First Report of Food Poisoning Due to Staphylococcal Enterotoxin Type B in Döner Kebab (Italy).

Staphylococcal food poisoning results from the consumption of food contaminated by staphylococcal enterotoxins. In July 2022, the Turin local health board was notified of a suspected foodborne outbreak involving six children who had consumed döner kebab purchased from a takeaway restaurant. The symptoms (vomiting and nausea) were observed 2-3 h later. A microbiological analysis of the food samples revealed high levels (1.5 × 107 CFU/g) of coagulase-positive staphylococci (CPS). The immunoassay detected a contamination with staphylococcal enterotoxins type B (SEB). The whole genome sequencing of isolates from the food matrix confirmed the staphylococcal enterotoxin genes encoding for type B, which was in line with the SEB detected in the food. This toxin is rarely reported in staphylococcal food poisoning, however, because there is no specific commercial method of detection. The involvement of enterotoxin type P (SEP) was not confirmed, though the corresponding gene (sep) was detected in the isolates. Nasal swabs from the restaurant food handlers tested positive for CPS, linking them to the likely source of the food contamination.

Investigation and Follow-Up of a Staphylococcal Food Poisoning Outbreak Linked to the Consumption of Traditional Hand-Crafted Alm Cheese.

Staphylococcal food poisoning (SFP) is one of the most important foodborne diseases. This work describes a SFP event linked to the consumption of alm cheese and involved three people belonging to the same family. Leftovers of the consumed cheese, samples from the grocery store and the producing alm were collected and tested for Coagulase positive staphylococci (CPS) enumeration and for the presence of staphylococcal enterotoxins (SEs). Isolates were typed with MLST, spa typing, and tested for SEs and methicillin resistance genes. An in vitro test evaluated SEs production in relation to bacterial growth. The presence of CPS and SEs was detected in all cheese samples and all isolates belonged to the same methicillin sensitive ST8/t13296 strain harbouring sed, ser and sej genes. The in vitro test showed the production of enterotoxins started from 105 CFU/mL. The farmer was prescribed with corrective actions that led to eradication of the contaminating strain.

Genome-Wide Profiling of Enterotoxigenic Staphylococcus aureus Strains Used for the Production of Naturally Contaminated Cheeses.

Staphylococcus aureus is a major human pathogen and an important cause of livestock infections. More than 20 staphylococcal enterotoxins with emetic activity can be produced by specific strains responsible for staphylococcal food poisoning, one of the most common food-borne diseases. Whole genome sequencing provides a comprehensive view of the genome structure and gene content that have largely been applied in outbreak investigations and genomic comparisons. In this study, six enterotoxigenic S. aureus strains were characterised using a combination of molecular, phenotypical and computational methods. The genomes were analysed for the presence of virulence factors (VFs), where we identified 110 genes and classified them into five categories: adherence (n = 31), exoenzymes (n = 28), genes involved in host immune system evasion (n = 7); iron uptake regulatory system (n = 8); secretion machinery factors and toxins' genes (n = 36), and 39 genes coding for transcriptional regulators related to staphylococcal VFs. Each group of VFs revealed correlations among the six enterotoxigenic strains, and further analysis revealed their accessory genomic content, including mobile genetic elements. The plasmids pLUH02 and pSK67 were detected in the strain ProNaCC1 and ProNaCC7, respectively, carrying out the genes sed, ser, and selj. The genes carried out by prophages were detected in the strain ProNaCC2 (see), ProNaCC4, and ProNaCC7 (both positive for sea). The strain ProNaCC5 resulted positive for the genes seg, sei, sem, sen, seo grouped in an exotoxin gene cluster, and the strain ProNaCC6 resulted positive for seh, a transposon-associated gene. The six strains were used for the production of naturally contaminated cheeses which were tested with the European Screening Method for staphylococcal enterotoxins. The results obtained from the analysis of toxins produced in cheese, combined with the genomic features represent a portrait of the strains that can be used for the production of staphylococcal enterotoxin-positive cheese as reference material.

Evaluation of the efficacy of a training course in food safety addressed to food charity volunteers.

In Italy, the Banco Alimentare Onlus manages a network of 8,000 charitable organizations that distribute 67,000 tons of foodstuffs to 1.6 million needy persons. To provide their volunteers with the required food safety knowledge, the Banco Alimentare del Piemonte Onlus commissioned the Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta to hold training courses in food safety. Before and after each session, the participants completed a questionnaire to evaluate their knowledge on the topic of food safety. The responses were entered in a dedicated database and analyzed using STATA ver. 15.1. Comparison of the scores for each participant before and after training revealed a considerable discordance [ICC 0.06, 95% confidence interval (CI) 0.00-0.18]. Analysis of the post-training questionnaires showed that the number of questions left unanswered decreased and the number of correct answers increased. The difference between the percentage of correct and incorrect responses before and after the training course was statistically significant (P<0.001). Comparison of responses to the pre- and post-training questionnaires provided the data for statistical evaluation of the efficacy of the training course.

