Survival of Escherichia coli O157:H7 during the manufacture and ripening of Cacioricotta goat cheese.

The aim of this study was to assess the growth and survival of Escherichia coli O157:H7 during the manufacturing and ripening of Cacioricotta goat cheese. Goat milk was artificially contaminated with E. coli O157:H7 and the bacterial load was monitored from production up to 90 days of ripening. Goat milk was inoculated with 102 cfu ml-1 of E. coli O157:H7 and the bacterial count of the curd at time zero was 2.31 log10 cfu g-1. During the first day of ripening, the bacterial load has increased to 5.73 log10 cfu g-1 to more than 6.20 log10 cfu g-1 during the first week. The bacterial load remained constant up to 28 days and then slightly decreased until the end of ripening, with values of aw and pH of 0.88 and 5.41 respectively. The results of this study highlighted that E. coli O157:H7 is able to survive the manufacturing process and they suggest that the 90-day period of ripening alone is insufficient to remove E. coli O157:H7 in contaminated Cacioricotta goat cheese. Moreover, these results support the assumption that the presence of a low contamination of milk with E. coli O157:H7 could represent a potential source of infection and a threat to consumers.

Microbiological survey of raw and ready-to-eat leafy green vegetables marketed in Italy.

The presence of foodborne pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7, thermotolerant Campylobacter, Yersinia enterocolitica and norovirus) in fresh leafy (FL) and ready-to-eat (RTE) vegetable products, sampled at random on the Italian market, was investigated to evaluate the level of risk to consumers. Nine regional laboratories, representing 18 of the 20 regions of Italy and in which 97.7% of the country's population resides, were involved in this study. All laboratories used the same sampling procedures and analytical methods. The vegetable samples were screened using validated real-time PCR (RT-PCR) methods and standardized reference ISO culturing methods. The results show that 3.7% of 1372 fresh leafy vegetable products and 1.8% of 1160 "fresh-cut" or "ready-to-eat" (RTE) vegetable retailed in supermarkets or farm markets, were contaminated with one or more foodborne pathogens harmful to human health.

Low prevalence of Listeria monocytogenes in foods from Italy.

Listeria monocytogenes is an important foodborne pathogen that causes gastrointestinal disorders, and, especially in immunocompromised people, serious extraintestinal diseases, such as septicemia and meningitis, as well as abortion in pregnant women. Many foods, from both plant and animal origin, have been involved in listeriosis outbreaks. This article reports the results of a 12-year survey (1993 through 2004) on the presence of L. monocytogenes in several kinds of food marketed in Italy. Of 5,788 analyzed samples, 121 (2.1%) were contaminated with L. monocytogenes. The highest prevalence was found in smoked salmon (10.6%) and in poultry meat samples (8.5%) and the lowest in red meat (0.3%). L. monocytogenes was not found in 154 samples of fresh seafood products. Fifty-two isolates were also serotyped by the agglutination method. The most common serotypes detected in the 52 strains tested were 1/2a (36.5%), followed by 1/2c (32.8%), 1/2b (13.5%), 4b (11.5%), 3a (3.8%), and 3b (1.9%). The results of the present study showed low levels of L. monocytogenes in the analyzed samples. A total of 61.5% of the 52 L. monocytogenes strains analyzed belonged to serotypes 1/2a, 1/2b, and 4b, namely the serovars that are most commonly involved in extraintestinal human listeriosis outbreaks. In the ready-to-eat samples, these three serotypes were 40.0% (1/2a), 17.1% (1/2b), and 14.3% (4b). This finding highlights the need to implement strict hygienic measures during the production, distribution, and sale of foods to reduce the risk of foodborne listeriosis in humans to an acceptable level.

Comparison of different biochemical and molecular methods for the identification of Vibrio parahaemolyticus.

