L’ostréiculture française repose essentiellement sur l’élevage de l’huître creuse, Crassostrea gigas confronté cependant à des épisodes de mortalités anormales, touchant les différents stades de vie de l'huître. Plusieurs études ont démontré l’implication d’agents infectieux comme des bactéries du genre Vibrio dans ces mortalités. En France, V. aestuarianus est une bactérie connue depuis les années 2000 pour impacter la survie des huîtres. Sa fréquence de détection dans les cas de mortalités d’huîtres adultes analysés par le réseau REPAMO (REseau de PAthologie des Mollusques) est cependant en augmentation depuis 2011. Dans ce contexte, afin d’étudier le développement de la maladie induite par V. aestuarianus chez C. gigas, un modèle d’expérimentation par balnéation dans de l’eau de mer contenant des bactéries fraichement excrétées, au plus proche des modes de contaminations naturelles, a été développé. Le suivi de la présence de la souche 12/016 (souche virulente) et son mutant 12/016ΔvarS (souche non-virulente) dans l’eau de mer, dans les différents tissus et dans l’hémolymphe des animaux vivants et moribonds a montré que le cycle infectieux est constitué de I) une phase de pénétration rapide de la bactérie dans l’hôte (moins de 24h) et de colonisation initiale de l’hémolymphe et des branchies, II) une phase d’incubation de 3-4 jours au cours de laquelle la souche virulente se multiplie dans l'ensemble des tissus d'huître et III) une phase de mortalités aiguës (mort de l'animal par septicémie). A ce stade, le recrutement et la lyse hémocytaire ainsi que différentes lésions tissulaires comme la lyse du tissu conjonctif sous-épithélial au niveau du manteau et l’atrophie de diverticules digestives ont été observés. D'autre part, l'étude d’expression relative de 18 gènes de virulence connus chez d’autres Vibrion a montré que l’expression des facteurs de virulence de V. aestuarianus est régulée différemment au cours de différentes étapes de l'infection et nous avons observé que la métalloprotéase vam est significativement sur-exprimée dans l’hémolymphe des animaux contaminés à j4 post infection (étape intermédiaire de l’infection) par rapport à son niveau d’expression au premier jour de l’infection (étape précoce). / Oyster-farming in France is mainly based on pacific cupped oysters, Crassostrea gigas culture. Currently, oyster culture is confronted by several abnormal episodes of mass mortality affecting all life stages. These outbeaks involve, among other factors, infectious agents including bacteria of the genus Vibrio. In France, since 2000, V. aestuarianus is known as a bacterium that impacts the survival of C. gigas. Since 2011, its detection frequency in adult oyster mortalities cases reported by REPAMO network (REseau de PAthologie des Mollusques), is constantly increasing. In this context, to study V. aestuarianus disease development in C. gigas, an experimental infection model based on immersion in sea water containing freshly shed bacteria was developed. By monitoring the presence of strain 12/016 (virulent strain) and its mutant 12/016 ΔvarS (non-virulent strain) in the seawater, in the different tissues and in the haemolymph of live and moribund animals, we showed that the infectious cycle consists of several successive phases: I) rapid penetration of the bacterium into the host (less than 24 hours) and initial colonization of the haemolymph and gills, II) 3-4 days of incubation during which the virulent strain multiplies in whole oyster tissues and III) acute mortalities (animal death due to septicemia). At this stage, recruitment and haemocyte lysis as well as different tissue lesions such as lysis of the sub-epithelial connective tissue in the mantle and atrophy of digestive diverticula were observed. On the other hand, relative expression of 18 virulence genes (known in other Vibrion) were analyzed by RT-QPCR. Virulence factors are regulated differently during different stages of infection and vam metalloprotease is significantly over-expressed in the haemolymph of infected animals at day 4 post infection (intermediate stage of infection) compared to its level of expression at day 1 post infection (early stage).

