Aeromonads-induced Alterations in Activities of Catalase, Alkaline Phosphatase and Acid Phosphatase in the Freshwater Fish Channa punctatus (Bloch)

The short-term effects of intramuscular inoculation of sublethal dose (4 x 107 cfu /ml) of aeromonads viz. Aeromonas salmonicida and Aeromonas hydrophila on the activities of catalase, alkaline phosphatase and acid phosphatase in adult Channa punctatus (Bloch) were investigated. Spectrophotometric method was used to determine the activities of these enzymes from haemolysate, liver and kidney of fish. We found site-specific variations in the activities of these enzymes in aeromonad-treated fish groups compared to sham-injected control groups over the study periods. The comparative infectivity of two strains of aeromonads could be understood by analyzing these enzymatic parameters as biomarkers during initial stages of infection. T widespread distribution of bacteria including aeromonads and other pathogens in the aquatic environment and the stress induced by intensive culture practices predisposes fish to infections. Ichthyopathology for health care of wild and farmed fish has developed in recent years with extensive research inputs in the field of clinical haematology and biochemistry as diagnostic tool. Attempts have been made to use the determination of enzyme activities in blood and other organs in toxicological studies on fishes1,2,3,4,5. The study of the influence of aeromonad infection as one of the environmental stressors on the enzyme profile of fishes becomes important6,7. Therefore, the present experiment was designed to study some enzyme profiles in haemolysate, liver and kidney of spotted murrel Channa punctatus (Bloch), a tropical freshwater fish. Adult C. punctatus were injected intramuscularly (im) with sublethal dose (4 x 107 cfu /ml) of Aeromonas salmonicida and Aeromonas hydrophila, one at a time @ 0.5 ml /100g body weight of fish. The activity of catalase, alkaline phosphatase and acid phosphatase in haemolysate, liver and kidney of both aeromonad-treated fish groups were observed after 3 and 7 days of injection and compared with corresponding sham-injected control at each interval. The experiment was also performed to understand a comparative infectivity of two potent bacterial populations ubiquitously present in aquatic system by analyzing some enzymatic parameters as biomarkers during initial stages of infection. Materials and Methods : A. Fish stock acclimatization and maintenance : Seventy five adult spotted murrels, C. punctatus, were obtained from a local fish farm of Kolkata, West Bengal, India and acclimatized for one week in 15 glass aquaria (size: 0.6m x 0.3m x 0.3m) with continuous aeration for further studies (5 fish/ aquarium). The fish were fed ad libitum with Tubifex sp. and larvae of Culex sp. during this acclimatization period. The specimens selected for study were 81.73 ± 0.49 g in weight and 19.93 ± 0.17 cm in length. B. Bacterial culture collection and maintenance: Microbial cultures of two bacterial strains viz. Aeromonas hydrophila (MTCC 646) and Aeromonas salmonicida (MTCC 1522) were collected from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, (IMTECH), Chandigarh, India and were used for infecting fish. C. Artificial inoculation of aeromonads on C. punctatus : Both A. hydrophila and A. salmonicida were cultured in nutrient broth (NB) and incubated at 370C and 250C respectively for 24 h prior to infectivity testing. Bacterial cells were harvested by centrifugation at 5000 x g for 5 min and washed in PS (0.85 % NaCl). The strains were enumerated by correlating the OD values (600 nm) of the growing culture with the corresponding colony forming units (cfu) obtained by spread plate dilution method (OD 600nm 1 = 2 x 10 9 cfu /ml). Subsequently, the LD50 doses were determined for both the strains8 and a sublethal concentration (4 x 107 cfu /ml) from each strain of aeromonads was selected for intramuscular (im) inoculation. Changes in the enzyme profile in aeromonad-treated fish groups (10 fish per group) were recorded after 3 and 7 days of exposure and compared with corresponding shaminjected control (injected with sterile PS only) at each interval (10 fish per group). D. Preparation of enzyme extract : Free flowing blood (0.6 ml) was collected by severing the caudal peduncle of live healthy sham-injected control fishes as well as from aeromonad-treated fish groups. The blood containing citrate was centrifuged for 6 min at 6000 rpm and plasma was immediately removed. The erythrocyte sediment was washed three times with isotonic NaCl solution. A stock haemolysate was prepared by the addition of 1 ml chilled distilled water to the washed erythrocytic sediment. Then it was thoroughly vortexed, incubated in ice for 30 min and then centrifuged for 20 min at 12,000 rpm in a