This work is concerned with a study of the role of complement and antibodies in the serum of rabbits and of a non-susceptible strain of mice in the protection of Akr mice injected with active Akr tumor cells.

This thesis has been an attempt to observe the role antibodies play in extending the life span of tumor infected Akr mice.

White clover mosaic virus (WClMV) virions were purified using a modified method of Wetter (1960). Antisera were produced by rabbit immunisation and virus-specific polyclonal antibodies were obtained. Suitable conditions were optimised for serological detection of virus by DAS-ELISA and compared with a commercial kit.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-13Bitstream added on 2014-06-13T20:35:43Z : No. of bitstreams: 1 pereira_acb_me_sjrp.pdf: 828257 bytes, checksum: de1b44b38dfac1b95be8ef5a683e7543 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O lenho estriado de rupestris ou cascudo (Rupestris stem pitting – RSP), um dos componentes do Complexo do lenho rugoso (“Rugose wood” - RW), é considerado uma das doenças de videira transmitidas por enxertia de grande relevância econômica para a viticultura. O Rupestris stem pitting associated virus – RSPaV foi associado com a doença do lenho estriado ou cascudo, sendo classificado como espécie do gênero Foveavirus, pertencente a família Flexiviridae. No presente trabalho, descrevem-se o sequenciamento do gene da proteína capsidial (CP) de um isolado brasileiro do RSPaV (RSPaV-SP), sua expressão em Escherichia coli, purificação da proteína capsidial recombinante e a produção de anti-soro policlonal em coelho. O sequenciamento do gene resultou em uma seqüência de 780 nucleotídeos e 259 aminoácidos deduzidos com massa molecular estimada de 28 kDa. A análise filogenética, entre a seqüência correspondente à CP do RSPaV-SP e outras variantes do mesmo vírus, evidenciou a formação de 4 grupos distintos, sendo o isolado brasileiro incluído no grupo da variante BS do RSPaV. A proteína capsidial recombinante foi purificada em coluna de afinidade e apresentou massa molecular estimada de 32kDa (4kDa da seqüência do vetor e 28kD da CP do RSPaV-SP). O anti-soro produzido apresentou-se específico na detecção da proteína capsidial recombinante purificada por “Western-blot”, sem reação com proteína heteróloga a partir da diluição 1:4000. Nesta diluição, o anti-soro foi efetivo na detecção do vírus em extratos de plantas infectadas, sendo que nenhuma reação foi observada com extratos de plantas sadias. Considerando-se que este vírus apresenta variações de concentração na planta durante as estações do ano, e que, os testes sorológicos foram realizados durante a estação de baixa concentração do vírus, os resultados... / Rupestris stem pitting (RSP), a component of the rugose wood (RW) complex, is one of the most graft-transmissible grapevine virus diseases with great economic importance for viticulture . Rupestris stem pitting-associated virus (RSPaV), genus Foveavirus within the family Flexiviridae, has been associated with this disease. This work reports the sequencing of the coat protein (CP) gene of a brazilian an isolate of RSPaV (RSPaV-SP), its expression in Escherichia coli, purification of the recombinant coat protein and production of a polyclonal antiserum in rabbit. CP gene was found to be 780nt long, with a 256 deduced amino acid sequence encoding a predicted protein of 28 kDa. In filogenetic analysis, with RSPaV-SP and other variants of the virus, four groups were found and the sequence of RSPaV-SP showed the highest identity with the variant RSPaV-BS. The recombinant coat protein was purified by affinity chromatography and showed a molecular weight of 32kDa (4 kDa from a small vector sequence plus 28 kDa for the CP of RSPaV-SP). The antiserum proved specific for detection of the recombinant protein by Western Blot, and did not react with heterologous proteins starting at a dilution of 1:4000. At this dilution, the antiserum was effective in the virus detection of leaf extracts of infected plants and no reaction was observed with extracts from healthy grapevines. Considering that the virus is found at low concentrations in the plants during the seasons of the year, the results obtained so far were highly satisfactory for RSPaV detection. Serological methods have advantages over the biological indexing method, since they are cheaper and can be used in large-scale tests such as ELISA. Experiments using the ELISA technique were not successful. Purification of the native recombinant protein would be an alternative more efective to detect the virus using these technique.

