traT and CNF2 genes of Escherichia coli isolated from milk of healthy cows and sheep M.N. Ac ßik, N.E. Yurdakul, L. C ß akici, N. Onat, € O. Dogan, B. C ß etinkaya * Department of Microbiology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey Accepted 7 January 2004 Abstract The objectives of the present study were to isolate Escherichia coli from milk of apparently healthy cows and sheep and to in- vestigate the presence of traT and cytotoxic necrotising factor-2 (CNF2) virulence genes by multiplex polymerase chain reaction (PCR). Milk samples collected from a total of 1028 apparently healthy ruminants (737 cows and 291 sheep) in eastern Turkey were streaked onto 5% sheep-blood agar. E. coli was isolated and identified by biochemical tests in 5.9% (61/1028) of milk samples. Correct amplification with the molecular length of 232 bp was obtained from all E. coli isolates by the species-specific PCR. The isolation rates of the agent were calculated to be 7.6% (56/737) in cows and 1.7% (5/291) in sheep. The difference between these proportions was statistically significant (p<0.001). Multiplex PCR showed that traT and CNF2 genes were present in 62.3% and 6.6% of all isolates, respectively. Both genes were present in 16.4% of the isolates, with only 14.7% having neither gene. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: E. coli; traT; CNF2; Milk; Cow; Sheep 1. Introduction Mastitis is the inflammation of mammary gland as- sociated mostly with microbial infections. The eradica- tion of mastitis in cows and sheep is difficult owing to its complex aetiology. It is of great economic significance particularly in the dairy industry worldwide, causing millions of dollars in losses due to reduced milk pro- duction, treatment costs, culling and death. An annual cost of approximately £170 million has been estimated in the dairy cow population of the UK due to clinical mastitis (Bradley, 2002). Losses associated with sub- clinical mastitis, which are difficult to estimate, should also be considered in order to have a better under- standing of the economical impact of mastitis, which has been reported to be responsible for nearly 40% of total costs of common production diseases (Kossaibati and Esslemont, 1997). It must be underlined that mastitis is important for public health too, as the massive use of antibiotics in the treatment of food producing animals may contribute to the emergence of resistant bacteria entering the food chain (White and McDermott, 2001). In recent years, the incidence and prevalence of contagious mastitis has been reduced due to strict con- trol programmes, but environmental mastitis continues to cause serious problems in the dairy industry throughout the world (Bradley, 2002). Escherichia coli is the main causative agent of environmental mastitis and has been reported to be responsible for 35% of clinical cases in bovine mastitis compared to 10% caused by contagious pathogens in the UK (Bradley and Green, 2001). In Israel, coliform mastitis has been reported to account for more than 60% of clinical mastitis (Shpigel et al., 1998). The increase in the incidence of E. coli associated mastitis is thought to be due to routine use of dry-cow therapy and to post-milking teat dipping which is considered as an effective method to reduce mastitis caused by Gram positive microbes but is not equally efficient against Gram negative bacteria including E. coli (Erskine and Eberhart, 1991; Fang and Pyorala, 1996). In contrast to the UK and Israel, the prevalence of en- vironmental mastitis associated with E. coli has been * Corresponding author. Tel.: +90-424-237-0000; fax: +90-424-238- 8173. E-mail address: bcetinkaya@firat.edu.tr (B. C ß etinkaya). 0034-5288/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2004.01.003 Research in Veterinary Science 77 (2004) 17–21 www.elsevier.com/locate/rvsc