Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/6/2008
Publication Date: 1/1/2009
Citation: Casey, T., Bosworth, B.T. 2009. Design and Evaluation of a Multiplex PCR Assay for the Simultaneous Identification of Genes for Nine Different Virulence Factors Associated with Escherichia coli that Cause Diarrhea and Edema Disease in Swine. Journal of Veterinary Diagnostic Investigation. 21(1):25-30. Interpretive Summary: Diarrhea in newborn and recently weaned pigs caused by Escherichia coli is an important cause of economic loss for swine producers. These E. coli adhere in the small intestine and produce toxins that cause fluid secretion which leads to diarrhea and weight loss in young pigs. Rapid and simple diagnostic test are needed to identify the toxins and adherence factors produced by E. coli for epidemiological studies and for developing vaccines. We developed and tested a rapid and specific diagnostic polymerase chain reaction (PCR) assay which, in a single reaction, detects five different adherence factors and four different toxins in E. coli pathogenic for swine. This multiplex PCR assay will be useful in veterinary diagnostic laboratories for identification and characterization of E. coli isolates from swine, in studies of the epidemiology of porcine diarrheal disease, and for veterinary researchers designing vaccines to prevent E. coli infection, a significant cause of diarrhea in young pigs.
Technical Abstract: A multiplex PCR assay was developed for detection and characterization of pathogenic E. coli that cause diarrhea and Edema Disease in swine. This PCR assay was designed as a single reaction for detecting five different adhesins (K88, K99, 987P, F41 and F18), three enterotoxins (LT, STaP, STb), and the Shiga toxin (Stx2e) associated with porcine pathogenic E. coli. The specificity of the multiplex PCR assay was evaluated by comparison with results from previous analysis of porcine isolates characterized by colony blot hybridization with DNA probes for the five adhesins and four toxin genes. There was complete agreement between the two methods. The multiplex PCR assay for E. coli pathogens isolated from swine was further evaluated by examination of strains containing virulence factors that are known to have different antigenic subtypes or DNA sequence variations. We found that the K88 and F18 primers, with DNA from strains containing genes for different K88 and F18 antigenic subtypes, amplified products with the appropriate sizes regardless of the subtype. The STaP primers amplified product from only STaP positive isolates, not STaH positive strains, and the Stx2e primers amplified products from only Stx2 positive and not Stx1 positive isolates. The LT primers did not produce the appropriate product from strains containing genes for LTII. This multiplex PCR assay is simple to perform, and should be useful for diagnosis of porcine colibacillosis, including the genotypic characterization of E. coli isolates from pigs with diarrhea or Edema Disease.