Made available in DSpace on 2015-03-26T13:37:48Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1056837 bytes, checksum: 147af3aea5c0fd4d6961921a210a152c (MD5) Previous issue date: 2012-02-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cowpea mild mottle virus (CpMMV), the causal agent of stem necrosis disease, has drawn attention of soybean producers in recent years due to productivity losses in the main producing regions of Brazil. The disease was first recorded in the Midwest region of Brazil in the 2000/01 soybean season and rapidly spread throughout the country in the following years. CpMMV is usually hard to diagnose due to its wide range of symptoms while the occurrence of asymptomatic soybean cultivars complicates genotypic selection in breeding programs. Serological methods for viral detection require the use of an antiserum of good quality to achieve specificity and sensitivity. The entire coat protein sequence of a Brazilian CpMMV isolate was cloned into a bacterial expression vector and transformed into Escherichia coli BL21::DE3 for in vitro expression. The coat protein, fused to a 6 His-tag, was purified under denaturing conditions by affinity chromatography using a Ni-NTA resin. After renaturation, the integrity and identity of purified recombinant protein was confirmed by SDS-Page and MALDI-ToF/Tof mass spectrometer analyses. New Zealand rabbits were immunized with increasing amounts of the recombinant protein. The specificity and sensitivity of the antisera was shown by Western blot and indirect ELISA assays. The polyclonal antisera raised against recombinant coat protein proved to be a reliable tool for CpMMV detection. / O Cowpea mild mottle virus (CpMMV), agente causal da necrose da haste, tem chamado a atenção dos produtores de soja nos últimos anos devido às perdas de produtividade geradas nas principais regiões produtoras do Brasil. A doença foi relatada pela primeira vez na região Centro-Oeste do Brasil, na safra de 2000/01, e rapidamente se espalhou por todo o país nos anos seguintes. Além disso, tem sido relatada a ocorrência de cultivares de soja assintomáticas, o que dificulta a seleção de genótipos para programas de melhoramento. Os métodos sorológicos para a detecção viral requerem o uso de um antissoro de boa qualidade para alcançar especificidade e sensibilidade. A sequência completa da capa proteica de um isolado brasileiro de CpMMV (Bahia) foi clonado em um vetor de expressão bacteriano e transformado em Escherichia coli BL21: DE3 para expressão in vitro. A proteína capsidial ligada a uma cauda de histidina foi purificada sob condições desnaturantes através de cromatografia de afinidade, utilizando uma resina Ni-NTA. Depois da renaturação, a integridade e a identidade da proteína recombinante purificada foi confirmada por SDS-PAGE e análise de espectrometria de massa (MALDI-ToF/ToF). Dois coelhos da raça Nova Zelândia foram imunizados com quantidades crescentes da proteína recombinante. A especificidade e sensibilidade do antissoro foram avaliadas por Western blot e ELISA indireto. O antissoro policlonal a partir da proteína recombinante da capa proteica provou ser uma ferramenta confiável e eficiente para a detecção CpMMV.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Passion fruit (Passiflora edulis) and cowpea (Vigna unguiculata) are important crops of economical impact for the Northeast of Brazil. Fruit woodiness is an important virus disease of passion fruit, caused by Passionfruit woodiness virus (PWV) and Cowpea aphid-borne mosaic virus (CABMV), both from the genus Potyvirus. CABMV is also responsible for important disease on cowpea. The present research had the objective to study and compare the biological, serological and molecular properties of isolates of CABMV obtained from