characterization of bacteria isolated from cobia (rachycentron canadum) cultured in nha trang bay, vietnam

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES Department of Aquatic Pathology CHARACTERIZATION OF BACTERIA ISOLATED FROM COBIA (Rachycentron canadum) CULTURED IN NHA TRANG BAY, VIETNAM By VO LE THANH TRUC A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Can Tho, December 2013 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES Department of Aquatic Pathology CHARACTERIZATION OF BACTERIA ISOLATED FROM COBIA (Rachycentron canadum) CULTURED IN NHA TRANG BAY, VIETNAM By VO LE THANH TRUC A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Supervisor Ass. Prof. DANG THI HOANG OANH Can Tho, December 2013 ACKNOWLEDGEMENT First of all, the author thanks her supervisor, Ass. Prof. Dang Thi Hoang Oanh for her invaluable supporting, guiding and encouraging during the research. Many thanks also give to other doctors and masters of college of aquaculture and fisheries, especially to members in department of aquatic pathology for kindly help and support good conditions for working and learning. The author would like to express her sincere to Ms. Tran Viet Tien, Ms. Bui Thi Diem My, Ms. Au Thi Kim Ngoc, Mr. Le Thanh Can and Nguyen Trong Nghia for their kindly help throughout the experimental period. Last but not least, the author wants to give many thanks to her academic advisers Mrs. Duong Thuy Yen, who always guiding and give useful advice during academic years. The author, Vo Le Thanh Truc i ABSTRACT The purpose of this study was to isolate and characterize bacterial isolates which were recovered from diseased Cobia cultured in Nha Trang Bay, Khanh Hoa province. A total of eights diseased pathogens which were collected in October, 2012. Diseased fish displayed lethargic, hemorrhagic and whitish granules in internal organs. Bacterial isolates were examined for morphology, selected biochemical characteristics as well as susceptibility to common used antibiotics in aquaculture. These isolates were identified as P. damselae subsp. Piscicida by using API 20E test kit. Result of antibiotic sensitivity tests showed that there were two out of eight isolates were resistant to doxycyline and tetracycline. All isolates were completely susceptible to ampiciline, bicomarin and amoxyciline. Enrofloxacine and ciprofloxacin are the antibiotics that all of the isolates were susceptible with the largest zone. In another hand, eight isolates was susceptible to erythromycin and Trim/sulfa with the intermediate inhibition zone range from 12 - 15mm. ii TABLE OF CONTENTS ACKNOWLEDGEMENT . i ABSTRACT ii LIST OF TABLES v LIST OF FIGURES . vi CHAPTER . INTRODUCTION 1.1 1.2 1.3 GENERAL INTRODUCTION RESEARCH OBJECTIVES RESEARCH ACTIVITIES . CHAPTER . LITERATURE REVIEW . 2.1 COBIA (RACHYCENTRON CANADUM) AND AQUACULTURE OF COBIA IN VIETNAM 2.2 SOME COMMON DISEASES IN RACHYCENTRON CANADUM (LINNAEUS, 1766) . 2.2.1 Streptococcal infection 2.2.2 Vibriosis 2.2.3 Photobacterium damselae sbsp. piscicida 2.3 SOME COMMON ANTIBIOTICS USE IN AQUACULTURE . 2.3.1 Oxytetracycline . 2.3.2Beta-lactams . 2.3.3 Florfenicol . CHAPTER . 11 3.1 TIME AND PLACES 11 3.2 MATERIALS 11 3.3 METHODS . 11 3.3.1 Bacterial isolation . 11 3.3.2 Bacterial identification . 11 3.3.3 Antibiotic susceptibility test 12 3.4 DATA COLLECTION, CALCULATION AND ANALYSIS . 13 CHAPTER . 14 iii RESULTS AND DISCUSSION . 14 4.1 FISH SAMPLING AND CLINICAL SIGNS . 14 4.2 BACTERIAL IDENTIFICATION 15 4.2.1 Morphological observation . 15 4.2.2 Physiological and biochemical characterization 16 4.2.3 Bacterial pathogen analysis by API 20E test kit ( BioMrieux, France) 16 4.3 ANTIBIOTIC SENSITIVITY TEST . 18 CHAPTER . 21 CONCLUSIONS AND RECOMMENDATIONS . 21 5.1 Conclusions . 21 5.2 Recommendations . 21 REFERENCES . 22 APPENDIX . 