Multiplex polymerase chain reaction for simultaneous detection of Lawsonia intracellularis, Serpulina hyodysenteriae, and salmonellae in porcine intestinal specimens.
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.
Elder
R O
RO
Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln 68583-0905, USA.
Duhamel
G E
GE
Mathiesen
M R
MR
Erickson
E D
ED
Gebhart
C J
CJ
Oberst
R D
RD
D000818:Animals; D017401:Brachyspira hyodysenteriae / Q000302:isolation & purification*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology; D006090:Gram-Negative Bacteria / Q000302:isolation & purification*; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000662:veterinary*; D007413:Intestinal Mucosa / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012475:Salmonella / Q000302:isolation & purification*; D012481:Salmonella Infections, Animal / Q000175:diagnosis*; D013145:Spirochaetales Infections / Q000175:diagnosis / Q000662:veterinary*; D013552:Swine; D013553:Swine Diseases
D017931:DNA Primers; D004269:DNA, Bacterial