AIMS: Multicentre evaluation of biochemical and molecular methods for the identification of Vibrio parahaemolyticus. METHODS AND RESULTS: For the biochemical identification methods, API 20E and API 20NE and Alsina's scheme were evaluated in intra- and interlaboratory tests in order to determine the accuracy and concordance of each method. Both in intra- and interlaboratory tests, the Alsina's scheme showed the highest sensitivity (86% of correct identifications in the interlaboratory test). False-positive results were obtained by all methods (specificity was 95% for API 20E, 73% for API 20NE and 84% for Alsina's scheme) and concordance varied from 65% of API 20NE to 84% of API 20E. For the molecular identifications, polymerase chain reaction (PCR) for the detection of toxR gene, tl gene and pR72H fragment were tested on 30 strains by two laboratories. The PCR for toxR showed the highest inclusivity (96%), exclusivity (100%) and concordance (97%). CONCLUSIONS: Among the biochemical identification methods tested, the Alsina's scheme gave more reliable results; however, in order to avoid false-positive results, all the biochemical identifications should be confirmed by means of molecular methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Availability of an efficient identification method of Vibrio parahaemolyticus to use in official control of fisheries products.

Verocytotoxin-producing Escherichia coli O157 in minced beef and dairy products in Italy.

A total of 3879 samples of foodstuffs were examined for the presence of Verocytotoxin-producing Escherichia coli O157 (VTEC O157). The survey was conducted by 9 of the 10 Italian Veterinary Public Health Laboratories. Samples were collected between May 2000 and September 2001 in 14 regions and comprised 931 minced beef specimens and 2948 dairy products (DP) with less than 60 days of ripening. The DP included 657 pasteurised and 811 unpasteurised bovine DP, 477 pasteurised and 502 unpasteurised ovine DP, and 501 water-buffalo's milk mozzarella cheese. Samples were collected at retail level, from plants processing minced beef and dairy plants and from farms directly manufacturing cheeses. All the samples were tested using a sensitive procedure based on ISO/DIS 16654:1999 (later ISO 16654:2001), which includes an immunomagnetic separation step. A preliminary inter-laboratory trial was organised with artificially contaminated samples to assess the ability of all the participating laboratories to isolate E. coli O157 by the established procedure. VTEC O157 was isolated from four (0.43%) of the minced beef samples, collected in four different regions and during different months, but was not detected in any of the dairy products. E. coli O157 VT-eae+ was isolated from one raw cow's milk cheese. This survey provided national data on the presence of VTEC O157 in foodstuffs, demonstrating a low prevalence of the organism. The survey also encouraged updating of knowledge and procedures on VTEC O157 in laboratories with official responsibility for microbiological testing of foods of animal origin.

Fate of Escherichia coli O157:H7 during the manufacture of Mozzarella cheese.

AIMS: The fate of Escherichia coli O157:H7 was investigated during the manufacture of Mozzarella cheese. METHODS AND RESULTS: The Mozzarella cheese was made from unpasteurized milk which was inoculated to contain ca 10(5) cfu ml(-1)E. coli O157:H7. Two different heating temperatures (70 and 80 degrees C), commonly used during curd stretching, were investigated to determine their effects on the viability of E. coli O157:H7 in Mozzarella cheese. Stretching at 80 degrees C for 5 min resulted in the loss of culturability of E. coli O157:H7 strains, whereas stretching at 70 degrees C reduced the number of culturable E. coli O157:H7 by a factor of 10. CONCLUSIONS: The results show that stretching curd at 80 degrees C for 5 min is effective in controlling E. coli O157:H7 during the production of Mozzarella cheese. Brining and storage at 4 degrees C for 12 h was less effective than the stretching. SIGNIFICANCE AND IMPACT OF THE STUDY: Mozzarella cheese should be free of E. coli O157:H7 only if temperatures higher than or equal to 80 degrees C are used during milk processing.

Drug resistances in salmonella isolates from animal foods, italy 1998-2000.

We investigated the distribution of serotypes and patterns of drug resistance of 206 strains of salmonella isolated in southern Italy in the years 1998-2000 from raw food of animal origin, faeces of food animals and animal feed. To improve knowledge of mobile genetic elements carrying the resistance genes, some molecular features were also investigated within isolates resistant to three or more antibiotics. A high proportion of isolates, 52.2% and 37.7%, respectively, belonging to both Typhimurium and other serotypes of animal origin, proved to be multidrug resistant. The DT104 complex specific multidrug pattern of resistance was quite infrequent among isolates other than Typhimurium, but resistances to nalidixic acid and kanamycin were more frequent within these last ones (36.9% vs. 11.4% and 56.5% vs. 2.2%, respectively). Class I integrons were detected in isolates of Typhimurium and seven different serotypes. The relevance of food animal environment as a drug resistance reservoir and animal food as a potential resistance gene vehicle between the farm and human ecological niches is confirmed by our findings.

