Polymyxin-cloth Enzyme Immunoassay Research Articles

Enrichment of injuredSalmonellain poultry products for detection by polymyxin–cloth enzyme immunoassay

Because injuredSalmonellacells have the potential to cause salmonellosis, their detection in foods is important to food safety testing programs. In pure cultures, a small number of heat-injuredSalmonella enteritidiscells were detected by the polymyxin–cloth enzyme immunoassay after 2-h static pre-enrichment in brain–heart infusion (BHI) medium supplemented with 0.05% yeast extract and 0.05% cholate (BYC), followed by overnight static enrichment in the presence of 0.3% selenite. However, in chicken carcass rinse, heat-injuredS. enteritidiscells (400 cfu ml–1) could not be detected even after 6-h pre-enrichment in BYC, while uninjured cells were detectable. Prolonged (20 h) pre-enrichment in BYC, followed by 20-h incubation of a 10-fold dilution with BYC with addition of 0.03% selenite, permitted the detection of heat-injuredS. enteritidis(less than 7 cfu ml–1) in the chicken carcass rinse.

A liquid medium for growth of the thermophilic campylobacters in a carbon dioxide air mixture as studied by polymyxin-cloth enzyme immunoassay

A new liquid medium containing casein hydrolysate, beef extract, yeast extract, biocarbonate and an erythrocyte digest was developed for growth ofCampylobacter jejuniand other thermophilic campylobacter species. 26 strains, consisting of six Penner serotypes and the 20 most common Lior serogroups, were evaluated and all grew well in this medium at 40°C in an atmosphere of 5% CO2moist air. Furthermore, this medium allowed growth of these bacteria without exogenous CO2at 40°C. After incubation for 28h in 5% CO2or 36h without exogenous CO2, cultures inoculated with approximately 102colony forming units (CFU)/ml reached cell densities of 106to 109(mean 108) CFU/ml and produced positive signals in the polymyxin-cloth enzyme immunoassay. This medium therefore has the potential to provide rapid and affordable isolation of these pathogens for a Hazard Analysis Critical Control Point program.

Assay of Salmonella in enrichment cultures of foods, feeds and environmental samples by the polymyxin-cloth enzyme immunoassay

A variety of foods, animal feeds and environmental samples were analyzed for the presence of Salmonella using the polymyxin-cloth enzyme immunoassay (p-CEIA) system. Salmonella lipopolysaccharide (LPS) antigens were captured from test samples on polymyxin-coated polyester cloth, followed by immunoenzymatic detection of bound antigens using a monoclonal antibody recognizing an LPS common core oligosaccharide. Dairy and egg products, animal feeds and environmental samples from food processing plants were pre-enriched for 24 h, followed by selective enrichment for a further 24 h in either tetrathionate brilliant green (TBG), selenite cystine (SC) or brain–heart infusion broth containing 0.5% yeast extract, 0.5% cholate and 0.3% selenite (BYCS). The samples were assayed by the p-CEIA after each stage of enrichment. After selective enrichment, the p-CEIA gave results which were in complete agreement with the standard culture technique in the analysis of all foods examined. On the other hand, a combination of selective enrichment and the p-CEIA out-performed the Modified Semi-Solid Rappaport Vassiliadis (MSRV) method in screening pre-enrichment cultures of feeds and environmental samples. Use of the new selective medium BYCS prior to performing the p-CEIA gave the highest recovery of Salmonella from feeds and environmental samples.

A polymyxin-cloth enzyme immunoassay system for detection of Escherichia coliO157 in ground beef

A polymyxin-cloth enzyme immunoassay (polymyxin-CEIA) was developed for the detection of the foodborne pathogen Escherichia coliO157. This simple assay utilized inexpensive polymyxin-coated polyester cloth as an adsorbent for E. colilipopolysaccharide (LPS) antigens present in test samples such as enrichment cultures of ground beef. Captured LPS was immunoenzymatically detected using commercially available anti-O157 polyclonal antibodies. The assay was sensitive and highly specific for strains bearing the O157 and related antigens, and permitted the detection of c. 1–10 cfus spiked into ground beef samples after a brief enrichment in double modified trypticase soy broth. We suggest that the polymyxin-CEIA will provide a simple, rapid and inexpensive screening method for E. coliO157 in ground beef.

