An investigation into the ecological niches and seasonal nature of Clostridium estertheticum and Clostridium gasigenes in the Irish beef farm environment.

Blown pack spoilage (BPS) of vacuum packaged beef is caused by psychrotolerant and psychrophilic Clostridium species, primarily Clostridium estertheticum and Clostridium gasigenes. The aim of this study was to investigate the environmental niches and impact of season on these BPS Clostridium spp. on Irish beef farms. On each of five different beef farms, faecal (10), soil (5), silage (5), air (5), bedding straw (5), drinking water (5) and puddle/ditch water (5) samples were collected during Spring, Summer, Autumn and Winter and tested for C. estertheticum and C. gasigenes using culture (direct plating and enrichment) and molecular (conventional PCR and quantitative PCR (qPCR)) based techniques. C. estertheticum and C. gasigenes were detected in all sample types, with qPCR detection rates ranging from 4% to 50% and at concentrations of up to 1·5 log10 CFU per g and 3·5 log10 CFU per g, respectively. The impact of season was not clear as the results were mixed depending on the detection method used. It was concluded that BPS-causing C. estertheticum and C. gasigenes are widely distributed in the beef farm environment.

Coupling the dynamics of diffused gases and microbial growth in modified atmosphere packaging.

Coupling microbial dynamics with the complete dynamics of the packaging gases is still a challenge. In this work the microbial growth kinetic parameters for Pseudomonas and Lactic Acid Bacteria (LAB) in MAP are identified based on accurate estimation of diffusivity of gases and parameter scaled sensitivity approaches. The microbial dynamics are also compared with those estimated based on partial pressure measurement. Scaled sensitivity coefficient analysis using dissolved gases as variable inputs, shows that in most cases the only coefficients large enough for estimation were those for CO2max-diss, and for µmax. The current data showed that dissolved gases led significant differences on the microbial parameter of CO2max values when compared with the headspace gases. On the other hand, the (so-called) dissolved specific growth rate follows a clear trend down for both microorganisms in relation to the increase of the initial headspace CO2. Finally, current results indicate a possible correlation between CO2max-diss, CO2max-headspace, and µmax as functions of CO2init.

Thermal Inactivation of Listeria monocytogenes in Crab Meat.

Listeria monocytogenes is an important bacterial pathogen in seafood products, but limited information is currently available on the thermal resistance of relevant isolates in seafood. Thermal inactivation studies were undertaken (i) to provide much needed thermal inactivation data for L. monocytogenes in crab meat and (ii) to investigate whether tryptone soya broth (TSB) is representative of crab meat in thermal inactivation studies involving L. monocytogenes. D-values were obtained for a cocktail of two crab isolates (serotypes 1/2a and 4b) at 50, 55, and 60°C. In crab meat, D-values were 174.4, 28.2, and 1.6 min, respectively. Similar D-values of 176.4, 28.8, and 1.4 min were obtained in TSB. The corresponding z-values were 4.9°C (crab meat) and 4.8°C (TSB), respectively. The conclusions were that (i) current pasteurization conditions (e.g., 70°C for 2 min) would achieve complete destruction of any L. monocytogenes present in crab meat and (ii) TSB could be used as a model matrix for assessing the thermal inactivation of L. monocytogenes in crab meat.

The pattern of Campylobacter contamination on broiler farms; external and internal sources.

AIM: The aim of this study was to apply the most sensitive molecular techniques in combination with culture-based methods to characterize broiler farms in terms of the timeline ('appearance' and 'pattern') of Campylobacter contamination prior to and post detection in the birds. METHODS AND RESULTS: Faecal and environmental samples were collected from three broiler farms (two flocks per farm). Real-time PCR was used to test for the presence of Campylobacter. Culture-based methods (enrichment and direct plating) were also applied and isolates were subject to a range of confirmatory tests before speciation (multiplex PCR). All flocks were colonized by Campylobacter before first thin and a similar pattern of Campylobacter contamination was observed; (day -1) a range of external and internal samples real-time PCR positive but culture negative; (day 0) chicks negative; (6-9 days pre-detection in the birds) internal samples (feeders, drinkers, barrier and/or bird weigh) culture positive and (post broiler infection) increasing concentrations of Campylobacter in internal samples but also on the tarmac apron and anteroom. CONCLUSION: It was concluded that; (i) vertical transmission did not occur; (ii) the environment was a potential source of Campylobacter; (iii) testing areas frequented by all birds (e.g. feeders and drinkers), may offer an opportunity for early Campylobacter detection and (iv) once the broilers are infected with Campylobacter, these bacteria are spread from the birds, through the anteroom to the areas surrounding the broiler house, highlighting the need for improved biosecurity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has established the pattern of Campylobacter contamination on broiler farms, identified an early detection opportunity, highlighted the need to better understand the role of viable but nonculturable Campylobacter in the ecology of Campylobacter on broiler farms and demonstrated the need for improved biosecurity to prevent the spread of Campylobacter from within the house to the surrounding environment.

