Subtyping of a large collection of historical Listeria monocytogenes strains from Ontario, Canada, by an improved multilocus variable-number tandem-repeat analysis (MLVA).

Listeria monocytogenes is responsible for severe and often fatal food-borne infections in humans. A collection of 2,421 L. monocytogenes isolates originating from Ontario's food chain between 1993 and 2010, along with Ontario clinical isolates collected from 2004 to 2010, was characterized using an improved multilocus variable-number tandem-repeat analysis (MLVA). The MLVA method was established based on eight primer pairs targeting seven variable-number tandem-repeat (VNTR) loci in two 4-plex fluorescent PCRs. Diversity indices and amplification rates of the individual VNTR loci ranged from 0.38 to 0.92 and from 0.64 to 0.99, respectively. MLVA types and pulsed-field gel electrophoresis (PFGE) patterns were compared using Comparative Partitions analysis involving 336 clinical and 99 food and environmental isolates. The analysis yielded Simpson's diversity index values of 0.998 and 0.992 for MLVA and PFGE, respectively, and adjusted Wallace coefficients of 0.318 when MLVA was used as a primary subtyping method and 0.088 when PFGE was a primary typing method. Statistical data analysis using BioNumerics allowed for identification of at least 8 predominant and persistent L. monocytogenes MLVA types in Ontario's food chain. The MLVA method correctly clustered epidemiologically related outbreak strains and separated unrelated strains in a subset analysis. An MLVA database was established for the 2,421 L. monocytogenes isolates, which allows for comparison of data among historical and new isolates of different sources. The subtyping method coupled with the MLVA database will help in effective monitoring/prevention approaches to identify environmental contamination by pathogenic strains of L. monocytogenes and investigation of outbreaks.

Cloth-based hybridization array system for the detection of Clostridium botulinum type A, B, E, and F neurotoxin genes.

A simple cloth-based hybridization array system was developed for the characterization of Clostridium botulinum isolates based on the botulinum neurotoxin serotype. Bacterial isolates were subjected to a multiplex PCR incorporating digoxigenin-dUTP and primers targeting the four botulinum neurotoxin gene serotypes (A, B, E, and F) predominantly involved in human illness, followed by hybridization of the amplicons with an array of toxin gene-specific oligonucleotide probes immobilized on polyester cloth and subsequent immunoenzymatic assay of the bound digoxigenin label. This system provided sensitive and specific detection of the different botulinum neurotoxin gene markers in a variety of C. botulinum strains, exhibiting the expected patterns of reactivity with a panel of target and nontarget organisms.

Antibody-dependent cell-mediated cytotoxicity directed by a human monoclonal antibody reactive with gp120 of HIV-1.

We used a human monoclonal antibody (MAb; 15e) to identify an antibody-dependent cell-mediated cytotoxicity (ADCC) epitope on HIV-1 gp120. 15e has been shown to recognize a conformation-dependent epitope on gp120 which is important in both CD4 binding and neutralizing of HIV-1 infection. 15e binds to gp120 of HIV-1IIIB but not HIV-1RF. Using a standard ADCC assay, 15e was found to mediate ADCC against cells infected with HIV-1IIIB but not HIV-1RF. 15e did not mediate ADCC against cells with recombinant gp120 bound to surface CD4, indicating that 15e does not mediate innocent bystander ADCC against uninfected CD4 cells. To better define the 15e epitope, we performed ADCC against target cells infected with a vaccinia vector which expresses processed HIV-1IIIB gp160 from which the third variable region was deleted (amino acids, 312-328). MAb 15e efficiently mediated ADCC against cells expressing this altered form of gp120, indicating that this region is not contributing to the conformational epitope defined by 15e. 15e defines an important epitope in the human immune response to HIV-1 infection. Antibodies with 15e-like activity may be useful in immunoprophylaxis or immunotherapy of HIV-1 infection.

Application of polymyxin-coated polyester cloth to the semi-quantitation of Salmonella in processed foods.

