Isolation and Identification of Probiotic Bacteria for the Management of Epizootic Shell Disease

Abstract

Epizootic shell disease (ESD) is an emerging disease in the American lobster (Homarus americanus) characterized by lesions, mainly on the carapace. The diseased appearance of the shell due to the lesions has had significant negative impact upon the lobster fisheries in New York, Connecticut, Rhode Island, and Massachusetts, since fishers are not able to sell affected lobsters to the more lucrative live market. ESD lesions are different from other shell diseases found in the American lobster populations because they are due to a poly-microbial infection that degrades the epicuticle layer, while the pillars of the chitin matrix remain intact in the lesions. ESD was first described in the 1980’s, and has shown the highest prevalence in Southern New England since the late 1990’s. In inshore waters, ESD was estimated to affect 10-40% of the lobsters with 50-80% of the ovigerous females affected by ESD. Additionally, ESD has expanded to lobster populations outside the original geographic range. New or consistent disease observations have been seen in Maine at low levels of disease prevalence. The increase in ESD has generated concern for the health of the lobsters and the economic status of the fishery. It has been shown that probiotics are an effective way to prevent infectious diseases in a variety of animals, including fish and shellfish by inhibition or exclusion of the pathogenic bacteria. The goal of this research was to isolate commensal, potential probiotic, bacteria from the shells of healthy looking lobsters and characterize their ability to reduce or eliminate the ESD-causing organisms. Twenty-four out of 217 isolates from lobsters were characterized as potential probiotic organisms based on their ability to inhibit the growth of putative ESD pathogens Thalassobius sp. I31.1 or Aquimarina macrocephali I32.4 (formerly known as Aquimarina homaria), ability to form strong biofilms, and their effect on Thalassobius sp. I31.1 growth and biofilm formation. While twenty-four isolates exhibited activity against at least one of the target organisms, but only two potential probiotic organisms, Bacillus sp. 06-YP001, and Pseudoalteromonas sp. 10B-YPO11, had inhibitory activity against both pathogens. Biofilm formation on polystyrene, sterilized lobster shell fragments or glass coverslips was variable in strength across isolates. The competition assays demonstrated that four isolates, Loktanella maritima 06-YPC210, Bacillus sp. 06-YP001, Pseudoalteromonas sp. 03-YP014, Pseudoalteromonas sp. 08-YPC21, and Phaeobacter inhibens S4Sm were effective in reducing the growth of Thalassobius sp. I31.1. These results demonstrate that potential probiotic organisms can be isolated from the