Comparison of culture, polymerase chain reaction, and fluorescent in situ hybridization for detection of Brachyspira hyodysenteriae and "Brachyspira hampsonii" in pig feces.

Swine dysentery is characterized by mucohemorrhagic diarrhea and can occur following infection by Brachyspira hyodysenteriae or "Brachyspira hampsonii ". A definitive diagnosis is often based on the isolation of strongly beta-hemolytic spirochetes from selective culture or by the application of species-specific polymerase chain reaction (PCR) assays directly to feces. While culture is highly sensitive, it typically requires 6 or more days to complete, and PCR, although rapid, can be limited by fecal inhibition. Fluorescent in situ hybridization (FISH) has been described in formalin-fixed tissues; however, completion requires approximately 2 days. Because of the time constraints of available assays, a same-day FISH assay was developed to detect B. hyodysenteriae and "B. hampsonii " in pig feces using previously described oligonucleotide probes Hyo1210 and Hamp1210 for B. hyodysenteriae and "B. hampsonii", respectively. In situ hybridization was simultaneously compared with culture and PCR on feces spiked with progressive dilutions of spirochetes to determine the threshold of detection for each assay at 0 and 48 hr. The PCR assay on fresh feces and FISH on formalin-fixed feces had similar levels of detection. Culture was the most sensitive method, detecting the target spirochetes at least 2 log-dilutions less when compared to other assays 48 hr after sample preparation. Fluorescent in situ hybridization also effectively detected both target species in formalin-fixed feces from inoculated pigs as part of a previous experiment. Accordingly, FISH on formalin-fixed feces from clinically affected pigs can provide same-day identification and preliminary speciation of spirochetes associated with swine dysentery in North America.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid identification of Brachyspira species isolated from swine, including the newly described "Brachyspira hampsonii".

The Brachyspira species traditionally associated with swine dysentery and other diarrheal diseases in pigs are Brachyspira hyodysenteriae, Brachyspira pilosicoli, and, to a lesser extent, Brachyspira murdochii. "Brachyspira hampsonii" is a recently proposed novel species that causes clinical disease similar to that caused by B. hyodysenteriae. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems are increasingly available in veterinary diagnostic laboratories, are less expensive, and are faster than traditional microbiological and molecular methods for identification. Thirty-three isolates associated with Brachyspira species of importance to swine were added to an existing MALDI-TOF MS database library. In total, species included in the library were: B. hyodysenteriae, "B. hampsonii" clades I and II, Brachyspira innocens, Brachyspira intermedia, B. murdochii, and B. pilosicoli. A comparison between MALDI-TOF MS and nox sequencing was completed on 176 field isolates. Of the 176 field isolates, 174 (98.9%) matched species identification by both methods. Thirty field isolates were identified by both methods as "B. hampsonii". Twenty-seven of the 30 (90%) "B. hampsonii" field isolates matched clade designation in both assays. The nox sequencing identified 26 as "B. hampsonii" clade I and 4 as clade II. Comparatively, MALDI-TOF MS identified 25 of the 30 as "B. hampsonii" clade I and 5 as clade II. The current study indicates MALDI-TOF MS is a reliable tool for the identification of swine Brachyspira species; however, final clade designation for "B. hampsonii" may still require molecular techniques.

Identification of Mycoplasma suis antigens and development of a multiplex microbead immunoassay.

The aims of the current study were to identify Mycoplasma suis antigens and develop a multiplex microbead immunoassay (MIA). A M. suis-expression library was screened for immunogens using sera from infected pigs. Based on bioinformatics, putative antigens were identified within positive inserts; gene fragments were expressed and purified as polyhistidine fusion proteins, and immunoreactivity was confirmed by Western blot. Selected antigens were used to develop a MIA. Sera from noninfected and infected pigs were used to set the median fluorescent intensity (MFI) cutoffs and as positive controls, respectively. Assay specificity was tested using sera from pigs seropositive for other pathogens (2 different pigs seropositive for each pathogen). Samples from 51 field pigs and 2 pigs during the course of acute (pig 1) and chronic (pig 2) infections were tested using MIA, indirect hemagglutination assay (IHA), and quantitative polymerase chain reaction (qPCR). Sixteen reactive plaques (52 genes) were detected. A heat-shock protein (GrpE), a nicotinamide adenine dinucleotide-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPN), and 4 proteins from paralogous gene families (PGFs) were identified as antigens by Western blot. While GrpE, GAPN, and 1 PGF protein were strong antigens, the others were not suitable as MIA targets. A MIA using GrpE, GAPN, and the strongly reactive PGF protein was developed. Cross-reactivity with sera from pigs infected with Mycoplasma hyopneumoniae, Porcine circovirus-2, Porcine parvovirus, Porcine reproductive and respiratory syndrome virus, and Porcine respiratory coronavirus with this MIA was not observed. Pig 2 was consistently positive by MIA and qPCR, whereas pig 1, initially negative, seroconverted before becoming qPCR positive. Only 2 samples (from pig 1) were IHA positive. Five (9.8%) field samples were qPCR positive and 40 (78.43%) were positive for all 3 MIA antigens; however, all were IHA negative. In summary, the MIA is specific and more sensitive than qPCR and IHA, providing simultaneous evaluation of antibody response to M. suis antigens.

