Mycotoxin Biomarkers: 5 Challenges in Going from Lab to Farm

• Great strides have been made in researching the applications of mycotoxin biomarkers at the farm level.
• Challenges to on-farm utilization of biomarkers include timing of sampling; species-dependent differences in mycotoxin metabolism; unknowns in the field; variability in laboratory experience; and current lack of biomarker reference values.

Mycotoxins are well known to impair animal health and cause economic losses in livestock production. In the field, diagnosis of mycotoxin-induced disorders in animals is often challenging, as relevant feed lots are no longer available or feed analysis results are not conclusive. Driven by advances in analytical techniques, the application of mycotoxin biomarkers – assessing mycotoxin exposure directly in the animal by analyzing blood or other body fluids – is evolving at the farm level. Despite its potential, the on-field application of mycotoxin biomarkers still has major limitations. We discuss why it is currently tricky to interpret results and which aspects to keep an eye on.

Mycotoxins – toxic secondary metabolites of fungi – are natural contaminants of foods and feeds. A plethora of mycotoxins have been identified that pose a risk for human and animal health. Due to their frequent occurrence worldwide and their toxic effects, mycotoxins require regular monitoring.

Analysis of mycotoxins in feed and food commodities represents the most traditional monitoring approach. Many different methods have been developed to detect mycotoxins in feed, ranging from time-efficient lateral flow devices to comparably more sophisticated, but also more expensive, spectrometry-based methods. For the analysis of mycotoxins addressed by regulations or recommendations, laboratories can verify the accuracy of their method via reference materials or participation in proficiency tests.

However, even the most accurate analysis method can only provide meaningful results if the sampling procedure has been performed correctly – a potential pitfall of mycotoxin analysis in feed. Mycotoxins are not distributed homogeneously within feed lots, but they occur in hotspots. Therefore, the sampling process is crucial to obtain a representative sample and thus meaningful information about the mycotoxin contamination. The multitude of scientific data and field reports on the relationship between mycotoxin levels in feed and negative health effects in animals facilitates interpretation of data. Yet, under field conditions the time of sampling can be challenging the diagnosis of mycotoxicosis: feed lots might have been exchanged between the time of sampling and the onset of clinical signs and are therefore no longer available for analysis.

To circumvent the above challenges, the measurement of mycotoxins biomarkers in biological matrices of animals (e.g. blood, bile, urine, feces or tissue) has evolved. The underlying concept of assessing mycotoxin exposure directly in the animal is fascinating and would allow significant advances in the diagnosis of mycotoxicosis.

Mycotoxin biomarkers can be classified into two categories: mechanisms-based biomarkers and exposure-based biomarkers. Mechanism-based biomarkers refer to a biological response caused by mycotoxins, such as alterations in protein, enzyme or gene expression levels. This is exemplified by the effects of fumonisins on the sphinganine-to-sphingosine ratio, which is currently the best-known mechanism-based mycotoxin biomarker in livestock species. Exposure-based biomarkers describe the measurement of the mycotoxin itself and/or its metabolites in biological matrices, for example analysis of aflatoxin M1 in milk. Driven by the inherent specificity of exposure-based biomarkers (i.e. aflatoxin M1 presence in milk can only result from aflatoxin B1 ingestion) and significant advances in the field of mass-spectrometry, research on these biomarkers has intensified in the last years. New analytical techniques and instruments with high sensitivity has allowed scientists to unravel the fate of major mycotoxins in animals, i.e. how rapidly they are absorbed into the blood stream or to which metabolites they are transformed in the body. The next – and probably most eagerly anticipated – step is to transfer the knowledge gained under experimental settings to farms. However, the in-field application of mycotoxin biomarkers is currently not without major limitations. With an emphasis on exposure-based biomarkers in blood, the following aspects must be considered for the in-field application of mycotoxin biomarkers.

1. The right timepoint of sampling

First, mycotoxins represent a heterologous group of contaminants. Therefore, mycotoxins show considerable differences in their kinetic profiles, e.g. to which extent they are absorbed, how quickly they appear in the circulation and how fast they are eliminated from the body via urine or feces. In other words, a given timepoint might be ideal to detect one mycotoxin in blood but might be too early or even too late to catch another one. Unfortunately, the situation is complicated by the fact that the same mycotoxin can show different characteristics in different animal species. For example, for deoxynivalenol the extent of absorption as well as the timepoint when it reaches maximum levels in blood varies between pigs and poultry. This implies that – depending on the mycotoxin and species of interest – biomarker analysis should be planned and /or interpreted in relation to last feed intake, which is often impractical on the farm. Otherwise, biomarker analysis in blood bears the risk of underestimating mycotoxin exposure.

2. The right biomarker and matrix

Usually, the concentrations of exposure-based mycotoxin biomarkers are low in blood, i.e. in the low ng/mL range. For very poorly absorbed mycotoxins, such as fumonisins, it is practically impossible to detect them in blood. Here, analysis of feces would be more promising. In addition, mycotoxins are extensively metabolized after absorption, with the respective metabolite(s) often exceeding the levels of the parent toxin in biological matrices. Hence, it is essential to identify the right biomarker for each mycotoxin, taking into account species-dependent differences in metabolism. As such, the biomarker-matrix combination should be adapted to the mycotoxin of interest and target species. Unfortunately, the kinetics of many mycotoxins have not been fully elucidated in livestock species. This impedes, for example, the selection of suitable biomarkers for emerging mycotoxins.

3. Knowing the unknown

Even for major mycotoxins, such as deoxynivalenol, zearalenone or ochratoxin, factors influencing the levels of exposure-based biomarkers are poorly explored. Obviously, the amount of ingested feed (and therefore ingested toxin) affects biomarker concentrations. Consequently, sick or weak animals with reduced feed intake might show comparably low biomarker levels. There are indications that other factors, such as sex or age of the animal as well as co-exposure to other mycotoxins or feed contaminants, impact biomarker levels. Further research in this field will help explain intra-individual differences in the mycotoxin biomarker response. For example, even when exposed to the same feed lot and sampled at the same time point, individual animals of a group can show marked variations in biomarker levels. These variations currently limit the comparison of biomarker results among groups, production cycles or farms.

4. The right lab

Any biological fluid or tissue represents a complex matrix, and that can interfere with the analytical measurement. In contrast to mycotoxin analysis in feed, no reference materials or proficiency tests exist for the evaluation of mycotoxin biomarker methods. Thus, the chosen analytical method and the experience of the laboratory is of utmost importance for reliable mycotoxin biomarker analysis.

5. The right interpretation

As described, many factors need to be considered when analyzing mycotoxin biomarkers on farms. Perhaps the biggest hurdle to take is the establishment of reference values for mycotoxin biomarkers. Such reference or cut-off values are essential to interpret biomarker results adequately and to correctly deduce the health risk for animals. So far, research has failed to correlate levels of exposure-based biomarkers to clinical signs or the severity of mycotoxicosis. For example, the concentration of zearalenone and its metabolites in bile did not reflect the morphological changes in the reproductive organs of gilts.

Conclusion

• As analytical methods for the assessment of mycotoxin biomarkers have become more time- and cost-effective, scientific progress in this field will hopefully accelerate. Filled knowledge gaps and availability of larger datasets might facilitate the application of mycotoxin biomarkers at the farm level in future.  However, current limitations still impede the usefulness of this application. 

References

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Dänicke, S. & Brezina, U. 2013. Kinetics and metabolism of the Fusarium toxin deoxynivalenol in farm animals: Consequences for diagnosis of exposure and intoxication and carry over. Food and Chemical Toxicology, 60, 58-75.

Dänicke, S. & Winkler, J. 2015. Invited review: diagnosis of zearalenone (ZEN) exposure of farm animals and transfer of its residues into edible tissues (carry over). Food and Chemical Toxicology, 84, 225-249.

Den Hollander, D., Croubels, S., Lauwers, M., Caekebeke, N., Ringenier, M., De Meyer, F., Reisinger, N., Van Immerseel, F., Dewulf, J. & Antonissen, G. 2021. Applied Research Note: Biomonitoring of mycotoxins in blood serum and feed to assess exposure of broiler chickens. Journal of Applied Poultry Research, 30, 100111.

Renaud, J. B., Miller, J. D. & Sumarah, M. W. 2019. Mycotoxin Testing Paradigm: Challenges and Opportunities for the Future. Journal of AOAC International, 102, 1681-1688.