Multiplex Polymerase Chain Reaction for Simultaneous Detection of Lawsonia Intracellularis, Serpulina Hyodysen Teriae, and Salmonellae in Porcine Intestinal Specimens

Abstract

Abstract. Proliferative enteritis, swine dysentery, and porcine salmonellosis are the most common enteric bacterial diseases affecting pigs in the growing and finishing stages of production. Currently, diagnoses of these diseases by standard cultural techniques of intestinal specimens can be laborious, time consuming, and expensive (swine dysentery, porcine salmonellosis) or impossible (proliferative enteritis). Amplification by polymerase chain reaction (PCR) of DNA sequences specific for each bacterial agent is a highly sensitive and specific method that overcomes the limitations associated with standard detection methods. A multiplex PCR (M-PCR) assay was developed for simultaneous detection and identification of the etiologic agents associated with proliferative enteritis, swine dysentery, and porcine salmonellosis in a single reaction using total DNA obtained directly from intestinal specimens. Purified DNA obtained from pure cultures of each bacterial agent alone or mixed in different combinations and concentrations and total DNA from intestinal specimens were amplified using the Lawsonia intracellularis-, Serpulina hyodysenteriae-, and salmonellae-specific M-PCR assay. Intestinal specimens consisted of feces and mucosal scrapings obtained from field cases of each disease alone or in combinations and feces obtained from pigs challenged with S. hyodysenteriae. The banding pattern of the amplified PCR products, after agarose gel electrophoresis and staining, indicated the presence of individual or combinations of etiologic agents in each specimen. Results from this study indicated that simultaneous amplification of L. intracellularis-, S. hyodysenteriae-, and salmonellae-specific DNA sequences by M-PCR can be used for specific detection and identification of three major enteric bacterial pathogens associated with proliferative enteritis, swine dysentery, and porcine salmonellosis occurring alone or in combinations. Also, the M-PCR assay can be done using DNA obtained directly from intestinal specimens submitted for diagnostic investigation.A key element to rational therapy and effective conThe diseases caused by L. intracellularis, S. hyodystrol of porcine enteric bacterial diseases is rapid and enteriae, and salmonellae are similar in several reaccurate detection and identification of etiologic agent(s) spects: 1) these bacteria cause diarrhea at a similar age in intestinal specimens submitted for diagnostic in-in the swine production cycle, 2) transmission of these vestigation. Proliferative enteritis (PE) caused by Law-bacteria is by the fecal-oral route, and 3) diagnoses of sonia intracellularis, 15,16 swine dysentery (SD) caused PE, SD, and PS by standard laboratory techniques reby Serpulina hyodysenteriae, 8 and porcine salmonel-quires special handling of intestinal specimens and can losis (PS) caused by salmonellae 25 are the most com-take up to several days or weeks and even then the mon bacterial diarrheal diseases affecting pigs during results are not always specific or sensitive enough for the gr...

Detection ofLawsonia Intracellularisin Swine using Polymerase Chain Reaction Methodology

“…1 The amplified sequences of Salmonella, invE and invA, are conserved throughout S. typhimurium, S. choleraesuis, S. derby, S. brandenburg, and S. cubana. 1 The veterinary diagnostic laboratories at the University of Nebraska-Lincoln and the University of Minnesota, livestock auction markets, and experimental pigs were the sources of specimens (mucosal scrapings and feces). The sensitivity and specificity for the multiplex assay was tested by using spiked cultures of various concentrations of bacteria.…”

“…All of the results were correlated with those of the conventional diagnostic procedures except for 1 specimen that was M-PCR positive for a Salmonella invA sequence even though the culture was negative. 1 One advantage of the PCR test is that live organisms are not required. A multiplex PCR assay can provide the diagnostic results in a timely fashion and perhaps more economically than traditional diagnostic methods.…”

Prevalence of selected enteropathogenic bacteria in Hungarian finishing pigs

“…Previously obtained type strains were used as positive controls for these reactions [Serpulina (Brachyspira) hyodysenteriae ATCC 27164, Serpulina pilosicoli ATCC 51139; Serpulina intermedia: ATCC 51140; Serpulina innocens ATCC 29796]. For the Lawsonia PCR (Elder et al, 1997), previously isolated L. intracellularis DNA was used as positive control (Biksi et al, 1998). For the Salmonella PCR (Elder et al, 1997), a previously isolated Salmonella enterica serovar Choleraesuis strain was used as positive control, identified by the Salmonella Laboratory of the National Food Investigation Institute, Budapest, Hungary.…”

A preliminary study of the molecular epidemiology of Brachyspira hyodysenteriae isolates in Australia

“…In this study, a multiplex PCR for B. hyodysenteriae, B. pilosicoli, L. intracellularis and Salmonella spp. including primers described by Elder et al (1997) was compared with the PCR targeting NADH oxidase (nox) as described by La et al (2006). Multi-locus sequence typing (MLST) was used to determine the relatedness of the B. hyodysenteriae isolates (La et al 2009).…”