The aim of this study was to evaluate new methods to improve detection and investigation of the effects of chronic or subclinical infection with Trypanosoma evansi in various mammalian species. Some of the more resistant host species, including pigs and buffaloes, are present in large feral populations in the northern parts of Australia, the area where T. evansi is most likely to gain entry to the country. Existing tests are not sufficiently reliable to detect all cases of disease and they cannot distinguish acute from chronic infections. Furthermore, the tests have different sensitivities in different host species. Surveillance for trypanosomiasis in Australia is problematic because of the need to work in remote parts of northern Australia where provision of a cold-chain for traditional blood and serum storage is difficult. An existing dried blood storage system was modified by treating cotton lint filter paper (Whatman #903) with a commercial post coating buffer (TropBio, Queensland). This treatment increased the longevity of antibodies to T. evansi in serum and blood stored on the paper (detected using an antibody-detection ELISA) compared to samples stored on plain paper, especially when the papers were stored under humid conditions and at high ambient temperatures. Attempts were made to improve the diagnostic utility and repeatability of antibody-ELISAs through the use of 2 recombinant T. brucei antigens (PFRA and GM6) and to optimize a competitive ELISA using RoTat 1.2 variable surface antigen and its monoclonal antibody. Antibody-detection using the two recombinant proteins was not sufficiently specific to enable their use for the detection of T. evansi. The RoTat 1.2 cELISA had good sensitivity and specificity (75% and 98% respectively) when used to test serum from cattle and buffaloes experimentally infected with T. evansi and uninfected animals. However, the test was not able to detect anti-T. evansi antibodies in serum from wallabies, pigs, a dog or a horse that were experimentally infected with T. evansi. The inability of the cELISA to detect anti-T. evansi antibodies may be due to the small number of samples tested or the lack of RoTat 1.2 specific antibodies in the animals tested. The feasibility of using an enzymatic test to detect trypanosome aminotransferase or antibodies to this enzyme was evaluated. Prior publications suggested that the detection of TAT was an appropriate diagnostic tool for the detection of T. evansi infection in camels. However, the results from this study did not support the use of this test for the detection of T. evansi infection in cattle or buffaloes with low to moderate parasitaemia. Trypanosomiasis is an immunological disease that affects most of the body’s organs, with more severe disease developing over time. Attempts were made to determine key cytokine and biochemical patterns that would distinguish infected from uninfected animals and acute from chronic infections. The results from this study showed that there was no specific pattern in serum cytokines or serum biochemistry that could be used to distinguish infected from uninfected animals, or different stages of disease. Immunohistochemistry was used on tissues from buffaloes and mice experimentally infected with T. evansi and T. brucei gambiense respectively to characterise the cellular immune response that was present. The immune response was predominantly cell mediated, with CD3+ T lymphocyte and macrophage infiltration occurring in most tissues. In end stage disease there was often suppression of the immune system with disruption of the architecture of the spleen and a decrease in B lymphocytes in the circulation. Trypanosomes were rarely visible in the tissues and were only seen in those animals with high parasitaemia. Lesions generally became more severe over time, but there was a large variation between animals, which suggests that immunohistochemistry is unsuitable as a diagnostic tool.

