Relationship Of Fimbriae F107 And Variant Of The SLT â…¡ Toxin And Establishment Of Preliminary Diagnostic Method To Edema Disease

Edema Disease (ED) of weaning piglets is caused mainly by Verotoxigenic EscheHchia coli (VTEC) which lead to edema in various tissues including the central nervous system. The name 'Edema Disease' was first used because edema of the stomach submucosa and the mesocolon were often a prominent feature of the disease. Fimbriae F107 and variant of the SLT II toxin were two factors of VTEC, so researches of relations between them has importance to diagnosis and prevention of ED.Two Strains 107/86 and G of E.coli from weaned pigs with ED which possess fimbriae F107 were cultured in trypticase soy broth(TSB) .As shown by electron microscopy ,the strain expressed fimbriae after growing for24hr, at 37 . The strain 107/86 as immunogen immunized BALB/C mice and the strain G was absorbed in ELISA plate to detect hybrid cell supernatants. Three positive clones F7-1, 2F7-2, H7 were obtained by ELISA and slide agglutination. Through SDS-PAGE, the crude bacterial extract showed one major protein band of 15kDa,our Mabs reacted with the 15 kDa protein band specifically in a Western-blot. 18 strains isolated from ED were tested with anti-F107 fimbriae Mab ,results shows 12 strains express fimbriae F107.The toxin SLT-â…¡e was difficult to purify, so we expressed fusion protein GST-SLT-â…¡eB in recombinant E.coli ppSLT-â…¡eB. The fusion protein purified by Glutathione Sephrose 4B as antigen immunized BALB/C mice. GST expressed in recombinant E.coli ppGEX-6P-l as negative antigen and SLT-â…¡eB as positive antigen absorbed in ELISA plate. SP2/0 myelomacells and spleen cells of the immunized BALB/C mice were fused by PEG-2000. The hybridoma culture supernatants were screened for anti-SLT-â…¡eB antibodies by indirect-ELISA and cytotoxicity neutralization. One monoclonal antibody 7C3-1 was detected and only reacted with a 33KDa fusion protein band in Western-blot. The Mab as probe has detected 18 strains of ED by Dot-ELISA, which shows 13 strains were positive.The procedure of detecting SLT-â…¡e was very complex. Strains of ED must be purified and cultured in SB, then these bacterial were lysated or induced by polymyxin B, it would take 3-4 days. So diagnosis of ED could not be completed quickly byidentifying SLT-â…¡e. Strains of ED colonized on the mucosal surface of the small intestine generally in vivo, for this reason, the strains should express fimbriae F107 as we expected. According to the adhesive character, we isolated strains from the mucosal of the small intestine and identified these bacterium with anti-fimbriae F107 antibodies by slide agglutination , indirect-ELISA and dot-ELISA. The results showed that dot-ELISA could detect the strains that expressed fimbriae sensitively. Major advantage of the technique was sensitive and prompt. The dot-ELISA with Mab had potential uses as a powerful diagnostic technique to ED.