Enumeration of Escherichia coli and determination of Salmonella spp. and verotoxigenic Escherichia coli in shellfish (Mytilus galloprovincialis and Ruditapes decussatus) harvested in Sardinia, Italy.

The aim of the present study was to evaluate the occurrence of Salmonella spp., Verotoxigenic E. Coli (VTEC) and enumerate E. coli in shellfish (Mytilus galloprovincialis and Ruditapes decussatus) collected before and after depuration from two class B harvesting areas located in Sardinia (Italy). All the samples were analyzed for Salmonella spp. detection according to European Commission Regulation (EC) 2073/2005 and examined using the five tube Most-Probable-Number (MPN) method for enumeration of E. coli in accordance with the European Union reference method ISO 16649-3:2015. E. coli VTEC was investigated following a direct multiplex Polymerase Chain Reaction (PCR) screening test. The enumeration of E. coli met the European law limit for Class A areas of 230 MPN/100g. The averaged enumeration of E. coli in samples of M. galloprovincialis and R. decussatus collected at the harvesting time was 39 and 37 MPN/100 g respectively. The average contamination levels in samples collected after purification were 58 MPN/100g (M. galloprovincialis) and 32 MPN/100 g (R. decussatus). E. coli VTEC was not detected, on the contrary, Salmonella ser. Typhimurium was detected in one sample of M. galloprovincialis and in one sample of R. decussatus collected at the harvesting time. No significant associations were observed between E. coli levels in shellfish and environmental parameters of water or with the detection of Salmonella ser. Typhimurium in M. galloprovincialis and R. decussatus samples. Nevertheless, the occurrence of Salmonella ser. Typhimurium, involved in human infection outbreaks, should be considered a potential risk for consumers.

Occurrence, Seasonal Distribution, and Molecular Characterization of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in Shellfish (Mytilus galloprovincialis and Ruditapes decussatus) Collected in Sardinia (Italy).

In this study, we investigated the occurrence, seasonal distribution, and molecular characterization of pathogenic vibrios in Mediterranean mussels (Mytilus galloprovincialis) and grooved carpet shells (Ruditapes decussatus) from two harvesting areas of Sardinia (Italy). Samples collected before and after depuration were submitted for qualitative and quantitative determination of Vibrio spp. Vibrio spp. isolates were presumptively identified by means of biochemical methods. Identification and virulence profile of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus were performed by molecular methods. The prevalence of Vibrio spp. in M. galloprovincialis and R. decussatus was, respectively, 96 and 77%. The averaged enumeration (mean ± standard deviation) of Vibrio spp. in samples of M. galloprovincialis and R. decussatus collected at the harvesting time was 2.04 ± 0.45 and 2.51 ± 0.65 log CFU/g, respectively. The average contamination levels in samples collected after purification were 2.28 ± 0.58 log CFU/g (M. galloprovincialis) and 2.12 ± 0.67 log CFU/g (R. decussatus). Four potentially pathogenic V. parahaemolyticus isolates (tdh+ or trh+) were recovered from grooved carpet shells samples. No isolate was tdh+/trh+. The presence of potentially pathogenic vibrios in Sardinian waters strengthens the need for rational purification practices under controlled conditions to guarantee the protection of consumers.

Clonal relationship among Vibrio parahaemolyticus isolated from Mediterranean mussels (Mytilus galloprovincialis) and grooved carpet shells (Ruditapes decussatus) harvested in Sardinia (Italy).

The aim of the present study was to investigate the genetic variability of Vibrio parahaemolyticus strains isolated from naturally contaminated Mediterranean mussels (Mytilus galloprovincialis) and Grooved carpet shells (Ruditapes decussatus) from three harvesting areas of Sardinia (Italy) using a combination of different typing methods: traditional phenotypic systems and molecular techniques. Ninety-nine putative V. parahaemolyticus strains isolated from shellfish collected before and after purification were included in the study. Seventy-two isolates were confirmed as V. parahaemolyticus and were submitted to REP, ERIC and BOX PCRs. The combined dendrogram showed the similarity of the data set of the three typing methods and demonstrates how the different techniques grouped the strains in two clusters in accordance with each singular dendrogram. Several strains rendered a unique pattern regardless of the typing method, which indicates the high discriminatory power of the methods. Moreover, the use of multiple typing methods allowed a more accurate characterization of the genetic profiles of isolates and the identification of clones hardly revealed through the common techniques. The intraspecific typing of environmental V. parahaemolyticus can be of great interest in order to recognize clonal relationships between environmental contamination, foodborne disease, and geographical/temporal distribution of this pathogen. The comparative analysis focusing on the obtained genetic profiles supports the possibility for typing methods to discriminate strains with similar phenotypic profile, identifying the level of genetic correlation among the strains and the presence of genetic clones.

Hygiene and Welfare Evaluation of Pigs Slaughtered in Agritourisms.

The slaughtering procedures at agritourism farms must be carried out in accordance with the general and hygiene requirements of Regulations (EC) No 852 and 853/2004. In addition, regional laws define minimum requirements allowing some flexibility. Piglets and finishing pigs are the most frequently slaughtered animal in Sardinian agritourism farms. The aim of the present survey was to evaluate: the general and hygiene requirements of outbuilding slaughterhouses in agritourisms; the animal welfare indicators; the microbial contamination of piglets and finishing pigs carcasses. Six agritourisms outbuilding slaughterhouses - EU-approved - were investigated. General and hygiene requirements of outbuilding slaughterhouses and animal welfare indicators of 68 piglets and 5 finishing pigs were evaluated by mean of a checklist. The following parameters were determined on 45 piglets and 5 finishing pigs carcasses: i) pH 1 and 24 h after slaughter, and ii) carcass surface microbial contamination by non destructive method (sponge) on the following sampling sites: ham; back (adults); belly; jowl (adults). Aerobic colony count (ACC; ISO 4833:2003), Enterobacteriaceae (EB; ISO 21528-2:2004), Salmonella spp. (ISO 6579:2002), Listeria monocytogenes (ISO 11290-1:1996 and 11290-2:1998) were also tested. All the plants except one have two separate rooms, for clean and dirty zones, stunning and bleeding operations being frequently carried out on open air. The piglet scalding was carried out in hot water bowls, and hair removal by singeing. Animal welfare signs revealed the following aspects: handling: hoisting prior to stunning, vocalizations (41%); stunning: not individual access to box, repeated shocks (4%), mean voltage 135.6 V, mean current for head-only electrical stunning 0.78 A; indicators of not effective stunning: palpebral reflex (24.2%), corneal reflex (12.8%), vocalizations (15.4%); bleeding: conscious and sensitive animal shackling (53.8%). Results of carcass evaluation showed, for piglets and adult pig respectively: i) pH: pH1=6.21±0.25 and 6.18±0.22; pH24=5.66±0.17 and 5.49±0.11; ii) ACC: 4.11±0.64 and 4.63±0.42 (log10 CFU/cm2, mean±standard deviation); iii) Enterobacteriaceae prevalence of 81.6% (2.55±0.80) in piglets and 100% (3.22±0.90) in adults. Salmonella spp. and Listeria monocytogenes were not detected in any of the samples. General requirements of outbuilding slaughterhouses in agritourisms are suitable to produce meat in compliance with hygienic rules, considering the low risk level. Results of Enterobacteriaceae levels of finishing pig carcasses were not in compliance with the EC Regulation No 2073/2005. Training of personnel is compulsory and can improve the stunning and bleeding procedures.