CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERY

NGUYEN NHUT THANH

CHARACTERIZATION OF PATHOGENIC BACTERIA FROM SILVER POMFRET (Pampus argenteus Euphrasen, 1788) CULTURED IN NHATRANG BAY, VIETNAM

GRADUATION THESIS ADVANCED AQUACULTURE

Supervisors name Dr. Dang Thi Hoang Oanh Ms. Tran Thi My Duyen

2013

Table content
CHAPTER 1 INTRODUCTION ..............................................................................................1 1.1 1.2 1.3 Introduction ..................................................................................................................1 Research objective ........................................................................................................2 Research activities ........................................................................................................2

CHAPTER 2: LITERATURE REVIEW ................................................................................3 2.1 Silver pomfret....................................................................................................................3 2.2 Cause of diseases ...............................................................................................................3 2.3 Some common bacterial diseases in brackish and marine fishes .........................................4 2.3.1 Vibriosis .....................................................................................................................4 2.3.2 Bacterial hemorrhagic septicaemia ..............................................................................4 2.3.3 Streptococcosis ...........................................................................................................5 2.4 Antibiotics in aquaculture ..................................................................................................6 2.4.1 Oxytetracycline...........................................................................................................6 2.4.2 Enrofloxacin ...............................................................................................................6 2.4.3 Ciprofloxacin ..............................................................................................................7 2.4.4 Florfenicol ..................................................................................................................7 CHAPTER 3 MATERIALS AND METHODS ........................................................................9 3.1 Time and sites of study ......................................................................................................9 3.2 Materials ...........................................................................................................................9 3.3 Methods ............................................................................................................................9 3.3.1 Fish sampling .............................................................................................................9 3.3.2 Bacterial isolation .......................................................................................................9 3.3.3 Bacterial identification .............................................................................................. 10 3.3.4 Antimicrobial susceptibility testing ........................................................................... 10 TIME TABLE ......................................................................................................................... 11 COST ESTIMATION ............................................................................................................. 11 REFERENCE .......................................................................................................................... 12 APPENDIX .............................................................................................................................. 14

CHAPTER 1 INTRODUCTION 1.1 Introduction With more than 3200 kilometers of coastline and large area of water surface, Vietnam has great conditions to develop aquaculture (both fresh and marine aquaculture). In fact, aquaculture in Vietnam is developing year by year, especially farmed stripped catfish (Pangasianodon hypophthalmus) and penaeid shrimp (tiger shrimp and white leg shrimp), Vietnam was ranked the worlds third of aquaculture (FAO 2010). Besides, marine aquaculture is contributed an important part for aquaculture with high value species such as sea bass, cobia (Rachycentron Canadum), grouper (Serranidae), silver pomfret (Pampus argenteus) Silver pomfret is one of a new cultured species is Vietnam as it has high nutrition contain and quality of flesh meat. Silver pomfret is easy to find in Northern gulf and central southern of Vietnam, in the past, people mainly catch silver pomfret in the wild. In recent years, people started to culture silver pomfret in cages with intensive model. In this system, fish are stocked at high density, usually over feeding. This can make the fish get stress and diseases, especially this can lead to disease spread out easily on the sea to wild fish, or the others cages. Bacterial diseases have been reported as one of the worst problems causing up to 100% mortality of cultured fishes (Bui Quang Te, 2008). This is the new specie, there is a few research about silver pomfret are conducted on over the world. Antibiotics are known as the useful treatment for the bacterial disease, but can make bad result if not use at correct ways or dosage. Bacterial resistance often happens and cause treatment failure. People prefer using higher dosages of antibiotics when the previous dosages do not have effect; they hope to heal illness completely, but residues in flesh product are not favorable to consumers. In addition, resistance characteristic could readily and quickly spread out in bacterial populations (Kumarasamy et al., 2010). The uncontrolled of using antibiotics can make the presence of antibiotic residues in fish meat and fish products and this also lead to a disease which cause by resistance bacteria and hard to treat. Thus, this thesis Characterization of pathogenic bacteria from silver pomfret (Stromateoides argenteus Euphrasen, 1788) cultured in Nhatrang bay, Vietnam is carried out to provide more information about disease on this species.
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1.2 Research objective The aim of this research is to investigate the bacterial pathogen which are isolated from silver pomfret (Pampus argenteus) in Nhatrang bay, then find down the most effective antibiotic to treat the disease. 1.3 Research activities This thesis will focus on the following contents: 1. Classification to species level of bacteria that was isolated from silver pomfret. 2. Antibiotics susceptibility testing and determination of concentration of those antibiotics to isolated bacterial strains. minimal inhibitory