Vibrio parahaemolyticus is a Gram-negative, halophilic pathogen that occurs naturally in coastal and estuarine environments. This human pathogen is frequently isolated from a variety of seafood, particular oysters, and is the leading cause of gastroenteritis associated with seafood consumption. Several outbreaks of V. parahaemolyticus infections linked to consumption of raw oysters have been documented. Contamination of oysters with V. parahaemolyticus is a concern for public health. This study investigated the efficacy of high pressure processing (HPP) in inactivating V. parahaemolyticus in raw Pacific oysters (Crassostrea gigas) and identified a process condition capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in raw oysters for commercial application. Raw Pacific oysters were inoculated with a clinical strain of V. parahaemolyticus 10293 (O1:K56) to levels of 10⁴⁻⁵ cells per gram and processed at 293 MPa (43K PSI) for 90, 120, 150, 180 and 210 s. Populations of V. parahaemolyticus in oysters after processes were analyzed with the 5-tube most probable number (MPN) method. A minimum HPP of 293 MPa for 120 s at groundwater temperature (8±1 °C) was identified capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in Pacific oysters. The HPP (293 MPa for 120 s at 8±1 °C) was validated at a commercial scale according to the FDA's National Shellfish Sanitation Program Post Harvest Processing (PHP) Validation/Verification Interim Guidance for Vibrio vulnificus and Vibrio parahaemolyticus. Negative results obtained by the MPN method were confirmed with a multiplex PCR detecting genes encoding thermolabile hemolysin (tl), thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). Oysters processed at 293 MPa for 120 sec had a shelf life of 6-8 days when stored at 5 °C or 16-18 days when stored in ice. This validated HPP was accepted by the FDA as a post harvest process to eliminate V. parahaemolyticus in raw oysters. / Graduation date: 2012

Gulf oysters are harvested and consumed year-round, with more than 90% consumed in a raw, unprocessed state. A chief concern of policymakers in recent years is the incidence of Vibrio vulnificus infection following raw seafood consumption. V.vulnificus refers to a halophilic bacterium naturally occurring in brackish coastal waters, which concentrates in filter-feeding oysters. Proposed FDA legislation requiring processing of all raw Gulf oysters sold during warmer summer months threatens the Gulf oyster industry, as little to no research regarding demand for post-harvest processing (PHP) has preceded the potential mandate. This research endeavors to examine the relationship between oyster consumers' fears of V.vulnificus infection and their willingness-to-pay (WTP) for processing of an oyster meal. The psychological model of Protection Motivation Theory (PMT) is employed alongside the economic framework of contingent valuation (CV) to result in an analysis of oyster processing demand with respect to threats and efficacy. A survey administered to 2,172 oyster consumers in six oyster producing states elicits projected consumption and PMT data. Principal Component Analysis is used to reduce the number of PMT variables to a smaller size, resulting in five individual principal components representing the PMT elements of source information, threat appraisal, coping appraisal, maladaptive coping, and protection motivation. Using survey data, the marginal willingness-to-pay (MWTP) for PHP per oyster meal is also calculated, and the five created PMT variables are regressed on this calculation using four separate OLS models. Results indicate significant correlation for four of the five created PMT variables. In addition, a mean MWTP for PHP of $0.31 per oyster meal is determined, contributing to the demand analysis for processing of Gulf oysters. The findings suggest a strong relationship between the fear elements and the demand for processing, and support arguments in favor of further research on specific PHP treatments and the necessity for a valid PMT survey instrument.

