Effects of Aflatoxins and Fumonisins, Alone or in Combination, on Performance, Health, and Safety of Food Products of Broiler Chickens, and Mitigation Efficacy of Bentonite and Fumonisin Esterase.

The current study evaluated the effects of feeding diets contaminated with aflatoxin B1 (AFB1), fumonisins (FBs), or both on the performance and health of broiler chickens and the safety of their food products as well as the efficacy of bentonite and fumonisin esterase to mitigate the effects of these mycotoxins under conditions representative for sub-Saharan Africa (SSA). Four hundred one-day-old Cobb 500 broiler chickens were randomly assigned to 20 treatments with either a control diet, a diet with moderate AFB1 (60 µg/kg feed) or high AFB1 (220 µg/kg feed), or FBs (17,430 µg FB1+FB2/kg feed), alone or in combination, a diet containing AFB1 (either 60 or 220 µg/kg) and/or FBs (17,430 µg FB1+FB2/kg) and bentonite or fumonisin esterase or both, or a diet with bentonite or fumonisin esterase only. The experimental diets were given to the birds from day 1 to day 35 of age, and the effects of the different treatments on production performance were assessed by feed intake (FI), body weight gain (BWG), and feed conversion ratio (FCR). Possible health effects were evaluated through blood biochemistry, organ weights, mortality, liver gross pathological changes, and vaccine response. Residues of aflatoxins (AFB1, B2, G1, G2, M1 and M2) were determined in plasma, muscle, and liver tissues using validated UHPLC-MS/MS methods. The results obtained indicated that broiler chickens fed high AFB1 alone had poor FCR when compared to a diet with both high AFB1 and FBs (p = 0.0063). Serum total protein and albumin from birds fed FBs only or in combination with moderate or high AFB1 or detoxifiers increased when compared to the control (p < 0.05). Liver gross pathological changes were more pronounced in birds fed contaminated diets when compared to birds fed the control or diets supplemented with mycotoxin detoxifiers. The relative weight of the heart was significantly higher in birds fed high AFB1 and FBs when compared to the control or high AFB1 only diets (p < 0.05), indicating interactions between the mycotoxins. Inclusion of bentonite in AFB1-contaminated diets offered a protective effect on the change in weights of the liver, heart and spleen (p < 0.05). Residues of AFB1 were detected above the limit of quantification (max: 0.12 ± 0.03 µg/kg) in liver samples only, from birds fed a diet with high AFB1 only or with FBs or the detoxifiers. Supplementing bentonite into these AFB1-contaminated diets reduced the levels of the liver AFB1 residues by up to 50%. Bentonite or fumonisin esterase, alone, did not affect the performance and health of broiler chickens. Thus, at the doses tested, both detoxifiers were safe and efficient for use as valid means of counteracting the negative effects of AFB1 and FBs as well as transfer of AFB1 to food products (liver) of broiler chickens.

Development of High-Throughput Sample Preparation Procedures for the Quantitative Determination of Aflatoxins in Biological Matrices of Chickens and Cattle Using UHPLC-MS/MS.

Aflatoxins (AFs) frequently contaminate food and animal feeds, especially in (sub) tropical countries. If animals consume contaminated feeds, AFs (mainly aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and their major metabolites aflatoxin M1 (AFM1) and M2 (AFM2)) can be transferred to edible tissues and products, such as eggs, liver and muscle tissue and milk, which ultimately can reach the human food chain. Currently, the European Union has established a maximum level for AFM1 in milk (0.05 µg kg-1). Dietary adsorbents, such as bentonite clay, have been used to reduce AFs exposure in animal husbandry and carry over to edible tissues and products. To investigate the efficacy of adding bentonite clay to animal diets in reducing the concentration of AFB1, AFB2, AFG1, AFG2, and the metabolites AFM1 and AFM2 in animal-derived foods (chicken muscle and liver, eggs, and cattle milk), chicken and cattle plasma and cattle ruminal fluid, a sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed. High-throughput sample preparation procedures were optimized, allowing the analysis of 96 samples per analytical batch and consisted of a liquid extraction using 1% formic acid in acetonitrile, followed by a further clean-up using QuEChERS (muscle tissue), QuEChERS in combination with Oasis® Ostro (liver tissue), Oasis® Ostro (egg, plasma), and Oasis® PRiME HLB (milk, ruminal fluid). The different procedures were validated in accordance with European guidelines. As a proof-of-concept, the final methods were used to successfully determine AFs concentrations in chicken and cattle samples collected during feeding trials for efficacy and safety evaluation of mycotoxin detoxifiers to protect against AFs as well as their carry-over to animal products.

