Hepatitis E Virus RNA Detection in Liver and Muscle Tissues Sampled from Home Slaughtered Domestic Pigs in Central Italy.

Hepatitis E virus is a worldwide emerging foodborne pathogen; raw or undercooked meats and liver pork products can cause infection through the orofecal route. In Central-Southern Italy, small traditional farming method, associated with the possibility of environmental sharing with wild species, can facilitate HEV diffusion and persistence. The aim of this study was to determine HEV genotype and subtype in Marche region from home slaughtered domestic pigs involved in small and traditional food chains. A total of 236 liver and muscle tissues and 6 pooled salami samples were screened. Laboratory workflow started with homogenization, followed by RNA extraction. Nested reverse transcription PCR and qRT-PCR were used to amplify specific parts of overlapping open reading frames belonging to the HEV genome. A total of 42/236 (17.79%) liver and 8/236 (3.39%) diaphragm specimens were positive; none of the pooled salami specimens showed positive HEV signal. The discovered HEV3c presented high nucleotide similarities with ones amplified from wild boar populations hunted in the same province. Extensive farming methods and environmental sharing with wild animal species support cross-infection infections, as observed in the present study. Although salami resulted negative for HEV RNA detection, the effects of food technologies on viral loads remain unclear. Therefore, further scientific investigations coupled with efficacious standardized laboratory procedures will be the next challenge.

Multidrug resistant Vibrio spp. identified from mussels farmed for human consumption in Central Italy.

AIMS: This study investigated phenotypic and genotypic antimicrobial resistance profiles of Vibrio strains identified from Mytilus galloprovincialis farmed for human consumption in the Adriatic Sea Central Italy. METHODS AND RESULTS: A total of 475 mussels (M. galloprovincialis) were involved in the present study, and culture-dependent microbiological methods permitted to identify a total of 50 Vibrio strains that were tested for antibiotic susceptibility followed by the genetic determinant detections. Antibiograms showed resistance against ampicillin (36.0%), amoxicillin-clavulanic acid (30.0%), gentamycin (14.0%), and imipenem (18.0%). Biomolecular assays amplified a total of 264 antibiotic resistance genes harbored by both susceptible and resistant Vibrio species. Among resistance genes, aacC2 (62.0%) and aadA (58.0%) for aminoglycosides, blaTEM (54.0%) for beta-lactams, qnrS (24.0%) for quinolones, tetD (66.0%) for tetracyclines, and vanB (60.0%) for glycopeptides were mainly amplified by PCR assays. CONCLUSIONS: Vibrio genus is involved in the antibiotic resistance phenomenon diffusion in the aquatic environments, as demonstrated by the harboring of many genetic determinants representing a kind of genetic "dark world".

Hepatitis E Virus RNA Detection from Hunted Wild Boars in Central Italy: an Epidemiological Investigation.

Every year, foodborne pathogens, including the hepatitis E virus (HEV), cause thousands of infections in different continents. Final consumers become infected through the ingestion of contaminated animal origin foodstuffs. Generally, in industrialized countries, HEV genotype 3 is involved in sporadic outbreaks. Infections have been described, in Europe and Japan as consequence of pork products and contaminated wild boar's primary or processed products (liver and muscle tissues) consumption. In Central Italy, hunting activities are largely practiced. In these small and rural communities, game meat and liver are ingested by hunters' families or at local and traditional restaurants. Therefore, these food chains can be considered critical HEV reservoirs. In this study, 506 liver and diaphragm tissues were collected from hunted wild boars in the Southern Marche region (Central Italy) and were screened for HEV RNA detection. From the 10.87% of liver and 2.76% of muscle samples, HEV3 subtype c was discovered. The observed prevalence values resulted in line with previous investigations performed in other Central Italian regions, but higher than Northern ones (3.7% and 1.9% from liver tissue). Therefore, the obtained epidemiological data highlighted the wide occurrence of HEV RNA circulation in a low-investigated area. Basing on results, a One-health approach was adopted due to the sanitary relevance of this Public Health concern.

