Methods for improving diagnostic techniques used for the identification and isolation of Brachyspira species from swine

Warneke, Hallie
Major Professor
Eric R. Burrough
Committee Member
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Veterinary Microbiology and Preventive Medicine

Brachyspira hyodysenteriae is the main etiological agent of swine dysentery and is an important species for ongoing research into the field. Swine dysentery (SD) is also caused by Brachyspira hampsonii and Brachyspira suanatina, which have recently been accepted as new species. These three species are all classified as strong beta species with a positive ring phenomenon. Brachyspira hampsonii includes 4 genetically distinguishable groups. Weak beta Brachyspira species include Brachyspira pilosicoli (the agent of porcine intestinal spirochetosis), Brachyspira intermedia, Brachyspira murdochii, and Brachyspira innocens that all have a negative ring phenomenon. Techniques used for diagnostic and research testing have been consistent but limited to PCR and sequencing that is expensive to producers and researchers. With the advent of MALDI-TOF MS systems in veterinary laboratories, adding Brachyspira species to the database would be beneficial. A total of 33 Brachyspira species were added to a MALDI-TOF MS database, which included the strains: B. hyodysenteriae, B. hampsonii clades I and II, B. pilosicoli, B. intermedia, B. murdochii, and B. innocens. After addition to the database, 176 field isolates were identified and compared using MALDI-TOF MS and nox sequencing, the gold standard for Brachyspira identification. From the field isolates, 98.9% matched species identification by both methods. Additionally, 92% of the B. hampsonii isolates matched clade designation by both methods.

In addition to improving identification techniques for Brachyspira, sampling techniques are another potential area for improvement. The gold standard for Brachyspira sampling is enteric samples. The prevalence of SD is low, therefore many animals have to be sampled for detection of Brachyspira. The introduction of oral fluids as an environmental sample has led to improvements in sampling for other organisms, and could be potentially applied to detecting the agents of swine dysentery and other Brachyspira species. To establish if oral fluids could be used as a sample matrix for Brachyspira species, in vitro and in vivo investigations were completed. The first such investigation enlisted spiked samples of oral fluids in comparison to runny feces, solid feces, and a phosphate buffered saline control. In this study, Brachyspira was able to be isolated from oral fluids. Coincidentally, oral fluids allowed better survival of the organism over a 72 hour period when kept at refrigerated temperatures compared to runny feces and solid feces. Two additional investigations were performed to determine if Brachyspira could be recovered from animals that were experimentally infected with pre-determined species of Brachyspira. Again, all Brachyspira spp. inoculated were recovered from oral fluids. Finally, a field trial was completed to see if oral fluids could be used to isolate Brachyspira from animals of unknown infection status. A total of 20 pens were tested with feces and oral fluids by both culture and PCR. Five pens were culture positive for B. hyodysenteriae (4 via feces and 1 via oral fluid), and surprisingly, all 5 pens were positive for B. hyodysenteriae by PCR of the oral fluid.

The experiments described herein provide an expanded database for identification of porcine Brachyspira using MALDI TOF-MS and suggest that oral fluids are not suitable for culture of Brachyspira from field samples but could be used for PCR. Accordingly, fecal cultures can be supplemented with PCR of oral fluids for detection of Brachyspira from group-housed swine.