CHAPTER 2: LITERATURE REVIEW 2.1 Silver pomfret Silver pomfret (Pampuss argenteus) is a valuable food fish with a wide geographical distribution from East China Sea to Southest Asia, the India Ocean, Arabian Gulf and the North Sea (Davis and Wheeler 1985). Silver pomfret is found in coastal water from 5 to 100 meters depth, it has maximum size 80 centimeters but usually see at 30 centimeters (FAO). In the past, it was only caught on the sea, but in recent years, it is stared being culture in cages. Cages are placed in clean area, with viscosity about 0.20.6m/s. This is new species cultured in Vietnam, it has high economic value base on its nutrition value, high flesh meat quality. There is not many researches done about this species have been reported in Vietnam.

2.2 Cause of diseases Aquatic organisms are sensitive with the diseases, because they exposure directly with the water, which they live in. Disease can be caused by an etiological (specific cause) or a nonetiological (contributing cause) agent. Etiological agents can be classified as either inanimate or animate. Inanimate etiological agents are factors without life of their own and can originate within a host (endogenous) or outside of a host (exogenous). Endogenous, inanimate factors are those associated with genetic and/or metabolic disorders of the host. Exogenous, inanimate agents include trauma, temperature shock, electrical shock, chemical toxicity, and dietary deficiencies. These etiological agents may serve as sublethal stressors that predispose fish to infectious disease. Animate etiologies are living communicable infectious agents, which include viruses, bacteria, fungi, protozoa, helminthes and copepods (Plumb, 1999). Nonetiological causes of disease are characterized as extrinsic (from outside the body) or intrinsic (within the body). Extrinsic factors are usually associated with environmental conditions or dietary problems, and intrinsic factors include age, gender, heredity, and fish species. Both fish species and strain of fish are important because all are not equally susceptible to a specific disease organism. Feed quality,
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water quality factors, and water temperature extremes can be classified as either etiological or nonetiological extrinsic factors and can contribute to infectious disease (Plumb, 1999). 2.3 Some common bacterial diseases in brackish and marine fishes Bacteria are one of the most important-causative agents causing adverse diseases to fish. Most bacterial agents causing diseases to fish are Gram-negative; some of them are Gram-positive. They are ubiquitous in the environment (sea, lakes, rivers, canals, ponds) and are considered as the primary pathogen or the opportunistic pathogen. They are usually chronic, acute or subacute diseases. In some cases, bacterial diseases can cause 100% of mortality (Bui Quang Te, 2006). 2.3.1 Vibriosis Vibriosis, also known as salt-water furunculosis, boil-disease, or ulcer-disease (Austin and Austin, 2007), is among the most prevalent fish diseases caused by bacteria of genus Vibrio (Woo and Bruno, 1998).This type of disease is commonly considered stress mediated with the predisposing factors of handling, moving from fresh to salt water (Plumb and Hanson, 2011), high temperature, crowding, and organic pollution (Noga, 2010). Vibriosis has been reported on many fish species, including salmon, rainbow trout, turbot, sea bass, sea bream, striped bass, cod, and eel (Actis et al., 1999 