27 iv LIST OF TABLES Table 4.1. Morphological, physical and biochemical characterization of the eight isolates of bacteria from diseased cobia samples. Table 4.2. Result from API 20E biochemical test strip. Table 4.3. Sensitivity of bacterial isolates to different antibacterial drugs (data are the diameters of the antibacterial area in the plate, the unit is mm). v LIST OF FIGURES Figure 4.1. Lethargic fish. Figure 4.2. Hemorrhage in body cavity and white spot on internal organs. .Figure 4.3 A Morphology of isolated bacteria on NA+. Figure 4.3 B Gram stain of isolated bacteria. Figure 4.4 O/F test. Figure 4.5 API 20E test result for P. damselae sub sp. Piscicida after 24hours incubation. Figure 4.6 Antibiotic sensitivity test. vi CHAPTER INTRODUCTION 1.1 General introduction Cobia, Rachycentron canadum, is considered one of the most suitable candidates for warm, open-water of aquaculture in the world. They contribute widely in warm marine water to tropical waters of the West and East Atlantic, through Caribbean and IndoPacific region. Cobia have good traits, most important traits are growing rapidly and high flex quality. They reach harvest sizes (6kg - 8kg) after one and half years (FAO, 2009). Furthermore, they can tolerate in wide range of salinities (5ppt – 44.5ppt) and temperatures (1.60C – 32.20C). Aquaculture research with cobia was first reported in 1975 but there were any large-scale commercial production of cobia until 2006. With the production report to FAO in 2004, China and Taiwan are two main production countries of cobia over the world. In Vietnam, farming of cobia is a new species to aquaculture, which are cultured by small-scale to medium-scale family farms as well as cooperative farms. The main factor constraining development of cobia culture in Vietnam is a shortage of quality fingerlings, although hatchery production in Vietnam is increasing at a rapid rate (Nhu et al., 2010) Like other species, cobia also gets diseases during culture either ponds, tanks or cages. Managing disease and parasite issues has been identified, particularly by the Taiwanese, to be one of the major challenges with regard to cobia culture so far (FAO, 2013). Bacteria cause diseases by some clinical signs: whitish, granulomatous deposits on kidney, liver and spleen, ascites in peritoneal cavity. Disease during culturing of cobia is one of the most concerns of farmer and producer because of high mortality. Cobia is the new species for aquaculture not only in Vietnam but also in the world. So, this thesis: “Characterization of bacteria isolated from cobia (Rachycentron canadum) (Linaeus, 1766) cultured in Nha Trang Bay, Vietnam” was conducted to update information of diseases in other to identify common bacterial pathogens in cukturedcobia. Hence, recommending treatments and antibiotics, increasing productivity, profitability for farmers. 1.2 Research objectives The research is aimed to identify isolated bacteria form diseased Cobia cultured in Nha Trang Bay, Vietnam and their susceptibility to commonly used antibiotics. 1.3 Research activities 1. The first activity is to identification of isolated bacteria to species level to find out which bacterial pathogen caused diseases on cobia. 2. The second activity is to antibiotic sensitivity tests which aim to figure out the susceptibility to common used antibiotics. CHAPTER RESULTS AND DISCUSSION 4.1 Fish sampling and clinical signs From each cage, fish were collected both healthy and diseased one. The infected fish vary from no external indicators through had some gross signs which can see easily by snake eyes such as lethargic, abnormal swimming and skin lesioning. Internally, compare to normal fish, ascites may be present in the peritoneal cavity and the host liver and kidney may pale in color while the spleen may have white tubercules present. Healthy cobia had clear internal body cavity, whereas the diseased body cavity with hemorrhage. (Figure 4.1 and 4.2). Figure 4.1. Lethargic fish. Figure 4.2. Hemorrhage in body cavity and white spot on internal organs 14 Bacteria were isolated from brain, kidney and liver and streak on nutrient agar (Merck) supplemented with 1.5% NaCl. Eight dominant bacterial isolates (isolate CB 2-2_N, CB 2-2_T, CB 2-2_G, CB 2-4_T, CB 2-4_N, CB 1-2_N, CB 1-4_G and CB 1-4_T) were recovered from kidney, brain and liver of the cobia suffering from diseases. 