Mycotoxins in ruminant production:Part 2: Improving hazard assessment
Accurate hazard analysis must extend beyond feed test concentrations for a limited number of individual toxins, and recognize the limitations of commercial feed analysis
By Cathy Bandyk, PhD, PASAssessment of mycotoxin risk is frequently limited to sampling one or more feedstuffs of possible concern, having them analyzed for a standard package of toxins at a commercial lab, and comparing the results for individual compounds to a chart that assigns an assessment rating (e.g., low, medium or high risk) to concentrations above or below specific break points. These ratings are often accompanied by feeding recommendations or limitations. While this may be a valid starting point, it does not account for all potential sources of mycotoxins, for limitations of feed testing, for the damage caused by chronic (low-level, long-term) exposure, for the additive or synergistic impact of exposure to multiple toxins, or the stacking of mycotoxin challenges on top of other stressors.
Overlooked hazardsMost mycotoxin exposure is via feed, but animals can also take in these substances through physical contact and inhalation (Whitlow and Hager, 2007; Gallo et al., 2015). There may be instances where these delivery routes help push the total toxin load above the safety threshold for detoxification or other natural defenses. Recent work (Rangel-Muñoz et al., 2023) has also demonstrated that flies can serve as vectors for toxigenic fungi, contaminating both grains and forages. In this way these ubiquitous pests can set the stage for creation of mycotoxins, even in feedstuffs that were previously determined to be “clean.”
Feed testing: An imprecise and partial pictureA major challenge, especially when testing individual ingredients, is obtaining a representative sample. Mold typically grows in isolated pockets, so any mycotoxins formed will be highly concentrated in those areas. This makes it relatively easy to collect samples that test much higher or lower than the actual average for the lot, bin, pit, or load, even when following recommended sampling protocols. Additionally, all major dietary components are potential mycotoxin carriers, and need to be considered. Both these concerns can be addressed by testing mixed rations, but that removes any chance of utilizing results to modify feeding strategies, or, often, of even receiving results before the feed in question is used and gone. With grazing cattle, it can be especially difficult to collect samples that accurately reflect the plants and plant parts that animals are selecting and consuming.
Analytical options for detecting mycotoxins in feed fall into two main categories: rapid screening and confirmatory analysis. As their name suggests, rapid methods can provide nearly real-time information, but this may be no more than a yes/no determination of whether certain compounds are present in a feed sample or not. Most of these methods are only suitable for certain sample types, are limited to detection of a limited number of mycotoxins they are designed to recognize, and they carry a greater risk of false positive or negative results. Technologies utilized include immunoassay-based methods (e.g., enzyme immunoassay [ELISA], fluorescence immunoassay [FIA], flow-injection liposome immune-analysis, and lateral flow devices); sensors and biosensors (e.g., molecularly imprinted polymers); thin-layer chromatography (TLC); Fourier transform infrared spectroscopy (FTIR); and, near-infrared spectroscopy (NIR) (University of Kentucky, 2022). These tests are best used as a relatively low-cost opportunity to screen for a potential problem before conducting more comprehensive testing.
The standard confirmatory methods include high performance liquid chromatography (HPLC) paired with various detection methods such as fluorescence or mass spectrometric (LCMS or LC-MS/MS), and gas chromatography for a few select mycotoxins. They offer high specificity and sensitivity, and provide quantitative information on individual toxins. However, these analyses involve expensive instrumentation, skilled technical operation, a longer turn-around time, and greater cost.
These tests are calibrated to determine concentrations of a standardized list of specific mycotoxins, which can vary somewhat between labs. They may not pick up toxins that are masked, and they cannot provide information on any of the emerging mycotoxins that are not screened for. Some feed mycotoxins may be conjugated -- that is, bound to another substance such as a sugar – and therefore ‘masked’ and undetected by methodologies that are specifically looking for pure substances (Whitlow and Hagler, 2007). The so-called ‘emerging’ mycotoxins are not a newly emerging problem per se, but are fungal toxins that have been more recently described and studied.
Several hundred different mycotoxins have been identified, but little is known about most. Beauvericin and enniatins were being discussed in the literature as early as 1996 (Fink-Gremmels 2018), but a lack of data and understanding of their mode of action precluded routine consideration of their possible role in mycotoxicosis. In his 2015 review, Gallo emphasized the diversity of fungal species that can produce mycotoxins, citing a total of 15 potentially toxigenic genera; most research efforts have focused on just three: Aspergillus, Fusarium, and Penicillium. Recent research does show that at least some of these compounds can be as dangerous as those that have been more thoroughly studied and regulated (Chiminelli et al., 2022; Xu et al., 2023; Vidal, 2023).
