PESQUISA VETERINÁRIA BRASILEIRA

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:

Experimental neonatal colibacillosis, in newbom piglets was attempted using 4 groups of enterotoxigenic Escherichia coli (ETEC) strains, as follows: 1) Two strains from serogroup 0149:K91, both producing thermolabile enterotoxin (LT) and K88 colonization factor; 2) Two strains from serogroup 0101:K30, producing thermostable enterotoxin (STa) and K99 colonization factor; 3) One strain from serogroup 0157:K?, producing thermostable enterotoxin of the STb type and K88 antigen, and 4) One strain from serogroup 08:K?, producing STa enterotoxin anda new colonization factor, named F42. All fourteen piglets inoculated orally with these strains of ETEC developed clinical disease and died up to 42 hours after inoculation, being possible to visualize, by indirect fluorescent antibody technique, in all of them, that colonization of small intestine by the inoculated ETEC had occurred. The production of STa "in vivo", into the gut, by strains from group 2 and 4 was an important factor to prove that experimental colibacillosis did occur. In fact, coprocultures either from the diarrheic stools or from the gut contents revealed a high rate of LT+-K88+ and STa+ -K99+ colonies recovery. Though some quantitative differences among the examined materiais have been observed, the recovery of STa + -F42 + colonies was lower than in the former groups of ETEC strains. However clinical symptoms, production of STa "in vivo" and colonization of the gut of inoculated piglets proved that F42 antigen is undoubtedly a new colonization factor among ETEC involved in porcine colibacillosis.

• Porcine colibacillosis experimental reproduction virulence factors LT STa K88 K99 and F42

Abstract in Portuguese:

Foi tentada a reprodução experimental da colibacilose suína neonatal, em leitões recém-nascidos, usando-se para tal 4 grupos de amostras de Escherichia coli enterotoxigênicas (ETEC), a saber: 1) Duas amostras do sorogrupo 0149:K81, produtoras da enterotoxina termolábil (L T) e do fator de colonização K88; 2) Duas amostras do sorogrupo 0101:K30, produtoras da enterotoxina termoestável (STa) e do fator de colonização K99; 3) Uma amostra do sorogrupo 0157:K?, produtora da enterotoxina termoestável do tipo STb e do fator de colonização K88, e 4) Uma amostra do sorogrupo 08:K?, produtora da enterotoxina termoestável (STa) e de um novo fator de colonização, denominado F42. Todos os 14 leitões inoculados por via oral com estas amostras de ETEC desenvolveram doença clínica com morte até 42 horas após a inoculação, tendo sido possível detectar em todos eles a colonização do intestino delgado pelas amostras de ETEC inoculadas, através da técnica de imunofluorescência indireta. A produção de STa "in vivo", por amostras dos grupos 2 e 4 foi um fator importante na comprovação de que a reprodução experimental da doença por estas amostras realmente ocorreu. De fato, a coprocultura, quer das fezes diarréicas, quer do conteúdo intestinal dos animais, revelou um alto índice de recuperação de colonias LT+ -K88+ e STa+ -K99+. Embora tenham ocorrido entre os diversos materiais examinados algumas diferenças quantitativas, a recuperação de colônias STa=+ -F42+ foi menor do que nos casos anteriores, porém os achados referentes a doença clínica, produção de STa "in vivo" e colonização do intestino delgado dos leitões inoculados, comprovaram que o antígeno F42 é, sem dúvida, um novo fator de colonização em amostras de ETEC envolvidas na colibacilose suína.

