- Email: [email protected]
Occurrence of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella and Campylobacter spp. on beef carcasses in Northern Ireland
Meat Science 58 (2001) 343±346
www.elsevier.com/locate/meatsci
Occurrence of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella and Campylobacter spp. on beef carcasses in Northern Ireland Robert H. Madden a,b,*, W. Eric Espie a, Lynn Moran a, Jane McBride a, Pam Scates a a
Food Science Division, (Food Microbiology), Department of Agriculture and Rural Development, Newforge Lane, Belfast, Northern Ireland, UK b Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, Northern Ireland, UK Received 14 August 2000; received in revised form 8 November 2000; accepted 2 December 2000
Abstract A survey of beef carcasses was conducted in all 10 European community approved abattoirs in Northern Ireland to determine the incidence of Escherichia coli O157:H7. Analyses were based on excised samples of neck meat taken less than 48 h post-kill. Overall, 780 carcasses were sampled and all were negative for E. coli O157:H7. A sub-set of samples was analysed for the presence of Listeria monocytogenes (n=200), Salmonella (n=200) and Campylobacter spp.(n=100). L. monocytogenes was not detected but Listeria innocua was found on ®ve carcasses and Listeria seeligeri on one. Three carcasses carried salmonellas; Salmonella Mbandaka was found on two and Salmonella Thompson on one. Campylobacter spp. were not detected on any carcasses. The results indicate that very few beef carcasses in Northern Ireland appear to carry any of the four pathogens sought, and this may help explain the low incidence of E. coli O157:H7 in the Northern Ireland human population, relative to the rest of the UK. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Beef; Campylobacter; Listeria; Salmonella; O157
1. Introduction The global spread of the pathogen Escherichia coli O157:H7 and its association with cattle has been well documented (Anon., 1993; Nataro & Caper, 1998). The incidence of human E. coli O157:H7 infection in Scotland is the highest in the UK and one major outbreak, resulting from contaminated beef, caused 21 deaths (Pennington, 1997). In contrast, Northern Ireland, which is less than 20 miles from Scotland at the closest point, has the lowest level of human illness due E. coli O157:H7 in the UK (Anon., 1999). To investigate possible reasons for the disparate rates of infection, a survey of Northern Ireland beef cattle carcasses was instigated to determine the level of contamination of beef carcasses in the chill, prior to boning out. Sampling was based on the excision of neck meat in the chill. Considerable resources were required to collect * Corresponding author. Tel.: +44-28-90255-312; fax: +44-2890668-376. E-mail address: [email protected] (R.H. Madden).
the samples and supply them to the laboratory, therefore, additional microbiological analyses were conducted on some samples for the presence of Listeria monocytogenes, Salmonella spp. and Campylobacter species. This ensured as much information as possible was provided by the survey, within the constraints of available resources. The latter analyses were intended to determine if the levels of contamination of beef carcasses by any of these pathogens merited further study. Overall, it was intended that the survey would provide an insight into the potential microbiological hazards presented by Northern Ireland beef carcases. 2. Materials and methods 2.1. Abattoir survey The survey was designed to detect contamination by E. coli O157:H7 at a prevalence rate of 0.5% with 98% con®dence of detecting at least one positive carcass. This required 780 carcasses to be sampled. Based on
0309-1740/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00153-4
344
R.H. Madden et al. / Meat Science 58 (2001) 343±346
their relative throughput of beef cattle for human consumption, a proportional sampling scheme for all 10 European Community approved abattoirs in Northern Ireland was prepared. To avoid sampling bias, no more than six carcasses were sampled in each abattoir chill per visit, hence, each abattoir was visited 13 times. 2.2. Carcass sampling Carcasses were sampled in the abattoir chill less than 48 h post-kill and a section of neck meat at least 10 cm10 cm3 mm was aseptically removed using a sterile boning knife and placed in a stomacher bag. The sample was held in a cool box during transport to the laboratory and analysed within 24 h of excision. 2.3. Microbiological analyses All media used were Oxoid (Oxoid, Basingstoke, UK) unless otherwise stated. Appropriate positive and negative control cultures were included with all microbiological assays. 2.3.1. E. coli O157:H7 In the laboratory, the neck sample was trimmed to a section 10 cm10 cm3 mm thick using appropriate aseptic techniques. Normally, the samples obtained were signi®cantly larger than required, therefore, after trimming, sucient meat was available for additional analyses. The trimmed meat section was added to 225 ml of buered peptone water supplemented with vancomycin (8 mg/l), ce®xime (0.05 mg/l) and cefsulodin (10 mg/l) (BPW-VCC) and incubated (6 h, 37 C). One millilitre was then transferred to a microtube containing 20 ml of paramagnetic beads coated with antibodies to E. coli O157 (Dynabeads type 710.04, Dynal, Oslo). After mixing (30 min, room temperature), the tube was placed in a magnetic separator, the magnet inserted and the whole inverted three times and left for 5 min. The liquid was then aspirated from the tubes, the magnet removed and the beads resuspended in 1 ml phosphate buered saline plus 0.05% Tween 20 by inverting the tube four to ®ve times. The magnet was then replaced and the tube left for 2 min. This washing procedure was carried out twice more then the liquid removed. The magnet was then removed and the beads suspended in phosphate buered saline (PBS; 30 ml) and spread onto sorbitol MacConkey agar supplemented with ce®xime (0.05 mg/l) and potassium tellurite (2.5 mg/l; CT-SMAC). After incubation (37 C, 18 h), non-sorbitol fermenting colonies were picked from each plate and streaked to obtain pure cultures. Suspect colonies were then checked using the Oxoid latex agglutination kit. 2.3.2. Salmonella spp. Duplicate samples (25 g) were added to buered peptone water (225 ml) and incubated overnight (37 C).
