Evaluation of Mycobacterium avium subsp. paratuberculosis survival during the manufacturing process of Italian raw milk hard cheeses (Parmigiano Reggiano and Grana Padano).

Mycobacterium avium subsp. paratuberculosis (MAP), the agent of paratuberculosis in ruminants, is suspected to be involved in the aetiology of some human diseases. Notably, the consumption of milk and dairy products is considered to be the main route of human exposure to MAP because of its ability to survive during pasteurization and manufacturing processes. The aim of this study was to investigate, through a microbiological challenge test, the survival of MAP during the manufacturing and ripening period of two Italian hard cheeses, Parmigiano Reggiano and Grana Padano, made from raw bovine milk. The challenge test was performed in two different phases: the creaming phase and the manufacturing phase. The creaming phase, which is the first step of cheese production, was reproduced in the laboratory employing raw cow's milk spiked with a MAP reference strain at a final concentration of 5.58 log10 CFU/mL. After the creaming at 18 °C and 27 °C for 12 h, a decrease of 0.80 log10 and 0.77 log10 was observed in partially skimmed milk, respectively. In the second phase, two batches of raw cow's milk (1000 L each) were inoculated with MAP reference and wild strains, respectively. Then, the entire manufacturing process for Parmigiano Reggiano and Grana Padano, both of Protective Designation of Origin (PDO), was reproduced in an experimental cheese factory, starting from a concentration in milk of 5.19 ±â€¯0.01 and 5.28 ±â€¯0.08 log10 CFU/mL of MAP reference and wild strains, respectively. Heating the curd at 53 °C for 20 min did not affect MAP survival, however a significant decrease (p < 0.05) in MAP viability was observed during the moulding phase and after salting in brine, regarding the wild strains and the reference strain, respectively. In addition, a significant decrease was observed during the ripening period, at which time the MAP concentration dropped below the limit of detection from the second and the third month of ripening, for the wild and reference strains, respectively. Taking into account the poor data availability about MAP survival in hard cheeses, this study may improve the knowledge regarding the effect of the cheese manufacturing process on the MAP dynamics, supporting also the safety of traditional raw milk hard cheeses.

Occurrence of hepatitis A and E and norovirus GI and GII in ready-to-eat vegetables in Italy.

Fresh vegetables and their ready-to-eat (RTE) salads have become increasingly recognized as potential vehicles for foodborne diseases. The EU Reg. 1441/2007 establishes microbiological criteria for bacterial pathogens for products placed on the market during their shelf-life (i.e. Salmonella spp., Listeria monocytogenes) for pre-cut fruits and vegetables (RTE) whilst it does not address the problem of contamination by enteric viruses. In this study we investigated the contamination by hepatitis A virus (HAV), hepatitis E virus (HEV) and norovirus (NoV) in 911 ready-to-eat vegetable samples taken from products at retail in Apulia and in Lombardia. The vegetable samples were tested using validated real-time PCR (RT-qPCR) assays, ISO standardized virological methods and ISO culturing methods for bacteriological analysis. The total prevalence of HAV and HEV was 1.9% (18/911) and 0.6% (6/911), respectively. None of the samples analysed in this study was positive for NoV, Salmonella spp. or Listeria monocytogenes. The detection of HAV and HEV in RTE salads highlights a risk to consumers and the need to improve production hygiene. Appropriate implementation of hygiene procedures is required at all the steps of the RTE vegetable production chain and this should include monitoring of emerging viral pathogens.

Paediatric HUS Cases Related to the Consumption of Raw Milk Sold by Vending Machines in Italy: Quantitative Risk Assessment Based on Escherichia coli O157 Official Controls over 7 years.

A quantitative risk assessment (RA) was developed to estimate haemolytic-uremic syndrome (HUS) cases in paediatric population associated with the consumption of raw milk sold in vending machines in Italy. The historical national evolution of raw milk consumption phenomenon since 2008, when consumer interest started to grow, and after 7 years of marketing adjustment, is outlined. Exposure assessment was based on the official Shiga toxin-producing Escherichia coli O157:H7 (STEC) microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2014, microbial growth during storage, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time-temperature field handling conditions detected. In case of boiling milk before consumption, we assumed that the risk of HUS is fixed at zero. The model estimates clearly show that the public health significance of HUS cases due to raw milk STEC contamination depends on the current variability surrounding the risk profile of the food and the consumer behaviour has more impact than milk storage scenario. The estimated HUS cases predicted by our model are roughly in line with the effective STEC O157-associated HUS cases notified in Italy only when the proportion of consumers not boiling milk before consumption is assumed to be 1%. Raw milk consumption remains a source of E. coli O157:H7 for humans, but its overall relevance is likely to have subsided and significant caution should be exerted for temporal, geographical and consumers behaviour analysis. Health education programmes and regulatory actions are required to educate people, primarily children, on other STEC sources.

