An in-vitro model for studying the interaction of Escherichia coli O157:H7 and other enteropathogens with bovine primary cell cultures.

Sections of kidney, trachea, ileum, colon, rectum and rumen were removed at post mortem from a neonatal calf and, with the exception of the rumen, primary cell lines were established for each of the cell types. The adherence of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, enteropathogenic E. coli (EPEC) serotype O111, E. coli K12 (a laboratory adapted non-pathogenic strain) and Salmonella enterica serotype Typhimurium was assayed on each cell type. For all adherence assays on all cell lines, EHEC O157:H7 adhered to a significantly greater extent than the other bacteria. S. Typhimurium and EPEC O111 adhered to a similar extent to one another, whereas E. coli K12 was significantly less adherent by 100-fold. In all cell types, >10% of adherent S. Typhimurium bacteria invaded, whereas c. 0.01-0.1% of adherent EHEC O157:H7 and EPEC O111 bacteria invaded, although they are regarded as non-invasive. EHEC O157 generated actin re-arrangements in all cell types as demonstrated by fluorescent actin staining (FAS) under densely packed bacterial micro-colonies. EPEC O111 readily generated the localised adherent phenotype on bovine cells but generated only densely packed micro-colonies on HEp-2 cells. The intensity of actin re-arrangements induced in bovine cells by EPEC O111 was less than that induced by EHEC O157:H7. The intimate attachment on all cell types by both EHEC O157:H7 and EPEC O111 was clearly demonstrated by scanning electron microscopy.

Contribution of fimbriae and flagella of Salmonella enteritidis to colonization and invasion of chicks.

Isogenic mutants of Salmonella enteritidis defective for the elaboration of fimbrial types SEF14, SEF17, SEF21 and flagella were used to study the contribution these organelles made to colonization, invasion and lateral transfer in young chicks. The caecum, liver and spleen were colonized within 24 h following oral inoculation of 1-day-old chicks with 10(5) wild-type S. enteritidis strain LA5. However, for some mutants, the numbers of organisms recovered from internal organs was reduced significantly, particularly at 24h post-inoculum, which supported the hypothesis that the organelles contribute to invasion and dissemination to internal organs. Specifically, mutations affecting SEF17, SEF21 and flagella contributed to a delay in colonization of the spleen, and those affecting SEF21 and flagella delayed colonization of the liver. Lower numbers of bacteria were recovered from the caecum with mutants deficient in elaboration of SEF21. Sentinel birds were colonized by LA5 or EAV40 (14s(-), 17(-), 21(-), fla(-)) directly from the environment within 2 days, although a consistent slight delay was observed with the multiple mutant. Overall, our data suggest a collective role for SEF17, SEF21 and flagella, but not SEF14, in the early stages of colonization and invasion of young chicks by S. enteritidis, but these surface appendages appear unnecessary for colonization of birds from their immediate environment.

Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis.

Salmonella enteritidis expresses flagella and several finely regulated fimbriae, including SEF14, SEF17 and SEF21 (type 1). A panel of mutants was prepared in three strains of S. enteritidis to elucidate the role of these surface appendages in the association with and invasion of cultured epithelial cells. In all assays, the naturally occurring regulatory-defective strain 27655R associated with tissue culture cells significantly more than wild-type progenitor strains LA5 and S1400/94. Compared with wild-type strains, SEF14 mutants had no effect on association and invasion, whereas SEF17, SEF21 and aflagellate mutants showed significant reductions in both processes. Histological examination suggested a role for SEF17 in localized, aggregative adherence, which could be specifically blocked by anti-SEF17 sera and purified SEF17 fimbriae. SEF21-mediated association was neutralized by mannose and a specific monoclonal antibody, although to observe enhanced association it was necessary for the bacteria to be in fimbriate phase prior to infection. Additionally, aflagellate mutants associated and invaded less than motile bacteria. This study demonstrated the potential for multifactorial association and invasion of epithelial cells which involved SEF17 and SEF21 fimbriae, and flagella-mediated motility.

SEF17 fimbriae are essential for the convoluted colonial morphology of Salmonella enteritidis.