Sub-samples (0.1 ml) were then transferred to RappaportVassiliadis broth (4.5 ml; Lab M, Bury, UK) and incubated (41 C, 24 h) in an indirect impedance cell in a rapid automated bacterial impedance tester (RABIT; Don Whitley Scienti®c Ltd, Shipley, Yorkshire, UK; Donaghy & Madden, 1993). Conductance values were logged every 6 min, and after incubation samples showing characteristic signs of growth were streaked onto brilliant green agar and incubated (37 C, 24 h). Typical salmonella colonies were puri®ed on nutrient agar and identi®ed by normal phenotypic tests before being serotyped. 2.3.3. Listeria spp. The method of Harvey and Gilmour (1993) was used. Supplements, nalidixic acid (40 mg/10 ml), cycloheximide (50 mg/10 ml) and acri¯avine hydrochloride (10 mg/10 ml), were prepared separately and ®lter sterilised. Listeria enrichment broth (LEM; Oxoid CM862) was prepared in 225 ml volumes, autoclaved (121 C, 15 min) and 2.5 ml of each supplement added when cooled. Duplicate samples (25 g) were added to supplemented LEM and incubated (30 C) with subcultures taken after 24 and 48 h and streaked onto Oxford agar plates (Oxoid CM856 plus SR140) and incubated (30 C). Plates were examined after 24 and 48 h. Typical colonies were restreaked onto tryptone soy agar with yeast extract and incubated (37 C, 24 h) then pure cultures were subjected to con®rmation by standard methods. 2.3.4. Campylobacter spp. Duplicate samples (10 g) were added to 90 ml of Preston broth (Bolton & Robertson, 1982) supplemented with FBP (iron, bisulphite, pyruvate, George, Homan, Smibert & Krieg, 1978) then incubated (24 h, 42 C) in a Don Whitley Mk III anaerobic cabinet (Don Whitley Scienti®c Ltd, Shipley, Yorkshire, UK) using a gas mix of 5% O2, 10% CO2, 85% N2. Subcultures were then streaked onto Preston agar and incubated as above. Plates were examined after 24 and 48 h and hanging drops prepared from characteristic campylobacter-like colonies. When no typical colonies were seen, up to ®ve other colonies were selected for microscopic examination. If typical spiral morphology and corkscrew motility were displayed, colonies were streaked onto blood agar and incubated as above to obtain pure cultures for speciation. After initial characterisation, presumptive Campylobacter spp. were con®rmed using standard biochemical tests (Bolton, Holt, & Hutchinson, 1982). 3. Results A total of 780 carcasses was sampled for E. coli O157:H7 but no positive samples were found (Table 1). In addition none of the samples analysed for Campylobacter spp. (n=100) and L. monocytogenes (n=200)
R.H. Madden et al. / Meat Science 58 (2001) 343±346 Table 1 Occurrence of four types of pathogenic bacteria on beef carcasses in Northern Ireland Organism
Number of Positive Species/serovar samples samples isolated analysed
Escherichia coli O157:H7 Listeria spp.
780 200
0 6
Salmonella
200
3
Campylobacter spp.
