Tài liệu 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 4 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 1 ........................................................................................................... 1 INTRODUCTION .................................................................................................. 1 1.1 1.2 1.3 GENERAL INTRODUCTION .......................................................................... 1 RESEARCH OBJECTIVES .............................................................................. 2 RESEARCH ACTIVITIES ............................................................................... 2 CHAPTER 2 ........................................................................................................... 3 LITERATURE REVIEW ....................................................................................... 3 2.1 COBIA (RACHYCENTRON CANADUM) AND AQUACULTURE OF COBIA IN VIETNAM .............................................................................................................. 3 2.2 SOME COMMON DISEASES IN RACHYCENTRON CANADUM (LINNAEUS, 1766) . 6 2.2.1 Streptococcal infection ............................................................................ 7 2.2.2 Vibriosis .................................................................................................. 7 2.2.3 Photobacterium damselae sbsp. piscicida .............................................. 8 2.3 SOME COMMON ANTIBIOTICS USE IN AQUACULTURE ..................................... 8 2.3.1 Oxytetracycline ....................................................................................... 8 2.3.2Beta-lactams ............................................................................................. 8 2.3.3 Florfenicol ............................................................................................... 9 CHAPTER 3 ......................................................................................................... 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 4 ......................................................................................................... 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 5 ......................................................................................................... 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 1 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 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 do antibiotic sensitivity tests which aim to figure out the susceptibility to common used antibiotics. 2 CHAPTER 2 LITERATURE REVIEW 2.1 Cobia (Rachycentron canadum) and Aquaculture of cobia in Vietnam Rachycentron canadum, commonly is a marine finfish species with high potential for aquaculture in Vietnam and all over the world. They are also called black kingfish, is a fast growing pelagic fish found in the tropical/subtropical seas all over the world. Cobia has large body with a maximum length of 2 meters and maximum weight of 60 kilograms. After spawning, larvae need 24 hours of fertilization for hatching in high salinity water and juveniles usually swim to warmer offshore water. Cobia broodstocks are captured by professional fishermen and transported to hatcheries. Larvae and juveniles are stocked in ponds or tanks and grow-out one are cultured in open ocean cages. Sets in clear water, far away from industrial zones, vessels and water current of 0.2 – 0.6m/s are suitable for cage construction as well as salinity from 27ppt to 33ppt and dissolve oxygen are 4mg/L to 6mg/L for growth out culture. Production, while still small, has increased significantly over the past three years. Most production currently comes from China and Taiwan Province of China and totaled around 20,000 tonnes in 2003 (FAO, 2006). Production of this fast-growing (to 6 kg in the first year) species is set to expand rapidly, 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 has become more and more popular. To date, research and development of cobia aquaculture has been initiated in over 23 countries and territories, half of them in the Asian-Pacific region. Statistics of FAO (2009) show that the global aquaculture production of cobia has been increasing rapidly from only 9 tons in 1997 to nearly 30,000 tons in 2007. Meanwhile, the volume from capture fisheries has remained stable, around 10,000 tons annually. It is estimated by the authors that in 2008, Vietnam has produced 1500 tons, thus, being the third largest cobia producer in the world. The cobia, Rachycentron canadum, is an important marine fish first artificially propagated and cultured in Taiwan for one decade, especially in sea-cage farming (Ku 3 and Lu 2000, Liu et al. 2003b). The farming of cobia will presumably become an emerging aquaculture industry in the near future since the industry is now being carried out in some tropical and subtropical regions of the Far East such as Hainan, China, Okinawa, Japan and Viet Nam. However, the industry faces various threats including viral, bacterial and parasitic diseases (Ku and Lu 2000, Liu et al. 2003b). History of cobia aquaculture in Vietnam dates back to 1997 when research on cobia reproduction started, leading to the first successful 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 provinces such as Ba Ria – Vung Tau and Kien Giang. Marine fish in Viet Nam are grown in cages and ponds. The farms tend to be small family-owned operations, although industrial-scale developments are also starting. Marine aquaculture