Masked Mycotoxins: Unveiling the Hidden Threat to Poultry Health

Dr. Md. Emdadul Haque, Dr. Prateek Shukla, Dr. Venket Shelke & Dr. Partha Das
Kemin Industries South Asia Pvt. Ltd.
Introduction:
Mycotoxins, secondary metabolites produced by fungi such as Aspergillus, Fusarium, and Penicillium, pose a major challenge in poultry production. Common toxins like aflatoxin B1 (AFB1), ochratoxin A (OTA), T-2 toxin, deoxynivalenol (DON), and zearalenone (ZEN) frequently contaminate feed ingredients, leading to reduced feed intake, poor growth, organ damage, immunosuppression, and even mortality.

While free mycotoxins are well-studied, masked mycotoxins, modified forms created by plants or during processing, are often overlooked. These conjugated toxins evade standard detection methods but can revert to their toxic parent form during digestion, posing a hidden risk to poultry and potentially humans.

This review aims to consolidate current knowledge on the occurrence, toxicity, detection, and effects of masked mycotoxins, highlighting the importance of understanding both free and masked forms and their impact on poultry.

Masked mycotoxin (Conjugated or hidden mycotoxin)

Masked mycotoxins are structurally modified derivatives of parent toxins, typically formed by plants as a defense mechanism. Common modifications include glucoside or sulfate conjugation, which changes their molecular mass, solubility, and polarity, making them undetectable by routine tests, exploring the different mycotoxins and their Masked versions in poultry in Figure 1.

Examples:

• DON-3-glucoside (D3G): a plant-conjugated form of DON.
• ZEN-14-sulfate: a modified form of ZEN.

Despite being less toxic initially, these compounds can hydrolyze back to their parent toxins in the gut, restoring full toxicity.

Formation and Plant Defense

Plants have special systems to protect themselves from harmful chemicals, including natural toxins like mycotoxins (toxins from fungi). They mainly use two strategies:

1. Chemical Modification (Phase I, II & III reactions):

• Phase I reactions: Break down toxic substances, usually by oxidation or hydrolysis (breaking chemical bonds using water). This is done by enzymes like cytochrome P-450. However, sometimes Phase I products can be more toxic than the original toxin.

• Phase II reactions: Attach (conjugate) small, water-soluble molecules like glucose, malonic acid, or glutathione to the toxin. This reduces toxicity and makes it easier for the plant to move the toxin for storage or removal. Schematic representation of the biochemical pathway leading to masked zearalenone formation in plants and its subsequent hydrolysis within the gastrointestinal tract is shown in Figure 2.

 Figure 1: Exploring the Different Mycotoxins and Their Masked Versions in Poultry

 Compartmentation:

• After chemical modification, toxins are moved into safe storage areas like vacuoles or the apoplastic space using membrane transporters.

Breeding for Fusarium resistance often increases DON conversion to D3G, linked to genes like Fhb1 (Fusarium head blight resistance gene 1) and UGT (UDP-glucosyltransferase). 

Figure 2: Scheme of formation of masked zearalenone in plants and zearalenone release in the digestive tract

Toxicological Considerations of Masked Mycotoxins:

• Lower toxicity in masked form: D3G is less harmful than DON; ZEN-glucoside is less estrogenic than ZEN.
• Risk during digestion: Gut microbes can break down masked forms, releasing parent toxins.
• ZEN-glucoside → ZEN (rapid absorption and excretion).
• D3G → DON (partial hydrolysis by gut bacteria).

Thus, masked toxins can increase total toxin load despite being undetectable in feed analysis. Impact of mycotoxin on poultry in Figure 3.

Figure 3: Impact of Mycotoxin in Poultry

Analytical Aspects of Masked Mycotoxins:

Plant-modified or conjugated forms of toxins in poultry feed are a significant analytical challenge. These compounds often evade conventional detection because their chemical structure differs from that of the parent toxin. Analytical approaches vary depending on whether the toxin is free, masked, or bound to plant matrices, and each method has inherent limitations.

Key Analytical Approaches

1. Direct Methods (Detection of Masked Forms Without Breakdown)

• LC-MS/MS (Liquid Chromatography–Tandem Mass Spectrometry):
  Considered the gold standard for accuracy and sensitivity. LC-MS/MS typically employs simple “dilute-and-shoot” sample preparation combined with reversed-phase chromatography for separation. This technique is highly effective for identifying masked mycotoxins in complex feed matrices.
• TLC (Thin Layer Chromatography):
  A rapid and cost-effective screening tool widely used in resource-limited settings and for fungal culture analysis. However, TLC lacks sensitivity and is unsuitable for detecting masked mycotoxins present at low concentrations.
• GC (Gas Chromatography):
  Applicable for quantifying certain mycotoxins such as trichothecenes, ZEN, OTA, and fumonisins. However, GC is impractical for masked forms because derivatization is required to make these compounds volatile, adding complexity and cost.
• Fluorescence Detection:
  Useful for specific derivatives (e.g., ZEN conjugates), but its application is limited to certain toxin classes.

