Identification of Phenotypic and Genotypic Characteristics Diversity of Isolated Flavobacterium psychrophilum in Rainbow Trout Farms in Mediterranean Region

Abstract

In this study, it was aimed to investigate the phenotypic and genotypic characteristics with presence of Flavobacterium psychrophilum in fourteen rainbow trout farms in Mediterranean Region. For this purpose, sperm, eyed egg, egg, yolk-sac larvae and 0,2-5 g fry were sampled from commercial trout farms between November 2012- March 2013. Isolation of potential F.psychrophilum from samples was made using conventional microbiological methods and a total of 172 obtained strains. Enzyme activity of all isolates was identified according to the API ZYM test system. As a result of the phenotypic tests, 65 isolates thought to be F. psychrophilum were identified. PCR assay was used in molecular confirming of these strains. It was determined that, 17 strains isolated from diseased fish (11 of them liver, 3 of them spleen, 3 of them kidney) gave specific 971 bp band which was specific to F.psychrophilum. RAPD-PCR assay was used to identify the clonal relationship between strains. F. psychrophilum isolates were yielded five different RAPD band patterns (F1-F5) according to this method. This strains detected to be grouped into 1 cluster and according to similarity coefficient %85 "unique" type (F1 the genotype). According to antibiotic sensitivity tests, isolates were found to be sensitive to trimetroprim/sulfamethoxazole, clindamycin, ampicillin, tetracycline, enrofloxacin, chloramphenicol, oxytetracycline, florfenicol and tobramycine. In conclusion, the findings from this study showed that the M13 primer used in RAPD-PCR can be used to determine the clonal relationship between F. psychrophilum isolates 

Keywords

• Oncorhynchus mykiss,Flavobacterium psychrophilum,identification,API ZYM,PCR

Ege ve Akdeniz Bölgesindeki Gökkuşağı Alabalıklarından İzole Edilen Flavobacterium psychrophilum Suşlarının Fenotipik Ve Genetik Farklılıklarının Belirlenmesi

Öz

Bu çalışma ile Akdeniz bölgesinde bulunan 14 farklı gökkuşağı alabalığı (Oncorhynchus mykiss Walbaum, 1792) işletmesinde Flavobacterium psychrophilum’un varlığı ile fenotipik ve genotipik özelliklerinin araştırılması hedeflenmiştir. Bu amaçla Kasım 2012-Mart 2013 tarihleri arasında ticari alabalık işletmelerinden sperm, yumurta, gözlenmiş yumurta, keseli larva ve 0.2-5 gr ağırlığındaki larvalardan örnekler alınmıştır. Örneklerden olası F. pyschrophilum’un izolasyonu konvansiyonel mikrobiyolojik metotlar kullanılarak yapılmış ve toplamda 172 adet izolat elde edilmiştir. Elde edilen tüm izolatların enzim aktivitesi API ZYM test sistemine göre tespit edilmiştir. Yapılan fenotipik testler sonucunda F. psychrophilum olduğu düşünülen 65 izolat belirlenmiştir. Bu izolatların moleküler olarak doğrulanmasında F. pyschrophilum spesifik Polimeraz Zincir Reaksiyonu (PCR) metodu kullanılmıştır. Hasta balıklardan izole edilen 17 izolatın (11 karaciğer, 3 dalak ve 3 böbrek) F. psychrophilum’a özgü 971 bp’lik bant verdiği saptanmıştır. İzolatlar arası klonal ilişkinin belirlenmesi amacıyla RAPD-PCR metodu kullanılmıştır. Bu metoda göre F. psychrophilum izolatlarının beş farklı RAPD bant paterni (F1-F5) verdiği saptanmıştır. İzolatların % 85 benzerlik katsayısına göre bir “unique” tip (F1 nolu genotip) ve bir küme içerisinde gruplandığı belirlenmiştir. Yapılan antibiyogram testi sonucu, izolatların trimetroprim/sulfametakzol, klindamisine, ampisillin, tekrasiklin, enrofloksasin, kloramfenikole, oksitetrasiklin, florfenikol ve tobramisin’e duyarlı olduğu belirlenmiştir. Sonuç olarak, bu çalışmadan elde edilen bulgular RAPD-PCR'da kullanılan M13 primerinin F. psychrophilum izolatları arasındaki klonal ilişkinin belirlenmesinde kullanılabilir olduğunu göstermiştir