passion fruit (CABMV-P Pet, CABMV-P Gua, CABMV-P Sb and CABMV-P Uba) and from cowpea (CABMV-C Fort and CABMV-C Bv). CABMV-C Fort and CABMV-C Bv were purified from infected cowpea cv. Pitiuba systemically infected. The obtained purified preparations presented concentrations of 16.4 mg of virus per mL (CABMV-C Bv) and 15.9 mg of virus per mL (CABMV-C Fort). Both purified virus preparations were used for rabbit immunizations to produce polyclonal antisera with reactive titers of 1:128.000 in PTA-ELISA. Electrophoresis analyses of the virus purified preparations revealed the presence of only one capsidial protein with molecular weight of 34 kDa for both virus isolates. Parts of the genomes, corresponding to the coat protein gene (cp), from the virus isolates obtained from cowpea and from passion fruit were amplified by RT-PCR. The philogenetic analyses of the amplified cDNA fragments grouped with the CABMV isolates sequences deposited in the GenBank, according to their original host. Based on the biological, serological and molecular results, the virus isolates studied were classified into two biotypes: Biotype Cowpea (CABMV-C) including CABMV-C Bv and CABMV-C Fort obtained from cowpea, and biotype Passion fruit (CABMV-P) including CABMV-P Pet, CABMV-P Gua, CABMV-P Sb and CABMV-P Uba obtained from passion fruit. Source of immunity to CABMV-C Bv and CABMV-C Fort was identified in cowpea and the genotype was designate Lab-Poty. Studies of virus interaction in cowpea demonstrated strong synergistic effect among CABMV-C, Cucumber mosaic virus (CMV) and Cowpea severe mosaic virus (CPSMV). / O maracujazeiro (Passiflora edulis) e o feijoeiro caupi (Vigna unguiculata) sÃo culturas de elevada importÃncia econÃmica para o Nordeste brasileiro. Entre as viroses que se manifestam no maracujazeiro, destaca-se o endurecimento dos frutos causado por Passionfruit woodiness virus (PWV) e Cowpea aphid-borne mosaic virus (CABMV), ambos pertencentes ao gÃnero Potyvirus, sendo o CABMV responsÃvel por importante doenÃa do feijoeiro caupi. A presente pesquisa teve como objetivo estudar e comparar as propriedades biolÃgicas, sorolÃgicas e moleculares de isolados de CABMV obtidos de maracujazeiro (CABMV-P Pet, CABMV-P Gua, CABMV-P Sb e CABMV-P Uba) e isolados obtidos de feijoeiro caupi (CABMV-C Fort e CABMV-C Bv). Os isolados CABMV-C Fort e CABMV-C Bv foram purificados a partir de plantas de feijoeiro caupi cv. Pitiuba sistemicamente infetadas. As preparaÃÃes purificadas obtidas apresentaram concentraÃÃes de 16,4 mg de vÃrus por mL (CABMV-C Bv) e 15, 9 mg de vÃrus por mL (CABMV-C Fort), as quais foram usadas na imunizaÃÃo de coelhos, para a produÃÃo de antissoros policlonais, com tÃtulo de 1:128.000 em PTA-ELISA. AnÃlises eletroforÃtica das preparaÃÃes virais revelaram uma Ãnica proteÃna capisidial com peso molecular de 34 kDa. Partes do genoma correspondente ao gene da capa protÃica (cp) dos isolados virais obtidos de feijoeiro caupi e de maracujazeiro foram amplificadas por RT-PCR. AnÃlises filogenÃticas dos fragmentos dos cDNA amplificados se agruparam com sequencias de isolados do CABMV depositadas no GenBank, de acordo com os hospedeiros originais. Com base nos resultados de estudos biolÃgicos, sorolÃgicos e moleculares, os isolados virais estudados foram classificados em dois biÃtipos: BiÃtipo Cowpea (CABMV-C) incluindo CABMV-C Bv and CABMV-C Fort obtidos de feijoeiro caupi e biÃtipo Passion fruit (CABMV-P) incluindo CABMV-P Pet, CABMV-P Gua, CABMV-P Sb e CABMV-P Uba obtidos de maracujazeiro. Fontes de imunidade a CABMV-C Bv e CABMV-C Fort foi identificada em feijoeiro caupi e o genÃtipo foi designado de Lab-Poty. Estudos de interaÃÃo viral em feijoeiro caupi, mostraram forte sinergismo entre CABMV-C, Cucumber mosaic virus (CMV) e Cowpea severe mosaic virus (CPSMV).

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Plant virus identification and characterization can be achived by several methods based in the biological, morphological, cytological, serological and molecular virus properties. The molecular properties have been used with frequency for vÃrus identification and characterization and the reverse transcription polymerase chain reaction (RT-PCR) has constituted an efficient and precise method for researches with RNA plant viruses. On the other hand, the enzyme-linked immunosorbent assay (ELISA) is the most used serological method for virus detection in plant tissues. A techncal innovation developed for plant virus identification represents a great technological development and support for plant virus research. A new approach involving virus particle immune precipitation to be used for RNA amplification by RT-PCR named IP-RT-PCR was sucessefuly used for amplification of RNA fragments from five virus species from the genera Comovirus, Cucumovirus, Potyvirus and Sobemovirus. Considering that the immune biological Companies have developed several DAS-ELISA kits, but neither of them produce and commercialize PTA-ELISA kits, a simple and practical PTA-ELISA kit was developed and validated for plant virus detection. The second part of the research had the objective to study, comparatively, the biological, serological and molecular properties of four plant virus isolates obtained from naturally infected Passiflora edulis (PWV-PET and PWV-GUA) and from naturally infected Vigna unguiculata (CABMV-BV and CABMV-FOR) with the objective to elucidate the identity of the causal agent of passion fruit woodiness in Brazil. In host range studies onle Canavalia ensiformes and Macroptilium lathyroides were infected by virus isolates obtained from cowpea and from passion fruit. The isolate PWV-GUA was purified from systemically infected M. lathyroides plants and the virus purified preparation (18.24 mg of virus.ml-1) was used for rabitt immunization for polyclonal antiserum production, which showed a title of 1:128,000 in PTA-ELISA. The electrophoresis analysis of the purified virus showed a unique capsidial protein with 34 kDA. Plant virus interaction studies in C. ensiformis indicated unilateral cross protection between PWV-GUA and CABMV-FOR. On the other hand, the isolate PWV-PET did not cross protect passion fruit plants against PWV-GUA. Filogenetic analysis of nucleotiode sequencies from cDNA fragments corresponding to coat protein (CP) genes amplified by IP-RT-PCR from the genomic virus isolates compared with virus sequencies from the Genbank grouped according to the host specifities. Based on the biological, serological and mainly molecular results, the virus isolates studied were classified into two biotypes: Biotype CABMC-C (Cowpea) to include isolates obtained from cowpea that do not infect passion fruit, and biotype CABMV-P (Passion fruit) to include the virus isolates responsible for the passion fruit woodiness in Brazil. / A identificaÃÃo e a caracterizaÃÃo de vÃrus de planta podem ser realizadas por vÃrios mÃtodos envolvendo propriedades morfolÃgicas, biolÃgicas, citolÃgicas, moleculares e sorolÃgicas. As tÃcnicas moleculares tÃm sido usadas com frequencia para identificaÃÃo e caracterizaÃÃo de vÃrus, e a tÃcnica de âreverse transcription polymerase chain reactionâ (RT-PCR) tem se constituÃdo em mÃtodo eficiente e preciso para pesquisas com vÃrus de planta com genoma de RNA. De outra parte, a tÃcnica de enzyme-linked immunosorbent assay (ELISA) constitui o mÃtodo sorolÃgico mais usado para detecÃÃo de vÃrus em tecidos vegetais. Uma inovaÃÃo tecnolÃgica desenvolvida nesta pesquisa para diagnose de vÃrus de planta representa grande avanÃo tecnolÃgico e suporte para pesquisa em virologia vegetal. A inovaÃÃo envolvendo a imunoprecipitaÃÃo (IP) de partÃculas de vÃrus para uso na RT-PCR denominada de IP-RT-PCR foi usada com sucesso para amplificaÃÃo de fragmentos de RNA de cinco espÃcies de vÃrus dos gÃneros Comovirus, Cucumovirus, Potyvirus e Sobemovirus. Considerando que kits de DAS-ELISA tÃm sido produzidos e comercializados por companhias de imunobiologicos, mas nenhuma companhia produz kits de PTA-ELISA, um kit