Engormix/Mycotoxins/Technical articles

Better scrutiny of mycotoxins with holistic approach of feedstuffs

Published on: 8/17/2012
Author/s : Swamy Haladi (Alltech Canada) and Luann Breeding, Lewis Jackson, and Alexandros Yiannikouris (Alltech Research HQ, Nicholasville, KY)
The significant research focus on mycotoxins in recent years has dramatically increased our awareness and understanding of mycotoxin nature, occurrence and impact on modern animal production. Mycotoxins in feedstuffs represent an unavoidable risk due largely to environmental factors. As a result, analytical tools using a holistic approach are needed to understand multiple mycotoxin contamination patterns. Application of Alltech's 37+ Program can be used as surveillance radar to monitor the overall level of mycotoxins present and estimate the level of risk associated with contamination.
Scientific literature, together with the analytical advances made to date, have shown that 500 metabolites can be referenced as mycotoxins. However, in practical terms, it is often the case that as few as between one and six major mycotoxins are monitored or analysed for, because the analytical approach used does not cover the analysis of a wider range of toxins. In Europe, aflatoxins, deoxynivalenol (a.k.a. vomitoxin or DON), fumonisins, T-2 toxin and zearalenone (ZEA) are often tested for in order to comply with regulatory limits or recommendations.
Thin Layer Chromatography (TLC) and ELISA are the common methods employed for these tests. These rapid techniques evaluate the contamination of raw material according to defined sampling plans. Although these methods are useful for the instant monitoring of the incoming raw materials, they do not provide an accurate representation of the overall mycotoxin challenge coming from the simultaneous presence of multiple groups of mycotoxins in a feed material. Estimation of the microbial population has also been investigated in order to evaluate the overall presence of toxinogenic species associated with mycotoxin production. However, poor correlations were found between mould and mycotoxin presence and contamination levels. 
Holistic approach
Understanding mycotoxin occurrence in a more holistic manner has obvious advantages. Since one mould can produce several mycotoxins and several mould species can be present in a given feedstuff, it is expected that there are, potentially, a substantially larger variety of mycotoxins present than being tested. To give an example, if a sample contains DON, it is likely that it may also contain several other DON-related metabolites including 3-acetyl-DON, 15-acetyl-DON and fusarenon-X, as well as masked forms of DON such as DON-3-glucoside. These toxins contribute to the toxicity of DON since deacetylation or deglycosilation occur in vivoand, consequently, increase the absolute amount of DON molecules present.
Not taking into account the incidences of these metabolites could result in underestimation of the DON contribution levels and associated toxicity. Ultimately, omitting such determination can provide an inaccurate estimation of the true level of mycotoxin contamination in feed and contribute to the appearance of unsuspected and/or unaccounted for animal production issues or pathologies. The ability to precisely analyse as many toxins as possible at a reasonable cost and in a timely manner can assist producers in better understanding and addressing the mycotoxin challenges they face.
Better scrutiny of mycotoxins with holistic approach of feedstuffs - Image 1
Better scrutiny of mycotoxins with holistic approach of feedstuffs - Image 2
 Alltech's 37+ Program
The newly developed Alltech 37+ Program can assist with the detection of over 37 mycotoxins using a holistic approach for the identification of the mycotoxin challenge. The objective of the 37+Program is to evaluate European feedstuffs for multiple mycotoxins using ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) methodology developed at Alltech's Global Research Headquarters, KY, USA. This approach represents a real breakthrough compared to other commercial methods that have a very narrow window of mycotoxin targets compared to the important variety of mycotoxins that can potentially contaminate a feed material.
More essentially, the methodological advances using UPLC-MS/MS can account for mycotoxin presence simultaneously in a more selective and sensitive way and in multiple feed matrices. Of course the dynamic range of activity for each toxin present in feed varies quite significantly. The mycotoxin levels need thus to be placed in the context of the practical concentrations that can induce a decrease in animal performances and/or pathological issues. 
Sample and analysis details
One hundred and four samples, from the 2011 harvest, collected from different regions of Europe were subjected to the analysis of 38 mycotoxins (Table 1). The major criteria for selecting these mycotoxins include their prevalence in the field as well as their established toxicological impact on animals. For ease of interpretation of the total toxicity to the animals, toxins were placed into groups according to their chemical properties and effects.
Only eleven samples out of 104 tested presented non-detectable values (below limit of detection) for all the mycotoxins tested - 89.5% of samples tested showed levels above the quantification limits. Type B trichothecenes were detected in 70% of the samples, followed by fumonisins (46%), Type A trichothecenes (22%) and Penicillium mycotoxins (22%). Aflatoxins, ochratoxins, ergot toxins and zearalenone toxins were detected between 4 and 16% of the samples tested.
When the averages for the entire dataset were calculated, fumonisins were the mycotoxins present at the highest concentrations (1,039 ppb) followed by Type B trichothecenes (760 ppb) and Penicillium toxins (179 ppb). The maximum concentration in a sample was recorded for fumonisins (40,000 ppb) followed by Type B trichothecenes (5,923 ppb) and Penicillium toxins (5,736 ppb). Although the concentrations of other groups of toxins were lower, it should be noted that the concentrations at which each group of toxins become toxic varies quite significantly. For example, the EU regulatory limit for aflatoxin B1is 20 ppb in pigs whereas the recommendation for DON is 900 ppb. 
The presence of Type B trichothecenes was expected due to temperate weather conditions in Europe favouring the growth of Fusarium species of moulds. Type A trichothecenes and Penicillium mycotoxins are also common to some parts of Europe (especially Eastern Europe). Fumonisins detection, however, was not expected. Fusarium moulds capable of producing fumonisins exclusively grow in the field especially following a warm summer in temperate areas. The presence of predisposing factors such as insect or pest damage, hail, rain etc. at harvest can further increase the incidence of all mycotoxins. These findings further exemplify the need for analysing feedstuffs for multiple mycotoxins rather than restricting the analysis to only aflatoxins and/or vomitoxin. The findings also support the need for the implementation of suitable strategies to counteract multiple mycotoxin challenges and not just one or two. 
Multiple mycotoxin profile
Only 10.5% of the samples tested contained no mycotoxins at quantifiable levels. Only one mycotoxin was detected in 24% of samples tested. The highest percentage of samples, 35.58%, contained three to five mycotoxins followed by 13.46% of samples containing five to 10 mycotoxins. Table 2 provides detailed mycotoxin analysis for various samples tested. Corn, corn silage, DDGS, barley and wheat were all predominantly contaminated with Type B trichothecenes and fumonisins. While small grain silage was contaminated with Type B trichothecenes and Penicillium mycotoxins, the major group of mycotoxins found in grass silage was Type B trichothecenes. This information will not only assist in understanding the mycotoxin contribution to final feed/TMR from various ingredients, but also helps in determining the safer inclusion levels of such ingredients. 
The use of Alltech's 37+ Program has allowed for an insight into the mycotoxin profile of European feedstuffs. Further accumulation of data will improve our understanding. The presence of multiple mycotoxins is a common phenomenon in the field and this can lead to mycotoxin interactions and, ultimately, increased risk for animal health and performance. The implementation of a prevention and detection program (based on HACCP principles) on farms and feed mills represents an integrated approach to control mycotoxin challenges.
This article was first published in All About Feed, Vol. 20 - N 3 - 2012. 
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