Additional evidence that juvenile oyster disease is caused by a member of the Roseobacter group and colonization of nonaffected animals by Stappia stellulata-like strains

Abstract

Juvenile oyster disease (JOD) causes significant annual mortalities of hatchery-produced Eastern oysters, Crassostrea virginica, cultured in the Northeast. We have reported that a novel species of the alpha-proteobacteria Roseobacter group (designated CVSP) was numerically dominant in JOD-affected animals sampled during the 1997 epizootic on the Damariscotta River, Maine. In this study we report the isolation of CVSP bacteria from JOD-affected oysters during three separate epizootics in 1998. These bacteria were not detected in nonaffected oysters at the enzootic site, nor in animals raised at a JOD-free site. Animals raised at the JOD enzootic site that were unaffected by JOD were stably and persistently colonized by Stappia stellulata-like strains. These isolates (designated M1) inhibited the growth of CVSP bacteria in a disk-diffusion assay and thus may have prevented colonization of these animals by CVSP bacteria in situ. Laboratory-maintained C. virginica injected with CVSP bacteria experienced statistically significant elevated mortalities compared to controls, and CVSP bacteria were recovered from these animals during the mortality events. Together, these results provide additional evidence that CVSP bacteria are the etiological agent of JOD. Further, there are no other descriptions of specific marine alpha-proteobacteria that have been successfully cultivated from a defined animal host. Thus, this system presents an opportunity to investigate both bacterial and host factors involved in the establishment of such associations and the role of the invertebrate host in the ecology of these marine alpha-proteobacteria.

FIG. 1

Results of bacteriological analysis of oysters from the first deployment. (A) Total heterotrophic bacteria recovered from animals (data from 2 July to 8 August acquired from meat samples; data from 12 August to 30 September acquired from swab samples). (B) Estimated abundance of M1 bacteria expressed as a percentage of total CFU. Symbols represent the means of results from three bags. ●, Damariscotta River samples; ○, Maquoit Bay samples.

FIG. 2

Results of bacteriological analysis of oysters from the second deployment. (A) Total heterotrophic bacteria recovered from animals. (B) Estimated abundance of CVSP bacteria expressed as a percentage of total CFU recovered. Symbols: Damariscotta River samples, Tray 1 (▴), Tray 2 (■), Bag 3 (●); Maquoit Bay samples, ○.

FIG. 3

Cumulative weekly mortalities observed in laboratory-maintained animals that had been injected either with a culture of CVSP bacteria (solid symbols) or with an equivalent amount of medium alone (open symbols). Triangles, circles, and squares indicate aquaria no. 1, 2, and 3 in each group, respectively.