Bacterial food-borne zoonoses.

In many countries of the world, bacterial food-borne zoonotic infections are the most common cause of human intestinal disease. Salmonella and Campylobacter account for over 90% of all reported cases of bacteria-related food poisoning world-wide. Poultry and poultry products have been incriminated in the majority of traceable food-borne illnesses caused by these bacteria, although all domestic livestock are reservoirs of infection. In contrast to the enzootic nature of most Salmonella and Campylobacter infections, Salmonella Enteritidis caused a pandemic in both poultry and humans during the latter half of the 20th Century. Salmonella Typhimurium and Campylobacter appear to be more ubiquitous in the environment, colonising a greater variety of hosts and environmental niches. Verocytotoxin-producing Escherichia coli O157 (VTEC O157) also emerged as a major food-borne zoonotic pathogen in the 1980s and 1990s. Although infection is relatively rare in humans, clinical disease is often severe, with a significant mortality rate among the young and elderly. The epidemiology of VTEC O157 is poorly understood, although ruminants, especially cattle and sheep, appear to be the major source of infection. The dissemination of S. Enteritidis along the food chain is fairly well understood, and control programmes have been developed to target key areas of poultry meat and egg production. Recent evidence indicates that these control programmes have been associated with an overall reduction of S. Enteritidis along the food chain. Unfortunately, existing controls do not appear to reduce the levels of Campylobacter and VTEC O157 infections. Future control strategies need to consider variations in the epidemiologies of food-borne zoonotic infections, and apply a quantitative risk analysis approach to ensure that the most cost-effective programmes are developed.

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.

Characterisation of epitopes of type 1 fimbriae of Salmonella using monoclonal antibodies specific for SEF21 fimbriae of Salmonella enteritidis.

Monoclonal antibodies (mAbs) were used to identify and characterise epitopes of type 1 (SEF21) fimbriae of Salmonella enteritidis. The distribution of the epitopes among salmonellas and other enterobacteria was investigated, as well as the influence of growth media and temperatures on their expression. At least four different epitope clusters were identified on SEF21 fimbriae of S. enteritidis. Two of these clusters were associated with fimbrial haemagglutinins that were either common to all salmonellae tested, or restricted only to S. enteritidis and S. dublin. The four epitope clusters were identified on type 1 fimbriae of most Salmonella serotypes, as well as non-haemagglutinating type 2 fimbriae of S. pullorum and S. gallinarum, and on many other enterobacterial species. The expression of the epitopes was affected by growth conditions.

Discrimination of strains of Salmonella enteritidis with differing levels of virulence by an in vitro glass adherence test.

The objective of this study was the in vitro differentiation of isolates of Salmonella enteritidis whose virulences differed in a chick model. A total of 14 strains of S. enteritidis were isolated from either the environment, dairy products, or infected patients. The isolates could be divided into two groups on the basis of their virulence (50% lethal dose) in chickens infected intraperitoneally. When the strains were incubated in adherence test medium (Spanish patent 9700408), only the virulent strains produced aggregates and formed visible filaments attached to the glass tube. These results suggest, although for a limited number of strains, that aggregation in such a medium could be used as a diagnostic tool to discriminate virulent strains of S. enteritidis.

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.

Salmonella enterica var Typhimurium and Salmonella enterica var Enteritidis express type 1 fimbriae in the rat in vivo.

In a series of experiments rats were dosed with purified type 1 fimbriae from Salmonella enterica var Enteritidis or with fimbriated cultures of either S. enterica var Typhimurium or S. enterica var Enteritidis. Paraffin-wax embedded histological sections of jejunal and ileal tissue were taken and stained by the streptavidin biotin complex (sABC) staining technique for the detection of salmonella and type 1 fimbriae. On oral infection with Enteritidis and Typhimurium both bacteria were shown to be closely associated with the rat ileal epithelium and expressed type 1 fimbriae, thus clearly demonstrating that type 1 fimbriae are expressed by salmonellae in vivo. Moreover, association with the ileum was also shown to occur when purified type 1 fimbriae were orally administered to rats. Our results suggest that type 1 fimbriae alone or in combination with other fimbriae may play an important role in the early stages of infection with these pathogenic bacteria.

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.

Seroreactivity of Salmonella-infected cattle herds against a fimbrial antigen in comparison with lipopolysaccharide antigens.