Recent research has focused on the non-lethal effects of predator intimidation and fear, dubbed non-consumptive effects (NCEs), in which prey actively change their behavior and habitat use in response to predator chemical cues. Although NCEs can have large impacts on community structure, many studies have ignored differences in predator population structure and properties of the natural environment that may modify the magnitude and importance of NCEs. Here, I investigated the roles of predator size and density (i.e. biomass), as well as habitat characteristics, on predator risk assessment and the magnitude of consumptive and NCEs using blue crabs, mud crabs, and oyster prey as a model system. Predation experiments between blue crabs and mud crabs demonstrated that blue crabs consume mud crabs; however, the consumptive effects were dependent upon blue crab body size and habitat type. When mud crabs were exposed to chemical cues from differing biomasses of blue crabs in laboratory mesocosms, mud crab activity and predation on oysters was decreased in response to high biomass treatments (i.e. large and multiple small blue crabs), but not to low biomass predators (i.e single small blue crab), suggesting that risk associated with predator size is perceptible via chemical cues and is based on predator biomass. Further experiments showed that the perception of risk and the magnitude of the NCEs were affected by the sensory cues available and the diet of the blue crab predator. The NCE based on blue crab biomass was also demonstrated in the field where water flow can disperse cues necessary for propagating NCEs. Properties of water flow were measured within the experimental design and during the experiment and confirmed cage environments were representative of natural conditions and that patterns in NCEs were not associated with flow characteristics. These results affect species conservation and commercial fisheries management and demonstrate that we cannot successfully predict NCEs without considering predator size structure and the contexts under which we determine predator risk.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Vírus causadores de gastroenterite, descritos como pequenos vírus de estrutura arredondada (Small Round Structured Viruses-SRSV), foram detectados em extratos de ostras Crassostrea spp., coletadas em regiões distintas do litoral do Estado de São Paulo, utilizando a Reação em Cadeia por Polimerase com transcrição reversa (RT-PCR).Treze lotes de amostras, contendo 10 g de estômagos e divertículos, foram processados para extração e concentração das partículas virais, mediante adsorção-eluição e precipitação por polietilenoglicol (PEG 6000), e purificação com fluorocarbono (Freon, Dupont Co.) para eliminação prévia dos inibidores da reação de RT-PCR. A extração do ácido nucleico viral, foi realizada com uma mistura de isotiocianato de guanidina e fenol (TRIzol ®, Gibco) e clorofórmio, sendo completada com uma purificação em coluna spin, com membrana de polissulfona (Millipore,Corp.). A reação de RT-PCR foi realizada em uma única etapa, utilizando o kit \'\'Super Script ONE-STEP RT-PCR System\'\'® (Gibco). A reação de amplificação enzimática revelou, por eletroforese em gel de agarose (2%), corada com brometo de etídio, produtos de 123 bp, em 3 (23%) dos 13 lotes de amostras coletadas durante o verão de 1998, sendo verificadas em uma delas por imunomicroscopia eletrônica (IME),partículas de SRSV. Os produtos encontrados pertencem ao grupo genômico G2(Snow Mountain e outros vírus). A aplicação deste método possibilitou pela 1ª vez a detecção molecular de SRSVs no Brasil em ostras naturalmente contaminadas. / The Small Round Structured Viruses (SRSVs) have been associated with gastroenteritis in humans, and were detected in extracts of oysters Crassostrea spp., by Reverse Transcriptase Polymerase Chain Reaction (RT - PCR). Thirteen lots of samples, originated from different regions of the State of São Paulo, were processed for viral particles extraction. The samples, containing 10 g of stomach and diverticula, were extracted by adsorption - elution and polyethylene glycol (PEG 6000) precipitation, and fluorocarbon (Freon, Dupont Co.) was used for purification and previous elimination of RT - PCR inhibitors. For the viral nucleic acid extraction, guanidine isothiocyanate - phenol mixture (TrizolÒ, Gibco ) and chloroform were used, followed by a polysulfone spin column (Millipore, Corp.) purification. The RT-PCR was done in one step, using the \'\'Super Script ONE-STEP RT-PCR System\'\'® (Gibco). The enzimatic amplification reaction run in 2% agarose gel electrophoresis stained with ethidium bromide, revealed 123 bp products in 3 (23%) of the 13 lots of samples collected during the summer of 1998. One of these three samples was positive for SRSV particles by Immune Electron Microscopy (IEM). These 3 products were classified in the Genogroup G2 (Snow Mountain and others viruses). The application of this methodology made it possible to detected, in a molecular basis, the first cases of SRSVs in environmentally contaminated oysters in Brazil.

