Brachyspira testing update  

Durda Slavic

The AHL currently offers culture and real-time PCR (rt-PCR) detection of Brachyspira spp. Culture is done on request only; it is time consuming, and one week notice to the lab is recommended because selective media need to be made. In contrast, rt-PCR can be done within a day or two and no advance notification is required. More importantly, in our hands rt-PCR appears to be more sensitive than culture.

Since 2013, 93 samples were tested in the AHL bacteriology section by culture and PCR for the presence of Brachyspira spp. We tested 64 fecal samples and 29 tissue samples. For culture, we used 2 different selective media and 2 different incubation temperatures (37° C and 42° C).  For rt-PCR, Brachyspira spp. was detected by using a genus-specific probe whereas species-specific probes were used to detect Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira hampsonii clade I, and B. hampsonii clade II. Of 64 fecal samples, 23/64 (34%) were positive for the presence of Brachyspira spp. by rt-PCR, whereas only 9/64 (14%) samples were positive on culture. All fecal samples that were positive on culture were also positive on PCR. In total, 29 tissue samples were tested and only 3/29 (10%) samples were positive on both PCR and culture. The rest of the samples were negative by both methods. Based on these limited data , it appears that our rt-PCR will detect Brachyspira spp. in 20% more fecal samples than culture. No differences were observed for tissue samples, but that could be attributed partially to the smaller sample size.

To confirm that our samples are truly positive for the presence of Brachyspira spp., we attempted sequencing of rt-PCR products from the randomly chosen 12 positive samples. Six of these samples were positive for Brachyspira spp. by both culture and rt-PCR and 6 samples were positive by rt-PCR only. All sequencing was done on PCR products obtained from nucleic acid extracted from original samples (e.g., feces and tissue). The sequencing results confirmed the presence of Brachyspira spp. in all 12 samples, but in 6 samples more than 1 species of Brachyspira appeared to be present.

To further explore the robustness of our rt-PCR and to resolve issues with a mixed Brachyspira population being present in the samples, 17 samples were randomly chosen for next generation sequencing by the GS Junior. The advantage of this type of sequencing over gel-based sequencing is that it can detect multiple species of Brachyspira in the same sample simultaneously. The results of next generation sequencing were then compared to the individual rt-PCR results, which included results for 5 different Brachyspira spp. (Table 1). There was good agreement between GS Junior sequencing and our rt-PCR results.

For diagnostic purposes, our Brachyspira panel is used for detection of Brachyspira spp. at the genus level and B. hyodysenteriae, B. pilosicoli, B. hampsonii clade I, and B. hampsonii clade II at the species level. If samples are positive for Brachyspira spp. at the genus level but negative for pathogenic species currently tested, it is most likely that other species of Brachyspira of questionable clinical significance are present, such as B. murdochii, B. intermedia, or B. innocens. If submitting samples for culture, please call the laboratory a week in advance to allow time for selective media to be made.   

Table 1.  Comparison of next generation sequencing (GS Junior) and rt-PCR results.  POS=positive, ND=not detected, INC=inconclusive.

Comparison of next generation sequencing (GS Junior) and rt-PCR results.  POS=positive, ND=not detected, INC=inconclusive