Outbreak of febrile gastroenteritis caused by Listeria monocytogenes 1/2a in sliced cold beef ham, Italy, May 2016.

In May 2016, two separate clusters of febrile gastroenteritis caused by Listeria monocytogenes were detected by the local health authority in Piedmont, in northern Italy. We carried out epidemiological, microbiological and traceback investigations to identify the source. The people affected were students and staff members from two different schools in two different villages located in the Province of Turin; five of them were hospitalised. The epidemiological investigation identified a cooked beef ham served at the school canteens as the source of the food-borne outbreak. L. monocytogenes was isolated from the food, the stools of the hospitalised pupils and the environment of the factory producing the cooked beef ham. All isolates except one were serotype 1/2a, shared an indistinguishable PFGE pattern and were 100% identical by whole genome sequencing (WGS). By combining a classical epidemiological approach with both molecular subtyping and WGS techniques, we were able to identify and confirm a Listeria gastroenteritis outbreak associated with consumption of sliced cold beef ham.

Molecular Typing of Staphylococcus Aureus Isolate Responsible for Staphylococcal Poisoning Incident in Homemade Food.

In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA) and D (SED), respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus, and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa-type. Showing a high heterogeneity of isolates, spa-type t13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves.

A Salmonella Enterica Subsp. Enterica Serovar Enteritidis Foodborne Outbreak after Consumption of Homemade Lasagne.

In the latest year, and also in 2013, Salmonella was the most frequently detected causative agent in foodborne outbreaks (FBOs) reported in Europe. As indicated in EFSA report (2015) the serotypes mostly associated to FBOs are S. Typhimurium and Enteritidis; while Salmonella Typhimurium is generally associated with the consumption of contaminated pork and beef, FBOs due to Salmonella Enteritidis are linked to eggs and poultry meat. In this study it is described the investigation of a domestic FBO involving four adults and linked to homemade lasagne. Investigations were performed to determine the relatedness of Salmonella strains, identify the sources of infection, and trace the routes of Salmonella contamination in this FBO. Salmonella strains were isolated in 3 out of 4 patient stool samples and from lasagne and all of them were serotyped as S. Enteritidis. Pulsed-field gel electrophoresis (PFGE) analysis revealed the genotypical similarity of all the strains. Although serotyping and PFGE analysis identified the common food source of infection in this FBO, it was not possible to determine how or at what point during food preparation the lasagne became contaminated with Salmonella.

Evaluation of Hygiene and Safety Criteria in the Production of a Traditional Piedmont Cheese.

Traditional products and related processes must be safe to protect consumers' health. The aim of this study was to evaluate microbiological criteria of a traditional Piedmont cheese, made by two different cheese producers (A and B). Three batches of each cheese were considered. The following seven samples of each batch were collected: raw milk, milk at 38°C, curd, cheese at 7, 30, 60, 90 days of ripening. During cheese making process, training activities dealing with food safety were conducted. Analyses regarding food safety and process hygiene criteria were set up according to the EC Regulation 2073/2005. Other microbiological and chemical-physical analyses [lactic streptococci, lactobacilli, pH and water activity (Aw)] were performed as well. Shiga-toxin Escherichia coli, aflatoxin M1 and antimicrobial substances were considered only for raw milk. All samples resulted negative for food safety criteria; Enterobacteriaceae, E.coli and coagulase-positive staphylococci (CPS) were high in the first phase of cheese production, however they decreased at the end of ripening. A high level of CPS in milk was found in producer A, likewise in some cheese samples a count of >5 Log CFU/g was reached; staphylococcal enterotoxins resulted negative. The pH and Aw values decreased during the cheese ripening period. The competition between lactic flora and potential pathogen microorganisms and decreasing of pH and Aw are considered positive factors in order to ensure safety of dairy products. Moreover, training activities play a crucial role to manage critical points and perform corrective action. Responsible application of good manufacturing practices are considered key factors to obtain a high hygienic level in dairy products.

Occurrence of Thermotolerant Campylobacter in Raw Poultry Meat, Environmental and Pigeon Stools Collected in Open-Air Markets.

Campylobacteriosis was the most commonly reported zoonosis for confirmed human cases in European Union during 2011. Poultry meat was very often implicated in Campylobacter infections in humans. In Italy commerce of raw poultry meat is common in open-air markets: these areas can be considered at high risk of bacterial contamination due to the high presence birds like pigeons. The aim of this study was to collect data about the contamination by thermotolerant Campylobacter of raw poultry meat commercialised in open-air markets, of work-surfaces in contact with poultry meat and of pigeon stools sampled in the market areas in Turin, Northern Italy. Between September 2011 and December 2012, 86 raw poultry meat samples, 86 environmental swabs and 108 animal samples were collected in 38 open-air markets. Analysis were carried out according to ISO10272-1:2006 standard. C.coli was detected in 2.3% (2/86) of raw poultry meat samples, whereas no swab (0/86) resulted positive. Of pigeon stool 28% (30/107) was positive for C.jejuni (83.3% C.jejuni subsp. jejuni and 16.7% C.jejuni subsp. doylei). C.jejuni subsp. jejuni was isolated from 1 dead pigeon. Our results showed lower rates of contamination than those reported at retail in Europe. Although samples were collected in areas at high risk of contamination, raw poultry meat and work surfaces reported a low level of presence of thermotolerant Campylobacter. The high percentage of C.jejuni isolated from pigeon stools showed the importance of a continuous application of preventive measures by the food business operators and the surveillance activity by the Competent Authority.

