Publications by authors named "Mélanie Gay"

14 Publications

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Optimization of tools for the detection and identification of Cryptocotyle metacercariae in fish: Digestion method and viability studies.

J Fish Dis 2021 Jul 21. Epub 2021 Jul 21.

ANSES, Laboratory for Food Safety, Boulogne-sur-Mer, France.

Some trematode metacercariae, including marine digeneans belonging to the genus Cryptocotyle, induce black spots in target tissues due to the attraction of fish host melanophores. To promote precise quantification of infection, the counting of black spots has to be confirmed by reliable quantification of metacercariae after tissue digestion. This process ensures the isolation of undamaged parasites for morphological and molecular identification. The aim of this work was to optimize the pepsin digestion protocol and to assess the duration of viability of Cryptocotyle metacercariae in fish post-mortem (pm). Four digestion protocols were compared by measuring the viability rate of metacercariae. The present study shows that the orbital digestion method was the least destructive for metacercariae and allowed better quantification of Cryptocotyle infection. Moreover, morphological identification seemed reliable up to 8 days pm for Cryptocotyle infection.
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http://dx.doi.org/10.1111/jfd.13495DOI Listing
July 2021

A review of molecular identification tools for the opisthorchioidea.

J Microbiol Methods 2021 Aug 1;187:106258. Epub 2021 Jun 1.

ANSES, Laboratory for Food Safety, F-62200 Boulogne-sur-Mer, France. Electronic address:

The superfamily Opisthorchioidea encompasses the families Cryptogonimidae, Opisthorchiidae and Heterophyidae. These parasites depend on the aquatic environment and include marine and freshwater species. Some species, such as Clonorchis sinensis and Opisthorchis viverrini, have a high impact on public health with millions of infected people worldwide and have thus been the object of many studies and tool developments. However, for many species, tools for identification and detection are scarce. Although morphological descriptions have been used and are still important, they are often not efficient on the immature stages of these parasites. Thus, during the past few decades, molecular approaches for parasite identification have become commonplace. These approaches are efficient, quick and reliable. Nonetheless, for some parasites of the superfamily Opisthorchioidea, reference genomic data are limited. This study reviews available genetic data and molecular tools for the identification and/or the detection of this superfamily. Molecular data on this superfamily are mostly based on mitochondrial and ribosomal gene sequence analyses, especially on the cytochrome c oxidase subunit 1 gene and internal transcribed spacer regions respectively.
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http://dx.doi.org/10.1016/j.mimet.2021.106258DOI Listing
August 2021

First Report on the Prevalence and Subtype Distribution of sp. in Edible Marine Fish and Marine Mammals: A Large Scale-Study Conducted in Atlantic Northeast and on the Coasts of Northern France.

Microorganisms 2020 Mar 24;8(3). Epub 2020 Mar 24.

Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 9017 - CIIL - Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France.

is frequently identified in humans and animal hosts and exhibits a large genetic diversity with the identification of 17 subtypes (STs). Despite its zoonotic potential, its prevalence and ST distribution in edible marine fish and marine mammals remain unknown. A large-scale survey was thus conducted by screening 345 fish caught in Atlantic Northeast and 29 marine mammals stranded on the coasts of northern France for the presence of the parasite using real-time Polymerase Chain Reaction PCR. The prevalence of the parasite was about 3.5% in marine fish. These animals were mostly colonized by poikilotherm-derived isolates not identified in humans and corresponding to potential new STs, indicating that fish are natural hosts of . Marine fishes are also carriers of human STs and represent a likely limited source of zoonotic transmission. 13.8% of the marine mammals tested were colonized and 6 different STs were identified including 3 potential new STs. The risk of zoonotic transmission through marine mammals is insignificant due to the lack of repeated contact with humans. The present survey represents the first data regarding the prevalence and ST distribution of in marine fish and marine mammals and provides new insights into its genetic diversity, host range and transmission.
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http://dx.doi.org/10.3390/microorganisms8030460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144014PMC
March 2020

Prevalence, Molecular Identification, and Risk Factors for Infection in Edible Marine Fish: A Survey Across Sea Areas Surrounding France.

