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P A Chapman

Publications and source records attributed to P A Chapman.

At least 19 recordsLinked to original sources
PubMed2003

An evaluation of rapid methods for detecting Escherichia coli O157 on beef carcasses.

Numbers of Escherichia coli O157 in food may be low and sensitive techniques are therefore needed for its detection. The objectives of this study were to use carcass meat samples artificially inoculated with various strains of E. coli O157 to compare the sensitivity of enrichment in three different media and to compare immunomagnetic separation followed by culture of magnetic beads to cefixime tellurite sorbitol MacConkey agar with three immunoassays for the detection of E. coli O157 in the enrichment cultures. Duplicate 250, 25 and 2-3 CFU of each of 16 strains of E. coli O157 added to 25-g samples of beef carcass meat were used to compare the sensitivity of (1) enrichment in supplemented tryptone soya broth (sTSB), Reveal 8-h and Reveal 20-h media, and (2) immunomagnetic separation and culture to cefixime tellurite sorbitol MacConkey agar (IMS/CT-SMAC) with Reveal, VIP and STAT immunoassays for detecting the organism. An initial inoculum of 250 CFU/25 g meat was detected in all 32 samples by IMS/CT-SMAC performed on all enrichment media and by Reveal performed on Reveal 8-h and Reveal 20-h media, but in only 30, 19 and 9 samples by Reveal, VIP and STAT, respectively, performed on sTSB medium. An initial inoculum of 25 CFU/25 g meat was detected in 28, 32 and 30 of 32 samples by IMS/CT-SMAC performed on sTSB, Reveal 8-h and Reveal 20-h media, respectively, and in 32 and 30 samples by Reveal performed on Reveal 8-h and Reveal 20-h media, but in only 22, 11 and 2 samples by Reveal, VIP and STAT, respectively, performed on sTSB medium. An initial inoculum of 2-3 CFU/25 g meat was detected in 25, 31 and 28 of 32 samples by IMS/CT-SMAC performed on sTSB, Reveal 8-h and Reveal 20-h media, respectively, and in 25 and 23 samples by Reveal performed on Reveal 8-h and Reveal 20-h media, but in only 14, 1 and 0 samples by Reveal, VIP and STAT, respectively, performed on sTSB medium.

Animals↗
PubMed2001

Comparison of culture, PCR and immunoassays for detecting Escherichia coli O157 following enrichment culture and immunomagnetic separation performed on naturally contaminated raw meat products.

The aims of this study were (i) to evaluate the specificity and sensitivity of three previously described PCR assays for the detection of Escherichia coli O157 and, (ii) to compare PCR, culture, and two visual immunoassays (VIAs), BioSign and Path-Stik, for detecting E. coli O157 after enrichment culture and immunomagnetic separation (IMS) performed on various naturally contaminated raw beef, lamb and mixed meat products. Twelve sorbitol nonfermenting (SNF) verocytotoxin-producing (VT +) E. coli O157, 6 SNF VT - E. coli O157, 4 sorbitol fermenting (SF) VT + E. coli O157, 3 SF VT - E. coli O157, 23 E. coli belonging to 17 other serogroups and 12 organisms of other species were used to check the specificity of PCR reactions. Only one primer pair generated amplimers only with E. coli O157 and was used for all subsequent work. After enrichment culture and on inoculated minced beef samples, PCR was as sensitive as culture for detecting 9 of the 12 strains of E. coli O157, but up to 4 log10 more sensitive than culture for detecting three strains. Of the 120 samples of naturally contaminated meat products examined, 80 (67%) were positive by PCR, 70 (58%) were positive by BioSign, 69 (58%) were positive by culture and 67 (56%) were positive by Path-Stik. Although both VIAs lacked sensitivity when compared to PCR, both compared favourably with culture and both were extremely rapid and easy to perform, giving a result in less than 15 min. Eleven samples were positive by PCR and both VIAs, but negative by culture because culture plates were heavily overgrown with SF organisms, making detection of any E. coli O157 present impossible.

Animals↗
PubMed2001

A field survey of Escherichia coli O157 ecology on a cattle farm in Italy.