Short communication: Arcobacter butzleri and Arcobacter cryaerophilus survival and growth in artisanal and industrial ricotta cheese.

Ricotta cheese is a ready-to-eat product with properties (pH >6.0, aw >0.98-0.99) and moisture content (75-80%) that may pose a risk to public health due to postprocess contamination by several bacterial pathogens, including Arcobacters. The objective of the study was to evaluate the behavior of Arcobacter butzleri and Arcobacter cryaerophilus in ricotta cheese during its shelf life assuming postprocessing contamination. Two types of ricotta cheese, artisanal water buffalo (WB) and industrial cow milk ricotta cheese, were experimentally contaminated with A. butzleri and A. cryaerophilus and the count was monitored at 2 different temperatures (6°C and 12°C) during shelf life of 5 d for WB cheese and 22 d for industrial ricotta cheese. In WB ricotta cheese the A. butzleri count remained stable during the 5 d of storage at 6°C, whereas a moderate but significant decrease was observed in A. cryaerophilus count. The counts of both species increased when WB ricotta cheese was stored at 12°C. In industrial ricotta cheese stored at 6°C, a significant reduction was observed both in A. butzleri and A. cryaerophilus counts during the 22-d storage period; at 12°C storage, a count increase was observed for both Arcobacter species up to d 14 of storage after which the log cfu/g count resulted constant until d 22 of storage. The ability of A. butzleri and A. cryaerophilus to survive at 6°C and to grow at 12°C in ricotta cheese has significant food safety implications.

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.

Quantitative risk assessment of verocytotoxin-producing Escherichia coli O157 and Campylobacter jejuni related to consumption of raw milk in a province in Northern Italy.

A quantitative risk assessment was developed to describe the risk of campylobacteriosis and hemolytic uremic syndrome (HUS) linked to consumption of raw milk sold in vending machines in Northern Italy. Exposure assessment considered the microbiological status of dairy farms, expected milk contamination, storage conditions from bulk tank to home storage, microbial growth during storage, destruction experiments, consumption frequency of raw milk, age of consumers, serving size, and consumption preference. The differential risk between milk handled under regulation conditions (4°C throughout all phases) and the worst field handling conditions was considered. The probability of Campylobacter jejuni infection was modeled with a single-hit dose-response beta-Poisson model, whereas for HUS an exponential dose-response model was chosen and two probabilities were used to model the higher susceptibility of children younger than 5 years old. For every 10,000 to 20,000 consumers each year, the models predicted for the best and worst storage conditions, respectively, 2.12 and 1.14 campylobacteriosis cases and 0.02 and 0.09 HUS cases in the 0- to 5-year age group and 0.1 and 0.5 HUS cases in the >5-year age group. The expected pediatric HUS cases do not differ considerably from those reported in Italy by the Minister of Health. The model developed may be a useful tool for extending the assessment of the risk of campylobacteriosis and HUS due to raw milk consumption at the national level in Italy. Considering the epidemiological implications of this study, the risk of illness linked to raw milk consumption should not be ignored and could be reduced by the use of simple measures. Boiling milk before consumption and strict control of temperatures by farmers during raw milk distribution have significant effects on campylobacteriosis and HUS and are essential measures for risk management.

Behaviour of Listeria monocytogenes in packaged water buffalo mozzarella cheese.

AIMS: The aim of the study was to evaluate the behaviour of Listeria monocytogenes in the conditioning liquid of packaged water buffalo mozzarella cheese (WBMC). METHODS AND RESULTS: The conditioning liquid was contaminated with L. monocytogenes, and the contaminated samples were stored at four different storage temperatures: 5 and 10 °C for 22 days; 20 °C for 9 days; 20 °C for 3 days and then at 5 °C for 6 days. The results showed that L. monocytogenes concentration decreased when contaminated samples were stored at 5 °C. When WBMC was stored at 20 °C and at 10 °C, L. monocytogenes started to grow after a lag phase of 3 and 10 days, respectively. When samples were stored at variable temperature conditions, L. monocytogenes numbers showed a lag phase of 5 days. CONCLUSIONS: Use of a conditioning liquid characterized by acidity and a correct storage temperature is able to counteract pathogen replication during shelf life. A high concentration of lactic acid bacteria was associated with effective control of L. monocytogenes but the role of lactic acid bacteria in WBMC conditioning liquid requires further investigation. SIGNIFICANCE AND IMPACT OF THE STUDY: According to European regulations, food producers should be able to justify decision-making on the shelf life assigned to their products, taking into account reasonable storage conditions and use by consumers. The results of the trial yielded information for producers of WBMC and similar cheeses for decision-making on product shelf life.

Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors.

