A metabonomic approach to identify biomarkers for bovine mycotoxicosis

Mycotoxins are fungal metabolites that can be present on grain, cereals, grass and conserved forage. Several risk factors increase the likelihood of mycotoxins and mycotoxicosis (disease in animals caused by mycotoxin ingestion), including high rainfall at crop flowering and during pre-harvest, hence wet summers such as 2012 result in high mycotoxin levels in grain. Although there is compulsory testing and legislative limits to concentrations of mycotoxins in grain destined for human consumption, there are no legal requirements for livestock feeds. The EU guidelines suggest maximum limits for Fusarium mycotoxins - Deoxynivalenol, and Zearalenone - for grains and complete feeds. These guidelines have been produced as a result of the major research area in mycotoxins concentrating on grain based mycotoxins (mainly Fusarium) using monogastric animal models e.g. pigs and poultry. However, this does not cover the broad spectrum of mycotoxins nor is it applicable to ruminants. Recent research has reported higher concentrations of mycotoxins can occur in straw than the associated grain samples. Silage (whole crop (grain and straw), maize and grass) the main forage source for dairy and beef cattle represents a significant source of mycotoxins, for which there is comparatively little research. This project will address this gap in scientific knowledge by investigating a broader spectrum of mycotoxins from the main fungal genera with a ruminant model system.

Due to the resilient microorganism diversity in the rumen, ruminants can withstand the effects of mycotoxins better than monogastrics, but this capacity may be compromised during times of stress (e.g. diet change or disease). In particular high production dairy cows are offered diets (cereals and grains rich in starch) which result in a change in the standard rumen microflora populated by fibrolytic (fibre degrading) bacteria in favour of more starch degrading organism which ultimately reduces the pH of the rumen. At this point the ability of the rumen to detoxify mycotoxins is substantially reduced. Guideline tolerance levels for different species have been proposed, but it is unknown how reliable these guidelines are. A recent veterinary survey in the UK showed a high incidence (10 - 80%) of mycotoxicosis in dairy and beef herds associated with sub-standard aerobically spoiled maize and grass silage when fed with cereal based rations.

The severity of mycotoxicosis depends on the mycotoxin type, animal health, stage of production and dose eaten. Some types damage organs directly (e.g. liver, kidney and rumen), whilst others impair reproduction or cause cancer. Physical effects range from performance loss to mortality. Different mycotoxins can interact in the rumen to exacerbate the effect and some are known to suppress immune function. Signs of mycotoxicosis in ruminant animals include loss of appetite, reduced milk yield or poor weight gain. Early veterinary diagnosis of mycotoxicosis is difficult due to a lack of specific symptoms and overlapping symptoms of other metabolic diseases such as acidosis in cattle. The problem does not end in animal disease or production losses as mycotoxins in the feed of dairy animals can lead to their metabolites appearing in dairy products, which pose a risk in human health, particularly for infants. A rapid early detection method of mycotoxicosis for ruminants is therefore required.

This project will produce a metabolite profile from urine, saliva and plasma of animals suffering mycotoxicosis to be used as biomarkers of the disease. This information in future more applied projects could be developed into a rapid diagnosis tool for veterinarians. As an industrial partner award project key industry members associated with animal feed, biochemistry and pharmaceutical products have already signed up to develop the basic science from this project into industrial development for maximum impact of the research.

