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Salmonellacontamination of pooled raw shelled egg mix (RSEM) used as an ingredient in lightly cooked or uncooked foods and high-risk kitchen hygiene practices in United Kingdom food service establishments using RSEM were investigated. Samples were collected from 934 premises. Salmonellawas found in 1 (0.13%) of 764 RSEM samples, 2 (0.3%) of 726 samples from surfaces where ready-to-eat foods were prepared, and 7 (1.3%) of 550 cleaning cloths. Poor RSEM storage and handling practices were highlighted. Workers in 40% of the premises sampled failed to use designated utensils when RSEM was added to other ingredients, workers in 17% of the premises did not clean surfaces and utensils thoroughly after use with RSEM and before preparing other foods, only 42% of workers washed and dried their hands after handling eggs or RSEM, workers in 41% of the premises did not store RSEM at refrigeration temperature before use, and workers in 8% of the premises added RSEM to cooked rice at the end of cooking when preparing egg fried rice. Take-away premises, especially those serving Chinese cuisine, were least likely to have a documented food safety management system and awareness of the key food safety points concerning the use of RSEM compared with other food service premises (P <0.0001). Food service businesses using RSEM must be aware of the continuing hazard from Salmonella,must adopt appropriate control measures, and must follow advice provided by national food agencies to reduce the risk of Salmonellainfection.

Although listeriosis is a rare cause of human disease in the United Kingdom, an increase in the number of cases has been observed since 2001, almost exclusively in persons older than 60 years. This increase prompted this study on the microbiological safety of ready-to-eat (RTE) foods, which included those types potentially linked to cases of listeriosis. Between May 2006 and April 2007, 6,984 RTE foods were sampled (2,168 sliced meats, 1,242 hard cheese, 1,088 sandwiches, 878 butter, 725 spreadable cheese, 515 confectionery products containing cream, and 368 probiotic drinks). The food types with the highest prevalence of Listeria monocytogeneswere sandwiches (7.0%) and sliced meats (3.7% within shelf life, 4.2% end of shelf life). L. monocytogenesat >100 CFU/g (exceeding the European Commission's food safety criteria limit) only occurred in sandwiches (0.4%) and sliced meats (0.7% within shelf life, 1.0% end of shelf life). Contamination with L. monocytogenesat >100 CFU/g was more frequent in meats that were prepacked and/or of pack size ≥300 g and in sandwiches that were supplied prepacked that contained salad vegetables as an ingredient. Satisfactory microbiological quality was associated with premises on which the management was trained in food hygiene and those that complied with hazard analysis and critical control point principles. This study provides important information about the microbiological safety of RTE foods and demonstrates that the control of L. monocytogenesin such foods, and in particular sandwiches and sliced meats, is essential in order to minimize the risk of this bacterium being present at levels hazardous to health at the point of consumption.

In the United Kingdom between 1999 and 2004, there were four outbreaks of Listeria monocytogenesinfection associated with sandwiches purchased from or provided in hospitals. Elderly or immunocompromised individuals and pregnant women are particularly vulnerable to infection; therefore, the focus of this study was on sandwiches served in health care establishments. Of 3,249 sandwich samples collected between April 2005 and March 2006, 3.3% were of unsatisfactory microbiological quality because of high levels of Enterobacteriaceae(2.0%; ≥104CFU/g for sandwiches not containing salad), Escherichia coli(0.8%; ≥102CFU/g), Staphylococcus aureus(0.6%; ≥102CFU/g), and/or Listeriaspp. (0.1%; two samples with L. welshimeriat 1.8 × 102and 7.4 × 103CFU/g and one sample with L. seeligeriat 1.8 × 103CFU/g). Overall, 7.6% of sandwiches were contaminated with Listeriaspp. L. monocytogeneswas detected in 2.7% (88) of samples: 87 samples at <10 CFU/g and 1 sample at 20 CFU/g. More frequent contamination with Listeriaspp. and L. monocytogeneswas found in sandwiches collected from hospital cafeterias, shops, or wards and in sandwiches stored and/or displayed at temperatures higher than 8°C. The presence of Listeriaspp. and L. monocytogenesalso was associated with sandwiches that (i) were supplied from outside the establishment, (ii) were prepacked, (iii) had a main sandwich filling of poultry meat, or (iv) contained salad ingredients, soft cheese, and/or mayonnaise. Control of L. monocytogenesin sandwich manufacturing and within health care establishments is essential to minimize the potential for consumption of this bacterium at levels hazardous to health. The findings from this study support the proposal that manufacturers supplying sandwiches to health care establishments should follow the British Sandwich Association recommended guidelines of complete absence of L. monocytogenesin sandwiches at the point of production.