A four-year survey of blown pack spoilage Clostridium estertheticum and Clostridium gasigenes on beef primal cuts.

UNLABELLED: The objective of this study was to determine the incidence of Clostridium estertheticum and Clostridium gasigenes on beef primals taking sample type and season into account. Molecular methods using direct extraction of DNA without enrichment and based on the polymerase chain reaction (PCR) amplification of 16S rDNA fragments were used to test for the presence of Cl. estertheticum and Cl. gasigenes in 4826 beef primal samples (1967 drip, 1896 wet swab and 963 dry swab) provided by 10 commercial beef abattoirs over a 4-year period. Overall 1·5% of samples were PCR positive with the incidence of Cl. estertheticum and Cl. gasigenes being 0·8 and 0·7%, respectively. Although the highest incidence of Cl. estertheticum (4·0%) and Cl. gasigenes (5·1%) was observed in June and November, respectively, seasonal differences were not significant (P < 0·05). Drip samples yielded more positive results than swab samples. It was concluded that a low but persistent percentage of beef primal cuts are contaminated with blown pack spoilage Clostridia. There was no seasonal effect and drip may be a more effective test sample than swabs. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on blown pack spoilage contamination rates of beef primal cuts (pieces of meat initially cut from the carcass during butchering) over an extended period of time. The results show the risk of contamination is low but persistent throughout the year necessitating continuous sporicidal treatment of plant and equipment. Moreover, the higher prevalence of positive meat drip samples suggests sampling regimes should be based on this sample type.

Effect of different modified atmospheric packaging (MAP) gaseous combinations on Campylobacter and the shelf-life of chilled poultry fillets.

Studies were undertaken to investigate the effect of different modified atmospheric packaging (MAP) gaseous combinations on Campylobacter and the natural microflora on poultry fillets. Skinless chicken fillets were stored in gaseous mixtures of 10%, 30%, 50%, 70% and 90% CO2 balanced with N2, 80:20% O2:N2 and 40:30:30% CO2:O2:N2 and control conditions (air) at 2 °C. Samples were analysed periodically for (previously inoculated) Campylobacter, total viable counts (TVC) (mesophiles), TVC (psychrophiles), Enterobacteriaceae, Pseudomonas and lactic acid bacteria (LAB) over 17 days of storage. The carbon dioxide solubility was determined by monitoring the changes in the headspace volume over time using a buoyancy technique and performing calculations based on volumetric measurements and the Henry's constant. Henry's constant was also used to estimate the oxygen solubility in the chicken fillets. The presence of O2 in the MAP gaseous mixtures increased the rate of Campylobacter decline on poultry fillets but in general the counts obtained in aerobic versus anaerobic packs were not significantly (P > 0.05) different. CO2 inhibited the growth of TVC, TEC, LAB and Pseudomonas but only at MAP gaseous combinations containing 50-90% CO2 where concentrations of up to 2000 ppm CO2 were recorded in the fillets after 5 days. Under these conditions a shelf-life in excess of 17 days at 2 °C was obtained. Although, dissolved O2, at levels of 33 ppm in 80:20% O2:N2 packs after 3 days, reduced Campylobacter, it also favoured the growth of the other microbes on the chicken. The optimum gaseous mixture for achieving the combined objectives of reducing Campylobacter and extending shelf was therefore 40:30:30 CO2:O2:N2, which achieved a shelf-life in excess of 14 days.

An investigation of broiler caecal Campylobacter counts at first and second thinning.

