The search for biomarkers of facial eczema, following a sporidesmin challenge in dairy cows, using mass spectrometry and nuclear magnetic resonance of serum, urine, and milk : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Sciences at Massey University, Palmerston North, Manawatu, New Zealand

Abstract

Facial eczema (FE) is a secondary photosensitisation disease of ruminants that is significant in terms of both its economic importance to New Zealand and its impact on animal welfare. The clinical photosensitivity signs, caused by the retention of phytoporphyrin, occur secondarily to hepatobiliary damage caused by the mycotoxin sporidesmin. Currently it is difficult to diagnose subclinical animals and those in the early stages of the disease. The project was aimed at applying new analytical and statistical techniques, to attempt the early diagnosis of FE in dairy cows following the administration of a single oral dose (0.24 mg/kg) of sporidesmin. Well-established traditional techniques including production parameters, liver enzyme (GGT, GDH) activity measurements, as well as measurements of phytoporphyrin by fluorescence spectroscopy were made for comparison. Serum, urine, and milk were analysed using 1H Nuclear Magnetic Resonance (NMR), multivariate analysis (MVA), and time series statistics. Urine and milk did not prove useful for identification of sporidesmin intoxication. Serum metabolites differed between treated cows before and after administration of the toxin, and could distinguish samples belonging to the clinical group. The metabolites that were identified as being relevant to this classification were a mixture of glycoproteins, carboxylic acids, ketone bodies, amino-acids, glutamate, and glycerol, which were elevated for treated cattle, and acetate, choline, isoleucine, trimethylamine N-oxide, lipids, lipoproteins, cholesterol, and -glucose, which showed decreased concentrations. Citrate was found to be at higher concentration in non-responders and subclinicals only. When serum was analysed using ultra performance liquid chromatography electrospray ionisation mass spectrometry (UPLC/ESI-MS) and UPLC tandem MS (MS/MS), only samples from clinical cows could be discriminated. The molecular ions involved could be tentatively identified as a combination of taurine- and glycine-conjugated bile acids. These bile acids all became elevated. This study confirmed that liver enzyme activities (GGT, GDH) and phytoporphyrin concentrations are not effective as markers of early stage sporidesmin damage. Additionally, the new techniques were unable to detect early stage FE. However, some markers of treated cows were identified. The research does provide a strong foundation for future applications of metabolomics analysis, with MVA and time series statistics, for early stage FE diagnosis