CARPATHIAN JOURNAL OF FOOD SCIENCE AND TECHNOLOGY Journal home page: http://chimie-biologie.ubm.ro/carpathian_journal/index.html SEASONAL IMPACT ON THE RISK ASSESSMENT RELATED TO THE SPATIAL PREVALENCE OF ENTEROVIRUS IN OYSTERS FROM OUALIDIA LAGOON IN MOROCCO Rihabe Boussettine1, Najwa Hassou1, Nabil Abouchoaib2, Hlima Bessi1, Moulay Mustapha Ennaji1* 1Team of Virology, Oncology, Total Quality and Medical Biotechnologies Laboratory of Virology, Microbiology, Quality, Biotechnologies/Ecotoxicoloy and Biodiversity (LVMQB-ETB), Faculty of Sciences & Technologies - Mohammedia (University Hassan II of Casablanca - Morocco). 2Casablanca Regional Research and Analysis Laboratory of National Food Safety Office (ONSSA), Province Nouaceur – Casablanca - Morocco. *[email protected] https://doi.org/10.34302/crpjfst/2020.12.2.7 Article history: ABSTRACT Received: Enteroviruses are ubiquitous pathogens that are present worldwide and 3 Febryuary 2020 they are able to survive for long periods in a marine environment. They are Accepted: transmitted through water and food contaminated with stools. The purpose 2 June 2020 of this study was to evaluate the prevalence of enteroviruses in oysters Keywords: collected from Oualidia lagoon, using real-time RT-PCR. To achieve this Enterovirus; objective, 624 oysters representing 52 batches were collected and tested for Oysters; viral contamination using real-time polymerase chain reaction. The results viral contamination; show that 32.69% of the samples tested were positive for enteroviruses. real-time PCR; The spatial distribution of the enteroviruses was statistically significant. water resources. Moreover, a prominent seasonal variation of enterovirus contamination was observed in this study. The consumption of oysters contaminated with this type of virus presents a major risk to human health, By causing serious illnesses such as gastroenteritis, hepatitis, and poliomyelitis, the presence of enteroviruses in oyster production areas represents a potential health risk. 1.Introduction The surveillance systems implemented by indicators of viral presence has been widely the majority of the countries for assessment of questioned (Xavier Abad et al., 1997). the microbiological quality of shellfish and Because shellfish are filter-feeding shellfish harvesting waters are mainly based on organisms, they can accumulate foodborne bacteriological parameters as indicators of fecal viruses, bacteria, and heavy metals in the contamination (Anonymous, 2004, 2005). digestive gland, the mucosa of the gills, and However, several studies have demonstrated other tissues. As a consequence, they have the inadequacy of bacterial indicators for frequently been implicated as major vectors in assessment of viral contamination in shellfish the transmission of many enteric diseases (Croci et al., 2000). In recent years, other (Greening, 2006; Lees, 2000; Dong Joo Seo et putative indicators have been proposed, such as al., 2014). Indeed, several viral outbreaks have bacteriophages, in particular, the B40-8 phage been associated with the consumption of of the Bacteroides fragilis (Havelaar, 1987). bivalve mollusks that had nonetheless met the However, the use of these microorganisms as bacteriological standards (Sugieda et al., 1996). 64 Boussettine et al./ Carpathian Journal of Food Science and Technology, 2020, 12(2), 64-74 The frequency of foodborne viral infections In the past, the method for detecting associated with shellfish consumption is enteroviruses has been based on virus increasing worldwide. This is a public health infectivity of cell lines, but this method is issue that results in a lack of public confidence expensive, time-consuming, and difficult to in shellfish and, therefore, in high economic perform due to the absence of a susceptible cell losses for the seafood industry. line for each enteric virus. Human enteric viruses are present in large Nucleic acid-based methods have proven to numbers in feces and sewage and they can be be highly specific and they can detect even very found in surface waters, groundwaters, and small amounts of viral particles in mussel seawaters. homogenates (LeGuyader et al., 2001). The viruses most often transmitted by However, successful application of molecular contamination of the marine water are methods for enteric virus identification has noroviruses (NoV), hepatitis A virus (HAV), been limited by the presence of RT-PCR hepatitis E virus (HEV), adenovirus (AdV), inhibitors in shellfish. astrovirus (AV), rotavirus (RV), and Dissection of the digestive tract and enteroviruses (EV) (poliovirus, coxsackievirus, diverticula (hepatopancreas) appears to reduce echovirus) (Griffen et al., 2003; Le Guyader et the presence of such inhibitors and it increases al., 2009). They