Global change poses new threats for life in the oceans forcing marine organisms to respond through molecular acclimatory and adaptive strategies. Although bivalve molluscs are particularly tolerant and resilient to environmental stress, they must now face the challenge of more frequent and severe Harmful Algal Blooms (HABs) episodes. These massive outbreaks of microalgae produce toxins that accumulate in the tissues of these filter-feeder organisms, causing changes in their gene expression profiles, which in turn modify their phenotype in order to maintain homeostasis. Such modifications in gene expression are modulated by epigenetic mechanisms elicited by specific environmental stimuli, laying the foundations for long-term adaptations. The present work aims to examine the links between environmental stress in bivalve molluscs (with especial emphasis on Harmful Algal Blooms) and specific epigenetic marks triggering responses through modifications in gene expression patterns. Overall, a better understanding of the molecular strategies underlying the conspicuous stress tolerance observed in bivalve molluscs will provide a framework for developing a new generation of biomonitoring strategies. In addition, this strategy will represent a valuable contribution to our knowledge in acclimatization, adaptation and survival. With that goal in mind, the present work has generated transcriptomic data using RNA-Seq and microarray technologies, facilitating the characterization and investigation of the epigenetic mechanisms used by the Mediterranean mussel Mytilus galloprovincialis during responses to HAB exposure. That information was made publicly available through a specialized online resource (the Chromevaloa Database, chromevaloa.com) assessing the response of chromatin-associated transcripts to Okadaic Acid. Specific epigenetic marks have been assessed under lab-controlled exposure experiments simulating the natural development of the HAB Florida Red Tide (FRT). Results demonstrate a role for the phosphorylation of histone H2A.X and DNA methylation in the response to FRT in the Eastern oyster Crassostrea virginica. Lastly, the study of co-expression networks based on RNA-Seq data series from the Pacific oyster Crassostrea gigas reveals dynamic transcriptomic patterns that vary with time, stressor and tissue. However, consistent functional profiles support the existence of a core response to general conditions of environmental stress. Such response involves metabolic and transport processes, response to oxidative stress and protein repair or disposal, as well as the activation of immune mechanisms supporting a tightly intertwined neuroendocrine-immune regulatory system in bivalves.

Στα πλαίσια αυτής της διδακτορικής εργασίας μελετήθηκαν οι εμπορικά σημαντικότερες περιοχές καλλιέργειας και συγκομιδής οστρακοειδών του Ελλαδικού χώρου. Κατά τη διάρκεια περιόδου 18 μηνών πραγματοποιήθηκε μηνιαία συλλογή δειγμάτων στρειδιών (Οstrea edulis) και μυδιών (Mytilus galloprovincialis), τα οποία συλλέχθηκαν από έξι (6) διαφορετικά σημεία του Ελλαδικού χώρου και αναλύθηκαν για τους εντεροϊούς (EV), τους αδενοϊούς (Adv), τον ιό της ηπατίτιδας Α (HAV), τους ιούς Noro I και II (NLVI και NLVII), για το βακτήριο Ε. coli, καθώς και για σωματικούς κολιφάγους, τους F-sperific RNA βακτηριοφάγους και τους βακτηριοφάγους του Β. fragilis. Επιπλέον αναπτύχθηκαν μέθοδοι τόσο για την ανίχνευση παθογόνων ιών ανθρώπινης προέλευσης στα οστρακοειδή, όσο και για την ανίχνευση των "πιθανών δεικτών" αυτών των ιών. Οι μέθοδοι εξετάστηκαν προκειμένου να αξιολογηθεί η απόδοση καλής ποιότητας από όλα τα εργαστήρια μέσω διεργαστηριακών αναλύσεων. Η μέθοδος που εφαρμόστηκε σε αυτή τη μελέτη για την ανίχνευση των ιών στα οστρακοειδή βασίζεται στην εξαγωγή και την ομογενοποίηση του πεπτικού αδένα με χρήση διαλύματος γλυκίνης, pH 10, απομόνωση των νουκλεϊνικών οξέων και ενίσχυση του γονιδιώματος των ιών που αναλύονται. Για την ανίχνευση του βακτηρίου E. coli χρησιμοποιήθηκε η μέθοδος των πολλαπλών σωλήνων, ενώ για την ανίχνευση των βακτηριοφάγων χρησιμοποιήθηκε η μέθοδος καλλιέργειας διπλοστιβάδας. Για το βακτήριο E. coli, σε σύνολο 138 δειγμάτων, 110 δείγματα (ποσοστό 79,7%) βρέθηκαν να ανήκουν στην κατηγορία Α (MPN/100g σάρκας = <20 έως 220), δηλαδή χαρακτηρίζονται σαν δείγματα χαμηλής μόλυνσης, 25 δείγματα (ποσοστό 18,1%) βρέθηκαν να ανήκουν στην κατηγορία Β (MPN/100g σάρκας = 220 έως 3500), οπότε χαρακτηρίζονται σαν δείγματα μεσαίας μόλυνσης, ακατάλληλα προς κατανάλωση χωρίς να προηγηθεί διαδικασία εξυγίανσης, ενώ μόνο 3 δείγματα (ποσοστό 2,2%) βρέθηκαν να ανήκουν στη κατηγορία C (MPN/100g σάρκας =3500 έως >18000), δηλαδή είναι δείγματα υψηλής μόλυνσης. Οι ιοί που εμφανίζονται με μεγαλύτερη συχνότητα στα οστρακοειδή της Ανατολικής Μεσογείου είναι οι αδενοϊοί (34% των δειγμάτων βρέθηκαν θετικά για τους αδενοϊούς) και ακολουθούν οι εντεροϊοί (16,7% των δειγμάτων βρέθηκαν θετικά για τους εντεροϊούς). Αντίθετα, ο ιός της ηπατίτιδας Α (ποσοστό θετικών δειγμάτων = 4,34%), καθώς και οι ιοί Noro I (ποσοστό θετικών δειγμάτων = 2,1%) και Noro II (ποσοστό θετικών δειγμάτων = 1,47%%) εμφανίζονται σε μικρό ποσοστό δειγμάτων. Τέλος, 80 δείγματα (58%) βρέθηκαν θετικά (παρουσία πλακών βακτηριοφάγων) για τους σωματικούς κολιφάγους, με τον αριθμό των πλακών να κυμαίνεται από 71,4 έως 584800 pfp/100g, 52 δείγματα (37,7%) βρέθηκαν θετικά για τους F-specific RNA βακτηριοφάγους (αριθμός των πλακών από 76,2 έως 17051 p100g) και 33 δείγματα (24%) βρέθηκαν θετικά για τους βακτηριοφάγους του Bacteroides fragilis (αριθμός των πλακών από 194.5 έως 5266,25 pfp/100g). Τόσο για το βακτήριο E. coli όσο και για τους βακτηριοφάγους πραγματοποιήθηκαν διεργαστηριακές αναλύσεις προτύπων, οι οποίες οδήγησαν στο συμπέρασμα ότι οι αντίστοιχες μέθοδοι χαρακτηρίζονται ως αξιόπιστες. Η στατιστική ανάλυση έδειξε ότι το βακτήριο E. coli παρουσιάζει θετική συσχέτιση με τους σωματικούς κολιφάγους, αλλά δεν δείχνει στατιστικά σημαντική συσχέτιση ούτε με τους F-specific RNA βακτηριοφάγους, ούτε με κανέναν από τους ιούς εντερικής προέλευσης. Επίσης, θετική συσχέτιση παρουσίασαν οι αδενοϊοί με τους εντεροϊούς, καθώς και οι σωματικοί κολιφάγοι με τους βακτηριοφάγους του B. fragilis. Η μοναδική συσχέτιση μεταξύ ιών εντερικής προέλευσης και βακτηριοφάγων βρέθηκε για τους αδενοϊούς και τους βακτηριοφάγους του B. fragilis. Εάν αυτό επιβεβαιωθεί σε περαιτέρω μελέτες, τότε η συγκεκριμένη κατηγορία βακτηριοφάγων θα μπορούσε να αποτελέσει έναν καλό δείκτη πρόβλεψης της παρουσίας αδενοϊών σε δείγματα οστρακοειδών. Επιπλέον μελετήθηκε η σχέση που μπορεί να υπάρχει μεταξύ των φυσικοχημικών παραμέτρων και των μικροοργανισμών που εξετάστηκαν. Η επεξεργασία αυτή οδήγησε στο συμπέρασμα ότι το βακτήριο E. coli ανιχνεύεται σε μεγαλύτερα ποσά όταν το διαλυμένο οξυγόνο και η περιεκτικότητα σε άλας του ύδατος είναι αυξημένα. Αντίθετα, αύξηση της θερμοκρασίας οδηγεί σε μείωση της ανίχνευσης του βακτηρίου. Επίσης, η περιεκτικότητα σε άλας φαίνεται να επηρεάζει θετικά και τον ιό της ηπατίτιδας