Metabolomics in veterinary medicine: Untargeted metabolomic analysis of porcine reproductive and respiratory syndrome virus and targeted metabolomic analysis of Salmonella Enteritidis

Ophoff, Aislinn
Major Professor
Paul J. Plummer
David J. Borts
Committee Member
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Veterinary Microbiology and Preventive Medicine

Metabolomics is an emerging diagnostic method within veterinary medicine used to examine small molecule metabolites within bodily samples. Neither targeted nor untargeted metabolomic methods have been widely used in veterinary medicine, especially in the areas of PRRSV vaccination biomarkers or targeted metabolite kits in Salmonella Enteritidis pathogenicity.

In order to evaluate the utility of untargeted metabolomics, the use of readily collectable serum and oral fluid samples were compared to characterize the metabolic changes in response to PRRSV vaccination. The samples were extracted using an RPLC method on a Thermo Fisher Dionex Ultimate 3000 UHPLC and Q Exactive Focus Quadrupole-Orbitrap mass spectrometer instrument. Mass to charge ratios (m/z) were analyzed and found to be significantly different across the experimental time course. M/z were compared to known metabolomic databases and several metabolites were identified as putative biomarkers for disease status including cytidine for serum metabolites, and protocatechuic acid and guanine for oral fluid samples. This project provides a basic proof-of-concept for the application of untargeted metabolomics in veterinary diagnostic medicine, including the application of these approaches to novel oral fluid samples. However, a number of challenges were identified that will limit the approach and additional research is required to validate the utility and reliability of these metabolites as a biomarker diagnostic test.

In order to demonstrate an alternate approach to veterinary infectious disease monitoring, a targeted metabolomic analysis method was used to examine poultry plasma prior to and following inoculation with Salmonella enterica serovar Enteritidis. The targeted metabolomic kit, Biocrates AbsoluteIDQ® p400 HR Kit, was used to process poultry plasma samples and ran on similar mass spectrometer instrumentation to that of the untargeted approach. The metabolite kit successfully provided the concentrations of known metabolites within the samples. Data analysis determined a significant difference in the metabolite profiles when comparing control and inoculated chicks, as well as between the different lines of chicks. The following metabolites were found to be of major significance between the samples by PLSDA and sPLSDA analysis: Cholesteryl ester 20:4, Hexoses, Glutamine, Cholesteryl ester 18:1, Lysophosphatidylcholine 18:2, Proline, and Phosphatidylcholine 34:2. Further analysis is required to determine the metabolic function of each of these metabolites, as well as their role in Salmonella infection.

These studies confirmed that metabolomics approaches have utility in veterinary diagnostics as a tool of preventative medicine. However, the studies also identified some limitations with each method including the difficulty in matching untargeted metabolomic data to database networks, in addition to overall cost of metabolomic analysis. The outcomes demonstrate the need for researchers to carefully evaluate the goals of their proposed metabolomic projects and select the correct platform that is most likely to yield actionable data.