KÜLTÜR GÖKKUŞAĞI ALABALIĞI (Oncorhynchus mykiss)’NDAN İZOLE EDİLEN GRAM-NEGATİF PATOJENLERİN LİPOPOLİSAKKARİT PROFİLLERİ

Öz

Bu çalışma, Türkiye’nin farklı bölgelerindeki kültür gökkuşağı alabalıkları (Oncorhynchus mykiss)’ndan izole edilen patojenik Gram-negatif bakterilerin lipo­polisakkarit (LPS) profillerinin belirlenmesi amacıyla yürütülmüştür. Bu amaçla, hasta balık örneklerinden izole edilen Vibrio anguillarum, Aeromonas hydrophi­la, A. schubertii, Pseudomonas fluorescens ve Yersi­nia ruckeri izolatlarından LPS numuneleri elde edil­miştir. LPS profilleri, Sodyum Dodesil Sülfat-Poliakrilamid Jel Elektroforezi (SDS-PAGE) ve gümüş-nitrat boyama metodu ile belirlenmiştir. Bu çalışmadan elde edilen sonuçlar, LPS analizi çalışma­larının bu bakterilerin karakterizasyonunda ve değişik coğrafik bölgelerden izole edilen suşlar arasındaki farklılıkların tespit edilmesinde kullanılabilecek fay­dalı araçlar olduğunu göstermiştir.

Anahtar Kelimeler

• Gökkuşağı alabalığı,O. mykiss,SDS-PAGE,LPS

LIPOPOLYSACCHARIDE PROFILES OF GRAM-NEGATIVE PATHOGENS RECOVERED FROM CULTURED RAINBOW TROUT (Oncorhynchus mykiss)

Abstract

The aim of this study was to determine the lipopoly­saccharide (LPS) profiles of pathogenic Gram-negative bacteria recovered from cultured rainbow trout (Oncorhynchus mykiss) in different regions of Turkey. For this purpose, LPS samples were obtained from 10 bacterial isolates of Vibrio anguillarum, Aer­omonas hydrophila, A. schubertii, Pseudomonas fluo­rescens and Yersinia ruckeri recovered from diseased fish. LPS profiles were determined by using Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE) and silver-nitrate staining method. Re­sults of the study demonstrated that LPS profiling is a useful tool for characterization of these bacteria and geographic discrimination of their isolates.

Keywords

• Rainbow trout,O. mykiss,SDS-PAGE,LPS

References

1. Acosta, F., Lockhart, K., Gahlawat, S.K., Real, F., Ellis, A.E. (2004): Mx expression in Atlantic salmon (Salmo salar L.) parr in response to Listonella anguillarum bacterin, lipopolysaccharide and chromosomal DNA. Fish & Shellfish Immunology, 17: 255-263.
2. Akaylı, T. (2001). Kültür Çipura Balıklarında (Sparus auratus, L 1758) Vibriosis’in ELISA ve Bakteriyolojik Yöntemlerle Teşhisi. Doktora Tezi, İstanbul Üniversitesi Fen Bilimleri Enstitüsü.
3. Akaylı, T., Çanak, Ö., Başaran, B. (2011a): Gökkuşağı alabalıklarında (Oncorhynchus mykiss Walbaum, 1792) görülen Aeromonas schubertii enfeksiyonu üzerine bir çalışma. Biyoloji Bilimleri Araştırma Dergisi, 4(1): 99-106.
4. Akaylı, T., Çanak, Ö., Başaran, B. (2011b): Yavru kültür gökkuşağı alabalıklarında (Oncorhynchus mykiss Walbaum, 1792) görülen yeni bir Pseudomonas türü: Pseudomonas plecoglossicida. Biyoloji Bilimleri Araştırma Dergisi, 4(1): 107-111.
5. Alsina, M., Martinez-Picado, J., Jofre, J., Blanch, A.R. (1994): A medium for presumptive identification of Vibrio anguillarum. Applied and Environmental Microbiology, 60(5): 1681-1683.
6. Altınok, İ., Balta, F., Çapkın, E., Kayış, Ş. (2007): Diseases of rainbow trout caused by Pseudomonas luteola. Aquaculture, 273(4): 393-397.
7. Altınok, İ., Kayış, Ş., Çapkın, E. (2006): Pseudomonas putida infection in rainbow trout. Aquaculture, 261(3): 850-855.
8. Altun, S., Kubilay, A., Diler, Ö. (2010): Yersinia ruckeri suşlarının fenotipik ve serolojik özelliklerinin incelenmesi. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 16(Suppl-B): 223-229.