Rapid and simple method for detecting Salmonellain chicken feces using polymyxin-cloth enzyme immunoassay

The presence of three strains ofSalmonella cells in chicken feces was detected by polymyxin-cloth enzyme immunoassay. A cotton swab-full ofSalmonella -free chicken feces (containing an aerobic count of about 3×10 7cfu) was mixed with a small number of Salmonella typhimurium (6 or 12 cfu),Salmonella enteritidis (6 or 15 cfu) orSalmonella hadar (5 or 10 cfu) in 5 ml of brain–heart infusion medium supplemented with 0.5% yeast extract, 0.3% sodium hydrogen selenite, and 0.5% sodium cholate. After 20 h static incubation at 37°C, heat-extracted lipopolysaccharide (LPS) antigens were captured by polymixin B adsorbed onto macroporous polyester cloth. The LPS antigens were then detected via sequential reactions with a mixture of mouse anti-Salmonella LPS antibody and rabbitSalmonella O antiserum followed by a mixture of horseradish peroxidase conjugates of antibody to mouse and rabbit immunoglobulin G. The total procedure took about 22 h. This method was also used to detect endogeneousSalmonella in feces of chicken inoculated withSalmonella and to measure the level of fecalSalmonella . This simple, rapid and sensitive method forSalmonella detection in feces should be useful not only for routine screening ofSalmonella shedding in poultry, but also forSalmonella testing of stool from human patients and food handlers.

Salmonella detection by the polymyxin-cloth enzyme immunoassay using polyclonal and monoclonal detector antibodies

Several commercially available O-antigen polyclonal antisera and a monoclonal antibody to the core region of lipopolysaccharide (LPS) were examined as sources of detector antibodies in a polymyxin-cloth enzyme immunoassay (polymyxin-CEIA) for Salmonella. In this assay, polymyxin-coated polyester cloth captured the LPS antigens from Salmonella broth cultures, followed by immunoenzymatic detection of the captured LPS using specific antibodies. Pools of polyvalent antisera reacted with all of the Salmonella strains tested, but also gave cross-reactions with some non- Salmonella bacteria. On the other hand, the monoclonal antibody gave positive reactions with all of the Salmonella tested except serogroup O-strains, but did not react with any of the non- Salmonella bacteria. The monoclonal antibody supplemented with a single factor serogroup O:35 rabbit antiserum was able to detect the serogroup O-strains without causing any cross-reactions with the non- Salmonella bacteria. As an example of the applicability of this assay system, low levels of Salmonella cells spiked into various food samples were successfully detected after an overnight enrichment in broth.

Immunological detection of lipopolysaccharide antigens of thermophilic campylobacters captured on polymyxin-coated polyester cloth.

Cholate-extracted lipopolysaccharide (LPS) antigens from thermophilic campylobacters were captured on polymyxin-coated polyester cloth. The captured antigens were detected by sequential reactions with rabbit anti-Campylobacter antibody, anti-rabbit IgG peroxidase conjugate and chromogenic peroxidase substrate. A polyclonal rabbit antibody elicited against a single Campylobacter-jejuni strain detected the reference strains of the twenty most frequently isolated thermophilic campylobacters in the Lior serotyping scheme. Moreover, LPS antigens of six C. Jejuni Penner serotypes fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting were recognized by four antisera prepared against homologous and heterologous Penner serotypes. The results suggest the potential application of polymyxin-cloth enzyme immunoassay for rapid detection of thermophilic campylobacters where monoclonal antibodies can be raised to possible common LPS epitopes.

Rapid confirmation of polymyxin-cloth enzyme immunoassay for group D salmonellae including Salmonella enteritidis in eggs by polymerase chain reaction

A rapid, simple and inexpensive combined screening test and confirmation procedure was developed for group D Salmonellae (e.g. Salmonella enteritidis) in eggs. S. enteritidis inoculated into egg samples was pre-enriched in a minimal volume of enrichment broth, which was then assayed by the polymyxin-cloth enzyme immunoassay (polymyxin-CEIA): cholate-extracted lipopolysaccharide antigens were captured on polymyxin-coated polyester cloth and detected by sequential reactions with factor 0:9 rabbit antiserum, anti-rabbit antibody-peroxidase conjugate and peroxidase substrate. Presumptive positive results obtained in the polymyxin-CEIA were confirmed by subjecting pre-enriched samples directly to a polymerase chain reaction (PCR) technique targeting Salmonella-specific invA gene sequences. The polymyxin-CEIA gave positive results in the assay of 18 different S. enteritidis strains inoculated into eggs, all of which were confirmed by the invA PCR. This entire procedure, combining the steps of sample pre-enrichment in a minimal volume of broth, polymyxin-CEIA screening and rapid confirmation by PCR, gave a short turn around time from sample collection to completion of the analysis of within 26 h.

