PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Vilela S.M.O., Pinheiro Júnior J.W., Silva J.S.A., Pace F., Silveira W.D., Saukas T.N., Reis E.M.F. & Mota R.A. 2012. Research of Salmonella spp. and evaluation of pathogenicity, cytotoxicity of Escherichia coli isolates proceeding from sparrows (Passer domesticus). Pesquisa Veterinária Brasileira 32(9):931-935. Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Rua Dom Manuel de Medeiros s/n, Recife, PE 52171-900, Brazil. E-mail: rinaldo.mota@hotmail.com The aim of this study was to research the occurrence of Salmonella spp. and Escherichia coli in feces samples of sparrows, as well as to identify the pathogenicity, cytotoxicity and sensitivity profile of the isolates to antimicrobial use. Two hundred and twenty eight sparrows were captured in eight farms. The in vitro pathogenicity test was performed by the isolates culture on congo red-magnesium oxalate Agar, whilst the in vivo pathogenicity test was performed in one day-old chicks. In order to study the cytotoxic effects of indicators, samples were inoculated into Vero cells. The results obtained for Escherichia coli isolation confirmed the presence of this microorganism in 30 (13.2%) of the evaluated samples. Out of those isolates, 10 (33.3%) presented the capacity of absorbing ongo red. As for in vivo pathogenicity a 68.0% of mortality rate of the evaluated samples was observed. Out of 20 isolates tested for cytotoxin production, none of them presented cytotoxic effect in the Vero cells. The Salmonella spp was isolated only in one sample (0.04%), and it was identified as Salmonella enterica subspecies houtenae. Results obtained through this research indicate the need for new studies to identify other virulence factors of E. coli samples and to delineate the phylogenetic profile of the isolates in order to establish a relation with colibacillosis outbreaks in chickens and broilers in the studied region, as well as to analyze the critical points in the aviculture productive chain to identify the source of Salmonella enterica subspecies houtenae.

• Salmonella spp. escherichia coli passer domesticus

Abstract in Portuguese:

RESUMO.- Vilela S.M.O., Pinheiro Júnior J.W., Silva J.S.A., Pace F., Silveira W.D., Saukas T.N., Reis E.M.F. & Mota R.A. 2012. Research of Salmonella spp. and evaluation of pathogenicity, cytotoxicity of Escherichia coli isolates proceeding from sparrows (Passer domesticus). Pesquisa Veterinária Brasileira 32(9):931-935. Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Rua Dom Manuel de Medeiros s/n, Recife, PE 52171-900, Brazil. E-mail: rinaldo.mota@hotmail.com Objetivou-se com este estudo pesquisar a ocorrência de Salmonella spp. e Escherichia coli em amostras de fezes de pardais, além de avaliar a patogenicidade, citotoxicidade e perfil de sensibilidade dos isolados frente a antimicrobianos. Foram capturados 228 pardais em oito granjas. O teste de patogenicidade in vitro foi realizado por meio do cultivo dos isolados em ágar oxalato de magnésio acrescido de vermelho de congo, enquanto o teste de patogenicidade in vivo foi realizado em pintos de um dia. Para o estudo dos indicadores dos efeitos citotóxicos, as amostras foram inoculadas em células Vero. Os resultados obtidos quanto ao isolamento de Escherichia coli confirmaram a presença deste microorganismo em 30 (13,2%) amostras analisadas. Destes isolados, dez (33,3%) apresentaram capacidade de absorção do vermelho congo. Quanto à patogenicidade in vivo observou-se uma taxa de mortalidade de 68,0% das amostras analisadas. Dos 20 isolados testados quanto à produção de citotoxina, nenhum apresentou efeito citotóxico nas células Vero. Obteve-se o isolamento de Salmonella spp. em apenas uma amostra (0,04%), sendo tipificada em Salmonella enterica subespécie houtenae. Os resultados obtidos nesta pesquisa indicam a necessidade da realização de novos estudos para identificar outros fatores de virulência das amostras de E. coli e traçar o perfil filogenético dos isolados para estabelecer uma relação com surtos de colibacilose em galinhas e frango de corte na região estudada, além de analisar os pontos críticos na cadeia produtiva da avicultura para identificar a origem da Salmonella enterica subespécie houtenae.