Α, αν και ο μικρός αριθμός θετικών δειγμάτων γι’αυτόν τον ιό δεν μπορεί να επιτρέψει την εξαγωγή ασφαλών συμπερασμάτων. Το pH και το διαλυμένο οξυγόνο των υδάτων οδηγεί σε αύξηση της ανίχνευσης των βακτηριοφάγων του B. fragilis, χωρίς όμως να μπορούμε να ισχυριστούμε ότι κάτι τέτοιο ισχύει, λόγω του μικρού αριθμού θετικών δειγμάτων γι’αυτούς τους βακτηριοφάγους. Τέλος, η αύξηση της θερμοκρασίας των υδάτων φαίνεται να οδηγεί και σε αύξηση της παρουσίας των F-specific RNA βακτηριοφάγων, και το ίδιο παρατηρήθηκε και με την αύξηση του διαλυμένου οξυγόνου στο νερό. Η παρούσα μελέτη αποτελεί την πρώτη διεξοδική έρευνα για την ιογενή κοπρανώδη μόλυνση τον οστρακοειδών στην Ελλάδα. Επιπλέον, αντιπροσωπεύει την πρώτη μελέτη σχετικά με τη αποτελεσματικότητα των οργανισμών - δεικτών ιϊκής μόλυνσης, καθώς και για τη συσχέτιση της μικροβιολογικής επιβάρυνσης των οστρακοειδών με τις φυσικοχημικές παραμέτρους του περιβάλλοντος ύδατος. Η μελέτη κατάλληλων δεικτών που σχετίζονται με την παρουσία εντερικών ιών στα οστρακοειδή οδήγησε σε χρήσιμα συμπεράσματα για τη χρήση της ανίχνευσης των βακτηριοφάγων ως δεικτών ιϊκής μόλυνσης. Εντούτοις, απαιτείται περαιτέρω μελέτη προκειμένου να προσδιοριστεί και η χρήση των βακτηριοφάγων ως δεικτών που θα μαρτυρούν την προέλευση (ανθρώπινη ή ζωική) των εντερικών ιών που ανιχνεύονται στα οστρακοειδή. / In this doctorate investigation, important shellfish growing areas of Greece have been defined and studied. Oysters (Ostrea edulis) and mussels (Mytilus galloprovincialis) were obtained on a monthly basis over an 18 month sampling period. These samples were collected by six (6) different points of Greece and were analyzed for enteroviruses (EV), adenoviruses (Adv), virus of hepatitis A (HAV), Noro viruses I and II (NLVI and NLVII ), bacterium E. coli, as well as for somatic coliphages, F-sperific RNA bacteriophages and bacteriophages of B. fragilis. Moreover, methods were developed for the detection of pathogenic viruses of human origin in the shellfish, as well as for the detection of potential "viral indicators". The methods were examined in order to validate the good quality performance from all the laboratories via interlaboratory analyses. The method that used in this study for the detection of human enteric viruses in the shellfish is based on the export and homogenisation of digestive gland with glycine buffer at pH 10, viral nucleic acid extraction and amplification of the genomes of the analysed human viruses. The procedure applied for detection of E. coli consists on a five tube, three dilution most probable number (MPN) method, while the method for the detection of bacteriophages was the double-agar-layer method. For E. coli analysis, in a total number of 138 samples, 110 samples (79,7%) were found to belong in category A (MPN / 100 g of flesh = < 20 until 220), that it means these samples are characterized as samples of low pollution, 25 samples (18,1%) were found to belong in category B (MPN / 100 g of flesh = 220 until 3500), therefore are characterized as samples of intermediate pollution, inadequate to consumption without precedes process of cleansing, while only 3 samples (2,2%) were found to belong in category C (MPN / 100 g of flesh = 3500 until > 18000), that it means they are samples of high pollution. The viruses that are presented with higher frequency in the shellfish of Eastern Mediterranean are the adenoviruses (34% of samples were found positive for adenoviruses) and follow enteroviruses (16,7% samples