simples e prÃtico de PTA-ELISA foi desenvolvido e validado para detecÃÃo de vÃrus de planta. A segunda etapa da pesquisa teve como objetivo estudar as propriedades biolÃgicas, sorolÃgicas e moleculares de isolados de vÃrus do gÃnero Potyvirus obtidos de maracujazeiro (Passiflora edulis) (PWV-PET e PWV-GUA) e isolados de Cowpea aphid-borne mosaic virus (CABMV-FOR e CABMV-BV) obtidos de feijoeiro caupi (Vigna unguiculata), visando elucidar a identidade do agente causal do endurecimento dos frutos do maracujazeiro no Brasil. Em estudos de gama de plantas hospedeiras, somente Canavalia ensiformis e Macroptilium lathyroides foram infetadas por isolados obtidos de maracujazeiro e de feijoeiro caupi. O PWV-GUA foi purificado a partir de plantas de M. lathyroides sistemicamente infetadas e a preparaÃÃo viral purificada (18,24 mg de vÃrus.ml-1) foi usada para imunizaÃÃo de coelho com a produÃÃo de antissoro policlonal com tÃtulo de 1:128.000 em PTA-ELISA. AnÃlise eletroforÃtica da preparaÃÃo viral purificada revelou uma Ãnica proteÃna capisidial com peso molecular de 34 kDa. Experimentos de interaÃÃo entre os isolados virais em C. ensiformis indicaram proteÃÃo unilateral entre PWV-GUA e CABMV-FOR. De outra parte, o isolado PWV-PET nÃo protegeu plantas de maracujazeiro contra a super infecÃÃo de PWV-GUA. AnÃlises filogenÃticas das seqÃÃncias dos fragmentos de cDNA correspondentes Ãs capas protÃicas (CP), amplificados a partir dos genomas dos isolados virais de maracujazeiro e de feijoeiro caupi por IP-RT-PCR, agruparam-se com as seqÃÃncias de isolados virais de referidas culturas depositadas no GenBank, apresentando um agrupamento em funÃÃo da especificidade de hospedeiros. Com base nos resultados dos estudos biolÃgicos, sorolÃgicos e, sobretudo moleculares, os isolados virais estudados foram classificados em dois biÃtipos: BiÃtipo CABMV-C (Cowpea) incluindo os isolados obtidos de feijoeiro caupi e biÃtipo CABMV-P (Passion fruit) para incluir os isolados responsÃveis pelo endurecimento dos frutos do maracujazeiro no Brasil.

Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V.cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V.cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease. / Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V.cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V.cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.

Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V.cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V.cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease. / Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V.cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V.cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.

Les entérocoques font partie de la flore normale intestinale des animaux et des humains. Plusieurs études ont démontré que les entérocoques d’origine animale pouvaient représenter un réservoir de gènes de résistance aux antibiotiques pour la communauté humaine et animale. Les espèces Enterococcus faecalis et Enterococcus faecium sont importantes en santé publique; elles sont responsables d’environ 12% de toutes les infections nosocomiales aux États-Unis. Au Canada, les cas de colonisation et/ou d’infections à entérocoques résistants à la vancomycine ont plus que triplé de 2005 à 2009. Un total de 387 isolats E. faecalis et E. faecium aviaires, et 124 isolats E. faecalis porcins ont été identifiés et analysés pour leur susceptibilité aux antibiotiques. De hauts pourcentages de résistance envers les macrolides et les tétracyclines ont été observés tant chez les isolats aviaires que porcins. Deux profils phénotypiques prédominants ont été déterminés et analysés par PCR et séquençage pour la présence de gènes de résistance aux antibiotiques. Différentes combinaisons de gènes de résistance ont été identifiées dont erm(B) et tet(M) étant les plus prévalents. Des extractions plasmidiques et des analyses par hybridation ont permis de déterminer, pour la première fois, la colocalisation des gènes erm(B) et tet(M) sur un plasmide d’environ 9 kb chez des isolats E. faecalis porcins, et des gènes erm(B) et tet(O) sur un plasmide de faible poids moléculaire d’environ 11 kb chez des isolats