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 dissertacao_marcelo_gotze.pdf: 556489 bytes, checksum: cb0ceeb57d3c58da117f1a655a510d0d (MD5) Previous issue date: 2010-03-18 / Bovine babesiosis caused by Babesia bovis and Babesia bigemina, is the most important disease transmitted by Rhipicephalus (Boophilus) microplus in tropical and subtropical areas in South America. Definitive diagnosis can be made by detecting infected erythrocytes in blood smears. However, the parasitemia in peripheral blood is often too low for this method to be used for diagnostic purposes. For this reason, several serological tests, including complement fixation, indirect hemagglutination and indirect immunofluorescence (IIF) have been used to detect antibodies in infected cattle. Although these tests allow the detection of persistently infected animals, they have limitations in specificity and/or sensitivity. The IIF has been the most sensitive, but cross-reactivity between species, subjective interpretation, and low production has limited its usefulness. The enzyme linked immunosorbent assay (ELISA) have found wide application in the diagnosis of infectious diseases. The cELISA format (competitive) can provide an additional level of specificity, because the antibody is directed to a single epitope, specific for the organism to be detected. For these reasons, this study aimed to evaluate the sensitivity and specificity of cELISA compared to IIF and nested PCR (nPCR) for diagnosis babesiosis caused by B. bovis. Therefore, blood samples were collected from cattle in Brazil and Argentina, and processed for the diagnosis of B. bovis. The nPCR was used as the gold standard to validate the cELISA and the IIF as a comparative test. The cELISA for the diagnosis of B. bovis presented is easily processed with high levels of sensitivity and specificity. It is easily performed in a high number of samples, making it useful in cases of outbreaks of bovine babesiosis. / A babesiose bovina, causada por Babesia bovis e Babesia bigemina, é a doença mais importante transmitida por carrapatos Rhipicephalus (Boophilus) microplus em áreas tropicais e subtropicais da América do Sul. O diagnóstico definitivo pode ser feito através da detecção de eritrócitos infectados em esfregaços sanguineos, porém a parasitemia em sangue periférico é frequentemente muito baixa para que esse método seja utilizado de forma confiável para fins de diagnóstico. Por esse motivo, vários testes sorológicos, incluindo a fixação de complemento, hemaglutinação indireta e imunofluorescência indireta (IIF) têm sido usados para detectar anticorpos em bovinos infectados. Embora estes testes permitam a detecção de animais persistentemente infectados, eles têm limitações na especificidade e/ou sensibilidade. O IIF tem sido o mais sensível, mas a reatividade cruzada entre as espécies, interpretação subjetiva, e baixa produção tem limitado a sua utilidade. Os ELISA têm encontrado ampla aplicação no diagnóstico de doenças infecciosas. O formato cELISA (competitivo) pode fornecer um nível adicional de especificidade, pois o anticorpo é dirigido para um epitopo único específico para o organismo a ser detectado. Por estas razões, este estudo teve como objetivo avaliar a sensibilidade e especificidade do cELISA comparado com IIF e nested PCR (nPCR) para diagnóstico de babesiose causada por B. bovis. Para tanto, amostras de sangue bovino foram coletadas no Brasil e na Argentina e processadas para o diagnóstico de B. bovis. Utilizou-se o nPCR como teste padrão para a validação do cELISA, e a IIF como teste comparativo. O cELISA para diagnóstico de B. bovis apresentou-se de fácil processamento, com altos níveis sensibilidade e especificidade, além da rapidez no processamento de amostras em larga escala, sendo de grande utilidade para casos de surtos de babesiose bovina

Com o objetivo de avaliar os equinos alojados no Jóquei Clube de São Paulo, Brasil, quanto a presença de anticorpos contra Theileria equi e Babesia caballi, foram testadas 180 amostras de soro sanguíneo por meio da técnica de C-ELISA (Competitive Enzyme-Linked Immunosorbent Assay), metodologia atualmente recomendada pela OIE (Organização Internacional de Epizootíases) por ter alta sensibilidade e especificidade. A frequência de animais com sorologia positiva para Theileria equi foi de 6,66% (12/180), para Babesia caballi foi de 22,3% (40/180) e para infecção concomitante foi de 6,66% (12/180). Os resultados sorológicos obtidos por este estudo revelam que 35,5% (64/180) dos animais possuem anticorpos contra a babesiose equina sendo que a maioria dos animais acometidos tem dois e três anos de idade e portanto estão há menos tempo no hipódromo. Fatores como a ausência de carrapatos vetores, o uso de terapias babesicidas repetidas e o longo tempo de permanência dos animais no Jóquei após o tratamento, favorecem a diminuição dos títulos de anticorpos sem que ocorra reinfecção. Esses fatores podem justificar o menor número de animais