A multiplex PCR assay was developed for the simultaneous detection of the etiologic agents associated with porcine proliferative enteropathies (PPE), swine dysentery (SD)and porcine salmonellosis (PS)in a single reaction using DNA from swine intestinal samples. Single and multiplex PCR amplification of DNA from Lawsonia intracellularis, Salmonella typhimurium and Brachyspira hyodysenteriae with each primer set produced fragments of the predicted size without any nonspecific amplification, 210-bp, 298-bp and 403-bp bands, respectively. The single PCR assay could detect as little as 100 pg of purified DNA of S. typhimurium and L. intracellularis, and 50 pg of B.hyodysenteriae, respectively. However, multiplex PCR turned out to be 10 times lower sensitivity with S. typhimurium compared with single PCR. With 23 swine intestinal specimens suspected of having PPE, SD and/or PS, the multiplex PCR assay showed identical results with conventional methods except one. In conclusion, this multiplex PCR is a feasible alternative to standard diagnostic methods for detection of L. intracellularis, B. hyodysenteriae and Salmonella spp. from swine intestinal specimens.
Animals ; Desulfovibrionaceae Infections/microbiology/veterinary ; Intestines/microbiology ; Lawsonia Bacteria/*isolation&purification ; Polymerase Chain Reaction/*methods/veterinary ; Salmonella/*isolation&purification ; Salmonella Infections, Animal/diagnosis ; Sensitivity and Specificity ; Spirochaetales/*isolation&purification ; Spirochaetales Infections/microbiology/veterinary ; Swine ; Swine Diseases/*diagnosis/*microbiology

The prevalence of Lawsonia intracellularis, Brachyspira hyodysenteriae and Salmonella spp. were investigated by multiplex PCR using fecal samples of pigs with diarrhea or a history of diarrhea. The overall herd prevalence of L. intracellularis, B. hyodysenteriae and Salmonella spp. were 46.5%, 37.2% and 51.1%, respectively. Also, the prevalence of L. intracellularis, B. hyodysenteriae and Salmonella spp. among all sampled pigs were 19.9%, 10.8% and 17.7%, respectively. Seventeen of 43 herds were positive with 2 enteric organisms, and 2 herds were positive with L. intracellularis, B. hyodysenteriae and Salmonella spp. simultaneously. It was notable that 11 of 12 herds with more than 2, 000 pigs were affected with Salmonella spp., and that only 2 of 12 the herds were affected with B. hyodysenteriae. This study suggested that herds positive for L. intracellularis, B. hyodysenteriae and Salmonella spp. were distributed throughout Korea, although the relationship among other pathogens such as viral or parasitic ones and/or with metabolic disorders was not determined.
Animals ; DNA, Bacterial/isolation&purification ; Desulfovibrionaceae Infections/epidemiology/microbiology/*veterinary ; Diarrhea/microbiology/veterinary ; Korea/epidemiology ; *Lawsonia Bacteria/isolation&purification ; Polymerase Chain Reaction/veterinary ; Prevalence ; *Salmonella/isolation&purification ; Salmonella Infections, Animal/*epidemiology/microbiology ; *Serpulina hyodysenteriae/isolation&purification ; Spirochaetales Infections/epidemiology/microbiology/*veterinary ; Swine ; Swine Diseases/*epidemiology/microbiology

Lawsonia intracellularis is not culturable with a standard bacteriologic culture. One step PCR assay as a clinical diagnostic method was developed for the rapid detection of porcine proliferative enteritis (PPE) caused by L. intracellularis. Primers were designed based on the p78 DNA clone of L. intracellularis. The one step PCR resulted in the formation of a specific 210-bp DNA product derived from L. intracellularis. The nonspecific amplification product was not detected with swine genomic DNA or other bacterial strains causing similar symptoms to L. intracellularis infection. The one step PCR was as sensitive as 100 pg of L. intracellularis genomic DNA. We applied this method to field specimens diagnosed as PPE by macroscopic observation. Of 17 mucosal scraping specimens, 16(94%) were identified as positive to PPE and 15(88%) of 17 feces specimens. These results suggest that the one step PCR can be used as a rapid diagnostic method for L. intracellularis infection.
Animals ; Base Sequence ; DNA Primers ; Desulfovibrionaceae Infections/diagnosis/*veterinary ; Ileum/microbiology/pathology ; Intestinal Mucosa/microbiology/pathology ; Lawsonia Bacteria/genetics/*isolation & purification ; Polymerase Chain Reaction/*methods ; Reproducibility of Results ; Sensitivity and Specificity ; Swine ; Swine Diseases/*diagnosis/microbiology