4.2 Bacterial identification 4.2.1 Morphological observation Bacterial isolates (CB 2-2_N, CB 2-2_T, CB 2-2_G, CB 2-4_T, CB 2-4_N, CB 1-2_N, CB 1-4_G and CB 1-4_T) from diseased cobia displayed colonies on nutrient agar supplemented with 1.5% NaCl media with rounded, slightly convex, smooth and milky color (Figure 4.3 A and B). After 24 hours of incubation, those strains presented in green color on TCBS. Those isolates indicated the characteristics of Gram negative bacteria, short rod shape, positive mobility, positive oxidase and catalase, they displayed the ability of fermentation and oxidation of glucose. A B Figure 4.3 (A) Morphology of isolated bacteria on NA+. (B) Gram stain of isolated bacteria. Figure 4.4 O/F test 15 Table4.1. Morphological, physical and biochemical characterization of the eight isolates of bacteria from diseased cobia samples. Isolates Items tested CB 12N CB 14T CB 14G CB 22N CB 22T CB 22G CB 24T CB 24N Gram stain Morphology r r r r r r r r Color of milky milky milky milky milky milky milky milky pigment Motility Oxidase + + + + + + + + reaction Catadase + + + + + + + + reaction Aerobic + + + + + + + + fermentation Anaerobic + + + + + + + + fermentation Gas production 0/129 + + + + + + + + resistance Notes: ( r ): rod shape; ( - ): negative; ( + ): positive P. damselae subsp. Piscicida (Magarinõs et al., 1992) r milky + + + + + 4.2.2 Physiological and biochemical characterization Isolates CB 2-2_N, CB 2-2_G, CB 2-2T, CB 2-4_T, CB 2-4_N, CB 1-2_N, CB 1-4_G and CB 1-4_T were characterized as genus Pasteurellosis because all of them were bipolar rod shape, positive for the oxidase, catadase reaction, non-motile. Moreover, fermentation in O/F media also gave positive result without gas production and sensitive to the vibrio static agent O/129. In the test with blood media, these isolates give the result that they not have the ability to dissolve blood after 24 hours incubation. 4.2.3 Bacterial pathogen analysis by API 20E test kit ( BioMrieux, France) Identification by API 20E test kit aimed to identify correctly bacterial pathogens causing diseases on cobia. A profile number of 201500457 obtained from API 20E system corresponded to Photobacterium damselae sub sp. Piscicida 16 Table4.2. Result from API 20E biochemical test strip: No. Indicators 10 11 12 13 14 15 16 17 18 19 20 ONPG ADH LDC ODC CIT H2S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA CB 1-2N + + + + - CB CB CB CB CB CB CB 1-4T 1-4G 2-2N 2-2T 2-2G 2-4T 2-4N + + + + - + + + + - + + + + - + + - + + + + - + + - + + + + - P. damselae subsp. Piscicida (Magarinõs et al., 1992) + + + - Note: ( - ): negative result; ( + ): positive result ; ONPG: β-galactosidase (o-nitrophenyl-βDgalactopyranoside); ADH: arginine dihydrolase; LDC: lysine decarboxylase; ODC: ornithine decarboxylase; CIT: citrate; H2S: hydrogen sulfide; URE: urease; TDA: tryptophane deaminase; IND: indole; VP: Voges-Proskauer reaction; GEL: gelatin; GLU: glucose; MAN: mannitol; INO: inositol; SOR: sorbitol; RHA: rhamnose; SAC: sucrose; MEL: melibiose; AMY: amygdaline; ARA: arabinnose The results of eight isolates bacterial pathogen shows positive responses for arginine dihydrolase (ADH), lipase activities , and is negative for indole (IND), nitrate reduction, gelatinase (GEL), amylase (AMY), tryptophane deamynase (TDA) and hydrogen sulfide (H2S) production as well as create β-galactosidase (ONPG). They also had negative 17 result with citrate (CIT) but gave positive one with Voges-Proskauer reaction (VP). Moreover, the result also indicated these bacterial pathogens cannot utilize sucrose (SAC) and mannitol (MAN). 10 11 12 13 14 15 16 17 18 19 20 Figure 4.5 API 20E test result for P. damselae sub sp. Piscicida after 24hours incubation Except arginine dihydrolase(ADH) indicator gave a negative result, and producing urea (URE) in isolates CB 2-4T and CB 2-2T are different from other six isolates. Others indicators are all the same in eight isolates bacterial pathogen and were identified as P. damselae sub sp. Piscicida. In agreement with Kent, although the pathogen is not included in the API-20E code index, the system is valuable for a rapid presumptive identification of the bacterium, because all strains have a similar pattern (2005004), which neither false-positive nor false-negative reactions being detected. 