Immunochemical Methods [e.g., ELISA (Enzyme-Linked Immunosorbent Assay)]:

• ELISA offers a quick and economical screening option. However, cross-reactivity of antibodies with masked forms or structurally similar compounds can lead to overestimation of toxin levels. Furthermore, the lack of reference standards for most masked toxins complicates accurate quantification.

2. Indirect Methods (Conversion of Masked Forms to Parent Toxins)

• Hydrolysis Techniques:
  Acid, alkaline, or enzymatic hydrolysis is employed to cleave conjugated bonds, releasing the parent toxin. Post-hydrolysis, conventional detection methods such as LC-MS/MS (Liquid Chromatography–Tandem Mass Spectrometry or ELISA can quantify the total toxin load (free + masked).
• Limitations:
  While hydrolysis enables estimation of overall contamination, it cannot differentiate individual masked forms. Additionally, standardized hydrolysis protocols and certified reference materials are lacking for most masked mycotoxins, except for a few, such as DON-3-glucoside.

Direct methods provide specificity but are hindered by the absence of reference standards and high costs. Indirect methods offer a practical approach for total toxin estimation but sacrifice structural detail. For comprehensive risk assessment, a combination of advanced LC-MS/MS techniques and hydrolysis-based screening is recommended.

Mitigating the Risk of Masked Mycotoxins in Poultry Diets

Masked mycotoxins—structurally modified forms of parent toxins—pose a hidden challenge because they often escape conventional detection yet can revert to their toxic forms during digestion, impacting poultry health and productivity^5,6. Effective mitigation requires a multi-layered approach combining prevention, advanced feed additives, and supportive strategies.

1. Preventive Measures

The first line of defense is reducing fungal contamination at the source:

• Pre-harvest: Use resistant crop varieties and proper agronomic practices to limit fungal colonization.
• Post-harvest: Ensure thorough drying, maintain low moisture, and store grains under controlled conditions.
• Physical cleaning: Sorting and removing visibly contaminated grains reduces overall toxin load.

2. Rigorous Quality Control

Routine testing of raw materials and finished feed using advanced analytical techniques such as LC-MS/MS is essential for detecting both free and masked mycotoxins. This enables early intervention and corrective action.

3. Mycotoxin Binders

Since masked mycotoxins can hydrolyze back to their toxic parent forms in the gut, good-quality broad-spectrum binders are critical for reducing bioavailability.

Ideal binder characteristics include high binding capacity, stability under digestive conditions, minimal nutrient interaction, and proven in vivo efficacy.

Advanced solutions like TOXFIN™ 360° utilize patented STS technology for:

• Nano-porous structure enabling rapid and strong binding.
• pH-resistant performance for consistent efficacy.
• Negligible nutrient interaction.

4. Supportive Nutritional Strategies

While binders reduce absorption, supportive additives help minimize secondary effects and enhance detoxification:

1. a) Antioxidants

• Vitamins C & E, selenium, and zinc counter oxidative stress caused by mycotoxins.
• Plant extracts like silymarin support liver health and reproductive performance.

1. b) Probiotics & Prebiotics

• Improve gut integrity and reduce toxin bioavailability.
• Beneficial microbes such as Lactobacillus spp. alleviate the harmful effects of AFB1, ZEN, and DON.
• Bacillus subtilis fermentation extracts detoxify OTA into non-toxic OTα, improving immunity and kidney health.

1. c) Enzyme-Based Detoxification: Enzymes play a critical role in breaking down complex mycotoxin structures, including masked forms:

• β-glucosidase cleaves ZEN-glucosides (Z14G), releasing ZEN.
• Amylolytic enzymes (α-amylase, amyloglucosidase) and proteolytic enzymes (papain) enhance DON release from bound forms.
• Cell wall-degrading enzymes (cellulase, xylanase) assist in breaking plant matrices, improving toxin accessibility.

Impact on DON release⁵:

• Amylases increased DON release by 28%.
• Papain increased DON release by 19%⁵.
• Cellulase had only a minor effect⁵.

These enzymes help convert masked mycotoxins into free, more detectable forms, reducing their toxicity and improving feed safety.

Key Takeaways

• Masked mycotoxins, modified forms of toxins produced by fungi, pose a hidden threat to poultry health. These toxins, often undetectable by traditional methods, can be converted back into their toxic forms during digestion. Hence, they require integrated control strategies: prevention, advanced binders, and supportive additives.
• Broad-spectrum toxin binders remain the primary defense, while enzymes, antioxidants, and probiotics provide additional protection.
• Combining these approaches ensures better bird health, improved performance, and reduced economic losses.

 References are available upon request