9. Austin, B., Austin, D.A. (2012). Bacterial Fish Pathogens: Disease of Farmed and Wild Fish, 5th edition. Springer, New York. 978-94-007-4884-2.
10. Baran, İ., Timur, M., Aydın, N., İstanbulluoğlu, E., Aydıntuğ, M.K. (1981): Çifteler-Sakaryabaşı balık üretim ve araştırma istasyonunda alabalıklarda (Salmo gairdneri irideus) görülen bakteriyel hemorajik septisemi hastalığı üzerine incelemeler. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 27: 467-473.
11. Bastardo, A., Sierralta, V., Leon, J., Ravelo, C., Romalde, J.L. (2011): Phenotypical and genetic characterization of Yersinia ruckeri strains isolated from recent outbreaks in farmed rainbow trout Oncorhynchus mykiss (Walbaum) in Peru. Aquaculture, 317: 229-232.
12. Boesen, H.T., Pedersen, K., Larsen, J.L., Koch, C., Ellis, A.E. (1999): Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide. Infection and Immunity, 67(1): 294-301.
13. Bos, M.P., Tomassen, J. (2004): Biogenesis of the Gram-negative bacterial outer membrane. Current opinion in Microbiology, 7: 610-616.
14. Buller, N. (2004). Bacteria from Fish and other Aquatic Animals: a practical identification manual. CABI Publishing, Oxford, U.K., 0-85199-738-4.
15. Chart, H., Trust, T. J. (1984): Characterization of the surface antigens of the marine fish pathogens Vibrio anguillarum and Vibrio ordalii. Canadian Journal of Microbiology, 30(5): 703-710.
16. Çağırgan, H., Yüreklitürk, O. (1991): First isolation of Yersinia ruckeri from a rainbow trout farm in Turkey. In: EAFP 5th International Conference on Diseases of Fish and Shellfish 1991, Budapest, Book of Abstracts, pp.131.
17. Çelikkale, M.S., Düzgüneş, E., Okumus, İ. (1999). Türkiye Su Ürünleri Sektörü Potansiyeli, Mevcut Durumu, Sorunları ve Çözüm Önerileri. İstanbul Ticaret Odası, Yay. No. 1999-2. İstanbul. 975-512-321-0.
18. Davies, R.L. (1989). Biochemical and Cell-Surface Characteristics of Yersinia ruckeri in Relation to the Epizootiology and Pathogenesis of Infections in Fish. Doktora tezi, Institute of Aquaculture, University of Stirling.
19. Diler, O., Altun, S. (1994): Gökkuşağı alabalıklarından (Oncorhynchus mykiss) hemorajik septisemi etkeni olarak izole edilen bazı Aeromonas hydrophila suşlarının biyokimyasal özelliklerinin belirlenmesi. Süleyman Demirel Üniversitesi Eğirdir Su Ürünleri Fakültesi Dergisi, 4: 169-178.
20. Dooley, J.S., Trust, T.J. (1988): Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein. Journal of Bacteriology, 170(2): 499-506.
21. Dooley, J.S.G., Lallier, R., Shaw, D. H., Trust, T.J. (1985): Electrophoretic and immunochemical analyses of the lipopolysaccharides from various strains of Aeromonas hydrophila. Journal of Bacteriology, 164(1): 263-269.
22. Fulop, M., Manchee, R., Titball, R. (1995): Role of lipopolysaccharide and a major outer membrane protein from Francisella tularensis in the induction of immunity against tularemia. Vaccine, 13(13): 1220-1225.
23. Hitchcock, P.J., Brown, T.M. (1984): Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. Journal of Bacteriology, 154: 269-277.
24. Ingram, G.A. (1993). Diffusion in Gel Techniques. In: Stolen, J. S., Fletcher, T. C., Anderson, D. P., Roberson, B. S., van Muiswinkel, W. B., (Eds.), Techniques in Fish Immunology: FITC1, 2nd edition. SOS Publications, New Jersey. 0-9625505-0-7.
25. Kayış, Ş., Çapkın, E., Balta, F., Altınok, İ. (2009): Bacteria in rainbow trout (Oncorhynchus mykiss) in the southern Black Sea region of Turkey. The Israeli Journal of Aquaculture – Bamidgeh, 61(4): 339-344.
26. Kırkan, S., Göksoy, E.O., Kaya, O.(2003): Isolation and antimicrobial susceptibility of Aeromonas salmonicida in rainbow trout (Oncorhynchus mykiss) in Turkey hatchery farms. Journal of Veterinary Medicine, Series B, 50(7): 339-342.
27. Kimberley, A. W., Macnair, N.G. (2004). Finfish and Shellfish Bacteriology Manual: Techniques and Procedures. Iowa State Press, Iowa, USA. 0-8183-1952-0.
28. Knappskog, D.H., Rodseth, O.M., Slinde, E., Endersen, C. (1993): Immunochemical analyses of Vibrio anguillarum strains isolated from cod, Gadus morhua L., suffering from Vibriosis. Journal of Fish Diseases, 16: 327-338.