Two recent listeriosis outbreaks involving butter prompted this first cross-sectional study on the prevalence, levels, and types of Listeriaspecies in 3,229 samples of butter from production, retail, and catering premises in the United Kingdom during May and June 2004. When the criteria of the Microbiological Guidelines were used, 99.4% of samples were found to be of satisfactory microbiological quality, 0.5% were of acceptable quality, and 0.1% were of unsatisfactory quality as a result of high levels (>100 CFU/g) of Listeriaspp. The butter samples with Listeriaspp. present at more than 100 CFU/g were negative for L. monocytogenes. L. monocytogeneswas detected in 0.4% (n= 13) of samples, all at levels of less than 10 CFU/g, and were therefore of acceptable quality. Butter was contaminated more frequently with Listeriaspp., including L. monocytogenes, when packed in plastic tubs, when in pack sizes of 500 g or less, when stored or displayed above 8°C, when a hazard analysis system was not in place, and when the manager had received no food hygiene training. This study demonstrates that although butter is regarded as a low-risk product, it may provide an environment for the persistence and growth of Listeriaspp., including L. monocytogenes. The control of L. monocytogenesin food processing and supply systems is critical in order to minimize the potential for this bacterium to be present in foods at the point of consumption at levels hazardous to health.

Real-time PCR assays for detection of Clostridium botulinum neurotoxin (BoNT) gene fragments specific to BoNTA, B, and E were developed as alternatives to the mouse bioassay. The expected specificities of the PCR assays were demonstrated by in silico analysis as well as empirical testing of target DNA extracted from 83 pure cultures of C. botulinum, and 44 bacteria from other species. The sensitivities of the assays were found to be equivalent to 16, 10, and 141 genomes for BoNT A, B, and E, respectively. The assays were shown to be applicable to both purified DNA, as well as crude DNA extracted from cultures and enrichment broths. The assays were evaluated using DNA extracted directly from clinical and food specimens as well as from inoculated broths using material collected from seven confirmed and one suspected case of botulism. The appropriate BoNT genes were detected in material from seven of the eight cases of botulism and provided a supportive diagnosis faster than the conventional bioassay. These assays have already proven useful for pubic health microbiological investigation of suspected cases of human botulism by substantially improving the diagnostic process.

Recovery of Cryptosporidium parvum oocysts in a fecal suspension that experimentally contaminated onto lettuce leaves was investigated. Material recovered from the lettuce samples by washing in detergent solutions were concentrated by filtration using the Envirochek Sampling Capsule. Oocysts were concentrated by immunomagnetic separation (IMS) and detected by microscopy following modified Ziehl-Neelsen (MZN) staining. Cryptosporidal DNA was detected using a nested-PCR assay for amplification of a fragment of the Cryptosporidium oocyst wall protein (COWP) gene, which was applied to DNA extracted from both filtrates, and material recovered from MZN stained smears on glass slides after microscopy. No Cryptosporidium were detected by microscopy or by PCR of un-inoculated lettuce leaves. After IMS, means of 0–6.5% of the total numbers of oocysts inoculated were recovered and detected by microscopy. Detection by PCR was less sensitive than microscopy. There was a strong association between successful PCR amplification, the numbers of oocysts detected by microscopy and the numbers of oocysts in the inoculum. This study confirms that C. parvum oocysts can be recovered from contaminated lettuce using filtration and IMS, and detected by microscopy and PCR. However, further evelopments are required to improve recovery of this parasite.