Anahtar Kelimeler

• Oncorhynchus mykiss,Flavobacterium psychrophilum,identifikasyon,API ZYM,PCR

References

1. Austin, B., Austin, D.A. (2007).Bacterial Fish Pathogens: Disease in Farmed and Wild Fish. 4th ed., Springer-Praxis publishing, Chichester, p. 594, UK.Balta, F. (1997). Kültürü yapılan alabalıklarda (Oncorhyncus mykiss) görülen Flexibacter psychrophila enfeksiyonu. IX. Ulusal Su Ürünleri Sempozyumu, 19 Eylül 1997, Cilt II Eğirdir-ISPARTA, 621-648.
2. Bauer, A.W., Kirby, W.M., Sherris, J.C., & Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. American journal of clinical pathology, 45 (4), 493-496.
3. Beaz-Hidalgo, R., Lopez-Romalde, S., Toranzo, A.E., & Romalde, J.L. (2008). Polymerase Chain Reaction Amplification of Repetitive Intergenic Consensus and Repetitive Extragenic Palindromic Sequences for Molecular Typing of Pseudomonas anguilliseptica and Aeromonas salmonicida. Journal of aquatic animal health, 20(2),75–85.
4. Boyacıoğlu, M. (2007). Gökkuşağı alabalıklarında (Oncorhynchus mykiss) RTFS’ye (Rainbow Trout Fry Syndrome) neden olan Flavobacterıum psychrophilum etkeninin izolasyonu ve antibakteriyel sağaltım seçeneğinin belirlenmesi. T.C. Adnan Menderes Üniversitesi, Sağlık Bilimleri Enstitüsü, Farmakoloji ve Toksikoloji Anabilim Dalı, AYDIN.
5. Bruun, M. S., Madsen, L., & Dalsgaard, I. (2003). Efficiency of oxytetracycline treatment in rainbow trout experimentally infected with Flavobacterium psychrophilum strains having different in vitro antibiotic susceptibilities. Aquaculture, 215(1-4), 11-20.
6. Chen, Y. C., Davis, M. A., Lapatra, S. E., Cain, K. D., Snekvik, K. R., & Call, D. R. (2008). Genetic diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow trout, Oncorhynchus mykiss (Walbaum), and spawning coho salmon, O. kisutch (Walbaum). Journal of fish diseases, 31(10), 765-773.
7. Cipriano, R. C., & Holt, R. A. (2005). Flavobacterium psychrophilum, cause of bacterial cold-water disease and rainbow trout fry syndrome. US Department of the Interior, US Geological Survey, National Fish Health Research Laboratory.
8. Çağırgan, H., Tanrıkul, T. T., & Balta, F. (1997, September). Characteristics of yellow pigmented bacteria isolated from diseased rainbow trout (Oncorhyncus mykiss). In Eighth International Conference Diaseases of Fish and Shell Fish (pp. 73-81).

