Short Communication Coagulase gene polymorphisms detected by PCR in Staphylococcus aureus isolated from subclinical bovine mastitis in Turkey Murat Karahan, Burhan C ¸ etinkaya * Department of Microbiology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey Accepted 27 May 2006 Abstract The genetic relatedness of coagulase (coa) positive Staphylococcus aureus isolated from cows with subclinical mastitis in Turkey was investigated by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Among 700 milk samples positive in the California Mastitis Test (CMT), species specific PCR identified 200 (28.6%) isolates as S. aureus and 161 (80.5%) of these isolates were positive for the 3 0 end of the coa gene by PCR. Most isolates (n = 135, 83.9%) produced a single band on coa PCR, with molecular sizes ranging from 500 to 1400 bp, whereas a small number of isolates (n = 26, 16.1%) yielded two ampli- fication products. Coa RFLP analysis using AluI and Hin6I revealed 23 and 22 band patterns, respectively. The detection of double bands by coa PCR, previously reported in human isolates, suggests that milking personnel can play a role in the transmission of S. aureus. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Staphylococcus aureus; Cattle; Mastitis; coa gene; PCR-RFLP Bovine mastitis is responsible for reduced milk produc- tion, decreased milk quality and increased labour costs, resulting in significant economic losses worldwide (Beck et al., 1992). Subclinical and clinical Staphylococcus aureus mastitis is recognised as one of the most important diseases affecting the dairy industry (Bramley, 1992). Until recently, conventional methods, such as phage typing, serotyping, biotyping and antimicrobial susceptibility testing, have fre- quently been used to demonstrate epidemiological types of S. aureus (Lange et al., 1999; Su et al., 2000). However, the discriminatory power of these tests is low and more recent molecular methods, such as random amplified polymorphic DNA, ribotyping, multilocus enzyme electrophoresis, plas- mid profiling and coagulase gene polymorphism (coa RFLP) have improved the typing of S. aureus strains (Ten- over et al., 1994). Guler et al. (2005) previously typed coa genes of S. aur- eus isolates from bovine mastitis in the Central Anatolia region of Turkey. The aim of the present study was to iden- tify S. aureus in milk samples from dairy herds in the East and South-east of Turkey and to characterise coagulase positive isolates using coa RFLP. Milk samples were collected from 700 randomly-cho- sen California Mastitis Test (CMT)-positive cows, with- out clinical evidence of mastitis, in 32 locations in the East and South-east of Turkey. Approximately 10 mL of milk from one quarter of each cow were collected aseptically. Milk samples (100 lL) were inoculated onto Blood Agar Base (Merck) supplemented with 7% defi- brinated sheep blood. After aerobic incubation for 48 h at 37 °C, colonies yielding Gram-positive cocci were sub- jected to routine biochemical tests (i.e. coagulase, cata- lase, oxidase, oxidation–fermentation) for further identification. Isolates identified biochemically as Staphy- lococcus spp. were stored in Trypticase Soy Broth (Merck) containing 15% glycerol at À20 °C until used for polymerase chain reaction (PCR). Each isolate was subcultured overnight on Trypticase Soy Agar (Merck) before DNA extraction. 1090-0233/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2006.05.016 * Corresponding author. Tel.: +90 424 237 0000; fax: +90 424 238 8173. E-mail address: bcetinkaya@firat.edu.tr (B. C ¸ etinkaya). www.elsevier.com/locate/tvjl The Veterinary Journal 174 (2007) 428–431 The Veterinary Journal