Detection and characterization of pathogenic and non-pathogenic Brachyspira

Abstract

Brachyspira is associated with mucohaemorrhagic diarrhea in pigs, though the pathogenesis and virulence factors behind the disease are not currently understood. This thesis aimed to identify putative virulence factors of Brachyspira through a comparative genomics approach, and to determine the frequency of the occurrence of mixed Brachyspira species infections within clinical case material. “B. hampsonii” strain 30446 was originally isolated from colon contents of a pig with mucohaemorrhagic diarrhea, and through inoculation trials has been demonstrated to be pathogenic in swine. “B. hampsonii” strain KL-180 was isolated from a Lesser Snow Goose, and is remarkably genetically similar to strain 30446. However, inoculation trials indicate “B. hampsonii” strain KL-180 is not pathogenic in swine. Their differing pathogenicity, despite their genetic similarity, allows comparison to identify gene content differences, which may correspond to putative virulence factors. Genome sequences for each strain were available, and were annotated. The basic genome features of strain 30446 and strain KL-180 were similar, with an average nucleotide identity (ANI) of 97%. Comparison of the annotated genomes revealed genes unique to each strain. Any gene differences could be important to disease outcome, including hypothetical proteins. “B. hampsonii” strain 30446, contained a set of genes absent in strain KL-180 which correspond to the Streptolysin S (sag) operon. The presence of this operon was confirmed with PCR and primers designed for each gene. The presence of this operon in pathogenic Brachyspira species, and absence in the non-pathogenic “B. hampsonii” strain KL-180 supports the possibility of the operon being a virulence factor and contributing to disease outcome. Current diagnostic methods implemented on clinical case material often indicate the presence of more than one Brachyspira species. It is not currently known if co-infection with multiple species is common, or if the presence of multiple species contributes to Brachyspira pathogenesis. To investigate the frequency of the occurrence of multiple species in Brachyspira clinical cases, deep sequencing of the nox gene target was used on clinical case material from Western Canada, Mexico and Brazil. Synthetically created communities were also sequenced, to provide insight as to what levels of Brachyspira can be detected through this method. Pigs with clinical disease were very frequently colonized by multiple species of Brachyspira. “B. hampsonii” was detected in pigs from both Mexico and Brazil, though often at very low levels. Canadian clinical cases were more diverse than those from Mexico and Brazil, and were dominated by a range of Brachyspira species. Sequencing of the synthetic communities revealed a negative primer bias towards “B. hampsonii” strain 30599 (Clade I). Strain 30599 may therefore be present at higher levels and be more widely distributed than demonstrated by current diagnostic methods based on PCR of the nox gene. The results of this thesis demonstrate that the sag-like operon identified through comparative genomics and found in pathogenic strains of Brachyspira may be a virulence factor. Additionally, mixed Brachyspira infections are commonplace within clinical case material, and multiple species may be involved in disease outcome.