108 Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014 www.world-food.net Journal of Food, Agriculture & Environment Vol.12 (3&4): 108-114. 2014 WFL Publisher Science and Technology Meri-Rastilantie 3 B, FI-00980 Helsinki, Finland e-mail: info@world-food.net Shigatoxin-producing Escherichia coli in raw cow milk from small farm producers and phylogenetic subtype determination Ivo Sirakov, Ralitsa Popova, Hristo Daskalov *, Iskra Slavcheva, Eva Gyurova and Boyko Mitov National Diagnostic and Research Veterinary Institute, Blvd. Pencho Slaveykov 15, 1606 Sofia, Bulgaria. *e-mail: hdaskal@hotmail.com Received 30 April 2014, accepted 10 September 2014. Abstract Cases of unauthorised direct sale of raw cow milk from small farms do exist in Bulgaria. In 2012, we tested 80 samples of raw cow’s milk from small farms in south-western Bulgaria for the presence of shigatoxin-producing Escherichia coli strains. Twenty-three of these samples were taken from cows with subclinical mastitis. The tests included isolation on selective solid medium, biochemical detection and conventional PCR for detection of the shigatoxin genes (stx1, stx2), the intimin gene (eae) and the enterohemolysin gene (hlyA). The stx1 gene was detected in three Escherichia coli isolates from three cow’s milk samples from animals with subclinical mastitis. An Escherichia coli strain with the intimin eae gene was isolated from a sample of normal cow’s milk from a small farm. Two of the isolates with the stx1 gene were also found to harbour the enterohemolysin hlyA gene. The presence of virulence factors in the four Escherichia coli isolates was additionally validated using Real-time PCR. The amplified virulence genes were sequenced and a phylogenetic analysis based on amino acid sequences was performed. The cytotoxic effects of the E. coli isolates were studied on Vero cells. The potential role of shigatoxin-producing E.coli strains for provoking food-borne illness is discussed. Key words: Escherichia coli, raw cow milk, mastitis milk, PCR, virulence factors. Introduction Raw milk produced in very small farms which do not meet the EU quality and safety requirements could be a risk factor for consumer health. Raw cow’s milk from such farms is on the market in violation of the regulations for the sale of raw milk. There is also a hazardous practice of mixing milk from healthy animals and subclinical mastitis milk. Escherichia coli is a microbial agent which is often found in raw milk, especially in cases of poor hygiene during milking and primary processing. E. coli causes mastitis or concomitant infections in lactating cows. According to Schoonderwoer 1 , E. coli is naturally found in the gastrointestinal tract in animals, but some strains, such as shiga-toxin-producing E. coli, are associated with diarrhea in humans and animals. Other authors 2, 3 emphasise that verotoxin-producing E. coli (VTEC) can cause serious illness in humans, beginning with mild diarrhea and leading to hemorrhagic colitis and hemolytic uremic syndrome. Mainil 4 point out that a key factor for E.coli infections in humans are ruminants excreting E.coli in their feces. VTEC may be transmitted through consumption of undercooked meat, unpasteurised milk products, vegetables and water contaminated with the feces of carrier animals. Cases of person-to-person transmission are also known 3, 5, 6 . Animals intended for food are considered to be the main source of VTEC/ EPEC (enteropathogenic E.coli) strains. In humans, life-threatening infections associated with consumption of milk and milk products contaminated with VTEC/EPEC have been reported 7 . The main etiological agent in infections with enterohemorrhagic E.coli is generally thought to be Escherichia coli O157:H7. It is still unclear, which other factors in addition to verotoxin production are involved in the transformation of shigatoxin-producing-E.coli (VTEC) into enterohemorrhagic E. coli (EHEC). Each VTEC isolate should be considered potential EHEC and the detection methods should be targeted at detection of verotoxin rather than of the serotype 8 . Another key factor in the virulence of VTEC isolates is enterohemolysin (Ehly), which is coded by the enterohemolysin ehxA gene, also known as EHEC-HlyA. The relation of EHEC hemolysin and verotoxin production in pathogenic E.coli describes the possible role of EHEC hemolysin in bacterial virulence 9 . Subclinical mastitis caused by E. coli brings about substantial economic losses in dairy cattle herds because of reduced amount and quality of the produced milk, incurred veterinary treatment costs and in some cases death of the diseased animal 10 . Milk can be contaminated with pathogenic microorganisms through fecal contamination as well as from colonized teats or from an infected udder in the case of clinical or subclinical mastitis during the milking process from the environment or from contaminated water 11 . Polymerase chain reaction (PCR) is an effective method for detection of extremely low quantities of nucleic acids in various types of samples. That is why PCR is a common technique used for detection of various food-borne pathogens. The aim of the present study was to analyse samples of raw cow’s milk, including milk taken from cows with subclinical mastitis, for the presence of E. coli isolates with specific virulence markers (stx1, stx2 and eae) by means of PCR and to evaluate their possible role in provoking food-borne illness in consumers. Materials and Methods A total of 80 raw cow milk samples were analysed. The milk was collected from January to December 2012 from unauthorised sources (small farms or individual sellers).