Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-06-27T15:06:48Z No. of bitstreams: 1 texto completo.pdf: 717860 bytes, checksum: d71c1053a82b292b3ab10f06c3e68ce6 (MD5) / Made available in DSpace on 2016-06-27T15:06:48Z (GMT). No. of bitstreams: 1 texto completo.pdf: 717860 bytes, checksum: d71c1053a82b292b3ab10f06c3e68ce6 (MD5) Previous issue date: 2004-04-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A avaliação de viabilidade de uso da técnica de immuno-dot blot para a detecção do vírus da dengue em homogenatos de larvas e de mosquitos Aedes aegypti e larvas de Aedes albopictus foi realizada utilizando-se soros humanos reativos para dengue e soros policlonais antidengue, produzidos em coelhos da raça Nova Zelândia imunizados com suspensões de vírus DEN-1 (Havaí) e DEN-2 (Nova Guiné) purificados e emulsificadas em adjuvante incompleto de Freund. Os resultados demonstraram que a técnica apresenta sensibilidade adequada para a detecção do vírus da dengue em homogenatos de larvas e de mosquitos A. aegypti e larvas de A. albopictus ainda que os títulos dos anti- soros sejam baixos. A presença do vírus foi detectada em larvas do mosquito A. albopictus, o que pode contribuir para o monitoramento do vírus mesmo na ausência de surtos da doença. Pode, inclusive, contribuir para estudos epidemiológicos mais amplos desse vírus por fornecer ferramenta para esses estudos. / The immuno-dot blot technique was tested and standardized for detection of dengue viruses in Aedes aegypti and Aedes albopictus larvae and mosquitoes homogenates. The experiments were carried out with dengue-reactive human sera and with anti-dengue polyclonal antisera prepared in New Zealand male rabbits immunized with DEN-1 (Hawai) and DEN-2 (New Guinea) purified virus suspensions emulsified in Freund incomplete adjuvant. Results showed that the virus can be easily detected by immuno-dot blot in A. aegypti and A. albopictus larvae and mosquitoes homogenates and suggest that this technique, for its simple procedure and easy reading, could be useful for field virus detection. Although it was recently established that these viruses can multiply in A. albopictus larvae, it is not known whether A. albopictus can indeed vector the DEN-1 and DEN-2 viruses in Brazil and, if so, under what percentages, relatively to A. aegypti mosquitoes. This study provides a tool for broader epidemiological studies of these viruses. / Dissertação antiga

A relatively high percentage of porcine <i>Escherichia coli</i> isolates from cases associated with neonatal and post-weaning diarrhea are positive for the gene encoding the adhesin involved in diffuse adherence I (AIDA-I). This gene and its corresponding protein were first identified and characterized in <i>E. coli</i> strain 2787 isolated from human infantile diarrhea. Little is known about the role of the AIDA-I protein in pathogenesis of porcine enteric disease caused by AIDA-I positive E. coli and the properties of AIDA-I protein expressed by porcine AIDA-I positive <i>E. coli</i> isolates and its receptors. <p>In this study, we demonstrated that AIDA-I adhesin isolated from porcine AIDA-I positive <i>E. coli</i> PD20 and PD58 is an acidic protein consisting of five isoforms. It has a molecular weight (100 kDa) similar to the AIDA-I adhesin expressed by human AIDA-I positive <i>E. coli</i> strain 2787 and has a relatively high amino acid homology (78-87%) with it. Immunodetection of AIDA-I positive <i>E. coli</i> strains using polyclonal anti-AIDA-I antibodies had relatively low sensitivity and specificity, accordingly these tests are unlikely to be used for regular diagnostic detection. <p>Using affinity chromatography, we isolated from porcine intestinal mucus proteins that bind to purified AIDA-I adhesin. These proteins were separated by one- and two-dimensional electrophoresis and subjected to overlay Western blot with purified AIDA-I adhesin and AIDA-I positive <i>E. coli</i> to demonstrate 65 and 120 kDa (p65 and p120) proteins as AIDA-I binding proteins. The identity of p65 was not determined based on LCMS/MS data, whereas p120 was matched to two nuclear proteins (namely, DNA damage binding protein and splicing factor 3b) and one cytoplasmic protein, which is an IgG Fc binding protein. Based on similar amino acid homology, molecular weight, structural similarity to mucin and reported evidence of being secreted by goblet cells into the intestinal lumen, we think that the IgG Fc binding protein is the most likely candidate to serve as a potential receptor in intestinal mucus for AIDA-I adhesin.

A relatively high percentage of porcine <i>Escherichia coli</i> isolates from cases associated with neonatal and post-weaning diarrhea are positive for the gene encoding the adhesin involved in diffuse adherence I (AIDA-I). This gene and its corresponding protein were first identified and characterized in <i>E. coli</i> strain 2787 isolated from human infantile diarrhea. Little is known about the role of the AIDA-I protein in pathogenesis of porcine enteric disease caused by AIDA-I positive E. coli and the properties of AIDA-I protein expressed by porcine AIDA-I positive <i>E. coli</i> isolates and its receptors. <p>In this study, we demonstrated that AIDA-I adhesin isolated from porcine AIDA-I positive <i>E. coli</i> PD20 and PD58 is an acidic protein consisting of five isoforms. It has a molecular weight (100 kDa) similar to the AIDA-I adhesin expressed by human AIDA-I positive <i>E. coli</i> strain 2787 and has a relatively high amino acid homology (78-87%) with it. Immunodetection of AIDA-I positive <i>E. coli</i> strains using polyclonal anti-AIDA-I antibodies had relatively low sensitivity and specificity, accordingly these tests are unlikely to be used for regular diagnostic detection. <p>Using affinity chromatography, we isolated from porcine intestinal mucus proteins that bind to purified AIDA-I adhesin. These proteins were separated by one- and two-dimensional electrophoresis and subjected to overlay Western blot with purified AIDA-I adhesin and AIDA-I positive <i>E. coli</i> to demonstrate 65 and 120 kDa (p65 and p120) proteins as AIDA-I binding proteins. The identity of p65 was not determined based on LCMS/MS data, whereas p120 was matched to two nuclear proteins (namely, DNA damage binding protein and splicing factor 3b) and one cytoplasmic protein, which is an IgG Fc binding protein. Based on similar amino acid homology, molecular weight, structural similarity to mucin and reported evidence of being secreted by goblet cells into the intestinal lumen, we think that the IgG Fc binding protein is the most likely candidate to serve as a potential receptor in intestinal mucus for AIDA-I adhesin.