Detection and characterization of vancomycin-resistant enterococci in farm animals and raw meat products in Italy.

The emergence of vancomycin-resistant enterococci (VRE) in Europe has been ascribed to the long-time use of the glycopeptide antibiotic avoparcin as feed additive in food animals, until its ban in 1997 in EU. The pres- ence of VRE in food of animal origin is believed to represent a potential risk for the consumer. We studied the fecal carriage of VRE in broiler chickens and slaughter pigs in Italy before the avoparcin ban and eval- uated the impact of avoparcin withdrawal on the presence of VRE in raw meat products. Broilers and pigs were both found to be frequently colonized by VRE, as 36% and 24.6% of the flocks or the herds, respec- tively, were positive. Molecular typing of VRE strains by PFGE showed that animals housed in different pens within the same farm were colonized by clonally related strains. After the avoparcin ban, a decrease in the rate of VRE contamination in meat products was observed. Such a decrease was statistically significant in poultry (from 18.8% to 9.6%) but not in pork products (from 9.7% to 6.9%). The majority of VRE from all sources carried the vanA resistance gene and included Enterococcus faecium, E. faecalis, E. hirae, E. durans, and E. gallinarum. None of the strains carried the vanB gene, whereas constitutively resistant vanC-positive strains were frequently found. Our results show that avoparcin withdrawal has been successful in reducing VRE contamination in meat products. However, this measure needs to be complemented by a prudent use of glycopeptide antibiotics in human medicine.

Predilection sites of Trichinella spiralis larvae in naturally infected horses.

A total of 120 muscle tissues from three horses naturally infected with Trichinella spiralis were examined. The head was the most infected site. In particular, the muscles harbouring the highest number of larvae were: musculus buccinator (12, 411 and 1183 larvae g-1), the tongue (11, 615 and 1749 larvae g-1), m. levator labii maxillaris (17,582 and 1676 larvae g-1), and the masseter (4.9, 289 and 821 larvae g-1). Compared with the diaphragm, the number of larvae per gram was from 3.5 to 6.8 times higher in the tongue, from 3.5 to 6.5 higher in m. levator labii maxillaris, and from 2.5 to 4.6 higher in m. buccinator. Of the examined muscles, the diaphragm had from the 6th to the 15th highest level of infection (3.1, 166 and 256 larvae g-1). Published data from experimentally infected horses confirm these results, suggesting that efforts to detect predilection sites should focus on the head muscles.

Fate of Escherichia coli O157:H7 in unpasteurized milk stored at 8 degrees C.

Escherichia coli O157:H7 and verocytotoxins were not found in any of 100 unpasteurized milk samples obtained from the bulk tanks of eight dairy farms located in the Puglia and Basilicata areas. Seven E. coli O157:H7 (EHEC) strains were inoculated separately into raw milk samples and then examined periodically to determine the fate of EHEC as influenced by the storage temperature (8 degrees C) and time. There was essentially no change in the viable population of three EHEC strains for up to 14 d. The remaining four strains showed an increase in population from < 2 log to 3 log cfu ml-1 in a time period of between 9 and 17 d. The results indicate good survival or even multiplication of E. coli O157:H7 in raw milk when stored at 8 degrees C and reaffirm the need for pasteurization and holding the milk at < or = 5 degrees C.

Detection of Trichinella spiralis in a horse during routine examination in Italy.

Routine examination for Trichinella infection by artificial digestion of 5-g samples of muscle tissue revealed the presence of muscle larvae in one out of 28 horses imported from Romania to an abattoir in Italy. The parasite, identified as Trichinella spiralis by the polymerase chain reaction, showed a reproductive capacity index of 68 in Swiss mice. Light microscope examination of 200 nurse cell-larva complexes showed that 22% of them were calcified and that the capsules of the non-calcified nurse cells were 17.5-27.5 microns (s = 22.67 microns) thick and had very few cellular infiltrates. The serum samples from the parasitologically positive horse and from three other horses of the same stock, from which Trichinella larvae were not recovered by digestion, showed a low level of positivity as determined by ELISA and Western blot analyses using a crude antigen, whereas negative results were observed in both tests when an excretory-secretory antigen was used. The results, together with data from the literature, suggest that the horse had acquired the infection 8-10 months previously and confirm earlier observation obtained from experimental infections, which showed that muscle worm burden and specific circulating antibodies were lost several months after infection.