Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 56-59). Also available on the World Wide Web.

Thesis (MSc Food Sc )--Stellenbsosch University, 2003. / ENGLISH ABSTRACT: Kepi is a fermented milk beverage that originated in Eastern Europe. Traditional Kepi is a lightly acidic, carbonated beverage, with a slight yeasty taste. The starter used to produce this beverage is an irregularly shaped, yellowish-white grain-like structure similar in appearance to a cauliflower floret. The characteristic flavour of Kepi is produced by a complex spectrum of microbial species that include species of yeasts, lactic acid bacteria, acetic acid bacteria and mycelial fungi. At the end of the fermentation process the grainy starter can be recovered and re-used, since the microbes can easily be recovered as a solid matrix. The microbes comprising Kepi grains have only been identified using classical identification techniques such as selective growth media, morphological, physiological and biochemical characteristics. In this study, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis was used to typify and identify the complex microbial consortium present in the Kepi grains. A part of the 168 ribosomal RNA (rRNA) gene from the microbial population in mass-cultured, traditionally cultured and Irish Kepi grains were amplified using 'Eubacterial' specific primers and a part of the 268 rRNA gene was amplified using yeast specific primers. The PCR fragments were resolved by DGGE, resulting in unique fingerprints for the Eubacteria and yeasts present in the different Kepi grain types. The traditionally cultured Kepi grains were found to incorporate the most Eubacteria and yeast species, while the mass-cultured Kepi grains contained the lowest number of Eubacteria and yeast species. The different Eubacteria and yeast species were identified by cloning the PCR products and sequencing the cloned inserts. The obtained DNA sequences were compared to sequences available on the NCBI website. 8ix lactobacilli were identified: Lb. crispatus (KC-4); three Lb. species (KC-36, KC-38 and KC-43); and two unculturable lactobacilli (KC-2 and KC-3). The yeasts were identified as Saccharomyces cerevisiae (KC-y18) and Candida lambica (KC-y1). Unidentified isolates from kefiran strings that could not be identified using traditional methods were also identified by cloning the PCR products and sequencing the cloned inserts. The four isolates were identified as Lb. kefiri (KGI-A), Lb. parakefiri (KGIB), Lb. gallina rum (KGI-D) and an unculturable Lactobacillus (KGI-5). The phylogenetic relationship between the identified lactobacilli and the lactobacilli commonly found in Kepi grains was determined. The identified lactobacilli were grouped together in a clade with a bootstrap support value of 84%. The clade also contained representatives of Lb. delbrueckii subsp. lactis, Lb. acidophilus, Lb. gallinarum, Lb. helveticus, Lb. crispatus, Lb. species and unculturable lactobacilli. The bands in the peR-based DGGE fingerprints of the Eubacteria and the yeasts were identified, and a DGGE marker was subsequently constructed for the rapid identification of the Eubacteria present in mass-cultured Kepi grains. The data obtained in this study clearly showed that Kepi grains that are cultured differently, as well as Kepi grains from different origins have unique peRbased DGGE banding patterns for both the Eubacteria and yeasts present in the grains. The complex microbial consortium comprising Kepi grains could be typified and identified using PeR-based DGGE, DNA cloning and sequencing. The identification of the members of the microbial consortium is of importance for the future commercialisation of the mass-cultured Kepi grains. / AFRIKAANSE