4.3 Antibiotic sensitivity test Eight bacterial isolates were tested for their sensitivity to 11 antimicrobial agents. Out of 11 antibiotics used, the isolates were completely resistant to Amoxycilline and Ampicilline. Figure 4.6Antibiotic sensitivity test 18 Besides, their sensitivity of Erythromycin and Trim/sulfa was intermediate range (around 12mm – 15mm). They also were susceptible to Norfloxacin, Florfenicol and Enrofloxacine and give the best result of sensitivity to Ciprofloxacin and Enrofloxacine with the largest zone. Table 4.3 Sensitivity of bacterial isolates to different antibacterial. (µg/ dics) Amoxycilline 25 Erythromycin 15 Norfloxacin Ciprofloxacin 30 Florfenicol 30 Trim/sulfa 25 Tetracyline 30 Neomycin 30 Ampicilline 35 Doxycyline 30 Enrofloxacine Antibiotic CB 1-2N 14 31 28 27 18 25 13 24 26 CB 1-4G 14 29 30 27 17 25 14 23 28 CB 1-4T 13 26 31 28 24 29 22 24 32 CB 2-2G 13 30 30 27 19 25 12 23 29 CB 2-2T 14 30 30 32 15 30 15 25 28 CB 2-2N 12 22 22 25 13 25 13 24 22 CB 2-4T 14 35 35 30 20 29 12 26 35 CB 2-4N 13 28 31 26 20 25 15 23 29 Unit: mm Cobia now are cultured widely in warm, coastal zone around the world. As the result, the more cobia are grown, the more diseases will spread through. It is widely accepted that intensive fish culture, particularly of non-native species, is involved in the introduction and/or amplification of pathogens and disease in wild populations (Sziezko 1974; van Muiswinkel et al. 1999; Blazer and LaPatra 2002; Naylor and Burke 2005). A review by McLean et al. (2008) finds that wild cobia are affected by many of the same diseases and parasites that infect other warm water species. Outbreaks of Vibrio alginolyticus, Vibrio parahaemolyticus and Photobacterium damselae (subsp. piscicida) have produced mortality rates of up to 80% or more in young cobia (Lin et al. 2006). The results of this experiment show that main type of bacterial can infect to cobia (Rachycentron canadum) are Photobacterium damselae subsp. Piscicida. All of isolated bacteria have characteristics of genus Pasteurellosis. They are Gram-negative, non motile, bipolar baccilus, positive result of oxidase and catadase. Moreover, they have ability to ferment in both aerobic and anaerobic condition. Although some indicators not completely match with data studied by Janssen and Surgalla (1968), they were re19 identify by PCR 16s rRNA technique for confirmation of Photobacterium damselae subsp. Piscicida. Around the world, disease epidemics are responsible for billions of dollars in losses to the aquaculture industry every year. Cobia is no exception to this general rule and it would appear as time passes the variety of diseases encountered with this species increases. Further researches and studies should concentrate in a new generation of vaccines and probiotics for reducing dependence on antibiotic also on chemical treatments to control diseases. It is imperative that appropriate measures should be taken to develop more rigorous disease prevention programs as well as develop a disease – specific diagnostic tools. 20 CHAPTER CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions Bacterial isolates recovered from diseased cobia which displayed clinical signs as lethargic, hemorrhagic and whitish granules in internal organs were identified as Photobacterium damselae sub sp. Piscicida through the basic biochemical and the API 20E tests. All of isolates were resistant to amoxycilline and ampicilline but give high susceptible to ciprofloxacin and enrofloxacine. 5.2 Recommendations Further research should continue to go through this study for more information about pathogenicity of bacterial isolates from diseased cobia. Besides, later research and study also more experiments and more antibiotics on the antibiotic sensitivity and minimal inhibitory concentration to survey the antibiotic resistance comprehensively in many antibiotics. 21 REFERENCES Barrow, G. I., and R. K. A. Feltham, 1993. Manual for the identification of medical bacteria. Cambridge University Press, Cambridge (1993). Bermúdez-Almada, M. C., and A. Espinosa-Plascencia. The Use of Antibiotics in Shrimp Farming. Blaylock, Reginald B., and Daryl S. Whelan, 2004. Fish health management for offshore aquaculture in the Gulf of Mexico. In: Efforts to develop a responsible offshore aquaculture industry in the Gulf of Mexico. CJ Bridger Ed: 129-161. Bùi Kim Tùng, 2001. Thuốc kháng sinh. Sở Khoa Học Công Nghệ Môi Trường tỉnh Bà Rịa – Vũng Tàu. 225 trang. Bunkley-Williams, Lucy, and Ernest H. Williams Jr, 2006. New records of parasites for culture Cobia, Rachycentroncanadum (Perciformes: Rachycentridae) in Puerto Rico. In: Revista de Biología Tropical 54: 1-7. Bunkley-Williams, Lucy, and Ernest H. Williams Jr, 2010. New records of parasites for Cobia, Rachycentroncanadum (Linnaeus)(Perciformes: Rachycentridae), from an aquaculture operation off northeastern Puerto Rico. Catherine Latanich, 2009. Farmed cobia (Rachycentron canadum) worldwide (United States, Belize, and Asia Regions). Seafood watch farmed Cobia report. De la Federación, Diario Oficial. "acuerdo mediante el cual se aprueba la actualizacion de la carta nacional acuicola." Deng, Huan et al, 2009. Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicas. Aquaculture 287.1 (2009): 18-27. Depaola, Angelo, James T. Peeler, and Gary E. Rodrick, 1995. Effect of oxytetracycline-medicated feed on antibiotic resistance of gram-negative bacteria in catfish ponds. In: Applied and Environmental Microbiology 61.6: 2335-2340. Deveney, Marty R., Leslie Anne Chisholm, and Ian David Whittington, 2001. First published record of the pathogenic monogenean parasite Neobenedeniamelleni (Capsalidae) from Australia. In: Diseases of aquatic organisms 46.1: 79-82. 22 FAO, 2009. Fishstat plus Vers. 2.3.2000: Universal software for fishery statistical time series: Aquaculture production 1950–2007; Capture production 1950–2007. FAO Fisheries and Aquaculture Department, Fishery Information, Data and Statistics Unit. G. Gopakumar and A.K. Abdul Nazar, 2012. Broodstock development, Breeding & larval rearing of cobia and pompano. Handbook on open sea cage culture, 61 – 62. Gopakumar, G., et al, 2010. Successful seed production of cobia Rachycentroncanadum and its prospects for farming in India. In: Marine Fisheries Information Service; Technical and Extension Series 206: 1-6. Guerra-Santos, Bartira, et al, 2012. Parameters hematological and histopathologic alterations in cobia (Rachycentroncanadum Linnaeus, 1766) com amyloodiniose. In: PesquisaVeterináriaBrasileira 32.11: 1184-1190. Hsu, Po‐Yuan et al, 2013. Purification and characterization of a phospholipase by Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum. Journal of Basic Microbiology (2013). Huy, N.Q., Sveier, H., Hung, B.V., Tuan, L.A., Can, N.V., Thien, T.M., and Svennevig, N. 2008. Growth Performance of Cobia, Rachycentron canadum, in sea cages using extruded fish feed or trash fish. In: Cage Aquaculture in Asia, Eds Yi Yang, Xinzhong Wu and Yingqi Zhou. Proceedings of the Second International Symposium on Cage Aquaculture in Asia, 34d – 8th July, 2006, Zhejiang, China. Asian Fisheries Society and Zhejiang University K.K. Philipose, S.R.Krupesha Sharma, N.Sandu, 2012. Diseases and their management in cage culture. In: Hand book on Open Sea Cage Culture: 129 – 131. Kent ML, 1982. Characteristics and identification of Pasteurella and Vibrio pathogenic to fishes using API-20E (AnalytaProducts) multitube test trips. Can J FishAquat Sci 39:1725–1729. Le, T.L., Svennevig, N., 2005. Second annual progress report of project SVR 0330: Building advanced research, education and extension capacity of the Research Institute for Aquaculture No. 1, Phase II. Research Institute for Aquaculture No1, Bac-ninh. 24p. Le, T.L., Svennevig, N., 2006. Third annual progress report of Project SVR 0330: Building advanced research, education and extension capacity of the Research Institute for Aquaculture No. 1, Phase II. Research Institute for Aquaculture No1, Bac-ninh. 19p. 23 León Comte, Juan Carlos de, 2008. Estudio técnico y financiero para la exportación de cobia (Rachycentroncanadum) de Guatemala a Estados Unidos. Li, Guifeng, et al, 2006. Identification and phylogenetic analysis of Vibrio vulnificus isolated from diseased Trachinotusovatus in cage mariculture. In: Aquaculture 261.1: 17-25. Liao, I. Chiu, et al, 2004. Cobia culture in Taiwan: current status and problems. In: Aquaculture 237.1: 155-165. Liu, P.; Lin, J.; Hsiao, P.; and Lee, K., 2004. Isolation and characterization of pathogenic Vibrio anginolyticusfrom diseased cobia Rachycentron canadum. Journal of Basic Microbiology.44:23-28. Liu, P.C., Lin, J.Y. and Leek. K., 2003 b. Virulence of Photobacterium damselaesubsp. Piscicida in cultured cobia Rachycentron canadum. J. Basic Microbiol., 43, 499 – 507. enetti, D.D., Orhun, M.R., OHanlon, B., Zink, I., Cavalin, F.G., Sardenberg, B., Palmer, K., Denlinger, B., Bacoat, D., 2007. Aquaculture of cobia (Rachycentron canadum) in the Americas and the Caribbean. In: Liao, I.C., Leano, E.M. (Eds.), Cobia Aquaculture: Research, Development and Commercial