Bacterial disease is still a big concern in intensive catfish culture in Indonesia. The aim of this research was

to find out causative agent on cat fish based on the 16S rDNA gene sequences. This research combined

between exploratory in the field and experimental method in Laboratory. Causative agents diversity of bacterial

diseases of catfish was isolated from Kendal Regency, Indonesia and based on postulat Koch results. Twenty

bacteria (K01–K20) were isolated from external wound and kidney of moribound catfish with TCBS, NA and

GSP medium. The postulat results showed that three isolates (K6, K14 and K19) were weaken up to 60% of

fishes and caused 10–20% mortality. On the other hand, there were 14 isolates that did not demosntrated their

virulence. Based on 16S rDNA sequence analysis, strain K6, K14 and K19 were closely related to Aeromonas

sobria (97%.), Pseudomonasplecoglossicida (96%.), Aeromonas caviae (96%.) respectively. Sensitivity test to

all isolates showed that these causative agents resistant to some fish drugs

Aryati, Restiana Wisnu

Haditomo, AH Condro

Prayitno, Slamet Budi

Sarjito, Sarjito

English

Diponegoro University Institutional Repository

Delivered by Ingenta to: ?IP: 180.246.41.222 On: Sat, 09 Dec 2017 11:09:45Copyright: American Scientific PublishersCopyright © 2017 American Scientific PublishersAll rights reservedPrinted in the United States of AmericaR E S E A R CH AR T I C L EAdvanced Science LettersVol. 23, 6479–6482, 2017The Diversity of Causative Agent Associated withBacterial Diseases on Catfish (Clarias gariepinus)with Molecular Based from Kendal, IndonesiaSarjito∗, A. Harjuno Condro Haditomo, Restiana W. Ariyati, and S. Budi PrayitnoDepartment of Aquaculture, Fisheries and Marine Science Faculty, Diponegoro University, Tembalang, Semarang, IndonesiaBacterial disease is still a big concern in intensive catfish culture in Indonesia. The aim of this research wasto find out causative agent on cat fish based on the 16S rDNA gene sequences. This research combinedbetween exploratory in the field and experimental method in Laboratory. Causative agents diversity of bacterialdiseases of catfish was isolated from Kendal Regency, Indonesia and based on postulat Koch results. Twentybacteria (K01–K20) were isolated from external wound and kidney of moribound catfish with TCBS, NA andGSP medium. The postulat results showed that three isolates (K6, K14 and K19) were weaken up to 60% offishes and caused 10–20% mortality. On the other hand, there were 14 isolates that did not demosntrated theirvirulence. Based on 16S rDNA sequence analysis, strain K6, K14 and K19 were closely related to Aeromonassobria (97%.), Pseudomonasplecoglossicida (96%.), Aeromonas caviae (96%.) respectively. Sensitivity test toall isolates showed that these causative agents r sistant to some fish drugs.Keywords: Causative Agent, Bacterial Diseases, Catfish, 16S rDNA.1. INTRODUCTIONCatfish (Clariasgariepinus) is the one of important aquacul-ture commodity in Kendal Regency. Kendal is one of catfish(c. gariepinus) producer in central java, producing approximately21.967,06 tonnes in 2015.1 Increasing of catfish demand fordomestic markets encourages the fish farmers to increase theirproduction. This was done through intensification and extensi-fication. Unappropriate management of intensive culture wouldcause a negative impacts, such as bacterial disease outbreaks.23Bacterial disease is still a big concern in catfish culture. Bacte-rial infection in the catfish was characterized by pale