OPSOMMING: Kepi is 'n gefermenteerde melkdrankie wat sy oorsprong het in Oos Europa. Tradisionele Kepi is 'n effens suur, gekarboneerde drankie wat effens na gis smaak. Die beginkultuur wat gebruik word om dié drankie te maak is 'n oneweredige, geel-wit korrelagtige struktuur wat baie lyk soos 'n blomkoolkoppie. Die karakteristieke smaak van Kepi word geproduseer deur 'n komplekse spektrum mikrobiese spesies wat giste, melksuur- en asynsuurbakterieë en ~. misillêre fungi insluit. Aan die einde van die fermentasieproses kan die korrelagtige beginkultuur herwin word en weer gebruik word, aangesien die mikrobes maklik herwin kan word as 'n soliede matriks. Die mikrobes waaruit Kepikorrels bestaan, is nog slegs met behulp van klassieke identifikasiemetodes soos selektiewe groeimedia, morfologiese, fisiologiese and biochemiese eienskappe geïdentifiseer. In hierdie studie is polimerase kettingreaksie (PKR)-gebaseerde denaturerende gradiënt jelelektroforese (DGGE) analise gebruik om die komplekse mikrobiologiese konsortium in die Kepikorrels te tipeer en te identifiseer. 'n Gedeelte van die 16S ribosomale RNS (rRNS) geen van die mikrobiologiese populasie in massagekweekte, tradisioneel gekweekte en Ierse Kepikorrels is geamplifiseer met 'Eubakferiële' spesifieke peilers en In gedeelte van die 26S rRNS geen is geamplifiseer met gis spesifieke peilers. Die PKR fragmente is onderskei deur DGGE, wat unieke vingerafdrukke vir die Eubakteriële- en gisspesies in die verskillende Kepikorrel tipes gelewer het. Die tradisioneel gekweekte Kepikorrels het die meeste Eubakteriële- en gisspesies geïnkorporeer, terwyl die Ierse Kepikorrels die minste Eubakteriële- en gisspesies geïnkorporeer het. Die verskillende Eubakteriële- en gisspesies is geïdentifiseer deur klonering van die PKR produkte en deur die gekloneerde insetsels se volgordes te bepaal. Die ONS volgordes is dan vergelyk met volgordes wat op die NCSI webwerf beskikbaar is. Ses lactobacilli is geïdentifiseer: Lb. ctispetus (KC-4); drie Lb. spesies (KC-36, KC-38 en KC-43); en twee onkultiveerbare lactobacilli (KC-2 en KC-3). Die giste is geïdentifiseer as Saccharomyces cerevisiae (KC-y18) en Candida lambica (KC-y1). Ongeïdentifiseerde isolate van kefiranstringe is ook geïdentifiseer deur klonering van die PKR produkte en deur die gekloneerde insetsels se volgorde te bepaal. Dié vier isolate is geïdentifiseer as Lb. kefiri (KGIA), Lb. parakefiri (KGI-B), Lb. gallina rum (KGI-D) en 'n onkultiveerbare Lactobacillus (KGI-5). Die filogenetiese verwantskap is bepaal tussen die geïdentifiseerde lactobacilli en lactobacilli wat geredelik in Kepikorrels gevind word. Die geïdentifiseerde lactobacilli was saam in 'n groep gegroepeer met 'n bootstrap waarde van 84%. Die groep het ook verteenwoordigers van Lb. delbrueckii subsp. lactis, Lb. acidophilus, Lb. gallina rum, Lb. helveticus, Lb. crispatus, Lb. species en 'n onkultiveerbare laktobacilli ingesluit. Die bande in die PKR-gebaseerde DGGE vingerafdrukke van die Eubakterieë en die giste is geïdentifiseer, en 'n DGGE merker is gemaak vir die vinnige identifikasie van die Eubakterieë wat in die massagekweekte Kepikorrels teenwoordig is. Die data wat in die studie verkry, is wys duidelik dat Kepikorrels wat op verskillende maniere gekweek is, en wat verskillende oorspronge het, unieke PKR-gebaseerde DGGE bandpatrone het vir beide die Eubakterieë en giste wat in die korrels teenwoordig is. Die komplekse mikrobiologiese konsortium waaruit Kepikorrels bestaan kon getipeer en geïdentifiseer word deur PKR-gebaseerde DGGE, klonering van DNS en volgordebepaling. Die identifikasie van lede van die mikrobiologiese konsortium is belangrik vir die toekomstige kommersialisasie van die massagekweekte Kepikorrels.