cited by Toranzo, 2005).Within Vibrionaceae, the species causing the most economically serious diseases in marine culture are Listonella Vibrio) anguillarum, Vibrio ordalii, Vibrio salmonicida, Vibrio vulnificus biotype 2 (Toranzo et al., 2004), and Vibrio harveyi (FAO, 2006). V.anguillarumis the most common fish-pathogenic vibrio (Noga, 2010). Infected fish may havered areas on body, skin ulcers, depression, exophthalmos, corneal ulcers, and swollen abdomen (Noga, 2010).Vibriosis, as with other bacterial septicaemias, can be controlled by maintaining good water quality, but where outcreaks occur, treatment with an oral antibiotic is the only option (Woo and Brono, 1998) 2.3.2 Bacterial hemorrhagic septicaemia Bacterial hemorrhagic septicaemia, also referred to as motile aeromonad infection, infectious dropsy, red pest, reddisease, red sore, rubella, and others (Plumb and
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Hanson, 2011), has been associated with several members of genus Aeromonas such as A. hydrophila,A. sobria, A. caviae, and Pseudomonas sp.(FAO, 2006). Disease syndromes may include lethargy, anorexia, irregular reddened skin ulcerations, reddened abdominal fluid, and pale gills (Austin and Austin, 2007). By far the most significant fish pathogen is A. hydrophila (Noga, 2010). A. hydrophila is widely distributed in the aquatic environment (Roberts, 2012). This species is a pathogenic species mainly to fresh water fish, and occasionally to marine fish (Austin and Austin, 2007). They occur as Gram-negative, motile, straight rods (0.3-1.0 x 1.03.5m) (Roberts, 2012). This bacteria species showed resistant to many types of antibiotics, including ampicillin, chloramphenicol, erythromycin, nitrofurentoin, novobiocin, streptomycin, sulphonamides, tetracycline, oxytetracycline (Aoki, 1988; De Paoplaet al., 1988, cited by Austin and Austin, 2007), but were very sensitive to enrofloxacin (Brag and Todd, 1988, cited by Austin and Austin, 2007). 2.3.3 Streptococcosis Streptococcosis is sometimes called popeye because exophthalmos (exophthalmia) is very common. This disease can cause darkening, pale gill, reddened fluid and organs in infected fish (Noga, 2010).Diverse host species have been reported with streptococcus infection, including rainbow trout, tilapia, hybrid triped bass (Eldar et al., 1994) and sea bass (Bromage et at., 1999; Creeper and Buller, 2006). Even though, many species of Streptococcosis, including S. agalactiae, S. iniae, S. dysqalactiae, S. dysgalactiae, S. pyogenes, S. parauberis, and S. equi, have been reported from fish,S. iniaeand S. agalactiaeare the two that mostfrequently cause serious disease in tilapia. Streptococcus iniae are small, Gram-positive, facultative anaerobic cocci, appearing in chains (Roberts, 2012). Although S.iniae can affect various freshwater and coastal fish species (Austin and Austin, 2007), this bacterial species is more commonly isolated from fresh-water fish such as rainbow trout and tilapias than from marine fish such as flounders and sardines (Kusada and Salati, 1999, cited by Roberts, 2012).Fish infected by this species often get damaged brain, exophthalmia, surface and internal. More importantly, fish pathogen S.iniae can cause disease in human hemorrhaging (Austin and Austin, 2007). This bacterial agent can be treated with fluoroquinolone compound, enrofloxacin (Stoffregenet al., 1996, cited by Austin and Austin, 2007). Besides, laboratory studies also showed the efficacy of oxytetracycline and