nor blackenof the body, haemorrhagic surrounds the mouths, fins, and tails,exopthalmia, fin root, body wounds and pale; dark color on liverand kidneys; red and root antenna.45 Aeromonashidrophylla,A. caviae and A. sobria;45 A. salmonicida;6 Vibrio sp.;7 Pseu-domonas spp.8 Edwardsiella ictaluri,9 were reported as causativeagents of bacterial diseases on catfish. These pathogens causedhigh mortality in larvae, fingerling, adult and broodstock,10 andin catfish larvae up to 70%.4Several researches had been performed to find out bac-terial pathogen on cat fish based on the 16S rDNA genesequences.2411 However, to our knowledge, there were limitedreports being documented so far describing the application ofpolymerase chain reaction on the diversity of causative agent∗Author to whom correspondence should be addressed.associated with bacterial diseases on cat fish derived from inten-sive pond of Kendal Regency, Central Java, Indonesia. Thisresearch is important in designing disease prevention strategy andsupporting the catfish production.2. MATERIAL AND METHODSThe moribound catfish were collected from intensive cultureponds of central production in Kendal Regency on May to August2016. Moribound catfish were kept in the container and immedi-ately brought to Integrated Laboratory of Diponegoro Universityfor bacterial isolation. Bacteria were isolated from Kidney andthe wound by streak method then cultured onto Thio-SulphateCitrate Bile Salt Sucrose (TCBS), TrypticSoybean Agar (TSA)and Glutamate Starch Penicillin (GSP) medium. Morphologicalfeatures of the colonies were randomly picked and purified by asingle colony to the plate.12Postulate Koch test was conducted to twenty isolates by using420 healthy catfish as an experimental animals. During accli-matitation experimental catfish were divided into 42 aquariums.Twenty isolates were cultured in zobell liquid medium, thenintramuscularly injected on healthy catfish with 0.1 mL of inoc-ulants bacterial density of 108 CFU/mL. Clinical signs and mor-tality of catfish were observed for 96 hours.Molecular characterization 16S rDNA of three isolateswere carried out.13–15 They further explained that theAdv. Sci. Lett. Vol. 23, No. 7, 2017 1936-6612/2017/23/6479/004 doi:10.1166/asl.2017.9659 6479Delivered by Ingenta to: ?IP: 180.246.41.222 On: Sat, 09 Dec 2017 11:09:45Copyright: American Scientific PublishersR ES E A R CH AR T I C L E Adv. Sci. Lett. 23, 6479–6482, 2017Polymerase Chain Reaction (PCR) was conducted withEppendorf Mastercycler (Eppendorf Inc. Germany). Twoprimers, GM3F (5′AGAGTTTGATCMTGGC-3′) and GM4R(5′-TACCTTGTTACGACTT-3′) were used to amplify nearlycomplete 16S rDNA gene. Prior to PCR, genomic DNA of threecausative agent associated with bacterial diseases on the cat-fish for PCR analysis were extracted from cell materials takenfrom agar plate, suspended in steril water (Sigma, Germany) andsubjected to five cycles of freeze (−80 C) and thaw (95 C).Then, PCR amplification of partial 16S rDNA gene of bacteriaand purification of PCR products were performed based on themethod of Ref. [13]. Sequencing were commenced to gain thealkaline composition that made up DNA sequences. The sequenc-ing was done using the BigDye Terminator V 3.1 dyes and auto-matic DNA sequencer ABI3130 Genetic Analyzer XL AppliedBiosystems at Macrogen Korea. Whereas, the determined DNAsequences of causative agent were then compared for homol-ogy to the BLAST (Basic Local Alignment Search Tool) onNational Center for Biotechnology Information, National Insti-tute for Health