100
0
a
Listeria innocua (5)a Listeria seeligeri (1) Mbandaka (2) Thompson (1)
Number found.
were positive, however, three carcasses (n=200) carried Salmonella serovars (Table 1). 4. Discussion Cattle are known to carry E. coli O157:H7 (Chapman, Wright, Norman, Fox, & Crick, 1993; Chapman, Wright, & Siddons, 1994) but the carcasses of carrier animals should be free of the organism unless abattoir practices permit cross-contamination from the hide or visceral contents to the carcass. Carriage rates in beef cattle at slaughter appear to vary markedly, with values being reported of 3.6% in Italy (Conedera, Marangon, Chapman, Zuin, & Caprioli, 1997), 10.4% in the Netherlands (Heuvelink et al., 1998), 13.4% in England (Chapman, Siddons, Malo, & Harkin, 1997) and 28% in the USA (Elder, Keen, Siragusa, Barkocy-Gallagher, Koohmaraie, & Laegreid, 2000). Seasonal variation from 4.8 to 36.8% was also reported by Chapman et al. (1997) indicating that carriage rates, and information derived from them, should be interpreted with caution. In Northern Ireland, a survey of cattle faeces submitted for diagnostic investigation (i.e. enteric disease was either present or suspected) showed that 1.97% (n=508) were positive for E. coli O157:H7 (Anon., 1997). This carriage rate is signi®cantly less than those reported above. With good hygienic practices during skinning and evisceration, the rate of carcass contamination should be signi®cantly below the carriage rate and the failure of this study to detect the organism on 780 carcasses means that the local carcass contamination rate was below 0.5% (P<0.02). In comparison, Vanderlinde, Shay, and Murray (1998) found 0.5% of beef carcasses (n=732) in Australian export meat plants carried E. coli O157:H7. Elder et al. (2000) noted that whilst 28% of cattle presented for slaughter in the Midwestern USA carried E. coli O157, only 2% of carcasses sampled in the chill were positive. Thus, approximately 7% of the animals infected with E. coli O157 gave rise to contaminated carcasses. Using this proportion, and the
345
incidence determined in the faeces locally (Anon., 1997), the prevalence in chilled carcasses in Northern Ireland would be 0.14%. This value corresponds to a single positive carcass out of the 780 sampled which is within the experimental error of the study. Thus, both the carcass survey and the survey of faeces support the premise that the prevalence of E. coli O157:H7 on beef carcasses in Northern Ireland is very low. None of the 100 carcasses sampled yielded Campylobacter spp., therefore, the contamination rate is 3.0% or less (P<0.05). In the USA, a survey of 2089 steer/heifer carcasses (McNamara, 1995) found that 4.0% carried Campylobacter jejuni or Campylobacter coli, but in Australia, Vanderlinde et al. (1998) found only 1/124 carcasses positive in domestic meat plants. The latter group found the contamination rate was even lower in export meat plants with 1/533 carcasses positive. The Northern Ireland survey suggests that Campylobacter contamination is less common than that found in the USA, but that further sampling would be required to determine if levels were as low as in Australian export meat plants. Listeria monocytogenes was also absent from Northern Ireland carcasses (n=200), but a British survey of cattle carcasses (n=29) found 7% positive for L. monocytogenes (Fenlon, Wilson, & Donachie, 1996) whilst in the USA, Karr et al. (1996) found L. monocytogenes on 2.5% of carcasses (n=40). However, in a major survey of 2089 steer and heifer carcasses in the USA, 4.1% were positive for L. monocytogenes (McNamara, 1995). In Australia, Vanderlinde et al. (1998) found contrasting results for the presence of Listeria spp. in domestic and export meat plants with 15% of carcasses in the former positive (n=20) but only 0.77% (n=130) in the latter. These results suggest Northern Ireland beef carcasses are produced to a high standard with regard to L. monocytogenes. However, the Salmonella analyses showed that some pathogens of faecal origin were present on Northern Ireland beef carcasses, with 1.5% positive. This rate is the same as that reported in the USA by Sofos et al. (1999) for steer/heifer carcasses (n=2168), and similar to the 1.4% (n=144) found in Australian domestic meat plants by Vanderlinde et al. (1998). Again, the latter group reported markedly lower recoveries in export meat plants with 0.27% of carcasses positive. Thus, whilst salmonellas were found on Northern Ireland beef carcasses, the incidence was comparable with major beef producing countries. Overall, a major survey for E. coli O157:H7 on beef carcasses found none. Sub-sets of samples yielded no Campylobacter spp. or L. monocytogenes but 1.5% of carcasses carried salmonellas. Given the survey results, and the very low incidence of infection in the local human population (Anon., 1999), beef appears to be of low signi®cance as a potential source of human infection
346
R.H. Madden et al. / Meat Science 58 (2001) 343±346
with E. coli O157:H7 in Northern Ireland at the present time. The precise reasons for this would appear to merit further study.