AIMS: The objectives of this study were the following: (i) to investigate if Campylobacter negative flocks at first thinning remain negative at second thinning; (ii) to determine if the caecal counts in birds infected during first thinning remain lower than in birds that were positive at first thinning; and (iii) to determine if reducing the time between first and second thinning to a maximum of 4 days would reduce both the incidence and prevalence of broiler caecal contamination. METHODS AND RESULTS: Twenty-two flocks were tested at first and second thinning using ISO methodologies. Of the 14 that had a 4-day duration between first and second thinning, nine flocks were Campylobacter negative at first thinning. By second thinning, all 14 flocks were positive and Campylobacter counts ranged from 5·5 to 6·6 log(10) CFU g(-1) regardless of the status at first thinning. The other eight flocks were all Campylobacter positive at first thinning with counts ranging from 0·8 to 6·1 log(10) CFU g(-1) which increased to 5·1 to 6·9 log(10) CFU g(-1) by second thinning (3-10 days). PCR speciation and MLST genotyping suggested the majority of isolates were Camp. jejuni belonging to STs 257, 814, 6763 and 6764. CONCLUSIONS: It was concluded that; thinning introduces Campylobacter into broiler flocks; caecal counts in birds at second thinning are similar, regardless of flock status at first thinning and reducing the time between first and second thinning to a maximum of 4 days is not an effective control strategy. SIGNIFICANCE AND IMPACT OF THE STUDY: This study informs Campylobacter control strategy at primary production, suggesting all post-first thinning broilers should be treated as 'high risk' regardless of Campylobacter status at first thinning or duration between first and second thinning.

Airborne Salmonella and Listeria associated with Irish commercial beef, sheep and pig plants.

Air samples from lairage, hide/fleece pulling or dehairing/scraping, evisceration and chilling areas in commercial beef, sheep and pig plants were examined for Salmonella spp. and Listeria monocytogenes, by impaction or sedimentation onto selective (Brilliant Green Agar, BSA; Listeria Selective Agar, LSA) and non-selective (Plate Count Agar, PCA) media. Both pathogens were frequently detected in all three plants. Improved recoveries were achieved by combining sedimentation, and broth based resuscitation, suggesting cell injury. Salmonella were recovered from all three plants, with the highest counts on BGA in the pig plant. The most common serotypes were S. Typhimurium in the beef/sheep plants and S. Derby in the pig plant. Very low counts of L. monocytogenes (e.g. 2.6CFUm(2)) were detected at hide removal on LSA sedimentation plates in the beef plant. These included serogroup 1/2a-3a and 1/2b-3b-7. Pathogen counts in the three plants were generally very low, suggesting that air is unlikely to be a significant source of carcass or plant surface contamination.

Occurrence, virulence genes and antibiotic resistance of enteropathogenic Escherichia coli (EPEC) from twelve bovine farms in the north-east of Ireland.

Cattle faecal samples (n = 480) were collected from a cluster of 12 farms, and PCR screened for the presence of the intimin gene (eae). Positive samples were cultured, and colonies were examined for the presence of eae and verocytotoxin (vtx) genes. Colonies which were positive for the intimin gene and negative for the verocytotoxin genes were further screened using PCR for a range of virulence factors including bfpA, espA, espB, tir ehxA, toxB, etpD, katP, saa, iha, lpfAO157/OI-141 and lpfAO157/OI-154. Of the 480 faecal samples, 5.8% (28/480) were PCR positive, and one isolate was obtained from each. All 28 isolates obtained were bfpA negative and therefore atypical EPEC (aEPEC). The serotypes detected included O2:H27, O8:H36, O15:H2, O49:H+, O84:H28, O105:H7 and O132:H34 but half of the isolates could not be serogrouped using currently available antisera. Twenty-two (79%) of the isolates carried the tir gene but only 25% were espB positive, and all other virulence genes tested for were scarce or absent. Several isolates showed intermediate resistance to ciprofloxacin, kanamycin, nalidixic acid, minocycline and tetracycline; full resistance to nalidixic acid or tetracycline with one isolate (O-:H8) displaying resistance to aminoglycosides (kanamycin and streptomycin), quinolones (nalidixic acid) and sulphonamides. This study provides further evidence that cattle are a potential source of aEPEC and add to the very limited data currently available on virulence genes and antibiotic resistance in this pathogenic E. coli group in animals.