A field survey was performed in a heifer raising operation in Northern Italy to study the introduction, maintenance and dissemination of Escherichia coli O157 in the herd and to identify possible control measures at the farm level. Rectal swabs from two different groups of animals (surveys 1 and 2) were tested for E. coli O157 by an immunomagnetic separation technique. In survey 1, a group of female calves (341 animals initially) introduced from 30 dairy herds during April 1996 to March 1997 were tested for E. coli O157 on arrival from the original herd when housed in individual hutches, 2-3 days after completion of weaning (which was associated with grouping) and 2 months after weaning. No statistically significant difference between excretion rates (3.8%, 4.2%, 4.4%, respectively) was found. Calves from which E. coli O157 was isolated on arrival came from 6 of the 30 dairy herds. Strains isolated during survey 1 belonged to seven different pulsed field gel electrophoresis (PFGE) profiles. In survey 2, a group of young animals aged, at the beginning of the study, between 2 1/2 and 7 1/2 months (median = 124 days) was tested monthly for E. coli O157 for 11-15 months from May 1996 to July 1997. The group included 92 animals for 11 months and then gradually decreased to 59 animals. Overall, E. coli O157, belonging to six different PFGE profiles, were isolated from 138 (10.7%) of 1293 rectal swabs. Monthly excretion rates ranged from 2.7% to 23.7%, with summer peaks in both years. Fifty-nine (64.1%) of the 92 heifers were positive at least once: of these 59 animals, 22 (37.3%) were positive on only one occasion, 23 (39%) were positive on two occasions and 14 (23.7%) were positive on three or more occasions. From two heifers positive on 9 out of the 15 sampling visits, strains with the same PFGE profile were isolated, respectively, on seven and eight occasions while strains with only one band difference were isolated on the remaining occasions. E. coli O157 was also isolated from 6 of 16 samples of bedding, two of two samples of slurry and one of five samples from water troughs collected during survey 2.

Animals↗
PubMed2001

Escherichia coli O157 in cattle and sheep at slaughter, on beef and lamb carcasses and in raw beef and lamb products in South Yorkshire, UK.

A 1 year study of Escherichia coli O157 in cattle and sheep at slaughter, on beef and lamb carcasses and in raw beef and lamb products from retail butchers' shops was performed in the Sheffield area. Each month, samples of rectal faeces were collected immediately after slaughter from 400 cattle and 600 sheep, and 400-430 samples of raw meat products were purchased from butchers' shops. Meat samples were also obtained from 1500 beef and 1500 lamb carcasses. All samples were examined for E. coli O157 by enrichment culture, immunomagnetic separation and culture of magnetic particles onto cefixime tellurite sorbitol MacConkey agar. Raw meat products were also examined for numbers of generic E. coli by a standard membrane culture method. E. coli O157 was isolated from 620 (12.9%) of 4800 cattle, 100 (7.4%) of 7200 sheep, 21 (1.4%) of 1500 beef carcasses, 10 (0.7%) of 1500 lamb carcasses and from 22 (0.44%) of 4983 raw meat products. E. coli O157 was isolated more frequently from lamb products (0.8%) than from beef products (0.4%). Numbers of generic E. coli in meat products reached seasonal peaks in July and August with counts of > 10(4)/g occurring more frequently in lamb products (50.8 and 42.4%, respectively) than in beef products (19.3 and 23.8%, respectively). The majority of E. coli O157 strains, from animals, carcasses and meat samples, were isolated during the summer. Most were verocytotoxigenic as determined by Vero cell assay and DNA hybridisation, eaeA gene positive and contained a 92 kb plasmid. The isolates were compared with 66 isolates from human cases over the same period. A combination of phage type, toxin genotype and plasmid analysis allowed subdivision of all the E. coli O157 isolates into 96 subtypes. Of these subtypes, 53 (55%) were isolated only from bovine faecal samples. However, 61 (92%) of the 66 isolates from humans belonged to 13 subtypes which were also found in the animal population.

Animals↗
PubMed2001

A comparison of immunomagnetic separation and culture, Reveal and VIP for the detection of E. coli O157 in enrichment cultures of naturally-contaminated raw beef, lamb and mixed meat products.