The IgG seroreaction of Salmonella-infected cattle herds against a fimbrial antigen (SEF14) was compared with that against lipopolysaccharide (LPS) antigens. Sera from 23 dairy herds (n = 205) from an island with no occurrence of salmonellosis, four herds (n = 303) with recent outbreaks of S. dublin and four herds (n = 168) with recent outbreaks of S. typhimurium, were tested in a SEF14-ELISA, S. dublin LPS (0:1, 9, 12) ELISA and S. typhimurium LPS (0:1, 4, 5, 12) ELISA. At a cut-off OD of 0.5, only one of the animals tested from the salmonellosis-free island showed significant seroreaction against the SEF14 antigen, which was confirmed in a Western-blot analysis. Three out of the four S. dublin-infected herds had several seroreactors in the SEF14-ELISA, whereas all the four herds were positive in the 0:1, 9, 12-ELISA. All but two samples (both from the same herd) in the four S. typhimurium-infected herds, positive in the 0:1, 4, 5, 12-ELISA, had OD values below 0.5 in the SEF14-ELISA. The results indicate that cattle can produce detectable specific antibodies against fimbrial antigens which may be used for screening of S. dublin-infected herds, particularly in areas with low prevalence of salmonellosis, increasing the predictive value of serology.

World Health Organisation--supervised interlaboratory comparison of ELISAs for the serological detection of Salmonella enterica serotype Enteritidis in chickens.

A collaborative exercise, supervised by the World Health Organisation, was set up to compare ELISAs used for the serological detection of Salmonella enteritica serotype Enteritidis in chickens. The aim was to ascertain how far agreement could be reached on the interpretation of optical density readings for high titre, intermediate titre and low titre sera. Two sets of sera were sent to 14 participants. The first set compared high, medium and low titre sera raised in specified-pathogen-free and commercial broiler breeder chickens. The second set comprised 20 sera of different antibody titres raised in commercial birds reared under laboratory conditions and sent blind. Both indirect and double-antibody sandwich blocking ELISAs were used with a number of different detecting antigens. With a few exceptions good agreement was reached on the interpretation of results obtained from high and low titre sera from the optical density obtained with a single serum dilution. Differences were observed in the interpretation of medium titre sera. The results suggested that most ELISAs produce reasonably comparable results and that practical problems may arise from interpretation of the results mainly as a result of the choice of the criteria used for differentiating sera obtained from infected and uninfected chickens. These problems are discussed.

Development and application of enzyme-linked immunosorbent assay for specific detection of Salmonella enteritidis infections in chickens based on antibodies to SEF14 fimbrial antigen.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to the SEF14 fimbrial antigen (SEF14-DAS ELISA) and was evaluated for its use in the specific detection of chicken flocks infected with Salmonella enteritidis. The SEF14-DAS ELISA successfully discriminated between chickens experimentally infected with S. enteritidis and those infected with S. panama or S. typhimurium, although the SEF14 responses in adult birds infected with S. enteritidis were detectable but low. In contrast, ELISAs used to detect antibodies to lipopolysaccharide (LPS) and flagella were unable to discriminate between the infected groups of chicks and adult birds infected with different Salmonella serotypes. LPS and flagellar responses were low and variable in chicks, whereas in adult hens they were found to be consistently strong. When flocks naturally infected with S. enteritidis were tested by the SEF14-DAS ELISA and ELISAs to detect LPS and flagellar antibodies, it was found that they could all identify the infected flocks, although there was little correlation between individual serum samples. The study shows that the SEF14-DAS ELISA may offer advantages over existing assays with comparable sensitivities coupled with higher specificities for the serological detection of S. enteritidis-infected chicken flocks.

Studies into the role of the SEF14 fimbrial antigen in the pathogenesis of Salmonella enteritidis.

To investigate the role of the SEF14 fimbrial antigen in pathogenesis, a single defined sefA (SEF14-) inactivated mutant of Salmonella enteritidis strain LA5 was constructed and tested in a number of biological assay systems. There was no significant difference between the wild-type strain and the isogenic SEF14- mutant in their abilities to adhere to and invade HEp-2 epithelial cells or their survival in mouse peritoneal macrophages, whereas the SEF14- mutant was ingested more rapidly by isolated human PMN. Both the strains colonized the intestine, invaded and spread systemically in 1 day-old chicks, laying hens and BALB/c mice equally well. A significantly greater number of chicks excreted the wild-type SEF14+ strain during the first week following infection as compared to those infected with the SEF14- mutant. However, similar numbers of chicks excreted the two strains between 2 and 7 weeks after infection. These results indicate that possession of SEF14 fimbriae alone do not appear to play a significant role in the pathogenesis of S. enteritidis although its contribution to virulence may be dependent on the host species infected.

Evaluation of SEF14 fimbrial dot blot and flagellar western blot tests as indicators of Salmonella enteritidis infection in chickens.

