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

Kaynakça

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