AIMS: The aims of this study were (i) to evaluate the specificity and sensitivity of three previously described PCR assays for the detection of E. coli O157 and, (ii) to compare PCR, culture, and two visual immunoassays (VIAs), BioSign and Path-Stik, for detecting E. coli O157 after enrichment culture and immunomagnetic separation (IMS) performed on various naturally contaminated raw beef, lamb and mixed meat products. METHODS AND RESULTS: Twelve sorbitol non fermenting (SNF) verocytotoxin-producing (VT+) E. coli O157, 6 SNF VT- E. coli O157, 4 sorbitol fermenting (SF) VT+ E. coli O157, 3 SF VT- E. coli O157, 23 E. coli belonging to 17 other serogroups and 12 organisms of other species were used to check the specificity of PCR reactions. Only one primer pair generated amplimers only with E. coli O157 and was used for all subsequent work. After enrichment culture and on inoculated minced beef samples, PCR was as sensitive as culture for detecting 9 of the 12 strains of E. coli O157, but up to 4 log10 more sensitive than culture for detecting three strains. Of the 120 samples of naturally contaminated meat products examined, 80 (67%) were positive by PCR, 70 (58%) were positive by BioSign, 69 (58%) were positive by culture and 67 (56%) were positive by Path-Stik. Eleven samples were positive by PCR and both VIAs, but negative by culture because culture plates were heavily overgrown with SF organisms making detection of any E. coli O157 present impossible. CONCLUSIONS: PCR and both VIAs compared well with culture of beads to CT-SMAC for detecting E. coli O157 after enrichment culture and IMS. PCR appeared to be the most sensitive method, but needed specialised equipment and was also the most expensive, laborious and technically demanding technique. Although lacking the sensitivity of PCR, the VIAs were of comparable sensitivity to culture and were extremely quick and easy to perform giving a result in less than 15 minutes. SIGNIFICANCE AND IMPACT OF THE STUDY: Culture techniques may fail to detect E. coli O157 retrieved by IMS due to overgrowth with other organisms.

Cell Culture Techniques↗
PubMed2000

Infection with verocytotoxin-producing Escherichia coli O157 during a visit to an inner city open farm.

Two cases of Escherichia coli O157 infection occurred in children after visiting an inner city open farm. Subsequently faecal samples collected from animal pens and samples of composted mixed animal manure and vegetable waste were examined for E. coli O157 by enrichment culture, immunomagnetic separation and culture of magnetic beads to cefixime tellurite sorbitol MacConkey agar. Strains of E. coli O157 were characterized by hybridization with DNA probes for VT1, VT2 and eaeA, plasmid profile analysis, phage typing and pulsed field gel electrophoresis (PFGE). Verocytotoxin-producing E. coli O157 strains were isolated from faecal samples from a cow, a horse, 3 breeds of pigs, 2 breeds of sheep and 2 breeds of goats and from 2 samples of compost which had been processed for 3 months. All strains were phage type 21, hybridized with probes for VT2 and eaeA but not with one for VT1, harboured 92 and 2 kb plasmids and gave indistinguishable banding patterns with PFGE. Although only two culture-confirmed cases of infection had been identified, the farm had over 100,000 visitors per year and so it was closed as a precaution both to allow a thorough investigation and to prevent further cases. The investigation identified many factors which may have contributed to transmission of E. coli O157 infection. Most of these were readily resolved by appropriate corrective measures and as there were no further cases associated with the farm during the ensuing 4 weeks it then re-opened. These cases highlight the risk, especially to young children, of acquiring zoonotic infections during visits to open farms and emphasize the need for adequate guidance and supervision before and during such visits.

Agriculture↗
PubMed2000

A one year study of Escherichia coli O157 in raw beef and lamb products.

