Laboratory approaches for expanding the understanding of an emerging strain of Erysipelothrix rhusiopathiae in the Arctic

Recent investigations into mass mortality events in the Canadian Arctic Archipelago have discovered that one singular ‘Arctic clone’ (Ac) of Erysipelothrix rhusiopathiae has been causing a large number of muskox dieoffs. There is limited understanding of how this strain of the bacterium is maintained and transmitted, or why it is causing such high mortality in this wildlife population. Evidence suggests that this strain could be more virulent than other strains of E. rhusiopathiae. The aim of this project was to contribute to the further understanding of the Ac in the Canadian Arctic. Advances in animal welfare have redefined the ethics behind the use of mammalian models for scientific research. An emerging invertebrate disease model, using Galleria mellonella larvae as a host, has gained increasing recognition. The immune system of the larvae is similar to the mammalian innate immune system. Several successful examples using this model in studies of bacterial virulence suggest that this could be a useful tool to test the virulence of the Ac compared to nonArctic clones (Nac) of E. rhusiopathiae. Since there were no prior investigations conducted on E. rhusiopathiae using this model, my first data chapter serves as an initial evaluation of its use. The experiments included the measurement of the growth kinetics and the inoculation of G. mellonella larvae in biological triplicate. Kaplan-Meier survival curves were generated to illustrate the larvae mortality over the course of the experiment. Up to 2.08 × 106 colony forming units of either Ac or Nac were injected into each larva but did not cause significant mortality during the 9-day observation period. The only experimental group that showed a significant difference was the undiluted Nac group (p-value of 0.049). The limited mortality observed across the majority of the test groups, and limited statistically significant differences in mortality in the treatment versus control groups (as shown using a Cox proportional hazards model) suggests that the G. mellonella is not a suitable model for studying the virulence of E. rhusiopathiae or the Ac. My results suggest that future studies to assess virulence of E. rhusiopathiae would best utilise other in vivo models. The outbreak investigation of E. rhusiopathiae in muskoxen in the Canadian Arctic Archipelago has raised the need for a rapid and sensitive detection assay, particularly for identifying strains belonging to the Ac. I evaluated the design and implementation of a triplex qPCR for simultaneous identification of three strains of interest. Signal leakage and limit of detection testing was conducted. An investigation was conducted to determine the cause of apparent false amplification of the Ac target in four outlier isolates. These ‘imposter’ isolates had cycle threshold (Ct) higher than other Ac isolates on the yellow channel; this observation was used to establish a trend between amplification of the E. rhusiopathiae and Ac targets to help spot future false positives or co-infections. Digital PCR established that true Ac isolates had around 500-600 copies/µL of the Ac target while the imposters had around 0.3 copies/µL and sequencing results from whole genome sequencing of the ‘imposter’ isolates were used to confirm the findings (i.e., that these isolates lacked the Ac-specific sequence) and concluded that the imposters were likely showing late amplification due to earlier contamination of the DNA extracts. The triplex qPCR I developed was further used for strain identification in DNA extracted directly from 367 samples collected from animal carcasses and carcass sites on Ellesmere Island and Axel Heiberg, where recent muskox die-offs occurred. Nearly 90% (n=329) of the samples tested were positive for Ac. Further optimisation is necessary for validation of the assay in different sample types and target concentrations, especially for samples with lower concentrations of the pathogen’s DNA. Overall, my Masters work has demonstrated that the G. mellonella larvae model is not suitable for E. rhusiopathiae infections at the studied conditions, helping redirect future virulence studies of the Ac towards other models. The development of the new triplex qPCR assay has provided a rapid, sensitive and cost-effective alternative to identify Erysipelothrix species and distinguish between Ac and non-Ac strains in infections in the Canadian Arctic.

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