References Anon. (1993). Multistate outbreak of Escherichia coli O157:H7 infections from hamburgers Ð Western United states, 1992±1993. Morbidity and Mortality Weekly Reports, 42, (pp. 258-263). Center for Disease Control and Prevention, Atalanta, USA. Anon. (1997). Escherichia coli O157 in cattle in Northern Ireland. In E. Mitchell, Northern Ireland Surveillance Bulletin. Belfast, Northern Ireland: Department of Health and Social Services, and Department of Agriculture for Northern Ireland. Anon. (1999). Review of Communicable Diseases 1999, Northern Ireland. Communicable Diseases Surveillance Centre Services for Northern Ireland, Belfast, Northern Ireland. Bolton, F. J., & Robertson, L. (1982). A selective medium for isolation of Campylobacter jejuni/coli. Journal of Clinical Pathology, 35, 462± 467. Bolton, F. J., Holt, A. V., & Hutchinson, D. N. (1984). Campylobacter biotyping scheme of epidemiological value. Journal of Clinical Microbiology, 37, 677±681. Chapman, P. A., Siddons, C. A., Malo, A. T. C., & Harkin, M. A. (1997). A 1-year study of Escherichia coli O157 in cattle, sheep, pigs and poultry. Epidemiology and Infection, 119, 245±250. Chapman, P. A., Wright, D. J., Norman, P., Fox, J., & Crick, E. (1993). Cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infections in man. Epidemiology and Infection, 111, 439±447. Chapman, P. A., Wright, D. J., & Siddons, C.A (1994). A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from bovine faeces. Journal of Medical Microbiology, 40, 424±427. Conedera, G., Marangon, S., Chapman, P. A., Zuin, A., & Caprioli, A. (1997). Atypical strains of verocytotoxin-producing Escherichia coli 0157 in beef cattle at slaughter in Veneto region, Italy. Journal of Veterinary Medicine, 44, 301±306. Donaghy, J. A., & Madden, R. H. (1993). Detection of Salmonella in
animal protein by Rappaport-Vassiliadis broth using indirect impediometry. International Journal of Food Microbiology, 17, 281±288. Elder, R. O., Keen, J. E., Siragusa, G. R., Barkocy-Gallagher, G. A., Koohmaraie, M., & Laegreid, W. W. W. (1997). Correlation of enterohemorrhagic Escherichia coli O157 in feces, hides and carcasses of beef cattle during processing. Proceedings of the National Academy of Sciences of the USA, 97, (pp. 2999±3003). Fenlon, D. R., Wilson, J., & Donachie, W. (1996). The incidence and level of Listeria monocytogenes contamination of food sources at primary production and initial processing. Journal of Applied Bacteriology, 81, 641±650. George, H. A., Homan, P. S., Smibert, R. M., & Krieg, N. R. (1978). Improved media for growth and aerotolerance of Campylobacter fetus. Journal of Clinical Microbiology, 8, 36±41. Harvey, J., & Gilmour, A. (1993). Occurrence and characteristics of Listeria in foods produced in Northern Ireland. International Journal of Food Microbiology, 19, 193±205. Heuvelink, A. E., Van den Biggelaar, F. L. A. M., DeBoer, E., Herbes, R. G., Melchers, W. J. G., Huis In'T Veld, J. H. J., & Monens, L. A. H. (1998). Isolation and characterization of verotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep. Journal of Clinical Microbiology, 36, 878±882. Karr, K. J., Boyle, E. A. E., Kastner, C. L., Marsden, J. L., Phebus, R. K., Prasai, R. K., Pruett, W. P., & Zepeda, C. M. G. (1996). Standardized microbiological sampling and testing procedures for the beef industry. Journal of Food Protection, 59, 778±780. McNamara, A. M. (1995). Establishment of baseline data on the microbiota of meats. Journal of Food Safety, 15, 113±119. Nataro, J. P., & Caper, J. B. (1998). Diarrheagenic Escherichia coli. Clinical Microbiological Reviews, 11, 142±201. Pennington, T. H. (1997). The Pennington Group: report on the circumstances leading to the 1996 outbreak of infection with Escherichia coli 0157 in central Scotland, the implications for food safety and the lessons to be learned. The Stationery Oce Ltd., Edinburgh, UK. Sofos, J. N., Kochevar, S. L., Bellinger, G. R., Buege, D. R., Hancock, D. D., Ingham, S. C., Morgan, J. B., Reagan, J. O., & Smith, G. C. (1999). Sources and extent of microbiological contamination of beef carcasses in seven United States slaughtering plants. Journal of Food Protection, 62, 140±145. Vanderlinde, P. B., Shay, B., & Murray, J. (1998). Microbiological quality of Australian beef carcass meat and frozen bulk packed beef. Journal of Food Protection, 61, 437±443.