Between April 1996 and March 1997 we examined 5093 samples of raw beef and lamb products for the presence of E. coli O157. Samples were purchased from 81 small butchers' shops in south Yorkshire. In March 1997 we also examined five samples of dried mint for the presence of E. coli O157. Strains of E. coli O157 were isolated by enrichment culture in modified buffered peptone water followed by immunomagnetic separation and culture of magnetic beads onto cefixime tellurite sorbitol MacConkey agar. Strains were characterized by phage typing, toxin genotyping and plasmid analysis. Strains of E. coli O157 were isolated from 72 (1.4%) of 5093 samples; it was isolated from 36 (1.1%) of 3216 samples of beef products and from 29 (2.9%) samples of lamb products. The highest prevalence was found in lamb sausages and lamb burgers where E. coli O157 was isolated from 3 (4.1%) of 73 and 18 (3.7%) of 484 samples respectively. Strains of E. coli O157 were isolated most frequently during early summer. Strains of E. coli O157 were also isolated from 2 of 5 samples of dried mint although we did not determine how the mint had become contaminated. All isolates of E. coli O157 were Verocytotoxin-producing as determined by both Vero cell assay and DNA hybridization for the genes encoding Verocytotoxin and all were positive for the eaeA gene. A combination of phage typing, toxin genotyping and plasmid profile subdivided the 72 strains of E. coli isolated into 20 different subtypes, of which 18 were indistinguishable from strains isolated previously from cattle and sheep; of these 18 strains, 8 were indistinguishable from strains isolated from human cases of infection during the study period.

Animals↗
PubMed2000

Sources of Escherichia coli O157 and experiences over the past 15 years in Sheffield, UK.

In the first documented outbreak of HC caused by Escherichia coli O157, which occurred in the North-west USA in 1982, there was a strong association between infection and prior consumption of ground beef from a chain of fast food restaurants. Foods of bovine origin, including beef, milk and dairy products, have since been implicated in many outbreaks of infection world-wide. Investigations during the course of outbreaks, or at random, have shown that cattle are a major reservoir of E. coli O157. E. coli O157 was isolated from cattle at slaughter in Sheffield in 1987, this being the first isolation from cattle in the UK. Following a cluster of cases in May/June 1992, an abattoir study showed the organism to be present in 4% of cattle at slaughter and on up to a third of carcasses from rectal swab-positive animals. E. coli O157 was isolated from a food source (unpasteurized milk), for the first time in the UK, in Sheffield in May 1993. During surveillance in 1995-6, E. coli O157 was isolated from 15.7% of cattle, with a monthly prevalence which varied from 5 to 37%. E. coli O157 was also isolated from 2.2% of sheep. During surveillance in 1996, E. coli O157 was isolated from 5.9% of samples of lamb products and from 1.5% of samples of beef products, despite the prevalence in cattle being much higher than in sheep. Work is in progress to try to explain this higher prevalence in lamb products. During 1997 in Sheffield, the only cases of E. coli O157 for which a confirmed source was established were associated with direct animal contact on farm visits. During on-farm investigations of these cases, E. coli O157 was isolated from faecal samples from adult cattle, calves, three different breeds of sheep, two different breeds of pigs, goats and a pony.

Abattoirs↗
PubMed1998

A case-control study of true-positive versus false-negative cervical smears in women with cervical intraepithelial neoplasia (CIN) III.

Cervical smears (n = 150) from five departments showing high-grade dyskaryosis were examined by three cytologists. All the smears came from patients with biopsy-proven CIN III. One hundred had been correctly reported (true positives) but 50 had originally been reported as negative and had been found to be positive only on review (false negatives). There were significant differences between the two sets in the characteristics of the dyskaryotic cell population. The false-negative smears tended to have fewer than 200 dyskaryotic cells. The nuclei of the dyskaryotic cells tended to have fine rather than coarse nuclear chromatin. A smear with fewer than 50 dyskaryotic cells is 26 times more likely to be reported as negative than one with more than 200 dyskaryotic cells. The results suggest that there is a type of severely dyskaryotic smear that is inherently likely to be missed on routine screening.

Case-Control Studies↗
PubMed1997

A fifteen month study of Escherichia coli O157:H7 in a dairy herd.

