Streptococcus parauberis’in Türkiye’deki Tatlısu Balıklarından İzolasyon ve İdentifikasyonu

Öz

Streptococcus parauberis alfa hemolitik, gram pozitif kokoid bir bakteri olup Streptococcaceae familyasına aittir. Bu bakteri kültür balıklarında yoğun üretimden kaynaklı çok önemli bir hastalık olan streptokokozise neden olmaktadır ve balık çiftliği endüstrisinde önemli bir ekonomik kayba neden olmaktadır.  Çalışmamızda Eğirdir gölü balıklarından (Sander lucioperca, Carassius gibelio, Cyprinus carpio) ve kültür balığı türü olan (Oncorhynchus mykiss, Walbaum)’dan 37 tane laktik asit bakterisi izole edilmiştir. Laktik asit bakterilerinin tanımlanması için MRS, TSA ve M17 agardan izole edilen 37 izolatın fenotipik ve morfolojik karakterleri koloni morfolojisi, hücre morfolojisi, hareketlilik, gram boyama, katalaz ve oksidaz üretimiyle yapılmıştır. Diğer biyokimyasal özellikler de Bergey’s Manual of Systematic Bacteriology’e göre yapılmıştır. İzole edilen bakterilerinden 25 tanesi S. parauberis, 2 tanesi Vagococcus sp., 2 tanesi Lactococcus garvieae ve 5 tanesi de Lactococcus lactis olarak biyokimyasal yöntemler ve 16Sr RNA gen sekansıyla tanımlanmıştır fakat 3 tanesi (F65, F49 ve F50) tanımlanamamıştır. Çalışmamızda S. parauberis’in izolasyon ve identifikasyonu ilk olarak Türkiye’deki Eğirdir gölü balıklarından ve gökkuşağı alabalığından gerçekleştirildi. 

Anahtar Kelimeler

• 16Sr RNA,Tatlısu,moleküler tanımlama,balık hastalığı,yetiştiricilik

Isolation and Identification of Streptococcus parauberis From Freshwater Fish in Turkey

Öz

Streptococcus parauberis is an alpha-hemolytic gram positive coccoid bacterium belonging to the Streptococcaceae family. This bacterium cause streptococcosis is a major disease in cultured fish due to the intensification of aquaculture and causes significant economic losses in fish farm industry. In our study, we isolated a total of 37 lactic acid bacteria from wild fish (Sander lucioperca, Carassius gibelio, Cyprinus carpio) of Lake Eğirdir and cultured fish (Oncorhynchus mykiss, Walbaum) of farm in Turkey. For the isolation of bacteria phenotypic and biochemical characteristics of the 37 isolates obtained from the colonies grown on MRS, TSA and M17 agar characterized by determining colony morphology, cell morphology, motility, gram staining and the production of cytochrome oxidase and catalase. Further biochemical characteristics were determined using conventional tests according to Bergey’s Manual of Systematic Bacteriology. From the isolated lactic acid bacteria 25 of were identified as S. parauberis, 2 of as Vagococcus sp., 2 of as Lactococcus garvieae and 5 of as Lactococcus lactis by culture-based, biochemical test and 16Sr RNA gene sequencing techniques but 3 of them couldn’t (F65, F49 and F50) identified.  In this study, the main objective of us is to identify S. parauberis with conventional and 16Sr RNA gene sequencing techniques from wild fish in Lake Eğirdir and in cultured fish in fish farm of Egirdir-Turkey. 

