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Available online at http://www.urpjournals.com International Journal of Research in Biological Sciences Universal Research Publications. All rights reserved ISSN 2249–9687 Original Article PATHOGENIC BACTERIA ISOLATED FROM TIGER PRAWN PENAEUS MONODON IN SHRIMP CULTURE PONDS AT EAST COAST OF THANJAVUR DISTRICT TAMIL NADU, INDIA. N.NARASIMHAN *1, T.RAVIMANICKAM *3, M.SUKUMARAN *1, R.RAVICHELVAN *2, R.RAVICHANDRAN *2, D.MADHAVAN *2 *1 PG and Research Department of Zoology, Rajah Serfoji Government College, Thanjavur - 613 005, Tamil Nadu, India. *2 PG Department of Zoology, Govrnment Arts College, Ariyalur - 621 704, Tamil Nadu, India. *3 PG Department of Bio –Technology and Gene Technology, JJ College,Pudukkottai ,Tamilnadu, India Received 25 April 2013; accepted 20 May 2013 Abstract Pathogenic bacterial flora was isolated from muscle tissues of black tiger shrimp, Penaeus monodon. The infected shrimp samples were collected from Omsakthi Aquafarm at Sedhubhavasatram in Thanjavur District, Tamil Nadu, India, during December 2012 to February 2013. Bacterial species were isolated by serial dilution technique method were plated in nutrient agar medium (Jolt et al., 1994). Totally eleven different bacterial strains such as Aureobacterium faciens, Aeromicrobium erythreum, Bacillus subtilis, Escherichia coli, Vibrio cholarae, Enterobacter aerogens, Micrococcus Luteus, Pseudomonas putida, Pseudomonas aeruginosa and Enterococcus pseudo avium were isolated from muscle tissues of P.monodon. The culture plates were incubated at 37°C for 24 to 48 hours. The different species of bacteria were identified using the biochemical tests namely, Motility test, Indole test, Methyl red test, Voges - proskauer test, Citrate utilization test, Catalase test, Oxidase test, Triple sugar Iron test, Urease test and Nitrate test. Broth cultures were observed the colour formation, ring formation and gas production which indicated positive and no colour change, no ring formation and no gas production which indicated nagative results. © 2013 Universal Research Publications. All rights reserved Key Words : Penaeus monodon, Biochemical tests, Pathogenic bacteria, Shrimp culture ponds. INTRODUCTION Bacterial infection is one of the major disease problems in shell fish and fin fish aquaculture. Disease cause the largest economic losses in aquaculture and bacterial infections are second only to fungal diseases in economic importance. Bacterial infections are generally restricted to chronic, steady losses (Intesar, 2003). Vibrio cholerae is one of the important etiological agents in mass mortalities of Penaeus monodon rearing systems (Tanasomwang et al., 1998.) Gram positive bacteria is based on the general models of infection and disease that are caused by the pathogenic bacteria can be grouped into three categories, extra cellular invasive pathogens produce toxin, but the disease that these pathogen cause are primary due to their invasion and growth within tissue, rather than production and dissemination of a toxin. Food security is a complex issue, where shell fish and fishery products are generally 98 regarded as high risk commodity is respect of pathogen contents, natural toxins and other possible contaminants and adulterants. Although there has been a significant increase in the knowledge of shrimp disease (Nash, 1988 ; Mayer, 1991 ; Maeda and Liao, 1992). Gram negative bacteria are cocci or coccibacilli whose associated disease usually involve the accumulation of copious amounts to pus frequently affect the respiratory tract. Although studies have been performed on microbiological disease in Indian prawns, there is a paucity of detailed investigations on the effects of microbial pathogens. The research in shrimp pathology has been with reference to disease in shrimps collected from the shrimp farms at the time of occurrence of disease. There have been laboratory, based studies on shrimps (Brock and Lightner, 1990). Shrimp affected by this bacterial disease were than taken from the culture ponds and microbial pathogens was controlled in the laboratory conditions. International Journal of Research in Biological Sciences 2013; 3(2): 98-101 Vibrio sp., Bacillus sp., Pseudomonas sp., E.coli., and Enterobacter sp., were the common bacterial pathogens (Moriarty, 1998). The bacteria causing the most serious diseases of the post larval and adult stages of P.Monodon of the genus Vibrio, Bacillus, Pseudomonas and Aeromonas (Lightner and Redman, 1998). This paper reports