Hemocyte Viability Assay as an Alternative Method for Testing Bacterial Pathogenicity in Bivalves

Bacterial pathogens cause disease outbreaks in hatcheries, leading to larval mortality and significant economic losses. One of the major challenges in studying bacterial pathogenesis in larvae is the limited availability of healthy larvae throughout the year. This seasonal constraint delays testing and impedes progress in understanding bacterial infections, disease management, and the development of effective treatments for bivalve aquaculture. To address this challenge, a hemocyte viability assay was developed as an alternative for testing bacterial pathogenesis. A resazurin-based assay was optimized using eastern oyster hemocytes, and lethal concentration (LC)50 values were determined for the bacterial pathogen Vibrio coralliilyticus RE22 (121 {+/-} 2 multiplicity of infection, MOI), probiont Phaeobacter inhibens S4 (2,157 {+/-} 56 MOI), and several bacterial isolates from shellfish hatcheries. The most virulent isolates had LC50 values ranging from 53 to 195 MOI. To validate the assay, the isolates were tested on oyster larvae, confirming that bacterial isolates with LC50 <200 MOI against hemocytes also caused larval mortality, while isolates exhibiting LC50 values [&ge;]690 MOI showed no adverse effects on oyster larvae. This high-throughput hemocyte viability assay provides a rapid and reliable alternative to larval assays, particularly during the off-season for hatcheries, facilitating year-round research on bacterial pathogenicity in bivalves.