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Animal Nutrition Conference of Canada 2021
The following technical article is related to the event::
Animal Nutrition Conference of Canada 2021

Calls for a Transformation in Measuring and Combatting Mycotoxin Related Stress: New Evidence using Biomarkers in Biological Fluids and Mycomarker®: A Novel, Data-driven, Mycotoxin Diagnostic Service in Feed and Animal Blood

Published on: 10/21/2021
Author/s : Dr. Christos Gougoulias / Innovad SA, Postbaan 69, 2910 Essen, Belgium.
Summary

Mycotoxins in feed and relevant crop (field) surveys have been major focuses within livestock production until today. However, the lack of evaluation of the true impact of detoxifier products on systemic levels of mycotoxins in animals and possible correlation to their health status is evident.

We here report significant systemic reduction of aflatoxin B1 (AFB1-40%) and deoxynivalenol (DON-50%) in broiler plasma, under concomitant administration of high concentrations of a mycotoxin blend [DON at 5 mg/kg feed i.e. the maximum permitted level by the European Food Safety Authority (EFSA), AFB1 at 20 mg/kg feed i.e. 1000x higher, and ochratoxin A (OTA) at 2.5 mg/kg feed i.e. 25x higher than the maximum safe EFSA guidelines] and detoxifier dose (2.4g/kg feed) studied. A similar trend of systemic reduction of DON-Glc-A in plasma and DON in urine was seen in pigs, under concomitant administration of DON (0.9 mg/kg feed i.e. the maximum permitted level by EFSA) and zearalenone (ZEN at 75 mg/kg feed i.e. 750x higher than the maximum safe EFSA guidelines) suggesting thus, significant beneficial bioactivity. Additionally, we report the successful transfer of the analytical methodology from plasma to dried blood spots (r>0.947), whereby a drop of blood is collected on a filter paper. Advantages include the sampling itself, reduced animal invasiveness, transportation and increased storage. This has now been transferred in real field conditions with a novel service ‘Myco-marker®’. This supportive diagnostic tool assesses, both in feed & blood, the overall impact of mycotoxins on animal health and performance with high sensitivity (LC-MS/MS).

Introduction
Globally, the ever-increasing demand for protein consumption (via meat production) in a sustainable manner, requires further reduction of the overall production cost. Feed cost remains key to this. However, variability of raw material costs and the complex load of mycotoxins contamination continue to negatively impact productivity. 
On top of that, animal health and productivity are affected by a number of other stress factors including low quality of feed, diet composition changes, heat, vaccinations, infections etc. In addition, modern farming animals possess limited natural resistance and immunity against such stresses. Mycotoxins worsen the impact of such stress factors and tend to impair key metabolic organs such as the liver and kidneys. The negative impact of mycotoxins expands to defence mechanisms and the immune system. For example, DON and fumonisins increase the paracellular gut permeability and predispose to the development of Clostridium perfringens-induced necrotic enteritis in broilers (Antonissen et al. 2014; Antonissen et al. 2015) whereas, DON in pigs inhibits vaccination efficiency of PRRSV live vaccines (Savard, Gagnon, and Chorfi 2015).
Presence of mycotoxins in grain is widespread and it is estimated that ~90% of samples contain mycotoxins and nearly 40% contain multiple mycotoxins, which can exert a synergistic negative effect to animals (Kovalsky et al. 2016; Eskola et al. 2019).
For appropriate feed risk assessment, several mycotoxins and their fungal and plant metabolites need to be quantified with high precision. In that respect, quick and often cheap analyses tools such as Lateral Flow Devices (LFDs) and ELISAs are considered incomplete approaches, when used in isolation. The problematic sampling of raw materials with the known ‘hot spots’ adds dramatically to this distorted picture. More importantly, inherently, the feed risk assessment approach per se, does not provide a true representation of the true exposure of animals to mycotoxins.
Therefore, the primary aims here were: a) the development and validation of biomarkers of exposure i.e. several mycotoxins and their Phase I and Phase II metabolites, detected with high analytical approaches (LC-MS/MS and HRMS), b) in different biological fluids in broiler chickens and pigs, c) the application of these biomarkers of exposure as a tool for testing the efficacy of a mycotoxin detoxifier (Escent® S) in broilers and pigs and, d) the transfer of the validated methodology into a practical, user-friendly as well as easy to sample and transport system, namely in dried blood spots (DBS).
Results
Chicken
Fig. 1 Study design of the trials in broilers indicating number of animals per treatment, dose of detoxifier and dose of multiple mycotoxins.
Fig. 1 Study design of the trials in broilers indicating number of animals per treatment, dose of detoxifier and dose of multiple mycotoxins.
Fig. 2 Mean HRMS peak area-time curves (+ SD) of deoxynivalenol-sulphate (DON-S (a) and mean concentration-time curves (+ SD) of AFB1 (b) in plasma of broiler chickens after oral administration of a bolus of DON (0.5 mg/kg BW), OTA (0.25 mg/kg BW) and AFB1 (2 mg/kg BW), either with Escent® S (treatment group, n=8, blue curve) or without (control group, n=8, orange curve).
Fig. 2 Mean HRMS peak area-time curves (+ SD) of deoxynivalenol-sulphate (DON-S (a) and mean concentration-time curves (+ SD) of AFB1 (b) in plasma of broiler chickens after oral administration of a bolus of DON (0.5 mg/kg BW), OTA (0.25 mg/kg BW) and AFB1 (2 mg/kg BW), either with Escent® S (treatment group, n=8, blue curve) or without (control group, n=8, orange curve).
The detoxifier technology Escent® S showed a statistically significant positive effect on chicken systemic detoxification compared to the control treatment, when the mean percentage difference of the corresponding areas under the concentration-time curve were calculated (p<0.05). Namely, it was shown that the Escent® S treatment reduced by 50% the exposure of DON and by 40% the exposure to AFB1 (Aflatoxin B1) in chicken blood (Lauwers, Croubels, Letor, et al. 2019). It should be noted that the animals were contaminated with a blend of three mycotoxins (DON, OTA and AFB1) with the levels of OTA being 25 times higher and AFB1 1,000 times higher than the EFSA guidelines. The findings are of great importance taking into consideration the direct toxicity of DON (and that of other mycotoxins) and its predisposing role in Clostridium perfringens-induced necrotic enteritis in chickens. 
Pigs
Fig. 3 Study design of the trials in pigs indicating number of animals per treatment, dose of detoxifier and dose of multiple mycotoxins.
Study design of the trials in pigs indicating number of animals per treatment, dose of detoxifier and dose of multiple mycotoxins.
Pigs treated with the detoxifier technology Escent® S exhibited a numerically reduced systemic concentration of DON and ZEN in plasma and urine and an increased concentration in faeces (Table 1) (Lauwers, Croubels, Letor, et al. 2019). In other words, Escent® S demonstrated a positive effect on pig detoxification despite the multi-mycotoxin contamination, as it succeeded in lowering the systemic exposure to DON and ZEN and it increased the amount of ZEN that was excreted via faeces. It should be noted that due to human error during the experiment, Escent® S was not applied at the recommended dose. Namely, Escent® S was applied only at 1kg/ton instead of the recommended 3kg/ton. 
Table 1 Percentage (%) difference of the mean toxicokinetic area under curve determined after single oral administration of DON (0.9 ppm) and ZEN (75 ppm) to pigs, either with Escent® S (n=8) or without (Control, n= 8).
Percentage (%) difference of the mean toxicokinetic area under curve determined after single oral administration of DON (0.9 ppm) and ZEN (75 ppm) to pigs, either with Escent® S (n=8) or without (Control, n= 8).
The findings are of particular importance taking into consideration the direct toxicity of DON and ZEN in pigs, their predisposing role in a spectrum of pig diseases and their negative impact on live PRSS vaccines.