amoxicillin in controlling S.iniae (Darwishet al., 2002; Darwish and Ismaiel, 2003, cited by Austin and Austin, 2007). 2.4 Antibiotics in aquaculture Antibiotics are known as the best treatment when fish get bacterial diseases. People usually take over dosages for sure that they treat the disease completely; this is not only wasting of money but also make opportunities for bacterial resistance. The residual of antibiotic in fish (aquatic species) could affect to human health. The antibiotics most frequently used in aquaculture to combat bacterial diseases include oxytetracycline, florfenicol, sarafloxacin, and enrofloxacin (Roque et al., 2001; SotoRodrguez et al., 2006). Globally, other antibiotics such as chlortetracycline, quinolones, ciprofloxacin, norfloxacin, oxolinic acid, perfloxacin, sulfamethazine, gentamicin, and tiamulin are used (Holmstrom et al., 2003). 2.4.1 Oxytetracycline Oxytetracycline is widely employed to treat bacterial infections in aquaculture farms, such as vibriosis and furunculosis (Capone et al., 1996; Prescott et al., 2000; Reed et al., 2006; Wang et al., 2004). It belongs to the tetracycline group, which exerts antimicrobial action against both Gram (-) and (+) bacteria, ricksettsias, mycoplasmas, and others (Gmez-Gil et al., 2001). Tetracyclines are produced by Streptomyces spp., which possess determinants for resistance to this class of antibiotics. Oxytetracycline is a bacteriostatic antibiotic that exerts its antimicrobial effect against protein synthesis, by bonding directly to the S7 protein of the 30S subunit of the bacterial ribosome, thereby impeding the bonding of aminoacyl-tRNA (aminoacyl transfer RNA) to the A-site of the ribosome. This prevents the addition of amino acids to the growing peptide chain (Chambers, 2004; Isidori et al., 2005; Jara, 2007). In order for oxytetracycline to interact with its target site, it needs to pass through the external membrane via passive diffusion through the OmpF and OmpC pores, and through the cytoplasm membrane via an energy dependent process (Jara, 2007). 2.4.2 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. Enrofloxacin is a derivative of nalidixic acid. It has a basic dihydroquinoline (4-quinolone ring) chemical core with an ethyl group at the 4th position, favoring its absorption and availability. It is primarily
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lipophilic and has a low molecular weight, favoring tissue penetration. 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. None of the fluoroquinolones included enrofloxacin is approved for use in shrimp in the United States. Their potential use in other countries, such as Mexico, as well as the potential for extra-label use in the United States provides a need for efficient methods to monitor food supplies for the presence of fluoroquinolones residues (Schneider et al., 2005). 2.4.3 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. 2.4.4 Florfenicol

This fluorinated antibiotic, derived from thiamphenicol, is a potent and broadly acting bacteriostatic agent. It is effective in the treatment of infections caused by Pasteurella piscicida, Aeromonas salmonicida, Vibrio anguillarum, and Edwardsiella tarda. Its chemicalstructure is very similar to that of chloramphenicol, and florfenicol is effective against bacteria that have developed the ability to deactivate other drugs, such as thiamphenicol and chloramphenicol. Pharmacokinetically, florfenicol use has been reported among some species of fish such as Atlantic salmon (Salmo salar), in which a bioavailability of more than 95% is present, exhibiting a good distribution among all of the organs and tissues. Its halflife in fish is less than 15 h (Yanong & Curtis, 2005). However, published information for shrimp is scarce, meaning that the kinetic behavior of this compound among these crustaceans has not yet been completely elucidated.

CHAPTER 3 MATERIALS AND METHODS 3.1 Time and sites of study

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Time: January to June , 2013. Locations: Fish are sampled at Nhatrang bay, Vietnam. Place for conducting experiments: Department of Aquatic Biology and Pathology, College of Aquaculture and Fisheries, Cantho University.

3.2 Materials The media, chemicals, antibiotics and consumables used for this study are listed below:
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Nutrient agar (NA) Tryotic soy agar (TSA). Crystal violet (color Index No.42555) Ammonium oxalate Iodine Potassium iodide Safranin (Color Index No.50240) Chemicals for biochemical tests: O/F, motility, Indole, Oxidase, Catalase Antibiotics: Enflorxacin (ENR/5g), Florfenicol (FFC/30g,).

3.3 Methods 3.3.1 Fish sampling Silver pomfret samples are collected from culture cages in Nhatrang bay, Nhatrang. Samples of 2-3 diseased fish are collected from each cage, then sampled on-cages and keep the samples to transport to the laboratory of the College of Aquaculture and Fisheries, Cantho University for analysis. 3.3.2 Bacterial isolation Fish samples were first put on clean trays for observing and recording of external signs into a sampling sheet as shown Appendix 2. They were disinfected with 700 alcohols, and then carefully dissected to avoid damaging internal organs, and reduce the risk of contamination. Internal signs of fishwere also observed and noted.

