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

Abstract

Brachyspira (B.) hyodysenteriae is widespread globally, and can cause mucohaemorrhagic colitis (swine dysentery, SD) with 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. However, recently there have been reports of atypical weakly haemolytic isolates of B. hyodysenteriae (whBh). In this study, 34 European whBh and 82 strongly haemolytic isolates were subjected to comparative genomic analysis. A phylogenetic tree constructed using core single nucleotide polymorphisms showed that the whBh formed a distinct sub-clade. All eight genes previously associated with haemolysis in B. hyodysenteriae were present in the whBh. No consistent patterns of amino acid substitutions for all whBh were found in these genes. In contrast, a genome region containing six coding sequences (CDSs) had consistent nucleotide sequence differences between strongly and whBh isolates. Two CDSs were predicted to encode ABC transporter proteins, and a TolC family protein, which may have a role in the export of haemolysins from B. hyodysenteriae. Another difference in this region was the presence of three CDSs in whBh that are pseudogenes in strongly haemolytic isolates. One of the intact CDSs from whBh 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.

Figure 1

A phylogenetic construction of B. hyodysenteriae isolates using a maximum-likelihood tree. The 39 field isolates sequenced in this project are indicated with red font and have been included together with 77 published B. hyodysenteriae genomes. The sub-clade of weakly haemolytic isolates is indicated by red branches and for each weakly haemolytic isolate, the farm of origin (inner circle), sequence type (middle circle), and clone (outer circle) are presented in a colour-coded manner, as indicated in the figure legend and summarised in Table 1. The respective tree node of the sub-clade of weakly haemolytic isolates was supported with a bootstrap value of 100%. Image generated using the interactive Tree Of Life (iTOL) [53].

Figure 2

In vitro haemolytic capacity of representative B. hyodysenteriae field strains. Isolates were grown to an OD₆₀₀ of 0.250 ± 0.05 (A) and field strains JR11 and JR72 and type strain B78^T were also grown to an OD₆₀₀ of 0.550 ± 0.05 (B). Haemolysis is presented as the mean value of absorption at 450 nm after incubation of the red blood cell suspension with the supernatant of representative B. hyodysenteriae isolates. B. hyodysenteriae strain B78^T served as positive control and B. innocens strain C336 was the negative control. Isolates with a significant difference in haemolytic capacity compared to B78^T (p < 0.05 after Bonferroni correction) are indicated with an asterisk (*).

Figure 3

Chromosomal arrangement of genes in B. hyodysenteriae showing differences between strongly and whBh. B. hyodysenteriae genes are labelled according to the locus tag in the reference strain B. hyodysenteriae WA1 (Accession number NC_012225). Genes have been coloured to indicate intact genes in blue and pseudogenes in red. CDSs identified as significantly associated with haemolysis phenotype by gene-based and SNP-based genome-wide association studies are indicated with an asterisk (*) and dagger (†) respectively. Isolates WA1, JR20, and BH30 are presented as exemplar strongly haemolytic isolates. D28, BH23 and JR63 are presented as exemplar weakly haemolytic isolates. Regions of sequence similarity between isolates are shown by grey shading. Image generated using EasyFig [54].