E. faecalis aviaires. De plus, nous avons démontré, grâce à des essais conjugatifs, que ces plasmides pouvaient être transférés. Les résultats ont révélé que les entérocoques intestinaux aviaires et porcins, lesquels peuvent contaminer la viande à l’abattoir, pouvaient représenter un réservoir de gènes de résistance envers la quinupristine-dalfopristine, la bacitracine, la tétracycline et les macrolides. Afin d’évaluer l’utilisation d’un antisérum polyclonal SA dans l’interférence de la résistance à de fortes concentrations de bacitracine (gènes bcrRAB), lors d’un transfert conjugatif répondant aux phéromones, un isolat multirésistant E. faecalis aviaire a été sélectionné. Après induction avec des phéromones produites par la souche réceptrice E. faecalis JH2-2, l’agrégation de la souche donatrice E. faecalis 543 a été observée ainsi que des fréquences de transfert élevées en bouillon lors d’une courte période de conjugaison. Le transfert conjugatif des gènes asa1, traB et bcrRAB ainsi que leur colocalisation a été démontré chez le donneur et un transconjugant T543-1 sur un plasmide de 115 kb par électrophorèse à champs pulsé (PFGE) et hybridation. Une CMI de > 2 048 µg/ml envers la bacitracine a été obtenue tant chez le donneur que le transconjuguant tandis que la souche réceptrice JH2-2 démontrait une CMI de 32 µg/ml. Le séquençage des gènes asa1, codant pour la substance agrégative, et traB, une protéine régulant négativement la réponse aux phéromones, a révélé une association de cet élément génétique avec le plasmide pJM01. De plus, cette étude présente qu’un antisérum polyclonal SA peut interférer significativement dans le transfert horizontal d’un plasmide répondant aux phéromones codant pour de la résistance à de fortes doses de bacitracine d’une souche E. faecalis aviaire multirésistante. Des isolats cliniques E. faecium d’origine humaine et canine ont été analysés et comparés. Cette étude rapporte, pour la première fois, la caractérisation d’isolats cliniques E. faecium résistants à l’ampicilline (EFRA) d’origine canine associés à CC17 (ST17) au Canada. Ces isolats étaient résistants à la ciprofloxacine et à la lincomycine. Leur résistance envers la ciprofloxacine a été confirmée par la présence de substitutions dans la séquence en acides aminés des gènes de l’ADN gyrase (gyrA/gyrB) et de la topoisomérase IV (parC/parE). Des résistances élevées envers la gentamicine, la kanamycine et la streptomycine, et de la résistance envers les macrolides et les lincosamides a également été observées. La fréquence de résistance envers la tétracycline était élevée tandis que celle envers la vancomycine n’a pas été détectée. De plus, aucune résistance n’a été observée envers le linézolide et la quinupristine-dalfopristine. Les données ont démontré une absence complète des gènes esp (protéine de surface des entérocoques) et hyl (hyaluronidase) chez les isolats canins EFRA testés tandis qu’ils possédaient tous le gène acm (adhésine de liaison au collagène d’E. faecium). Aucune activité reliée à la formation de biofilm ou la présence d’éléments CRISPR (loci de courtes répétitions palindromiques à interespaces réguliers) n’a été identifiée chez les isolats canins EFRA. Les familles de plasmide rep6 and rep11 ont significativement été associées aux isolats d’origine canine. Les profils PFGE des isolats d’origine humaine et canine n'ont révélé aucune relation (≤ 80%). Ces résultats illustrent l'importance d'une utilisation judicieuse des antibiotiques en médecine vétérinaire afin d’éviter la dissémination zoonotique des isolats EFRA canins. Nous pensons que ces résultats contribueront à une meilleure compréhension des mécanismes de résistance aux antibiotiques et de leurs éléments mobiles ainsi qu’à de nouvelles stratégies afin de réduire le transfert horizontal de la résistance aux antibiotiques et des facteurs de virulence. / Enterococci are part of normal intestinal gut flora of animals and humans. Many studies have shown that enterococci from animal origin could represent an antimicrobial resistance genes reservoir for the human community. The two species Enterococcus faecalis and Enterococcus faecium are important in public health; they are responsible for approximately 