Detecting enterotoxigenic Escherichia coli in animal production: method development and validation

Abstract

Swine enteric colibacillosis is a disease characterized by an intestinal infection caused by enterotoxigenic Escherichia coli (ETEC). This infection mostly causes illness or death in neonatal and weaned pigs making it responsible for significant economic losses. Bacterial fimbriae (F4/F5/F6/F18/F41) are responsible for the adhesion to epithelial cells, and when both the immunological systems and the gut microbiota are poorly developed, ETEC colonizes and produces one or more enterotoxins (LT/STa/STb/Stx2e) that can have local and systemic effects. Therefore, it is of prime importance to monitor and characterize ETEC in the swine industry to develop mitigation strategies. In this study, our aim was to develop a methodology to detect ETEC and its major virulence factors (i.e., toxins/fimbriae) from swine. Firstly, we optimized a qPCR methodology using ETEC control strains for the screening of ST/LT/stx2 toxins. Thus, the efficiency of primers, sensitivity, and specificity were determined. Also, the limit of detection was performed in artificially contaminated samples. This methodology is intended to be applied to the initial screening of enriched liquid cultures. Secondly, to better characterize the virulence features of ETEC isolates, we developed a multiplex PCR to specifically determine both the fimbriae (F4/F5/F6/F18/F41), and toxins (LT/Sta/STb/Stx2e). To validate our method, we collected rectal swabs from pigs. Our results showed that the qPCR approach can detect between 20-400 DNA copies/L depending on the toxin tested and is also highly specific and sensitive. Furthermore, we also optimized the multiplex PCR technique using the ETEC control strains. Regarding its validation, our preliminary data with porcine rectal samples showed a high prevalence of the above-mentioned toxins (namely ST) as well as the F4 and F18 fimbriae. In sum, this methodology has the potential to be adopted as a routine technique for the rapid detection of ETEC strains in livestock, since the method exbibit a robust performance.