An investigation of the immediate and storage effects of chemical treatments on Campylobacter and sensory characteristics of poultry meat.

Campylobacteriosis is the most common foodborne bacterial infection in developed countries and many cases are associated with poultry. This study investigated the immediate and storage effect of dipping inoculated poultry skin samples in trisodium phosphate (TSP, 10 & 14%, w/v), lactic acid (LA, 1 & 5%, v/v), citric acid (CA, 1 & 5%, w/v), peroxyacids (POA, 100 & 200 ppm) and acidified sodium chlorite (ASC, 500 & 1200 ppm). Spray application was also tested using the higher concentrations in the laboratory. In a broiler processing plant the efficacy of using TSP (14%) and CA (5%) applied by immersion and spray was investigated using naturally contaminated carcasses and the effect of these treatments on the sensory attributes of a skin-on (drumstick) and skin-off (fillet) raw and cooked product was assessed using descriptive sensory analysis. In the laboratory, immersion in TSP (14%), LA (5%), CA (5%) and ASC (1200 ppm) significantly (P<0.05) reduced the Campylobacter counts and a 2.5 to 3 log10 cfu/cm(2) reduction was observed within the shelf-life (3-5 days) of poultry meat. Spraying was ineffective even after storage. In the broiler processing plant, immersion in TSP (14%) or CA (5%) achieved Campylobacter reductions of 2.49 and 1.44 log10 cfu/cm(2), respectively. There were no significant differences between the treatments for any of the attributes measured in either raw or cooked drumsticks. The 'colour' of raw chicken fillets treated with both TSP (14%, w/v) and CA (5%, w/v) was significantly (P≤0.05) lighter than that of control samples. The 'intensity of chicken odour' and the perception of 'salt' in cooked chicken fillets treated with CA (5%, w/v) were also significantly (P≤0.05) higher than that of either control or TSP (14%, w/v) treated samples. It was concluded that TSP (14%) or CA (5%) could be applied to significantly reduce Campylobacter contamination of broilers without adversely affecting the sensory quality of the product.

The effect of urea and ammonia treatments on the survival of Salmonella spp. and Yersinia enterocolitica in pig slurry.

AIMS: The objective of this study was to investigate the survival of Salmonella and Yersinia enterocolitica strains in pig slurry and evaluate urea and ammonia as disinfection strategies. METHODS AND RESULTS: Salmonella Anatum, Salmonella Derby, Salmonella Typhimurium DT19 and Y. enterocolitica bioserotypes 4, O:3, 2, O:5,27 and 1A, O:6,30 were selectively marked by insertion of the plasmid, pGLO encoding for green fluorescent protein and for ampicillin resistance. Strain cocktails were inoculated into fresh pig slurry (control), slurry treated with urea [final concentration 2% w/w, (0.33 mol l(-1) )] and slurry treated with ammonia [final concentration 0.5% w/w, (0.3 mol l(-1) )] and stored at 4, 14 and 25°C. Bacterial counts were determined at regular intervals on xylose lysine deoxycholate agar (XLD), and XLD supplemented with ampicillin (0.1 mg ml(-1) ) and arabinose (0.6 mg ml(-1) ) for Salmonella and cefsulodin-irgasan-novobiocin agar (CIN) and CIN supplemented with ampicillin and arabinose for Y. enterocolitica. The pH of the control-, urea- and ammonia-treated samples ranged from 7.1 to 7.7, 8.8 to 8.9 and 8.0 to 8.3, respectively. Salmonella D(4) values ranged from 2.71 to 21.29 days, D(14) values from 2.72 to 11.62 days and D(25) values from 1.76 to 6.85 days. The equivalent D values ranges for the Y. enterocolitica strains were 3.7-19.23, 1.8-16.67 and 1.63-7.09 days, respectively. Treatment significantly (P < 0.01) affected D values with control > ammonia > urea, as did incubation temperature; 4 > 14 > 25°C. CONCLUSIONS: Urea and to a lesser extent ammonia may be used to disinfect Salmonella- and/or Y. enterocolitica-contaminated pig slurry, decreasing the storage time required while increasing its fertilizer value. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents data supporting the treatment of pig slurry to kill important zoonotic agents, thereby reducing environmental contamination, cross-infection of other animals and decreasing zoonotic disease in the food chain.