on pathogenic bacteria associated with mortalities of P.monodon in rearing ponds, with a view to provide potential approaches for improving the quality assurance and create awareness among the consumers. MATERIALS AND METHODS Study area: Infected shrimp Penaeus monodon were collected from Omsakthi Aquafarm at Sedhubhavasatram, Thanjavur district, Tamil Nadu, India. Study period: Samples were collected from December 2012 to February 2013. To avoid further contamination, during transportation from the source to laboratory, Samples were carried by special sterile bags packed in insulated box with ice to maintain the temperature around 5 to 6 C. Isolation of shrimp bacteria: The muscle samples of affected Penaeus monodon were collected and homogenized were used as a sample for bacterial isolation (Aneja, 2001). The homogenated each tissue sample was serially diluted from 10-1 from 10-9 from the diluted sample taken 0.1 ml of sample from 10-5, 10-6 and 10-7 and spread over on nutrient agar medium separately, one plate maintain as a control without sample. The plates were incubated at 37 C for 24 to 48 hours observed the bacterial colonies. Identification of shrimp bacteria The isolated bacterial species were identified by the following the morphological and biochemical characteristics of the individual colony was recorded. The individual colony was transferred to nutrient agar. The isolates were subjected to following different biochemical test for example Gram staining, Motility test, Indole test, Methyl red test, Voges Proskauer test, catalase test, Nitrate test and Carbohydrate fermentation test as described by Jolt et al., (1994). RESULTS AND DISCUSSION The different species of pathogenic bacteria were identified using the physical and chemical tests. They are presented in Table -1. Physical Analysis: Motility test: Hanging drop method was carried out for motility test. The bacterial culture plate BCP2 and BCP8 resulted were showed non motile and other cultures were motile. Chemical Analysis Gram staining: All the bacterial cells have been stained by counter stain safranin and appeared red in colour. The culture BCP1, BCP2, BCP3 and BCP7 were resulted in gram positive and other cultures were negative. Among the species five bacterial were rod shaped (BCP3, BCP4, BCP5, BCP8 and BCP9) two bacterial species (BCP1 and BCP2) were rod or cocci shaped and four bacterial were cocci shaped (BCP6, BCP7, BCP10 and BCP11). 99 Indole test: Ring formation was observed only BCP2, BCP4, BCP6, BCP8, BCP 10 and BCP11 and other culture were indicate no ring formation. Broth cultures showed no ring formation so it indicates the negative result. Methyl red test. Addition of methyl red indicated to the MR broth cultures of such as BCP2, BCP4 and BCP7 showed the presence of pink colour, which indicated positive results and there was no colour change and other broth culture were showed negative results. Voges Proskauer test: Addition of methyl red indicated to the VP broth cultures of such BCP2, BCP3, BCP6 and BCP8 showed the presence of pink red colour, which indicated positive results and there was no colour change in other culture strains which showed negative results. Citrate Utilization test: The broth cultures such as BCP4 showed negative results and other culture showed green to blue colour, which indicated positive results. Urease test: The both cultures BCP1, BCP2, BCP6, BCP10 and BCP11 showed yellow to pink colour, which indicated positive results and other cultures were showed no colour changed, which indicated negative results. Catalase test: The appearance of gas bubbles was observed in all the broth cultures, which indicated positive results. The negative results were not showed. Oxidase test: The broth cultures such as BCP4, BCP6 and BCP9 were showed negative results and other cultures were showed positive results. Nitrate test: After incubation few drops of a Naphthylamine and Sulphanilic acid were added. The red colour formation was indicated as positive results. The broth cultures were showed the red colour in BCP2, BCP3, BCP8 and BCP9. The absence of red colour were showed BCP1, BCP4 and BCP7 indicates the negative results and the red colour was reductase in BCP5, BCP6, BCP10 and BCP11. Carbohydrate fermentation test: All the broth cultures were showed positive results. This indicated the gas production. Identification of bacteria: The isolated different bacterial strains were identified based on the culture morphology and biochemical characteristics. The table results were compared