Isolation and identification of Vibrio spp. in diseased Channa punctatus from aquaculture fish farm

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1 Indian Journal of Geo-Marine Sciences Vol. 41(2), April 2012, pp Isolation and identification of Vibrio spp. in diseased Channa punctatus from aquaculture fish farm Sankar Ganesh. P 1, Saravanan.J 1, Krishnamurthy. P 1, Chandrakala. N 2, & Rajendran.K* 1 1 Department of Biotechnology, Periyar Maniammai University, Periyar Nagar, Vallam, Thanjavur , Tamil Nadu, India. 2 Department of Zoology, Kundavai Nachiyar Govt. Arts College for Women, Thanjavur , Tamil Nadu, India. *[ nkraj64@yahoo.co.uk] Revised 07 February 2011 ; revised 06 June 2011 During the present study Vibrio spp. were isolated from infected Channa punctatus and physico-chemical properties were analyzed from various ponds. Among the twenty ponds under study, four were contaminated by agricultural wastewater. The microbial communities in all these ponds appeared to be dominated by V. harveyi, V. parahaemolyticus, V. fischeri and V. cholerae. Out of these C. punctatus was mostly infected by V. cholerae, V. parahaemolyticus and V. harveyi. It can be transmitted to all fishes and infect fishes of other ponds. [Keywords: Channa punctatus, Vibrio spp, aquaculture] Introduction Bacteria can have an impact in quality of water, by controlling the amount of oxygen and other elements and by causing diseases in aquatic organisms and humans 1. Both beneficial (nutrients recycling, organic matter degrading, etc.) and harmful role (as parasites) are being caused by bacteria in the pond ecosystem. The dominant roles in the spoilage of marine product are due to bacteria 2. In environment as well as in culture ponds Vibrio spp. are responsible for disease outbreak. Microbiological analysis of water quality has proved to be a very useful and significant method of biological assessment of new or intermittent water pollution. The assessment of bacterial types in a monitoring programme will provide protection to the diversity of ecosystem 3. The diseases commonly result from nutritional and environmental problems as well as infections by parasites, viruses, bacteria and fungal agents 4. Some fish diseases can be correctly diagnosed only by fish body inspection and anatomization. However, most fish diseases can be diagnosed by microscopic examination 5. A suitable environment is necessary for any organism, since life depends upon the continuance of a proper exchange of essential substances and energies between the organism and its surroundings 6. *Author for correspondence The physico-chemical analysis of water is of great significance in removing the constraints, which impede the production of inland fish. Fish production is closely correlated with the biological production, which in turn depends upon the ecological and physico-chemical conditions of water body. Research studies on limnological aspects are of great significance in developing fresh water fisheries 7. Prior to all, an effective management of the environment yields better control of diseases. This is possible through understanding the parameters causing the proliferation of pathogenic bacteria. The present investigation focused to evaluate the prevalence of Vibrio spp. in a pond culture system rearing C. punctatus. Materials and Methods The infected Channa punctatus were collected, aseptically from Maanal fish farm at Orathanadu, Thanjavur, India. Water samples were collected from the pond in sterile conical flasks and ph of the samples was recorded immediately. Flasks were covered with sterilized polythene and stored in a refrigerator at 4 C. Samples were serially diluted by serial dilution technique. 1 ml of the sample taken in the sixth position of test tube were plated on a Thiosulphate Citrate Bile salt Sucrose (TCBS) plates and incubated at 30 C for 48 hrs. The infected region showed the symptoms of Vibriosis were aseptically

