Microbial safety of meat in the European Union.

The two most frequently reported zoonotic diseases in humans in the EU in 2005 were Campylobacter and Salmonella infections with incidences of 51.6 and 38.2 cases per 100,000 population, respectively. Reported human infections caused by Yersinia spp., Verocytotoxigenic Escherichia coli, and Listeria monocytogenes had comparably lower incidences of 2.6, 1.2 and 0.3 cases per 100,000 population, respectively. Meat and meat products are important sources for these infections but knowledge on exactly how important they are compared with other types of food, drinking water and environmental exposure is quite limited. Occurrences of zoonotic pathogens in raw meat are variable, although most often are between 1% and 10%, depending on the organism, geographical factors, farming and/or meat production practices, etc. Zoonotic pathogens in meat have to be controlled through a complete, continuous farm-to-fork system. It is of utmost importance to control faecal contamination of carcasses through efficient HACCP-based process hygiene management systems.

Evaluation of a monoclonal antibody able to detect live Listeria monocytogenes and Listeria innocua.

A monoclonal Listeria antibody, designated B4, was evaluated. The ability of the antibody to bind to viable bacteria belonging to Listeria spp. compared to bacteria of the same species killed by heat treatment, acid or base treatment, sanitizers, and irradiation was examined. The antibody was found to react with viable L. monocytogenes and L. innocua, but not with heat-killed (72 degrees C, 5 min) strains of these organisms. When L. monocytogenes and L. innocua were killed by methods other than heat treatment, it was ambiguous whether the antibody detected the organism or not. It was concluded that the B4 antibody has potential to be used in an immuno capture step to capture live L. monocytogenes and L. innocua from foods prior to identification of L. monocytogenes by polymerase chain reaction (PCR).

Variations in virulence between different electrophoretic types of Listeria monocytogenes.

A total of 245 strains of Listeria monocytogenes, representing 33 different electrophoretic types (ETs), were examined quantitatively for haemolytic activity. No significant difference was observed in the mean haemolytic activity between different ETs. Eighty four out of 91 strains examined were found to be virulent for chick embryos. Strains belonging to ET 2 and ET 4 were found to be less virulent than strains of other ETs (P = 0.0447). Furthermore, strains from clinical cases were found to be more virulent (P = 0.0002) than strains from foods (the MTD among clinical strains was 2.46 in mean compared with 3.64 among food isolates). The explanation for this may be that more virulent strains are more prone to cause human infection. It is, however, also possible that strains of L. monocytogenes may become more virulent while multiplying in a living organism compared with multiplying in foods.

Microbiological criteria for Listeria monocytogenes in foods under special consideration of risk assessment approaches.

This paper shortly summarizes data related to risk assessment of Listeria monocytogenes. From available data on risk assessment, it is concluded that the levels of L. monocytogenes consumed is an important factor affecting the incidence of listeriosis. Foods that do not support the growth of L. monocytogenes are unlikely to be a source of listeriosis, whereas foods that support the growth to high levels, should be the target of risk management efforts. Based on current epidemiological information from several countries, a concentration of L. monocytogenes not exceeding 100/g of food at the time of consumption is of low risk to the consumers. In order not to exceed these levels at the point of consumption, lower levels may need to be applied at the port of entry, for those foods in which growth can occur within the shelf life. In order to establish such levels, knowledge of the shelf life and behaviour of L. monocytogenes in the food during prevailing storage and distribution conditions is needed.

Incidence and control of Listeria monocytogenes in foods in Denmark.

