F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

Fímbrias do tipo IV estão associadas a diversos fenótipos em bactérias gram-negativas, e o presente estudo consistiu na caracterização da fímbria Pil e investigação de seu papel na adesão bacteriana de isolados de EPEC atípica. Por PCR e RT-PCR foram investigadas a presença e a funcionalidade do operon Pil e os resultados demonstraram que este está sendo transcrito somente nos isolados BA558 e BA956. Os genes pilS e pilV foram clonados em vetor de expressão para obtenção das proteínas Pil recombinantes e produção de anticorpos policlonais. A análise qualitativa dos testes de inibição da adesão utilizando os soros anti-PilS e anti-PilV juntos demonstraram que o isolado BA558 apresentou mudança de fenótipo de adesão. Esses resultados nos permitem concluir que o operon Pil está funcional em BA558 e BA956, e a expressão da fímbria Pil nessas cepas não está relacionada à formação de biofilme e autoagregação, porém a proteína fimbrial PilS juntamente com a adesina PilV parecem exercer uma função acessória importante na interação de BA558 às células HEp-2. / Type IV fimbriae are associated with several phenotypes in gram-negative bacteria. The aim of this study was the characterization of the Pil fimbria and its role in the interaction of atypical EPEC isolates in bacterial adhesion. Using PCR and RT-PCR, we investigated the presence and functionality of the pil operon genes. The results showed that these genes are transcribed only in the BA558 and BA956 isolates. The pilS and pilV genes were cloned into an expression vector for recombinant proteins and polyclonal antibodies production. Qualitative analysis of the adherence inhibition assays using both rabbit sera changed to localized-like the phenotype of BA558 isolate adhesion. Together, these results allow us to conclude that the Pil operon is functional only in the BA558 and BA956 isolates and that the expression of Pil fimbriae in aEPEC is not related to biofilm formation and autoaggregation but, the fimbrial PilS protein together with PilV adhesin seem to play an important accessory function in the interaction between the BA558 and epithelial cells in vitro.

Fímbrias do tipo IV estão associadas a diversos fenótipos em bactérias gram-negativas, e o presente estudo consistiu na caracterização da fímbria Pil e investigação de seu papel na adesão bacteriana de isolados de EPEC atípica. Por PCR e RT-PCR foram investigadas a presença e a funcionalidade do operon Pil e os resultados demonstraram que este está sendo transcrito somente nos isolados BA558 e BA956. Os genes pilS e pilV foram clonados em vetor de expressão para obtenção das proteínas Pil recombinantes e produção de anticorpos policlonais. A análise qualitativa dos testes de inibição da adesão utilizando os soros anti-PilS e anti-PilV juntos demonstraram que o isolado BA558 apresentou mudança de fenótipo de adesão. Esses resultados nos permitem concluir que o operon Pil está funcional em BA558 e BA956, e a expressão da fímbria Pil nessas cepas não está relacionada à formação de biofilme e autoagregação, porém a proteína fimbrial PilS juntamente com a adesina PilV parecem exercer uma função acessória importante na interação de BA558 às células HEp-2. / Type IV fimbriae are associated with several phenotypes in gram-negative bacteria. The aim of this study was the characterization of the Pil fimbria and its role in the interaction of atypical EPEC isolates in bacterial adhesion. Using PCR and RT-PCR, we investigated the presence and functionality of the pil operon genes. The results showed that these genes are transcribed only in the BA558 and BA956 isolates. The pilS and pilV genes were cloned into an expression vector for recombinant proteins and polyclonal antibodies production. Qualitative analysis of the adherence inhibition assays using both rabbit sera changed to localized-like the phenotype of BA558 isolate adhesion. Together, these results allow us to conclude that the Pil operon is functional only in the BA558 and BA956 isolates and that the expression of Pil fimbriae in aEPEC is not related to biofilm formation and autoaggregation but, the fimbrial PilS protein together with PilV adhesin seem to play an important accessory function in the interaction between the BA558 and epithelial cells in vitro.