AIMS: To investigate the presence of enteric viruses [hepatitis A (HAV) and norovirus (NoV)] in shellfish harvested from the deltaic area of the Po river in relation to environmental factors. METHODS AND RESULTS: Fortnightly sampling of shellfish was carried out in two lagoon areas (category B production areas) and one sea area (category A). Environmental parameters in the lagoon and hydrometric level of the tributary river were monitored throughout the sampling period. Samples (n = 120) were analysed for bacterial (E. coli and Salmonella) and viral (HAV and NoV) contamination; samples from category B areas were analysed before and after purification treatment. All the samples were negative for HAV whereas 10 samples (8.3%), all harvested in the lagoon areas, were positive for NoV. Sequencing identified the strains as genotypes II.4 and II.b. None of the samples was found to be contaminated after depuration. CONCLUSIONS: The monitoring showed a low frequency of NoV presence; viral contamination, detected exclusively in shellfish collected from the deltaic area (category B), could be influenced by the flow of the tributary river. The data collected are useful for the design of targeted prevention strategies and for the modulation of control plans after meteorological events.

Evaluation of the protective effects of alpha-tocopherol and retinol against ochratoxin A cytotoxicity.

Ochratoxin A (OTA), a mycotoxin frequently present in food and feedstuffs, produces a wide range of toxic effects, including cell death via lipid peroxidation. In one human and four animal cell lines we determined the half lethal concentration (LC50) of OTA, its effect on reactive oxygen species (ROS) production, and its ability to induce cytochrome p450 activity. We also examined the protective effect of alpha-tocopherol and all-trans-retinol in the most sensitive cell lines (i.e. bovine mammary epithelia, for which LC50 was 0.8 microg/ml (24 h), and Madin Darby canine kidney, for which LC50 was 4.3 microg/ml (48 h)). Pre-incubation for 3 h with either antioxidant significantly (P<0.05) ameliorated the OTA-induced reduction in cell viability and significantly decreased (P<0.05) ROS production. These findings indicate that oxidative stress is an important factor in OTA cytotoxicity. Supplementation with antioxidant molecules may counteract the short-term toxicity of this mycotoxin.

Molecular detection of cell line cross-contaminations using amplified fragment length polymorphism DNA fingerprinting technology.

We have tested amplified fragment length polymorphism (AFLP) technology, in comparison with isoenzyme analysis, for the simultaneous detection of inter- and intraspecific cell line cross-contaminations (CCCs) in the cell line collection held at the Istituto Zooprofilattico della Lombardia e dell'Emilia Romagna. Isoenzyme analysis identified four cases of interspecific CCCs. In a single experiment, AFLP was able to identify the species of origin of all cell lines for which a reference genomic deoxyribonucleic acid was available and to detect five interspecific contaminations. Four CCCs confirmed data on isoenzymes, whereas the fifth CCC was detected in a species for which isoenzyme analysis was noninformative. In addition, AFLP was able to identify the putative source of the contaminations detected. The utility of the technology in the detection of intraspecific cell line contaminations depends on the number of cell lines that have to be distinguished in a specific species and on the availability of highly informative fingerprinting systems. In mice, a single AFLP primer pair produced 16 polymorphisms and distinguished all the 15 strains of mouse cell lines analyzed. In humans, 18 AFLPs identified 83 different profiles in the 159 cell lines analyzed. Amplified fragment length polymorphism can conveniently be applied for cell line fingerprinting in species for which hypervariable markers are not available. In species for which a highly informative multiplex of microsatellite markers is available, AFLP can still provide a useful and cheap tool for simultaneously testing inter- and intraspecific contaminations.

Establishment and characterization of two new pig cell lines for use in virological diagnostic laboratories.

Two pig cell lines derived from kidney and trachea tissues and referred to as newborn swine kidney (NSK) and newborn pig trachea (NPTr) were established following serial culture of primary cells. They were characterized by an epithelial-like morphology, high capacity to replicate and stability of the cell monolayer for several days after seeding. Their modal chromosome number was modified in comparison to that of primary swine cells and they both displayed a transforming potential in vitro and displayed oncogenicity in nude mice. Infection with pig endogenous retroviruses was detected. Almost all the swine viruses tested, i.e., pseudorabies virus, pig parvovirus, hog cholera virus, transmissible gastroenteritis virus of swine, encephalomyocarditis virus, swine vesicular disease virus and the enteroviruses, except pig reproductive respiratory syndrome virus, were capable of replicating in the new cell lines with titres similar to the ones detected in the reference culture systems. Furthermore, all the selected influenza virus sub-types isolated from human, swine and avian species replicated with cytopathic effect in NSK and NPTr cells, whereas, of all the equine influenza viruses tested only the Miami and Suffolk sub-types replicated.

