H.Hüseyin HADİMLİ, Kürşat KAV

The Molecular characterization of Arcanobacterium pyogenes strains isolated from samples of sheep and cattle

Bu çalışmanın amacı, Arcanobacterium pyogenes saha izolatlatlarının karakterizasyonu için virulens genlerinin varlığını belirlemek, tiplendirmek ve klonal tipleri, virülens genleri ve hastalık oluşumu arasındaki ilişkiyi incelemektir. Bu çalışmada, farklı örneklerden izole edilen A. pyogenes izolatı (51) kullanıldı: koyun (n=4) ve sığır (n=25) apseli karaciğerlerinden, süt ineklerine ait akciğerler (n=5), bronko-alveolar yıkantılardan (n=3), mastitisli sütlerden (n=12), suppuratif doku örneğinden (n=1) ve buzağı eklem sıvısından (n=1). Bütün A. pyogenes izolatlarında, tavşan, koyun, sığır, tavuk, köpek ve insan ertitrositleri ile hemolitik aktivite varlığı belirlendi. Aynı zamanda, vero hücre kültüründe sitotoksik etkileri incelendi. Klonal analiz için genomik DNA parmak izi BOX-PCR tiplendirme ile yapıldı. Sekiz virulens faktör genlerinin varlığının değerlendirilmesi için klasik PCR kullanıldı: nanH (nöraminidaz H), nanP (nöraminidaz P), plo (pyolysin-PLO), cbpA (kollejen bağlayan protein A) ve fimA, fimC, fimE, fimG (4 farklı fimribanın önemli fimbrial yapılar). Aynı zamanda, klonal tipleri, virulens faktörleri ve hastalık varlığı arasında bir ilişki incelendi. Bütün suşların hemolizinleri, tavşan, koyun, sığır, tavuk, köpek ve insan eritrositlerine hemolitik etkisine sahipti. Ayrıca, bütün suşların vero hücresine sitotoksik olduğu bulundu. Klonal analiz sonuçlarına göre; A. pyogenes izolatlarının 12 farklı tipte olduğu belirlendi. Bütün suşlar plo geni pozitif iken, nanH geni %62, nanP geni %84, cbp geni %58, fimA geni %96, fimC geni %66, fimE geni %42 ve fimG geni %10’du. Klonal tipler, virulens faktörler ve hastalık varlığı için A. pyogenes izolatları arasında bir ilişki bulunmadı.

Anahtar Kelimeler:

hemolisin, koyun, sığır, Arcanobacterium pyogenes, virülans, genler, gen ifadesi, alyuvarlar, DNA parmakizi, DNA, teşhis, süt ineği, süt sığırı, sitotoksisite, buzağı, inek, doku dağılımı, bronkoalveoler lavaj, bağlayıcı proteinler, bakteriyel hastalıklar, eklem sıvısı, sialidaz, süt, akciğerler, karaciğer, kan yıkımı

Koyun ve sığırlardan izole edilen Arcanobacterium pyogenes suşlarının moleküler karakterizasyonu

The purpose of this study was to determine the presence of virulence genes, perform typing for the characterization of Arcanobacterium pyogenes field strains, and investigate the correlation between clonal types, virulence genes and occurence of disease. The isolates (n=51) used in this study were isolated from different sources (liver (n=4) of sheep; liver (n=25), lungs (n=5), broncho-alveolar lavage (BAL) fluid (n=3), milk (n=12) and suppurative tissue (n=1) of dairy cows and synovial fluid (n=1) of a calf). The presence of haemolytic activity in A. pyogenes isolates was determined using rabbit, sheep, cattle, chicken, dog and human erythrocytes. Also, the presence of any cytotoxic effect was investigated by growth in Vero cell cultures. Genomic DNA fingerprinting for clonal analysis was generated by BOX-PCR typing. Conventional PCR was used for the determination of the presence of eight A. pyogenes virulence factor genes, namely, nanH (encoding neuraminidase H), nanP (encoding neuraminidase P), plo (encoding pyolysin-PLO), cbpA (encoding collagen-binding protein A) and fimA, fimC, fimE and fimG (encoding the major fimbrial subunit of four different fimbriae). Furthermore, the correlation between clonal types, virulence factors and the occurence of disease was investigated. The haemolysins of all strains had haemolytic effect on rabbit, sheep, cattle, chicken, dog and human erythrocytes. In addition, all strains were found to be cytotoxic to Vero cells. According to clonal analysis results, the A. pyogenes isolates were determined to belong to 12 different types. While all A. pyogenes strains were positive for the plo gene, the positivity rate was 62% for the nanH gene, 84% for the nanP gene, 58% for the cbp gene, 96% for the fimA gene, 66% for the fimC gene, 42% for the fimE gene and 10% for the fimG gene. It was determined that no correlation existed between the clonal types and virulence factors of A. pyogenes isolates and occurence of disease.

Keywords:

haemolysins, sheep, cattle, Arcanobacterium pyogenes, virulence, genes, gene expression, erythrocytes, DNA fingerprinting, DNA, diagnosis, dairy cows, dairy cattle, cytotoxicity, calves, cows, tissue distribution, bronchoalveolar lavage, binding proteins, bacterial diseases, synovial fluid, sialidase, milk, lungs, liver, haemolysis,

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