Screening of Vibrio Isolates to Develop an Experimental Infection Model in the Pacific Oyster Crassostrea Gigas
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DISEASES OF AQUATIC ORGANISMS Vol. 59: 49–56, 2004 Published April 21 Dis Aquat Org Screening of Vibrio isolates to develop an experimental infection model in the Pacific oyster Crassostrea gigas Mélanie Gay, Franck C. J. Berthe, Frédérique Le Roux* Laboratoire de Génétique et Pathologie, Institut français de recherche pour l’exploitation de la mer (IFREMER), 17390 La Tremblade, France ABSTRACT: 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 out- breaks 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 experi- mentally 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 sim- ple 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 experi- mental disease for further studies and better understanding of bacterial interaction and pathogenesis in C. gigas. KEY WORDS: Vibrio splendidus · Virulence · Agonism · Antagonism Resale or republication not permitted without written consent of the publisher INTRODUCTION riosis has also been reported in juvenile and adult mol- luscs. For example, Vibrio tapetis is the aetiological While experimental transmission of mollusc diseases agent of brown ring disease in the Japanese clam under laboratory conditions has sometimes been Ruditapes philippinarum (Paillard & Maes 1990, Bor- achieved (Bachère et al. 1986, Le Deuff et al. 1994, Chu rego et al. 1996), and V. harveyi has been associated 1996, Hervio et al. 1996, Audemard et al. 2002), the with a severe epizootic outbreak in the abalone Halio- lack of a reproducible disease model is broadly recog- tis tuberculata (Nicolas et al. 2002). In the Pacific oys- nised as an impediment to scientific investigations ter Crassostrea gigas,2 potentially pathogenic strains (Mialhe et al. 1995, Berthe et al. 1998). Diseases caused were identified as V. splendidus (Lacoste et al. 2001, by non-cultivable pathogenic agents, unknown agents Waechter et al. 2002, Le Roux et al. 2002), and were or agents having a complex life-cycle present the most associated with summer mortality syndrome outbreaks difficulties. Considering this, bacterial infection has (Goulletquer et al. 1998). the potential for further development of an experimen- Vibrio pathogenesis has only been documented in a tal disease model. limited number of molluscan diseases. To date, descrip- Bacterial diseases in molluscs have been described tions were made of V. tapetis infection in Ruditapes mainly for the larval stage and in hatcheries, with a philippinarum, V. anguillarum in Crassostrea gigas marked predominance of the genus Vibrio (Tubiash et larvae, and V. lentus in Octopus vulgaris (Elston & al. 1965, Brown & Losee 1978, Disalvo et al. 1978, Leibovitz 1980a,b, Elston et al. 1982, Oubella et al. Elston & Leibovitz 1980a,b, Jeffries 1982, Hada et al. 1994, Paillard et al. 1994, Allam et al. 2000, Farto et al. 1984, Lambert et al. 1998, Sugumar et al. 1998). Vib- 2003). In adult oysters, vibriosis-induced host tissue *Corresponding author. Email: [email protected] © Inter-Research 2004 · www.int-res.com 50 Dis Aquat Org 59: 49–56, 2004 alteration and immune response, as well as virulence Bacterial isolation. Isolation was performed from the mechanism and regulation, are still poorly understood. haemolymph in order to avoid contamination from the This could be explained by the absence of clinical signs digestive tract flora where Vibrio is predominant preceding death of shelled molluscs, the high diversity (Prieur 1982). Samples of haemolymph were pooled of the Vibrio species involved and the opportunistic from 3 oysters, serial dilutions spread onto thiosulfate status of different strains (Le Roux et al. 2002, Waechter citrate bile sucrose agar (TCBS, Diagnostic Pasteur) et al. 2002). The development of a robust experimental and incubated for 48 h at 20°C. Three colonies of each model would therefore represent major progress. macroscopic type were selected and isolated for sub- The main objective of this study was to isolate Vibrio culture. The origin and source of the isolations are strains and screen them for virulence in order to develop given in Table 1. All strains were further sub-cultured an experimental model of vibriosis in Crassostrea gigas. on tryptic soy agar (TSA) supplemented with 2% NaCl The first step of this work was to establish a collection of (Difco) at 20°C, and were stored at –80°C in basal Vibrio potentially pathogenic to C. gigas. To achieve this, medium (1% w/v peptone, 0.1% w/v MgSO4 ·7H2O, live oysters from very localised sub-populations suffering 10% v/v glycerol; pH 7.2) in the collection of the EU summer mortality outbreaks were collected in different Community Reference Laboratory for Mollusc