Fluorescent in situ hybridization for detection of "Brachyspira hampsonii" in porcine colonic tissues.

Swine dysentery is classically associated with infection by the strongly beta-hemolytic Brachyspira hyodysenteriae; however, the proposed novel species "Brachyspira hampsonii" has also been isolated from clinical cases of dysentery in the United States and Canada. Microbial culture is highly sensitive for detecting Brachyspira in clinical samples but requires several days for completion and is often followed by molecular testing for speciation. Alternatively, in situ hybridization using molecular probes applied to sections of formalin-fixed tissue can provide rapid, culture-independent identification of agents observed histologically. Accordingly, a fluorescent in situ hybridization assay was developed for confirmation of a clinical diagnosis of swine dysentery associated with infection by "B. hampsonii." An oligonucleotide probe (Hamp1210) targeting a specific 23S ribosomal RNA sequence of "B. hampsonii" was developed following sequence analysis and comparison of numerous Brachyspira spp. clinical isolates with reference sequences available in GenBank. The application of Hamp1210 and a previously published probe for B. hyodysenteriae (Hyo1210) to diseased colonic tissues successfully detected the target species in both experimentally infected pigs and naturally infected pigs from field cases, and the Hamp1210 probe consistently detected both clade I and clade II isolates of "B. hampsonii"; however, a strong positive signal was also observed in a single case where the Hamp1210 probe was applied to tissues infected with Brachyspira intermedia. In situ hybridization incorporating the Hamp1210 probe can reduce the delay from sample submission to pathogen identification in cases of swine dysentery associated with "B. hampsonii" infection where formalin-fixed tissues are available.

Comparative virulence of clinical Brachyspira spp. isolates in inoculated pigs.

Classical swine dysentery is associated with the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae. However, multiple Brachyspira spp. can colonize the porcine colon. Since 2008, several Brachyspira spp. not identified as B. hyodysenteriae by genotypic and/or phenotypic methods have been isolated from the feces of pigs with clinical disease typical of swine dysentery. In the current study, 8 clinical isolates, including 5 strongly beta-hemolytic and 3 weakly beta-hemolytic Brachyspira strains, and a reference strain of B. hyodysenteriae (B204) were inoculated into pigs (n = 6 per isolate) to compare pathogenic potential following oral inoculation. Results revealed that strongly beta-hemolytic isolates induced significantly greater typhlocolitis than those that are weakly beta-hemolytic, regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as "Brachyspira sp. SASK30446" and Brachyspira intermedia by polymerase chain reaction (PCR) produced lesions similar to those caused by B. hyodysenteriae. The results suggest that phenotypic culture characteristics of Brachyspira spp. may be a more sensitive indicator of potential to induce dysentery-like disease in pigs than molecular identification alone based on currently available PCR assays. Additionally, culture of mucosal scrapings obtained at necropsy was more sensitive than direct PCR on the same samples for detection of Brachyspira spp.

Evaluation of FOXC2 as a candidate gene for chronic progressive lymphedema in draft horses.

Chronic progressive lymphedema (CPL) is a debilitating condition identified in Clydesdales, Shires and Belgian draft horses and results in progressive swelling of the lower legs associated with the development of thick skin folds, ulcerations, fibrosis and marked hyperkeratosis. The result is severe discomfort and recurrent secondary infection, often requiring euthanasia. Due to the delayed onset, many horses are bred prior to diagnosis. CPL has only been documented in three related draft horse breeds, suggesting a genetic cause. Determining the molecular basis would enable owners to test horses prior to breeding and facilitate the elimination of CPL. Mutations in the FOXC2 gene cause a comparable condition in humans, lymphedema-distichiasis. This gene was sequenced in affected and unaffected draft horses and a control horse. Four single nucleotide polymorphisms (SNPs) were identified in unaffected draft horses and the control horse, indicating that they were not associated with CPL. A fifth SNP was seen in a single affected draft horse and the control horse. Since it was not seen in all affected draft horses, this SNP is not associated with the CPL phenotype.