Anahtar Kelimeler

• freshwater,molecular identification,fish disease,aquaculture,16Sr RNA

Kaynakça

1. Baeck G.W, Kim J.H, Gomez D.K, Park S.C. 2006. Isolation and characterization of Streptococcus sp. from diseased flounder (Paralichthys olivaceus) in Jeju Island. J Vet Sci. 7(1):53–58. doi:10.4142/jvs.2006.7.1.53
2. Bondad-Reantaso M.G, Subasinghe R.P, Arthur J.R, Ogawa K, Chinabut S, Adlard R, Tan Z, Shariff M. 2005. Disease and health management in Asian aquaculture. Vet Parasitol. 132(3-4) 249-272. doi:10.1016/j.vetpar.2005.07.005
3. Didinen B.I, Kubilay A, Diler O, Ekici S, Onuk E.E, Findik A, 2011. First isolation of Vagococcus salmoninarum from cultured rainbow trout (Oncorhynchus mykiss, walbaum) broodstocks in Turkey. Bull Eur Ass Fish Pathol. 31(6): 235-243.
4. Didinen B.I, Yardımcı B, Onuk E.E, Metin S, Yıldırım P. 2014. Naturally Lactococcus garvieae infection in rainbow trout (Oncorhyncus mykiss Walbaum, 1792): new histopathological observations, phenotypic and molecular identification, Revue Méd Vét. 165(1-2): 12-19.
5. Diler O, Altun S, Adiloglu A, Kubik A, Isikli B.T. 2002. First occurrence of streptococcosis affecting farmed rainbow trout (Oncorhynchus mykiss) in Turkey. Bull Eur Ass Fish Pathol. 22(1): 21.
6. Domenech A, Ferna´ndez Garayza´bal J.F, Pascual C, Garcia J.A. Cutuli M.T, Moreno M.A, Collins M.D, Dominguez L. 1996. Streptococcosis in cultured turbot, Scophthalmus maximus (L.), associated with Streptococcus parauberis. J Fish Dis. 19(1): 33-38. doi: 10.1111/j.1365-2761.1996.tb00117.x
7. Eldar A, Y Bejerano, A Livoff, A Horovitcz, Bercovier H. 1995. Experimental streptococcal meningo-encephalitis in cultured fish. Vet Microbiol. 43(1): 33-40. doi:10.1016/0378-1135(94)00052-X
8. Facklam RR, Elliott JA. 1995. Identification, classification and clinical relevance of catalase-negative, gram-positive cocci, excluding the streptococci and enterococci. Clin Microbiol Rev. 8(4): 479–495.