Made available in DSpace on 2014-08-13T14:50:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2011Bitstream added on 2014-08-13T18:01:18Z : No. of bitstreams: 1 ribeiro_es_me_araca.pdf: 554726 bytes, checksum: 120b6f528464317c0d52e748f3f81855 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A importância da leishmaniose visceral no contexto da saúde pública tem aumentado na ultima década. A manifestação da leishmaniose visceral nos cães é variável e estudos sobre a genética e a sua relação com a leishmaniose visceral têm indicado alguns genes envolvidos na susceptibilidade e resistência a esta doença. O objetivo deste estudo foi avaliar a relação entre as variantes alélicas dos gene Slc11a1 (solute carrier family 11 member 1) e DLA-DRB1 (dog leukocyte antigen) com a positividade para leishmaniose, determinada por meio da amplificação de DNA de cinetoplasto de Leishmania sp., por PCR, e pela presença de anticorpos anti-Leishmania sp. ao teste ELISA indireto. Foram observadas associações estatisticamente significantes entre a positividade para leishmaniose e: i. a presença do alelo (141, 145 ou 149) da região microssatélite (TAAA)n localizada no íntron 1 do gene Slc11a1 (P< 0,0001) ii. a variação do número de guaninas (8 ou 9 G) da região promotora (P=0,0280) (em ambos os casos quando analisados separadamente) e iii. a presença de qualquer dos alelos da região microssatélite associado a 8 ou 9 G da região promotora (P= 0,0025). A presença do alelo 141 foi estatisticamente associada à negatividade em cães (P<0,0001). Não foi observada associação estatisticamente significante entre a positividade à leishmaniose e a freqüência dos alelos do exon 2 do gene DLA- DRB1, embora tenha sido observada associação significante entre a freqüência dos alelos DRB1-W, DRB1*00101 ou DRB1-T e o resultado negativo para leishmaniose, podendo ser estes potenciais marcadores para resistência à leishmaniose em cães / The importance of visceral leishmaniasis in the context of public health has increased in the last decade. The manifestation of visceral leishmaniasis in dogs is variable and genetic studies and its relationship with visceral leishmaniasis have shown some candidate genes involved in susceptibility and resistance to this disease. This study aimed at evaluating the relationship between polymorphism of the genes Slc11a (solute carrier family 11 member 1), and DLA-DRB1 (dog leukocyte antigen) and the positivity to leishmaniasis determined by the amplification of Leishmania sp. kinetoplast DNA by PCR and the presence of anti-Leishmania sp. the indirect ELISA. We observed statistically significant associations between the leishmaniasis positive diagnosis and i. the presence of intron 1 microsatellite region alleles (141, 145 or 149) (P<0.0001), ii. the variation on the guanine number (8 or 9) in the promoter region (P=0.0280) (in both cases when analyzed separately), iii. the presence any of the microsatellite alleles associated to 8 or 9 G in the promoter region (P=0.0025). Microsatellite allele 141 was associated to negativity to leishmaniasis (P<0.0001). There was no association between the presence of any of the exon 2 DLA-DRB1 alleles and the leishmaniasis positive animals, although significant association between the frequency of the alleles DRB1-W, DRB1*00101 or DRB1-T and negative results to leishmaniasis were observed. These might be potential markers for dog’s resistance to visceral leishmaniasis

Resumo: Os resultados da presença do papilomavírus humano (HPV) em relação ao câncer das vias aerodigestivas superiores são muito controversos. Destas vias, a orofaringe tem sido a localização com maior prevalência de HPV, o que desperta grande interesse dos pesquisadores sobre a real participação do vírus na carcinogênese desta região. Este trabalho teve como objetivo verificar a prevalência de HPV em pacientes com carcinoma espinocelular de orofaringe, através da reação em cadeia polimerase (PCR) e correlacioná-la com dados demográficos, clínico-patológicos e de sobrevida. O estudo foi realizado através da análise de 27 peças oriundas de blocos de parafina obtidos de pacientes portadores de carcinoma espinocelular de orofaringe diagnosticados e tratados no Centro de Oncologia Bucal da Faculdade de Odontologia de Araçatuba - UNESP. Foram realizadas extrações do DNA com o QIAamp DNA minikit, conforme instrução do fabricante. Após confirmar a presença e integridade do DNA, foi realizada a nPCR para detecção do HPV. Dois grupos foram formados considerando-se a presença ou ausência do HPV. Foram amplificadas 26 amostras para o gene -globina sendo que o HPV foi detectado em 50% dos casos estudados. Não houve diferença estatisticamente significativa entre os grupos em relação as variáveis clínico-patológicas e a sobrevida. / Abstract: Reports on the presence of human papillomavirus (HPV) in upper aerodigestive tract are very controversial. In this tract, the oropharynx has been the site with the highest prevalence of HPV, which encourage the great interest of researchers for the real role of the virus in the carcinogenesis of this area. This study had the objective of verifying the HPV prevalence in patients with oropharyngeal squamous cell carcinoma using polymerase chain reaction (PCR) and its correlation with demographic data, clinicopathological aspects and survival. This study was accomplished by the analysis of embedded paraffin tissues of oropharyngeal squamous cell carcinoma from 27 patients diagnosed and treated in São Paulo State University, Dentistry College of Araçatuba - UNESP. DNA extraction was accomplished with QIAamp DNA minikit, according to the manufacturer's protocol. After confirming the presence and integrity of DNA, nPCR for HPV was performed. Two groups were formed, considering patients with HPV and without HPV. -globin gene was PCR amplified in 26 samples and the HPV detected in 50% of the studied cases. There was no statistically significant difference between the groups in relation of clinicopathological variables and survival. / Orientador: Glauco Issamu Miyahara / Coorientador: Eder Ricardo Biasoli / Banca: José Fernando Garcia / Banca: Elaine Maria Sgavioli Massucato / Banca: Denise Tostes Oliveira / Banca: Norberto Nobuo Sugaya / Doutor

Orientador: Marcelo Vasconcelos Meireles / Banca: Flávia Lombardi Lopes / Banca: Alex Akira Nakamura / Resumo: O objetivo deste estudo foi avaliar a ocorrência e os métodos de diagnóstico para Cryptosporidium spp. em psitacídeos exóticos de cativeiro provenientes das regiões sul e sudeste do Brasil. A purificação dos oocistos nas amostras fecais de 463 psitacídeos foi realizada por meio de centrifugo-flutuação em solução de Sheather. Para análise microscópica, nós utilizamos a coloração negativa de verde malaquita. A amplificação de um fragmento parcial do gene 18S rRNA de Cryptosporidium spp. foi feita usando-se nested PCR seguida de sequenciamento dos fragmentos amplificados (nPCR/S). As amostras também foram testadas por meio de PCR duplex em tempo real, visando-se amplificar um fragmento do gene 18S rRNA de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A ocorrência de Cryptosporidium spp. pela microscopia e nested PCR (nPCR) foi de 3, 02% (14/463) e 4, 97% (23/463), respectivamente. A nPCR/S demonstrou positividade de 1, 73% (8/463) para Cryptosporidium genótipo III de aves, 0, 86% (4/463) para Cryptosporidium parvum e 0, 22% (1/463) para Cryptosporidium canis. A PCR duplex em tempo real demonstrou positividade de 9, 50% (44/463) para as criptosporidiose gástrica, sendo 1, 94% (9/463) para C. galli, 5, 83% (27/463) para Cryptosporidium genótipo III de aves e 1, 73% (8/463) para infecções mistas. Não houve diferença estatística significante entre a positividade pela nPCR e microscopia (p = 0. 1237) e houve concordância justa entre elas (Kappa = 0. 242). Diferença es... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the prevalence and diagnostic methods for Cryptosporidium spp. in caged adult exotic parrots from Southern and Southeastern Brazil. The oocyst purification in fecal samples from 463 psittacines was performed by centrifugal-flotation in Sheather's sugar solution. For microscopic analysis, we used malachite green negative staining. Amplification of a partial fragment of the 18S rRNA gene of Cryptosporidium spp. was accomplished using nested PCR (nPCR) followed by sequencing of the amplified fragments (nPCR/S). Samples were also tested by duplex real-time PCR targeting the 18S rRNA gene of Cryptosporidium galli and Cryptosporidium avian genotype III. The prevalence rates of Cryptosporidium spp. by microscopy and nPCR was 3. 02% (14/463) and 4. 97% (23/463), respectively. The nPCR/S showed positivity of 1. 73% (8/463) for Cryptosporidium avian genotype III, 0. 86% (4/463) for Cryptosporidium parvum and 0. 22% (1/463) for Cryptosporidium canis. Duplex real-time PCR showed a positivity of 9. 50% (44/463) for gastric cryptosporidiosis, 1. 94% (9/463) for C. galli, 5. 83% (27/463) for Cryptosporidium avian genotype III and 1. 73% (8/463) for mixed infections. There was no statistically significant difference between positivity for nPCR and microscopy (p = 0. 1237) and fair agreement between them (Kappa = 0. 242). A significant statistical difference (p <0. 0001) and fair agreement (Kappa = 0. 317) were obtained between nPCR and duplex real-time PCR. We found out that duplex real-time PCR is the best option for the diagnosis of gastric cryptosporidiosis and that Cryptosporidium avian genotype III is the most common Cryptosporidium species /genotype in psittacines / Mestre