database USA.6Three causative agent were tested their sensitivity to four fishdrugs (A™, B™, C™ and D™) in vitro based on method.17 Inhibi-tion of the causative agents by each drugs were demonstrated byproduction of clear zone in the bacterial colonies area. Sensitiv-ity test was evaluated according standard developed by NationalCommittee for Clinical Laboratory Standards.183. RESULTS AND DISCUSSIONClinical signs of moribound catfish which affected by bacterialdiseases from intensive pond of Kendal were haemorrhagic sur-rounds the mouth, fins, and tail. Exopthalmia; fin root; bodywounds and pale; dark color in liver and kidney. This morpho-logical symptom (body wound and pale, haemorrhagic surroundsthe mouths, fins, and tails; red and root antenna), the behaviourabnormalities such as body upsite down, lethargy, decreasedTable I. Characteristic of isolates bacteria associated on cat fish fromintensive pond of Kendal, Indonesia.ColonyNo. Isolate code Media Source Colour Form Characteristic1 K.1 TCBS Kidney Green Rounded Convex2 K.2 TCBS Kidney White Rounded Convex3 K.3 TCBS Fins root Cream Rounded Convex4 K.4 TCBS Anal fin Yellow Rounded Convex5 K.5 TCBS fins root Green Rounded Convex6 K.6 TCBS fins root Yellow Rounded Convex7 K.7 TCBS Tail fin Yellow Rounded Convex8 K.8 TCBS Fin Dorsal Yellow Rounded Convex9 K.9 GSP Fins root Yellow Rounded Convex10 K.10 GSP Tail fin Cream Rounded Convex11 K.11 GSP Kidney Pink Rounded Convex12 K.12 GSP Kidney White Rounded Convex13 K.13 GSP Anal fin Yellow Rounded Convex14 K.14 GSP Kidney Yellow Rounded Convex15 K.15 NA Fins root Yellow Rounded Convex16 K.16 NA Dorsal fin Yellow Rounded Convex17 K.17 NA Kidney White Rounded Convex18 K.18 NA Kidney Cream Rounded Convex19 K.19 NA Fins root White Rounded Convex20 K.20 NA Tail fin Yellow Rounded ConvexTable II. Percentage of sick and mortality of cat fish (C. gariepinus)during postulates koch’s performed.No. Isolates code Sick cat fish (%) Total mortality of cat fish (%)1. K6 60 202. K14 35 103. K19 35 10Table III. Three isolates selected of causative agent associated withbacterial disease at cat fish (C. gariepinus).ColonyNo. Isolates code Media Source Color Form Characteristic1 K.6 TCBS Fins root Yellow Rounded Convex2 K14 GSP Kidney Yellow Rounded Convex3 K19 TCBS Wound Yellow Irregular Convexappetite, increased mucus production, swimming imbalance, defi-ciency in oxygen uptake were also found in experimental catfish.The clinical signs above have been reported.41920 These clini-cal signs observed in the present study has also been observedin naturally-diseased catfish cultured.19 These clinical signs wereobserved in the present study may due to attachment and colo-nization of many opportunistic pathogens to fish skin of cat fish.4Twenty bacterial isolates were obtained from kidney, fins root,and body wound of moribound catfish (Table I). Postulate Kochtest results indicated that three isolates namely K6, K14 and K19were able caused disease symptom up to 60% of the challengedcatfish, whilst the three isolates was caused mortality range of10–20% (Table II). He present study also found that experi-mentally catfish were injected by others isolates and PBS had100% survival rate and normal behaviour. Therefore, these iso-lates (K6, K14 and K19) were positively confirmed as a causativeagent associate bacterial diseases in catfish from intensive cul-ture pond that is located at Kendal Regency. These mortality ratewas lower than that reported,4 in catfish A. sobria infected (30%)and A. caviae-infected (20–30%). Moreover, A. sobria was lesspathogenicity compare to A. hydrophila on Stinging CatfishShing (Heteropneustes fossilis)6 The occurance pathogenicityof these bacteria on the fish was mainly contributed by thehaemolysin and aerolysin.2122 Based on the postulat Koch results(Table II), three isolates (K6, K14 and K19) out of 22 isolates(K1–K20) were choosen for further investigation (Table III).On the basis of sequence 16S rDNA analysis using BlastSystem (Table IV), the results showed that causative agentsof bacterial diseases on catfish from pond of kendal, i.e., K6,K14 and K19 were closely related to Aeromonas sobria (97%.),Pseudomonas plecoglossicida (96%.), Aeromonas caviae (96%.)respectively. Both of Pseudomonas spp. and Aeromonas spp. wasnaturally found in the aquatic environment, represent the Gram-negative commensal bacteria and include in pathogenic bacte-ria of fish.712 The presents study revealed that the causativeTable IV. Analysis of three isolates compared with BLAST system.No. Isolates code Results Homologi (%) No. access1. K6 Aeromonas sobria 97 KC210798.12. K14 Pseudomonasplecoglossicida96 KC431807.13. K19 Aeromonas caviae 96 JQ231158.16480Delivered by Ingenta to: ?IP: 180.246.41.222 On: Sat, 09 Dec 2017 11:09:45Copyright: American Scientific PublishersR E S E A R CH A R T I C L EAdv. Sci. Lett. 23, 6479–6482, 2017Table V. The result of sensitivity test three causative agent of bacterial diseases on catfish in kendal regency.A™ B™ Drug C™ D™Times (Hour)24 48 24 48 24 48 24 48Dosage (l)Drugs isolate code 6 8 10 6 8 10 2,5 5 7,5 2,5 5 7,5 2,5 5 7,5 2,5 5 7,5 2 4 6 2 4 6K06 R R R R R R R R R R R R R R R R R R R R R R R RK14 R R R R R R R R R R R R R R R R R R R R R R R RK16 R R R R R R R R R R R R R R R R R R R R R R R Ragents associated with bacterial diseases of catfish that wasintensively cultured at Kendal ponds, were closely related toA. sobria (K6), P. plecoglossicida (K14), and A. caviae (K19).A. sobria and A. caviae were commonly reported as a bacte-rial pathogen associated with motile aeromonas septicemia inAfrican cat fish,4 catfish, Clarias;11 haemorrhage of Tra catfish,Pangasianodon hypophthalmus,5 and walking cat fish culture,Clarias bratachus.19 A. sobria was also reported in walking cat-fishes, C. batrachus;21 Nordmann and Poirel,22 catfish in India,11and in China as a aquatic pathogenic bacteria. While, A. veroniibiovar sobria was also found as a causative agent of EpizooticUlcerative Syndromein fish in Bangladesh.23Pseudomonas spp. has been reported to be an important fishpathogen that has endangered aquaculture.8 P. plecoglossicidawas found as causative agent of bacterial haemorrhagic ascitesof ayu, Plecoglossus altivelis.2425 Out break of this diseaseoften occurs after seeds was introduced in culture ponds, andit also could found at any development stage during culture.24P. plecoglossicida was also found in natural environment.26On the other hand, P. plecoglossicida could become as a nonpathogenic bacteria that was potentially produce an anti-tumordrugs;27 and as a candidate of probiotic.24 However, in the presentstudy also confirmed that P. plecoglossicida was found as acausative agent associated with bacterial diseases on cat fish thatwas intensively cultured in pond of Kendal regency.The sensitivity test results (Table IV) revealed that threecausative agents associate with bacterial disease in catfish fromKendal, namely: A. sobria (K6), P. plecoglossicida (K14),A. caviae (K19), were not sensitive to drug A™, B™, C™ and D™.These were detected by the