Swine dysentery (SD) has historically been associated with the presence of Brachyspira hyodysenteriae, a strongly beta-hemolytic spirochete; however, beginning in the latter part of 2008 and early 2009 strongly beta-hemolytic Brachyspira spp. not identified as B. hyodysenteriae by polymerase chain reaction assays were recovered with increasing frequency from North American pigs with clinical SD. In 2012, biochemical and molecular characterization of these atypical and untypable Brachyspira revealed the emergence of a novel species and the name “Brachyspira hampsonii” has been proposed. The ultimate goal of this study was to develop a rapid, culture-independent assay to confirm the presence of virulent, non-B. hyodysenteriae spirochetes capable of producing SD within pig tissues. This study addressed the overall goal in two ways: 1) sequencing of the 23S rRNA gene was performed on numerous clinical isolates of “B. hampsonii” (clades I & II) along with multiple typical isolates of commonly recognized Brachyspira spp. infecting swine for comparison, and 2) based upon the results of 23S rDNA sequencing, an oligonucleotide probe specific for 23S rRNA of “B. hampsonii” was developed and evaluated for specificity and sensitivity in a fluorescent in situ hybridization (FISH) assay on formalin-fixed, paraffin-embedded colonic tissues from pigs experimental and naturally infected with a variety of Brachyspira spp.
The newly developed probe (Hamp1210) had a high degree of sensitivity on the tissues tested recognizing all tissues from which “B. hampsonii” was recovered by culture; however, a low percentage of B. hyodysenteriae infected samples had weak to moderate signal with this probe as well as a single case that was culture-positive for Brachyspira intermedia. The FISH assay in this study was designed to be performed with a 6 hour hybridization period making the turnaround time shorter than many previously described protocols. Additionally, a previously described probe for B. hyodysenteriae (Hyo1210) was validated under these same hybridization conditions and exhibited similar sensitivity. The observed cross-reactivity suggests that a second, more specific assay should be used to confirm positive results in index situations; however, this FISH assay, when incorporating both the Hamp1210 and Hyo1210 probes, can provide a rapid preliminary molecular identification of pathogenic spirochetes in cases with clinical signs of SD and reduce the time delay between sample submission, pathogen identification, and treatment initiation.
Iowa State University Veterinary Diagnostic Laboratory
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