Submitted by Elís Domingos Ferrari null (elisd.ferrari@yahoo.com.br) on 2017-10-31T11:41:02Z No. of bitstreams: 1 FINAL-Defesa Mestrado CORRIGIDO.pdf: 1177720 bytes, checksum: 7a08f0cef1e5a124653c8190f8178822 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-11-10T18:35:51Z (GMT) No. of bitstreams: 1 ferrari_ed_me_araca_par.pdf: 606404 bytes, checksum: 87114f79e3b3cffb7d3ab08cd9009eb6 (MD5) / Made available in DSpace on 2017-11-10T18:35:51Z (GMT). No. of bitstreams: 1 ferrari_ed_me_araca_par.pdf: 606404 bytes, checksum: 87114f79e3b3cffb7d3ab08cd9009eb6 (MD5) Previous issue date: 2017-10-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi avaliar a ocorrência e os métodos de diagnóstico para Cryptosporidium spp. em psitacídeos exóticos de cativeiro provenientes das regiões sul e sudeste do Brasil. A purificação dos oocistos nas amostras fecais de 463 psitacídeos foi realizada por meio de centrifugo-flutuação em solução de Sheather. Para análise microscópica, nós utilizamos a coloração negativa de verde malaquita. A amplificação de um fragmento parcial do gene 18S rRNA de Cryptosporidium spp. foi feita usando-se nested PCR seguida de sequenciamento dos fragmentos amplificados (nPCR/S). As amostras também foram testadas por meio de PCR duplex em tempo real, visando-se amplificar um fragmento do gene 18S rRNA de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A ocorrência de Cryptosporidium spp. pela microscopia e nested PCR (nPCR) foi de 3, 02% (14/463) e 4, 97% (23/463), respectivamente. A nPCR/S demonstrou positividade de 1, 73% (8/463) para Cryptosporidium genótipo III de aves, 0, 86% (4/463) para Cryptosporidium parvum e 0, 22% (1/463) para Cryptosporidium canis. A PCR duplex em tempo real demonstrou positividade de 9, 50% (44/463) para as criptosporidiose gástrica, sendo 1, 94% (9/463) para C. galli, 5, 83% (27/463) para Cryptosporidium genótipo III de aves e 1, 73% (8/463) para infecções mistas. Não houve diferença estatística significante entre a positividade pela nPCR e microscopia (p = 0. 1237) e houve concordância justa entre elas (Kappa = 0. 242). Diferença estatística significante (p <0. 0001) e concordância justa (Kappa = 0. 317) foram obtidas nas comparações entre nPCR e PCR duplex em tempo real. Nós concluímos que a PCR duplex em tempo real é a melhor opção para o diagnóstico de criptosporidiose gástrica e que Cryptosporidium genótipo III de aves é o mais comum dentre as espécies/ genótipos de Cryptosporidium que acometem psitacídeos. / The aim of this study was to evaluate the prevalence and diagnostic methods for Cryptosporidium spp. in caged adult exotic parrots from Southern and Southeastern Brazil. The oocyst purification in fecal samples from 463 psittacines was performed by centrifugal-flotation in Sheather's sugar solution. For microscopic analysis, we used malachite green negative staining. Amplification of a partial fragment of the 18S rRNA gene of Cryptosporidium spp. was accomplished using nested PCR (nPCR) followed by sequencing of the amplified fragments (nPCR/S). Samples were also tested by duplex real-time PCR targeting the 18S rRNA gene of Cryptosporidium galli and Cryptosporidium avian genotype III. The prevalence rates of Cryptosporidium spp. by microscopy and nPCR was 3. 02% (14/463) and 4. 97% (23/463), respectively. The nPCR/S showed positivity of 1. 73% (8/463) for Cryptosporidium avian genotype III, 0. 86% (4/463) for Cryptosporidium parvum and 0. 22% (1/463) for Cryptosporidium canis. Duplex real-time PCR showed a positivity of 9. 50% (44/463) for gastric cryptosporidiosis, 1. 94% (9/463) for C. galli, 5. 83% (27/463) for Cryptosporidium avian genotype III and 1. 73% (8/463) for mixed infections. There was no statistically significant difference between positivity for nPCR and microscopy (p = 0. 1237) and fair agreement between them (Kappa = 0. 242). A significant statistical difference (p <0. 0001) and fair agreement (Kappa = 0. 317) were obtained between nPCR and duplex real-time PCR. We found out that duplex real-time PCR is the best option for the diagnosis of gastric cryptosporidiosis and that Cryptosporidium avian genotype III is the most common Cryptosporidium species /genotype in psittacines. / FAPESP: 2015/26334-8