Salmonella enteritidis isolated from poultry infections generated a convoluted colonial morphology after 48 h growth on colonisation factor antigen (CFA) agar at 25 degrees C. A mutant S. enteritidis defective for the elaboration of the SEF17 fimbrial antigen, in which the agf gene cluster was inactivated by insertion of an ampicillin resistance gene cassette, and other wild-type S. enteritidis transduced to this genotype failed to produce convoluted colonies. However, growth of SEF17- mutants at 25 degrees C on CFA agar supplemented with 0.001% Congo red resulted in partial recovery of the phenotype. Immunoelectron microscopy demonstrated that copious amounts of the SEF17 fimbrial antigen were present in the extracellular matrix of convoluted colonies of wild-type virulent S. enteritidis isolates. Bacteria were often hyperflagellated also. Immunoelectron microscopy of SEF17- mutants grown on CFA agar+0.001% Congo red demonstrated the elaboration of an as yet undefined fimbrial structure. Isolates of S. enteritidis which were described previously as avirulent and sensitive to environmental stress failed to express SEF17 or produce convoluted colonies. These data indicate an essential role for SEF17, and possibly for another fimbria and flagella, in the generation of the convoluted colonial phenotype. The relationship between virulence and colonial phenotype is discussed.

Expression of SEF17 fimbriae by Salmonella enteritidis.

Specific immunological reagents were used to investigate the expression of SEF17 fimbriae by cultured strains of Salmonella enteritidis. Most strains of Salm. enteritidis tested expressed SEF17 when cultured at temperatures of 18-30 degrees C. However, two wild-type strains produced SEF17 when also grown at 37 degrees C and 42 degrees C. Colonization factor antigen agar was the optimum medium for SEF17 expression, whereas Drigalski and Sensitest agars poorly supported SEF17 production. Very fine fimbriae produced by a strain of Salm. typhimurium were specifically and strongly labelled by SEF17 monoclonal and polyclonal antibodies, indicating considerable antigenic conservation between the two. Curli fimbriae from Escherichia coli were similarly labelled. The production of these fimbriae correlated with the binding of fibronectin by the organism. Congo red binding by cultured bacteria was not a reliable criterion for the expression of SEF17 fimbriae.

Characterisation of monoclonal antibodies specific to SEF 21 fimbriae of Salmonella enteritidis and their reactivity with other Salmonellae and Enterobacteria.

A panel of monoclonal antibodies (mAbs) specific to type 1 (SEF 2) fimbriae of S. enteritidis was produced using crude and HPLC purified preparations of SEF 21 fimbriae. Sixteen mAbs were selected by indirect ELISA using both purified SEF 21 antigen and whole cells of S. enteritidis. Eight mAbs were confirmed by immunoprecipitation assay to react specifically with SEF 21 fimbriae. These mAbs were further characterised for their reactivity patterns by the "whole cell" ELISA and latex agglutination test with a number of strains of Salmonella and other enterobacteria. Not all SEF 21 mAbs reacted in both ELISA and latex agglutination tests with whole bacterial cells. mAb 611 was the only one suitable for use in both tests. Unexpectedly these mAbs reacted with the type 1 fimbriae of many of the tested strains of enterobacteria. mAb 721 reacted with most strains of Salmonella (89.1%) and enterobacteria (71.4%) tested. mAb 611 reacted with 61%-75% of strains of Salmonella and with 6.9%-17.6% of enterobacteria in ELISA and latex tests respectively. These mAbs will be useful reagents for further characterisation of type 1 fimbriae expressed by members of the family Enterobacteriaceae.

Characterisation of monoclonal antibodies against a fimbrial structure of Salmonella enteritidis and certain other serogroup D salmonellae and their application as serotyping reagents.

A panel of 13 monoclonal antibodies from different hybridomas was produced against a novel salmonella fimbrial antigen expressed predominantly by Salmonella enteritidis strains. The specificity of the monoclonal antibodies to this antigen (SEF14) was confirmed by enzyme-linked immunosorbent assay (ELISA) using purified SEF14, immune electron microscopy and, with 11 monoclonal antibodies, the identification of a repeating protein subunit (14,300kDa) on the antigen. Blocking-ELISA with the monoclonal antibodies identified epitopes in at least three, non-overlapping clusters which appeared evenly distributed on SEF14 in immune electron microscopy. The use of the monoclonal antibodies in direct-binding ELISA on a range of salmonella serotypes suggested that the epitopes on SEF14 are highly conserved and were expressed by all the S enteritidis strains examined; some strains of S dublin and the only strain of S moscow available were the only other serotypes that expressed SEF14. A latex agglutination reagent based on a monoclonal antibody was developed and used to test for SEF14 on 280 strains (representing 120 serotypes in 24 serogroups of salmonellae) that had been grown on Sensitest agar for 18 hours at 37 degrees C. All S enteritidis strains (64) and most S dublin strains (28 of 33) produced SEF14 as did the two strains representing S blegdam and S moscow. SEF14 was not detected in any other strains of serotypes from serogroup D or from any other serogroup examined.