LeapAgri: European-African partnership for safe and efficient use of mycotoxin-mitigation strategies in sub-Saharan Africa

MycoSafe-South, the so-called "European-African Partnership for the Safe and Effective Use of Mycotoxin Reduction Strategies in Sub-Saharan Africa", intended to leverage the expertise and infrastructure available in Europe to strengthen the capacity of the African partners to tackle the mycotoxin problem and the associated food and feed safety issues. The aim of the project was to find strategies to reduce exposure with two important types of mycotoxins - aflatoxins and fumonisins. The MycoSafe-South project was coordinated by Ghent University and included several partners from Europe and Africa. The aim of the project was to (1) be able to offer safe detoxification methods for aflatoxin- and fumonisin-contaminated food to children and adults through safe and effective post-harvest intervention strategies, (2) to develop intervention strategies to reduce human exposure to aflatoxins through animal products (i.e. milk, meat and eggs), and (3) to organize educational programs and awareness campaigns that will facilitate knowledge transfer and help interested parties understand mycotoxin-associated health risks. Most of the practical work was carried out by four doctoral fellows. The team at the Veterinary Institute was involved in the testing of a clay-based aflatoxin binder in a SHIME test system (Simulator of the Human Intestinal Microbial Ecosystem). Using this test system, we documented that the binder can adsorb aflatoxin, and that, on the other hand, it had no negative effects on microbial parameters. The same SHIME system was also used to test the effect of a fumonisin-degrading enzyme. This showed that the enzyme also had the desired effect, and the concentration of fumonisin B1 decreased significantly after adding the enzyme to the system while the concentration of hydrolyzed fumonisin increased. The enzyme was also tested in a study in piglets to simulate administration in a capsule format. The animals thus received the enzyme together with fumonisin B1 either via a bolus or intra-orally. Compared to an intra-oral administration of the enzyme, the results showed that the enzyme was not as effective for fumonisin degradation when given via a bolus. This suggests that providing the fumonisin-degrading enzyme in capsule form may not work for fumonisin detoxification. The binder and the fumonisin-degrading enzyme were planned to be tested in a larger human intervention study in Kenya or South Africa, and it was planned to analyze aflatoxin and aflatoxin metabolites in samples from the study at the Veterinary Institute. Due to the corona pandemic, the experiments were not possible to carry out, and it also proved difficult to get ethical approval to carry out the study. The Scottish company Harbro Ltd. was an industrial partner in the project and provided a feed additive ("Maxammon"), which in an earlier pilot trial was also able to document the removal of aflatoxins. MycoSafe-South investigated whether "Maxammon" can reduce aflatoxins and fumonisins in contaminated maize in model experiments in the laboratory. At the Veterinary Institute, we focused particularly on aflatoxin B1, and investigated what happens to the molecule in an alkaline environment ("Maxammon" is based on urea). The trials with contaminated maize proved to be highly variable and gave no clear indication of a mycotoxin reduction and "Maxammon" unfortunately did not prove to be an alternative product for aflatoxin and fumonisin detoxification. Under the leadership of the University of Johannesburg, a small intervention study was planned in Soweto/South Africa, where the effect of a so-called nixtamalisation process on aflatoxins and other mycotoxins during the preparation of maize-based dishes was to be tested. The process is known to to reduce aflatoxins. In that context, a method was developed at the Veterinary Institute in which, after a so-called micro-sampling of 10 microliters of blood from a finger prick, the levels of 28 different mycotoxins and mycotoxin detoxification products can be determined. The method can be used in new surveys where the mycotoxin exposure in the population is to be evaluated. Unfortunately, the intervention study was not yet carried out due to challenges in recruiting volunteers in Soweto, but can hopefully still be carried out in the near future. Another central aim of the project was to raise awareness of the mycotoxin problem in Africa and to teach important facts about mycotoxins to African local communities. In 2020, a LEPA-Agri MycoSafe-South workshop was therefore held at the University of Johannesburg, followed by a "Farmer's Lab" in Soweto where local people had the opportunity to learn about mycotoxins. The results from Mycosafe-South were presented at the third symposium of the African Society for Mycotoxicology in collaboration with Belgian MYTOX-South consortium in Stellenbosch in September 2022.

The MycoSafe-South outcomes and impacts reported here are limited to those that are directly related to activities and contributions of the Norwegian partners. • General outcomes: -Strengthening of European-African collaborations beyond the current MycoSafe-South project -Education of young scientists, i.e. graduation of four PhDs, with one candidate that was promoted and co-supervised by the Norwegian partner -Creating awareness for the mycotoxin-problems in Africa • Specific outcomes: -The efficacy of two mycotoxin detoxifying animal feed additives and their safety for the gut microbiota was shown in an in vitro human gut model, the “simulator of the human intestinal microbial ecosystem” (SHIME®) -The efficacy of two application modes for fumonisin esterase was compared in an in vivo piglet model, which showed that ingestion of the fumonisin detoxifying enzyme in a capsule is probably not effective -Development of a multi-mycotoxin liquid chromatography – high-resolution mass spectrometry method for the biomonitoring of multiple mycotoxin biomarkers of exposure, including several mycotoxin biotransformation products, in human blood using the Volumetric Absorptive Microsampling technique -Determination of a suitable single fumonisin B1 dose for humans necessary for quantification of the toxin and its (partially) hydrolyzed metabolites in feces; in order to study the efficacy of fumonisin esterase under controlled experimental conditions -Submission of ethical dossiers to Kenyan and South African authorities showed that the mycotoxin detoxifiers are likely to be classified as pharmaceutical drugs, not as food supplements, which makes it difficult to conduct human intervention studies

The project will address the above stated aims following an inter-institutional approach in the frame of four joint European-African PhD candidates that are interconnected and linked in five work packages. The safety and efficacy of two approaches for the detoxification of aflatoxins (AFs) and fumonisins (FBs) in food will be examined by in vitro and in vivo models. These imply (1) food processing techniques including nixtamalization, dehulling and fermentation; and (2) the application of mycotoxin binders/modifiers. Based on the in vitro and in vivo animal experiments , the best-treatment strategy will be selected for a randomized placebo-controlled human intervention study in Kenya and South-Africa. The interaction between the ruminal microbiome and its capacity to degrade AFs will be investigated in Holstein Friesian and Zebu cattle at ILRI. Furthermore, the impact of feeding a diet simultaneously contaminated with AFs and FBs on the formation of AFM1 will also be evaluated. Animals will be fed either a control diet, an AFs-contaminated diet or an AFs+FBs-contaminated diet. Subsequently, safety and efficacy of adding a mycotoxin detoxifying agent to these diets will be investigated. Mycotoxins in poultry feed can have serious effects on poultry health, which impacts the productivity, but in addition, secondary exposure to AFs through consumption of eggs, liver and meat derived from chickens fed AFs-contaminated feed, poses a risk to consumer health. In contrast, FBs residues in animal tissues are low12, but co-exposure with AFs may alter the disposition of FBs. The project will therefore provide more evidence on how AFs and FBs in poultry feed contribute to negative chicken health and production effects.