The development of a novel Diagnostic Tool: Myco-marker® 
The analytical methodology developed and validated for the detection of biomarkers of exposure in blood of chickens and pigs was then successfully transferred onto DBS with the application of mini volumes of blood on Whatman® 903 Protein Saver FTA paper cards. Table 2 shows a snapshot of the validation results for linearity across three different days of analysis of 23 mycotoxins of pig whole blood extracted from an 8 mm disk of dried blood spots (Lauwers, Croubels, De Baere, et al. 2019).
Myco-Marker® has now been translated into a new service (patent pending) to the industry for the evaluation of the overall risk of mycotoxins. Myco-Marker® is a diagnostic tool that combines the analytical detection of mycotoxins as well as phase I and phase II metabolites both in feed and in animal (chickens and pigs) blood. The DBS system offers great advantages to the end user including easy sampling as only a drop of blood is required (~60μl), reduced animal invasiveness, easier sample transportation without border restrictions and without any specific storage conditions, no need for plasma separation and no need for special procedures or tubes. The mycotoxin exposure data in animal blood allow, for the first time, the quantification with high accuracy the level of mycotoxins that enter and systemically expose the animal.
The Myco-Marker® diagnostic tool has the potential to evaluate not only the overall exposure of mycotoxins but also to assess their impact on animal health. It is only then when the producer can seek and assess appropriate mitigating strategies.
Table 2. Validation results for linearity shown as mean ± standard deviation of three curves across three different days of analysis (linear range, correlation coefficient (r) and goodness-of-fit coefficient (g)) and limit of quantification (LOQ) of 23 mycotoxins of pig whole blood extracted from an 8 mm disk of dried blood spots.
Validation results for linearity shown as mean ± standard deviation of three curves across three different days of analysis (linear range, correlation coefficient (r) and goodnessof-fit coefficient (g)) and limit of quantification (LOQ) of 23 mycotoxins of pig whole blood extracted from an 8 mm disk of dried blood spots.

Discussion 
Mycotoxins in feed may impact greatly animal health and productivity. Feed analysis provides only a rough estimate of the risk involved in relation to the true amount of mycotoxins that animals could be potentially exposed to. Additionally, feed analysis is prone to significant methodological errors. The development of cost effective and reliable methods for determining the true exposure of mycotoxins to animals in field conditions has been the desire for several years. 
The present evidence here relates to a) the evaluation of the efficacy of the detoxifier technology Escent® S with the use of biomarkers of exposure and b) the development of Mycomarker®, a novel diagnostic tool for enhancing productivity in the animal farming sector. In particular, Myco-marker® relates to a novel and inventive method for biomonitoring mycotoxins and their phase I and phase II metabolites, in an easy and user-friendly manner via accessible animal matrices, and more specifically in the blood of broiler chickens and pigs.
Published in the proceedings of the Animal Nutrition Conference of Canada 2020. For information on the event, past and future editions, check out https://animalnutritionconference.ca/.

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