gradually develops with expanding farming of this potential species, opens up a new direction for offshore and inland aquaculture. In 2002, the first commercial batches of more than 20,000 cobia fingerlings were produced under a project co-funded by the Government of Vietnam and the Government of Norway. Since then, research on improvement of larvi-culture technology of cobia has resulted in better growth, survival and production. During 2008, more than 400,000fingerlings were produced at a hatchery of the Research Institute for Aquaculture No1 (RIA-1) at Cat Ba Island in addition to a smaller number produced at a private hatchery at Khanh Hoa 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 availability 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 has now led to several larger fish 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 2002 when the 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 low 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 systems. Although this rearing method is relatively simple, low-cost and easy-copied, there are some uncontrolled factors and it has been experienced to result in relatively low survivals (Benetti et al., 2007; Liao et al., 2004; Weirich et al., 2004) and the cobia fingerlings obtained from these systems have been reported to be of unstable quality i.e. potential of size variation and parasite infection. Thus, the intensive production needs to be developed at appropriate proportion to reduce risk and to ensure sustainable development. However, the present intensive rearing method is relatively expensive and sophisticated and there is a need to simplify the protocol to reduce the production costs. In this regards, to shorten the live prey feeding period, improve nutritional condition and hatchery zoo-techniques will be elements to improve growth, survival and quality of cobiafingerlings. Disease outbreak is another challenge for sustainable development of cobia aquaculture in Vietnam. During larval rearing, infections of protozoa such as Vorticella sp., Epistylis sp., Pseudorhabdosynochus epinepheli, Benedenia and Trichodina have been detected (Le and Svennevig, 2005). Samples collected from sudden crashes of cobia larviculture in RIA-1's hatcheries revealed Viral Nervous Necrosis (VNN) infection of 20–30% (Le and Svennevig, 2005). The vertical transmission of VNN has been confirmed. The use of iodine and peroxide did not effectively eliminate VNN from fertilized eggs (Le and Svennevig, 2006). Therefore, quarantine and screening of the broodstock before the reproduction cycle is essential to prevent the VNN vertical transmission. In addition, 5 high mortality caused by Amyloodinium ocellatum attaching to gills and skin of cobia juveniles has been detected in RIA-1's hatcheries in 2005 and 2006. A high density of A. ocellatum in gills of cobia juveniles might inhibit breathing, leading to slow movement and finally cause high mortality. Formalin treatment at a concentration of 0.03–0.1 mL L−1 for 1 h with strong aeration or fresh water treatment can be effective in case the first symptom is detected in time. It is also important to mention the challenge of tropical typhoons in the areas. Vietnam is located in south-east Asia, situated in the western Pacific Rim exposed to the tropical typhoons from the Pacific Ocean during autumn. Large-scale farms need to be situated in fairly open sea areas to maximize the production, but can also be exposed to harsh weather conditions. The failure of some cobia farms in the Northern central region during a typhoon in 2005 showed to be caused by insufficient dimensioning of the mooring system. At the moment, the Government of Vietnam are supporting development of new semi-submersible cages (National project KC07/03-06/10), which can be controlled to sink temporarily to avoid surface damages during stormy conditions. This cage type can be installed in semi-open or open sea areas where a larger exposure resulting in high of water exchange will contribute to the maintenance of good water quality. Alternative grow-out systems such as land-based recirculation systems should also be considered to provide more options for the industry in regions haunted by typhoons. 2.2 Some common diseases in Rachycentron canadum (Linnaeus, 1766) Bacteria and parasites can cause serious sub-lethal impacts on production by reducing the growth and feeding efficiency of stocks as well as leaving fish more susceptible to other infections (Mustafa et al, 2001; Boxaspen, 2006; McLean et al, 2008). The reported bacterial diseases of cobia include mycobacteriosis, vibriosis, pasteurellosis, and streptococcosis, which are caused by pathogens including Mycobacterium marinum, Vibrio anguillarum and V. ordalii, Pasteurella piscicida and Streptococcus spp. (Liao et al., 2004, Lowery and Smith, 2006). The viral disease lymphocystis, and the parasitic diseases myxosporidosis, Trichodina spp., Neobenedenia spp., and Amylodinium spp. also can affect cobia (Kaiser and Holt, 2005). To the present, most study in cobia diseases focus on parasitic pathogens. In Taiwan, scientists are given some common bacterial pathogens that make high mortality up to 80% or more in young cobia in culture cobia (Lin et al., 2006). 