Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR

Abstract

PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of Hindlll-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 23-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 23-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using total DNA obtained from normal swine feces inoculated with decreasing concentrations of S. hyodysenterae

Simultaneous Detection of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in Porcine Faeces and Tissue Samples by Multiplex‐PCR

“…The specificity of the method for all three targets was in agreement with previous studies using the same primers (Jones et al, 1993;Elder et al, 1994;La et al, 2003). There was no difference in the size of the amplicons when primers were evaluated both under uniplex and multiplex conditions.…”

“…This multiplex-PCR showed sensitivity similar to another multiplex-PCR for the same agents but using primers targeting other regions of their genome of B. hyodysenteriae, B. pilosicoli and L. intracellularis (La et al, 2006). It is worth noting that the analytical sensitivity in the multiplex-PCR assay is very similar to or even better than those reported for uniplex-or duplex-PCR assays for the detection of such pathogens (Jones et al, 1993;Elder et al, 1994;La et al, 2003). There are some discrepancies in the literature about the detection limits of PCR assays using the same primers and conditions, which are probably due to differences in the quality of the DNA template used in the PCR.…”

Minimal prophylactic concentration of dietary zinc compounds in a mouse model of swine dysentery

“…Alternatively, reduced recovery rates of B. hyodysenteriae could be attibutable to Zn 2+ -induced inhibition of hemolysin production by the spirochetes. However, this possibility appears less likely since DNA extracted from the ceca of culture-negative inoculated mice yielded negative results for a B. hyodysenteriae-specific sequence by polymerase chain reaction amplification (data not shown) (Elder et al, 1994). Macroscopic and microscopic lesions in the ceca of mice fed the basal diet and challenge-exposed with B. hyodysenteriae were similar to those described previously (Zhang et al, 1995).…”

Immunoblot reactivity of polyclonal and monoclonal antibodies with periplasmic flagellar proteins FlaA1 and FlaB of porcine Serpulina species

“…Currently, swine dysentery is diagnosed on the basis of results from bacteriologic culture of feces or mucosal scrapings or based on demonstration of S. hyodysenteriae-specific products after amplification of DNA sequences by PCR (4,10). The PF of B. burgdorferi have been used as antigens for serologic diagnosis of Lyme disease in human beings; patients develop specific reactivity to the PF proteins early in the course of the infection (12,37).…”