www.elsevier.com/locate/meatsci
Occurrence of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella and Campylobacter spp. on beef carcasses in Northern Ireland Robert H. Madden a,b,*, W. Eric Espie a, Lynn Moran a, Jane McBride a, Pam Scates a a
Food Science Division, (Food Microbiology), Department of Agriculture and Rural Development, Newforge Lane, Belfast, Northern Ireland, UK b Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, Northern Ireland, UK Received 14 August 2000; received in revised form 8 November 2000; accepted 2 December 2000
Abstract A survey of beef carcasses was conducted in all 10 European community approved abattoirs in Northern Ireland to determine the incidence of Escherichia coli O157:H7. Analyses were based on excised samples of neck meat taken less than 48 h post-kill. Overall, 780 carcasses were sampled and all were negative for E. coli O157:H7. A sub-set of samples was analysed for the presence of Listeria monocytogenes (n=200), Salmonella (n=200) and Campylobacter spp.(n=100). L. monocytogenes was not detected but Listeria innocua was found on ®ve carcasses and Listeria seeligeri on one. Three carcasses carried salmonellas; Salmonella Mbandaka was found on two and Salmonella Thompson on one. Campylobacter spp. were not detected on any carcasses. The results indicate that very few beef carcasses in Northern Ireland appear to carry any of the four pathogens sought, and this may help explain the low incidence of E. coli O157:H7 in the Northern Ireland human population, relative to the rest of the UK. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Beef; Campylobacter; Listeria; Salmonella; O157
1. Introduction The global spread of the pathogen Escherichia coli O157:H7 and its association with cattle has been well documented (Anon., 1993; Nataro & Caper, 1998). The incidence of human E. coli O157:H7 infection in Scotland is the highest in the UK and one major outbreak, resulting from contaminated beef, caused 21 deaths (Pennington, 1997). In contrast, Northern Ireland, which is less than 20 miles from Scotland at the closest point, has the lowest level of human illness due E. coli O157:H7 in the UK (Anon., 1999). To investigate possible reasons for the disparate rates of infection, a survey of Northern Ireland beef cattle carcasses was instigated to determine the level of contamination of beef carcasses in the chill, prior to boning out. Sampling was based on the excision of neck meat in the chill. Considerable resources were required to collect * Corresponding author. Tel.: +44-28-90255-312; fax: +44-2890668-376. E-mail address: [email protected] (R.H. Madden).
the samples and supply them to the laboratory, therefore, additional microbiological analyses were conducted on some samples for the presence of Listeria monocytogenes, Salmonella spp. and Campylobacter species. This ensured as much information as possible was provided by the survey, within the constraints of available resources. The latter analyses were intended to determine if the levels of contamination of beef carcasses by any of these pathogens merited further study. Overall, it was intended that the survey would provide an insight into the potential microbiological hazards presented by Northern Ireland beef carcases. 2. Materials and methods 2.1. Abattoir survey The survey was designed to detect contamination by E. coli O157:H7 at a prevalence rate of 0.5% with 98% con®dence of detecting at least one positive carcass. This required 780 carcasses to be sampled. Based on
0309-1740/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00153-4
344
R.H. Madden et al. / Meat Science 58 (2001) 343±346
their relative throughput of beef cattle for human consumption, a proportional sampling scheme for all 10 European Community approved abattoirs in Northern Ireland was prepared. To avoid sampling bias, no more than six carcasses were sampled in each abattoir chill per visit, hence, each abattoir was visited 13 times. 2.2. Carcass sampling Carcasses were sampled in the abattoir chill less than 48 h post-kill and a section of neck meat at least 10 cm10 cm3 mm was aseptically removed using a sterile boning knife and placed in a stomacher bag. The sample was held in a cool box during transport to the laboratory and analysed within 24 h of excision. 2.3. Microbiological analyses All media used were Oxoid (Oxoid, Basingstoke, UK) unless otherwise stated. Appropriate positive and negative control cultures were included with all microbiological assays. 2.3.1. E. coli O157:H7 In the laboratory, the neck sample was trimmed to a section 10 cm10 cm3 mm thick using appropriate aseptic techniques. Normally, the samples obtained were signi®cantly larger than required, therefore, after trimming, sucient meat was available for additional analyses. The trimmed meat section was added to 225 ml of buered peptone water supplemented with vancomycin (8 mg/l), ce®xime (0.05 mg/l) and cefsulodin (10 mg/l) (BPW-VCC) and incubated (6 h, 37 C). One millilitre was then transferred to a microtube containing 20 ml of paramagnetic beads coated with antibodies to E. coli O157 (Dynabeads type 710.04, Dynal, Oslo). After mixing (30 min, room temperature), the tube was placed in a magnetic separator, the magnet inserted and the whole inverted three times and left for 5 min. The liquid was then aspirated from the tubes, the magnet removed and the beads resuspended in 1 ml phosphate buered saline plus 0.05% Tween 20 by inverting the tube four to ®ve times. The magnet was then replaced and the tube left for 2 min. This washing procedure was carried out twice more then the liquid removed. The magnet was then removed and the beads suspended in phosphate buered saline (PBS; 30 ml) and spread onto sorbitol MacConkey agar supplemented with ce®xime (0.05 mg/l) and potassium tellurite (2.5 mg/l; CT-SMAC). After incubation (37 C, 18 h), non-sorbitol fermenting colonies were picked from each plate and streaked to obtain pure cultures. Suspect colonies were then checked using the Oxoid latex agglutination kit. 2.3.2. Salmonella spp. Duplicate samples (25 g) were added to buered peptone water (225 ml) and incubated overnight (37 C).