Dis- areas. In order to enhance the chances of selecting eases (IFREMER, La Tremblade, France). oysters that carry pathogenic Vibrio spp., cohabitation Genotyping. Bacterial DNA was prepared according experiments were conducted under laboratory con- to Sambrook et al. (1989). PCR amplification of the ditions, and observed transmissible events enabled us to DNA gyrase subunit (gyrB) was achieved with primers identify candidates for bacterial isolation. During these and methods previously described (Yamamoto & trials, a collection of Vibrio strains was isolated from Harayama 1995). Amplified products were purified moribund and healthy animals followed by their char- using a gel extraction kit (Qiagen). Sequencing was acterisation by means of gyrase subunit (gyrB) gene carried out using the Sequitherm Excell II kit (Epi- sequencing (Le Roux et al. 2004). centre) and a Li-Cor DNA sequencer (ScienceTec) The second step of the study was to select virulent according to the manufacturer’s instructions. strains. The screening of strains—either individual or Phylogenetic analyses. GyrB sequences were pooled strains—was performed by injection challenges aligned and phylogenetic analyses were performed using Crassostrea gigas after pre-screening in clams using Seaview and Phylo-win programs (Galtier et al. Ruditapes philippinarum, given the higher susceptibil- 1996). Phylogenetic trees were built using the BIONJ ity of the latter species to bacterial infection, as has method (Gascuel 1987) applied to Kimura’s 2-para- been previously demonstrated (Le Roux et al. 2002). meter distances. Reliability of topologies was assessed by the bootstrap method with 1000 replicates. Experimental challenges. Bacteria were grown at MATERIALS AND METHODS 20°C for 36 h in marine broth (Diagnostic Pasteur) and harvested in sterile seawater (121°C for 15 min) at a Cohabitation experiments. During summer 2001, live oysters (neighbouring oysters displaying a mortal- Table 1. Crassostrea gigas. Number of moribund oysters ity rate of >20%), were collected from several beds obtained after 8 d of cohabitation experiments for field (n = along the Atlantic coast of France and from 1 batch 15) and sentinel (n = 15) batches. Sentinel batches for which from a hatchery (Table 1). These oysters (n = 15, bacteriological analysis was conducted are underlined referred to as ‘field batch’) were kept in aquaria along with healthy oysters from populations which had never Origin/ Cumulative mortality at 8 d suffered from mortality (IFREMER hatchery, n = 15, reference post cohabitation Field batch Sentinel batch referred to as ‘sentinel batch’). Experiments were per- formed for 1 wk in 2.5 l aquaria filled with 5 µm- Arcachon 01-84 15 15 filtered seawater, under static conditions at 20°C with Arcachon 01-88 15 18 aeration. Oysters were fed daily with a mixture of Arcachon 01-90 15 14 Fouras 01-92 15 10 Isochrysis galbana and Chaetoceros calcitrans. During Fouras 01-94 15 10 the experiment, sentinel oysters displaying a weakness Fouras 01-116 18 19 of adduction activity (moribund oysters) were sacri- Perquis J1 15 11 ficed, and the haemolymph was collected for bacterial Perquis J8 15 15 analysis. For each cohabitation experiment, controls Hatchery 15 12 Trinité 01-105 10 12 consisted of sentinel oysters maintained under the Trinité 01-106 16 13 same conditions; haemolymph samples were collected Trinité 01-111 14 15 from healthy animals as described above. Gay et al.: Virulent Vibrio isolated from Crassostrea gigas 51 concentration of 4 to 8 × 108 colony forming units (CFU) higher than 90%. In the sentinel batch, mortality ml–1 calculated by reading the optical density (OD) at occurred 3 to 5 d after the experiment started, with a 600 nm with a spectrophotometer (Biophotometer, cumulative mortality rate above 50% in 9/12 cases at Eppendorf). This bacterial concentration was con- 8d post cohabitation. No mortality was observed in the firmed by enumeration on marine agar plates using a aquaria containing only sentinel oysters (controls). conventional dilution plating technique with incuba- Sentinel batches used for bacterial isolation (Table 1) tion at 20°C for 2 d. were shown to be herpes virus free by PCR (data not A pre-screening of virulent strains was performed shown). Strains 1 to 82 were isolated from diseased using the clam model. Since we could not individually animals, whereas Strains 83 to 125 were isolated from test all 125 strains, bacterial isolates from a cohabitation healthy controls. experiment were injected from a pool of 3 to 5 strains. For this, equal volumes of each strain at the concentra- tion of 4 × 108 CFU ml–1 were mixed and 100 µl of this Genotypic characterisation of strains mixture (4 × 107 CFU) was injected into the adductor muscle of adult clams (n = 30, size 3 to 5 cm).