Development of an Early Mortality Syndrome Challenge Test for Shrimps Using Gnotobiotic Artemia sp.

Abstract

Acute hepatopancreatic necrosis disease (AHPND) also known as early mortality syndrome (EMS), is a recently emerged bacterial disease in shrimp farming, which causes a unique pattern of mortality in shrimp post larvae in newly stocked ponds. Investigations on the control of EMS/AHPND are still in progress and it would be interesting to develop a standard challenge model to establish a fast, reliable and systemic evaluation on EMS/AHPND. Hence, this study was designed to provide basic tools for development of a standardized challenge model, focusing on the role of toxins, using gnotobiotic Artemia, with different pathogenic strains of Vibrio parahaemolyticus. Experiments with gnotobiotic Artemia sp. were designed to check whether the virulence of V. parahaemolyticus is related to the secretion of exotoxins into the surrounding medium. Challenge tests were performed to check the effect of filter sterilized cell-free supernatant and filter sterilized suspension obtained from resuspension of different strains of V. parahaemolyticus in marine broth,on survival of gnotobiotic Artemia sp. It indicated that KM, RY and MO904 strains of V. parahaemolyticus may be capable of producing exotoxins that are secreted into the culture medium. Spent medium of strain RY,KM and MO 904 resulted in a significantly different mortality (p< 0.05) from control and other strains. These so-called cell-free spent media are causing mortality in gnotobiotic Artemia sp. showing toxin mediated etiology. The mortality depends on the type of culture medium in which pathogens are cultured and the virulence of each pathogenic strain. Based on the results, it can be suggested that release of exotoxins by V. parahaemolyticus are negatively affecting gnotobiotic Artemia and that effect may vary depending on the V. parahaemolyticus strain. This study provides a basis for the development of a reliable in-vivo Artemia-based assay for AHPND toxins screening.