6 2.2.1 Streptococcosis Streptococcal infection of fish is considered as re-emerging disease affecting a variety of wild and cultured fish throughout the world. Five different species are considered to be of significance as fish pathogens: Lactococcus garvieae, L. piscium, Streptococcus iniae, S. agalactiae, S. parauberisand, Vagococcus salmoninarum. Therefore, streptococcosis of fish should be regarded as a complex of similar diseases caused by different genera and species capable of inducing a central nervous damage characterized by supperative exophthalmia and meningoencephalitis. Warm water streptococcosis typically involves L. garvieae, S. iniae, S.agalactiaeanm, S. parauberis. It is important to report that the etiological agents of warm water streptococcosis are considered also as potential zoonotic agents capable to cause disease in humans. Among these fish streptococci, L. garvieae, S. iniaeand S. parauberis can be regarded as the main etiological agents causing diseases in marine aquaculture in both nursery and grow-out culture stages. 2.2.2 Vibriosis Vibriosis is a disease characterized by haemorrhagic septicaemia and caused by various species of Vibrio. It occurs in cultured and wild marine fish in salt or brackish water, particularly in shallow waters during late summer. Within the Vibrionaceae, this genus includes the human pathogens V. cholerae, V. mimicus, V. parahaemolyticus, and V. vulnificus, as well as fish pathogens Listonella anguillarum(formerly V. anguillarum), V. ordalii, V. damsela, V. carchariae, V. vulnificus, V. alginolyticus, and V. salmonicida (Reed and Francis-Floyd, 2002). Vibrio spp. pathogens also affect other species of marine fish, penaeid shrimp, as well as abalone (Liu et al., 2004). In addition, Vibrio spp. bacteria account for a significant portion of the food-borne infections from eating raw or undercooked shellfish (Thompson et al., 2004) and become the economically most important disease in marine fish culture, affecting a large number of species. It is also an important disease of many wild fish populations. Fish affected by vibriosis show typical signs of a generalized septicemia with hemorrhage on the base of fins, ulcers on body surface, swelling and boils, exophthalmia and corneal opacity. Moribund fish are frequently anorexic with pale gills, which reflect a severe anaemia. Oedematous lesions, predominantly centered on the hypodermis, are often observed. On the top of the boils, the epidermis is destroyed and the skin is greyish white. Around the boil, the skin is hemorrhaged. Internally there are hemorrhage in liver and intestine, and there is fluid in the heart lumen. Histologically, the muscle fibres are widely separated. 7 2.2.3 Pasteurellosis Photobacterium damselae ssp. piscicida (Gauthier et al. 1995) is a Gram-negative, nonmotile, bipolar coccobacillus (Snieszko et al. 1964), which was previously known as Pasteurella piscicida (Snieszko et al. 1964). It is the causative agent of the fish disease photobacteriosis, also known as pasteurellosis or pseudotuberculosis. High mortalities of P. piscicida infection were first observed in natural populations of white perch (Morone americanus) and striped bass (Morone saxatilis) in 1963 in Chesapeake Bay (USA) (Snieszko et al, 1964), and it has caused economic loss to the fish farming industries in Japan (Kusuda and Yamaoka, 1972). Several countries in the Mediterranean area (Toranzo et al, 1991; Bakopoulos et al, 1995, 1997; Baptista et al, 1996; Candan et al, 1996; Topic Popovic et al, 2001) have encountered similar high mortalities in cultured sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). The pathogen has also been isolated and caused high mortalities in farmed cobia (Rachycentron canadum) in Taiwan 2001 (Lopez et al, 2002). Photobacterium damselae ssp. damselae (Smith et al. 1991; Truper and De Clari 1997) which was formerly classified as Vibrio damsela is a halophilic bacterium causing skin ulcers in warm and cold water fish (Love et al. 1981; Sakata et al, 1989; Fouz et al, 1992a,b). 2.3 Some common antibiotics use in aquaculture 2.3.1 Oxytetracycline Oxytetracyline is also called tetracycline antibiotic which used to treat infections with bacteria in quaculture farms such as chlamydia, mycoplasma, protozoa and several ricketsiae. In other words, oxytetracyline is a broad spectrum antibiotic that is active against a wide variety of bacteria. However, resistance has been acquired by coliforms, mycoplasma, streptococci and staphylococci (Bui Kim Tung, 2001). 2.3.2Beta-lactams This group includes Amoxicillin and Ampicillin. Amoxicillin is active against penicillin-sensitive Gram-positive bacteria and some Gram-negative bacteria. Grampositive spectrum includes alpha- and beta- haemolytic Streptococci, some Staphylococci species and Clostridia species. Gram-negatives: Escherichia coli, many strains of Salmonell, and Pasteurellamultocida are susceptible to destruction by beta8 lactamases. Ampicillin is active against alpha- and beta- haemolytic streptococci,, including Streptococcus equi, non-penicillinase-producing Staphylococcus species, and most trains of Clostridia. Also effective against Gram-negative bacteria, such as Escherichia coli, Salmonella and Pasteurellamultocida (Bui Kim Tung, 2001). 2.3.3 Florfenicol This fluorinated antibiotic, derived from thiamphenicol, is a potent and broadly acting bacteriostatic agent. Activity similar tochloramphenicol, including many Gram-positives and Gramnegatives and without the risk of inducing human aplastic anaemia associated with chloramphenicol. It is effective in the treatment of infections caused by Pasteurella piscicida, Aeromonas salmonicida, Vibrio anguillarum, and Edwardsiella tarda. Pharmacokinetically, florfenicol use has been reported among some species of fish such as Atlantic salmon (Salmosalar), in which a bioavailability of more than 95% is present, exhibiting a good distribution among all of the organs and tissues. Its half-life in fish is less than 15hrs (Yanong and Curtis, 2005). 