Hepatitis A Virus PCR Analysis and E. coli Detection in Oysters at Oualidia Lagoon and Their Correlation

The present study aims to evaluate hepatitis A virus (HAV) prevalence and faecal contamination indicators Escherichia coli (E. coli) in oysters from Oualidia lagoon (Moroccan Atlantic coast) and to study the correlation between the two parameters. The survey was carried out on 87 samples of oysters (Crassostrea gigas) collected monthly between November 2015 and February 2017 from three sites corresponding to different oyster farms in the lagoon. Sanitary status of bivalve molluscs was assessed by E. coli enumeration using ISO 16649-3. Detection of hepatitis A virus, was carried out by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) according to ISO 15216-2 method. The prevalence of samples for which E. coli contamination exceeds the threshold of 230 E. coli/100g of flesh and intravalvular fluid (FIF) is 43%. HAV RNA was detected in 2% of the samples analyzed. This RNA was even detected in a sample meeting the bacterial criteria. Viral health surveillance of bivalve molluscs is therefore necessary before their delivery for human consumption.


Introduction
Contaminated shellfishes are frequently involved in foodborne diseases and infections. Coastal marine ecosystem is threatened by a wide range of pollutants that filter-feeding molluscs can accumulate in their tissues and are therefore likely Food and Nutrition Sciences to cause a serious risk for consumer's health [1] [2] [3]. Viral persistense has been demonstrated in bivalve molluscs and as they are often consumed raw or slightly cooked, the microbial risk is therefore increased. [3] [4] [5] [6].
Enteric viruses of human origin are very common in seawater where they are rejected in large amount, shed by infected people [7].
They belong to a large panel of family and genus and among this diversity, HAV, ranked as priority hazards by WHO/FAO, is known since antiquity to be responsible for many epidemics, causing acute inflammation of the liver [8]. Nowadays it is still implicated in several outbreaks around world as 1.4 millions clinical cases occur annually [9].
This illness is strongly related to age: if it is a mild disease in children under five, protective antibodies bestow lifelong immunity, in older children and adults may have acute inflammation of the liver with icteric symptomatology, though without involving chronic liver disease [10]. Healing is current; however, in rare cases, it can lead to fulminant hepatitis that progresses to a fatal outcome. The economic burden is significant as recovery can be long and disabling [11].
HAV is a member of the Picornaviridae family, genus Hepatovirus [12]. Although known as limited to man and some species of non-human primates [13], presence of HAV has been recently detected in animals other than primates as seals [14], woodchuck [15] and marsupials [16]. A single serotype is present in human and strains are classified into six genotypes [17]. Very resistant under various conditions of environment, it can survive, and remain infectious, for a long period outside a host [18]. Epidemics occur in a cyclical manner by fecal-oral transmission and close contact with infected persons and contaminated food and water [19].
HAV outbreaks linked to consumption of bivalve molluscs represent 17% of total foodborne outbreaks [5]. These outbreaks occur as a consequence of the contamination of surrounding water by pathogens.
Currently, microbiological surveillance of bivalve molluscs is based solely on bacteriological parameters.

Description of Study Area
Oualidia is a lagoon without outlet, located on the Atlantic coast, 168 km south of Casablanca, at (32˚46'N, 09˚01'W). It is listed as RAMSAR site (wetlands of international importance).
It communicates with the ocean through a major inlet of 150 m wide and a secondary one of 50 m. The sedimentation is sandy near the passes and silty upstream where turbidity is higher in the rainy season, between October and April [22]. An artificial dam contributes to trap sedimentary organic matters and protects this ecosystem from confinement. Anthropic pressure on this ecosystem is linked to agriculture, tourism, oyster farming and urbanization. Even if some residential on the shoreline only have septic tanks, the commissioning of the treatment plant in 2012 helped reduce this pressure.
Renowned for its oysters with a production close to 36 tons annualy, Oualidia lagoon is classified as a "B" harvesting area. Therefore, depuration processing is a statutory requirement before marketing for human consumption.

