Research carried out in Europe has shown the dominance of C. jejuni in animal intestinal tracts, for example, broiler chickens, cattle, and wild-living mammals and birds [2, 7, 8]. Pigs MAPK Inhibitor Library chemical structure are known to be frequently infected with HDAC activation Campylobacter (prevalence between 50% and 100%), to exhibit high counts of this pathogen in their faeces (ranging from 102 to 107 Colony Forming Units (CFU) of Campylobacter per gram), and to show a dominance of C. coli [9–11]. Nevertheless, some studies have found a dominance of

C. jejuni in pigs and of C. coli in chickens [12–15]. Given these contradictory data, the risk of foodborne disease associated with animal species is not clear. In terms of risk assessment, the ability to differentiate and quantify these two species is essential to describe more precisely the presence of Campylobacter in livestock animals. The identification of Campylobacter using conventional methods is slow (culture-based methods can take up to five days) and problematic due to their fastidious growth requirements and biochemical Akt inhibitor inertness [16, 17]. Moreover, the detection of C. coli and/or C. jejuni in complex substrates like faeces or environmental samples is difficult as the culture conditions have to be selective enough to avoid overgrowth from competiting organisms. Additionally these bacteria may enter into a viable but nonculturable state (VBNC) [18]. The correct differentiation

of thermophilic those Campylobacter spp., especially C. coli and C. jejuni, by phenotypic tests is difficult and hippurate hydrolysis test used to distinguish

these two species is often problematic [19]. Furthermore, C. jejuni may also coexist with C. coli in pigs, but at 10-100-fold lower numbers than C. coli [10, 11, 20], so C. jejuni will be less frequently isolated from such samples because only a few colonies are identified to the species level with conventional culturing and biochemical testing techniques. Molecular methods are an alternative to the bacteriological method for the detection of C. coli and C. jejuni in various substrates [1, 17, 21–24]. Real-time PCR has provided a reliable tool to detect and to quantify C. jejuni and/or C. coli in pure culture [25], in poultry, milk, or water [26, 27], and in complex substrates like food products [28–30] and faecal samples [20, 31–33]. However, of the real-time PCR techniques developed, none were capable of differentiating and quantifying C. coli and C. jejuni directly from pig faecal, feed, and environmental samples. The present study aimed to develop a species-specific real-time PCR method to detect and quantify C. coli and C. jejuni directly in pig faecal, feed, and environmental samples. The first step in the development of the assay was the definition of the multiplex PCR assay to quantify C. coli and C. jejuni isolates from bacterial cultures.