are commonly transmitted by the sensitivity of molecular methods (Le the fecal-oral route. Guyader et al., 2006). Enteroviruses are ubiquitous pathogens that The purpose of this study was to evaluate are present worldwide and they are able to the presence of enteroviruses in oysters survive for long periods in the marine collected for environmental monitoring and to environment (OMS, 2013). Furthermore, the analyze the seasonal variation of the resistance of these viruses to conditions such as contamination levels Additionally, our study acidic pH and extreme temperatures facilitates aimed to illustrate the importance of including a their transmission (Bouseettine et al., 2020). routine virological analysis of shellfish in the Enteroviruses are associated with a monitoring of shellfish harvesting areas in heterogeneous range of diseases. In addition to Morocco. poliomyelitis, they can cause severe acute diseases such as hepatitis, gastroenteritis, 2.Materials and Methods meningitis, and non-specific febrile illness 2.1.Sample collection and processing (Cristina and Costa-Mattioli, 2007; Gibson, Oualidia lagoon (34°47'N - 6°13'W and 2014; Shulman et al., 2006). 34°52'N - 6°14'W) is located on the Moroccan Studies have shown the involvement of a Atlantic coast in the province of Sidi Bennour, panel of enteroviruses, such as enterovirus 168 km south of the city of Casablanca. It types 68-71, in acute flaccid paralysis (AFP) extends parallel to the coast for a distance of (Bahri et al., 2005; Delpeyroux et al., 2013; approximately 8 km and it is 0.5 km wide. Due OMS, 2013). to its location between two hills that constitute Therefore, environmental monitoring can its watershed, Oualidia lagoon is a confluence provide an additional tool to determine the of runoff water during rainfall in the region. On different viruses present in a community (Pinto the other hand, fecal contamination (during the et al., 2007; Shulman et al., 2006) by spreading of animal manure) and wastewater examination of environmental samples, infiltration from septic tanks are the major particularly from bivalve mollusks. mainland inputs to the lagoon (El Himer et al., Enteroviruses are one of the most frequently 2013 ; Hassou et al., 2016). This lagoon is monitored viruses in environmental waterways facing various environmental problems (liquid and they are often used as a bio-indicator of and agricultural practices) that will destroy the viral contamination (Wurtzer et al., 2014). quality of the coast and threaten the collection 65 Boussettine et al./ Carpathian Journal of Food Science and Technology, 2020, 12(2), 64-74 of various aquatic products for March 2019, amounting to a total of 52 consumption (Hassou, 2014). Oyster samples. The sampling sites (S1 and S2) (Crassostrea gigas) samples (n=12 correspond to two oyster farms located in individuals/sample) were collected bimonthly Oualidia lagoon (Figure 1). over a 13-month period from March 2018 to Figure 1. Shellfish collection sites • S1, downstream of the lagoon (sandy 2018 and July-August - September 2018 and a sedimentary), rainy period from October 2018 to May 2019. • S2 in the middle of the lagoon (muddy sedimentary), 2.2.Viral RNA extraction: Bivalve mollusks were transported to the The tissues were homogenized with TRIzol laboratory under controlled temperature (+4 reagent (1/1 w/v), and the homogenates were °C) within 24 hours of being harvested. They incubated at room temperature with mild were processed immediately. The digestive agitation (200 rpm) for 20 minutes. After tissues were dissected, finely chopped, and centrifugation at 3000 x g for 10 minutes, the stored at -80 °C for further use. Aliquots of 2 supernatants were collected and mixed with 4 grams were prepared for extraction of viral μl of the internal extraction control RNA. RNA RNA. The pluviometry data for 2018-2019 was extracted from 500 µl of each supernatant were obtained from the local weather station. using a PureLink™ Viral RNA/DNA Mini Kit Based on the pluviometry data, the study period (Invitrogen), according to the manufacturer’s was divided into a dry period comprising June protocol. The RNA was recovered in DNase/RNase-free sterile water from the kit. 66 Boussettine et al./ Carpathian Journal of Food Science and Technology, 2020, 12(2), 64-74 The RNA quality (ratio of absorbance at 260 Positive control: A positive result indicates and 280 nm) was assessed (30 µl of each that the primers and the probes for detecting the eluate) using a NanoDrop 2000/2000c target enterovirus gene worked properly. In spectrophotometer (Thermo Fisher Scientific, case of a negative result, the test results are USA). invalid and must be repeated. Negative control: A negative result 2.3.One-Step qRT-PCR indicates that the reagents