Intradiaphragmatic abscesses in a wild boar (Sus scrofa): Inspective implications based on anatomopathological evidences.

The intradiaphragmatic localization of an abscess is rarely described in humans and in other animal domestic and wild species, and can be caused by penetrative traumas (i.e., firearm injuries). Here we describe two intradiaphragmatic abscesses in a hunted adult male wild boar (Sus scrofa) pluck, associated with adhesion phenomena with the contiguous anatomical structures (pleural, phrenic, and glissonian serosas) and observed during the post mortem inspection, in accordance with the Reg. EU 627/2019. One of these lesions also presented a phreno-abdominal fistula. We found in cytopathological evaluation of the neoformations' content the presence of spheroidal bacterial soma, characterized by linearly concatenated "Streptococcus-like" aggregation pattern. Furthermore, microbiological assays revealed a polymicrobial pattern characterized by the presence of telluric microorganisms, some of which have a marked pyogenic action (Streptococcus suis type I, Sphingomonas paucimobilis, Carnobacterium divergens, and Lactobacillus sakei). Our results and collected evidence demonstrate the pathogenetic hypothesis of bacterial contamination secondary to penetrative trauma caused by a not-mortal projectile's wound, defining the inspective behaviour according to the cogent legislation. These lesions, in the reason of their potential relation to toxemia, bacteremia and septicemia phenomena, represent a sanitary risk that impose, from a normative point of view, the total condemnation of the carcass. These inspective implications, originating from the observation and interdisciplinary description of the anatomopathological and microbiological features of the lesions, are directly projected on the normative scenario, providing useful elements to guarantee the health of the consumer.

Hepatitis E Virus Detection in Hunted Wild Boar Liver and Muscle Tissues in Central Italy.

In different European countries, including Italy, hepatitis E virus (HEV) has been recognized as an emerging public health concern. Humans are infected through the orofecal route by the ingestion of contaminated uncooked or undercooked animal-origin foodstuffs. Wild boars (Sus scrofa) have gained a crucial role as viral reservoirs. HEV-3 is the most frequently identified genotype from hunted wild boar liver and muscle tissues. The Marche region, more specifically Ascoli Piceno province, is characterized by a rooted hunting tradition and related product consumption. In this research study, 312 liver and 296 muscle specimens were screened using biomolecular assays, and HEV RNA was detected from 5.45% and 1.35% of liver and muscle samples, respectively. Phylogenetic analysis revealed that positive animals were infected by genotype 3 subtype c. Based on the environmental pathogen characteristics, HEV has also evolved to guarantee its survival in a wild environment. Therefore, wild boars and ruminants have a key role in its persistence. Epidemiological data regarding HEV circulation have resulted as necessary, and biomolecular analysis represents an important means of monitoring and establishing preventive measures. A multidisciplinary approach could provide a wide perspective regarding HEV and infectious implications on human, animal, and environmental health.

Hepatitis E virus detection in hunted wild boar (Sus scrofa) livers in Central Italy.

Hepatitis E virus (HEV) is a zoonotic pathogen, responsible for numerous cases of infection in humans. Transmission occurs through the orofecal route, and ingestion of contaminated foods represents an important risk factor for final consumer's health. Wild animal species, in particular wild boar (Sus scrofa), are the main virus reservoirs; liver is the target organ, from which, through the hematic diffusion, HEV reaches different tissues and organs, as muscular one. The hygienic-sanitary critical issues connected with game meat food chain in general, and particularly wild boar, with special regards to any geographical area where this animal species can be directly in contact with humans, domestic ones (i.e., domestic pig), and other wild reservoirs (i.e., wild ruminants), finds favorable environmental conditions, have induced us to conduce the present scientific investigation. During the hunting season 2019/2020, a total of 156 wild boar livers were collected from provided plucks at slaughterhouse in Ascoli Piceno. Nested RT-PCR was used for the viral RNA detection. Results demonstrated a positivity of 5.12% (8/156), and the circulation in the screened area of genotype 3 subtype c, which is frequently identified in Central Italy. HEV sanitary relevance and the emerging role of any food chains in its transmission impose further detailed studies. The molecular screening of hunted wild boars' livers can provide important information about virus's circulation in wild animal populations in a specific area.

