Species-specific virulence of Vibrio harveyi for black tiger shrimp is associated with bacteriophage-mediated hemocyte agglutination

Shrimp cultivation is an important Thai industry in which production losses can result from disease outbreaks caused by luminous Vibrio harveyi. In some cases, mortality from luminous vibriosis results from extracellular bacterial products (ECPs) such as proteases, phospholipases, hemolysins and cytotoxins that are mediated by specific bacteriophages. Increased virulence of V. harveyi lysogenized by a Siphoviridae-like bacteriophage (VHS1) was first confirmed and characterized. Then crude extracellular proteins from the lysogen culture broth were concentrated and shown to cause high and rapid mortality in the black tiger shrimp (Penaeus monodon) but not in the whiteleg shrimp (Penaeus vannamei). Sequential crude protein fractionation by fast protein liquid chromatography (FPLC) yielded a fraction selected for highest mortality in P. monodon. This fraction also agglutinated black tiger shrimp hemocytes and mud crab (Scylla serrata) hemocytes but not hemocytes of the whiteleg shrimp or human erythrocytes. By SDS-PAGE, this fraction contained 3 prominent protein bands. Mass spectrometry analysis and comparison to public databases revealed homology with hypothetical bacterial and bacteriophage proteins at low percent identity and coverage. Further work is needed to determine whether these proteins originate from VHS1 (bacteriophage) gene expression or from phage-mediated up-regulation of host gene(s) expression.