Food Allergens: State of the Art in Piedmont Region in the Period 2011-2012.

The US National Institutes of Allergy and Infectious Diseases defines food allergy as adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food. Undeclared allergens in food label represent a risk for consumers, as there is no therapy for food allergies. According to Directive 2003/89/EC, declaration of all ingredients and derived substances in the label is mandatory. In 2011-2012, in Piedmont region (North-western Italy) 285 food samples were analysed for ß-lactoglobulin and 234 for egg proteins. The aim of this work was to analyse 2 years data in order to assess the presence of undeclared milk and egg allergenic proteins in food placed on the market checking the compliance of labeling of food allergens. Analyses were carried out with ELISA tests, both for the detection of the egg and milk proteins. ß-lactoglobulin was found in 2.8% (8/286) of samples, while egg proteins in 4.7% (11/234).

Ruminant rhombencephalitis-associated Listeria monocytogenes strains constitute a genetically homogeneous group related to human outbreak strains.

Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cow's milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.

Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene.

Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP). Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type) and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002). Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.

Detection and phylogenetic analysis of an atypical pestivirus, strain IZSPLV_To.

Recently, atypical bovine pestiviruses (BVDV-3) have been identified in batches of contaminated foetal calf serum (FCS) and in naturally infected cattle. During routine screening of FCS by conventional panpestivirus PCR assay, one batch showed traces of pestivirus nucleic acids, and the contaminating virus was typed as BVDV-3-like. Phylogenetic analysis based on three genome regions (5'UTR, N(pro) and E2) showed that this strain, named IZSPLV_To, clusters in a separate clade with CH_KaHo/cont, a cell culture contaminant detected in Switzerland. This study is the first report of the detection in Italy of a FCS batch contaminated with BVDV-3 and adds more evidence that atypical pestiviruses represent a serious cause for concern in cell culture laboratories, with potential repercussions on BVD control and vaccine biosafety. Our findings suggest that the BE/B2 primers may be able to detect BVDV-3 in a panpestivirus assay, but testing of a larger number of strains is required.

Detection of a morphogenetically novel Sarcocystis hominis-like in the context of a prevalence study in semi-intensively bred cattle in Italy.

The aim of this study was to determine the prevalence of sarcosporidiosis in semi-intensively bred cattle in northwestern Italy. A diagnostic protocol was setup in which infected animals were identified by rapid histological examination of the esophagus, diaphragm, and heart and the detected Sarcocystis spp. were subsequently typed using conventional electron microscopy in combination with molecular techniques. Sarcosporidia cysts were detected in 78.1% of the animals and were seen most often in the esophagus. The cattle is intermediate host for Sarcocystis hominis (final host, humans and some primates), Sarcocystis cruzi (final host, domestic and wild canids), and Sarcocystis hirsuta (final host, wild and domestic cats).All these three species of Sarcocystis were identified, variously associated, with the following prevalence: S. cruzi (74.2%), S. hirsuta (1.8%), and S. hominis (42.7%). Furthermore, a new S. hominis-like (prevalence 18.5%), characterized by hook-like structures of villar protrusion and a different sequence of the 18S rRNA gene, was identified. The cattle sheds testing positive for zoonotic Sarcocystis were assessed for risk factors contributing to the maintenance of the parasite's life cycle. Significant associations emerged between consumption of raw meat by the farm owner, mountain pasturing, and absence of a sewerage system on the farm and cattle breed. Our study demonstrates that sarcosporidiosis may constitute a public health problem in Italy and indicates several issues to be addressed when planning surveillance and prevention actions. The applied diagnostic approach revealed that cattle can harbor a further type of Sarcocystis, of which life cycle and zoonotic potential should be investigated.

Histidine at codon 154 of the prion protein gene is a risk factor for Nor98 scrapie in goats.

Prion protein gene (PRNP) polymorphisms are involved in modulating the appearance of atypical/Nor98 scrapie in sheep, with the alleles AHQ and AF141RQ strongly associated with occurrence of the disease. The presence of histidine at codon 154 has also been detected in Nor98-affected goats, but statistical analysis of the association between Nor98 and goat PRNP polymorphisms has not been reported previously. Here, a case-control study was carried out on eight Nor98-positive goats and 246 negative herdmates belonging to eight Italian Nor98 scrapie outbreaks. The results revealed that histidine at codon 154 is also strongly associated with the disease in goats.