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

Abstract

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.

Rapid and visual detection of Lawsonia intracellularis with an improved recombinase polymerase amplification assay combined with a lateral flow dipstick

“…The above results suggest a decreased susceptivity to inhibition in RPA-FLD from feces in contrast to a greater susceptivity to inhibition in conventional PCR. Previous study has demonstrated that disease symptoms corresponded with higher fecal shedding of L. intracellularis detected by a SYBR green quantitative polymerase chain reaction (qPCR) assay after inoculating L. intracellularis -free pigs with 5 × 10 5 bacteria per pig [20]. Thus, the sensitivity in term of number of L. intracellularis in feces sample presented in this study provided a means to monitor L.intracellularis shedding from naturally or experimentally infected pigs.…”

Biology and Diseases of Rabbits

“…L. intracellularis can be cultured from homogenized intestinal tissue in cell lines including IEC-18 (rat small intestinal cells) and McCoy cells (mouse fibroblasts) (Lawson and Gebhart, 2000;Watarai et al, 2008). A quantitative PCR (qPCR) assay that is able to assess the growth of L. intracellularis in cultured cells has also been used to detect the organisms in pig fecal samples and could be used in other animal species (Drozd et al, 2010).…”

Association between faecal load of lawsonia intracellularis and pathological findings of proliferative enteropathy in pigs with diarrhoea

“…Subclinical infection is common [4], L. intracellularis can be demonstrated by PCR in animals vaccinated by an avirulent live vaccine [5] and the bacterial load in faeces could previously not be assessed in routine diagnostic work as the bacterium could only be cultivated and maintained in cell cultures [6]. Development of quantitative PCR (qPCR) tests for quantification of L. intracellularis in faeces [5,7-10] has now made it possible to determine the number of L. intracellularis bacteria in faeces on a routine basis. This may be useful for confirmation of L. intracellularis as the microbiological cause in porcine diarrhoea.…”