Production: Asian Fisheries Society, Manila, Philippines, World Aquaculture Society, Louisiana, USA, The Fisheries Society of Taiwan, Keelung, Taiwan, and National Taiwan Ocean University, Keelung, Taiwan, pp. 57–77. Liu, Ping‐Chung, et al, 2004. Isolation and characterization of pathogenic Vibrio alginolyticus from diseased cobia Rachycentron canadum. In: Journal of basic microbiology 44.1: 23-28. Machen, John W, 2008. Immune response of hybrid striped bass (Moronechrysops x M. saxtilis) to vaccination with a commercial vaccine against Vibrio anguillarum and V. ordalii. In: Vibrio spp. disinfection and immunization of cobia (Rachycentron canadum) for the prevention of disease in aquaculture facilities.(2008): 36. Magarinõs B, Romalde JL, Bandin I, et al., 1992. Phenotypic, antigenic, and molecular characterization of Pasterella piscicida strains isolated from fish. Applied and Environmental Microbiology, 58: 3316 – 3322. Magarinõs B, Toranzo AE, Romalde JL, 1996. Phenotypic and pathobiological characteristics of Pasteurellapiscicida. Annu Rev FishDis 6:41–64 24 McLean, Ewen, Guillaume Salze, and Steven Craig, 2008. Parasites, diseases and deformities of cobia. In: Croatian Journal of Fisheries 66.1: 1-16. Miao, S., Jen, C.C., Huang, C.T., Hu, S.-H., 2009. Ecological and economic analysis for cobia Rachycentron canadum commercial cage culture in Taiwan. Aquaculture International 17, 125–141. Ministry of Fisheries, 1994. Five years of extension activities for aquaculture (1993– 1998). Hanoi Agriculture Publisher, pp. 32–45 (in Vietnamese). Nguyen, Q.H. 2002. Current status of cobia (Rachycentron canadum) culture production in Vietnam. Vietnamese Fisheries Journal 7:14 – 16. In Vietnamese. Nhu, V.C., Nguyen, H.Q., Le, T.L., Tran, M.T., Sorgeloos, P., Dierckens, K., Reinertsen, H., Kjorsvik, E and Svennevig, N. 2010. Cobia Rachycentron canadum aquaculture in Vietnam: Recent developments and prospects. Aquaculture (in press). Nhu, V. C. 2005. Present status of hatchery technology for cobia in Viet Nam. Aquacult. Asia, 10(4): 32–35. Nhu, V. C., Dierckens, K., Nguyen, H. T., Hoang, T. M. T., Le, T. L., Tran, M. T. & Sorgeloos, P., 2010. Effect of early co-feeding and different weaning diets on the performance of cobia Rachycentron canadum larvae and juveniles. Aquaculture, 305(1), 52-58. Nhu, V.C., 2005. Present Status of Hatchery Technology for Cobia Rachycentron canadum in Vietnam, Aquaculture Asia Magazine, Oct.–Dec, pp. 32–35 Nhu, Van Can, et al, 2011. Cobia Rachycentron canadum aquaculture in Vietnam: Recent developments and prospects. Aquaculture 315.1: 20-25. P.R. Rajan, J.H.-Y. Lin, M.-S. Ho and H.-L. Yang, 2003. Simple and rapid detection of Photobacterium damselae ssp. piscicida by a PCR technique and plating method. Journal of Applied Microbiology 2003, 95, 1375–1380. Petersen, E. H., Luan, T. D., Chinh, D. T. M., Tuan, V. A., Binh, T. Q., Truc, L. V., & Glencross, B. D., 2011. Bioeconomics of cobia, Rachycentron canadum, culture in Vietnam. ACE Discussion Paper, 2. Reed, P. A. and Francis-Floyd, R., 2002. Vibrio Infections of Fish. Fisheries and Aquatic Sciences Department, University of Florida, IFAS Extension FA31. 25 Revez, Joana MarreirosCabrita, 2006. Faculdade de ciâncias mar e ambiente. Diss. UNIVERSIDADE DO ALGARVE. Romalde, Jesus L, 2002. Photobacteriumdamselae subsp. piscicida: an integrated view of a bacterial fish pathogen. In: International Microbiology 5.1 (2002): 3-9. Sun, Jun-Ren, et al, 2007. Invasive infection with Streptococcus iniae in Taiwan. In:Journal of medical microbiology 56.9: 1246-1249. Tang J, Yang XL, Zheng ZL, Yu WJ, Hu K, Yu HJ, 2006. Pharmacokinetics and the active metabolite of enrofloxacin in Chinese mitten-handed crab (Eriocheirsinensis). In: Aquaculture 260(1–4):69–76. Thompson, F., L.; Iida, T.; Swigs, J., 2004. Biodiversity of Vibrios. Microbiology and Molecular Biology Reviews.Sept. 2004. 