Characterization of a potentially novel 'blown pack' spoilage bacterium isolated from bovine hide.

AIMS: To characterize a psychrotrophic bacterium, designated TC1, previously isolated from a cattle hide in Ireland, and to investigate the ability of this strain to cause 'blown pack' spoilage (BPS) of vacuum-packaged beef primals. METHODS AND RESULTS: TC1 was characterized using a combination of phenotypic, chemotaxonomic and genotypic analyses and was assessed for its ability to spoil vacuum-packaged beef at refrigerated temperatures. TC1 was Gram-positive and formed elliptical subterminal endospores. The strain was able to grow between 0 and 33 °C, with optimal growth between 23 and 24 °C. TC1 could be differentiated from its phylogenetically closest neighbour (Clostridium lituseburense DSM 797(T)) by 16S rRNA gene sequencing, pulsed-field gel electrophoresis and cellular fatty acid composition. TC1 spoiled (BPS) beef within 42 days when inoculated in cold-stored (1 °C) vacuum-packed beef. CONCLUSIONS: The phenotypic, chemotaxonomic and genotypic characterization indicated that TC1 may represent a potentially novel, cold-tolerant, gas-producing bacterium of considerable economic significance to the beef industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports and characterizes an emerging BPS bacterium, which should be considered in future activities designed to minimize the psychrophilic and psychrotrophic spoilage of vacuum-packaged beef.

Serotypes and virulence profiles of atypical enteropathogenic Escherichia coli (EPEC) isolated from bovine farms and abattoirs.

AIMS: The objective of this study was to examine the prevalence of enteropathogenic Escherichia coli (EPEC) on beef and dairy farms and in beef abattoirs and to characterize the isolates in terms of serogroup and virulence markers. METHODS AND RESULTS: Bovine faecal samples (n = 1200), farm soil samples (n = 600), hide samples (n = 450) and carcass samples (n = 450) were collected from 20 farms and three abattoirs throughout Ireland over a 12-month period. After selective enrichment, samples testing positive for the intimin gene (eae) using PCR screening were cultured, and colonies were examined for the presence of the eae, vt(1) and vt(2) genes. Colonies that were positive for the intimin gene and negative for the verotoxin genes were further screened using PCR for a range of virulence factors including tir, espA, espB katP, espP, etpD, saa, sab, toxB, iha, lpfA(O157/OI-141) , lpfA(O113) and lpfA(O157/OI-154) . PCR screening was also used to screen for variations in the intimin gene (eae). Of the 2700 source samples analysed, 3.9% (47 of 1200) of faecal, 2% (12 of 600) of soil, 6.4% (29 of 450) of hide and 0.7% (3 of 450) of carcass samples were PCR positive (for the presence of the eae gene). All 140 isolates obtained were atypical EPEC (aEPEC), while θ and ß intimin types were common. The virulence factors hlyA, tir, lpfA (O113) , lpfA (O157/OI-154) , and iha were frequently detected, while lpfA(O157/OI-141) , saa, espA, espB and toxB were also present but to a lesser extent. CONCLUSIONS: It was concluded that cattle are a source of aEPEC, many of which have the virulence machinery necessary to be pathogenic to humans. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest the need for increased research on aEPEC with particular emphasis on food safety and public health risk.

Comparison of aerial counts at different sites in beef and sheep abattoirs and the relationship between aerial and beef carcass contamination.

The study examined and compared levels of aerial contamination in commercial beef and sheep plants at four sites, i.e. lairage, hide/fleece pulling, evisceration and chilling. Aerial contamination was determined by impaction and sedimentation onto Plate Count Agar to enumerate Total Viable Counts, MacConkey Agar to enumerate coliforms and Violate Red Bile Glucose Agar to enumerate Enterobacteriaceae. AS I cannot see any difference in the text here - I am not sure what the change is?. The levels of aerial contamination were similar at equivalent sites in beef and sheep plants, irrespective of the sampling method or the type of organisms recovered. Mean log counts recovered on each medium in the chillers were generally significantly lower (P < .05) than the corresponding mean log numbers recovered at the other three sites. The relationship between impaction (air) and sedimentation (surface) counts could be described by the surface to air ratio (SAR) which in this study had an R(2) of 0.77. Further studies in an experimental plant compared counts recovered from the neck of beef carcasses with aerial counts determined by impaction and sedimentation onto agar and irradiated meat pieces. A relationship between counts on beef carcasses and in the air could not be established, irrespective of the method used to compare counts.