Front Microbiol 2019 15;10:1037. Epub 2019 May 15.

CNRS, Inserm, CHU Lille, U1019 - UMR 8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Université de Lille, Lille, France.

, a zoonotic pathogen, is able to infect a wide range of hosts including wild and domestic animals, and humans. Although it is well known that some parasites are both fish pathogens and recognized agents of zoonosis with a public health impact, little information is available concerning the prevalence of in wild aquatic environments. To evaluate the prevalence of spp. in commercially important edible marine fish in different European seas (English channel, North sea, Bay of Biscay, Celtic sea and Mediterranean sea), 1,853 specimens were collected as part of two surveys. Nested PCR followed by sequence analysis at the 18S rRNA gene locus was used to identify spp. The overall prevalence of spp. in sampled fish reached 2.3% (35 out of 1,508) in a first campaign and 3.2% (11 out of 345) in a second campaign. Sequence and phylogenetic analysis of positive samples identified ( = 10) and seven genotypes which exhibited between 7.3 and 10.1% genetic distance from , with the exception of one genotype which exhibited only 0.5-0.7% genetic distance from Among 31 analyzed fish species, 11 (35.5%) were identified as potential hosts for A higher prevalence of spp. was observed in larger fish, in fish collected during the spring-summer period, and in those caught in the North East Atlantic. (saithe) was the most frequently positive species. In fish infected by other parasites, the risk of being positive increased 10-fold (OR: 9.95, CI: 2.32-40.01.04, = 0.0002). Four subtypes were detected among the positive samples: IIaA13G1R1, IIaA15G2R1, IIaA17G2R1, and IIaA18G3R1. These subtypes have been previously detected in terrestrial mammals and may constitute an additional source of infection for other animals and in particular for humans. Microscopical examination of histological sections confirmed the presence of round bodies suggestive of the development of within digestive glands. We report herein the first epidemiological and molecular data concerning the detection of in edible marine fish in European seas surrounding France broadening its host range and uncovering potential novel infection routes.
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http://dx.doi.org/10.3389/fmicb.2019.01037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530514PMC
May 2019

Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae, V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products.

Crit Rev Food Sci Nutr 2019 13;59(4):597-610. Epub 2017 Nov 13.

f French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Laboratory for Food Safety , Boulevard du Bassin Napoléon , Boulogne-sur-Mer , France.

Seafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872-1:2007 and TS 21872-2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood.
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http://dx.doi.org/10.1080/10408398.2017.1384715DOI Listing
August 2019

Metazoan parasite communities in Alosa alosa (Linnaeus, 1758) and Alosa fallax (Lacépède, 1803) (Clupeidae) from North-East Atlantic coastal waters and connected rivers.

Parasitol Res 2017 Aug 7;116(8):2211-2230. Epub 2017 Jun 7.

ESE Agrocampus-Ouest INRA, Ecologie Halieutique, Rue de Saint Brieuc, 35042, Rennes, France.

Metazoan parasites were studied in 96 Alosa alosa and 78 Alosa fallax from North-East Atlantic coastal waters and connected rivers (among them three sympatric sites) in order to increase knowledge on these anadromous endangered fish and measure the parasitic impact on host condition. All shads were infected by one to six metazoan parasite taxa among the 12 identified in the whole sampling, with a mean abundance of parasites higher for A. alosa (167 ± 10) than for A. fallax (112 ± 11). Helminths, mostly trophically transmitted, were the best represented (eight taxa, prevalence up to 99%) in contrast with crustaceans and Petromyzontidae that rarely occurred (four taxa, prevalence <6%). Despite some quantitative differences, metazoan parasite communities of A. alosa and A. fallax remained stable in composition whatever the host developmental stage, sex, sample site, and salinity. Among the nine parasite taxa harbored by each Alosa species, six were shared with some differences in distribution patterns including in sympatric conditions, suggesting increasing dissimilarities between A. alosa and A. fallax with the age. Information on feeding ecology provided by trophically transmitted helminths confirmed euryphagous opportunistic diet of immatures and adults of both shad species, and assessed feeding of adults during spawning migrations. Our study also revealed the significant negative impact of Hemiurus appendiculatus on A. alosa and Pronoprymna ventricosa on A. fallax. Because helminth parasites are omnipresent in the shads and decrease their fitness, parasitological data must be included in further investigations and management programs on A. alosa and A. fallax.
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http://dx.doi.org/10.1007/s00436-017-5525-8DOI Listing
August 2017