Sub-samples (0.1 ml) were then transferred to RappaportVassiliadis broth (4.5 ml; Lab M, Bury, UK) and incubated (41 C, 24 h) in an indirect impedance cell in a rapid automated bacterial impedance tester (RABIT; Don Whitley Scienti®c Ltd, Shipley, Yorkshire, UK; Donaghy & Madden, 1993). Conductance values were logged every 6 min, and after incubation samples showing characteristic signs of growth were streaked onto brilliant green agar and incubated (37 C, 24 h). Typical salmonella colonies were puri®ed on nutrient agar and identi®ed by normal phenotypic tests before being serotyped. 2.3.3. Listeria spp. The method of Harvey and Gilmour (1993) was used. Supplements, nalidixic acid (40 mg/10 ml), cycloheximide (50 mg/10 ml) and acri¯avine hydrochloride (10 mg/10 ml), were prepared separately and ®lter sterilised. Listeria enrichment broth (LEM; Oxoid CM862) was prepared in 225 ml volumes, autoclaved (121 C, 15 min) and 2.5 ml of each supplement added when cooled. Duplicate samples (25 g) were added to supplemented LEM and incubated (30 C) with subcultures taken after 24 and 48 h and streaked onto Oxford agar plates (Oxoid CM856 plus SR140) and incubated (30 C). Plates were examined after 24 and 48 h. Typical colonies were restreaked onto tryptone soy agar with yeast extract and incubated (37 C, 24 h) then pure cultures were subjected to con®rmation by standard methods. 2.3.4. Campylobacter spp. Duplicate samples (10 g) were added to 90 ml of Preston broth (Bolton & Robertson, 1982) supplemented with FBP (iron, bisulphite, pyruvate, George, Homan, Smibert & Krieg, 1978) then incubated (24 h, 42 C) in a Don Whitley Mk III anaerobic cabinet (Don Whitley Scienti®c Ltd, Shipley, Yorkshire, UK) using a gas mix of 5% O2, 10% CO2, 85% N2. Subcultures were then streaked onto Preston agar and incubated as above. Plates were examined after 24 and 48 h and hanging drops prepared from characteristic campylobacter-like colonies. When no typical colonies were seen, up to ®ve other colonies were selected for microscopic examination. If typical spiral morphology and corkscrew motility were displayed, colonies were streaked onto blood agar and incubated as above to obtain pure cultures for speciation. After initial characterisation, presumptive Campylobacter spp. were con®rmed using standard biochemical tests (Bolton, Holt, & Hutchinson, 1982). 3. Results A total of 780 carcasses was sampled for E. coli O157:H7 but no positive samples were found (Table 1). In addition none of the samples analysed for Campylobacter spp. (n=100) and L. monocytogenes (n=200)
R.H. Madden et al. / Meat Science 58 (2001) 343±346 Table 1 Occurrence of four types of pathogenic bacteria on beef carcasses in Northern Ireland Organism
Number of Positive Species/serovar samples samples isolated analysed
Escherichia coli O157:H7 Listeria spp.
780 200
0 6
Salmonella
200
3
Campylobacter spp.