Bacterial samples from liver, kidney, spleen, brain, and eyes were inoculated on agar plates already supplemented with sodium chloride to acquire the salinity of 15,and incubated at 280C. After 24 hours of incubation, bacterial growth was checked, and representative bacterial colonies were sub-cultured for purity. 3.3.3 Bacterial identification Pure culture of bacterial isolates after being obtained were used in primary tests, including Gram staining, motility, oxidase, catalase, oxidative-fermentative, O/129 tests, following the method of Frerichs and Millar (1993), and Buller (2004). Detailed procedures for each specific test are shown in Appendix 3.Bacterial strains were fully identified by using API 20E, following the instruction of the suppliers. Besides that, PCR technique also use to indentify the bacteria. 3.3.4 Antimicrobial susceptibility testing The susceptibility patterns of the identified isolates were made by using disk diffusion method described in Clinical and Laboratory Standards Institute document M2-A09 (CLSI, 2006). Incubating loops are used to take 1-2 colonies from pure culture, and put into bottle with about 30ml sterile BHI to vortex at 200 rounds/minutes for 24 hours. Bacterial solution is now transferred into 50ml falcon tube for centrifugation (4000 rounds/minute for 15 minutes). The upper solution part is eliminated, and bacteria are washed 2-3 times under sterile saline solution. After the final centrifugation, and elimination of the upper part, 25ml of saline solution is added, and the solution is well mixed. The density of bacteria is determined, using spectrophotometer (wavelength = 600nm). Bacterial solution is then diluted to the approximate concentration of 1-2 x 108 colony-forming units (CFU)/ml. The standardized bacterial suspension is evenly spreadon the surface of the agar plates with a cotton swab. The surface of the medium is letto dry for 3-5 minutes to allow for the absorption of excess moisture. Antibiotic disks are placed on the surface of the inoculated and dried plate with sterile forceps, and lightly pressed down to ensure complete contact between the disk and the agar surface. Position disks such that the minimum center-center distance is 24mm and no closer than 10-15mm from the edge of the petri dish. A maximum of six disks may be placed in a 9-cm petri dish and 12 disks on a 150mm plate.

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The zones of inhibition are observed after 24 hours of incubation at 280C. The zone of inhibition is the point at which no growth is visible to the unaided eye. Compare the diameter of the zone of inhibition of the test isolates with those in the chart of interpretative standard for veterinary pathogen.

TIME TABLE Time table of the research proposal Month No Research activities 1 Proposal preparation and defense 2 Title of research activities 1 3 Title of research activities 2 4 Data analysis and writing 5 Thesis defense April June July August Sept. Dec.

COST ESTIMATION No. Meterials

Nutrient agar (Merck) Nutrient broth (Merck) API 20 E (BioMeuriex, France)

Cost 1,905,200 3,124,000 3,630,000 1,098,240 242,560 Total 10,000,000 Students sign