Occurrence of the three major Vibrio species pathogenic for human in seafood products consumed in France using real-time PCR.

Int J Food Microbiol 2014 Oct 18;189:75-81. Epub 2014 Jul 18.

Institut Pasteur, Laboratoire des bactéries pathogènes entériques, Centre National de Référence des Vibrions et du Choléra, Paris, France.

Vibrio spp. have emerged as a serious threat to human health worldwide. Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus are of particular concern as they have been linked to gastrointestinal infections and septicemia associated with the consumption of raw or undercooked seafood. We developed hydrolysis probe-based real-time PCR systems with an internal amplification control for the detection of these species. We applied these systems to a total of 167 fresh or frozen crustacean, fish and shellfish samples consumed in France. Of them, 34.7% (n=58) were positive for Vibrio. V. parahaemolyticus was the most common, in 31.1% of samples, followed by V. vulnificus in 12.6% and V. cholerae in 0.6%. Furthermore, V. parahaemolyticus and V. vulnificus were present simultaneously in 9.6% of samples. Virulence genes (tdh and trh sequences) were present in 25% of the V. parahaemolyticus-positive samples. The V. cholerae strain detected was non toxigenic. The densities of V. parahaemolyticus and V. cholerae ranged from <10(2) to 10(4)bacteria/g of seafood. All samples positive for V. vulnificus displayed low-level contamination with fewer than 10(2)bacteria/g. Our findings indicate that seafood consumption presents a potential risk to human health in France and highlight the importance of tools for a preventive consumer protection policy.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2014.07.014DOI Listing
October 2014

Combination of a pesticide exposure and a bacterial challenge: in vivo effects on immune response of Pacific oyster, Crassostrea gigas (Thunberg).

Aquat Toxicol 2007 Aug 12;84(1):92-102. Epub 2007 Jun 12.

Ifremer La Tremblade, Laboratoire de Génétique et Pathologie (LGP), 17390 La Tremblade, France.

To assess the impact of pollution induced by pesticides on Pacific oyster, Crassostrea gigas, health in France, in vivo effects of combined pesticide exposure and bacterial challenge on cell activities and gene expression in hemocytes were tested using flow cytometry and real-time PCR. As a first step, an in vivo model of experimental contamination was developed. Pacific oysters were exposed to a mixture of eight pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) at environmentally relevant concentrations over a 7-day period. Hemocyte parameters (cell mortality, enzyme activities and phagocytosis) were monitored using flow cytometry and gene expression was evaluated by real-time PCR (RT-PCR). The expression of 19 genes involved in C. gigas hemocyte functions was characterized using RT-PCR. After 7 days of exposure, phagocytosis was significantly reduced and the 19 selected genes were down-regulated in treated animals. As a second step, the experimental contamination method previously developed was used to study interactions between pesticide exposure and bacterial challenge by intramuscular injection of two Vibrio splendidus-related pathogenic strains. Oyster mortality and expression of 10 of the 19 selected genes were followed 4 and 24h post-injection. Oyster mortality was higher in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. Gene expression was up-regulated in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. We hypothesize that gene over-expression due to an interaction between pesticides and bacteria could lead to an injury of host tissues, resulting in higher mortality rates. In conclusion, this study is the first to show effects of pesticides at environmentally relevant concentrations on C. gigas hemocytes and to hypothesize that pesticides modulate the immune response to a bacterial challenge in oysters.
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http://dx.doi.org/10.1016/j.aquatox.2007.06.002DOI Listing
August 2007

The effect of cohabitation of Tubifex tubifex (Oligochaeta: Tubificidae) populations on infections to Myxobolus cerebralis (Myxozoa: Myxobolidae).