formation of a clear zone around thepaper discs on all three bacteria with diameter of 0–1,3 mm. Thecriteria of resistant bacteria if they have inhibitory zone rangedbetween 0–10 m.18 The previous research also found that GenusAeromonas;2829 A. caviae;30 and A sobria,31 were resistence toantibiotic. Pseudomonas spp. and Aeromonas spp. was resistantto multiple drug for 96.6% and 61.9%.2 This resistance occursmay related to the using of fish drugs irrational dosage duringaquaculture process,32 and the content of the active compound(antibiotics) in fish drugs.33 This resistance occurs when bacte-ria mutate in one way or another, it will impact on decreasingor losing of effectiveness of the drug, chemical compound orother material to prevent or treat infection.2834 Therefore, thethree resistant causative agent associated with bacterial diseaseson catfish was also caused by the irrational drugs administrationduring the production process, i.e.,: the prevention and treatmentof disease. Moreover, bacterial resistance could also occur dueto mutate and selection randomly charge, in this case it act asan agent of antibiotic selection, so it was possible the groupmultiplication of resistant bacteria and suppressed the growth ofbacteria sensitively to the antibiotic properties.35 Furthermore,resistance to these fish drugs could be transmitted to other bac-teria through a group of antibiotic resistant genes, between samegenes locus with agents, such as plasmids, transposons, andintegrons.26334. CONCLUSIONBacterial causative agents in the catfish cultured in central pro-duction in Kendal with a molecular approach were Pseudomonasplecoglossicida, Aeromonasveronii and Aeromonas sobria. Theresult of sensitivity test obtained that three causative agents haveresistant character to four fish drugs. Clinical signs of moriboundcatfish were haemorhagic surrounds the mouth, fins, tail andwound, exopthalmia, fins root, body wound and pale, dark colorin liver and kidneys, red and root antenna.Acknowledgments: This study is part of research grantsfunded by Directorate for Research and Community Services,Directorate General of Education Strengthening Research Min-istry of Research, Technology and Higher Education. Fiscalyear 2016. Number: 022/SP2H/LT/DRPM/II/2016; February 17th2016. On this opportunity, the authors would like to thank toDean of Fisheries and Marine Sciences Faculty, UNDIP, Headof Integrated Laboratory of Diponegoro University, AquacultureLaboratory of Fisheries and Marine Sciences Faculty, UNDIPand Fish Quarantine, Quality Control and Safety of Fishery ClassI Semarang.References and Notes1. Marine and Fisheries Ministry of Central Java Province (2016).2. H. N. K. Nguyen, T. T. H. Van, H. T. Nguyen, P. M. Smooker, J. Shimeta, andP. J. Coloe, Veterinary Microbiology 171, 397 (2014).3. S. B. Sarjito and A. H. C. Prayitno dan Haditomo, UNDIP Press (2013).4. M. U. Anyanwu, K. F. Chah, and V. S. Shoyinka, International Journal of Fish-eries and Aquatic Studies 2, 93 (2015).5. L. T. Thanh Ly, D. N. Nguyen, P. H. Vo, and C. V. Doan, The Israeli Journal ofAquaculture–Bamidgeh 61, 215 (2009).6. M. D. Monir, T. Ahammed, S. C. Borty, N. Bagum, Md. A. Islam, andY. Mahmud, Trend in Fisheries Reasearch 4, 2319 (2015).7. B. Austin and D. A. Austin, Ellis Horword Limited, Chichester, England (2007),p. 552.8. Z. Mao, Y. Qiu, L. Zheng, J. Chen, and J. Yang, Journal of MicrobiologicalMethods 89, 179 (2012).9. M. Crumlish, T. T. Dung, J. F. Turnbull, N. T. N. Ngoc, and H. W. Ferguso,Journal of Fish Diseases 25, 733 (2002).10. M. R. Durborow, L. R. Thune, and C. A. Camus, SRAC Publication (1998),p. 479.11. D. Arunava, A. Rathore, C. Janani, C. Hemanth, and R. A. Balakrishnan, IOSRJournal of Agriculture and Veterinary Science (IOSR-JAVS) 2, 87 (2013).12. T. D. Brock and M. T. Madigan, Prentice Hall, Englewood Cliffs, New Jersey(1991).13. O. K. Radjasa, D. Nasima, A. Sabdono, K. Kita-Tsukamoto, and K. Ohwada,J. Biol. Sci. 7, 658 (2007a).6481Delivered by Ingenta to: ?IP: 180.246.41.222 On: Sat, 09 Dec 2017 11:09:45Copyright: American Scientific PublishersR ES E A R CH AR T I C L E Adv. Sci. Lett. 23, 6479–6482, 201714. O. K. Radjasa, H. Urakawa, K. Kita-Tsukamoto, and K. Ohwada, Mar. Biotech-nol. 3, 454 (2001).15. Sarjito, O. K. Radjasa, S. B. Prayitno, A. Sabdono, and S. Hutabarat, Curr.Res. Bacteriol. 14 (2009).16. S. F. Atschul, T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, andD. J. Lipman, Nucleid Acid Res. 25, 3389 (1997).17. O. K. Radjasa, T. Martens, H. P. Grossart, T. Brinkoff, A. Sabdono, andM. Simon, J. Biol. Sci. 7, 239 (2007b).18. National Committee for Clinical Laboratory Standards (NCCLS), ApproveStandars M100-S11, NCCLS, Wayne, Pa (2001).19. S. Areerat, Aquaculture 63, 355 (1987).20. B. Ahamad, D. Punniamurthy, N. S. Kumar, V. Malmarugan, R. Suresh, andV. Ranganathan, Proceedings of the National Seminar on Current Perspec-tives in Biolological Sciences (NSOCPIBS-2012) (2013), pp. 121–151.21. A. K. Chopra, C. W. Houston, J. W. Peterson, and G. F. Jin, Canadian Journalof Microbiology 39, 513 (1993).22. P. Nordmann and L. Poirel, Clinical Microbiology Infection 8, 321 (2002).23. M. Rahman, P. C. Navarro, I. Kühn, G. Huys, J. Swings, and R. Möllby, Appliedand Environmental Microbiology 68, 650 (2002).24. S. C. Park, I. Shimamura, M. Fukunaga, K. I. Mori, and T. Nakai, Applied andEnvironmental Microbiology 66, 1416 (2000).25. E. Nishimori, K. Kita-Tsukamoto, and H. Wakabayashi, Int. J. Syst. Evol.Microbiol. 50, 83 (2000).26. M. Mulet, A. Bennasar, J. Lalucat, and E. G. á-Valde, Molecular and CellularProbes 23, 140 (2009).27. Y. M. Liu, Z. H. Sun, Y. Ni, P. Zheng, Y. P. Liu, and F. J. Meng, World J.Microbiol. Biotechnol. 24, 2213 (2008).28. S. Shinha, T. Shimada, T. Ramamurthy, S. K. Bhattacharya, S. Yamasaki,Y. Takeda, and G. B. Nair, J. Med. Microbiol. 53, 527 (2004).29. A. W. Ashiru, P. O. Uaboi-Egbeni, J. E. Oguntowo, and C. N. Idika, PakistanJournal of Nutrition 10, 982 (2011).30. M. R. Motyl, G. Mckinley, and J. M. Jandal, Antimicrobial Agents andChemotherapy (1985).31. B. O. Mannin and J. Ransangan, Borneo Marine Research Institute(2010).32. L. J. Sukenda, D. Wahyuningrum dan, and A. Hasan, Jurnal AkuakulturIndonesia 7, 159 (2008).33. D. G. White and P. F. McDermott, Curr. Opin. Microbiol. 4, 313(2001).34. C. R. A. Sharma, C. R. Bora, R. K. Chaurasia, and V. Sahu, Curr. Res. Bac-teriol. 19, 1 (2009).35. R. M. Atlas, Mosby-Year Book, Inc., Missouri (1995), p. 374.Received: 5 September 2016. Accepted: 13 December 2016.6482

The Diversity of Causative Agent Associated with

Bacterial Diseases on Catfish (Clarias gariepinus)

with Molecular Based from Kendal, Indonesia

http://eprints.undip.ac.id/58286/1/The_diversity_of_Causative_Agent__Associateg_with_Bacterial_Diseases_on_Catfish_with_Molecular_Based_from__Kendal%2C_Indonesia.pdf

The Diversity of Causative Agent Associated with Bacterial Diseases on Catfish (Clarias gariepinus) with Molecular Based from Kendal, Indonesia

Abstract

Similar works

Full text

Available Versions

Diponegoro University Institutional Repository