On August 19, 2012, the Public Health Department of Galicia, Spain, was alerted to a possible outbreak of illness in O Grove (Pontevedra, NW Spain). All the cases were passengers travelling on a tourist cruise boat that included a visit to the rias (coastal inlets) followed by a dinner. At the time of the reported outbreak, warm weather conditions prevailed in this region of Galicia, with average air temperatures exceeding 25 °C.
Only nine cases were subjected to stool analysis. No left over food was available for analysis. The restaurant and origin of all the food consumed on the cruise were subsequently investigated. All the food consumed on the cruise was traced to its harvest site by the dealer records. The facility where the different items were stored and the premises used for processing and handling the prior the shipment to the restaurant were investigated, and the processing practices were also reviewed to assess the factors contributing to contamination, survival and proliferation of potentially causative microorganisms. Samples of raw product belonging to the same batch of suspected vehicle of infection were subjected to microbiological analyses.
Travellers were interviewed using epidemiological questionnaires instigated by the Regional Public Health Department (Conselleria de Sanidade, Xunta de Galicia). Questionnaires gathered a range of data, including age, gender, food consumption history, and date and type of symptoms. A case was defined as a person who consumed food during the cruise and presented with vomiting and/or diarrhea symptoms within 96 h. While a control was defined as a person who consumed food during the cruise and did not present with vomiting and/or diarrhea within 96 h. The restaurant staffs were also interviewed. Cases were defined as individuals who had dinner on the boat on Saturday 18th August 2013, and who reported symptoms of diarrhoea and/or vomiting during the following 72 h after the dinner (see above). A total of 54 persons belonging to the organized group and other 11 additional persons attending the dinner were identified. Epidemiological information from 65 people was subsequently available from follow-on interviews, of which 51 cases showed symptoms of illness and the remaining 14 people did not report associated symptoms. Travellers were interviewed using epidemiological questionnaires by the Regional Public Health Department (Conselleria de Sanidade, Xunta de Galicia). All the subject gave permission to use the questionnaire data for research publication.
Microbiological and molecular investigation
The local authority in Galicia test suspected gastrointestinal cases during the season of highest risk for Vibrio infections (April–August) using TCBSas an additional microbiological plating media. Stool specimens were collected from patients during the illness outbreaks and plated on Trypticase Soy Agar with 5 % sheep blood and McConkey Agar (Becton–Dickinson, Sparks, MD), and incubated at 37 °C for 18–24 h. Lactose negative colonies on McConkey Agar and/or beta-haemolytic and oxidase positive colonies on Trypticase Soy Blood Agar, were selected and subjected to species identification by biochemical tests on API 20E strips (BioMérieux, Marcy-l’Etoile, France). Serotyping. Lipopolysaccharide (O) and capsular (K) serotypes were determined by agglutination tests using specific antisera, essentially as described previously (Martinez-Urtaza et al. 2004). Presumptive identification of the isolates was confirmed by the presence of the species-specific toxR and tlh genes. The presence of the toxR gene was investigated by PCR as described previously (Kim et al. 1999). Additionally, presence of tlh, tdh and trh genes was determined by multiplex PCR according to the procedure described by Bej et al. (Bej et al. 1999). A GS-PCR assay to specifically detect the pandemic clone-specific nucleotide sequence in the toxRS operon of V. parahaemolyticus was also performed as described previously (Okuda et al. 1997). Isolates were tested against a battery of antibiotics.
Pulse Field Gel Electrophoresis (PFGE) was performed according to the “One-Day (24–28 h) Standardized Laboratory Protocol for Molecular Subtyping of Non-typhoidal Salmonella by PFGE” (Pulse-Net, CDC, Atlanta, USA) (Anonymous 2002) following a method described previously (Martinez-Urtaza et al. 2004). Chromosomal DNA was digested with 30 U of NotI (Promega, Southampton, United Kingdom) at 37° for 4 h. DNA macro-restriction fragments were resolved on 1 % SeaKem Gold Agarose (Cambrex, Baltimore, MD) in 0.5X TBE buffer. DNA from Salmonella Braenderup H9812 restricted with 50 U of XbaI (Promega, Madison, WI) at 37º for two h was used as a size marker. Pulse times were ramped from two to 40 s during a 18-h run at 6.0 V/cm. Restriction patterns were compared with the use of BioNumerics software (Applied Maths). Multi-locus sequence typing (MLST) analysis was performed as previously described (Gonzalez-Escalona et al. 2008) based on internal fragments of seven housekeeping genes: recA, gyrB, dnaE, dtdS, pntA, pyrC, and tnaA. Sequences of both strands were determined by custom sequencing (Macrogen Inc., Seoul, South Korea). All chromatograms were assembled, manually edited and trimmed in Bionumerics 5.1 (Applied-Maths, Kortrijk, Belgium). Allele numbers were assigned to each isolate by comparing the nucleotide sequence at each locus to all known corresponding alleles available at the V. parahaemolyticus MLST Database (http://pubmlst.org/vparahaemolyticus/).
Environmental and epidemiological surveillance
Following the Galician 2012 outbreak, several sentinel sites in NW Europe (2 along the southern coast of the UK, and 2 in Northern France) were selected to ascertain if this new V. parahaemolyticus variant had emerged in the region. The sites were chosen because they encompass commercially important shellfish harvesting areas that could be analysed practically by participating laboratories over a prolonged period of time in NW Europe. Bivalve shellfish produce monthly (Pacific oysters and Common Mussels), from the Autumn of 2012 until the Winter of 2015, were tested for V. parahaemolyticus in the facilities of IFREMER (France) and CEFAS (UK). Bivalve shellfish produce was processed and tested using molecular methods essentially as previously described (Powell et al. 2013). Strains that were identified possessing the virulence markers tdh and trh were subsequently screened using subtyping methods such as MLST and serotyping. Alongside field-based surveillance, we also contacted relevant health authorities as well as reference laboratories in Europe to identify any clinical cases of V. parahaemolyticus linked to the new variant identified from member state investigations.