9. FAO. 2016. Tha state of world fisheries and aquaculture, Rome, 200p.
10. Haines A, T.Gauthier D, E.Nebergall E, D.Cole S, M.Nguyen K, W.Rhodes M, K.Vogelbein W. 2013. First report of Streptococcus parauberis in wild finfish from North America. Vet Microbiol. 166(1-2):270-275 doi:10.1016/j.vetmic.2013.05.002
11. Hoshina T, Sano T, Morimot K, Jofre J. 1958. A streptococcus pathogenic to fish. J Tokyo Univ Fish. 44: 57-68.
12. Kesarcodi-Watson A, Kaspar H, Lategan M.L. J, Gibson L. 2008. Probiotics in aquaculture: The need, principles and mechanism of action and screening processes, Aquaculture, 274(1): 1-14. doi:10.1016/j.aquaculture.2007.11.019
13. Kia RE, Yadollah M. 2013. Detection and ıdentification of different Streptococcosis strains in farmed rainbow trout in Boyerahmad and Dena regions (North South of Iran), World Journal of Fish and Marine Sciences 5(3): 315-321. doi:10.5829/idosi.wjfms.2013.05.03.72123
14. Kitao T, T Aoki, R Sakoh. 1981. Epizootic caused by betahaemolytic Streptococcus species in cultured freshwater fish. Fish Pathol. 15(3-4): 301-307. doi:10.3147/jsfp.15.301
15. Kusuda K, Kawai K, Salati F, Banner CR, Fryer JL. 1991. Enterococcus seriolicida sp. nov., a fish pathogen Int. J Syst Bacteriol. 41(3-4): 406-409.
16. Lämmler Ch, Abdulmawjood A, Danic G, Vaillant S, Weig R. 1998. Differentiation of Streptococcus uberis and Streptococcus parauberis by restriction fragment length polymorphism analysis of the 16S ribosomal RNA gene and further studies on serological properties. Med Sci Res. 26, 177– 179.
17. Liu D. 2000. Rapid mini-preparation of fungal DNA for PCR. Journal of Clinical Microbiology. 38 (1): 471.
18. Nho LW, Shin GW, Park SB, Jang HB, Cha IS, Ha MA, Kim YR, Park YK, S. Dalvi R, Kang BJ, Joh SJ. 2009. Phenotypic characteristics of Streptococcus iniae and Streptococcus parauberis isolated from olive founder (Paralichthys olivaceus) Fems Microbiol Lett. 293(1): 20–27. doi:10.1111/j.1574-6968.2009.01491.x
19. Park-Ae M, Mun-Kwon G, Hwang-Jee Y, Jung-Sung H, Kim-Dong W, Jin-Young P, Kim J.S, Na Y.J, Kim M.Y, Kim D.S, Chae S.H, Seo J.S. 2013. Genome Sequence of Streptococcus parauberis Strain KCTC11980, Isolated from Diseased Paralichthys olivaceus, Genome Announc. 1(5):e00780-13. doi:10.1128/genomeA.00780-13
20. Pitkälä A, Koort J, Björkroth J. 2008. Identification and Antimicrobial Resistance of Streptococcus uberisand Streptococcus parauberis Isolated from bovine milk samples. J. Dairy Sci. 91(10):4075–4081 doi:10.3168/jds.2008-1040
21. Pourgholam R, Laluei F, Saeedi AA, Taghavi MJ, Safari R, Zahedi A. 2013. Identification of some streptococcus species isolated from rainbow trout (Oncorhynchus msykiss) in Iran by using molecular method. Journal of Novel Applied Sciences, 2 (S4): 1228-1233.
22. Romalde JL, Ravelo C, Valdes I. Magarinos B, de la Fuente E, Martin CS, Avendano-Herrera R, Toranzo AE. 2008. Streptococcus phocae, an emerging pathogen for salmonid culture. Vet Microbiol. 130(1-2): 198–207. doi: 10.1016/j.vetmic.2007.12.021
23. Salminen S. 1998. Demonstration of safety of probiotics. Int J Food Microbiol. 44: 93-106.
24. Pérez-Sánchez T, Balcázar JL, García Y, Halaihel N, Vendrell D, de Blas I, Merrifield DL, Ruiz-Zarzuela I. 2011. Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae. J Fish Dis. 34(7):499–507. doi: 10.1111/j.1365-2761.2011.01260.x
25. Schleifer, KH. 1986. Gram-positive cocci. In Bergey’s Manual of Systematic Bacteriology. Sharpe, M.E. and Holt, J.G. (Eds), The Williams and Wilkins Co., Baltimore, Md. p. 999–1103.
26. Soto LP. 2010. Molecular Microbial Analysis of Lactobacillus Strains Isolated from the Gut of Calves for Potential Probiotic Use. Vet Med Int. 2010: 1-7. doi:10.4061/2010/274987
27. Subasinghe R, Soto D, Jia J. 2009. Global aquaculture and its role in sustainable development Rev Aquacult. 1(1): 2-9. doi:10.1111/j.1753-5131.2008.01002.x
28. Toranzo A.E, Devesa S, Heinen P, Riaza A, Nu´n˜ ez S, Barja J.L. 1994. Streptococcosis in cultured turbot caused by an Enterococcus-like bacterium. Bull Eur Assoc Fish Pathol. 14(1): 19– 23.
29. Toranzo T, Alicia Magarin˜os Beatriz, L Romalde Jesu´ s. 2005. A review of the main bacterial fish diseases in mariculture systems. Aquaculture 246 (1-4): 37 – 61. doi:10.1016/j.aquaculture.2005.01.002
30. Williams A.M, Collins M.D. 1990. Molecular taxonomic studies on Streptococcus uberis types I and II. Description of Streptococcus parauberis sp. nov. J Appl Bacteriol. 68(5): 485–490. doi:10.1111/j.1365-2672.1990.tb02900.x
31. Yang W and Li A. 2009. Isolation and characterization of Streptococcus dysgalactiae from diseased Acipenser schrenckii, Aquaculture, 294(1-2): 14–17. doi:10.1016/j.aquaculture.2009.05.018
32. Zlotkin A, Hershko H, Eldar A. 1998a. Possible transmission of Streptococcus iniae from wild fish to cultured marine fish. Appl. Environ. Microbiol. 64(10): 4065– 4067.
33. Zlotkin A, Eldar A, Ghittino C, Bercovier H. 1998b. Identification of Lactococcus garvieae by PCR. J. Clin. Microbiol. 36(4): 983– 985.