J Invertebr Pathol 2006 Jan;91(1):1-8

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.

The competitive interactions between susceptible and resistant Tubifex tubifex (Oligochaeta: Tubificidae) exposed to Myxobolus cerebralis (Myxozoa: Myxobolidae) infections were investigated in two laboratory trials. Competition was assessed by the total parasite production over the course of the trials in mixed and pure cultures of M. cerebralis exposed worms, and by the genetic analyses of worms from the control and experimental groups at the beginning and end of the experiments. Mixed cultures of resistant and susceptible worms showed a 70% reduction in production of parasites released when compared with pure cultures of susceptible worms. In studies with laboratory and field-collected oligochaetes the mixed cultures at the end of the cohabitation experiments were dominated by resistant Tubifex from lineage V (HB strain) this strain of Tubifex has a competitive advantage over worms from other lineages. The results of this study suggest that certain species of Tubifex may be dead-end hosts to M. cerebralis by absorbing or inactivating the parasite and may also show greater survival compared to susceptible oligochaetes in certain whirling disease enzootic habitats.
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http://dx.doi.org/10.1016/j.jip.2005.08.006DOI Listing
January 2006

Two vibrio splendidus related strains collaborate to kill Crassostrea gigas: taxonomy and host alterations.

Dis Aquat Organ 2004 Nov;62(1-2):65-74

Laboratoire de Génétique et Pathologie, Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), 17390 La Tremblade, France.

For several years, strains phenotypically related to Vibrio splendidus have been associated with mortality outbreaks of molluscs. A former study on Crassostrea gigas demonstrated the genetic diversity of V. splendidus strains associated with diseased animals. Another study suggested that different strains may act in an additive/synergistic way leading to higher C. gigas mortality rates. Here, a strain pair (31+32) was characterised at taxonomic and virulence levels. Using a polyphasic approach, these strains were confirmed to be V. splendidus-related, without a clear discrimination between V. kanaloae and V. pomeroyi since hybridisation rates with both these strains were above 70 %. Following experimental infection of C. gigas by injection in the adductor muscle or in the pallial cavity, the host alterations induced were described. After injection of strains 31 and/or 32, bacteria were localised at the periphery of the muscle and induced extensive lesions of the translucent part of the adductor muscle. Muscle alterations were of 3 kinds: (1) presence of isolated rounded muscular fibres containing non-homogenous granular material and surrounded by a translucent halo; (2) presence of non-homogenous granular material in the cytoplasm of entire muscle bands; (3) affection of wide muscle areas with extremely condensed muscle fibres. Infiltration associated with these lesions was notably absent in the vast majority of the individuals.
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http://dx.doi.org/10.3354/dao062065DOI Listing
November 2004

Screening of Vibrio isolates to develop an experimental infection model in the Pacific oyster Crassostrea gigas.

Dis Aquat Organ 2004 Apr;59(1):49-56

Laboratoire de Génétique et Pathologie, Institut français de recherche pour l'exploitation de la mer (IFREMER), 17390 La Tremblade, France.

In an attempt to develop a reproducible experimental model of bacterial infection in Crassostrea gigas, oysters taken from very localised sub-populations suffering natural mortality outbreaks were used in cohabitation trials under laboratory conditions. From these trials, a collection of Vibrio strains was isolated from moribund and healthy oysters. In a second step, strains were experimentally tested for virulence by means of injection into healthy oysters. This screening revealed a span of virulence among isolated strains from none to medium. When pooling injected strains, results suggest increased virulence. Vibrio strains may have additive/synergistic action leading to higher C. gigas mortality rates in experimental challenges. Although the study initially aimed to develop a simple experimental model, a complex of interactions emerged between several bacterial strains during the pathogenic process in their molluscan host. Selected strains provide a suitable model of experimental disease for further studies and better understanding of bacterial interaction and pathogenesis in C. gigas.
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http://dx.doi.org/10.3354/dao059049DOI Listing
April 2004

Phylogenetic study and identification of Vibrio splendidus-related strains based on gyrB gene sequences.