ABSTRACTAn assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping of Giardia duodenalison the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containing G. duodenalisand other parasites, and their corresponding immunofluorescence-stained fecal smears on glass microscope slides. The assay was specific and discriminated between G. duodenalisassemblages A and B. RFLP analysis further distinguished two groups (designated groups I and II) within assemblage A. Among 35 DNA samples extracted from whole feces from patients with confirmed sporadic giardiasis, the tpigene was amplified from 33 (94%). Of these, nine (27%) samples contained assemblage A group II, 21 (64%) contained assemblage B, and 3 (9%) contained a mixture of assemblage A group II and assemblage B. The tpigene of G. duodenalisassemblage B was amplified from 21 of 24 (88%) DNA samples extracted from whole feces from patients with confirmed cases of infection in a nursery outbreak. No amplification was detected from the remaining three DNA samples. Overall, analysis of DNA extracted from material recovered from stained microscope slides identified identical G. duodenalisgenotypes in 35 (65%) of the 54 samples for which a genotype was established with DNA from whole feces. The heminested PCR method developed is sensitive, simple, and rapid to perform and is applicable for the analysis of other intestinal pathogens.

Between October 1998 and April 2000, 429 food samples were investigated for the presence ofListeriaspp. The foodstuffs included 138 ready-to-eat foods (68 traditional hard and semi-hard Portuguese cheeses, 23 salad vegetables and 47 cooked and/or cured meats) and 291 uncooked foods (14 raw vegetables, 65 raw chicken and 212 raw ewe's, cow's or goat's milk). Listeriaspp. were recovered from 63 samples (15%). Listeria monocytogeneswas present in 39 (9%) samples, Listeria innocuain 12 (3%), Listeria seeligeriin 23 (5%), Listeria ivanoviiin seven (2%) and Listeria grayiin two (0·5%). More than one Listeriaspecies was recovered from 18 samples. A combination of serotyping, phage-typing, cadmium and arsenic sensitivities were used to subtype 36 of the L. monocytogenesisolates: at least nine different strains were recognized. Four food samples yielded two different L. monocytogenesstrains.

ABSTRACTCryptosporidiumpresent in 1,705 fecal samples from humans and 105 from livestock animals were analyzed by PCR-restriction fragment length polymorphism of the Cryptosporidiumoocyst wall protein. Overall, genotype 1 (human exclusive type) was detected in 37.8% of the samples from humans, genotype 2 (broad host range) was detected in 61.5%, a third genotype designated genotype 3 (Cryptosporidium meleagridis) was detected in 0.3%, and both genotypes 1 and 2 were recovered from 0.4%. All samples from livestock yielded genotype 2. Among 469 patients infected during eight drinking water-related outbreaks, five outbreaks were predominantly due to genotype 1, and three were due to genotype 2. Fifty-four samples were collected from patients involved with five swimming pool-associated outbreaks: two outbreaks were due to genotype 1, one was due to genotype 2, and the remaining two involved both genotypes 1 and 2. Among 26 family outbreaks and 1 children's nursery outbreak (2 to 3 members per group), the same genotype was recovered from the different members of each outbreak: 13 were due to genotype 1, and 14 were due to genotype 2. In eighteen patients reporting contact with animals and/or farms, genotype 1 was recovered from one patient and genotype 2 was recovered from the remaining 17. Among the sporadic cases, there were distinct geographical and temporal variations in the distribution of the genotypes. The spring peak in cases was due to genotype 2. Genotype 1 was significantly more common in patients infected during the late-summer–autumn peak and in those with a history of foreign travel.

A simple amplified fragment length polymorphism method was developed for the epidemiological typing of Bacillus cereus. The method was applied to 21 cultures from seven food poisoning and eight non‐food poisoning incidents. Results were compared with those obtained by conventional serotyping using flagellar antigens and assessed in relation to epidemiological data. Amplified fragment length polymorphism was found to be highly reproducible and 16 different profiles (each unique to the 15 incidents) were recognized. The method was also able to discriminate three subtypes within serotype H1, which is responsible for the majority of the emetic type of B. cereusfood poisoning in England and Wales.

