Indian Journal of Experimental Bi ology Vol. 39, December 2001, pp. 1311 -l3l3 Edwardsiella tarda endotoxin as an immunopotentiator in singhi, Heteropneustes fossilis fingerlings B K Das, P Pattna ik, G Murjani & S C Mukherjee* Aquatic Animal Hea lth Division, Central Institute of Freshwater Aquaculture, Bhubaneswar, 751 002, India Received 4 January 2001; revised 7 August 200/ Endotox in of E. tarda grown in brain heart in fusion broth at 30°C for 18 hr was extracted by differential centrifugati on. Fingerlings of H. fossilis (weighing 1-2 g) were all owed for hyperosmotic infiltration in the endotoxin at the rate of 0,2,4,8, 16 and 20 mg/1. Mortality varied from 20-50% at 2 to 20 mg/ml. Toxin treated fishes were challenged 21 days post treatment wi th the same E. tarda strain containing 2.1x 10 9 CFU/ml. There was 80% mortality in the control group whereas onl y 20% mortality in toxin treated group at 2 mg/1 concentration after challenge with homologous E. tarda. Subsequently a seco nd challenge of E. tarda was given to th e survivors of fi sh one month after first challenge usin g same concentration where no mortality could be observed. It was concl ud ed th at the endotoxin could enhance percentage of survival against E. tarda infec ti on in Sin g hi . Edwardsiella tarda is a potential fi sh pathogen in many parts of the world 1.2. It has been associated with tilapia 3 ,channel catfish 4 , snake head 5 , sea bass 6 and magur 7 . Toxicity of extracellular products and intracellular components of E. tarda was tested in Japanese eel and flounder 8 . It has bec ome conventional to treat aquaculture ponds and fishes with chemicals and/or antibiotics to check the disease outbreaks all over the world. Frequent use of antibiotics for treatment against bacterial di seases, after a certain period of time leads to drug resistance in the fishes. The residual effect of th ese antibiotics also disturbs ecological balance whi ch ultimately affects the human being. In order to find an alte rn ative mea ns of therapy, the present study on e nd otoxin of E. tarda as an immunopotentiator was evaluated in Heteropneustes fossilis. As there is no report available on the effect of E. tarda toxin in Indi an catfis hes as we ll as other cultivable fishes, a base line experimental study was conducted. Indian catfish, si ng hi (Heteropneustes fossilis) is one of the major cultivable priced species in India and th e E. tarda in fection was found frequently in all stages during its culture. Hence in order to find efficacy and use as a toxoid the effect of endotoxin of E. tarda was observed via hyperosmotic infiltration and su bsequent cha ll enge experiments in H. fossilis duri ng it s ea rl y stages of lif e. Fingerlings (240) of H. fossilis weighing 1-2 g *Present address: Central In stitu te of Fi sheri es Educa ti on, Seven Bunglows. Versova. Mumbai 40006 1, India were obtained from the catfish hatchery of th e Institute. The fishes were maintained for 2 weeks in a 401 fiberglass container provided with freshwater at 26.5°-28°C and fed daily with pelleted feed at the rate of 6% of their body weight. Isolation of endotoxin a nd treatment--E. tarda used in this experiment was isolated from diseased singhi at the Institute. Pure culture of E. tarda was grown in BHI broth at 30°C for 18hr and collected. The bacterial concentration was adjusted to 2.1x l0 7 CFU/ml. The bacterial cells were then washed with phosphate buf fered saline (pH 7 .2) and the endotoxin was extracted from the cells u si ng centrifugation (12000 g for 15 min) at refrigerated temperature 9 . Th e cells were sonicated for 20 min using a so ni cator (Arteck model 150) and further centrifuged at 12000 g for 20 min and filtered with a 0.45jlm syringe filter. The toxin was then treated with 0.5% formaldehyde and the protein content of th e toxin was determined as per the methods of Lowry et a/ 10 • Before challenging the fis h with E. tarda endotoxin, 5 fish were selected at random for detecting the presence of E. tarda if any, using bra in heart infusion (BHI) broth and subsequently detection with Rimler-Shott's medium (RS) which did not show presence of any bacteria in their kidney, li ver and ski n surface. Fingerlings (40) in each group were subjected to hyperosmotic infiltration (0.85% normal saline) for 5 min in th e endotoxin prepared earlier at the rate of 0.1, 2, 6, 8, I Oml/1 adjusting the protein content at 2mg/ml.. Toxin treated fish and control fi shes were