The prevalence, distribution and characterization of Shiga toxin-producing Escherichia coli (STEC) serotypes and virulotypes from a cluster of bovine farms.

AIMS: To assess the prevalence of Shiga toxin-producing Escherichia coli (STEC) on a cluster of twelve beef farms in the north-east of Ireland. METHODS AND RESULTS: Samples were screened for stx1 and stx2 using PCR. Positive samples were enriched in mTSB and STEC O157 isolated using immunomagnetic separation. Enrichment cultures were plated onto TBX agar to isolate non-O157 STEC. All isolates were serotyped and examined for a range of virulence genes and their antibiotic resistance phenotype determined. Eighty-four isolates of 33 different serotypes were cultured from the 13·7% of samples that were stx positive. The most prevalent serotype was O157:H7, the most common Shiga toxin was stx(2) , and a variety of virulence factor combinations was observed. O-:H-, O26:H11, O76:H34, O157:H7, O157:H16 and OX18:H+ also carried eaeA and hlyA genes. Twenty-nine per cent of strains were resistant to at least one antibiotic, 48% of which had multiple drug resistance (MDR) with O2:H32 displaying resistance to five antibiotics. CONCLUSIONS: The ubiquitous nature of STEC on beef farms, the detection of stx(+) eaeA(+) hlyA(+) in the serotypes O-:H-, O157:H16 and OX18:H+ in addition to O157:H7 and O26:H11 and the widespread distribution of antibiotic resistance are of public health concern as new virulent STEC strains are emerging. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found no relationship between serotype and antibiotic resistance, therefore negating efforts to isolate serotypes using specific antibiotic supplemented media. The data presented provide further evidence of the emergence of new STEC virulotypes of potential public health significance.

A preliminary study of Salmonella, verocytotoxigenic Escherichia coli/Escherichia coli O157 and Campylobacter on four mixed farms.

The aims of this study were to investigate the incidence of Salmonella, verocytotoxigenic Escherichia coli (VTEC)/Escherichia coli O157 and Campylobacter on four mixed farms and to characterize the isolates in terms of a range of virulence factors. Eighty-nine composite (five different samples from the same animal species combined) faecal [cattle (24), pigs (14), sheep (4), poultry (4), horses (7), deer (4), dogs (9), rodents (2) and wild birds (20)] samples, 16 composite soil samples plus 35 individual water samples were screened using culture-based, immunomagnetic separation and molecular methods. Salmonella was detected in bovine faeces, cattle and poultry house water. Salmonella serotypes/phage types included Dublin, Kiel and Typhimurium DT193, and most isolates were spvC, invA and rck positive. The pefA and rck genes were found exclusively in the non-Typhimurium strains, while Salmonella Dublin and Salmonella Kiel strains carried Salmonella genomic island I marker(s). VTEC/E. coli O157 were found in deer and dog faeces only. The E. coli O157 isolate was an enteroinvasive E. coli, while the VTEC isolate was untypable but carried the vt1, eaeA, hlyA, tir and eptD genes. This article reports the first confirmed carriage of E. coli O157 in Irish deer. Campylobacter species were not detected over the course of this study. It was concluded that [1] Salmonella, VTEC and Campylobacter have low (<5%) prevalence or are absent on the farms in this study; [2] water was an important source of bacterial pathogens; [3] both dogs and deer may act as a source of pathogenic E. coli and [4] key virulence and resistance determinants are widespread in farm Salmonella strains. This study highlights the need to control water as a source of pathogens and suggests that the domestic pets and deer should be considered in any farm risk assessment.

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil.