Abstract in English:

ABSTRACT.- Barros M.R., Silveira W.D., Araújo J.M., Costa E.P., Oliveira A.A.F., Santos A.P.S.F., Silva V.A.S. & Mota R.A. 2012. [Antimicrobial resistance and plasmidial profile of Escherichia coli strain isolated from broilers and commercial layers in the state of Pernambuco, Brazil.] Resistência antimicrobiana e perfil plasmidial de Escherichia coli isolada de frangos de corte e poedeiras comerciais no estado de Pernambuco. Pesquisa Veterinária Brasileira 32(5):405-410.Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Av. Dom Manuel de Medeiros s/n, Recife, PE 52171-900, Brazil. E-mail: rinaldo.mota@hotmail.com Although exist poultry non-pathogenic Escherichia coli strains, many others have capacity to impose serious damages to this birds, being able to cause different infectious diseases. Pathogenic strains are termed Avian pathogenic Escherichia coli (APEC) strains. APEC strains harbor chromossomal and plasmid pathogenicity-related genes. The presence of resistance plasmids in avian E. coli strains could facilitate horizontal tranfer of virulence gene between pathogenic and non pathogenic strains. The aim of this paper was to determine the resistance level to 13 different antibacterial drugs of avian E. coli strains (35) isolated from commercial poultry of Pernambuco State, Brazil, and to correlate the detected resistance level to the presence of plasmids. The results show that 94.28% of strains were resistant to at least three different antibacterial drugs with the highest percentage to lincomycin. The Minimal Inibitory Concentration (MIC) showed that multi- resistance to various antibacterial drugs was present in these strains. Plasmids of several sizes, including plasmids of approximately 88Mda were detected in most of the studied strains. The results herein obtained suggest that the high resistance level observed could be due to the presence of plasmids, what could facilitate the transfer of pathogenicity related genes among pathogenic and non pathogenic strains; it is necessary to take a constant survey on the resistance level to antimicrobial drugs of avian E. coli strains to reach a better control of APEC strains and avoid transfer of pathogenicity related genes between strains.

• Escherichia coli plasmid plasmídio

Abstract in Portuguese:

RESUMO.- Barros M.R., Silveira W.D., Araújo J.M., Costa E.P., Oliveira A.A.F., Santos A.P.S.F., Silva V.A.S. & Mota R.A. 2012. [Antimicrobial resistance and plasmidial profile of Escherichia coli strain isolated from broilers and commercial layers in the state of Pernambuco, Brazil.] Resistência antimicrobiana e perfil plasmidial de Escherichia coli isolada de frangos de corte e poedeiras comerciais no estado de Pernambuco. Pesquisa Veterinária Brasileira 32(5):405-410.Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Av. Dom Manuel de Medeiros s/n, Recife, PE 52171-900, Brazil. E-mail: rinaldo.mota@hotmail.com Embora existam linhagens de Escherichia coli não patogênicas para aves, muitas outras possuem a capacidade de causar sérios danos à saúde das mesmas, sendo capazes de ocasionar diferentes tipos de processos infecciosos. As linhagens patogênicas são denominadas Avian Pathogenic Escherichia coli (APEC), possuindo genes relacionados ao processo de patogênese em epissomos (plasmídios) ou no cromossomo. A presença de plasmídios, contendo genes de resistência a antibióticos em linhagens aviárias, patogênicas ou não, indicam a possibilidade de transferência gênica lateral entre diferentes tipos de linhagens facilitando também a transferência de genes de patogenicidade ou virulência. Objetivou-se com este estudo avaliar o perfil de sensibilidade a antibióticos (13) de diferentes amostras (35) de E. coli isoladas de aves comerciais do Estado de Pernambuco apresentando, ou não, sinais clínicos de processos infecciosos e correlacionar esta resistência com a presença de plasmídios. Os testes utilizados demonstraram que 94,28% dos isolados foram resistentes a três ou mais antibióticos, com a lincomicina apresentando o maior percentual de resistência (100%). Na Concentração Inibitória Mínima (CIM) observou-se multirresistência a vários antimicrobianos. A presença de plasmídios foi detecada em 80,0% (28/35) dos isolados, com 16 isolados apresentando plasmídios com peso molecular aproximado de 88 MDa. Também foi verificada a presença de linhagens apresentando plasmídios de vários tamanhos. Concluiu-se que isolados de E. coli resistentes a antimicrobianos utilizados na avicultura estão presentes no Estado de Pernambuco, tanto em frangos de corte quanto em poedeiras comerciais. A presença de plasmídios detectados na maioria dos isolados pode estar associada à resistência aos antimicrobianos e sugere a presença de possíveis genes relacionados à patogenicidade. Monitorar a resistência a antibióticos em bactérias isoladas de animais torna-se um fator determinante para eleição e êxito do tratamento, bem como a possibilidade de eliminação daquelas que possuem plasmídios para se evitar a transferência de genes relacionados à patogenicidade.