Orientador: Marcos Rogério André / Banca: Rosangela Zacarias Machado / Banca: Darci Moraes Barros Battesti / Resumo: Embora novos genótipos de agentes Anaplasmataceae vêm sendo detectados em carnívoros selvagens, aves selvagens e cervídeos no Brasil, poucos são os estudos acerca da circulação destes patógenos em pequenos mamíferos em nosso território. O presente trabalho teve como objetivo investigar a presença de DNA de Ehrlichia e Anaplasma em roedores amostrados no Brasil. Entre 2000 e 2011, diferentes espécies de roedores [n = 60] foram capturadas em cinco biomas brasileiros. As amostras de baço de roedores foram submetidas a extração de DNA utilizando um kit comercial. A presença de DNA de Ehrlichia e Anaplasma foi investigada utilizando ensaios de PCR em tempo real e convencionais dirigidos a vários genes. Dentre as 458 amostras de baço de roedores testadas, 0,44% (2/458) mostraram-se positivas para Ehrlichia spp. e 2,40% (11/458) para Anaplasma spp., pelos ensaios de PCR em Tempo Real multiplex baseados no gene groESL. A quantificação média do número de cópias de um fragmento do gene groESL por microlitro variou de 1,071 x 102 a 2,808 x 102 cópias para Ehrlichia spp. e 1,123 x 100 a 1,310 x 102 para Anaplasma spp. Pelos ensaios de PCR convencional baseados no gene 16S rRNA, 1,09% (5/458) das amostras mostraram-se positivas para Ehrlichia sp. e 1,97% (9/458) para Anaplasma sp. Análises filogenéticas de máxima verossimilhança baseada em um fragmento de 546 pb do gene 16S rRNA posicionaram os genótipos de Anaplasma detectados em roedores próximos a A. phagocytophilum ou isolados de A. ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Although new genotypes of Anaplasmataceae agents have been detected in wild carnivores, wild birds and deer in Brazil, few reports have been done in order to assess the circulation of these pathogens in small mammals in our territory. The present work aimed to investigate the presence of Ehrlichia and Anaplasma in rodents sampled in Brazil. Between 2000 and 2011, different rodent species [n=60] were trapped in five Brazilian biomes. Rodents' spleen samples were submitted to DNA extraction using a commercial kit. The presence of Ehrlichia and Anaplasma DNA was investigated using real time and conventional PCR assays targeting several genes. Among 458 rodent tested spleen samples, 0.44% (2/458) were positive for Ehrlichia spp. and 2.40% (11/458) for Anaplasma spp., by a multiplex qPCR assay based on groESL gene. The mean quantification of the number of copies of 83pb groESL gene fragment per microliter ranged from 1,071 x 102 to 2,808 x 102 copies for Ehrlichia spp., and 1,123 x 100 to 1,310 x 102 for Anaplasma spp. Out of 458 samples tested, 1.09% (5/458) were positive for Ehrlichia sp. and 1.97% (9/458) for Anaplasma sp. by cPCR assays based on 16S rRNA gene. Maximum Likelihood phylogenetic analyse based on 546pb fragment of 16S rRNA gene positioned the Anaplasma genotypes detected in rodents near to A. phagocytophilum or A. marginale and A. odocoilei isolates. On the other hand, the Ehrlichia genotypes detected in sampled rodents were closely related to E. canis, according to the phylogenetic analyses based on 358pb 16S rRNA gene fragment. The present study showed a low occurrence of Anaplasma and Ehrlichia in rodents in Brazil. / Mestre

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-19T17:20:40Z No. of bitstreams: 1 Fred da Silva Juliao Uso de método de biologia molecular....pdf: 3387831 bytes, checksum: 0efc6396d21a6e8a634b8026665afe7c (MD5) / Made available in DSpace on 2012-07-19T17:20:40Z (GMT). No. of bitstreams: 1 Fred da Silva Juliao Uso de método de biologia molecular....pdf: 3387831 bytes, checksum: 0efc6396d21a6e8a634b8026665afe7c (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A leishmaniose visceral (LV) é uma zooantroponose sistêmica de importância reconhecida em saúde pública, 90% dos casos no novo mundo são oriundos do Brasil. Os cães domésticos e as raposas são considerados como os principais reservatórios. A persistência da Leishmania em áreas endêmicas e o insucesso das medidas de prevenção, dirigidas exclusivamente ao reservatório canino, sugerem que outros animais podem ter importância na manutenção do ciclo de transmissão da LV. OBJETIVO: Avaliar potenciais reservatórios para LV numa área endêmica, utilizando método de biologia molecular quantitativo (PCR real-time). MÉTODOS: Foram estudados animais domésticos (bovinos, equídeos, caprinos e ovinos) e animais silvestres (marsupial), no município de Salinas