Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae

Abstract

Bacterial diseases are a major cause of larval mortality in shellfish hatcheries. Even with proper sanitation measures, bacterial pathogens cannot be eliminated in all cases. The pathogenicity of bacteria isolated from Pacific Northwest shellfish hatcheries to Pacific oyster Crassostrea gigas larvae was investigated. We found 3 highly pathogenic strains and 1 mildly pathogenic strain among 33 isolates tested. These strains appear to be members of the genus Vibrio. Although there have been many studies of bivalve bacterial pathogens, a standard method to assess bacterial pathogenicity in bivalve larvae is needed. Thus, we developed 2 methods using either 15 ml conical tubes or tissue culture plates that were employed for rapidly screening bacterial strains for pathogenicity to Pacific oyster larvae. The tissue culture plates worked well for screening both mildly pathogenic strains and LD 50 (lethal dose) assays. This method allowed for non-intrusive and non-destructive observation of the oyster larvae with a dissecting microscope. The LD 50 for the 3 highly pathogenic strains ranged between 1.6 and 3.6 × 10 4 colony forming units (CFU) ml -1 after 24 h and between 3.2 × 10 2 and 1.9 × 10 3 CFU ml -1 after 48 h.KEY WORDS: Pacific oyster larvae · Vibrio · Bacteria · Pathogenicity testing · Shellfish hatchery Resale or republication not permitted without written consent of the publisherDis Aquat Org 58: [223][224][225][226][227][228][229][230] 2004 genicity of bacteria isolated from bivalve hatcheries during routine monitoring and disease outbreaks. MATERIALS AND METHODSIsolates and reference strains. Bacterial isolates collected from Pacific oyster Crassostrea gigas larvae (n = 16) and juveniles (n = 12), European flat oyster Ostrea edulis juveniles (n = 1), and hatchery environments (n = 4) from the Pacific Northwest of the United States (Table 1) were stored at -80°C in Marine 2216 broth (Difco Laboratories) with 10% (v/v) glycerol. Subsequently, they were shipped on dry ice from AquaTechnics (Sequim) to the University of Washington (Seattle, Washington). The following control strains were included in the pathogenicity assays: RE 14 (nonpathogenic or negative control), RE 82 (pathogenic or positive control), and previously described larval bivalve pathogens Vibrio alginolyticus American Type Culture Collection (ATCC) 19108 and V. tubiashii ATCC 19106 (Tubiash et al. 1965, 1970). All cultures were maintained at -80°C for long-term storage.Isolates were grown for 24 h on Marine 2216 agar and incubated at 20 and 26°C in the first and second set of experiments, respectively. Experiments were conducted using sand-filtered Puget Sound seawater collected at the Seattle Aquarium (Seattle, Washington). Suspensions of the bacterial strains in filter-sterilized (0.22 µm) seawater were adjusted to an optical density (600 nm) of 0.2 to attain a uniform concentration of bacteria for the experiments. Bacterial density was confirmed by standard plate-count assays using Marine 2216 agar.Pathogenicity testing. Experi...

The Extracellular Metalloprotease ofVibrio tubiashiiIs a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae

“…Previous studies identified extracellular products, such as a metalloprotease and a cytolysin/hemolysin, from culture supernatants of V. tubiashii strain ATCC 19105 (9,20). To determine if bacterial strains isolated from diseased Pacific oyster larvae also produce these secreted proteins, we grew V. tubiashii strains RE22 and RE98, which were previously described as pathogenic strains (14), and RE15, which was categorized as a nonpathogenic bacterial isolate. Harvested culture supernatants were filter sterilized and assayed for proteolytic and hemolytic activities.…”

“…Vibrio tubiashii strains RE22 and RE98 and an unknown bacterial isolate, RE15, isolated from a Pacific oyster (14), were grown on Luria-Bertani (LB) medium supplemented with 1% sodium chloride (LB-1% NaCl) at 30°C. For larval-toxicity assays, V. tubiashii strains were cultured overnight in 20 ml LB-1% NaCl at 25°C on a shaker.…”

“…Vibrio tubiashii, a bacterial species first reported by Tubiash et al (51), was identified as a causative agent of vibriosis (originally referred to as bacillary necrosis) in larval and juvenile bivalves of the hard clam (Mercenaria mercenaria) and Eastern oyster (Crassostrea virginica). Estes et al (14) characterized a number of pathogenic and nonpathogenic bacterial strains from diseased Pacific oysters (Crassostrea gigas) at shellfish hatcheries on the Pacific coast of North America and described some of the highly pathogenic bacterial isolates as V. tubiashii.…”

Mortalities of Eastern and Pacific Oyster Larvae Caused by the Pathogens Vibrio coralliilyticus and Vibrio tubiashii

“…These results raise the question of whether V. tubiashii has been problematic in West Coast hatcheries or whether the problems in hatcheries were principally or exclusively associated with V. coralliilyticus strains, such as RE98 and RE22, both of which were originally isolated from a West Coast shellfish hatchery during high-mortality events (3). Both RE98 and RE22 were previously shown to be highly pathogenic toward Pacific oysters but had not been tested against Eastern oysters (3,16). Comparison of the LD 50 results from Table 1 provides a simple means to assess the relative effects of these pathogens on healthy oyster larvae under the conditions of these assays (28-to 30-ppt salinity, 26°C).…”

Screening for and Identification of an Anti‐clam Vibrio Marine Bacterium from an Aquaculture Pond in the Yellow Sea

“…In Jiangsu Province, for example, M. meretrix production in 2008 was approximately 0.06 million tons, with an estimated first-sale value of US$ 62.6 million, representing nearly a half of the country's total [2]. Disease outbreaks caused by pathogenic bacteria, commonly of the genus Vibrio, are a major cause of mortality in shellfish larvi-culture and can result in financial losses for commercial growers [3]. A variety of antibiotics have been used for the prevention of diseases, but the overuse of antibiotics can lead to the emergence of resistant bacteria and environmental problems [4][5][6], such as: the abuse of antibiotics may remain in high concentration in aquatic products and in surface water, resulting in the high risk of these chemical to human health and the environment [7,8].…”