• Colibacilose suína reprodução experimental fatores de virulência LT STa K88 K99 e F42

Abstract in English:

Four hundred and seventy-seven cultures of Escherichia coli isolated in the region of Concórdia, Santa Catarina, Brazil, were examined for the presence of ''virulence factors", such as: thermolabile (LT) enterotoxin; thermostable (STa and STb) enterotoxins and the colonization factors K88, K99 and 987P. Paired samples of sera from some sick piglets· were also collected for the search of anti-LT antibodies. Eighty-six (18,02%) E. coli cultures produced ·STa enterotoxin whereas LT enterotoxin was detected in 8 (1,67%) only. Among 391 STa- cultures 49 (12,53%) were STb+. Among 28 K88+ cultures, 1 produced LT; 9 STa and the remaining were non-enterotoxigenic. None of the L T+ cultures codified for K99 antigen, which, on the other hand, was found in 5 STa+ and in 17 non-entérotoxigenic cultures. 987P antigen was found in 3 non-enterotoxigenic cultures. Four out of 10 samples of the examined paired sera showed serological conversion in relation to titres found in ·the passive immunehemolysis (PIH) test. The results of serogrouping revealed that all LT+ cultures belonged to serogroup 01.49. Among 86 STa+ cultures, 1 was grouped as serogroup 09, 2 as 010, 4 as 035; 2 as 064; 3 as 0108; 2 as 0138; 1 as 0149 and 1 as 0157. Seventy STa+ cultures were not classified.· Among 49 STb+ cultures 1 belonged to serogroup 09; 4 to 010; 1 to 035; 1 to 0139; 2 to 0149; 1 to 0157 and 39 cultures could not be serogrouped. These results suggest that in porcine colibacillosis of the region of Concórdia, SC, Brasil the thermostable enterotoxins (STa and STb) may play an important role. It could be possible that among these cultures other colonization factors, different from K88, K99 and 987P may occur. Also, most cultures examined could not be grouped among the serogroups generally accepted as enteropathogenic for swine.

• Virulence factors porcine colibacillosis Concórdia Brazil

Abstract in Portuguese:

Quatrocentos e setenta e sete amostras de Escherichia coli, isoladas na região de Concórdia, SC, Brazil, foram examinadas quanto a presença de fatores de virulência a saber: enterotoxína termolábil (LT), enterotoxinas termoestáveis (STa e STb) e os fatores de colonização K88, K99 e 987P. Amostras pareadas de soro de alguns leitões doentes foram também coletadas para a pesquisa de anticorpos anti-LT. Oitenta e seis (18,02%) amostras de E. coli produziram a enterotoxina STa enquanto a enterotoxina LT-foi detectada em apenas 8 (1.67%). Entre as-381 amostras STa-, 49 (12.53%) foram STb+. Entre 28 amostras K88+, uma produziu LT, 9 STa e as restantes eram não enterotoxigenicas. Nenhuma das amostras ·LT+ codificou para o antígeno K99, porém, este foi encontrado em 5 amostras STa+ e em 17 não entérotoxigênicas. Quatro entre os 10 soros examinados apresentaram conversão sorológica nos níveis de anticorpos anti-LT quando os soros foram examinados pelo teste da imunohemólise passiva (PIH). Os resultados dos exames sorológicos revelaram que todas as amostras LT+ pertenciam ao sorogrupo 0149. Entre 86 amostras STa+, 1 foi classificada como pertencendo ao sorogrupo 09, 2 ao 010; 4 ao 035; 2 ao 064; 3 ao 0108; 2 ao 0138; 1 ao 0149; 1 ao 0157 e 70 amostras não foram Classificadas. Entre as 49 amostras STb+ 1 pertencia ao sorogrupo 09; 4 ao 010; 1 ao 035; 1 ao 0139; 2 ao 0149; 1 ao 0157 e 39 não foram classificadas. Estes resultados sugerem que na colibacilose suína da região· de Concórdia as enterotoxinas termoestáveis (STa e STb) possam desempenhar um importante papel, existindo a possibilidade de que nelas possam ocorrer outros fatores de colonização que não K88, K99 e 987P. As amostras estudadas também, em sua maioria, não se enquadraram entre· aqueles sorogrupos geralmente aceitos como enteropatogênicos para suínos.

• Fatores de virulência colibacilose suína Concórdia Brazil