with Bergey’s manual of systemic bacteriology. Based on the results were confirmed as Aureobacterium faciens, Aeromicrobium erythreum, Bacillus subtilis, Escherichia coli, Vibrio cholarae, Enterobacter aerogens, Pseudomonas putida, Pseudomonas aeruginosa, Brucella canis and Enterococcus pseudo avium. In the present study bacterial species were isolated from infected shrimp muscle tissues. Totally eleven bacterial species were isolated by diluting plating technique. The isolated all the bacterial species was harmful pathogen. Bacterial infections are caused penaeid species and the overviews of diseases in cultured prawn were studied (Bright et al., 1988). Epizoobiology and pathogenicity of bacterial infections caused by many problems in cultured giant tiger prawn Penaeus monodon (Chen et al., 1992). Vibrio species are considered as part of the normal flora of seawater and can invade marine animals (Olsen et al., 1995). In general, the results of our study indicate that International Journal of Research in Biological Sciences 2013; 3(2): 98-101 VP TST G F Catalase + + + + - + + + + + - + + + + + + + - + - + - + + - + + + + + + + + + + + + + + + + + + + cocci + - + + + + + - + + + + + cocci - + - + - + + + + + + - - - Rod + - + + - + + + + + + + 9 - + Rod - - - + - + + - + + + 10 - + cocci + - - + + + + + + + + + 11 - + cocci + - - + + + + + + + + + Shape 2 + - 3 4 5 + - + + + 6 - + 7 + 8 = = = = Voges Proskauer Test Trible Sugar Iron Test Glucose Test Fructose Test bacterial load of shrimp pond water and sediment increases with increasing organic load, which is in agreement with previous reports by (Horsley, 1997; Dalmin et al., 2002). In the context of aquaculture, the muscle microbiota is constantly influenced by the aquatic microflora. Thus, a modified definition of probiotics for aquaculture has been proposed (Verschure et at, 2006). Experimental infections performed by Lavilla - pitogo et al., (1990) also showed high pathogenicity of the isolated strain. Usually in prawn culture water or even in entrophicated coastal water bacterial numbers are less than 10 6 cells /ml because protozoa feed on bacterial cells which results in maintaining a certain level of bacterial populations in water (Maeda and Nogami, 1989). Based on the present study, it is concluded that the pathogenic bacteria are perhaps that most important pathogens in shrimp culture ponds causing servere mortalities and financial losses. There is also a need to develop the shrimp culture practice and control the pathogenic microbes is very essential. ACKNOWLEDGEMENT The authors will express their gratitude for the staff of Gowri Biotech Research Laboratory for their help to this work. REFERENCES 1. Anaja, K.R., 2001. Experiments in microbiology plant pathology and biotechnology. 4th edn. New age International (p) Ltd. Publishers, 356 -360. 2. Bright Singh, I.S., Lakshmanaperuma, p., and Chandramohan, D., 1988. Bacterial infection of pond reared Penaeus indicus (H.milne Edwards). J.Aquatic. Trop. 13(2): 133 -142. 3. Brock, J.A., and Lightner, D. V., 1990. Microbial 100 + - Carbohydrate Fermentation G F S = = Nitrate + Oxidase + + TSI + + Urease + 1 Rod/cocc i Rod/cocc i Rod Rod Rod Citrate VP - Indole - Motility - Gram staining Methyl red Bacterial Culture Plate (BCP) Table.1. Bio-Chemical Characterization of bacterial isolates from infected shrimp Penaeus monodon. Identification of Bacteria. Aureobacterium faciens Aeromicrobium erythreum Bacillus subtilis Escherichia coli Vibrio cholerae Enterobacter aerogens Micrococcus luteus Pseudomonas putida Pseudomonas aeruginosa Brucella canis Enterococcus pseudo avium Positive Negative Pathogens and disease of marine crustaceans. Aquacult.1: 245-349. 4. Chen,S.N., Huang,S.L., and Kou, G.N., 1992. 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Intl. 15 (8) : 11-30 Olsen,J.E., Aabo,S., Hill, W., Notermans, S., Wernars, K., Granum, P.E., Popovic, T., Rasmussen, H.N., and Olsvik, O., 1995. Probes and polymerase chain reaction for detection of food borne bacterial pathogens. Inst.J.Food Microbiol.28:1-78. 17. Tanasomwang, V., Nakai, T., Nishimura, Y., and Muruga, K., 1998. Vibrio inhibiting marine bacteria isolated from black tiger shrimp hatchery. Fish Pathol. 33:459- 466. 18. Verschuere, L., Rombaul, G., Sorgeloos, P., and Verstraete, W., 2000. Probiotic bacteria as control agents in aquaculture, Microbiol, Mol. Biol.Rev.64: 655-671. Source of support: Nil; Conflict of interest: None declared 101 International Journal of Research in Biological Sciences 2013; 3(2): 98-101