com sorologia positiva para a doença nos cavalos com idade acima de quatro anos. Considerando-se esses resultados sugere-se que os animais sejam avaliados sorologicamente ao ingressarem no Jóquei Clube de São Paulo para que o uso de medicamentos contra a doença seja feito de forma adequada e para que os sinais clínicos compatíveis com babesiose equina em animais sorologicamente negativos sejam melhor avaliados e considerados em diagnósticos diferenciais. / In order to evaluate the presence of antibodies against Theileria equi and Babesia caballi in horses kept at the Jockey Club in São Paulo city, Brazil, a total of 180 samples of blood serum was tested using the Competitive Enzyme-Linked Immunosorbent Assay (C-ELISA test). This methodology has been recommended by the International Organization of Epizooties (IOE) due to its high sensitivity and specificity. The frequency of seropositive animals for Theileria equi, Babesia caballi and for both was 6.66% (12/180), 22.3% (40/180) and 6.66% (12/180), respectively. Serological results showed that 35.5% of the animals (64/180) had antibodies against equine piroplasmosis; they were from two to three years old and were at the Jockey Club for a shorter period of time. Factors such as absence of thick vectors, repeated therapy using babesicidal drugs and the long period of time that the animals stayed in the Jockey Club after treatment favoured the lowering of antibody titers with no reinfection. These factors might be responsible for the fewer number of animals with positive serology for the disease in horses over four years of age. Based on these findings, animals should be serologically evaluated at the time of entrance into the Jockey Club so that the use of drugs against the disease be performed properly and clinical signs suggestive of equine babesiosis in serologically negative animals be better evaluated and considered for differential diagnosis.

Com o objetivo de avaliar os equinos alojados no Jóquei Clube de São Paulo, Brasil, quanto a presença de anticorpos contra Theileria equi e Babesia caballi, foram testadas 180 amostras de soro sanguíneo por meio da técnica de C-ELISA (Competitive Enzyme-Linked Immunosorbent Assay), metodologia atualmente recomendada pela OIE (Organização Internacional de Epizootíases) por ter alta sensibilidade e especificidade. A frequência de animais com sorologia positiva para Theileria equi foi de 6,66% (12/180), para Babesia caballi foi de 22,3% (40/180) e para infecção concomitante foi de 6,66% (12/180). Os resultados sorológicos obtidos por este estudo revelam que 35,5% (64/180) dos animais possuem anticorpos contra a babesiose equina sendo que a maioria dos animais acometidos tem dois e três anos de idade e portanto estão há menos tempo no hipódromo. Fatores como a ausência de carrapatos vetores, o uso de terapias babesicidas repetidas e o longo tempo de permanência dos animais no Jóquei após o tratamento, favorecem a diminuição dos títulos de anticorpos sem que ocorra reinfecção. Esses fatores podem justificar o menor número de animais com sorologia positiva para a doença nos cavalos com idade acima de quatro anos. Considerando-se esses resultados sugere-se que os animais sejam avaliados sorologicamente ao ingressarem no Jóquei Clube de São Paulo para que o uso de medicamentos contra a doença seja feito de forma adequada e para que os sinais clínicos compatíveis com babesiose equina em animais sorologicamente negativos sejam melhor avaliados e considerados em diagnósticos diferenciais. / In order to evaluate the presence of antibodies against Theileria equi and Babesia caballi in horses kept at the Jockey Club in São Paulo city, Brazil, a total of 180 samples of blood serum was tested using the Competitive Enzyme-Linked Immunosorbent Assay (C-ELISA test). This methodology has been recommended by the International Organization of Epizooties (IOE) due to its high sensitivity and specificity. The frequency of seropositive animals for Theileria equi, Babesia caballi and for both was 6.66% (12/180), 22.3% (40/180) and 6.66% (12/180), respectively. Serological results showed that 35.5% of the animals (64/180) had antibodies against equine piroplasmosis; they were from two to three years old and were at the Jockey Club for a shorter period of time. Factors such as absence of thick vectors, repeated therapy using babesicidal drugs and the long period of time that the animals stayed in the Jockey Club after treatment favoured the lowering of antibody titers with no reinfection. These factors might be responsible for the fewer number of animals with positive serology for the disease in horses over four years of age. Based on these findings, animals should be serologically evaluated at the time of entrance into the Jockey Club so that the use of drugs against the disease be performed properly and clinical signs suggestive of equine babesiosis in serologically negative animals be better evaluated and considered for differential diagnosis.