Mycotoxicosis is a disease associated with livestock following ingestion of mycotoxins produced through fungal/mould contamination of feed. The disease manifests itself through reduced livestock performance reflecting in altered nutrient metabolism, endocrine malfunctions and immune-suppression. However it is difficult to diagnose at an early stage due to the commonality of symptoms with other production related diseases (e.g. SARA). Mycotoxin production is predominately associated with cereals and grains and hence there are current EU guidelines suggest maximum limits for two mycotoxins - deoxynivalenol and zearalenone - in feed grains and complete feeds. These specifically target reduction of mycotoxin contamination in human food and monogastric livestock feed (pigs and poultry) which are predominately fed on cereal and grains. However this does not cover the wider range of mycotoxins produced from not only fungal/mould contamination of grain and cereal but other feedstuffs e.g. straw and whole crop silage. It also does not investigate the impact of these mycotoxins on ruminants. A recent veterinary survey in the UK showed a high incidence of mycotoxicosis in dairy and beef herds associated with sub-standard aerobically spoiled maize and grass silage. The problem does not end in animal disease or production losses as mycotoxins in the feed of dairy animals can lead to their metabolic products in dairy products, which pose a risk in human health. This project will investigate the effect of mycotoxins associated with ruminant feed and mycotoxin ruminal metabolites on ruminal gastro-intestinal epithelial cells using metabonomic approaches (NMR). This will identify an array of key metabolites associated with mycotoxin poisoning of ruminants which will then be used in vivo to identify biomarkers in bodily fluid (plasma, urine and saliva) which could be used for early diagnosis of the disease.

Academic Impact -
As highlighted under academic beneficiaries the project will produce a high level of high impact academic findings. We will disseminate our results to the academic community by publications in high impact journals and presentations at conferences and meetings (in the areas of animal nutrition, cell biology, immunology, food research and microbial interactions). Duchy College are part of the project team who are experts in Knowledge transfer and outreach and will organise farm visit days and veterinary workshops where the latest more applied outputs from projects are disseminated to end users.

Economic and societal impacts -
It is envisaged that findings from this project will stimulate development of technology with support from UK and EU funding agencies, which will benefit our industry partners who are part of the Industrial Partnership Award as well as the wider industrial community.
The industry partners in the project include:

a) Micron-Biosystem Ltd. has a dedicated focus on researching techniques for the remediation of mycotoxin contamination in animal feedstuffs and holds patents and product registrations in this field. The company has recently completed the in-house development of methodologies based upon UPLC-MS/MS for the determination of a large suite of mycotoxins in a range of body fluids as well as feedstuffs. These techniques are being intensively utilised in both internal and collaborative R&D projects on three continents. Their involvement within the project will assist in the analysis of mycotoxins and their metabolites.

b) AB Vista Ltd. Is part of Associated British Foods (ABF) and is an international supplier of world leading feed ingredients and technical services. AB Vista's feed micro-ingredients are among the most advanced in the world and are sold across five continents. ABF is one of Europe's largest food companies employing 102,000 people in 46 countries around the world. Links with other companies in the ABF group allow consistent supply of reliable feed ingredients of the highest quality. These products help improve the nutritive value of animal feeds and help reduce the environmental impact while improving production efficiency, and end product quality. ABF's unrivalled expertise in animal feeds and experience of introducing new feed ingredients enables AB Vista to help customers optimise their returns when using its products.

c) Mole Valley Farmers Ltd. are a farmers co-operative which supply as well as feed and products on farm nutritional advice. Their nutritionists have significant experience of practical day to day on farm rationing, including issues such as mycotoxicosis and have regular veterinary contact. The nutritionist role also incorporates co-ordination of nutritional policy, evaluating new products and strategies, and liaising with Mole Valley Forage Services (MVFS). MVFS link forage production and rationing which have excellent geographical coverage.

The three industrial partners in the project will be instrumental in processing the information produced on mycotoxins biomarkers in ruminants into an applied project for industrial up-take. The industrial partners will each produce an exploitation plan which will form a key part of the projects pathway to impact

Further societal impacts from the project will involve improved farm animal health and reduced environmental pollution through increased knowledge at the farm level of the importance of high quality feed production. This will reduce feed waste and also reduce effluent loss from silage as farmers will be encouraged to produce high quality less mycotoxin containing silage. The findings will also ultimately benefit the general public by improving the safety of the food they consume as an early diagnosis of mycotoxicosis in animals will limit the chances of mycotoxins and their metabolites being transferred to animal products and subsequently into the human food chain.