Efficacy of Bentonite and Fumonisin Esterase in Mitigating the Effects of Aflatoxins and Fumonisins in Two Kenyan Cattle Breeds.

The objective of the study was to investigate the efficacy of bentonite and fumonisin esterase, separately or combined, in mitigating the effects of aflatoxins (AF) and fumonisins (FUM) in Boran and Friesian-Boran crossbreed cattle. These effects were studied by measuring mycotoxins, their metabolites, and biomarkers that relate to animal health, productivity, and food safety. The study was divided into three experiments each lasting for 2 weeks. Cows in experiment 1 received in random order aflatoxin B1 (AFB1) [788 µg/cow/day (69.7 µg/kg dry matter intake (DMI)) for Borans and 2,310 µg/cow/day (154 µg/kg DMI) for crossbreeds], bentonite (60 g/cow/day), or both AFB1 and bentonite. Boran cows in experiment 2 received in random order FUM (12.4 mg/cow/day (1.1 mg/kg DMI)), fumonisin esterase (120 U/cow/day), or both FUM and fumonisin esterase. Boran cows in experiment 3 received in random order AFB1 (952 µg/cow/day (84.2 µg/kg DMI)) + FUM (30.4 mg/cow/day (2.7 mg/kg DMI)), bentonite (60 g/cow/day) + fumonisin esterase (120 U/cow/day), or both AFB1 + FUM and bentonite + fumonisin esterase. Feeding AFB1 and/or FUM contaminated feed with or without the addition of the detoxifiers for 14 days did not affect DMI, milk composition, hematology, and blood biochemical parameters. The addition of bentonite in a diet contaminated with AFB1 led to a decrease in milk aflatoxin M1 (AFM1) concentration of 30% and 43%, with the carry-over subsequently decreasing from 0.35% to 0.20% and 0.08% to 0.06% for crosses and Borans, respectively. No significant change was observed in the sphinganine/sphingosine (Sa/So) ratio following feeding with FUM alone or in combination with fumonisin esterase; however, the ability of fumonisin esterase to hydrolyze FUM into less toxic fully hydrolyzed FUM and partially hydrolyzed FUM was evident in the rumen fluid and feces. These results indicate bentonite was effective in decreasing AFM1 concentration in milk, and AFB1 and AFM1 in plasma, while fumonisin esterase can convert FUM into less toxic metabolites and can be a suitable addition to feed cocontaminated with AFB1 and FUM.

Maximizing Laboratory Production of Aflatoxins and Fumonisins for Use in Experimental Animal Feeds.

Warm and humid climatic conditions coupled with poor agricultural practices in sub-Saharan Africa favor the contamination of food and feed by Aspergillus flavus and Fusarium verticillioides fungi, which subsequently may produce aflatoxins (AFs) and fumonisins (FBs), respectively. The growth of fungi and the production of mycotoxins are influenced by physical (temperature, pH, water activity, light and aeration), nutritional, and biological factors. This study aimed at optimizing the conditions for the laboratory production of large quantities of AFs and FBs for use in the animal experiments. A. flavus and F. verticillioides strains, previously isolated from maize in Kenya, were used. Levels of AFB1 and total FBs (FB1, FB2, and FB3) in different growth substrates were screened using ELISA methods. Maize kernels inoculated with three different strains of A. flavus simultaneously and incubated at 29 °C for 21 days had the highest AFB1 level of 12,550 ± 3397 µg/kg of substrate. The highest level of total FBs (386,533 ± 153,302 µg/kg of substrate) was detected in cracked maize inoculated with three different strains of F. verticillioides and incubated for 21 days at temperatures of 22-25 °C in a growth chamber fitted with yellow light. These two methods are recommended for the mass production of AFB1 and FBs for animal feeding trials.

Mycotoxins in Poultry Feed and Feed Ingredients from Sub-Saharan Africa and Their Impact on the Production of Broiler and Layer Chickens: A Review.

