Analysis of pathogen in an outbreak death of Marsupenaeus japonicus

In order to clarify the cause of sudden death that occurred in a shrimp (Marsupenaeus japonicus) farm in Weifang from July to August 2015, seven main shrimp pathogens including White spot syndrome virus (WSSV), Taura syndrome virus (TSV), Yellow head virus (YHV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Infectious myonecrosis virus (IMNV), Covert mortality nodavirus (CMNV) and Vibrio alginolyticus that causing acute hepatopancreatic necrosis disease (AHPND) were detected by Polymerase chain reaction (PCR) method. Meanwhile, histopathology of the diseased shrimp was observed. Furthermore, bacterial strains, isolated from the moribund shrimp and identified by the 16 SrDNA gene, were used for the evaluating of virulence by immersion challenge. The results showed that all the three shrimp samples gave strong positive PCR results (an amplicon of 941 bp) with primers WSSV 146 F2/R2, and two of three sample show weak positive results (an amplicon of 389 bp) with primers IHHNV 389 F/R and two of three sample show strong positive results (an amplicon of 167 bp) with primers CMNV-7 F2/R2. However the results of PCR amplification for the other four pathogens (TSV, YHV, IMNV and AHPND) show negative. Histopathology results show that there were inclusion body of WSSV in the epithelial tissue of stomach and gill, and the muscle fibres dispersed. Three dominant strains of bacterial microorganisms 2901, 2902, 2903 were isolated and 16 S rDNA sequencing indicated that they had higher similarity with Grimontia indica (99%)、Alteromonas sp. (99%) and Vibrio alginolyticus (100%) respectively. Challenge test show that LC₅₀ of bacteria 2901, 2902, 2903 were 9.8×10⁷ CFU/mL, 1.1×10⁸ CFU/mL and 2.3×10⁸ CFU/mL, which means the bacterial microorganisms had lower virulence and may not be the main pathogen of shrimp. Comprehensive analysis suggested that sudden death of M.japanicus might be caused by coinfection of WSSV, IHHNV and CMNV, in which WSSV infection might play a key role. The results of this study could provide strong support for clarifying the cause of outbreak death in shrimp M. japonicus.