403-431. Thyssen, An, et al, 1998. "Phenotypic characterization of the marine pathogen Photobacteriumdamselae subsp. piscicida. In: International journal of systematic bacteriology 48.4: 1145-1151. Trushenski, J., et al, 2010. Physiological responses of cobia (Rachycentron Canadum) following exposure to low water and air exposure stress challenges. In: Aquaculture 307.1: 173-177. Van Tuan Nguyen, 2009. An Evaluation of the Feeding Regime for Larval Mahimahi (Coryphaena Hippurus, Linnaeus 1758) and Cobia (Rachycentron Canadum, Linnaeus 1766). Diss. Curtin University of Technology. W. A. Janssen, M. J. Surgalla, November, 1968. Morphology, Physiology, and Serology of a Pasteurella Species Pathogenic for White Perch (Roccus americanus). Journal of Bacteriology: 96(5): 1606–1610. Weinstein, Mitchell R., et al, 1997. Invasive infections due to a fish pathogen, Streptococcus iniae. In: New England Journal of Medicine 337.9: 589-594. Won, K. M., & Park, S. I., 2008. Pathogenicity of Vibrio harveyi to cultured marine fishes in Korea. Aquaculture, 285(1), 8-13. 26 APPENDIX 1. Gram staining method A. Prepare a bacterial smear from a pure culture - Put a drop of saline on a clean glass slide Using a sterile loop touch an isolated colony and mix in the water drop. Mix until just slightly turbid (light inoculum is best, excess bacteria will not stain Let air dry and heat fix. Do not overheat; slide should not be too hot to touch. Allow to cool. B. Flood the slide with crystal violet, and allow to remain on the slide for 60 seconds. C. Wash off the crystal violet with running tap water. D. Flood the slide with Gram’s iodine, and allow to remain on the slide for 60 seconds. E. Wash off with running tap water. F. Decolorize with acetone solution until the solvent flows colorless from the slide (10 seconds). Excessive decolorization should be avoided since it may result in a false gram stain reading. G. Rinse immediately with running tap water. H. Counter stain with Safranin for 60 seconds. I. Rinse with tap water and allow to air dry properly. 2. Motility test - Ring the outside edge of a coverslip with Vaseline. - Place a loopful of sterile saline on the coverslip, using a sterile loop. - Sterilize the loop again, and take a tiny amount of bacteria to gently spread - it out in the saline. 27 - Lower a microscope slide onto the Vaseline and press lightly to ensure a good seal. - Carefully invert the slide and attached coverslip and examine under the microscope. Focus first onto the edge of the hanging drop with a low-power objectives before progressing through higher power objectives. - True motility is non-random and must not be confused with vibratory Brownian movement or convection currents. A motile organism is one which actively moves to change its position relative to other organisms present. 3. Catalase test - A plate of nutrient agar is streaked and incubated at the optimum temperature for 24 hrs (or longer if required to see good growth). - Scrape a sample of bacterial culture off the plate with a glass rod or platinum wire and transfer to a drop of 3% H2O2 on a clean glass slide. - A positive test is the almost immediate production of gas bubbles. 4. Oxidase test - Wet a small piece of filter paper with oxidase reagent. - The test organism (grown on a media free from glucose and nitrate) is removed with a platinum wire of glass rod and smeared across the surface of wet paper. - A positive reaction is shown by the development of a dark blue color within 10-30 sec. 5. Glucose Oxidation – Fermentation (O-F) test - Inoculate two tubes by stabbing with the loop carrying bacteria. - Overlay the medium in one tub with sterile liquid paraffin to a depth of cm. - Examine daily for up to days. Result Open tube Covered tube Oxidative Yellow (+)/ Green (-) Yellow (+)/ Green (-) Fermentative Yellow (+)/ Green (-) Yellow (+)/ Green (-) Note: some negative (-) samples have to wait until days. 28 6. Characteristics of isolated bacteria pathogens Organs Isolate no. Kidney Liver Bacterial affected and hemorrhage Bacterial affected Bacterial affected and hemorrhage and hemorrhage Bacterial affected and hemorrhage Bacterial affected and hemorrhage Bacterial affected Bacterial affected and hemorrhage and hemorrhage Bacterial affected Bacterial affected and hemorrhage and hemorrhage Bacterial affected and hemorrhage Bacterial affected and hemorrhage 29 Brain Bacterial affected and hemorrhage Bacterial affected and hemorrhage [...]