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

Abstract

Abstract. 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.

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.…”

Emergence of Brachyspira species and strains: reinforcing the need for surveillance

“…This series of events emphasizes the need to use both culture to obtain isolates and PCR methods for diagnosing Brachyspira infections, and to keep updating molecular diagnostic techniques. PCR methods based on the NADH oxidase ( nox ) gene are now available for “ B. hampsonii ” and “ B. suanatina ” [46, 47], and new rapid identification methods based on Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are being implemented for identification of isolates of Brachyspira species [48, 49]. …”

Cross-reactions in specific Brachyspira spp. PCR assays caused by “Brachyspira hampsonii” isolates

“…5,7,20 Although "B. hampsonii" has been isolated from Belgian pigs imported to Germany 19 and from 2 gilts brought from the Czech Republic to Belgium, 11 the actual distribution of the species in Europe is unknown. Several methods have been described for the detection or identification of "B. hampsonii" 22,23 ; however, in most laboratories, the diagnosis of swine dysentery is still supported by microbial culture and/or speciesspecific PCR for the identification of B. hyodysenteriae. 22,23 According to our results, some of the most commonly used In terms of the B. hyodysenteriae-specific PCR assays used on the "B. hampsonii" isolates, the most specific assay was based on the amplification of a 1,301-bp fragment located on the 23S rRNA gene, 10 and gave only 1 false positive.…”

“…Several methods have been described for the detection or identification of "B. hampsonii" 22,23 ; however, in most laboratories, the diagnosis of swine dysentery is still supported by microbial culture and/or speciesspecific PCR for the identification of B. hyodysenteriae. 22,23 According to our results, some of the most commonly used In terms of the B. hyodysenteriae-specific PCR assays used on the "B. hampsonii" isolates, the most specific assay was based on the amplification of a 1,301-bp fragment located on the 23S rRNA gene, 10 and gave only 1 false positive. The next best performance was given by an assay based on the amplification of a 821-bp fragment of the nox gene, 1 with 2 weak false positives.…”