Detection of pap, sfa, afa and fim Adhesin-Encoding Operons in Avian Pathogenic Escherichia coli

The occurrence of fim, pap, sfa, and afa genes was evaluated in 200 Escherichia coli strains isolated from chickens diagnosed with omphalitis, salpingitis, swollen head syndrome, or chronic respiratory disease. Analysis of the strains by colony hybridization tests demonstrated that 96% of the isolates were fim, 16% were pap, and 6% were sfa. None of the isolates was afa. The fim gene occurred in strains from all diseases evaluated, with no significant differences among the isolates. Conversely, significant differences were observed in relation to pap and sfa genes. Of the strains tested, 8% from either salpingitis or omphalitis were positive for pap gene, compared with 28% from swollen head syndrome and 20% from chronic respiratory disease. Evaluation of the sfa gene indicated its presence in 4% of the salpingitis and chronic respiratory disease isolates and 6% of omphalitis, but sfa was not observed in isolates from swollen head syndrome. An in vitro adherence test Intern J Appl Res Vet Med • Vol. 2, No. 2, 2004 Vol. 2, No. 2, 2004 • Intern J Appl Res Vet Med 136 ed by the fim gene cluster. This type of fimbria is common among Enterobacteriaceae, and several variants have been associated with avian pathogenic E. coli. Their role in infection is unclear, although it has been suggested that they may be involved in the initial stages of colonizing the upper respiratory tract. Studies indicate adhesin-encoding operons pap, sfa, and afa are prevalent in E. coli strains associated with urinary tract infections (pyelonephritis) in humans. P fimbriae consist of a major fimbrial subunit, PapA, which determines 11 different serogroups, and a terminally located adhesin, PapG. Receptor specificity of P fimbriae is conferred by PapG, which recognizes different receptors of the globosides GbO3 (globotriasylceramide), GbO4 (globotetraosylceramide), and GbO5 (globopentosylceramide) in P-blood group antigens of human and sheep erythrocytes. The role of P adhesins in avian pathogenic E. coli has not been fully elucidated, but they appear to be involved in the colonization of internal organs. The afimbrial adhesin is a mannoseresistant, P-independent, X-binding adhesin, expressed by the afa-1 operon. It mediates specific binding to uroepithelial celland human erythrocyte-receptors. The nature of the receptor on the eucaryotic cell surface is not yet known. The S fimbriae have a mannose-resistant adhesin, encoded by the sfa operon, that recognizes α-sialyl-β-2,3-galactose receptors, present on human and calf erythrocytes. The presence of S fimbriae is also correlated with pathogenicity of E. coli in human meningitis and septicemia. The role of afimbrial adhesin and S fimbriae in the pathogenicity of avian pathogenic E. coli remains unclear. The purpose of this study was to compare the occurrence of fim, pap, sfa, and afa genes in E. coli strains isolated from cases of omphalitis, salpingitis, swollen head syndrome, and chronic respiratory disease in poultry. MATERIALS AND METHODS Bacterial Strains and Growth Conditions A total of 200 strains of E. coli were isolated from poultry in the state of São Paulo in Brazil. The strains were isolated from oviducts of broiler breeders with salpingitis (n = 50), yolk sacs of one-day-old chicks with omphalitis (n = 50), subcutaneous facial tissue of chickens with swollen head syndrome (n = 50), and air sacs from broilers with chronic respiratory disease (n = 50). Standard bacteriologic methods were used for isolation and identification of the organisms. All strains were stored at -20 ̊C in brain heart infusion broth (Difco) to which 15% glycerol was added after incubation. For adherence assays, bacterial strains were grown on colonization factor antigen agar and incubated at 37 ̊C for 18 to 24 hours. Tracheal Ring Cell Preparation and Adherence Assay Tracheal sections were obtained from 10day-old specific-pathogen-free chicks (Granja Rezende, Brazil) and cut into 4-mm sections.Adherence tests were performed in 24-well, round-bottom microtiter plates. Three sections of trachea and Dulbecco’s Modified Eagle Medium without calf serum were added to each well. The material was examined by inverse light microscopy for evidence of ciliary motility that would indicate cell viability. Bacterial strains plus tracheal rings were incubated at 37 ̊C for 30 minutes, after which they were washed six times with 50 mM (pH 7.4) phosphatebuffered saline and incubated for an additional 4 hours. The rings were fixed in 10% buffered formalin. A strain of E. coli K-12 was used as a negative control. Colony Hybridization The test strains were examined by colony blot hybridization, using specific DNA probes labeled with [α-P]-dATP by nick translation. The DNA probe used to detect fim B-H genes was a 9.6-Kb Hind III-Sal I fragment from recombinant plasmid pIB254. Detection of pap, afa, and sfa