Shellfish Sampling
Oyster samples (n = 12 individuals/sample) were collected, over 16 months between November 2015 and February 2017, twice per month except for May and June where the sample has been taken only once, giving a total of 87 samples.
Sampling sites (S1, S2 and S3) correspond to three oyster farms located in Oualidia Lagoon. S1 is located downstream of the lagoon near the principal pass with a sandy sedimentary facies. S2 and S3 are further upstream, before inner dam location, and are characterized by muddy sediment (Figure 1).
Bivalve molluscs were transported to the laboratory under controlled temperature (+4˚C) within 24 hours. The samples were processed rapidly for Escherichia coli and for HAV or stored at −80˚C.

Viral RNA Extraction
To evaluate the presence of HAV, RNA extraction method included the use of guanidine isothiocyanate as chaotropic agent. Shellfish were thawed, cleaned and shukked in sterile conditions, the hepatopancreas was removed and chopped using a razor blade. 2.0 ± 0.2 g subsample of digestive tissues was transferred to clean tube and mixed with 10 μl of mengovirus as a process control of nucleic acid extraction (Ceeramtools ® ). 2 ml of Protéinase K solution (100 μl/ml) was added to digest the tissues that were incubated with shaking (37˚C ± 1.0˚C/320 ± 20 rpm) for 60 min followed by a second incubation (60˚C ± 2.0˚C/15min). Then samples were centrifuged at 3000 g for 5 min and the supernatant collected in a clean tube. RNA was extracted from 500 μl and eluted in 100 μl of elution buffer using commercial Nucleospin RNA virus kit (Macherey Nagel, Germany) following the manufacturer's instruction. RNA was tested for HAV immediately or stored at −20˚C until use.

E. coli Enumeration
Samples were analyzed for E. coli enumeration using most probable number

Evaluation of Viral Contamination in Shellfish Samples
Among the 87 samples analysed, 2% were HAV positive. The HAV-positive samples were collected during the winter after a rainy-period, in November 2015 and January 2016 (Table 2). Regarding the sampling sites, the two HAV-positive samples were from S3 located the most upstream.

E. coli Bacterial Contamination and Correlation with Viral Contamination
Analysis of oyster samples showed 43% are contaminated by E. coli and the legislation in force defines Oualidia lagoon as a "B" harvesting area (90% of results ≤ 4600 E. coli FIF and 100% ≤ 46,000 E. coli/100g of (FIF)) ( Figure 2). However, contamination with hepatitis A virus is variable as a sample HAV positive showed a low contamination with E. coli (not exceeding 230 E. coli/100g FIF) while the other HAV positive sample showed a high rate of contamination by E. coli.
Samples contaminated by HAV come from (S3) while RNA-HAV was not detected in samples from (S1) and (S2). Thus, our results showed no correlation between HAV and E. coli contamination p > 0.05.

Discussion
Shellfish viral contamination data are scarce in Morocco. This study was conducted to evaluate the presence of HAV in oysters collected from different sites of the Oualidia lagoon, classified as "B" harvesting area. We found that 2% of the samples are contaminated with this virus.
The prevalence of contaminated oysters found in our study is low given Moroccan epidemiological context. Thus the year 2016 when most of the samples were taken, was a year od drought which reduced the incidence of soil leaching, but similar finding was reported previously [24]. Yet in the mediterranean rim countries, incidence rates of HAV are quite different between regions. It was detected in 26% of shelfish samples in Tunisia [25], 10.1% in Spain [26] whereas all samples were HAV negative in south Italy [27]. The results obtained can be the consequence of the water renewal influenced by currents and other hydrographic variables [28] [29].
Cases of hepatitis A are reported throughout the year and seasonal pattern of HAV have not been clearly observed [30]. Although, positive samples in our study were taken during the winter period. Similar data were reported in Tunisia, Hong Kong and USA [31] [32] [33]. This result can be linked to rainfalls, sively to older ages but with more severe symptoms. Thereby decreased immunity will lead to more frequent outbreaks [34]. However, the long incubation period that separates the contamination and the onset of symptoms makes it diffi-

Conclusion
The absence of correlation between faecal bacteria as indicators of microbiological contamination and food-borne viruses highlights the need of additional con-

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.