Prevalence of shigatoxigenic Escherichia coli in bovine cattle: a survey in the north region of portugal

Abstract

Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens that can be transmitted to humans through many different routes, but mainly through consumption of contaminated foods. STEC strains are characterized by the production of Shiga toxins (Stx) that are encoded by two genes stx1 and/or stx2. Each toxin can be subdivided into subtypes and, currently, there are three known subtypes for stx1 (stx1a, stx1c, and stx1d) and seven for stx2 (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g). Others virulence factors, such the production of intimin protein, encoded by eae gene, are associated with severe outcomes of STEC-associated diseases. The most common STEC serotype implicated worldwide is E. coli O157:H7, but many other STEC strains are associated with severe human diseases. Ruminants, especially cattle, are a major reservoir for O157 and non-O157 STEC. Information on STEC prevalence in Portuguese dairy cattle is limited, so we analysed for the presence of STEC 329 faecal specimens collected from the rectum of healthy dairy cattle. The samples were collected from adults lactating cows (n=194) and from heifers (n=135) with ages among 6 to 18 months, between March and June 2019, at milk farms (n=17) in the North region of Portugal. After enrichment, in modified TSB with novobiocin, samples were analysed by real time PCR to detect the presence of stx1, stx2 and eae in accordance to ISO/TS 13136:2012(E). In addition, conventional PCR for the detection of sxt1 and stx2 gene subtypes was performed according to the guidelines of the VTEC European Union Reference Laboratory. A total of 139 isolates were recovered from 108 positive animals (dairy cows and heifers). The STEC prevalence was significantly higher in heifers (68/135; 50.4%) than in adult cows (40/194; 20.6%) (p < 0.05, Fisher exact test statistic value is <0.00001). STEC harbouring only stx2 (67/139; 48.2%) were the most common strains, followed by both genes stx1 and stx2 (40/139, 28.8%) and stx1 (32/139, 23%). Of the 139 STEC isolates, 35.3% have also the eae gene. Subtyping of stx1 (72) showed that stx1a was the most prevalent (100%), followed by stx1c (88.9%) and stx1d (6.9%). Stx1a and stx1c subtypes occurred alone or combination with other subtypes. However, subtype stx1d was only found in combination with subtype stx1a and or stx1c. Concerning strains with stx2 (107), the stx2a subtype was the most common and was found in 84.1% of the isolates, followed by stx2d (76.6%), stx2c (74.8%), stx2g (23.4%), stx2b (4.7%), stx2e (3.7%) and stx2f (0.9%). No strains carried stx2b or stx2f alone. For isolates carrying stx1 and stx2 simultaneously, combinations of subtypes stx1a, stx1c, stx2a, stx2c and stx2d were the most common. Furthermore, in 42 isolates (30.2%) 4 or more stx subtypes were detected simultaneously. These results show that STEC prevalence in dairy cattle is high, and that most isolates present a diverse combination of Shiga-toxin genes. Future strategies are needed to mitigate the presence of STEC in cattle and then reduce the possible contamination of food and, thus, humans.