Muller hinton agar (Merck) Stationary

Supervisors sign

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REFERENCE 1. Austin, B., and D. Austin, 2007. Bacterial Fish Pathogens Diseases of farmed and wild fish, Fourth edition. Praxis Publishing.United Kingdom. 552pp 2. Bi Kim Tng, 2001. Thuc khng sinh. S Khoa Hc Cng Ngh v Mi Trng tnh B Ra-Vng Tu. 225 trang. 3. Bi Quang T, 2006. Bnh hc thy sn. Vin nghin cu nui trng thy sn 1. 439 trang. 4. Bi Th Tho, 2003. Thuc khng sinh v nguyn tc s dng thuc trong chn nui. NXB H Ni. 323 trang. 5. Bullock, G.L., 1981. Streptococcal infections of fishes. US Fish & Wildlife Publications. Washington, D.C. Fish Disease Leaflet 63. 7 pp. 6. Bullock, G.L. and R.L. Herman, 1985. Eswardsiella infections of fishes. US Fish & Wildlife Publications. Washington, D.C. Fish Disease Leaflet 71. 6 pp 7. Clinical and Laboratory Standards Institute. Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Proposed Guideline.CLSI document M49-P [ISBN 1-56238-577-1]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005 8. Davis P, Wheeler A (1985) The occurrence of Pampus argenteus (Euphrasen, 1788) in the North Sea. J Fish Biol 26:105109 9. ng Th Hong Oanh, 2007. Gio trnh nguyn l v k thut chun on bnh thy sn. Khoa Thy Sn. i hc Cn Th. 87 trang. 10. Fontana, R., G.L. Cascio, M. Ligozzi, O. Friscia, T. Oldoni and the Italian Epidemiological Observatory Collaborative Group, 2001. Antimicrobial susceptibility of respiratory isolates of Enterobacteriaceae and Staphylococus aureus in Italy: Incidence and Trends over the period 1997-1999. Published online. Italia. Eur J Clin Microbiol Infect Dis. 20. 854863. 11. Frerichs, G.N. and S.D. Millar, 1993. Manual for the isolation and identification of fish bacterial pathogens. Institute of Aquaculture. Scotland. 60 pp. 12. Geert Huys, 2002. Antibiotic susceptibility testing of aquaculture associated bacteria with the dics diffusion method. Standard Operating Procedure (SOP). 13. Huys, G., K. Bartie, M. Cnockaert, D.T.T. Oanh, N.T. Phuong, T. Somsiri, S. Chinabut, F.M. Yusoff, M. Shariff, M. Giacomini, A. Teale, J. Swings, 2006. Biodiversity of chloramphenicol-resistant mesophilic heterotrophs from Southeast Asian aquaculture environments. Research in Microbiology. 158: 228-235. 14. Inglis, V, R.J. Roberts and N.R Bromage, 1994. Bacterial disease of fish. Institute of aquaculture. The University Press. Cambrige. 59-79. 15. Kumarasamy, K. K., M. A. Toleman, T. R. Walsh, J. Bagaria, F. Butt, R. Balakrishnan, U. Chaudhary, M. Doumith, C. G. Giske, S. Irfan, P. Krishnan, A. V. Kumar, S. Maharjan, S. Mushtaq, T. Noorie, D. L Paterson, A. Pearson, C. Perry, R. Pike, B. Rao, U. Ray, J. B. Sarma, M. Sharma, E. Sheridan, M. A. Thirunarayan, J. Turton, S. Upadhyay, M. Warner, W. Welfare, D. M. Livermore,
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N. Woodford, 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. 16. Nguyen Dai Duong, 2012 Flofenicol and enrofloxacin resistance in heterotrophic bacteria isolated from snake head fish (chana striatus) and climbing perch (anabes tedtudineus) farms in the Mekong river delta, A thesis submitted in partial fulfillment of the requirements forthe degree of Bachelor of Aquaculture 17. Plumb, J.A., 1999. Health maintenance and principle microbial diseases of cultured fishes. First edition. Iowa State University Press / Ames. 328 pp. 18. Noga, E. J., 2000. Fish disease: Diagnosis and Treatment. Second edition. Blackwell publishing. Iowa State University. 299 pp. 19. Serrano, P.H., 2005. Responsible use of antibiotics in aquaculture. FAO fisheries technical paper. 469. 97 pp. 20. Ti liu hng dn thc tp gio trnh chuyn mn Bnh Hc Thy Sn 1 nm 2007. Khoa Thy Sn. i hc Cn Th. 21. Tran Huu Tin, 2012, Antimicrobial susceptibility testing of bacterial agents isolated fromasian seabass (Latescalcarifer), A thesis submitted in partial fulfillment of the requirements forthe degree of Bachelor of Aquaculture 22. T Thanh Dung, ng Th Hong Oanh v Trn Th Tuyt Hoa, 2005. Gio trnh bnh hc thy sn. Khoa Thy Sn. i hc Cn Th. 123 trang. 23. Trn Hu Ti, 2011. Hin trng bnh v tnh hnh s dng thuc v ha cht trong nui c lc nng h ti tnh Hu Giang. Lun vn tt nghip i Hc. Khoa Thy Sn. i hc Cn Th. 24. Woo, P. T. K, and D. W. Bruno. 1998, Fish Diseases and Disorders, Volume 3.Viral, Bacterial, and Fungal Infections.CABI Publishing, United Kingdom.