Parasitic cysts in wild boars hunted in Central Italy: The sanitary controls in the wild game meats chain.

Seven hundred sixty-five (765) adult wild boars were examined during the 2016/2017 hunting season for the research of parasites. Post mortem inspection was carried out at the slaughterhouse by the Official Veterinarian on the plucks (heart, tongue, lungs, diaphragm, and liver) of the killed animals presented by hunters. Of these, 0.8% (6/765) were positive for Echinococcus granulosus sensu lato (s.l.), and 2.6% (20/765) were positive for the metacestode stage of Taenia hydatigena (Cysticercus tenuicollis), while 0.5% (4/765) animals showed a mixed infection (Echinococcus granulosus s.l. and Taenia hydatigena). Sixty-three (63) cystic lesions were found. Of these 25,4% (16/63) were caused by Echinococcus granulosus s.l. and 74,6% (47/63) were caused by Cysticercus tenuicollis. The more involved organs were liver and lungs, in a less extension omentum and diaphragm. Parasitological analyses showed an overall prevalence of 3.9% for metacestodes in the hunted animals examined (Paoletti et al., 2018). Hydatids were molecularly characterized as E. granulosus sensu stricto. Trichinella spp. examination results showed no evidences of parasitic cysts. The products of hunting used for own consumption and direct sale to the final consumer or retailer, according to Regulation (EC) No 853/2004, lack of overall control by the Competent Authority. This is a critical point in the food chain of the game meat. The data obtained show the importance of the post mortem inspection and the central role of the Competent Authority to ensure not only the food safety of game meat but also to collect data for extensive epidemiological investigations on live-stocks wildlife settings having a direct impact on public health.

Microscopic investigations as an aid in raw ham's ripening analysis.

The raw ham's ripening process contributes to the development of numerous biochemical reactions, mainly affecting proteins and lipids and allowing to obtain an adequate texture and a characteristic flavor. This article reports the results of histologic investigations carried out on 5 different anatomic regions from raw hams manufactured in the Fermo Province, Marche Region, Central Italy. Raw ham specimens were collected at the 10 following time intervals throughout the ripening process: 1) "Time 0", when ripening was started, 2) one month, 3) three months, 4) four months, 5) eight months, 6) nine months, 7) twelve months, 8) eighteen months, 9) twentythree months and 10) twenty-eight months after the ripening process began, respectively. Different microscopic findings of variable extension and degree were observed, with the vast majority of them being interpreted as dehydration- and proteolysisrelated modifications. In conclusion, morpho- histological investigations may represent a valuable aid in raw ham's ripening analysis.

Accreditation Procedure for Trichinella spp. Detection in Slaughterhouses: The Experience of an Internal Laboratory in Italy.

Trichinellosis is a severe foodborne zoonotic disease due to the consumption of undercooked meat containing Trichinella spp. larvae. According to Commission Regulation (EU) No 1375/2015, domestic pigs, farmed wild boar, and horses must be tested for the presence of the parasite in the muscles as part of post-mortem examination. In this study, the accreditation procedure and the maintenance of the certificate for internal laboratory attached to a slaughterhouse are described. The main advantages of such accreditation are represented by the possibility to obtain fast results in order to process carcasses quickly, whereas the difficulties for the technician are linked to performing proficiency testing and following training courses. This program can be considered particularly useful for surveillance and food safety purposes.

Epidemiological survey on cystic echinococcosis in wild boar from Central Italy.

