Mannheimia haemolytica serotip A1, A2, A6'nın Real Time PCR ile belirlenmesi, BOX ve RAPD PCR Yöntemleri ile Moleküler Karakterizasyonu

Öz

Bu çalışmada, koyunlardan elde edilen Mannheimia (M.) haemolytica izolatlarında serotip A1, A2 ve A6’nın Real-Time PCR yöntemiyle tespiti, ayrıca RAPD ve BOX PCR yöntemleri ile de izolatların genotiplendirilmesi amaçlandı. Araştırmada, Van YYÜ Veteriner Fakültesi Mikrobiyoloji Anabilim Dalı kültür koleksiyonunda bulunan ve koyunlardan izole edilen 44 adet M. haemolytica izolatı kullanıldı. Real-Time PCR yöntemiyle 44 adet M. haemolytica izolatının 27 (%61.4)’si serotiplendirilirken, 17 (%38.6) izolat serotiplendirilemedi. İzolatların %22.2’si serotip A1, %44.4’ü serotip A2, %33.4’ü de serotip A6 olarak belirlendi. RAPD PCR ile yapılan genotiplendirmede, OPA 4 primeri ile 18 farklı genotip, OPA 9 primeriyle 3 farklı genotip belirlenirken, BOX PCR yöntemiyle yapılan genotiplendirmede, izolatlarda 15 faklı genotip bulunduğu tespit edildi. Çalışmada, M. haemolytica serotipleri (A1, A2, A6) ile genotipler arasında istatistiksel olarak anlamlı bir ilişki olmadığı, serotip A1, A2 ve A6 izolatlarının moleküler identifikastonu için bu araştırmada uygulanan Real-Time PCR yönteminin uygun olduğu gözlendi. İzolatlar arasında RAPD PCR ile genotiplendirme için OPA 9 primerinin uygun olmadığı, OPA 4 primeri ve BOX PCR BOXA1R primerlerinin epidemiyolojik çalışmalarda genotiplendirme için kullanılabileceği, bunlardan BOXA1R primeri ile daha uygun bant profili elde edilebildiği tespit edildi. M. haemolytica kaynaklı pnömoni vakalarına yönelik yapılacak koruma-kontrol programlarında, bölgesel-ulusal düzeyde yaygın olan serotip ve genotiplerin değişken olması dolayısıyla yapılacak yeni çalışmalarda etkin koruma-kontrol sağlanabilmesi için yaygın serotip ve genotiplerin tespitinin epidemiyolojik açıdan önemli olduğu, bunun yanı sıra yapılacak aşı geliştirme çalışmalarında yaygın olan genotip ve serotiplerin dikkate alınarak hazırlanması gerektiği sonucuna varıldı.

Anahtar Kelimeler

• Real Time PCR
• Serotip
• Genotip

Destekleyen Kurum

Van Yüzüncü Yıl Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü

Proje Numarası

TYD-2021-9453

Etik Beyan

Bu çalışma, Van Yüzüncü Yıl Üniversitesi Hayvan Araştırmaları Yerel Etik Kurulu'nun (Karar No: 2025/10-17, 25 Eylül 2025 tarihli) yönetmeliklerine uygun olarak ölü hayvanlar veya hayvansal kaynaklı materyallerin kullanıldığı prosedürleri içerdiğinden, Hayvan Araştırmaları Etik Kurulu'nun onayı gerekmemiştir.

Teşekkür

TYD-2021-9453 numaralı projeye sağladıkları mali destekten dolayı Van Yüzüncü Yıl Üniversitesi Bilimsel Araştırma Projeleri (BAP) Koordinatörlüğüne teşekkür ederiz.

Determination of Serotypes A1, A2, and A6 in Mannheimia haemolytica Isolates by Real-Time PCR and Molecular Characterization by BOX and RAPD PCR Methods

Öz

In this study, the detection of serotypes A1, A2, and A6 in Mannheimia haemolytica isolates obtained from sheep was investigated using the Real-Time PCR method, and genotyping of the isolates was performed using RAPD and BOX PCR methods. A total of 44 Mannheimia haemolytica isolates isolated from sheep and preserved in the culture collection of the Department of Microbiology, Faculty of Veterinary Medicine, Van Yüzüncü Yıl University, were included. Using Real-Time PCR, 27 (61.4%) of the 44 isolates were successfully serotyped, while 17 (38.6%) could not be serotyped. Of the serotyped isolates, 22.2% were identified as serotype A1, 44.4% as serotype A2, and 33.4% as serotype A6. Genotyping by RAPD PCR revealed 18 different genotypes using the OPA 4 primer and three different genotypes using the OPA 9 primer, whereas BOX PCR analysis identified 15 different genotypes. No statistically significant association was found between Mannheimia haemolytica serotypes (A1, A2, A6) and genotypes. The Real-Time PCR method used in this study was found to be suitable for the molecular identification of these serotypes. For RAPD PCR genotyping, the OPA 9 primer was determined to be unsuitable, while the OPA 4 primer and BOX PCR BOXA1R primer were appropriate for epidemiological studies, with the BOXA1R primer providing clearer band profiles. In conclusion, serotype A2 was identified as the dominant serotype in the Van Province sheep population, followed by serotype A6, both of which should be prioritized in future regional vaccine formulations targeting ovine respiratory disease in eastern Turkey. Among the genotyping methods evaluated, BOX-PCR is recommended for routine epidemiological investigations. Identifying the prevalent serotypes and genotypes at regional and national levels is therefore of critical epidemiological importance for the development of effective prevention, control, and vaccination programs against Mannheimia haemolytica-associated pneumonia in small ruminants.

Anahtar Kelimeler

• Real-Time PCR
• genotype
• serotype

Destekleyen Kurum

Van Yuzuncu Yil University

Proje Numarası

TYD-2021-9453

Etik Beyan

The present study did not require approval from an Animal Research Ethics Committee, as it involved procedures using dead animals or animal-derived materials in accordance with the regulations of the Van Yüzüncü Yıl University Animal Researches Local Ethics Committee (Decision No: 2025/10-17, dated September 25, 2025).

Teşekkür

We gratefully acknowledge the financial support provided by the Scientific Research Projects (BAP) Coordination Unit of Van Yüzüncü Yıl University for the project TYD-2021-9453.

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