2 160 INDIAN J. MAR.SCI., VOL. 41, NO. 2, APRIL 2012 taken and plated on TCBS agar (Hi-Media, Mumbai) and incubated at 30 C for 48 h. The Channa punctatus infected red spot region were swabbed and plated on TCBS agar (Hi-Media, Mumbai), and incubated at 30 C for 48 h. Gram staining and cell arrangements were subjected on cells grown on TCBS agar after 48 hrs of growth at 30 C. Colony pigments, texture and other cultural characteristics were studied. Bacterial cultures were subjected to a battery of biochemical tests, which resulted in about 36 recordable characteristics compared with Bergey s manual of determinate bacteriology 8 and a set of keys for identification of environmental Vibrio sp 9 which include tests like Indole, Micrometry, Methyl red, Citrate utilization, Catalase, Oxidase, Urease, Dextrose, Sucrose, Maltose, Manitol, Growth at temperature such as 4 C, 20 C, 30 C, 40 C, Growth in NaCl (0% NaCl, 3% NaCl, 6% NaCl, 8% NaCl, 10% NaCl), Growth in ph (ph 4, ph 9), Gelatin hydrolysis, Luminescence and starch hydrolysis. Water samples were collected in cleaned polythene container and preserved according to standard methods Container was sterilized in autoclave at 121 C for 15 mins. The temperature of the samples was recorded immediately at the site of collection by a sensitive mercury thermometer graduated up to an accuracy of 0.1 C. For the analysis of dissolved oxygen, water samples were collected by BOD bottles of 300 ml capacity. Manganese sulphate and the alkali iodide azide reagent were added immediately at the collection site to fix the samples for studying dissolved oxygen. Measurement was carried out in the laboratory. The ph was measured at the place of sampling sites using standard microprocessor based pocket ph meter (Elico Li 120). Immediately after arrival in to the laboratory the conductivity of the samples was measured with the help of a digital conductivity meter (Digisun electronics model no. 909). Turbidity and total hardness of the samples were analyzed in the laboratory following standard methods of American Public Health Association 12. Results In the present study the diseased Channa punctaus and its rearing water samples were collected for the identification of pathogenic Vibrio spp. 20 different culture ponds were selected and few primary analysis were made on the ponds such as length and width of the ponds (Fig. 1) and the agricultural waste lying near, which contaminated the bore well water pumped to cultivate fishes in the ponds. By the primary analysis we short-listed four ponds (14, 16, 17 and 20) for further analysis. The diseased C. punctatus that showed melanisation and water samples were also aseptically collected from the selected ponds (Fig. 2). Vibrio spp. were isolated from the infected C. punctatus using Thiosulphate Citrate Bile Salt Sucrose (TCBS) agar (Fig. 3). The cells of all bacterial isolates were microscopically observed to be gram-negative curved rods of 2 µm to 3 µm in size. Isolated bacteria were subjected to a group of tests and the observation of 36 recordable characters is tabulated (Table 1). Physico chemical properties such as Temperature, ph, Dissolved oxygen (mg/l), Turbidity (NTU), Electrical conductivity, Hardness Fig. 1 Pond layout Fig. 2 Infected regions of Channa punctatus showed melanisation of infected region

3 GANESH et al.: VIBRIO SPP. IN DISEASED CHANNA PUNCTATUS 161 Fig. 3 The Vibrio spp. on Nutrient agar medium Table 1 Bio chemical characteristics of Vibrio spp. S.No Characteristics V. cholerae V. harveyi V. fischeri V. parahaemolyticus 1 Colour Y Y Y G 2 Morphology R.S R.S R.S S 3 Staining -R -R -R -R 4 Size of the colony (µm) Cell size (µm) Diameter (µm) Motility Indole Methyl red VP Citrate Catalase Oxidase Urease Dextrose Sucrose Lactose Maltose Manitol Arabinose Growth in 0%Nacl Growth in 3%Nacl Growth in 6%Nacl Growth in 8%Nacl Growth in 10%Nacl Growth at 4 0 C Growth at 20 0 C Growth at 30 0 C Growth at 35 0 C Growth at 40 0 C Growth in ph Growth in ph Gelatin hydrolysis Antibiotic Sensitivity (Ciprofloaxin) 35 Luminescence Starch hydrolysis Note: Y - Yellow, G - Green, R- - Negative Rod, R.S- Raised Smooth, + (Positive), - (Negative)