2.3.4 Enrofloxacin Enrofloxacin was developed as an antimicrobial agent during the 1980s for exclusive use in veterinary medicine and has proven to be effective in the treatment 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 from being copied, thus causing the bacteriocidal effect (Williams et al., 2002). The information related to this antibiotic for the most widely grown shrimp species such as Litopenaeus vannamei is scarce, but pharmacokinetic studies on enrofloxacin have been carried out using other species, such as crab (Scylla serrata), tilapia (Oreochromis niloticus), black shrimp (Penaeus monodon), Chinese shrimp (Penaeus chinensis), and European seabass (Dicentrarchus labrax) (Intorre et al., 2000; Tu et al., 2008; Wen et al., 2007; Xu et al., 2006). It is important to note that the pharmacokinetic results for enrofloxacin obtained for these species should not be extrapolated to other aquatic species, because each organism possesses a different metabolism, and the cultivation conditions may have a significant influence over the kinetic behavior displayed by the antibiotic. 9 2.3.5 Ciprofloxacin Ciprofloxacin is the main metabolite of Enrofloxacin and is active against a broad spectrum of aerobic Gram (-) bacteria, including enteric pathogens such as Pseudomonas and Serratia marcescens. It is also active against Gram (+) pathogens, even when these bacteria have developed resistance to other antibiotics, such as penicillin (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 (Banerjee et al., 2007). When these enzymes are inhibited, bacterial cell multiplication is interrupted. 10 CHAPTER 3 RESEARCH METHODOLOGY 3.1 Time and places - Time: The thesis was conducted from June to December, 2013. Place: Isolation, identification, PCR will be done at Department of Aquatic Pathology - College of Aquaculture and Fisheries, Can Tho University. 3.2 Materials - - Equipment: Tissues paper, gloves, aluminum foils, nylon bags, refrigerators, petri dishes, alcohol lamps, inoculating loops, 100 – 1000µl pipets, alcohol spray, API test kit. Chemicals: absolute alcohol, NaCl, distilled water, antibiotic samples, glycerol, crystal violet solution. Culture media: NA(Nutrient Agar) and TCBS (Thiosulfate Citrate Bile Salts Sucrose) with addition 1.5% NaCl, OF (Oxidative – Fermentative media), Blood Agar. 3.3 Methods 3.3.1 Bacterial isolation Diseased juvenile cobia (R. canadum) were sampled from disease cages in Nha Trang Bay, Vietnam during October, 2012. These fish displayed lethargic, whitish, granulomatous deposits on kidney, liver, hemorrhage in peritoneal cavity. Five samples were collected in each cage with the recording of health status, water conditions and feeding. Ten strains were stored at 70°C in nutrient broth (Merck) containing 15% glycerol and supplemented 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 times of culture and then inoculated on test tube slants as pure culture and stored on 4°C for further analysis of identification. Bacteria were inoculated in NA media for 24 – 48 hours. Shape and color of colonies were observed and recorded. Cell morphology was studied in Gram-stained 11 preparations from nutrient agar (Himedia) plates supplemented with 1% NaCl according to Hucker’s modification method (Barrow & Feltham, 1993). Bacterial identification procedure in this thesis followed Manual for the isolation and identification of fish bacterial pathogens (Frerichs and Millar, 1993). Bacterial pathogens were identified follow basis biochemical tests: Gram staining, motility, oxidase, catalase, hemolytic, API test. The specific steps for some of the biochemical tests were described in Appendix. Strains of bacteria were identified using API 20E test kit (BioMerieux, France) following the manufacture instruction. 3.3.3 Antibiotic susceptibility test Antibiogram test was carried out by the method of Geert Huys (2002). Bacterial cultivation and material preparations (DAY 1) - The organism to be tested was cultivated on TSA medium at 280C under aerobic atmosphere. Streak out the pure culture on TSA plate in a way that distinct colonies were obtained. - Prepare the desired volume of TSA medium according to the manufacturer’s instructions. Before pouring the agar medium, bottles cooled to 40-500C in room temperature. All plates were poured on a flat, horizontal surface to an identical depth of 5 mm + 1 mm (corresponding to 20 mL + 1 mL of medium in 10 cm radius petri dishes). - Prepare tubes containing 5 mL sterile NB (Nutrient Broth) solution (with 0.85% NaCl) Inoculation of the antibiogram (DAY 2) - Take a number of pure colonies from the fresh grown plate culture to suspend in a tube containing NB solution until turbidity (visually) corresponding to 1.0 McFarland standard is reached. It remained important to take more than one colony in order to obtain a representative sample. - Using a micropipet, spot 100 µL of the standardized suspension on the surface of TSA plates. Spread plate the suspension using a sterile glass triangle rod. Allow to dry the plates. Longer drying times allow pre-incubation of the cells which should be avoided. Manually using sterile forceps applied the discs onto the agar surface. Discs must not be relocated once they have made contact with the agar surface. Incubate the plates at 280C for 24 hours. 12