Hafize Tuğba YÜKSEL, Uğur PARIN, Şükrü KIRKAN, Erdem ÇİÇEK

Detection of Virulence Genes in Streptococcus uberis Isolated from Bovine Mastitis in Aydın Province by Multiplex Polymerase Chain Reaction

Sütçü ineklerde mastitise sebep olan etkenlerden olan Streptococcus uberis'in izolasyonu ve tür spesifik hedef genler ile identifikasyonu yapılarak, virulans genlerinin varlığı ve dağılımınının multipleks Polimeraz Zincir Reaksiyonuyla ortaya konması amaçlanmıştır. Araştırmada laboratuvara getirilen süt örneklerinden elde edilen izolatlar, koloni görünümü, Gram boyama ve katalaz testi ve biyokimyasal testler ile identifikasyonları sonucu S.uberis suşları ayrıldı ve moleküler çalışmalar için -20 °C'de saklandı. İzole edilen suşlardan DNA ekstraksiyonu, genomik DNA ekstraksiyon kiti (Fermentas®) ile prosedüre uygun olarak yapıldı. Fenotipik olarak S. uberis olduğu belirlenen tüm suşlarda 16S rRNA ve virulans ilişkili genlerin varlığı incelendi. İncelenen 200 süt örneğinin 35 (%17.5)'inden Streptococcus uberis izolasyonu gerçekleştirildi. Polimeraz zincir reaksiyonu ile, identifiye edilmiş S. uberis suşları incelendiğinde toplam 35 adet izolatın tamamında (%100) 1400 bp fragment aralığında16S rRNAgeni tespit edilmiştir. Virulans genleri tek olarak incelendiğinde, 32 (%91) suştan hasB, 32 (%91) suştan gapC, 32 (%91) suştan skc, 30 (%86) suştan cfu, 30 (%86) suştan hasC, 30 (%86) suştan hasA, 29 (%83) suştan sua, 28 (%80) suştan oppF, 25 (%71) pauA, 9 (%26) suştan Ibp geni identifiye edilmiştir. pauB genine ise rastlanmamıştır. Mastitis vakalarından izole edilen S. uberis etkenlerinin virulans genlerinin dağılımı incelendiğinde etkenin patojenitesi açısından kapsül formasyonunu eksprese eden genlerin önemli role sahip olduğu ortaya konulmuştur, ayrıca serin proteaz enzimi sentezinde görevli olan genlerin de dikkate değer oranda dağılım gösterdiği görülmüştür.

Aydın İlinde Bulunan Sığır Çiftliklerinde İzole Edilen Streptococcus uberis Kökenli Mastitislerin ve Virulens Genlerinin Multiplex Polimeraz Zincir Reaksiyonu ile Belirlenmesi

The objective of this study was to identify the presence and distribution of virulence genes via multiplex Polymerase Chain Reaction by identification of Streptococcus uberis, one of the agents which cause mastitis in dairy cows. S. uberis strains were identified from samples and stored at -20 °C deep freeze for molecular studies. DNA extraction of the isolated strains was performed according to the procedure with the genomic DNA extraction kit (Fermentas®). The presence of 16S rRNA and virulence-associated genes was examined in all strains identified as S. uberis phenotypically. S. uberis isolation was conducted in 35 (17.5%) of the 200 milk samples. After examination of the identified S. uberis strains by polymerase chain reaction, 16S rRNA gene was detected in a total of 35 isolates (100%) at 1400 bp fragment base pairs. When the virulence genes were examined individually, 32 (91%) strains expressed hasB, 32 (91%) strains expressed gapC, 32 (91%) strains expressed skc, 30 (86%) strains expressed cfu, 30 (86%) strains expressed hasC, 30 (86%) strains expressed hasA 29 (83%) strains expressed sua, 28 (80%) strains expressed oppF, 25 (71%) pauA, 9 (26%) strains expressed Ibp genes. The pauB gene was not identified. When the distribution of virulence genes of S. uberis strains isolated from mastitis cases are examined, it has been shown that the genes which exert capsular formation in terms of the pathogenity of the agent play important role and also the genes involved in the synthesis of serine protease enzymes are distributed in significant proportion.

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