100
0
a
Listeria innocua (5)a Listeria seeligeri (1) Mbandaka (2) Thompson (1)
Number found.
were positive, however, three carcasses (n=200) carried Salmonella serovars (Table 1). 4. Discussion Cattle are known to carry E. coli O157:H7 (Chapman, Wright, Norman, Fox, & Crick, 1993; Chapman, Wright, & Siddons, 1994) but the carcasses of carrier animals should be free of the organism unless abattoir practices permit cross-contamination from the hide or visceral contents to the carcass. Carriage rates in beef cattle at slaughter appear to vary markedly, with values being reported of 3.6% in Italy (Conedera, Marangon, Chapman, Zuin, & Caprioli, 1997), 10.4% in the Netherlands (Heuvelink et al., 1998), 13.4% in England (Chapman, Siddons, Malo, & Harkin, 1997) and 28% in the USA (Elder, Keen, Siragusa, Barkocy-Gallagher, Koohmaraie, & Laegreid, 2000). Seasonal variation from 4.8 to 36.8% was also reported by Chapman et al. (1997) indicating that carriage rates, and information derived from them, should be interpreted with caution. In Northern Ireland, a survey of cattle faeces submitted for diagnostic investigation (i.e. enteric disease was either present or suspected) showed that 1.97% (n=508) were positive for E. coli O157:H7 (Anon., 1997). This carriage rate is signi®cantly less than those reported above. With good hygienic practices during skinning and evisceration, the rate of carcass contamination should be signi®cantly below the carriage rate and the failure of this study to detect the organism on 780 carcasses means that the local carcass contamination rate was below 0.5% (P<0.02). In comparison, Vanderlinde, Shay, and Murray (1998) found 0.5% of beef carcasses (n=732) in Australian export meat plants carried E. coli O157:H7. Elder et al. (2000) noted that whilst 28% of cattle presented for slaughter in the Midwestern USA carried E. coli O157, only 2% of carcasses sampled in the chill were positive. Thus, approximately 7% of the animals infected with E. coli O157 gave rise to contaminated carcasses. Using this proportion, and the
345
incidence determined in the faeces locally (Anon., 1997), the prevalence in chilled carcasses in Northern Ireland would be 0.14%. This value corresponds to a single positive carcass out of the 780 sampled which is within the experimental error of the study. Thus, both the carcass survey and the survey of faeces support the premise that the prevalence of E. coli O157:H7 on beef carcasses in Northern Ireland is very low. None of the 100 carcasses sampled yielded Campylobacter spp., therefore, the contamination rate is 3.0% or less (P<0.05). In the USA, a survey of 2089 steer/heifer carcasses (McNamara, 1995) found that 4.0% carried Campylobacter jejuni or Campylobacter coli, but in Australia, Vanderlinde et al. (1998) found only 1/124 carcasses positive in domestic meat plants. The latter group found the contamination rate was even lower in export meat plants with 1/533 carcasses positive. The Northern Ireland survey suggests that Campylobacter contamination is less common than that found in the USA, but that further sampling would be required to determine if levels were as low as in Australian export meat plants. Listeria monocytogenes was also absent from Northern Ireland carcasses (n=200), but a British survey of cattle carcasses (n=29) found 7% positive for L. monocytogenes (Fenlon, Wilson, & Donachie, 1996) whilst in the USA, Karr et al. (1996) found L. monocytogenes on 2.5% of carcasses (n=40). However, in a major survey of 2089 steer and heifer carcasses in the USA, 4.1% were positive for L. monocytogenes (McNamara, 1995). In Australia, Vanderlinde et al. (1998) found contrasting results for the presence of Listeria spp. in domestic and export meat plants with 15% of carcasses in the former positive (n=20) but only 0.77% (n=130) in the latter. These results suggest Northern Ireland beef carcasses are produced to a high standard with regard to L. monocytogenes. However, the Salmonella analyses showed that some pathogens of faecal origin were present on Northern Ireland beef carcasses, with 1.5% positive. This rate is the same as that reported in the USA by Sofos et al. (1999) for steer/heifer carcasses (n=2168), and similar to the 1.4% (n=144) found in Australian domestic meat plants by Vanderlinde et al. (1998). Again, the latter group reported markedly lower recoveries in export meat plants with 0.27% of carcasses positive. Thus, whilst salmonellas were found on Northern Ireland beef carcasses, the incidence was comparable with major beef producing countries. Overall, a major survey for E. coli O157:H7 on beef carcasses found none. Sub-sets of samples yielded no Campylobacter spp. or L. monocytogenes but 1.5% of carcasses carried salmonellas. Given the survey results, and the very low incidence of infection in the local human population (Anon., 1999), beef appears to be of low signi®cance as a potential source of human infection
346
R.H. Madden et al. / Meat Science 58 (2001) 343±346
with E. coli O157:H7 in Northern Ireland at the present time. The precise reasons for this would appear to merit further study.