9. Del Cerro, A., Márquez, I., & Prieto, J. M. (2010). Genetic diversity and antimicrobial resistance of Flavobacterium psychrophilum isolated from cultured rainbow trout, Onchorynchus mykiss (Walbaum), in Spain. Journal of fish diseases, 33(4), 285-291.
10. Didinen, B. I., Diler, Ö., Ekici, S., & Altun, S. (2007). Flavobacterium psychrophilum izolatlarının teşhisinde API ZYM kullanımı ve ATB VET ile antimikrobiyal duyarlılığın belirlenmesi. Süleyman Demirel Üniversitesi Eğirdir Su Ürünleri Fakültesi Dergisi, 1(2), 62-68.
11. Diler, Ö., Altun, S., & Işıklı, B. I. (2003). Kültürü yapılan gökkuşağı alabalıkları (Oncorhynchus mykiss)’ndan izole edilen Flavobacterium psychrophilum’un fenotipik karakterleri. Süleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 7(1), 1-8.
12. Durmaz, Y., Onuk, E. E., & Ciftci, A. (2012). Investigation of the presence and antibiotic susceptibilities of Flavobacterium psychrophilum in rainbow trout farms (Oncorhynchus mykiss Walbaum, 1792) in The Middle and Eastern Black Sea Regions of Turkey. Ankara Universitesi Veteriner Fakültesi Dergisi, 59, 141-146.
13. Hesami, S., Allen, K. J., Metcalf, D., Ostland, V. E., MacInnes, J. I., & Lumsden, J.S. (2008). Phenotypic and genotypic analysis of Flavobacterium psychrophilum isolates from Ontario salmonids with bacterial coldwater disease. Canadian journal of microbiology, 54(8), 619-629.
14. İspir, Ü., Şeker, E., Sağlam, N., & Dörücü, M. (2004). Doğu Anadolu bölgesinde bazı gökkuşağı alabalığı (Oncorhynchus mykiss) işletmelerinde görülen Flavobacterium psychrophilum enfeksiyonunun araştırılması. FıratÜniversitesi Fen ve Mühendislik Bilimleri Dergisi, 16(4), 718-724.
15. Korun, J., & Timur, G. (2001). Gökkuşağı alabalıklarında (Oncorhyncus mykiss) fry mortalite sendromu (FMS) üzerinde bir çalışma. İstanbul Üniversitesi Su Ürünleri Dergisi, 12, 15-30.
16. Kubilay, A., Altun, S., Didinen, B. I., Ekici, S., & Diler, O. (2009). Gökkuşağı alabalığı (Oncorhynchus mykiss) işletmelerinde Flavobacterium psychrophilum izolasyonu. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 15(5), 709-715.
17. Lorenzen, E., Dalsgaard, I., & Bernardet, J. F. (1997). Characterization of isolates of Flavobacterium psychrophilum associated with coldwater disease or rainbow trout fry syndrome I: phenotypic and genomic studies. Diseases of aquatic organisms, 31(3), 197-208.
18. Lorenzen, E., Karas, N. (1992). Detection of Flexibacter psychrophilus by immunofluorescence in fish suffering from fry mortality syndrome: a rapid diagnostic method. Diseases aquatic organisims, 13, 231-234.
19. Madetoja, J., Hänninen, M. L., Hirvelä–Koski, V., Dalsgaard, I., & Wiklund, T. (2001). Phenotypic and genotypic characterization of Flavobacterium psychrophilum from Finnish fish farms. Journal of Fish Diseases, 24(8), 469-479.
20. Madetoja, J., & Wiklund, T. (2002). Detection of the fish pathogen Flavobacterium psychrophilum in water from fish farms. Systematic and applied microbiology, 25(2), 259-266.
21. Madsen, L., & Dalsgaard, I. (2000). Comparative studies of Danish Flavobacterium psychrophilum isolates: ribotypes, plasmid profiles, serotypes and virulence. Journal of fish diseases, 23(3), 211-218.
22. Madsen, L., & Dalsgaard, I. (1999). Reproducible methods for experimental infection with Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss. Diseases of aquatic organisms, 36(3), 169-176.
23. Magariños, B., Toranzo, A. E., Barja, J. L., & Romalde, J. L. (2000). Existence of two geographically-linked clonal lineages in the bacterial fish pathogen Photobacterium damselae subsp. piscicida evidenced by random amplified polymorphic DNA analysis. Epidemiology & infection, 125(1), 213-219.
24. Mancuso, M., Avendaño-Herrera, R., Zaccone, R., Toranzo, A. E., & Magariños, B. (2007). Evaluation of different DNA-based fingerprinting methods for typing Photobacterium damselae ssp. piscicida. Biological research, 40(1), 85-92.
25. National Committee for Clinical Laboratory Standards, (1999). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. National Committee for Clinical Laboratory Standards, Wayne, Pennsylvania, Approved Standard M 31-A19 (11).
26. Nematollahi, A., Decostere, A., Pasmans, F., & Haesebrouck, F. (2003). Flavobacterium psychrophilum infections in salmonid fish. Journal of fish diseases, 26(10), 563-574.
27. Onuk, E.E., Çiftçi, A., Fındık, A., Çiftçi, G., Altun, S., Balta, F., Özer, S., & Çoban, A.Y. (2011). Phenotypic and molecular characterization of Yersinia ruckeri isolates from rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) in Turkey. Berliner und münchener tierärztliche wochenschrift, 124 (7-8), 320-328.
28. Ostland, V. E., McGrogan, D. G., & Ferguson, H. W. (1997). Cephalic osteochondritis and necrotic scleritis in intensively reared salmonids associated with Flexibacter psychrophilus. Journal of fish diseases, 20(6), 443-451.
29. Özcan, M., Sarıeyyüpoğlu, M. (2013). Elazığ ilindeki bazı alabalık işletmelerinde izole edilen Flavobacterium psychrophilum'un antibakteriyel duyarlılıklarının incelenmesi. Yunus araştırma bülteni, 2, 11-19
30. Rangdale, R.E. & Way, K. (1995). Rapid identification of C. psychrophila from infected spleen tissue using an enzyme-linked immunosorbent assay (ELISA). Bulletin european association of fish pathologists, 15, 213-216.
31. Ravelo, C., Magarinos, B., López-Romalde, S., Toranzo, A. E., & Romalde, J. L. (2003). Molecular fingerprinting of fish-pathogenic Lactococcus garvieae strains by random amplified polymorphic DNA analysis. Journal of clinical microbiology, 41(2), 751-756.
32. Romalde, J. L., Magariños, B., Villar, C., Barja, J. L., & Toranzo, A. E. (1999). Genetic analysis of turbot pathogenic Streptococcus parauberis strains by ribotyping and random amplified polymorphic DNA. FEMS microbiology letters, 179(2), 297-304.
33. Valdebenito, S., & Avendaño‐Herrera, R. (2009). Phenotypic, serological and genetic characterization of Flavobacterium psychrophilum strains isolated from salmonids in Chile. Journal of fish diseases, 32(4), 321-333.
34. Versalovic, J., Koeuth, T., & Lupski, R. (1991). Distribution of repetitive DNA sequences in eubacteria and application to finerpriting of bacterial enomes. Nucleic acids research, 19(24), 6823-6831.
35. Wiklund, T., Madsen, L., Bruun, M. S., & Dalsgaard, I. (2000). Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification. Journal of applied microbiology, 88(2), 299-307.