The Danish regulatory policy on Listeria monocytogenes in foods is based on the principles of HACCP and was developed using a health risk assessment approach. The Danish policy focuses examinations and criteria for L. monocytogenes in ready-to-eat foods and is based on a combination of inspection and product-testing. Based on current epidemiological information from several countries, a concentration of L. monocytogenes not exceeding 100 cfu/g of food at the time of consumption, seems to be of low risk to the consumers. In Denmark, ready-to-eat foods have been placed into six categories where absence of L. monocytogenes in 25 g is required in foods heat treated in the final package and in heat-treated as well as preserved, non heat-treated foods which can support growth within the shelf life. This level is necessary in foods capable of supporting growth, in order not to exceed 100 L. monocytogenes per g at the point of consumption. In heat-treated and preserved foods, which are not supportive of growth within the shelf-life and for raw, ready to eat foods, a level below 10 L. monocytogenes per g is regarded acceptable. A level between 10 and 100 L. monocytogenes per g is not satisfactory and a level above 100/g is not acceptable. Data on the qualitative and quantitative occurrence of L. monocytogenes in foods in Denmark are presented and discussed. In 1997 and 1998, greater than 15,000 samples from different categories of food were examined (semi-quantitatively) for the presence of L. monocytogenes. A significant difference could be seen in the number of samples containing more than 100 L. monocytogenes per g, between different categories of foods (1997, P = 0.001; 1998, P = 0.016). In 1997, preserved meat products and preserved fish products and to a lesser extent vegetables and meat or vegetable mayonnaise were more likely to contain high numbers (i.e. above 100 cfu/g) of L. monocytogenes than other food categories. In 1998, preserved meat products, but also heat-treated meat products, vegetables and meat or vegetable mayonnaise had the highest frequency of samples with > 100 L. monocytogenes per g. In a survey performed in 1994 and 1995, 1.3% of ready-to-eat food samples (heat-treated meat products, preserved meat and fish products) were found to be contaminated with L. monocytogenes at a level above 100 cfu/g. The samples included in this survey were primarily products produced by authorized companies and were comprised mainly of vacuum packed products or products packed in modified atmosphere and with long shelf lives, typically above several weeks. The corresponding percentages of positive samples primarily processed in the retail outlets (heat-treated meat products, preserved meat and fish products) in 1997 and 1998 were 0.3% and 0.6%, respectively. The results suggest that ready-to-eat meat and fish products with extended shelf-lives produced by authorized companies are more likely to contain high numbers (> 100 cfu/g) of L. monocytogenes than products processed in the retail sector which often have a shorter shelf life.

Multilocus enzyme electrophoresis for characterization of Listeria monocytogenes isolates: results of an international comparative study.

Multilocus enzyme electrophoresis (MEE) is a standard technique that is used to elucidate the epidemiology of a variety of bacterial species. Recently, the method has been employed by several laboratories for investigations of clinical and foodborne isolates of Listeria monocytogenes. To assess the sensitivity and reproducibility of MEE in characterising L. monocytogenes isolates for epidemiological purposes and, ultimately, to agree on a standard protocol, seven laboratories participated in a blinded study of 80 strains. The strain collection included both epidemiologically related and unrelated isolates. Each laboratory used its own protocol for MEE. The number of enzymes that were assayed by the laboratories ranged from 8 to 23, and the total number of identified electrophoretic types (ETs) varied between 14 and 25. Of the II pairs of duplicate strains, the number of pairs recognised as identical by the seven laboratories ranged from 3 to 10 (median = 8). From 10 to 18 (median = 15) of the 22 groups of epidemiological related strains were recognised as homogeneous by the different laboratories. The discriminatory power of the method, calculated using Simpson's index of diversity for 69 strains (80 strains minus the 11 duplicates), ranged from 0.827 to 0.925. This relatively low discriminatory power is a consequence of a somewhat low genetic diversity of L. monocytogenes compared to other bacterial species. Efforts should be pursued to standardise the method in order to improve the intra- and inter-laboratory reproducibility.

Application of multilocus enzyme electrophoresis in studies of the epidemiology of Listeria monocytogenes in Denmark.

A total of 245 strains of Listeria monocytogenes were investigated. These strains were isolated from human and animal cases of listeriosis as well as from different kinds of raw and processed foods. Thirty-three electrophoretic types (ETs) were identified among the 245 strains. The strains investigated included all human clinical strains isolated in Denmark during 1989 and 1990. Seventy-three percent of the strains isolated in this period were assigned to one of only two ETs (ET 1 and ET 4). ET 1, which was found to be the most frequently occurring ET among strains isolated from human clinical cases, was also found to occur rather frequently in animal clinical cases. ET 1 was, however, found only sporadically among strains isolated from foods and food factories. The data indicate that there might be something distinctive about the physiology or ecology of the ET 1 clone which makes it more likely to bring about disease in human beings either because of high pathogenicity or because of a special ability to multiply to infectious doses in processed foods. Another type, designated ET 4, was found to be the next most frequently occurring ET, after ET 1, among human clinical isolates. This could be explained by the fact that ET 4 was found to be the most frequently occurring ET within food isolates.

