Fluorescent in situ hybridization for detection of “Brachyspira hampsonii” in porcine colonic tissues

Abstract

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.

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

“…Although a positive signal was not identified after hybridization of B. murdochii and B. pilosicoli with either probe, it bears noting that cross-reactivity with the Hamp1210 probe and Brachyspira intermedia in FISH on formalin-fixed tissues has been observed. 4 However, given the relatively high threshold of detection for FISH on formalin-fixed feces in the current experiment, it seems unlikely that a clinically insignificant infection would be detected by this method. The diagnostic specificity of FISH on formalinfixed feces under field conditions will become more apparent once it has been adapted into routine use.…”

“…3,12,14 A definitive diagnosis of SD is commonly based on the isolation of strongly beta-hemolytic, ring phenomenon-positive spirochetes from culture of mucohemorrhagic feces or colonic tissue and/or by species-specific polymerase chain reaction (PCR) assays run on extracts of such samples. 4 While Brachyspira culture using selective agars is a highly sensitive assay, it can be technically challenging, time consuming, typically requires speciation using PCR following isolation, and often requires 6 days or longer to complete, which can result in a delay in disease diagnosis. 4 Polymerase chain reaction assays, while rapid, can be limited by fecal inhibition, particularly in pigs.…”

“…4 While Brachyspira culture using selective agars is a highly sensitive assay, it can be technically challenging, time consuming, typically requires speciation using PCR following isolation, and often requires 6 days or longer to complete, which can result in a delay in disease diagnosis. 4 Polymerase chain reaction assays, while rapid, can be limited by fecal inhibition, particularly in pigs. 13 Fluorescent in situ hybridization (FISH) assays have been described for the identification of B. hyodysenteriae and "B. hampsonii " in formalin-fixed tissues.…”

“…13 Fluorescent in situ hybridization (FISH) assays have been described for the identification of B. hyodysenteriae and "B. hampsonii " in formalin-fixed tissues. 2,4 However, these assays require approximately 2 days or more to complete following fresh tissue collection. 4 Because of the time constraints of currently available assays, the current study reports the development of a same-day FISH assay to detect B. hyodysenteriae 563064V DIXXX10.1177/1040638714563064FISH for detection of "Brachyspira hampsonii" in fecesWilberts et al…”

Hibridização in situ fluorescente para diagnóstico de Brachyspira hyodysenteriae e B. pilosicoli em suínos

“…Também apresentaram-se em contato com células epiteliais necróticas ou em degeneração, invadindo o espaço intercelular e membrana basal. Pode-se observar também as bactérias no lúmen de criptas e no interior de células caliciformes ) e B. hampsonii (Burrough et al 2013, Wilberts et al 2015, não demonstrando reação cruzada entre essas espécies. Esses dados demonstram que a utilização do FISH pode ser uma ferramenta útil para a detecção de B. hyodysenteriae e B. pilosicoli no Brasil.…”