AIMS: This study estimated the incidence of non-O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non-O157 VTEC in clay and sandy loam soils. METHODS AND RESULTS: Twenty farms were tested over a 12-month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR-positive samples were plated onto Chromocult tryptone bile X-glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non-O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non-O157 serotypes had highest D-values in the sandy loam soil with D-values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60-48·25 days. CONCLUSIONS: This study shows that non-O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first such study of non-O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non-O157 VTEC should be seen as an emerging risk to be controlled within the food chain.

Diversity of culturable psychrophilic and psychrotrophic anaerobic bacteria isolated from beef abattoirs and their environments.

This study identified 431 psychrophilic or psychrotrophic isolates from commercial Irish beef abattoir environments and "blown packs" of vacuum-packed beef, using PCR and 16S rRNA sequencing, and estimated their intraspecies genetic diversity using restriction fragment length polymorphism (RFLP) analysis and spacer region PCR (SR-PCR). Twenty-five species were identified in the 431 isolates, with the most frequently recovered species being Clostridium gasigenes (n=315), Clostridium estertheticum (n=17), and a potentially novel species designated strain TC1 (n=52). These species were previously found to be associated with a particular type of spoilage known as blown-pack spoilage (BPS), which occurs in chilled-stored (i.e., -1.5°C to 4°C) vacuum-packaged meat within 2 to 4 weeks and involves the production of large volumes of gas. Overall, the study demonstrates the considerable and not previously reported diversity of the anaerobic microflora in abattoirs and the presence of a wide range of organisms capable of causing BPS at chilled temperatures.

The effect of bovine diet on Salmonella survival in synthetic abomasal fluid.

AIMS: To investigate the effect of diet on the survival of Salmonella in the bovine abomasum. METHODS AND RESULTS: Five fistulated cows were randomly assigned to one of five diets denoted as: (i) 100% grass, (ii) grass + 5·3 kg DM concentrate, (iii) 100% grass silage, (iv) 100% hay and (v) maize/grass silage plus concentrates. Rumen fluid was harvested from each dietary treatment and inoculated with nonacid (NA) and acid-adapted (AA) 5-strain Salmonella cocktails. After 24-h incubation period, Salmonella were acid challenged to synthetic abomasum fluid (SAF, pH 2·5) for 5 h to determine their resistance to low pH. The study found that the volatile fatty acids composition and the pH profile of bovine rumen fluid were significantly altered (P <0·05) by some of the dietary treatments but not others. Regression analysis found that significantly higher numbers of acid-adapted Salmonella survived in SAF after incubation in rumen fluid from diets 1, 2 and 4, but fewer significant differences were found between diets for nonacid-adapted Salmonella. The results suggest that the acid-adapted cells were subjected to a higher level of cell injury than the nonadapted cells. CONCLUSIONS: Pre-incubation in rumen fluid did influence the resistance of nonacid and acid-adapted Salmonella to SAF but it was dependant on the dietary treatment fed to the cows. SIGNIFICANCE AND IMPACT OF THE STUDY: This study examined the use of diet, as a modulating factor to limit the bovine excretion of Salmonella with a view to providing a scientific basis for the design of dietary management controls in the future.

The effect of storage temperature and inoculum level on the time of onset of 'blown pack' spoilage.

AIMS: To examine the effect of storage temperature and inoculum level on the time of onset of 'blown pack' spoilage (BPS) caused by psychrotolerant bacteria in vacuum-packed (VP) meats. METHODS AND RESULTS: Gas-producing species and strains (n = 11), recovered in our laboratory or reported as associated with BPS, were inoculated onto beef or lamb meat pieces at final levels of <10, 10, 10(2) and 10(3) CFU cm(-2), VP and stored at -1.5, 1 or 4 degrees C. Six strains produced observable amounts of gas within 42 days and a further four strains produced gas within 100 days. BPS was observed earliest in VP meats inoculated with Clostridium estertheticum ssp. estertheticum at all inoculum levels/storage temperature combinations examined. Storage temperature and inoculum level significantly affected (P < 0.001 and P < 0.05 respectively) the onset of BPS in all cases. CONCLUSIONS: Controlling contamination levels and lowering the storage temperature delay the onset of BPS. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the positive effects of low contamination-low temperature as control interventions preventing/delaying BPS in VP chilled meats and identifies some of the contaminants most likely to cause BPS in chilled stored VP meat products.