A dairy herd associated with Escherichia coli O157 infection in humans was studied for the 15 months following the outbreak to examine seasonal, age and management factors affecting faecal excretion of the organism and to determine the mode and frequency of milk contamination with the organism. Between May 1993 and July 1994, 28 visits were made to the farm to collect a total of 3593 rectal swabs from cows, heifers and calves and 329 milk samples. E. coli O157:H7 was isolated from 153 (4.3%) of 3593 bovine rectal swabs. The maximum prevalence at any one visit was 14% in lactating cows, 40% in non-lactating cows, 56% in calves and 68% in heifers. The prevalence in lactating cows, which was significantly lower than in the other groups, peaked during May-July 1993 and again briefly after the cattle were housed during November 1993 and then again during May 1994. Excretion rates of E. coli O157:H7 in lactating cows were highest during the first month after calving, falling during lactation and rising to another peak at 7 months postpartum. Between November 1993 and May 1994 there was no evidence of excretion in any group. Eighty-seven (74%) of the animals which excreted E. coli O157:H7 did so on only one occasion but 23 (32%) of 73 cows and heifers and 7 (16%) of 44 calves which excreted the organism did so on more than one occasion. E. coli O157:H7 was not isolated from milk taken from the bulk tank but it was isolated from individual milk samples (one milk jar and one fore-milk) from two animals previously shown to be faecal excretors of the organism. All isolates of E. coli O157:H7 obtained were of the same phage type, toxin genotype and plasmid profile.

Age Factors↗
PubMed1997

Outbreaks of food poisoning in adults due to Escherichia coli O111 and campylobacter associated with coach trips to northern France.

Thirty-seven out of 48 people on a coach excursion to northern France developed gastrointestinal symptoms within 4 days of the trip. Twenty-six had stool samples positive for Escherichia coli O111, 8 were also positive for Campylobacter species, and 1 was positive for campylobacter alone. Strains of E. coli were positive for the effacing and attaching protein (eaeA) gene, but negative for other E. coli virulence genes, and therefore belonged to the enteropathogenic E. coli (EPEC) group. Twenty-two out of 37 people in a second party which followed the same itinerary 2 weeks later also became ill. One had a stool sample positive for E. coli O111. Analytical epidemiology suggested that the source of infections was a restaurant in northern France at which both parties had eaten.

Adhesins, Bacterial↗
PubMed1997

A 1-year study of Escherichia coli O157 in cattle, sheep, pigs and poultry.

Samples of rectal faeces were collected immediately after slaughter from 400 cattle each month for a 1-year period and from 1000 each of sheep, pigs and poultry over the same period. Samples were examined for Escherichia coli O157 by enrichment culture in buffered peptone water with vancomycin, cefixime and cefsulodin followed by immunomagnetic separation and culture of magnetic particles onto cefixime tellurite sorbitol MacConkey agar. E. coli O157 was isolated from 752 (15.7%) of 4800 cattle, 22 (2.2%) of 1000 sheep and from 4 (0.4%) of 1000 pigs, but not from any of 1000 chickens. Of the cattle sampled. 1840 (38.4%) were prime beef animals, 1661 (34.6%) were dairy animals being culled and the status could not be determined for the other 1299 (27%) animals. E. coli O157 was found in 246 (13.4%) of the 1840 beef cattle and 268 (16.1%) of the 1661 dairy cattle. The monthly prevalence of E. coli O157 in cattle was 4.8-36.8% and was at its highest in spring and late summer. Seventeen of the 22 isolates from sheep were also made over the summer period. All E. coli O157 isolates from sheep and 749 (99.6%) of the 752 E. coli O157 isolates from cattle were verocytotoxigenic as determined by Vero cell assay and DNA hybridization, eaeA gene positive, contained a 92 kb plasmid and were thus typical of strains causing infections in man. In contrast isolates from pigs were non-toxigenic, eaeA gene negative and did not contain a 92 kb plasmid and would, therefore, be unlikely to be a source of infection for man.

Animals↗
PubMed1997

An outbreak of infection due to verocytotoxin-producing Escherichia coli O157 in four families: the influence of laboratory methods on the outcome of the investigation.

Three members of family A, who had diarrhoea on 20 October, lived on a small arable farm which had 10 cattle. Manure from the animals was used to fertilize the ground for growing potatoes which were then offered for retail sale, unwashed, directly from the farm. The mother from family B bought potatoes, which were covered with manure, from family A in early November and over the subsequent 10 days she became ill with diarrhoea and her daughter and son both became ill with bloody diarrhoea. The mother from family C visited family B while the daughter from the latter family was symptomatic; the mother developed diarrhoea several days later. The mother and two sons from family D visited family B while the son from the latter family was symptomatic; the first son developed bloody diarrhoea 6 days later which progressed to development of haemolytic-uraemic syndrome. Direct culture of faecal samples onto cefixime rhamnose sorbitol MacConkey agar failed to isolate E. coli O157 from any of the symptomatic patients, and direct culture onto cefixime tellurite sorbitol MacConkey agar isolated the organism from only one patient. In contrast, a combination of isolation of E. coli O157 by immunomagnetic separation and detection of E. coli O157-specific secretory IgA, suggested E. coli O157 infection in all eight symptomatic patients, but not in any of the family members who were not ill. Two children who excreted the organism for 60 and 89 days respectively were the only two patients who did not develop a secretory IgA response. E. coli O157 was not isolated from potatoes from the farm and faecal samples from the farm animals were not available for examination. The study illustrates the need to use the most sensitive methods available during the investigation and follow up of cases of E. coli O157 infection.

