Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs

Abstract

The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x10(3) CFU/ml and 6.5x10(1) CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd.