Mycotoxins are as ubiquitous as the fungi that produce them, and much more is now known about their geographic distribution, seasonality and multiple contamination risks. However, ensuring correct sampling, extraction and detection methods are crucial to the precise identification of contaminated feed materials.
By Dr Jules Taylor-Pickard, global Mycosorb manager, Alltech
Awareness of the inherent dangers that may potentially be found in any feedstuff is the key to ensuring these factors cause minimal impact on the animals consuming them. The previous decade has witnessed a great move forward in both the analysis and understanding of the risks posed by fungal toxins (mycotoxins) in animal feed.
Fungal spores colonise feedstuffs from a focal point and grow outwards from there, spreading through the material.
Most feed manufacturers, and, increasingly, those rearing animals, now understand that fungal contamination may occur at any level of feed production, ranging from infection of standing crops, poor harvest conditions, spoilage during storage, contamination from spores in feed processing machinery and in the finished feed. More research and the increased availability of data regarding the impact of both single and multiple mycotoxin contamination means that both veterinarians and farmers are now better equipped to recognise the often confusing symptoms displayed by affected individuals.
There is also the issue of toxins passing into animal-derived products, such as meat and liver. This has become an issue for human health, and some countries have already dealt with scares relating to toxins finding their way into the food chain.
Many countries in the world now routinely test imports for mycotoxins, and have set limits for acceptance (Table 1). Although it is clear that multiple mycotoxin contamination is the norm, and has a cumulative toxicity effectin vivo, most limits are determined by single toxin presence.
This is due to the expense and difficulty of analysis in most cases. There is also variation between the limits different countries set, with some not setting any, whilst others exercise ‘zero tolerance’, which, while laudable, can cause difficulties in feedstuff supply in poor harvest years where industry needs outstrip supply of suitable quality grain.
Because grain contaminated with more than one type of mycotoxin has a higher toxicity than single toxin presence, the limits should be adjusted downwards accordingly where multiple infections may be present.
The sampling and analysis of raw materials for fungal contamination can be difficult and expensive. In many global regions the responsibility for control of toxins in the food chain is in the hands of the importer or feed manufacturer. They have to determine the level of potential threat within a consignment or guarantee that the target animal (and the food chain as a whole) is protected from mycotoxicosis. Due to the amount of trade potentially affected by multiple contaminations, it is hardly surprising that single contamination is still the main assessment used.
Accurate analysis of mycotoxins is mainly governed not by methodology, but rather by difficulties in sampling. Fungal spores colonise feedstuffs from a focal point and grow outwards from there, spreading through the material. Sampling large grain consignments, for example, can be very difficult as these focal points may easily be missed. The best method of sampling for mycotoxins involves taking samples throughout a consignment, at all levels, as this is the best way to try to find any ‘hot spots’ of fungal growth.
Whitakeret al.(2005) discussed the importance of using correct sampling procedures, including defining suitable ‘sampling plans’ to reduce inaccuracies in detection of contamination. Random sampling, as well as adequate blending of the samples taken from consignments, is essential to obtain a true picture of potential contamination. The particle size of the material (e.g. kernel, grain or milled product) must be taken into account in order to ensure sampling probes collect a representative amount.
The larger the particle size (e.g. peanuts) the more difficult it is to obtain representative samples. Probes must also be suitable for the type of consignment, e.g. ship, HGV or tote bags. This ensures that every area of the batch can be reached and sampled suitably. Ideally, lots of small samples should be taken from as many locations within the batch as possible, as this will give a more accurate picture of contamination compared to a few, larger samples. These smaller samples can be combined together, finely ground and blended and then sub-sampled to give a representative test sample. This procedure will also minimise laboratory costs.
It is well known that visual assessment of feed materials is not a reliable method of gauging the presence of mycotoxins. Three methods of analysis, thin layer chromatography (TLC), high pressure liquid chromatography (HPLC) and ELISA, are now routinely used to screen feedstuffs for toxin contamination. These are used to test individual batches of feed materials on an accept/reject basis as well as for absolute levels in surveys to estimate relative risks from contamination worldwide. Of these methods, HPLC is more precise and sensitive than TLC, whereas ELISA offers rapid and relatively reliable yes/no detection.
More recently, combining gas chromatography with mass spectrometry has been used to detect specific individual toxins. Detection of low levels of toxins has been achieved by more novel methods, including antibodies, electrophoresis and immune-chemical methods, as well as via enzymes, bacteria, DNA, ‘electronic noses’ and genomic techniques. Choosing an appropriate method depends on the required results – hence quicker detection methods can be used to establish presence or absence, whereas more complex analyses may be used to determine the actual levels of contamination precisely.
The initial step in analysis involves solvent extraction of the feed material, which isolates the toxins. The choice of solvent is important in ensuring good recovery levels of mycotoxins. Other potentially interfering factors, such as oils and residues, must be removed by a process of centrifugation, filtration, drying and dilution before analysis for mycotoxins can be carried out. It must always be borne in mind that it is difficult to determine mycotoxin contamination with 100% certainty, due to sampling variation and the type of method used in analysis. Interference and the complexity of toxin breakdown products or other fungal metabolites within the sample can also make accurate analysis difficult.
Due to sampling errors, variation in detection and levels of contamination are common. For example, in a recent survey conducted in 2006-7, two commercial laboratories (Romer Lab and Alltech) analysed hundreds of samples of feedstuffs from Asia to evaluate the potential threat of contamination. Figure 1 shows the results for zearalenone, and the variation is clear in both the detection and relative levels of contamination found. The results for other single mycotoxins tested showed similar variability, highlighting the importance of sampling and that a low or ‘not detected’ result should not be taken as absence of risk. In practical terms, even though feed materials may have been analysed and found to be within safe limits, this cannot be guaranteed, and the use of an effective mycotoxin binder is still needed to minimise risk to livestock.
A truly global problem
Survey data from different global regions are now available and form a useful database in assessing relative risk, and also illustrate the different ways analytical methods may be applied to yield data relating to toxin exposure. Belgian laboratories published data in 2009 relating to levels of contamination in feedstuffs. They classified samples showing higher than 25 ppb for zearalenone and 100 ppb for DON as positive for contamination. In total, 35% of the samples tested positive for zearalenone (n=54) and 72% of samples tested positive for DON (n=87), with wheat and barley showing the highest levels of contamination.
In a Spanish study conducted in 2008 (Table 2), samples were assayed by ELISA to assess levels of contamination. The results were expressed as ‘positive for contamination’, i.e. where zearalenone, DON and T-2 levels exceeded 25 ppb.
Survey work with samples collected from across Asia in 2006-7 was completed by two laboratories using either ELISA or HPLC methods of detection. The results showed that multiple mycotoxin contamination was present in most of the feedstuffs analysed. The combined findings of the laboratories can be seen in Table 3.
The Asian surveys confirmed that many samples were contaminated with more than one toxin, which co-existed at high levels in some samples. By-products typically contained much higher levels of toxin contamination compared to whole raw materials.
The need for monitoring
Several factors regarding the global supply of feedstuffs in recent years have led to concerns regarding increased exposure to mycotoxin contamination. Unpredictable or severe weather (such as drought or heavy rain) can affect the production of mycotoxins from fungi growing on the plant material. Drought typically exacerbates toxin secretion, whereas grain harvested in wet conditions or inadequately dried following harvest is more likely to develop fungal growth, leading to subsequent toxin production.
As harvests and the price of feedstuffs have become more unpredictable, feed manufacturers and farmers have sought alternatives, often local solutions regarding feed ingredients. This has seen increased interest for by-products from the food and fuel industry, as well as copra and palm kernel meal, following oil extraction. Whilst useful as a feedstuff, care must be taken to ensure these materials have been suitably prepared and stored in order to minimise fungal growth. In many developing countries, which market the by-products of oil extraction locally or overseas, the material can be poorly processed, stored and handled, in a humid and hot environment which is ideal for fungal growth and cross contamination with toxins.
The manufacture of biofuels worldwide has lead to an increase in grain by-product availability as a feedstuff for animals. However, if these are inadequately dried after processing, they may harbour fungi leading to mycotoxin contamination. Biofuel by-products can be remarkably variable in their quality, due to both the way the fuel is produced and the care taken with the by-product afterward.
Methodology for mycotoxin analysis continues to evolve, making it possible to detect even small amounts in feed and animal products. It also enables a choice regarding what analysis is required, ranging from simple presence or absence to measured levels of individual toxins. Survey work regarding the types and levels of contamination in feedstuffs continues to evolve in its coverage and comprehensive nature, allowing traders, feed producers and farmers to more accurately identify risk elements in feed due to toxin contamination.