Breast cancer is the second leading cause of death in United States. Using 2D-HPLE, a novel separation technology, G-protein coupled receptor-associated sorting protein 1(GASP-1) was identified in sera of patients with early stage cancer, while it could not be detected in sera from healthy individuals. This was the first indication that GASP-1 was positively correlated with breast cancer. However, the function of GASP-1 in breast cancer was unknown. In this study, I verified the 2D-HPLE results by quantifying the expression level of GASP-1 in sera and tissue specimens of cancer patients using specific antibodies against GASP-1. A GASP-1 specific ELISA was developed and used to quantify GASP-1 levels in cancer patient sera. Immunohistochemistry was performed to verify and localize GASP-1 expression in tumor. I also characterized the tumorigenic potential of GASP-1 andidentified the signaling pathways mediated by GASP-1 in breast cancer cells in vitro.GASP-1 expression levelsin MDA-MB-231 cells were modified by transfecting cells with anti-GASP-1 shRNA and over-expression plasmids. Stable cell lines were prepared and their tumorigenic potential was evaluated using cell proliferation, migration, and colony formation assays. These cells were analyzed for markers used to identify epithelial to mesenchymal transition (EMT) using RT-PCR and western blot. They were also analyzed for NFkappaB activity, src phosphorylation, and GPR30 expression. The results showed that GASP-1 was over-expressed in sera and tissue specimens of breast cancer patients and other cancer types including brain, lung, liver and pancreatic cancer and that it correlated with early stage disease. GASP-1 positively regulated migration, and is required for cell proliferation and colony formation. GASP-1 is also necessary for the expression of EMT marker slug, increases NFkappaB activity and GPR30 expression level, while decreases the inhibitory phospho-src Tyr 530. I conclude that GASP-1 is a nearly marker for multiple cancer types. GASP-1 promotes tumorigenesis in breast cancer, possibly through multiple cancer related signaling pathways. These findings may contribute to our understanding of the mechanism of breast cancer tumorigenesis and identify new biomarkers that can be used for diagnosis and therapy of cancer. / Biology

Human pleasant touches promote feelings of security, supportiveness, and wellbeing. Conversely, human unpleasant touches promote the body for either “fight or flight” or “short term acute stress” during emergencies, feeling of stress or danger. The promoted stress response is released from the hypothalamus by the sympathetic nerve system further to the spinal cord to reach the signals to the adrenal medulla, where stress hormones adrenaline is released. Adrenaline, which is characterized by a mimic sympathetic nerve system, interacts with α and β receptors on different organs. The aim for this study was to investigate whether the stroker (partner/stranger) touch effects on adrenaline hormone releases. The null hypothesis for this study entails a significant adrenaline reduction in partners’ touches compared with strangers’ touches. Indirect competitive ELISA method was used, and concentration data of a total of sixteen participants was obtained. Whitney-U test was carried out to compare group differences within stroker (stranger/partner) touches and adrenaline releasing level. In addition, correlation in adrenaline with noradrenaline and oxytocin hormones was obtained using Spearman’s correlation test. The significant p-value 0.05 was conducted. The result of this study showed no differences between stroker (partner/stranger) associated with adrenaline hormone release. Correlation between partner maximum (max) concentration data for both oxytocin and adrenaline had significant differences. However, max variables for adrenaline and noradrenaline within stroker did not show significant differences. The conclusion of this study is that the gentle touch stimulus used in this study was not enough to detect stress hormone in adrenaline.