DETECTION OF PATHOGENIC BACTERIAL (EDWARDSIELLA TARDA AND VIBRIO ALGINOLYTICUS) ASSOCIATED WITH SWOLLEN ABDOMEN OF CULTURED TURBOT (SCOPHTHALMUS MAXIMUS) AND FLOUNDER (PARALICHTHYS OLIVACEUS) BY NESTED-PCR

Turbot(Scophthatmus maximus) and flounder(Paralichthys olivaceus) are rare and economic fish in the north of China.In recent years,they have become the dominant cultured varieties.Tianjin is the main cultural area of turbot and flounder.However,the prevalence of diseases of the cultured turbot and flounder has resulted in great loss to the cultural enterprise.Swollen abdomen disease was the serious threat to the turbot and flounder according to the investigation.The mortality can reach 80%-90% while the disease broke out.On the foundation of the former work,we have confirmed that Edwardsiella tarda and Vibrio alginolyticus were the pathogenic bacterial associated with swollen abdomen disease.E.tarda is an important pathogenic bacteria to the maricultured fish,furthermore,it can cause other diseases to birds,amphibian and mammals.V.alginolyticus is an opportunist pathogen,which exists extensively in seawater,and causes serious diseases to maricultured fish and even human being.The treatment of the aquatic animals' bacterial disease mostly depend on the antibiotics recently.The misuses of the antibiotics have given more pressures to the human's health.Meanwhile,the antibiotics have brought more opportunities to the pathogenic bacterial to adapt it.Hence,it is necessary to develop a sensitive and reliable method to detect the pathogenic bacterial.We expect to build a system which can forecast the prevalence of the swollen abdomen disease through some detecting methods.In this paper,a diagnostic method was first developed.Two pairs of primers,E781F and E781R,E485F and E485R,were designed for amplification of 781 bp and 485 bp DNA fragments of the hemolytic gene of E.tarda respectively.Similarly,two pairs of primers,V899F and V899R,V550F and V550R,were designed for amplification of 899 bp and 550 bp DNA fragments of the collagenase gene of V.alginolyticus respectively.No specific fragments were amplified when other principal oceanic fish bacterial pathogens were used as negative controls.The sensitivity of the detection of E.tarda and V.alginolyticus was 10fg and 1fg respectively,which are better than the other diagnostic methods.Nested-PCR detected the presence of E.tarda and V.alginolyticus in 32 samples which were the cultured turbot and flounder taken from the farms of Tianjin,23(23/32) of which were positive for E.tarda,11(11/32) of which were positive for V.alginolyticus,and 2(2/32) of which were positive for both organisms.The results showed that the nested-PCR was effective and reliable methods for the detection of E.tarda and V.alginolyticus.