Despite the wide distribution of wild boar populations in Italy and the increase of its diffusion in urbanized areas, only one case report has described the occurrence of Echinococcus granulosus s.l. in a wild boar from Marche (Central Italy). The present study investigated the presence of E. granulosus sensu lato with an epidemiological survey on wild boars from central Italy that had been killed during hunting season. Seven hundred sixty-five (765) adult wild boars were examined during the 2016-2017 hunting season. Of these animals, 1.0% (8/765) were positive to E. granulosus s.l. with a fertility of 0.3% (2/765), and 2.9% animals (22/765) were positive for the metacestode stage of Taenia hydatigena (Cysticercus tenuicollis), while 0.5% (4/765) showed mixed infection (E. granulosus s.l. + T. hydatigena). Sixteen hydatids were found, of which 12.5% were fertile, 37.5% were sterile, 31.3% were caseous, and 18.8% were calcified. Eight hydatids (two fertile and six sterile cysts) were molecularly characterized by analysis of the mitochondrial gene, cytochrome c oxidase subunit 1 (cox1), and the NADH dehydrogenase subunit 1 (ND1). Hydatids found in wild boars were characterized as E. granulosus sensu stricto (G1 genotype). The present survey represents the first epidemiological study on cystic echinococcosis in wild boar in Italy which highlights the need for more extensive epidemiological investigations to determine the causal factors, economic impact, and public health importance of the disease in this livestock-wildlife setting.

Characterization of A Staphylococcal Food Poisoning Outbreak in A Workplace Canteen during the Post-Earthquake Reconstruction of Central Italy.

In summer 2017, a foodborne outbreak occurred in Central Italy, involving 26 workers employed in the post-earthquake reconstruction. After eating a meal provided by a catering service, they manifested gastrointestinal symptoms; 23 of them were hospitalized. The retrospective cohort study indicated the pasta salad as the most likely vehicle of poisoning. Foods, environmental samples, and food handlers' nasal swabs were collected. Bacillus cereus (Bc) and coagulase-positive staphylococci (CPS) including S. aureus, together with their toxins, were the targets of the analysis. CPS, detected in all the leftovers, exceeded 105 CFU/g in the pasta salad, in which we found Staphylococcal Enterotoxins (SEs) (0.033 ng SEA/g; 0.052 ng SED/g). None of the environmental and human swabs showed contamination. We characterized 23 S. aureus from foods. They all belonged to the human biotype, showed the same toxigenic profile (sea, sed, sej, and ser genes), and had the same Pulsed Field Gel Electrophoresis (PFGE) pattern; none of them harbored mecA or mupA genes. We also detected Bc contamination in the pasta salad but none of the isolates harbored the ces gene for the emetic toxin cereulide. The EU Reference Laboratory for CPS confirmed the case as a strong-evidence outbreak caused by the ingestion of SEs produced by a single strain of S. aureus carried by the same human source. This outbreak was successfully investigated despite the emergency situation in which it occurred.

The hygiene-sanitary control in the wild game meats.

The use of game meat as a food source is currently a growing trend in our country. These products have strong and historic ties with cultural and culinary tradition, but are also appreciated for their sensory and nutritional characteristics. A major contributor to the supply of this type of product is hunting. Practiced since the dawn of time for survival, hunting has evolved into a recreational activity with substantial commercial interests. Of particular importance in this context is hunting of large ungulates. The progressive urbanization of the population has allowed for the re-establishment of bush and wooded areas that represent the ideal habitat of species such as the wild boar, whose numbers are increasing throughout the country. It is therefore clear that implementation of safety rules regarding the hunting and consumption of game meat needs to be urgently addressed. The understanding and application of rules isn't always easy since the health law is intertwined with that of hunting, and the decision- making power left to the different regions does not contribute to a uniform application throughout the country. The aim of this study was to examine the norms that regulate the use of large wild game meat intended for human consumption and their applicability in hunting activities. From the comparison of the data reported in the literature and our field experience the rules implementation and the problems are evaluated. Operational procedures are then proposed to simplify some of the most difficult aspects and fill in the gaps highlighted.