Isolation, Identification Of E. Tarda From Farmed Turbot And Establishment Of Rapid Detection Method

Turbot (Scophthalmus maximus L) is an economically important marine fish species and is valued for its rapid growth and good taste. Since introduced into China from Europe in the 1990s, the commercial culture develops very rapidly. However, with the rapid development of the turbot culture in China, several diseases occurred and it suggestes that they resulted from high-intensive cultivation and improper management since 2001. Edwardsiellosis is very common now, there were several reports between 2006 and 2007.Edwardsiella tarda is the causative agent of edwardsiellosis in many commercially important freshwater and marine fish such as channel catfish, eels, mullet, chinook salmon, flounder, carp, tilapia and striped bass. It causes septicemia with extensive skin lesions, affecting internal organs and leads to extensive losses in both freshwater and marine aquaculture. It reported in humans as the cause of gastroenteritis and generalized infections mainly among individuals with impaired immune systems.During the spring and summer of 2006, an epizootic occurred among cultured turbot (Scophthalmus maximus L.) in several fish farms in Qingdao, China. Eye tumefaction, inflammation, haemorrhages, ascites and the presence of a purulent fluid were the main macroscopic lesions observed. A Gram-negative, rod shaped bacterium (designated as LTB-4) was isolated from the infected fish. Isolate LTB-4 was gram-negative rod-shaped bacteria, occurring singly or in pairs with round-ends, (0.5～0.9)μm×(1.0～2.0)μm in size. Biochemically the isolate was positive for indole, H2S production, lysine decarboxylase, ornithine decarboxylase and D-glucose utilization, meanwhile negative for V.P. (voges proskauer) test, and malonate, sucrose, D-mannitol, D-sorbitol, L-rhamnose, and melibiose utilization. The LD50 was established as 3×103 cfu/g by intraperitoneal (i.p.) injection. Isolate LTB-4 is identified as E. tarda by morphology, physiological and biochemical tests and 16S rDNA sequence analysis. Unlike those commonly described E. tarda strains reported in China, no flagellum was observed. Partial gyrB genes was amplified from E. tarda using the universal primers of gyrB genes and sequenced. The PCR primers for the gyrB gene specific to E. tarda were designed. It revealed positive amplification of the gyrB fragment in E. tarda, whereas other bacterial species were negative; with the detection limit of 4.0×104 cfu/mL. Moreover, the technique enabled the recognition of E. tarda from diseased fish. An indirect Enzyme Linked Immunosorbent Assay (ELISA) for the rapid diagnosis of E. tarda, the pathogen of edwardsiellosis, is developed. The best working concentration of antigen and antiserum were 106 cfu/mL and 1∶10000 separately, determined by using checkerboard titration. The working concentration of HRP-labeled goat anti-bovine IgG was 1∶1000. The sensitivity of the serum was tested, and the lowest E. tarda suspension was 105 cfu/mL. Cross-reactions of antisera with other bacteria were detected, all results were negative.