The poultry industry in sub-Saharan Africa (SSA) is faced with feed insecurity, associated with high cost of feeds, and feed safety, associated with locally produced feeds often contaminated with mycotoxins. Mycotoxins, including aflatoxins (AFs), fumonisins (FBs), trichothecenes, and zearalenone (ZEN), are common contaminants of poultry feeds and feed ingredients from SSA. These mycotoxins cause deleterious effects on the health and productivity of chickens and can also be present in poultry food products, thereby posing a health hazard to human consumers of these products. This review summarizes studies of major mycotoxins in poultry feeds, feed ingredients, and poultry food products from SSA as well as aflatoxicosis outbreaks. Additionally reviewed are the worldwide regulation of mycotoxins in poultry feeds, the impact of major mycotoxins in the production of chickens, and the postharvest use of mycotoxin detoxifiers. In most studies, AFs are most commonly quantified, and levels above the European Union regulatory limits of 20 µg/kg are reported. Trichothecenes, FBs, ZEN, and OTA are also reported but are less frequently analyzed. Co-occurrences of mycotoxins, especially AFs and FBs, are reported in some studies. The effects of AFs on chickens' health and productivity, carryover to their products, as well as use of mycotoxin binders are reported in few studies conducted in SSA. More research should therefore be conducted in SSA to evaluate occurrences, toxicological effects, and mitigation strategies to prevent the toxic effects of mycotoxins.

Multi-Mycotoxin Occurrence in Dairy Cattle and Poultry Feeds and Feed Ingredients from Machakos Town, Kenya.

Mycotoxins are common in grains in sub-Saharan Africa and negatively impact human and animal health and production. This study assessed occurrences of mycotoxins, some plant, and bacterial metabolites in 16 dairy and 27 poultry feeds, and 24 feed ingredients from Machakos town, Kenya, in February and August 2019. We analyzed the samples using a validated multi-toxin liquid chromatography-tandem mass spectrometry method. A total of 153 mycotoxins, plant, and bacterial toxins, were detected in the samples. All the samples were co-contaminated with 21 to 116 different mycotoxins and/or metabolites. The commonly occurring and EU regulated mycotoxins reported were; aflatoxins (AFs) (70%; range 0.2-318.5 µg/kg), deoxynivalenol (82%; range 22.2-1037 µg/kg), ergot alkaloids (70%; range 0.4-285.7 µg/kg), fumonisins (90%; range 32.4-14,346 µg/kg), HT-2 toxin (3%; range 11.9-13.8 µg/kg), ochratoxin A (24%; range 1.1-24.3 µg/kg), T-2 toxin (4%; range 2.7-5.2 µg/kg) and zearalenone (94%; range 0.3-910.4 µg/kg). Other unregulated emerging mycotoxins and metabolites including Alternaria toxins, Aspergillus toxins, bacterial metabolites, cytochalasins, depsipeptides, Fusarium metabolites, metabolites from other fungi, Penicillium toxins, phytoestrogens, plant metabolites, and unspecific metabolites were also detected at varying levels. Except for total AFs, where the average contamination level was above the EU regulatory limit, all the other mycotoxins detected had average contamination levels below the limits. Ninety-six percent of all the samples were contaminated with more than one of the EU regulated mycotoxins. These co-occurrences may cause synergistic and additive health effects thereby hindering the growth of the Kenyan livestock sector.

A Review of the Impact of Mycotoxins on Dairy Cattle Health: Challenges for Food Safety and Dairy Production in Sub-Saharan Africa.

Mycotoxins are secondary metabolites of fungi that contaminate food and feed and have a significant negative impact on human and animal health and productivity. The tropical condition in Sub-Saharan Africa (SSA) together with poor storage of feed promotes fungal growth and subsequent mycotoxin production. Aflatoxins (AF) produced by Aspergillus species, fumonisins (FUM), zearalenone (ZEN), T-2 toxin (T-2), and deoxynivalenol (DON) produced by Fusarium species, and ochratoxin A (OTA) produced by Penicillium and Aspergillus species are well-known mycotoxins of agricultural importance. Consumption of feed contaminated with these toxins may cause mycotoxicoses in animals, characterized by a range of clinical signs depending on the toxin, and losses in the animal industry. In SSA, contamination of dairy feed with mycotoxins has been frequently reported, which poses a serious constraint to animal health and productivity, and is also a hazard to human health since some mycotoxins and their metabolites are excreted in milk, especially aflatoxin M1. This review describes the major mycotoxins, their occurrence, and impact in dairy cattle diets in SSA highlighting the problems related to animal health, productivity, and food safety and the up-to-date post-harvest mitigation strategies for the prevention and reduction of contamination of dairy feed.