Animals↗
PubMed1997

Atypical strains of verocytotoxin-producing Escherichia coli O157 in beef cattle at slaughter in Veneto region, Italy.

Cattle arriving for slaughter at abattoirs in the Veneto region of N. Italy were examined for intestinal carriage of Escherichia coli O157. Rectal swabs were cultured in modified buffered peptone water and E. coli O157 was concentrated by an immunomagnetic separation technique; the magnetic beads were cultured onto cefixime tellurite sorbitol MacConkey agar. Sorbitol non-fermenting E. coli O157 was isolated from 15 (3.6%) of 419 feedlot cattle but not from 437 veal calves or 65 culled cows. All strains of E. coli O157 hybridized with DNA probes specific for the VT1 or VT2 genes, but two strains did not produce toxin detectable by Vero cell assay. Six different plasmid profiles were observed with all strains harbouring the large 93 kb plasmid characteristic of VTEC. Six strains produced urease but otherwise strains were biochemically typical of E. coli O157. One strain was resistant to streptomycin, tetracycline and sulphonamides but the remainder were sensitive to all antimicrobials tested. This is the first description of the isolation of verocytotoxin-producing E. coli O157 from cattle in Italy. As the contamination of bovine carcasses with E. coli O157 during slaughter and processing has been demonstrated, the risk of transmission of this organism from beef cattle to the human population in the Veneto region, through foods of bovine origin or by other routes, should not be overlooked.

Abattoirs↗
PubMed1997

Use of commercial enzyme immunoassays and immunomagnetic separation systems for detecting Escherichia coli O157 in bovine fecal samples.

A commercial enzyme immunoassay (EIA) (E. coli O157 Visual Immunoassay; Tecra Diagnostics) performed on enrichment cultures in modified Escherichia coli broth (mECn) was compared with immunomagnetic separation (IMS) (Dynabeads anti-E. coli O157; Dynal) performed on enrichment cultures in modified buffered peptone water (BPW-VCC) for the detection of E. coli O157 in bovine fecal samples. Tests on fecal suspensions inoculated with each of 12 different strains of E. coli O157 showed that both the EIA and IMS methods were 10- to 100-fold more sensitive than direct culture or enrichment subculture methods for detection of the organism. EIA and IMS were then compared for detection of E. coli O157 in bovine rectal swabs. For confirmation of positive EIA tests, a commercial system (Immunocapture System [ICS]; Tecra Diagnostics) was compared with IMS; both were performed on mECn enrichment cultures. Of 200 rectal swabs examined, 17 gave positive results in the EIA which were confirmed by both confirmation systems, 2 gave positive results in the EIA which were confirmed by IMS but not by ICS, and 1 gave a positive result in the EIA which was confirmed by ICS but not by IMS. Of these 20, 15 were also positive by the BPW-VCC-IMS culture system; a further 3 samples were positive by this culture system but gave a negative result in the EIA. Eight samples were negative by the BPW-VCC-IMS culture system but gave a positive result in the EIA which could not be confirmed by either confirmation system. Further examination of the eight unconfirmed EIA-positive samples yielded sorbitol-fermenting E. coli O157 from three samples. Of the remaining five cultures, four were positive in an EIA for verocytotoxins (VT) and two were positive in a cell culture assay for VT1. The remaining 170 samples were negative by both EIA and BPW-VCC-IMS. The Tecra EIA and IMS are both technically simple and sensitive methods for detecting E. coli O157 in bovine fecal samples. There was no statistically significant difference between the numbers of positives detected by the different assays (P = 0.29).

Animals↗