they were found positive for enteroviruses). On the contrary, virus of hepatitis A (percentage of positive samples = 4,34%), as well as the Noro I viruses (percentage of positive samples = 2,1%) and Noro II viruses (percentage of positive samples = 1,47%%) are presented in small number of samples. Finally, 80 samples (58%) were found positive (presence of plaques of bacteriophages) for somatic coliphages, with the number of plaques between 71,4 and 584800 pfp / 100 g, 52 samples (37,7%) were found positive for F - specific RNA bacteriophages (number of plaques from 76,2 to 17051 pfp/ 100 g) and 33 samples (24 %) were found positive for the bacteriophages of B. fragilis (number of plaques from 194,5 to 5266,25 pfp / 100 g). Interlaboratory studies involved the testing of reference materials of E. coli and bacteriophages were used as part of the good quality performance assessment program to be applied all over the study, and led to the conclusion that the corresponding methods are characterized by good reliability. According to the statistical analysis of the results, the presence of E. coli seems to be significantly related with the presence of somatic coliphages. However, E. coli do not present significant statistical relation neither with F - specific RNA bacteriophages, nor with all of the viruses of intestinal origin. Also, adenoviruses were significantly related with enteroviruses, as well as somatic coliphages with the bacteriophages of B. fragilis. The unique significant relation between viruses of intestinal origin and bacteriophages was found for the adenoviruses and bacteriophages of B. fragilis. If this is confirmed in further studies, then this category of bacteriophages could constitute a good indicator of forecast of presence adenoviruses in samples of shellfish. Moreover, we studied the relation that can exist between the physic-chemical parameters and the micro-organisms that were examined. This analysis led to the conclusion that E. coli is detected in higher levels when the dissolved oxygen and the salinity of water are increased. On the contrary, increase of temperature leads to reduction of detection of E. coli. Also, the salinity appears to influence positively also virus of hepatitis A, even if the small number of positive samples of this virus cannot allow the export of sure conclusions. The pH and the dissolved oxygen of waters lead to increase of detection of bacteriophages of B. fragilis, but the small number of positive samples for these bacteriophages can’t give safe conclusions. Finally, the increase of temperature of waters appears to lead also to increase of presence of F - specific RNA of bacteriophages, and the same was also observed with the increase of dissolved oxygen in water. This study constitutes the first extensive research for the fecal viral pollution of shellfish in Greece. Moreover, it represents the first study with regard to the effectiveness viral indicators, as well as for the correlation of microbiological parameters of shellfish with the physical-chemical parameters of water. The study of suitable indicators that are related with the presence of enteric viruses in the shellfish led to useful conclusions on the use of detection of bacteriophages as indicators of viral pollution. Nevertheless, further study is required in order to determine also the use of bacteriophages as indicators that will testify the origin (human or animal) of the enteric viruses that are detected in the shellfish.