Effects of high levels of selenite and Escherichia coli on enrichment of Salmonella and detection of Salmonella by polymyxin-cloth enzyme immunoassay

The effect of various concentrations of selenite on the recovery of Salmonella typhimurium and Escherichia coli was examined in a rich medium containing 0.5% cholate. For heat-injured S. typhimurium, longer preincubation periods were required for recovery in higher concentrations of selenite. When 0.7% selenite was added to 6-h preincubated cultures of heat-injured cells, S. typhimurium was fully recovered while E. coli, whether heat-injured or uninjured, did not recover when present at numbers at less than 2 × 10 7 colony forming units (CFU). The polymyxin-cloth enzyme immunoassay detected S. typhimurium grown from a small number (as low as 1 CFU) of cells in the presence of a large excess of E. coli.

Effects of cholate and selenite on polymyxin-cloth enzyme immunoassay of Salmonella statically cultured in the presence of microflora from chicken faeces

Selective enrichment of Salmonella using a combination of 0·3% selenite, 0·5% cholate and static incubation was examined for five common serotypes in the presence of Salmonella -free chicken faeces. Enrichment cultures were then assayed by the polymyxin-cloth enzyme immunoassay (polymyxin-CEIA). This selective enrichment method was effective in suppressing large excesses of chicken faecal microflora, while permitting the growth of small numbers of Salmonella , which produced strong signals in the polymyxin-CEIA. When the medium contained no selective agents, the ability of the polymyxin-CEIA to detect Salmonella was greatly reduced in the presence of large excesses of chicken faecal microflora. The advantages of using static incubation and a dot-blot format for the polymyxin-CEIA are discussed for application in the hazard analysis critical control point programme.

Rapid and economical detection of Salmonella enteritidis in eggs by the polymyxin-cloth enzyme immunoassay

A rapid, simple and economical procedure for the detection of Salmonella enteritidis in eggs was developed. The contents of whole eggs inoculated with low numbers of S. enteritidis were mixed with a minimal volume of a nutrient-rich broth (1:2 ratio of egg to broth) and incubated overnight. The lipopolysaccharide (LPS) antigens of S. enteritidis were extracted by heating in the presence of cholate. The antigens were captured on polymyxincoated polyester cloth, and the captured antigens were detected by sequential reactions with anti-serogroup D1 rabbit antiserum, anti-rabbit antibody-peroxidase conjugate and tetramethylbenzidine substrate solution. This polymyxin-cloth enzyme immunoassay (polymyxin-CEIA) was highly specific for salmonellae bearing the factor 0:9 antigen, reacting in the assay of 19 S. enteritidis strains tested, including two rough isolates, but not with salmonellae lacking the factor 0:9 antigen or non- Salmonella bacteria. The threshold sensitivity of the polymyxin-CEIA for S. enteritidis suspensions was ca. 10 6 cfu ml . This combined enrichment culture and polymyxin-CEIA required less than 24 h to complete and detected as few as 1–2 S. enteritidis cfu inoculated into a whole egg. This procedure should facilitate the routine monitoring of S. enteritidis in large numbers of egg samples.

Use of inexpensive O antisera as the detecting antibodies for Salmonella antigens in the polymyxin-cloth enzyme immunoassay

The application of O antigen-specific antisera to the detection of Salmonella lipopolysaccharide (LPS) antigens was examined in an enzyme immunoassay using polymyxin-coated polyester cloth. The LPS antigens were extracted with deoxycholate and captured on polymyxin-cloth. A mixture of rabbit antisera to Salmonella O antigens was allowed to react with the captured antigens, and the reacted antibodies were detected by an anti-rabbit IgG-peroxidase conjugate. The assay gave positive results with 40 different Salmonella serotypes which represented more than 99.9% of the serogroups isolated from over 122000 food, feed and environmental samples analysed at Laboratory Services Division, Agriculture Canada, from 1975 to 1990. Strong cross-reactivity with Staphylococcus aureus was eliminated by pregrowth of the organisms in the presence of sodium deoxycholate. The O antisera were commercially available and are more economical than monoclonal or affinity purified polyclonal antibodies.