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Analysis of the Pimelea toxin simplexin for the development of a cattle microbial probiotic
Zhi Hung Loh * 1 , Natasha L. Hungerford 1 , Diane Ouwerkerk 1, 2 , Athol V. Klieve 1 , Mary T. Fletcher 1
1  Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Health and Food Sciences Precinct, Coopers Plains, QLD 4108, Australia
2  Agri-Science Queensland, Department of Agriculture and Fisheries (QDAF), Ecosciences Precinct, Dutton Park, QLD 4102, Australia.

Published: 14 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster
10.3390/IECT2021-09149 (registering DOI)
Abstract:

Pimelea poisoning of cattle (also known as St. George or Marree disease) is a poisoning unique to Australia and caused by inadvertent grazing of native Pimelea within pastures. The toxin responsible for the poisoning was previously isolated and identified as the novel diterpenoid orthoester simplexin, but no effective treatments for poisoned animals exist. A previous feeding trial reported that cattle fed daily with increasing low doses of simplexin showed reduced poisoning symptoms over time which suggested cattle developed resistance against the toxin, potentially via by the adaptation of rumen microorganisms. To date, there are no reports on simplexin degradation by rumen microorganisms.

This study aims to develop a microbial probiotic derived from the rumen fluid of field-exposed animals that is capable of detoxifying simplexin, thus allowing cattle to consume Pimelea with less adverse effects. Investigations are ongoing to identify rumen bacteria able to hydrolyse simplexin in in vitro mixed rumen-based anaerobic fermentations fed daily with Pimelea plant species (P. trichostachya) and to assess isolated rumen bacteria in in vitro incubation trials. Simplexin levels in both studies were analysed by ultra-performance liquid chromatography coupled with high-resolution, accurate mass spectrometry (UPLC-MS/MS) which allows simplexin quantification at ppb concentrations (ng/mL) on a Thermo Scientific Q-Exactive Orbitrap mass spectrometer. Results to date showed decreases in simplexin levels, suggestive of simplexin detoxification by rumen microorganisms. Simplexin acid hydrolysis studies were also performed to create a metabolite database to aid in future elucidation of potential simplexin degradation pathways. UPLC-MS/MS analysis based on predicted molecular formulae enabled identification of three hydrolysed simplexin products which also shared several fragment ions with simplexin. Future studies will include the identification and characterisation of simplexin metabolites from fermentation and incubation trials in which simplexin levels indicate that degradation has occurred.

Keywords: plant toxins; probiotic; rumen microorganisms; metabolism; degradation
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