A simple polymerase chain reaction (PCR)-based procedure was developed for the detection of fragments of staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, and SEI together with the toxic shock syndrome toxin (TSST-1) genes of Staphylococcus aureus. One hundred and twenty-nine cultures of S. aureuswere selected, 39 of which were recovered from 38 suspected staphylococcal food-poisoning incidents. The method was reproducible, and 32 different toxin genotypes were recognized. The presence of SE genes was associated with S. aureusstrains reacting with phages in group III, and the TSST-1 gene with phages in group I. There was a 96% agreement between the PCR results for detection of SEA–D and TSST-1 as compared with a commercial reverse passive latex agglutination assay for the detection of SEs from cultures grown in vitro. Enterotoxin gene fragments were detected in S. aureuscultures recovered from 32 of the 38 suspected staphylococcal food poisoning incidents, and of these, 17 were associated with SEE, SEG, SEH, and SEI in the absence of SEA–D. Simple PCR procedures were also developed for the detection of SE directly in spiked food samples, and this was most successfully achieved in mushroom soup and ham. Detection was less successful in three types of cheese and in cream. SEA or SEB were detected by enzyme-linked immunosorbent assay in three food samples (two of which were associated with food poisoning incidents) naturally heavily contaminated with S. aureus: the appropriate SEA or SEB gene fragments were detected directly in these three foods by PCR.

ABSTRACTSamples of whole feces in which Cryptosporidiumoocysts were recognized by hospital laboratories were collected from 218 patients with diarrhea. All samples were reexamined by light microscopy, and oocysts were detected in 211 samples. A simple and rapid procedure for the extraction of DNA from whole feces was developed, and this was used to amplify fragments of theCryptosporidiumouter wall protein (COWP), the thrombospondin-related adhesive protein C1 (TRAP-C1), and the 18S rRNA genes by PCR. For seven samples oocysts were not detected by microscopy and DNA failed to be amplified by the three PCR procedures. Among the 211 samples “positive” by microscopy, the sensitivities of PCRs for the 18S rRNA, COWP, and TRAP-C1 gene fragments were 97, 91, and 66%, respectively. The sensitivities of all three PCR procedures increased with increasing numbers of oocysts as observed by microscopy. Two genotypes of the COWP and TRAP-C1 genes can be detected by PCR-restriction fragment length polymorphism analysis. With this series of samples, the same genotypes of the COWP and TRAP-C1 genes always segregated together. A combined genotyping data set was produced for isolates from 194 samples: 74 (38%) were genotype 1 and 120 (62%) were genotype 2. Genotype 2 was detected in a significantly greater proportion of the samples with small numbers of oocysts, and genotype 1 was detected in a significantly greater proportion of the samples with larger numbers of oocysts. There were no significant differences in the distribution of the genotypes by patient sex and age. The distribution of the genotypes was significantly different both in patients with a history of foreign travel and in those from different regions in England.

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Human listeriosis is generally believed to be foodborne, although other routes of transmission occur. Foods associated with transmission have generally been highly processed, have extended shelf lives at refrigeration temperatures, capable of supporting the growth of L. monocytogenesand consumed without further cooking. In a small number of instances where data are available, these foods were contaminated with higher levels (> 103/g) of the bacterium. Changes in eating habits in the UK and the ways in which foods are produced has seen a growth in the sale of just such foods. Common features of foodborne human listeriosis are discussed.

An analysis was made of the distribution of serovars ofListeria monocytogenes isolated from 1363 patients with listeriosis. Overall, serovar 4b was found in 64 % of cases, serovar 1/2a in 15 %, serovar 1/2b in 10 %, and serovar 1/2c in 4 %. The patients were categorised as pregnancy associated cases, non-pregnant previously healthy cases, and non-pregnant cases with severe underlying illness. The serovars were unequally distributed between these three groups of patients. Serovar 1/2b occurred most often in the non-pregnant cases with severe underlying illness, and serovar 1/2c occurred least frequently in the pregnancy associated cases. Serovar 4b occurred more often in the pregnancy associated cases than in previously healthy non-pregnant cases, and more often in the latter than in those with underlying illness. Similar distributions of the serovars between the categories of patients occurred over different time periods. These results may be interpreted as indicating an association between virulence and serological type ofListeria monocytogenes.

ABSTRACTA method was developed for extracting cryptosporidial DNA from stained fecal smears on glass microscope slides. The correct genotype of Cryptosporidium parvumwas amplified by PCR from 89 (85%) of 105 smears following conventional staining but not from negative controls. This technique may have applications for analysis of other infectious agents.