Dis Aquat Organ 2004 Mar;58(2-3):143-50

Laboratoire de Génétique et Pathologie, Institut français de recherche pour l'exploitation de la mer, 17390 La Tremblade, France.

Different strains related to Vibrio splendidus have been associated with infection of aquatic animals. An epidemiological study of V. splendidus strains associated with Crassostrea gigas mortalities demonstrated genetic diversity within this group and suggested its polyphyletic nature. Recently 4 species, V. lentus, V. chagasii, V. pomeroyi and V. kanaloae, phenotypically related to V. splendidus, have been described, although biochemical methods do not clearly discriminate species within this group. Here, we propose a polyphasic approach to investigate their taxonomic relationships. Phylogenetic analysis of V. splendidus-related strains was carried out using the nucleotide sequences of 16S ribosomal DNA (16S rDNA) and gyrase B subunit (gyrB) genes. Species delineation based on 16S rDNA-sequencing is limited because of divergence between cistrons, roughly equivalent to divergence between strains. Despite a high level of sequence similarity, strains were separated into 2 clades. In the phylogenetic tree constructed on the basis of gyrB gene sequences, strains were separated into 5 independent clusters containing V. splendidus, V. lentus, V. chagasii-type strains and a putative new genomic species. This phylogenetic grouping was almost congruent with that based on DNA-DNA hybridisation analysis. V. pomeroyi, V. kanaloae and V. tasmaniensis-type strains clustered together in a fifth clade. The gyrB gene-sequencing approach is discussed as an alternative for investigating the taxonomy of Vibrio species.
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http://dx.doi.org/10.3354/dao058143DOI Listing
March 2004

Prevalence and susceptibility of infection to Myxobolus cerebralis, and genetic differences among populations of Tubifex tubifex.

Dis Aquat Organ 2002 Aug;51(2):113-21

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis 95616, USA.

The prevalence of infection and susceptibility of the aquatic oligochaete Tubifex tubifex to Myxobolus cerebralis, was examined in 2 studies on the upper Colorado River, Colorado, USA, where whirling disease occurs in wild trout populations. In the first study, the prevalence of infection ranged from 0.4 to 1.5%, as determined by counting the number of T. tubifex releasing triactinomyxons of M. cerebralis directly following their collection from the field. The susceptibility of those T. tubifex not releasing triactinomyxons was assessed by the number of these oligochaetes releasing triactinomyxons 3 mo following experimental exposures to spores of M. cerebralis. The prevalence of infection following experimental exposures of these T. tubifex ranged from 4.2 to 14.1%. In a second study, all T. tubifex collected at 2 different times directly from the 2 field sites in Colorado were exposed to spores of M. cerebralis. Individual oligochaetes representing those groups of T. tubifex releasing and those groups not releasing triactinomyxons at 3 mo were screened with molecular genetic markers. T. tubifex populations found at the 2 study sites consisted of 4 genetically distinct lineages that varied with respect to their susceptibility to experimental exposure to M. cerebralis. Lineages I and III contained the most oligochaetes susceptible to M. cerebralis and were the most prominent lineages at Windy Gap Reservoir, a site of high infectivity for wild rainbow trout on the upper Colorado River. In contrast, at the Breeze Bridge site which is below Windy Gap Reservoir and where M. cerebralis infections are less severe in wild trout, oligochaetes in lineages V and VI that are resistant to M. cerebralis were more prominent. These results suggest that certain habitats, such as Windy Gap Reservoir, are conducive to large and more homogenous populations of susceptible T. tubifex lineages that may serve as point sources of infection for M. cerebralis. Although not a direct objective of this study, there was no evidence of M. cerebralis infections among any oligochaetes other than those that would be classified as T. tubifex by standard morphological characteristics.
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http://dx.doi.org/10.3354/dao051113DOI Listing
August 2002
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