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.

Investigation of the impact of increased dietary insoluble fiber through the feeding of distillers dried grains with solubles (DDGS) on the incidence and severity of Brachyspira-associated colitis in pigs.

Diet has been implicated as a major factor impacting clinical disease expression of swine dysentery and Brachyspira hyodysenteriae colonization. However, the impact of diet on novel pathogenic strongly beta-hemolytic Brachyspira spp. including "B. hampsonii" has yet to be investigated. In recent years, distillers dried grains with solubles (DDGS), a source of insoluble dietary fiber, has been increasingly included in diets of swine. A randomized complete block experiment was used to examine the effect of increased dietary fiber through the feeding of DDGS on the incidence of Brachyspira-associated colitis in pigs. One hundred 4-week-old pigs were divided into five groups based upon inocula (negative control, Brachyspira intermedia, Brachyspira pilosicoli, B. hyodysenteriae or "B. hampsonii") and fed one of two diets containing no (diet 1) or 30% (diet 2) DDGS. The average days to first positive culture and days post inoculation to the onset of clinical dysentery in the B. hyodysenteriae groups was significantly shorter for diet 2 when compared to diet 1 (P = 0.04 and P = 0.0009, respectively). A similar difference in the average days to first positive culture and days post inoculation to the onset of clinical dysentery was found when comparing the "B. hampsonii" groups. In this study, pigs receiving 30% DDGS shed on average one day prior to and developed swine dysentery nearly twice as fast as pigs receiving 0% DDGS. Accordingly, these data suggest a reduction in insoluble fiber through reducing or eliminating DDGS in swine rations should be considered an integral part of any effective disease elimination strategy for swine dysentery.

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.