da Margarida, Bahia, de 2007 a 2009. Todos os animais domésticos de produção mantidos e/ou pernoitando nas áreas urbanas do município foram incluídos. Os marsupiais foram capturados com armadilha animal modelo Tomahawk colocadas no peridomicílio das residências onde ocorreram casos de LV humana e/ou canina na localidade de Encarnação. Nos animais domésticos de produção foi coletado apenas amostra de sangue periférico e nos marsupiais, além de sangue, foi obtida uma amostra de pele através de biópsia da orelha. Em todas as amostras foi realizado PCR real-time para investigar a presença de DNA do parasito e estimar a carga parasitária. Os primers e sondas utilizados foram selecionados em gene SSU rRNA, que aparece 160 vezes no genoma de Leishmania spp. e é altamente conservado entre as espécies de Leishmania. RESULTADOS: No total, foram avaliados 80 animais domésticos (20 bovinos, 33 equídeos, 20 caprinos e 7 ovinos) e 103 marsupiais, todos da espécie Didelphis albiventris. Cinco bovinos foram positivos no teste de PCR real-time com carga parasitária variando de 12,7 a 183,5 parasitos/mL. Apenas um marsupial apresentou amostra de sangue positiva (6,0 parasitos/mL). Todos os demais animais testaram negativo. CONCLUSÃO: A técnica de PCR real-time pode ser uma ferramenta útil para avaliar o papel de potenciais reservatórios domésticos e silvestres para LV. A execução do PCR real-time é menos trabalhosa e mais prática do que a realização do teste de xenodiagnóstico, além disso, ela poder ser automatizada, permitindo a análise de grande número de amostras em estudos epidemiológicos. A detecção de carga parasitária de Leishmania em sangue de bovinos, em quantidade comparável à encontrada em cães, sugere que eles podem ser reservatórios para LV. A relativa abundância de bovinos nas áreas endêmicas para LV, assim como as evidências da preferência alimentar do vetor por estes animais, ressaltam a importância do papel que os bovinos podem ter na transmissão da LV. Mais trabalhos são necessários para elucidar estas questões. / Visceral leishmaniasis (VL) is a systemic zooantroponose of public health relevance, 90% of cases in the new world are from Brazil. Domestic dogs and foxes are considered the main reservoirs. The persistence of Leishmania in endemic areas and the failure of preventive measures, directed exclusively to the canine reservoir, suggest that other animals may be important in maintaining the transmission cycle of Leishmania. OBJECTIVE: To investigate potential reservoirs for VL in an endemic area, using molecular biology quantitative method (real-time PCR). METHODS: We studied domestic animals (cattle, horses, goats and sheeps) and wildlife (marsupial), in the city of Salinas da Margarida, Bahia, from 2007 to 2009. All livestock animals maintained and/or staying overnight in the urban areas of the municipality were included. The marsupials were captured with Tomahawk model animal traps placed outside the home of homes where there were human and/or dog VL cases in the locality of the Encarnação. In livestock animals, we collected only a sample of peripheral blood and in marsupials, in addition to blood we also collected a sample of ear skin biopsy. In all samples we carried out real-time PCR to detect the presence of parasite DNA and to estimate the parasite load. The primers and probes used were selected on SSU rRNA gene, which appears 160 times in the genome of Leishmania spp. and is highly conserved among species of Leishmania. RESULTS: In total, 80 livestock animals were evaluated (20 cattle, 33 horses, 20 goats and 7 sheep), and 103 marsupials, all Didelphis albiventris. Five cattle were positive by real-time PCR with parasite load ranging from 12.7 to 183.5 parasites/mL. Only one marsupial had a positive blood sample (6.0 parasites/mL). All other animals tested negative. CONCLUSION: The real-time PCR technique can be a useful tool for assessing the potential role of domestic and wild reservoir for LV. The implementation of real-time PCR is less laborious and more practical than the test of xenodiagnosis, in addition, it can be automated, allowing for the analysis of large number of samples in epidemiological studies. Detection of Leishmania parasite load in the blood of cattle, in an amount comparable to that found in dogs, suggests that they may be reservoirs for VL. The relative abundance of cattle in endemic areas for VL, as well as evidence of vector feeding preference for these animals, highlight the important role that cattle may exert in the transmission of VL. More research is needed to clarify these issues.

