Rapid Diagnostic Test of Red Sea Bream Iridoviral Disease (RSIVD) in Grouper Epinephelus Sp. Based on Serological Co-Agglutination and Molecular Study

Abstract

Red sea bream iridoviral disease (RSIVD) infection is known as a contagious disease in the marine aquaculture commodities mainly on grouper (Epinephelussp.)which causes a high mortality rate. Symptoms of disease were weak, darker skin and swollen spleen of fish. The aim of this study was to create and apply a rapid diagnostic test supported by molecular analysis. Field trials on a mass mortality outbreaks were identified in the city of Tanjungpinang, Indonesia. Serum anti RSIV was obtained by immunizing of the vaccine RSIV intraperitoneally on rabbits with graded doses per week was 0.5, 1, 2 and3mL, to boost antibody titers. In the fifth week, serum was harvested via the auricular vein; serum was purified to obtain immunoglobulin G(IgG)then was coupling with protein A ofStaphylococcus aureus at the same volume(kit co-agglutination RSIVD). Field samples of spleen were taken from the normal fish and suspected fish then crushed and suspended with PBS pH 7.2, and centrifuged at 8.000 rpm for 15 min. Fifty microliters of RSIVDco-agglutination kit and 50 µL of spleen supernatant were reacted on the sterile glass object. The results showed sandy agglutination after 10 min for positively infected spleen, and no agglutination in the samples of healthy fish (negative) as well as in control with PBS (negative). Confirmation testing by polymerase chain reaction (PCR) using primer forward 1-F (5’-CTC-AAA-CAC-TCT-GGC-TCA-TC-3’) and reverse 1-R (5’-GCA-CCA-ACA-CAT-CTC-CTA-TC-3’) had a band of 570 bp. Sequencing results showed the similarity of 99% identity with RSIV. Testing with RSIVD co-agglutination kit showed the advantages such as cheap, fast and an accurate in diagnosing the red bream iridoviral disease (RSIVD).