Weakly haemolytic variants of Brachyspira hyodysenteriae newly emerged in Europe belong to a distinct subclade with unique genetic properties. Weakly haemolytic variants of Brachyspira hyodysenteriae

The spirochaete Brachyspira hyodysenteriae colonises the porcine colon and initiates a severe mucohaemorrhagic colitis called swine dysentery (SD). This pathogen is widespread globally, and can cause severe economic impact in infected herds. Typical strains of B. hyodysenteriae are strongly haemolytic on blood agar, and the haemolytic activity is believed to contribute to virulence in vivo. Recently there have been reports of atypical weakly haemolytic isolates of B. hyodysenteriae (whBh) recovered from pigs in herds where SD has not been diagnosed. The purpose of this study was to characterise a collection of European whBh isolates to determine their genetic relationships, and to look for possible explanations for the weakly haemolytic phenotype. Thirty whBh isolates were subjected to whole genome sequencing; these sequences, and available genomic sequences for four other whBh isolates and >80 strongly haemolytic isolates, were subjected to comparative genomic analysis. A phylogenetic tree constructed using core Single Nucleotide Polymorphisms showed that the whBh isolates formed a distinct sub-clade, and epidemiologically-related isolates clustered in 10 sub-groups. All eight genes previously associated with haemolysis in B. hyodysenteriae were present in the whBh isolates. Although various amino acid substitutions were identified in different haemolysin genes in different whBh isolates, no consistent patterns of substitutions were found. In contrast, a genome region containing six coding sequences (CDSs) had consistent nucleotide sequence differences between strongly and weakly haemolytic isolates. Two CDSs were predicted to encode ABC transporter proteins, and a TolC family protein was identified as consistently different in the whBh isolates. These proteins may have a role in the export of haemolysins from B. hyodysenteriae. Another difference in this region was the presence of three CDSs in the whBh isolates that are pseudogenes in strongly haemolytic isolates. Two of the intact CDSs from whBh isolates encoded hypothetical proteins, and one encoded a predicted PadR-like transcriptional repressor that may play a role in repression of haemolysis functions. In summary, a sub-clade of whBh isolates has emerged in Europe, and several genomic differences that potentially explain the weakly haemolytic phenotype were identified. These markers may provide targets for discriminatory molecular tests needed in SD surveillance.