Microbiological Quality of Red Meat Offal Produced at Australian Export Establishments.

A national baseline study of offal hygiene was undertaken at 17 Australian export establishments. A total of 1756 samples of different offal types were analysed for aerobic plate count (APC), generic Escherichia coli, and coliform bacteria. Average APC values varied from 1.51 to 5.26 Log10 CFU/g, depending on species and offal type. The average APC on beef, sheep, lamb, and goat offal was 3.25, 3.38, 3.70, and 2.97 Log10 CFU/g, respectively. There is a small but significant difference in APC on offal sampled frozen (3.26 Log10 CFU/g) and offal sampled fresh (3.73 Log10 CFU/g). Escherichia coli prevalence on beef, sheep, lamb, and goat offal was 15.4%, 28.1%, 17.5%, and 39.3%, respectively. The number of E. coli on positive offal samples ranged from 1.42 to 1.82 Log10 CFU/g. While the quality of some offal approach that of muscle meat, the hygienic quality of red meat offal can be understood by considering the anatomical site from which it is harvested, the usual bacterial levels found at that site, the difficulty in hygienically removing the offal from the carcase, the process prior to packing, and the chilling method used.

Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa).

Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory-secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value=0.66) that increased to very good (k-value=0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P<0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from seropositive animals did not detect Trichinella DNA in any mainland animals, but did reveal the presence of a second larvae-positive wild boar on Gabba Island, supporting its utility as an alternative, highly sensitive method in muscle examination. The serology results suggest Australian wildlife may have been exposed to Trichinella parasites. However, because of the possibility of non-specific reactions with other parasitic infections, more work using well-defined cohorts of positive and negative samples is required. Even if the specificity of the ELISAs is proven to be low, their ability to correctly classify the small number of true positive sera in this study indicates utility in screening wild boar populations for reactive sera which can be followed up with additional testing.

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife.

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.

Use of routine beef carcase Escherichia coli monitoring data to investigate the relationship between hygiene status of incoming stock and processing efficacy.

In Australian export-registered abattoirs microbiological monitoring is carried out within the E. coli and Salmonella Monitoring (ESAM) program. During the calendar year 2003, the ESAM database indicated a national prevalence of Escherichia coli of around 3.0% for steers/heifers and 7.1% for cows/bulls. An investigation was carried out to attempt to elucidate why some establishments had E. coli prevalence markedly higher or markedly lower than the national average. The investigation was based on a questionnaire completed by fifteen export establishments which provided data on livestock, processing, operator training and management. The responses were verified by site visits and then evaluated for their relationship with ESAM data on E. coli in two stages. In stage 1, E. coli prevalence for each abattoir was plotted against each variable recorded by the questionnaire; no single variable was a reasonable predictor for prevalence of E. coli on carcases. In stage 2, variables influencing contamination were grouped under two categories: contamination on incoming livestock (Problem variables) together with the ability of the plant's process to deal with such contamination (Process variables). The analysis prompted two main conclusions. Firstly, plants with a large incoming problem with livestock (long haul, high tag score and proportion of cows/bulls slaughtered) plus "poor" processes had higher than average E. coli prevalence. Secondly, plants with hot water decontamination systems had low E. coli prevalence even when there was a substantial incoming problem with livestock, such as a relatively high proportion of cows/bulls.

A comparison of selected methods for measuring the virulence properties of Listeria spp.

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.

Using national microbiological data to set meaningful performance criteria for slaughter and dressing of animals at Australian export abattoirs.

Slaughter establishments in Australia that export meat to the USA are required by the controlling authority, the Australian Quarantine and Inspection Service (AQIS), to test carcases under the Escherichia coli and Salmonella monitoring (ESAM) program and to use statistical process control techniques to ensure meat is produced hygienically. However, analysing the ESAM database for E. coli using standard statistical techniques proved difficult because of inter-plant variability and because the vast majority of results were below the limits of detection. As well, it is likely that, in slaughter and dressing, higher than normal microbiological counts can often be random events, for which there is neither logical explanation nor obvious management reaction. One approach to statistical process control is to set performance criteria so that a high proportion of establishments are likely to pass, while prompting individual plants to improve the process if they cannot meet the criteria. A spreadsheet-based tool was developed in Visual Basic in order to interrogate the ESAM database and to identify those plants with microbiological performance significantly different from the norm. The present paper describes how performance criteria for cattle, sheep, pigs and goats and for sub-categories within a species (e.g. sheep/lambs, cows/bulls) were established.

