Microevolution of Renibacterium salmoninarum: evidence for intercontinental dissemination associated with fish movements

Abstract

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive bacterium. We exploited recent advances in the next-generation sequencing technology to generate genome-wide single-nucleotide polymorphism (SNP) data from 68 diverse R. salmoninarum isolates representing broad geographical and temporal ranges and different host species. Phylogenetic analysis robustly delineated two lineages (lineage 1 and lineage 2); futhermore, dating analysis estimated that the time to the most recent ancestor of all the isolates is 1239 years ago (95% credible interval (CI) 444-2720 years ago). Our data reveal the intercontinental spread of lineage 1 over the last century, concurrent with anthropogenic movement of live fish, feed and ova for aquaculture purposes and stocking of recreational fisheries, whilst lineage 2 appears to have been endemic in wild Eastern Atlantic salmonid stocks before commercial activity. The high resolution of the SNP-based analyses allowed us to separate closely related isolates linked to neighboring fish farms, indicating that they formed part of single outbreaks. We were able to demonstrate that the main lineage 1 subgroup of R. salmoninarum isolated from Norway and the UK likely represent an introduction to these areas ~40 years ago. This study demonstrates the promise of this technology for analysis of micro and medium scale evolutionary relationships in veterinary and environmental microorganisms, as well as human pathogens.

Figure 1

Phylogenetic tree of the 68 isolates of R. salmoninarum included in this study, showing all lineages. The evolutionary history was inferred using a Bayesian Markov Chain Monte Carlo approach, with a generalized time-reversible model (Tavaré, 1986), through the MrBayes (Ronquist and Huelsenbeck, 2003) plugin in Geneious. The consensus tree is taken to represent the evolutionary history of the taxa analyzed. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All ambiguous positions were removed for each sequence pair. There were a total of 3600 positions in the final data set. The leftmost node represents a hypothetical most recent common ancestor. The above and bottom branches from this node represent lineages 1 and 2, respectively. Isolates are color coded according to the host: green, rainbow trout; red, Atlantic salmon; yellow, Chinook salmon; pink, pink salmon; teal, Grayling; gold, Coho salmon; orange, Eastern brook trout; brown, brown trout; gray, not known.

Figure 2

Detail of lineage 1. The phylogenetic tree was constructed as described under Figure 1. Posterior probability values are shown on each branch. For the detailed look at subgroup UK/NOR1, branches have been transformed so as to no longer represent evolutionary distance. Isolates are color coded according to their geographical origin: red, UK; blue, Norway; green, Canada; yellow, USA.

Figure 3

Ancestral state reconstruction of the geographical origin. Nodes in the trees have been estimated from a maximum parsimony evaluation of terminal values, where country of origin has been evaluated in an unordered, categorical matrix. The tree is not drawn to scale. The legend is as follows: red, UK; blue, Norway; green, Canada; yellow, USA. Nodes A, B and C are referenced in the text. The tree was calculated by using SNPs from open reading frames (ORFs) annotated as pseudogenes in the ATCC33209 genome.