Spirochetal diarrhea has low mortality but causes great economic losses to farmers due to high infectivity and negative impact on growth (1). The disease is caused by the bacterium Brachyspira pilosicoli and is treated with antibiotics. Today, we see an increased incidence of resistance in B. pilosicoli and multidrug-resistant strains have been found in Sweden (2), why the disease may be difficult to treat and control in the future. Therefore, the need for new ways to prevent the disease arises. In this study, we wanted to identify proteins of B. pilosicoli that the bacterium uses to attach to the intestinal mucosa. With knowledge of this process, we want to find ways to block the binding and thereby prevent onset of symptoms.
A shotgun phage display library (3) from B. pilosicoli strain P43/6/78T was used to isolate bacterial proteins by affinity selection against a complex ligand – the porcine intestinal epithelial cell line IPEC J2. The genes encoding these proteins are simultaneously isolated and Sanger sequencing combined with deep sequencing using Nanopore technology identified the proteins.
When selecting a phage library against a complex ligand there will be a lot of background. In this case, with the help of deep sequencing, a large number of the selected phage library clones could be identified and the majority of clones corresponded to a peptide ABC transporter substrate-binding protein, OppA. OppA homologues in B. pilosicoli have been evaluated as vaccine candidates (4) and a homologue in another spirochete, Treponema denticola, has been shown to have fibronectin-binding properties (5).
Deep sequencing may be a way of screening a large number of clones with high background resulting from selecting a phage library against a complex ligand. OppA may be one mean by which B. pilosicoli attaches to the intestinal epithelium but further confirmative studies are required.