A study of the prevalence and enumeration of Salmonella enterica in cattle and on carcasses during processing.

Salmonella prevalence and counts were estimated for samples from the oral cavity, hide, rumen, and feces of 100 cattle at slaughter and from the pre- and postchill carcasses of these cattle. Samples were collected from 25 consecutively slaughtered cattle from each of four unrelated groups slaughtered at a single abattoir on different days. Ten additional fecal samples from each group were collected from their respective abattoir holding pens prior to slaughter. The prevalence of Salmonella was estimated using automated immunomagnetic separation, and the counts were estimated using a combination of most probable number (MPN) and automated immunomagnetic separation. A total of 606 samples were collected with Salmonella isolated from 157 (26%), including 29% of oral cavities, 68% of hides, 16% of feces collected after evisceration, 25% of rumen samples, 2% of prechill carcasses, 3% of postchill carcasses, and 48% of feces collected from holding pens. The prevalence and count of Salmonella varied between the different groups of animals tested. The highest count obtained was from a rumen sample (1.1 x 10(4) MPN/g). Other counts were generally low, with a maximum count in feces collected after evisceration and in the abattoir holding pens of 93 and 23 MPN/g, respectively. The highest count on hides, in oral cavities, and on carcasses was 4.8 MPN/cm2, 23 MPN/g, and 0.31 MPN/cm2, respectively. Even though Salmonella was present on the hides and in the rumen and feces of at least one animal from each group of cattle, the processing of animals at this abattoir resulted in few contaminated carcasses, and when contamination occurred, Salmonella was detected at low numbers.

An investigation of Escherichia coli O157 contamination of cattle during slaughter at an abattoir.

The extent of contamination with Escherichia coli O157 was determined for 100 cattle during slaughter. Samples from 25 consecutively slaughtered cattle from four unrelated groups were collected from the oral cavity, hide, rumen, feces after evisceration, and pre- and postchill carcass. Ten random fecal samples were collected from the pen where each group of animals was held at the abattoir. E. coli O157 was detected using automated immunomagnetic separation (AIMS), and cell counts were determined using a combination of most probable number (MPN) and AIMS. E. coli O157 was isolated from 87 (14%) of the 606 samples collected, including 24% of 99 oral cavity samples, 44% of 100 hides, 10% of 68 fecal samples collected postevisceration, 6% of 100 prechill carcass swabs, and 15% of 40 fecal samples collected from holding pens. E. coli O157 was not isolated from rumen or postchill carcass samples. E. coli O157 was isolated from at least one sample from each group of cattle tested, and the prevalence in different groups ranged from less than 1 to 41%. The numbers of E. coli O157 differed among the animals groups. The group which contained the highest fecal (7.5 x 10(5) MPN/g) and hide (22 MPN/cm2) counts in any individual animal was the only group in which E. coli O157 was isolated from carcasses, suggesting a link between the numbers of E. coli O157 present and the risk of carcass contamination. Processing practices at this abattoir were adequate for minimizing contamination of carcasses, even when animals were heavily contaminated with E. coli O157.

Isolation and characterisation of Arcobacter butzleri from meat.

A survey was conducted to determine the prevalence of Arcobacter in ground chicken, pork, beef and lamb meats. Meat samples were enriched in Arcobacter broth (AB) containing cefoperazone, amphotericin and teicoplanin (CAT) supplement. Samples were screened for the presence of Arcobacter spp. using a multiplex polymerase chain reaction (PCR) followed by isolation on blood and selective agar. Arcobacter butzleri was the only species of Arcobacter isolated from 35% of 88 samples of ground meats. A. butzleri was more frequently isolated from poultry (73%) than pork (29%), beef (22%) or lamb (15%) samples. No significant differences were found in the isolation rates and from the different regions sampled. Isolates were characterised by pulsed-field gel electrophoresis (PFGE) using SacII, EagI and SmaI restriction endonucleases. A number of isolates with indistinguishable PFGE fingerprints were found to be epidemiologically related, which may indicate cross-contamination of common types of Arcobacter from different meat species or between meat species. The public health significance of Arcobacter in ground meat needs to be determined.

A probabilistic analysis of Clostridium perfringens growth during food service operations.

