準則提出嚴格之統計檢定結果。同時在實証研究中，我們考慮歐元兌美元及美元兌日圓的tick-by-tick匯率資料。故本研究主要的重點之一，乃是對於OGA演化投資策略，於這些模擬及實証資料上的有效性應用，作了深入且廣泛的探討。 / In this study, the performance of ordinary GA-based trading strategies are evaluated under five classes of time series model, namely, linear ARMA model, bilinear model, ARCH model, threshold model and chaotic model. The performance criteria employed are the winning probability, accumulated returns, Sharpe ratio and luck coefficient. We then provide the asymptotic statistical tests for these criteria. Unlike many existing applications of computational intelligence in financial engineering, for each performance criterion, we provide a rigorous statistical results based on Monte Carlo simulation. In the empirical study, two tick-by-tick foreign exchange rates are also considered, namely, EUR/USD and USD/JPY. As a result, this study provides us with a thorough understanding about the effectiveness of ordinary GA for evolving trading strategies under these artificial and natural time series data.

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.

The incidence of tick-borne zoonoses such as Ehrlichiosis, Rocky Mountain Spotted Fever, and Lyme disease has steadily increased in the southeastern United States in recent years. According to the Centers for Disease Control and Prevention (CDC), the southeastern states accounted for 1,200 of the 27,000 total cases of Lyme disease reported in the U.S. in 2007. Although Ixodes scapularis is the most commonly recognized vector for the Lyme disease spirochete Borrelia burgdorferi, Dermacentor variabilis (a common vector for Rocky Mountain Spotted Fever) also has been shown to be a viable host for this pathogen. The purpose of the present study was to use PCR and DNA sequencing technologies to determine if Borrelia burgdorferi sensu lato is present in ticks and whole blood samples removed from raccoons and opossums trapped in south-central Kentucky. Raccoons and opossums were trapped in Barren and Warren counties of Kentucky between June 2007 and June 2008. Ticks were removed and stored in 70% ethanol. Sterile blood samples were collected into three 10 ml tubes containing the anticoagulant K2EDTA and stored at 4°C. Genomic DNA was extracted from ticks and blood samples using a QIAamp DNA mini kit and a QIAamp DNA blood mini kit (Qiagen) respectively. DNA samples were analyzed by polymerase chain reaction (PCR) for the presence of B. burgdorferi using oligonucleotide primers specific for the OspA gene. A total of 976 ticks were collected. Three different species were obtained from raccoons; Dermacentor variabilis, Amblyomma americanum, and Ixodes sp. Dermacentor variabilis was the only tick species found on opossums. Twenty-five percent (163/642) of the tick DNA samples were positive for Borrelia burgdorferi. Prevalence of B. burgdorferi by tick species was 24.4% (141/577) in D. variabilis, 40.6% (13/32) in A. americanum, and 27.6% (8/29) in I. scapularis. In the present study, 15.7% (8/51) of the total raccoon blood samples examined by PCR were positive for B. burgdorferi, while no opossum blood samples were positive. The high prevalence of B. burgdorferi in ticks common to raccoons and opossums observed in this study, as well as in a tick species that aggressively bites humans in the southeast U. S. (A. americanum), creates concern that there are ample opportunities for people to come in contact with the infected ticks on these animals. Future studies are urgently needed to fully assess the presence and prevalence of B. burgdorferi in Kentucky and other southeastern states in the U. S.