Clearly, traditional feed testing has offered an incomplete evaluation of mycotoxin exposure, metabolism, and subsequent risk. Alternatively, the use of biomarkers offers the potential to directly measure animal exposure to a broad range of mycotoxins and their metabolites (Vidal, 2023). While not yet widely available, analysis of body fluids yields quantitative evidence of actual on-farm levels of toxin exposure. Researchers have evaluated plasma, urine, feces (Lauwers et al., 2019) and blood as sample matrices; this work led to development of a validated biomonitoring protocol combining feed and blood spot testing (MycoMarker®, Innovad; Lauwers et al., 2019b). Field application of this program has enabled producers in multiple countries to obtain reliable exposure information for 36 toxic compounds, including key emerging mycotoxins. It is worth noting that in a summary of more than 1,000 ruminant blood samples subjected to the MycoMarker analysis discussed above, emerging mycotoxins (Beauvericin, Enniatin B1, Enniatin B, Tenuazonic acid, and Enniatin A1) were the five most commonly detected (Vidal, 2023).
Chronic vs acute exposureAcute poisoning from mycotoxins is rare in ruminant livestock; health, reproduction and performance losses are most commonly due to long-term, low level intakes (Whitlow and Hagler, 2007; Jiang, 2021). This chronic exposure has been shown to take a toll on reproductive efficiency, liver health, immune function, disease susceptibility, and productivity (Jiang, 2021) as well as alter endocrine systems (Hernandez-Valdivia et al., 2021). In a 26-month study with dairy cows, consumption of a diet naturally contaminated with low levels of Aflatoxin (mean 8.1 μg/kg DM) led, over time, to changes in blood serum markers, increased incidence of abortions, and longer calving intervals (Hernandez-Valdivia et al., 2021) . The authors emphasized that the observed changes in biochemical parameters were seen in apparently healthy cows. When there is a similar lack of identifiable symptoms accompanying ongoing exposure at the farm level, it can set the stage for viewing mycotoxin-impaired health and performance levels as ‘normal.’ Additionally, diseases resulting from mycotoxin-compromised immune function are likely to be seen and managed as primary rather than secondary infections.
One plus one is greater than two Mycotoxin studies are one of the few areas of research where field challenges almost always elicit greater and more consistent responses than controlled research, even when comparing similar doses of a toxin of interest (Fink-Gremmels, 2008). This can be largely attributed to the fact that natural contamination is seldom limited to a single mycotoxin, and it is well established that exposure to multiple mycotoxins can alter their expected affect on animals (Whitlow and Hagler, 2007). The additive and synergistic action of co-exposure can lead to toxicosis, even when individual mycotoxins are present at “safe” levels (Grenier and Oswald, 2011).
This concept was demonstrated when mid-lactation Holstein cows were dosed with a precise amount of Aflatoxin B1 (AFB1), provided in a pure form or as unpurified Aspergillus parasiticus culture (Applebaum et al., 1982). The culture also contain lesser amounts of other aflatoxins and metabolites. While the ‘impure’ treatment resulted in a significant reduction in milk yield, the purified AFB1 did not.
Dry matter intake, milk production, and multiple blood parameters were evaluated in dairy goats fed a control diet, or the same diet plus purified AFB1, either alone or in combination with ochratoxin, zearalenone, or both (Huang et al., 2018). The three-way combination consistently led to more severe outcomes in the metrics measured, and was the only treatment that differed significantly from control for feed intake (lower), white blood cell count (higher) and red blood cell count (lower). Milk yield was decreased for the aflatoxin-only treatment, but was even lower for the two-way combinations, and lowest (a 13.7% reduction) with exposure to all three. Serum alkaline phosphatase, an indicator of liver damage, exhibited modest numerical increases in the 1- and 2-toxin treatments, but nearly doubled when all three were given.
Mycotoxins are capable of causing reductions in nutrient availability, immune status, liver function, and gut integrity, which can leave animals more susceptible to pathogenic and metabolic disease. Conversely, animals suffering from stress- or pathogen-induced dysbiosis or disease are less able to cope with mycotoxin challenges. Regardless of the sequence of these events, when mycotoxin exposure is stacked with other stressors (metabolic, environmental, infectious, and/or social) it poses a heightened risk.
The magnitude of mycotoxin-related challenges experienced by an animal is determined by multiple contributors and their interactions. Accurate hazard analysis must extend beyond feed test concentrations for a limited number of individual toxins, and recognize the limitations of commercial feed analysis and the potential contribution of chronic and co-exposure and influence of other stress factors.
References available upon request.
Part 2 of a 4 part series. The final articles in this series will look more specifically at the costs of mycotoxicosis, and review available mitigation strategies.