The serological responses to Salmonella enteritidis flagella (H: g,m) and its fimbrial antigen SEF14 were evaluated as indicators of infection in chickens and to confirm serological results obtained by an ELISA using S enteritidis lipopolysaccharide (LPS) (O: 9,12) as the detecting antigen. The SEF14 antigen and flagella were extracted from S enteritidis and transferred to nitrocellulose paper for use in Western and dot blot tests. Antisera to 19 salmonella serotypes including S enteritidis were raised in rabbits and their cross reactivity to the flagellar and SEF14 antigens was evaluated. Cross reactivity with the SEF14 antigen was found in one antiserum, raised against S blegdam, and to flagella in eight of 19 antisera raised against various salmonella serotypes, most of which shared the flagellar factors g or m with S enteritidis. The intensity of cross reaction to flagella was strongest in S derby and S blegdam antisera. Antisera raised in chickens against S typhimurium and S panama did not cross react in either test, and neither did pooled sera from eight-week-old salmonella-free, broiler breeder parent chickens. Field sera from two commercial flocks with no history of salmonella infection were negative when tested by the LPS ELISA. These sera were also negative when tested by the flagellar and SEF14 blots. S enteritidis infection in a commercial laying flock was detected initially when the sera were tested by the LPS ELISA and confirmed in individual and pooled sera by the SEF14 and flagellar tests. S enteritidis PT4 was isolated from this flock post mortem.

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.

Salmonella fimbriae: novel antigens in the detection and control of salmonella infections.

Fimbriae are thin, proteinaceous surface organelles produced by members of the Enterobacteriaceae, including most salmonellas. A number of fimbrial antigens expressed by strains of Salmonella enteritidis and S. typhimurium have now been described and characterized. However, their functions are still poorly understood, although some evidence indicates they have a role in bacterial survival in the host or external environment. Diagnostic tests based on the detection of fimbriae or specific antibodies against them have recently been developed and applied successfully to the rapid and specific identification of S. enteritidis infections. The role of salmonella fimbriae in future generations of live vaccines either as protective antigens or as the carriers of heterologous antigens is also discussed.

Serodiagnosis of Mycobacterium bovis infection in badgers: development of an indirect ELISA using a 25 kDa antigen.

A 25 kDa antigen of Mycobacterium bovis has previously been identified as immunodominant during badger infections. This 25 kDa antigen was partially purified from sonicated M bovis bacilli by using water precipitation and ion exchange chromatography, and its purification was monitored with a mouse monoclonal antibody, MBS43, which was specific for the antigen. The partly purified antigen was used to develop an ELISA for the assay of badger sera for the presence of specific antibodies. A presumed negative badger population was used to calculate the assay's threshold of seropositivity and using this value, its sensitivity (37 percent) and specificity (98 percent) were determined in a second population of known culture status. The results indicate that it may be possible to develop a specific and cost effective serological field assay for the diagnosis of M bovis infection in living badgers.

The use of latex particle agglutination to specifically detect Salmonella enteritidis.

This paper reviews the development and evaluation of a latex particle agglutination test to specifically identify cultured Salmonella enteritidis organisms. The test is based on the use of two monoclonal antibody-coated latex reagents, one of which detects the recently discovered SEF14 fimbriae expressed predominantly by S. enteritidis and S. dublin organisms, while the second reagent detects the H'p' antigen of S. dublin flagella. In a series of field trials 141 out of 142 strains of S. enteritidis from eighteen phage types were correctly identified by the latex test. A further 175 salmonella isolates representing 35 serotypes were tested and only two false-positives (S. dublin) in the latex test were recorded. This is the first rapid serotype specific test for S. enteritidis to be developed, and highlights the potential advantage of fimbrial antigens as novel diagnostic antigens of the future.

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.

An interlaboratory trial of a latex agglutination kit for rapid identification of Salmonella enteritidis.

An interlaboratory trial was conducted of a latex agglutination kit for the rapid identification of Salmonella enteritidis, to assess the stability of the components and its performance with respect to a panel of three coded salmonellas and 243 field isolates. Two of the components of the kit deteriorated with time. All 62 isolates of S enteritidis were correctly identified by the kit; only two false positives were recorded and no false negatives.

Development of monoclonal antibody ELISA for simultaneous detection of bovine coronavirus, rotavirus serogroup A, and Escherichia coli K99 antigen in feces of calves.

A rapid ELISA was developed for simultaneous detection of bovine coronavirus (BCV), rotavirus (RV) serogroup A, and Escherichia coli K99 antigen in feces of calves. A mixture of 3 monoclonal antibodies specific for BCV, RV, or K99 was used successfully to capture the antigens; the same antibodies labeled with peroxidase were used to detect BCV, RV, or K99. The triple ELISA was compared with standard reference diagnostic methods by examining feces from experimentally and naturally infected and healthy calves. All the components of the test were highly specific (greater than 90%) and sensitive (BCV, 77%; K99, 93%; RV, 100%) when used in a format requiring short incubation steps at 20 C and visual recording of results.

Enhanced expression of 987P (F6) fimbrial adhesin by cultured enterotoxigenic Escherichia coli.

The expression of the 987P fimbrial adhesin by strains of enterotoxigenic Escherichia coli was enhanced and, in some cases, restored by passaging the organisms through Craigie's tubes. In contrast, the fimbrial adhesins K99, K88 and F41 were not optimally expressed in this medium. The results suggest that Craigie's tubes should be used for the optimum expression of 987P.