... temperature during winter in the North, and tropical typhoons occurring especially in autumn in central Vietnam The main grow-out constraints would be parasites, bacteria and virus and feed quality and management to keep the FCR low Quality and quantity of cobia fingerlings affect the profit of cobia farming (Miao et al., 2009) At present, cobia fingerlings in Vietnam are produced mainly in the semi-intensive... production of about 12,000 fingerlings in 1999 at a marine hatchery located in Cat Ba Island, Hai Phong province (Van Can Nhu, 2010) Marine fish species are common in marine cages and ponds in Viet Nam’s coastal water, including cobia, which is increasingly popular in the north (from Vinh Ha Long Bay and Bai Tu Long Bay), centre (Van Phong Bay, Khanh Hoa) and also beginning to be cultured in the south-central... is not limiting factor (Nhu, 2005) For example, the Research Institute for Aquaculture No1 in Vietnam produced 400,000 fingerlings in 2007 and 900,000 in 2008 (Nhu et al, 2010) The industry still relies on fingerling imports from Taiwan and China (Hainan) (Huy, 2008) Earlier farming of cobia mainly in the southern Vung Tau region depended on imported fingerlings, but the more stable fingerling availability... locally produced fingerlings were available In a 2005 survey, there was a total of 16,319 marine cages producing approximately 3510 tons of marine aquaculture products (Ministry of Fisheries and The World Bank, 2006) The main constraint of cobia farming in Vietnam is market development In addition, insecurity in supply of high quality juveniles and then some geographical or climatic constraints such as... farms to grow cobia the Norwegian financed Marine Farms Vietnam being 4 the largest The production during 2009 from the latter company could reach 1000 tonnes Cobia farming in Vietnam was initially conducted in simple, small scale wooden raft cages installed in closed bays, using wild-captured fingerlings as seed and trash fish as feed The hatchery-fingerlings were imported from Taiwan or China before... province Production in Vietnam was estimated to be 2,600 tonnes in 2009 (Nhu et al, 2010) The main factor constraining development of cobia culture in Viet Nam is shortage of quality fingerlings although hatchery production in Vietnam is increasing at a rapid rate (Nhu et al, 2010) The cobia is the only emerging tropical mariculture species for which the life cycle has been fully closed and fingerling... (Wen et al., 2007) It is not active against anaerobic bacteria and may be used occasionally, in combination with other antibacterial agents, for the treatment of mycobacterial infections The antibacterial effects of ciprofloxacin arise from its inhibition of Topoisomerase IV and bacterial DNA gyrase, which act by cleaving the DNA of the bacterial chromosome and rejoining the ends once a superhelix is formed... of bacterial diseases that affect aquaculture organisms The mechanism of enrofloxacin acts at the level of the cellular nucleus, inhibiting DNA synthesis During the multiplication phase of the bacteria, the DNA folds and unfolds alternately This process is controlled by the enzyme DNA gyrase, which is inhibited by enrofloxacin, causing a collapse of bacterial metabolism and preventing the genetic information... not only in Asia but also in the Americas Cobia fingerlings used for aquaculture are mainly hatchery produced, with Taiwan Province of China being one of the first to establish hatchery production Seed production in 1999 was around three million fingerlings of about 10 cm with a market value of US$0.50 per fish Due to the rapid growth of cobia and its suitability for commercial production, cobia aquaculture... sensitivity of Erythromycin and Trim/sulfa was intermediate range (around 12mm – 15mm) They also were susceptible to Norfloxacin, Florfenicol and Enrofloxacine and give the best result of sensitivity to Ciprofloxacin and Enrofloxacine with the largest zone Table 4.3 Sensitivity of bacterial isolates to different antibacterial (µg/ dics) Amoxycilline 25 Erythromycin 15 Norfloxacin 5 Ciprofloxacin 30 Florfenicol . with 1.5 % sodium chloride. 3.3.2 Bacterial identification After being cultured for 24 hours at 28 C, the uniform colonies were removed for further culture. The isolates were purified after several. and material preparations (DAY 1) - The organism to be tested was cultivated on TSA medium at 28 0 C under aerobic atmosphere. Streak out the pure culture on TSA plate in a way that distinct. must not be relocated once they have made contact with the agar surface. Incubate the plates at 28 0 C for 24 hours.