The purpose of this study was threefold: first, the study was designed to illustrate the use of data and information collected in food safety surveys in a quantitative risk assessment. In this case, the focus was on the food service industry; however, similar data from other parts of the food chain could be similarly incorporated. The second objective was to quantitatively describe and better understand the role that the food service industry plays in the safety of food. The third objective was to illustrate the additional decision-making information that is available when uncertainty and variability are incorporated into the modelling of systems.

Aerobio Growth of Listeria monocytogenes on Beef Lean and Fatty Tissue: Equations Describing the Effects of Temperature and pH.

The aerobio growth rate and the duration of the lag period were determined for Listeria monocytogenes strain Murray B growing on ground beef lean and on pieces of fatty tissue. The organism grew at 0°C on lean tissue at pH ≥ 6 and on fatty tissue. It failed to grow at 0°C on lean at pH 5.6 but did grow at 2.5°C. The effect of temperature, between 0 and 30°C, on the growth rate on fatty tissue can be described by a modified Arrhenius equation Ln (gen/h) = -205.73 + 1.2939 × 105/K -2.0298 × 107/K2, where K = °Kelvin. This equation accounted for 99.7% of the variance. The combined effect of temperature and pH on the growth rate on beef lean was described by Ln (gen/h) = - 232.64 + 1.4041 × 105/K - 2.1908 × 107K2 + 1.1586 × 102/pH - 4.0952 × 102/pH2 (variance accounted for 99.5%). For lean at about pH 5.5-5.6, this equation applied between about 2.5 and 35°C; for lean of pH 6-7, it applied between about 0 and 35°C. Though the lag period increased with decrease in temperature and pH, measured lag times were more variable than generation times, and the goodness of fit of modified Arrhenius equations to lag times was relatively poor (variance accounted for 83-92%).

Occurrence, Numbers, and Growth of Listeria monocytogenes on some Vacuum-Packaged Processed Meats.

Listeriae were detected on 93 of 175 samples of vacuum-packed processed meats obtained from retail stores. More than 1000 colony forming units of listeriae per g were found on seven of 130 samples in which the numbers of listeriae were estimated. When sliced corned-beef and ham, from four manufacturers, were inoculated with Listeria monocytogenes and vacuum-packed, the growth rate of the organism varied with the composition of the product. High residual nitrite or lowered aw reduced growth, particularly when products were stored at 0 to 5°C. As the storage temperature increased from 0 to 15°C, the growth rate of L. monocytogenes increased more rapidly than that of the other flora (lactic acid bacteria and Brochothrix thermosphacta ). Growth rates on inoculated packs were similar to rates observed for packs contaminated with L. monocytogenes during commercial production.

Detection of Listeria spp. in Meat and Environmental Samples by an Enzyme-linked Immunosorbent Assay (ELISA).

An ELISA kit (TECRA™) for the detection of Listeria spp. was evaluated for its ability to detect these organisms in naturally contaminated meat and in environmental samples from meat processing plants. Of the 170 samples examined, Listeria monocytogenes and/or L. innocua were detected in 74 by enrichment and selective plating. Testing of the enrichment broths with the ELISA kit detected 72 of these positive samples and gave 2 false-negative and 2 false-positive reactions.

Growth of Listeria monocytogenes on Vacuum-packaged Beef.

Pieces of beef striploin (400 g) were inoculated with Listeria monocytogenes strain Murray B, vacuum packaged, and stored at either 0°C or 5.3°C. Growth of the organism on the beef depended on the temperature of storage, the pH of the lean, and the type of tissue. Growth was more rapid at 5.3°C than at 0°C, and faster on striploins of high pH (6.0-6.1) than on striploins of low pH (5.5-5.7). During storage, the population of L. monocytogenes was higher on fatty tissue than on lean principally because growth occurred earlier on the fat. When low pH striploins were held at 5.3°C, listeria grew from an initial count of 2-5×103 CFU/cm2 to 3×107 CFU/cm2 in 16 d on the fat, and in 20 d, to 106 CFU/cm2 on the lean and to 5×107 CFU/ml in the purge fluid. After storage at 0°C for 76 d, the populations reached were 106 CFU/cm2 on the fat, 104 CFU/cm2 on the lean, and 3×105 CFU/ml in the purge fluid. When high pH striploins were held at 0°C for 10 weeks, listeria grew from an initial population of 150-400 CFU/cm2 to just over 106 CFU/cm2 on the fat, 2×105 CFU/cm2 on the lean, and 4×106 CFU/ml in purge fluid.