References Anon. (1993). Multistate outbreak of Escherichia coli O157:H7 infections from hamburgers Ð Western United states, 1992±1993. Morbidity and Mortality Weekly Reports, 42, (pp. 258-263). Center for Disease Control and Prevention, Atalanta, USA. Anon. (1997). Escherichia coli O157 in cattle in Northern Ireland. In E. Mitchell, Northern Ireland Surveillance Bulletin. Belfast, Northern Ireland: Department of Health and Social Services, and Department of Agriculture for Northern Ireland. Anon. (1999). Review of Communicable Diseases 1999, Northern Ireland. Communicable Diseases Surveillance Centre Services for Northern Ireland, Belfast, Northern Ireland. Bolton, F. J., & Robertson, L. (1982). A selective medium for isolation of Campylobacter jejuni/coli. Journal of Clinical Pathology, 35, 462± 467. Bolton, F. J., Holt, A. V., & Hutchinson, D. N. (1984). Campylobacter biotyping scheme of epidemiological value. Journal of Clinical Microbiology, 37, 677±681. Chapman, P. A., Siddons, C. A., Malo, A. T. C., & Harkin, M. A. (1997). A 1-year study of Escherichia coli O157 in cattle, sheep, pigs and poultry. Epidemiology and Infection, 119, 245±250. Chapman, P. A., Wright, D. J., Norman, P., Fox, J., & Crick, E. (1993). Cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infections in man. Epidemiology and Infection, 111, 439±447. Chapman, P. A., Wright, D. J., & Siddons, C.A (1994). A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from bovine faeces. Journal of Medical Microbiology, 40, 424±427. Conedera, G., Marangon, S., Chapman, P. A., Zuin, A., & Caprioli, A. (1997). Atypical strains of verocytotoxin-producing Escherichia coli 0157 in beef cattle at slaughter in Veneto region, Italy. Journal of Veterinary Medicine, 44, 301±306. Donaghy, J. A., & Madden, R. H. (1993). Detection of Salmonella in
animal protein by Rappaport-Vassiliadis broth using indirect impediometry. International Journal of Food Microbiology, 17, 281±288. Elder, R. O., Keen, J. E., Siragusa, G. R., Barkocy-Gallagher, G. A., Koohmaraie, M., & Laegreid, W. W. W. (1997). Correlation of enterohemorrhagic Escherichia coli O157 in feces, hides and carcasses of beef cattle during processing. Proceedings of the National Academy of Sciences of the USA, 97, (pp. 2999±3003). Fenlon, D. R., Wilson, J., & Donachie, W. (1996). The incidence and level of Listeria monocytogenes contamination of food sources at primary production and initial processing. Journal of Applied Bacteriology, 81, 641±650. George, H. A., Homan, P. S., Smibert, R. M., & Krieg, N. R. (1978). Improved media for growth and aerotolerance of Campylobacter fetus. Journal of Clinical Microbiology, 8, 36±41. Harvey, J., & Gilmour, A. (1993). Occurrence and characteristics of Listeria in foods produced in Northern Ireland. International Journal of Food Microbiology, 19, 193±205. Heuvelink, A. E., Van den Biggelaar, F. L. A. M., DeBoer, E., Herbes, R. G., Melchers, W. J. G., Huis In'T Veld, J. H. J., & Monens, L. A. H. (1998). Isolation and characterization of verotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep. Journal of Clinical Microbiology, 36, 878±882. Karr, K. J., Boyle, E. A. E., Kastner, C. L., Marsden, J. L., Phebus, R. K., Prasai, R. K., Pruett, W. P., & Zepeda, C. M. G. (1996). Standardized microbiological sampling and testing procedures for the beef industry. Journal of Food Protection, 59, 778±780. McNamara, A. M. (1995). Establishment of baseline data on the microbiota of meats. Journal of Food Safety, 15, 113±119. Nataro, J. P., & Caper, J. B. (1998). Diarrheagenic Escherichia coli. Clinical Microbiological Reviews, 11, 142±201. Pennington, T. H. (1997). The Pennington Group: report on the circumstances leading to the 1996 outbreak of infection with Escherichia coli 0157 in central Scotland, the implications for food safety and the lessons to be learned. The Stationery Oce Ltd., Edinburgh, UK. Sofos, J. N., Kochevar, S. L., Bellinger, G. R., Buege, D. R., Hancock, D. D., Ingham, S. C., Morgan, J. B., Reagan, J. O., & Smith, G. C. (1999). Sources and extent of microbiological contamination of beef carcasses in seven United States slaughtering plants. Journal of Food Protection, 62, 140±145. Vanderlinde, P. B., Shay, B., & Murray, J. (1998). Microbiological quality of Australian beef carcass meat and frozen bulk packed beef. Journal of Food Protection, 61, 437±443.