Abstract in English:

ABSTRACT.- Nakazato G., Campos T.A., Stehling E.G., Brocchi M. & Silveira W.D. 2009. Virulence factors of avian pathogenic Escherichia coli (APEC). Pesquisa Veterinária Brasileira 29(7):479-486. Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas (Unicamp), Cidade Universitária Zeferino Vaz s/n, Cx. Postal 6109, Barão Geraldo, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br Avian pathogenic Escherichia coli (APEC) strains cause a great diversity of diseases in birds and are responsible for great economic losses in the avian industry. To date, several studies have been carried out to better understand the APEC pathogenesis for a possible development of tools which could prevent the economics losses caused by these strains. This review discusses the virulence factors described do date to be expressed by these strains and the advances made to understand and identify virulence determinants present in APEC.

• APEC avian Escherichia coli virulence factors

Abstract in Portuguese:

RESUMO.- Nakazato G., Campos T.A., Stehling E.G., Brocchi M. & Silveira W.D. 2009. Virulence factors of avian pathogenic Escherichia coli (APEC). [Fatores de virulência de Escherichia coli aviária patogênica (APEC).] Pesquisa Veterinária Brasileira 29(7):479-486. Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas (Unicamp), Cidade Universitária Zeferino Vaz s/n, Cx. Postal 6109, Barão Geraldo, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br Linhagens de Escherichia coli patogênicas para aves (APEC) causam uma grande diversidade de doenças em aves e são responsáveis por grandes prejuízos na indústria aviária. Nos últimos anos, vários estudos foram realizados para melhor entender a patogênese de linhagens APEC e para desenvolver ferramentas que podem prevenir as perdas econômicas causadas por estas linhagens. Esta revisão discute os fatores de virulência descritos nestas linhagens e os avanços realizados para entender e identificar os determinantes de virulência presentes em APEC.

Abstract in English:

ABSTRACT.- Campos T.A., Nakazato G., Stehling E.G., Brocchi M. & Silveira W.D. 2008. Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis. Pesquisa Veterinária Brasileira 28(10):508-514. Departamento de Microbiologia e Imunologia, Instituto de Biologia, Cx. Postal 6109, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz s/n, Barão Geraldo, Campinas, SP 3081-862, Brazil. *Corresponding author: wds@unicamp.br The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5’ and 3’regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.

• APEC clonal analysis fliC gene

Abstract in Portuguese:

ABSTRACT.- Campos T.A., Nakazato G., Stehling E.G., Brocchi M. & Silveira W.D. 2008. Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis. Pesquisa Veterinária Brasileira 28(10):508-514. Departamento de Microbiologia e Imunologia, Instituto de Biologia, Cx. Postal 6109, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz s/n, Barão Geraldo, Campinas, SP 3081-862, Brazil. *Corresponding author: wds@unicamp.br The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5’ and 3’regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.

Abstract in English:

ABSTRACT.- Campos T.A., Lago J.C., Nakazato G., Stehling E.G., Brocchi M., Castro A.F.P. & Silveira W.D. 2008. Occurrence of virulence-related sequences and phylogenetic analysis of commensal and pathogenic avian Escherichia coli strains (APEC). Pesquisa Veterinária Brasileira 28(10):533-540. Departamento de Microbiologia e Immunologia, Instituto de Biologia, Unicamp, Cidade Universitrária Zeferino Vaz s/n, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfAO157/O141, lpfAO157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfAO157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.

• Avian colibacillosis Escherichia coli APEC virulence related-genes pathogenic clones

Abstract in Portuguese:

ABSTRACT.- Campos T.A., Lago J.C., Nakazato G., Stehling E.G., Brocchi M., Castro A.F.P. & Silveira W.D. 2008. Occurrence of virulence-related sequences and phylogenetic analysis of commensal and pathogenic avian Escherichia coli strains (APEC). Pesquisa Veterinária Brasileira 28(10):533-540. Departamento de Microbiologia e Immunologia, Instituto de Biologia, Unicamp, Cidade Universitrária Zeferino Vaz s/n, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfAO157/O141, lpfAO157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfAO157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.

Abstract in English:

Abstract.- Brocchi M., Ferreira A., Lancellotti M., Stehling E.G., Campos T.A., Nakazato G., Pestana de Castro A.F. & Silveira W.D. 2006. Typing of avian pathogenic Escherichia coli strains by REP-PCR. Pesquisa Veterinária Brasileira 26(2):69-73. Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade de Campinas, Cx. Postal 6109, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

• Avian Escherichia coli typing REP-PCR.

Abstract in Portuguese:

Abstract.- Brocchi M., Ferreira A., Lancellotti M., Stehling E.G., Campos T.A., Nakazato G., Pestana de Castro A.F. & Silveira W.D. 2006. Typing of avian pathogenic Escherichia coli strains by REP-PCR. Pesquisa Veterinária Brasileira 26(2):69-73. Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade de Campinas, Cx. Postal 6109, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.