Os antígenos plaquetários humanos são estruturas imunogênicas resultantes de alterações pontuais (SNP) que levam a substituição de um aminoácido a nível proteico. O objetivo deste estudo foi determinar a frequência alélica e genotípica do sistema HPA-1 a -5 e -15 em doadores de plaquetas do Estado do RS e comparar as frequências alélicas encontradas com as observadas em outras populações. A genotipagem HPA foi realizada através do método de PCR-SSP. Um total de 201 doadores de plaquetas foram incluídos no estudo sendo 167 caucasoides e 34 não caucasoides. O alelo “a” foi o mais frequente nos sistemas HPA-1 a -5 em ambos grupos. O genótipo HPA-15AB foi predominante sobre os genótipos homozigotos para este sistema. O teste exato de Fisher revelou diferença estatisticamente significativa para o sistema HPA-5. Houve maior prevalência do alelo HPA-5B no grupo não caucasoide. Para o grupo caucasoide, o método de neighbor-joining e a PCA revelaram proximidade genética entre este grupo e as populações europeias. De um modo geral, concluímos que as frequências alélicas para os sistemas HPA-1 a -5 e -15 encontradas em nosso grupo caucasoide são similares às descritas em populações europeias. Estes dados corroboram a formação étnica da população do RS. A maior frequência do alelo HPA-5b encontrada no grupo não caucasoide de nosso estudo indica a possibilidade de alosensibilização para pacientes que recebem transfusões de plaquetas não compatibilizadas geneticamente. / Human platelet antigens are immunogenic structures that result from single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions. The present study sought to determine the allele and genotype frequencies of HPA-1 through 5 and HPA-15 in platelet donors from the state of Rio Grande do Sul, Brazil, and compare their allele frequencies to those observed in other populations. HPA genotyping was performed via the single specific primer-polymerase chain reaction (PCR-SSP) method. The study sample comprised 201 platelet donors (167 Caucasians and 34 non-Caucasians). Allele “a” was that most commonly found for HPA-1 through 5 in both groups. The HPA-15AB genotype predominated over homozygous genotypes of this system. Fisher’s exact test revealed statistically significant differences for the HPA-5 system, with a greater prevalence of the HPA- 5B allele in non-Caucasians. The neighbor-joining method and principal components analysis (PCA) revealed genetic proximity between the Caucasian group and European populations. We conclude that the allele frequencies of HPA-1 through 5 and HPA-15 found in our Caucasian sample are similar to those reported for European populations. These findings corroborate the ethnic makeup of the population of Rio Grande do Sul. The higher frequency of the HPA-5b allele found in the non-Caucasian group of our sample suggests the possibility of allosensitization in patients who receive platelet transfusions from genetically incompatible donors.

The aim of this thesis was to optimize the method of detection of phytoplasmas using DNA-microarray. It consisted of testing an appropriate method of genetic material isolation, development and optimization of PCR to amplify different groups of phytoplasmas, optimization of detection of DNA at a microarray, and sequence analysis of phytoplasma in order to design more suitable probes. PCR was first optimized for collection isolates, then also for natural samples. All 16Sr groups from the collection were sequenced and phytoplasmas were detected in them by hybridization. Phytoplasmas were detected also in natural samples: oilseed rape (species Brassica napus), red clover (Trifolium pretense), purple coneflower (Echinacea purpurea), and apple tree (Malus domestica). Using the DNA from insect vectors, only sample 202/6 from the group 16Sr-XII was positive. The sequence of red clover and oilseed rape correspond with the database samples in the group 16Sr-I "Aster yellows".