A rapid and economical semi-quantitative test for Salmonella cells in foods is proposed. Food samples containing different levels of Salmonella cells were homogenized and serially diluted in enrichment broths and then incubated for about 20 h at 37 degrees C. The presence of Salmonella cells in each dilution was assayed by capturing deoxycholate-extracted Salmonella lipopolysaccharides on a sheet of polymyxin-coated polyester cloth, followed by colorimetric detection with an anti-Salmonella antibody-enzyme conjugate. The minimum dilutions which resulted in no detectable growth were correlated with the extent of Salmonella contamination in the food samples.

Extraction of Salmonella antigens in non-sedimentable forms for enzyme immunoassay.

Heating Salmonella typhimurium in ethylenediaminetetraacetate was found to dissociate its antigens into forms that are non-sedimentable at 10,000 x g. The treatment caused a marked increase in the rate of immunoreaction and the sensitivity in an enzyme immunoassay for the detection of Salmonella antigens. The method permits the extraction of Salmonella antigens from solid-rich samples and the preparation of solid-free samples by means of centrifugation. When the level of the antigens in the supernatant was too low for the immunoassay, the antigens were readily concentrated by passing a large volume of the supernatant through a macroporous hydrophobic cloth coated with anti-Salmonella antibody. Using this method, the detection of as few as 10 Salmonella cells per gram of chicken meat was possible within a total of 18 h, which included 16 h of enrichment in either tetrathionate, selenite cystine, or nutrient broths.

Use of polymyxin-coated polyester cloth in the enzyme immunoassay of Salmonella lipopolysaccharide antigens.

Polyester cloth coated with polymyxin B was used to capture Salmonella typhimurium lipopolysaccharide antigens which were then quantitatively or qualitatively assayed using a specific antibody-peroxidase conjugate. This simple, rapid method can be used to assay a large number of samples by employing a large sheet of the polymyxin-coated cloth onto which multiple samples can be blotted. The method is reproducible and economical, since polymyxin B is relatively inexpensive, stable and available in pure form.

Use of detergents in the preparation of Salmonella samples for enzyme immunoassay on polymyxin-coated polyester cloth.

Heating Salmonella cells in the presence of detergents such as deoxycholate or Triton x-100 greatly increased the sensitivity of an enzyme immunoassay for lipopolysaccharide antigens using polymyxin-coated polyester cloth as the solid phase for antigen capture. The presence of a 100-fold excess of E. coli lipopolysaccharide in the test sample did not affect the detection of the Salmonella lipopolysaccharide. The sensitivity of the assay using deoxycholate-heat treatment of the antigens followed by detection on polymyxin-coated cloth was equivalent to that of an assay using antibody-coated polyester cloth for antigen capture.

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 polymyxin-coated 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 O:9 antigen, reacting in the assay of 19 S. enteritidis strains tested, including two rough isolates, but not with salmonellae lacking the factor O: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 122,000 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.

Effect of temperature and agitation on enrichment of Escherichia coli O157:H7 in ground beef using modified EC broth with novobiocin.

The effects of temperature and agitation on the enrichment of Escherichia coli O157:H7 in meat using modified EC broth with novobiocin (mEC + n) were studied. Enrichment at 37 degrees C was compared to 42 degrees C, both with and without shaking. Incubation at 42 degrees C without shaking effectively suppressed ground beef microflora while allowing good growth of E. coli O157:H7 cells. Cells inoculated into ground meats (beef, pork, turkey) were readily detected by enrichment for 24 h in mEC + n at 42 degrees C without shaking, followed by screening the enrichment cultures using a rapid and inexpensive commercially available enzyme immunoassay system, the E. coli O157 Rapitest.

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.

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.

Use of hydrophobic cloths as antibody adsorbents for enzyme immunoassay: detection of Brucella antigens.