12% of all nosocomial infections in the United States. In Canada, cases of colonization and/or infections to vancomycin resistant enterococci have more than tripled from 2005 to 2009. A total of 387 poultry E. faecalis and E. faecium isolates, and 124 porcine E. faecalis isolates were identified and analyzed for their antibiotic susceptibilities. High percentages of resistance to macrolides and tetracyclines were found in both avian and porcine isolates. Two predominant phenotypic profiles were determined and analyzed by PCR and sequencing for the presence of antimicrobial resistance genes. Various combinations of antibiotic resistance genes were detected; erm(B) and tet(M) were the most common genes. For the first time, plasmid extraction and hybridization revealed colocalization of erm(B) and tet(M) on a plasmid of ~9 kb in porcine E. faecalis isolates, and of erm(B) and tet(O) on a low-molecular-weight plasmid of ~11 kb in poultry E. faecalis isolates. Furthermore, we demonstrated, through mating experiments, these plasmids could be transferred. Results indicate that the intestinal enterococci of healthy pigs and poultry, which can contaminate meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes. To assess the use of a polyclonal antiserum AS on the contact interference of a high level bacitracin resistant (bcrRAB genes) pheromone-responsive plasmid, a multiresistant E. faecalis isolate of poultry origin was selected. After induction with pheromones produced by the recipient strain E. faecalis JH2-2, clumping of the donor E. faecalis strain 543 was demonstrated as well as high transfer frequencies in short time broth mating. Conjugative transfer of asa1, traB and bcrRAB genes and their co-localization were also demonstrated in the donor strain and a transconjugant T543-1 on a plasmid band of 115 kb by PFGE and Southern blotting. A MIC to bacitracin of > 2 048 µg/ml was obtained for both strains 543 and T543-1 whereas the recipient strain JH2-2 demonstrated a MIC of 32 µg/ml. Sequencing of the asa1 gene encoding for an AS, and traB for a pheromone shutdown protein, confirmed the association of this genetic element to the pheromone-responsive plasmid related to pJM01. More significantly, this study presents the evidence that a polyclonal antiserum AS can significantly interfere with the horizontal transfer of a pheromone-responsive plasmid encoding high-level bacitracin resistance of a poultry multidrug resistant E. faecalis strain. Clinical isolates of E. faecium of human and canine origin were analyzed and compared. This report describes for the first time the characterization of canine clinical ampicillin-resistant E. faecium (AREF) isolates related to CC17 (ST17) in Canada. These isolates were resistant to ciprofloxacin and lincomycin. Resistance to ciprofloxacin was confirmed by amino acid substitutions in DNA gyrase (gyrA/gyrB) and topoisomerase IV (parC/parE) genes. High-level gentamicin, -kanamycin and -streptomycin resistances and macrolides resistance were also observed. The frequency of tetracycline resistance was high whereas vancomycin resistance was not detected. Also, no resistance was observed to linezolid and quinupristin-dalfopristin antibiotics. Data demonstrated the complete absence of enterococcal surface protein (esp) and hyaluronidase (hyl) genes among the canine AREF isolates tested while all were acm (collagen adhesin from E. faecium) positive. However, most of them were shown to harbor efaAfm gene, encoding for a cell wall adhesin. No biofilm formation or clustered regularly interspaced short palindromic repeats (CRISPR) elements were identified in these canine AREF isolates. rep6 and rep11 families of plasmids were significantly associated with isolates from dogs. The PFGE patterns of human and dog isolates were considered unrelated (≤ 80%). These findings also support the importance of prudent use of antibiotics in veterinary medicine to avoid zoonotic spread of canine AREF isolates. We are confident that our results may help to better understand the mechanisms of antibiotic resistance and mobile element carrying them as well as new strategies to reduce the horizontal transfer of antibiotic resistance and virulence traits.