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APPENDIX 1. Plate inoculation procedure for culture and purification of bacteria

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Inoculate sample on a small segment of the surface of the culture medium. Flame inoculation loop until red hot, allow to cool and touch loop on edge of uninoculated area of medium to ensure coolness. Spread part of sample over about of the plate by making 3-4 parallel streaks with the loop. Repeat streaking procedure as shown, flaming and cooling the loop between each sequence. Label underside (not lid) of plate and incubate.

2. Gram-staining method:
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Apply ammonium oxalate/crystal violet solution to heat-fixed smear for 1 min. Wash with water. Apply iodine solution for 1 min. Tip off iodine solution. Decolorise with alcohol/acetone (95% :5%) until no more violet color emanates from the smear Wash thoroughly with water. Apply safranin solution for 2 min. Wash with water, drain and/or blot dry and examine.

Gram-positive organisms- blue/purple Gram-negative organisms pink/red. 3. Motility

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Ring the outside edge of a coverslip with Vaseline. Place a loopful of liquid culture on the centre of the coverslip within the vaseline ring. 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.

4. Catalase Test
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A plate of nutrient agar is streaked and incubated at the optimum temperature for 24 hr (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.

5. Oxidase Test
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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 purplecolor within 10-30 sec.

6. Glucose Oxidation-Fermentation (O-F) Test

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Inoculate two tubes by stabbing with needle carrying bacteria. Overlay the medium in one tub with sterile liquid paraffin to a depth of 1 cm. Examine daily for up ti 7days. Result Oxidative Fermentative No reaction Open tube Yellow (+) Yellow (+) Blue/Green (-) Covered tube Green (-) Yellow (+) Green (-)

Note: Negative (-) has to wait until 7 days. 7. Sugar Fermentation There are many different sugars can be metabolized by bacteria. The ability to ultilize sugar as a sole carbon source are characteristic for different bacteria within the Enterobacteriaceae, vibrios and aeromonads. The standard carbohydrate broth base consists of nutrient broth supplemented with a bromthymol blue (as a pH indicator) Nutrient broth Sugar 1.6% bromthymol blue Distilled water Adjust pH to 6.8
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8g 10g 4 ml 1000 ml

Dispense the 1% sugar medium into screw capped tube, 5 ml per tube. Autoclave the medium at 100oC for 10 minutes. 8. PCR procedure Tissue of liver store in 1.5ml tube with ethanol, eliminate ethanol and grin in 600l Lysis buffer (0.5M NaCl, 0.001 EDTA, 1% SDS, 0.8% Triton, 0.1M Tris-HCl), 40l SDS 10% and 2.5l Proteinase K (40mg/ml). Motioning reversal 25 times, incubating at 370C in 15 minutes. Add 2.5l RNase (2mg/ml), motioning reversal 25 times and incubating solution at 370C in 30 minutes. Continue adding to 600l Chloroform Isoamyl (24:1), reversal and centrifugal 13000 cycles in 15 minutes. Protein will settle down into one small layer between the two phases. Put 600l Phenol Chloroform Isoamyl (25:24:1) and reversal the tube to wash DNA. Centrifuge 13000 cycles in 10 minutes. Carefully suck the solution above into another 1.5ml tube. Add 600l cool Isopropanol and centrifuge 13000 cycles in 10 minutes. DNA will settle down to the bottom, release the solution above and add 600l cool alcohol 70%. Hand tightly to wash DNA. Carefully take away alcohol above. Repeat one more time. Reversal and dry the tube in few minutes. Put 50l TE buffer (10mM Tris, 1mM EDTA, pH = 7) into DNA tube and store in 40C.

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