Partial Biological Characterization Of E.coli Causing Geese Vitellary Peritonitis

1. Isolation and identification of E.coli causing geese salpingitis and peritonitisSamples from the geese died of salpingitis were collected from the veterinary stations. Bacteria were isolated and identified by biochemistry, O antigen test with monosera and 16S rDNA homologous analysis. The result showed that they were E.coli and named as E0238, E0239, E0240, E0453, E0454, E0241, E0245, respectively. The O serotyping of these E.coli were performed by slide agglutination and cuvette agglutination using the standard O antiserum. The results of O serotypes of strains were O2, O21, O37, O1, O24, O24,O148.2. Detection of Virulence-associated Genes in E.coli causing salpingitis and peritonitisAccording to the sequences of virulence-related genes published in the GenBank and some research reports, eleven pairs of specific primers for different virulence-associated genes of APEC were designed to amplify these genes in the isolates E0238,E0239 and E0240, which were isolated from the geese with salpingitis and peritonitis. The results showed that fimC gene was distributed among all of three strains, which was found only one virulence-associated gene in the isolates E0238(O2) and E0240(O37). Howerer, E0239(O21) harbored a combination of fimC-papC-fyuA-irp2-aer-iss-colV-vat-stx2f virulence associated genes, presumably rendering it is particularly virulent. Our results suggested that fim genes maybe some function in salpingitis and peritonitis.3 . fimFGH Sequence analysis of the E.coli strains causing salpingitis and peritonitisAccording to the fimFGH gene available in GenBank, specific primers were designed to amplify fimFGH gene by PCR from E0238,E0239 and E0240 strains which cause salpingitis and peritonitis. fimFGH genes were sequenced and analysed. The results demonstrated that fimF,fimG,fimH were highly conserved among all three E.coli strains (> 96% homology). The adhensin fimH and FimH protein deduced from the nucleotide sequences were the most conserved (> 98% homology) in all of the strains compared with that published in the GenBank. 4 major variable positions were found that distributed along the FimH (N91S99→S91N99 or N91N99;A48→V48;N156→K156,namely N70S78→S70N78或S70N78;A27→V27;N135→K135 , mature FimH) in E0239 strain. Basing on the multiple protein alignment, there was a variable region for FimF and FimG (AA 111→124 in FimF, AA 4→16 in FimG). A111→S111 mutation in FimF was found in both E0238 and E0239 strains. C4V9→R4L9 mutation was for E0238 and E0240 strains. The function of these mutations will be investigated in future.4. Identification of specific proteins of the E.coli strain causing geese salpingitis and peritonitisE. coli cells of E0238, E0239 and E0240 were cultured and inactivated with 1.5‰formaldehyde. Mice were immunized with these cells respectively for four times in interval 12-14 days. The serum was collected from the mouse and abosorbed with the same O serotyping E.coli isolated from chicken. Then the supernatant of E. coli cells (E0238, E0239 and E0240) sonicated was anlysed by SDS-PAGE and Western blot. The results showed that a distinguish protein band about 68kDa when anlysis with the absorbed serum, which was found only in E.coli strain causing geese salpingitis and peritonitis.5. OMP of the E.coli causing geese salpingitis and peritonitisThe OMP (outer membrane proteins) of the E.coli causing geese salpingitis and peritonitis were prepared and determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The results showed that the OMP band patterns of E0238(O2), E0239(O21) strains were different from the other E.coli strains from chickens. An unique protein band (about 68kDa) was found in E.coli causing geese salpingitis and peritonitis. The band was then cut out the gel and analyzed by MALDI-TOF MS. The typical mass spectrum of the protein was compared with that in genbank database. The result showed that there were two kinds of proteins, one was outer membrane cobalamin receptor protein and the other was flagellar biosynthesis protein. The function of thses two proteins will be further investigated.