A cloth-ELISA (C-ELISA) antigen capture assay for the detection of Brucella melitensis and B. abortus was developed. Segments (6-mm squares) of hydrophobic polyester cloth coated with diluted serum from a B. abortus-infected cow were incubated with saline suspensions of heat-killed Brucella cells, or with cultures of bovine or sheep blood or bovine tissue homogenates that had been inoculated with B. abortus or B. melitensis added to trypticase soy broth (TSB) and incubated for 2-3 days. The captured antigen was detected by a bovine anti-Brucella antibody-horseradish peroxidase conjugate system. The enrichment culture technique detected as few as three Brucella colony-forming units (c.f.u.) in 0.5 ml of bovine blood and was positive in cultures in which the Brucella concentration had reached 3 X 10(6) c.f.u. ml-1 (after 2 or 3 days incubation). The combined enrichment-cloth-ELISA method gave complete correlation with cultural isolation and results were available 3 days before colonies appeared in conventional culture. Hydrophobic cloths have potential use in diagnostic procedures since they provide simple, rapid and economical assays.

Discrimination against contact lens wearers.

Employers' attitudes toward the use of contact lenses at work have become less discriminatory as lenses have improved and numerous studies have demonstrated their safety, provided that additional personal protective equipment is used when necessary. In 1994, the Occupational Safety and Health Administration published its relevant Standard (29 CFR 1910), stating that "contact lenses do not pose additional hazards to the wearer...". Accommodations required by wearers of contact lenses must comply with Title I of the Americans with Disabilities Act. However, many companies still oppose their use. The recently published policy of the American College of Occupational and Environmental Medicine and the American Academy of Ophthalmology on the use of contact lenses should lead to their wider acceptance. Elements of a corporate contact lens policy are outlined. International aspects are summarized as well.

Swab-based enzyme immunoassay system for detection of meat residues on food contact surfaces as a hygiene monitoring tool.

An enzyme immunoassay system based on the use of a macroporous swab as a solid phase for the capture and subsequent immunoenzymatic detection of immunoglobulin G (IgG) from meat residues on food contact surfaces was developed as a hygiene-monitoring tool. Moistened polyester swabs coated with anti-bovine or anti-chicken IgG were rubbed on the test surface, and the captured IgG was subsequently detected directly on the swabs by brief sequential reactions with anti-bovine or anti-chicken IgG-peroxidase conjugate and chromogenic peroxidase substrate.

Detection of hazelnut proteins in foods by enzyme immunoassay using egg yolk antibodies.

An enzyme immunoassay (EIA) was developed for the detection of hazelnut proteins in foods. This assay used inexpensive chicken egg yolk antibodies in a sandwich EIA format for the immunospecific capture and detection of hazelnut proteins present in a variety of different food matrices. The assay was able to detect less than 1 ppm of hazelnut protein in most of the foods tested and did not exhibit any appreciable cross-reactivity with other nuts or food matrices. This assay will be a useful tool for the food industry and regulatory agencies that wish to test foods for the presence of undeclared hazelnut allergens.

Performance of the Dynabeads anti-Salmonella system in the detection of Salmonella species in foods, animal feeds, and environmental samples.

The immunomagnetic separation Dynabeads anti-Salmonella technique was evaluated for the detection of Salmonella species in a variety of foods, feeds, and environmental samples. Salmonella cells in preenrichment broths were captured using the Dynabeads anti-Salmonella system and were then washed and plated on indicator media. A total of 308 naturally contaminated samples were analyzed, including 46 cheese and egg products, 183 animal feeds, and 79 environmental swabs. The results of the Dynabeads method gave 100% correlation with the results of the standard culture technique used for foods and the modified semisolid Rappaport-Vassiliadis method used for feeds and environmental samples.

Polymacron enzyme immunoassay system for detection of naturally contaminating Salmonella in foods, feeds, and environmental samples.

A simple dot blot enzyme immunoassay was developed to screen enrichment broth cultures for the presence of Salmonella. This unique system utilizes macroporous polyester cloth (Polymacron) with an inexpensive hemoglobin coating to provide a high-affinity adsorbent for lipopolysaccharide (LPS) antigens in test samples. Bound LPS antigens are then detected using a monoclonal antibody conjugate recognizing a core oligosaccharide epitope common to all salmonellae frequently found in foods and related samples. The entire test (not including enrichment culture) could be completed in less than 1 h. The performance of this assay was evaluated in the analysis of enrichment broth cultures from a variety of egg and dairy products, chicken carcasses, animal feeds, and food-processing plant environmental samples for the presence of Salmonella.