29. Koebnik, R., Locher, K.P., Van Gelder, P. (2000): Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Molecular Microbiology, 37(2): 239-253.
30. Kokka, R.P., Lindquist, D., Abbott, S.L., Janda, J.M. (1992): Structural and pathogenic properties of Aeromonas schubertii. Infection and Immunity, 60(5): 2075-2082.
31. Köprücü, S., Sarıeyyüpoğlu, M. (2010): Aeromonas hydrophila ile enfekte edilen gökkuşağı alabalığında (Oncorhynchus mykiss) histopatolojik bir araştırma. Fırat Üniversitesi Fen Bilimleri Dergisi, 22(1): 11-17.
32. Kubilay, A., Timur, G. (2001): Yersinia ruckeri bakterini ile immunize edilen gökkuşağı alabalıklarında (Oncorhynchus mykiss) antikor üretiminin IFAT ve ELISA teknikleri ile saptanması. Turkish Journal of Veterinary and Animal Sciences, 25: 437-445.
33. Kubilay, A., Altun, S., Diler, O., Ekici, S. (2008): Isolation of Flavobacterium columnare from cultured rainbow trout (Oncorhynchus mykiss) fry in Turkey. Turkish Journal of Fisheries and Aquatic Sciences, 8: 165-169.
34. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
35. Lima De Faria, A. (1969). Handbook of Molecular Cytology. North-Holland Publications, Londra.
36. Pazos, F., Santos, Y., Magarinos, B., Bandin, I., Nunez, S., Toranzo, A.E. (1993): Phenotypic characteristics and virulence of Vibrio anguillarum related organisms. Applied and Environmental Microbiology, 59(9): 2969-2976.
37. Pedersen, K., Grisez, L., Houdt, R.V., Tiainen, T., Ollevier, F., Larsen, J.L. (1999): Extended serotyping scheme for Vibrio anguillarum with the definition and characterization of seven provisional O-serogroups. Current Microbiology, 38: 183-189.
38. Roberts, R.J. (2012). Fish Pathology 4th edition. Wiley-Blackwell. 978-1444332827.
39. Sakai, M. (1999): Current reseach status of fish immunostimulants. Aquaculture, 172: 63-92.
40. Santos, Y., Pazos, F., Bandin, I., Toranzo, A.E. (1995): Analysis of antigens present in the extracellular products and cell surface of Vibrio anguillarum serotypes O1, O2 and O3. Applied and Environmental Microbiology, 61(7): 2493-2498.
41. Sousa, J.A., Magarinos, B., Eiras, J.C., Toranzo, A.E., Romalde, J.L. (2001): Molecular characterization of Portuguese strains of Yersinia ruckeri isolated from fish culture systems. Journal of Fish Diseases, 24: 151-159.
42. Solem, S.T., Jorgensen, J.B., Robertsen, B. (1995): Stimulation of respiratory burst and phagocytic activity in Atlantic salmon (Salmo salar L.) macrophages by lipopolysaccharide. Fish & Shellfish Immunology, 5: 475-491.
43. Sorensen, U.B.S., Larsen, J.L. (1986): Serotyping of Vibrio anguillarum. Applied and Environmental Microbiology, 51(3): 593-597.
44. Swain, P., Nayak, S.K., Sahu, A., Meher, P.K., Mishra, B.K. (2003): High antigenic cross-reaction among the bacterial species responsible for diseases of cultured freshwater fishes and strategies to overcome it for specific serodiagnosis. Comperative Immunology, Microbiology & Infectious Diseases, 26: 199-211.
45. Tanrıkul, T.T. (2007): Vibriosis as an epizootic disease of rainbow trout (Onchorhynchus mykiss) in Turkey. Pakistan Journal of Biological Sciences, 10(10): 1733-1737.
46. Tinsley, J.W., Lyndon, A. R., Austin, B. (2011): Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhyncus mykiss (Walbaum). Journal of Applied Microbiology, 111:8-16.
47. Timur, G., Korun, J. (2004): First outbreak of vibriosis in farmed rainbow trout (Oncorhynchus mykiss) in Turkey. İ. Ü. Su Urunleri Fakultesi Dergisi, 18(18): 1-9.
48. Timur, G., Timur, M. (1991): An outbreak of enteric redmouth disease in farmed rainbow trout (O. mykiss) in Turkey. Bulletin of European Association of Fish Pathologists, 11(5): 182-183.
49. Timur, G., Timur, M. (2003). Balık Hastalıkları. İstanbul Üniversitesi Su Ürünleri Fakültesi, Yayın No. 5, İstanbul. 975-404-699-9.
50. Tsai, C.M., Frasch, C.E. (1982): A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Analytical Biochemistry, 119(1): 115-119.
51. Türkiye İstatistik Kurumu (TUİK) [Online]: İstatistiksel Tablolar ve Dinamik Sorgulama - Kültür Balıkları Üretim Miktarı tablosu, http://www.tuik.gov.tr/PreTablo.do?alt_id=1005 [Ziyaret Tarihi: 6 Kasım 2013]