Genomic characterization of porcine rotaviruses in Italy.

A total of 23 rotavirus strains isolated from pigs were analyzed. Twenty strains had been isolated from diarrheic piglets from an outbreak that occurred in northern Italy in 1983. Three strains had been isolated in 1984 from swine herds located in distinct areas of northern Italy. All 23 strains were characterized as type G6P[5] by PCR. The isolation from piglets of rotaviruses displaying typical bovine G- and P-type specificities points out the high frequency of rotavirus transmission between cattle and pigs.

Induction of BGT-1 and amino acid system A transport activities in endothelial cells exposed to hyperosmolarity.

We studied the responses to hypertonicity of cultured endothelial cells from swine pulmonary arteries. In 0.5 osmol/kgH(2)O medium, initial cell shrinkage was followed by a regulatory volume increase (RVI), complete after 1 h, concomitant with an increase in cellular K(+) content. Then the activity of amino acid transport System A increased, accompanied by an accumulation of ninhydrin-positive solutes (NPS), reaching a peak at approximately 6 h. The subsequent decline in System A activity was paralleled by an induction of the betaine-GABA transporter (BGT-1), detected as increases of BGT-1 mRNA and of transport activity, which peaked at approximately 24 h. Inhibitors of transcription or translation prevented induction of both transport activities. The increased expression of BGT-1, which involved activation of "tonicity-responsive enhancer," was inhibited by 5 mM extracellular betaine. Cellular K(+) concentration gradually declined after the accumulation of NPS and during the induction of BGT-1. This very effective adaptation to hypertonicity suggests it has a physiological role.

The response of pigs inoculated with a thymidine kinase-negative (TK-) pseudorabies virus to challenge infection with virulent virus.

12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK-) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK- mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK- mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK- mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK- mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK- mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK- mutant-inoculated pigs or from the controls.

A study of the ability of a TK-negative and gI/gE-negative pseudorabies virus (PRV) mutant inoculated by different routes to protect pigs against PRV infection.

The capacity of a TK-negative (TK-) and gI/gE-negative (gI/gE-) pseudorabies virus (PRV) mutant to protect pigs against Aujeszky's disease carried out by experimental infection with a virulent PRV strain, was tested. There were three groups, each of four susceptible pigs which were inoculated twice by two different schedules. Group 1 received the modified virus by the intradermal (first inoculation)-intramuscular (second inoculation) routes; group 2 was treated by the intranasal (first inoculation)-intramuscular (second inoculation) routes. The third group was left untreated as the control. All of the pigs were challenged intranasally with a virulent PRV strain and they were subsequently injected with dexamethasone. Two pigs in each group were necropsied on days 5 and 15 after dexamethasone inoculation. The challenge exposure resulted in mild clinical signs, increase in growth and a shorter period of virus shedding in vaccinated pigs, whereas the control group showed severe signs of Aujeszky's disease. No difference in the titre of the virulent virus which was excreted by pigs of all three groups, was observed and all animals seroconverted. Both the mutant strain and the wild-type virus established a latent infection although only the latter was reactivated and shed. Slight lesions were observed in target tissues of the vaccinated animals and no significant differences were detected between the two inoculation schedules.

A study on the long-term immunity induced by La Sota strain of Newcastle disease virus grown in a BS/BEK cell line of bovine embryo kidney origin.

Twenty-day-old susceptible chickens were divided into three groups; two were vaccinated with inactivated, water in oil emulsified La Sota strain of Newcastle disease virus (NDV) obtained from a bovine embryo kidney (BS/BEK) cell line and from chicken embryos, respectively. The third unvaccinated group represented the control. At 30-day intervals subgroups were exposed to the Herts 33 virulent NDV strain. Serological and clinical findings showed no appreciable difference in the immunogenicity of the antigen from either culture systems and no significant differences could be observed in its ability to protect against ND challenge.

A study on the characteristics of La Sota strain of Newcastle disease virus adapted to grow in a cell line from bovine embryo kidney (BS/BEK).

The lentogenic La Sota strain of Newcastle disease virus (NDV) was adapted to grow in the BS/BEK cell line of bovine embryo kidney origin with a infectious titre similar to that in chicken embryos. No modification in the biological properties was detected after serial passages in the cell line, as indicated by CPE and by size and shape of the plaques. By contrast, the intracerebral pathogenicity index test, determined in 1-day-old chicks, was lower than for La Sota grown in chicken embryos. However, the immunogenicity of the cell culture adapted virus did not show any variation as demonstrated by serological response and by protection following challenge with virulent NDV. Accordingly, it appears that La Sota grown in cell cultures has retained its biological and immunological characteristics and if these results are confirmed by field trials and long term protection tests, the use of the BS/BEK cell line could be an alternative to chicken embryos for the cultivation of NDV.