A Quantitative Polymerase Chain Reaction Assay for Detection and Quantification of Lawsonia Intracellularis

Abstract

Lawsonia intracellularis, a causative agent of porcine proliferative enteropathy, is an obligate intracellular bacterium that is difficult to culture, propagate, and quantify. The intestinal epithelial cell line (IEC-18, derived from rat small intestine crypt cells) has been used to isolate and propagate this pathogen. However, the lack of rapid and simple quantification methods has led to mixed results when using the rat cell line, complicating Lawsonia studies. To overcome these limitations, a SYBR green quantitative polymerase chain reaction (qPCR) assay targeting a unique hypothetical protein was developed for detecting and quantifying L. intracellularis in IEC-18 rat epithelial cells, porcine fecal samples collected from different farms, and experimentally infected pigs. The method was optimized to detect as few as 3 copies per qPCR reaction of the bacterium growing in IEC-18 cells, providing a new and necessary approach to assess the growth of L. intracellularis in these cell lines. Furthermore, the qPCR assay was successful in detecting L. intracellularis in fecal samples collected from pigs with and without a history of Lawsonia infections, as well as in experimentally infected pigs, which further confirmed the suitability of the qPCR assay for studying the epidemiology of this pathogen.