Comparison of multilocus enzyme electrophoresis (MEE), ribotyping, restriction enzyme analysis (REA) and phage typing for typing of Listeria monocytogenes.

The discriminatory power of four methods for typing of Listeria monocytogenes was compared. The four methods were multilocus enzyme electrophoresis (MEE), ribotyping, restriction enzyme analysis (REA), and a newly developed Danish phage typing system. Ninety-nine human clinical, food and slaughterhouse isolates of Listeria monocytogenes were typed by each method. The most discriminatory single typing method was phage typing with an overall discriminatory index (DI) of 0.88 followed by REA, MEE and ribotyping with DI-values at 0.87, 0.83 and 0.79 respectively. Considering strains from each of the two predominant O-serotypes alone, serotype 1 was best discriminated by the molecular typing methods, in particular REA, which showed a DI of 0.92. The serotype 4 strains were best discriminated by phage typing (DI = 0.78). If two or more typing methods were combined, the combination of REA and MEE were found to be the most discriminatory combination. The DI values were 0.96, 0.74 and 0.90 for serotype 1, 4, and both combined, respectively. Phage typing is a rapid and inexpensive typing method but not as reproducible as the molecular typing methods. It is the most suitable method for mass screening. In situations where results are required to be highly reliable, i.e. when studying the relationships between only a few strains, a single or a combination of molecular typing methods should be used, preferable MEE and REA.

Characterisation of Danish isolates of Listeria monocytogenes by multilocus enzyme electrophoresis.

A total of 84 strains of Listeria monocytogenes were analysed by multilocus enzyme electrophoresis at twelve enzyme loci. Eight enzyme loci were polymorphic with between 2 and 4 alleles per locus. Fourteen electrophoretic types (ETs) were identified. Among 62 human clinical isolates from Denmark, 8 different ETs were defined. Two ETs, designated ET 1 and ET 6, accounted for 77% of the human clinical isolates investigated. These ETs are identical with those responsible for several epidemics in Switzerland and in the United States. Comparison of 58 isolates of L. monocytogenes, typed by MEE, in relation to phage typing showed that phage typing was more discriminatory than MEE. The ability of MEE to distinguish between phage types of Epi-type and other phage types, however, was almost optimal. MEE typed 23 of 24 strains of Epi-type as belonging to ET 1. In contrast ET 1 was not found in 26 strains with phage types other than the Epi-type.

Evaluation of an ELISA Test for Detection of Listeria spp.

An ELISA test and two routine culture procedures have been compared in their ability to detect Listeria spp. in food products and swabs of pig tonsils. The culture procedures used were those recommended by the Food and Drug Administration (FDA) and United States Department of Agriculture (USDA). One hundred samples of minced beef, artificially inoculated with Listeria monocytogenes , together with 149 naturally contaminated samples of minced beef, pig tonsil, pig feed and soft, whitemolded, blue-veined cheese were tested by the three methods. The USDA procedure proved to be the most sensitive detection method when samples of artificially contaminated meat containing less than 3 colony forming units (CFU) of L. monocytogenes per gram were examined. In samples where the L. monocytogenes count was above 3 CFU per gram, the ELISA test and the USDA-detection procedure proved to be equally sensitive. When naturally contaminated samples were examined, the sensitivity of the ELISA test was 92% and the specificity 80%. The detection limit of the ELISA test for demonstration of L. monocytogenes in pure cultures was found to be approximately 106 CFU per ml. The corresponding detection limit of the culture procedure was calculated to be approximately 104 CFU per ml.

The incidence of Listeria spp. in faeces of Danish pigs and in minced pork meat.

One hundred and seventy-two samples of pig faeces, collected at a slaughterhouse, were investigated for Listeria species. Three of the samples (1.7%) contained L. monocytogenes, while L. innocua was demonstrated in four other samples. The 172 samples represented pigs from 72 herds, 14 (19%) of which were SPF herds. Listeria was not demonstrated in any of the samples from the SPF herds representing 24% of all samples investigated. This indicates that the incidence of L. monocytogenes in SPF herds might be very low. In 51 samples of minced pork, Listeria spp. were demonstrated in 32 samples, L. monocytogenes in six samples, L. innocua in 26 samples, and L. seeligeri in one sample. In comparison with similar findings in cattle faeces and minced beef, the conclusion was drawn that faecal contamination may play an important rôle in the dissemination of L. monocytogenes in raw meats.