4 162 INDIAN J. MAR.SCI., VOL. 41, NO. 2, APRIL 2012 Table 2 Physico-chemical Parameters Parameters Pond 14 Pond 16 Pond 17 Pond 20 Temperature 37 C 36 C 37 C 37 C ph Dissolved Oxygen (mg/l) Turbidity (NTU) Electrical Conductivity (mg/l) were also analyzed (Table 2). The Physicochemical observation confirms the drastic variations among diseased and disease free ponds. This characterization is taking to a suggestion that the increase in Vibrio community in these samples might be due to some kind of stress. Microbial communities in all these ponds appeared to be dominated by V. harveyi, V. parahaemolyticus, V. fischeri and V. cholerae. Out of these C. punctatus was mostly infected by V. cholerae, V. parahaemolyticus and V. harveyi. It can be transmitted to all fish s and deterioration of fishes of other ponds. Due to bacterial infection, population of Channa punctatus is reduced and it affects the economy of farmers. So there is hike to find remedy to reduce the Vibrio spp. infection in Channa punctatus. Discussion The Vibrio communities contaminate the pond water bodies and infect the Channa puctatus, which result in a very poor yield of Channa punctatus. Water temperature ranged between a minimum of C during winter to a maximum of C during summer. According to Sharma and Gupta 13 summer season is suitable for survival and multiplication of the Vibrio spp. due to which spreading of the fish infection increased drastically. According to Alikunhi 14, the suitable ph for the water bodies is to be in neutral but it may changes ( ) in winter, ( ) in summer and ( ) in monsoon. Fish have their own tolerable limits for ph fluctuation beyond which they cannot survive 13. The ph is slightly high during summer and is good enough for normal fish growth. The Vibrio spp. growth rate increases well during this season due to the contamination by wastewater and the increase in ph. Dissolved oxygen concentration is also another parameter used in judging the suitability of the water body to support fish community 15. Some fluctuation in the dissolved oxygen was noticed during monsoon, which is the result of higher photosynthetic activity which leading to prevention of microbial decomposition and provides a suitable environment for fish culture. Concentration of dissolved oxygen during summer is low due to decreased photosynthetic activity which leading to depletion of oxygen content. However, higher concentration of dissolved oxygen in winter would be due to enhanced photosynthetic activity accomplished by low temperature. In the initial stages, the disease was marked by little red spots on the skin surface, which progressed in size until a circular to oval shaped deep hemorrhagic ulcer exposing the skeletal musculature is visible. A diverse group of biotic agents such as viruses, bacteria, and cutaneous ectoparasites may initiate skin lesions, which are subsequently colonized by Aphanomyces invades and ultimately lead to EUS 16. In the present investigation, Vibrio spp. such as V. harveyi, V. parahaemolyticus, V. fischeri and V. cholerae were isolated from the infected fish samples. Out of these V. cholerae, V. parahaemolyticus and V. harveyi were present in high infected C. punctatus. Generally gram-negative bacteria are found to be the dominant forms in the shrimp culture ponds 17, and the same was noticed, in the present study also. Among the Vibrio cholerae, Vibrio parahaemolyticus seems to play a vital role in Channa punctaus and shrimp culture systems. Vibrio parahaemolyticus being the dominant species might have acted as a secondary pathogen in the viral disease outbreak. The present investigation revealed that the outbreak of bacterial infections occur at the time of increase in bacterial load, especially Vibrio spp. It suggested that the culture species become susceptible due to the increased bacterial load. Proper water quality management and continuous monitoring of benthic microbes are the key factors to avoid bacterial outbreaks in culture ponds. Acknowledgments Authors thank Prof. N. Ramachandran, Vice-Chancellor, Periyar Maniammai University for providing facilities and encouragement to carry out this work. Authors acknowledge Maanal Fish Farm, Orathanadu, Thanjavur, India for providing the samples and support to carry out this study. References 1 Hurst C J, Knudsen G R & Mclnerney M J, Manual of Environmental Microbiology, (American Society for Microbiology. Washington) 1997, pp Palaniappan, R., Studies on the microflora on Prawns Penaeus indicus (milne Edwards) (Crustacean decopoda,

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