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.

Seroprevalence and spatial distribution of livestock brucellosis using three serological tests in Kajiado County, Kenya.

Background: Brucellosis is a serious zoonotic infection with a global socioeconomic impact on both the livestock industry and human health. In Kenya, brucellosis is endemic but there is limited information on the true burden of the disease due to weak or peace-meal surveillance. The true burden and spread of animal brucellosis in Kajiado County is not known. Aim: The aim of the study was to determine the current seroprevalence and spatial distribution of livestock brucellosis in Kajiado County and also to compare the three serological tests, namely; Rose Bengal plate test (RBPT), indirect enzyme-linked immunosorbent assay (i-ELISA), and competitive ELISA (c-ELISA) in the detection of seropositive animals. Methods: A cross-sectional study was undertaken in 5 sub-counties and 13 wards, where a total of 782 serum samples from unvaccinated bovine (n = 278; 34 herds), ovine (n = 256; 25 flocks), and caprine (n = 248; 28 flocks), were screened for anti-Brucella antibodies using RBPT, i-ELISA, and c-ELISA tests, in parallel. Results: Overall animal seroprevalence was 6.91% (54/782); while that for bovine, ovine, and caprine was 18.35% (51/278), 0.78% (2/256), and 0.4% (1/248), respectively. Bovine seroprevalence was 2.2% (6/278), 14.4% (40/278), and 4.7% (13/278) in RBPT, i-ELISA, and c-ELISA tests, respectively; while ovine 0.78% (2/256) and caprine 0.4% (1/248) were positive only in c-ELISA. Bovine herd seropositivity was 67.65% (23/34), whereas ovine and caprine flock seropositivities were 8% (2/25) and 3.6% (1/28), respectively. Conclusion: The findings indicate a moderate seroprevalence of brucellosis in bovine, while that of ovine and caprine was low in Kajiado County. Indirect ELISA was found superior to both c-ELISA and RBPT in detecting bovine seropositive animals, while c-ELISA was superior to both RBPT and i-ELISA in detecting seropositive ovines and caprines. These results will contribute to baseline data for further study of Brucella infection and a starting point for the formulation of a strategy for the control of brucellosis in Kajiado County.

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.

Efficacy of Fumonisin Esterase in Piglets as Animal Model for Fumonisin Detoxification in Humans: Pilot Study Comparing Intraoral to Intragastric Administration.

Fumonisins, a group of highly prevalent and toxic mycotoxins, are suspected to be causal agents of several diseases in animals and humans. In the animal feed industry, fumonisin esterase is used as feed additive to prevent mycotoxicosis caused by fumonisins. In humans, a popular dosage form for dietary supplements, with high patient acceptance for oral intake, is capsule ingestion. Thus, fumonisin esterase provided in a capsule could be an effective strategy against fumonisin intoxication in humans. To determine the efficacy of fumonisin esterase through capsule ingestion, two modes of application were compared using piglets in a small-scale preliminary study. The enzyme was administered intraorally (in-feed analogue) or intragastrically (capsule analogue), in combination with fumonisin B1 (FB1). Biomarkers for FB1 exposure; namely FB1, hydrolysed FB1 (HFB1) and partially hydrolysed forms (pHFB1a and pHFB1b), were measured both in serum and faeces using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and toxicokinetic parameters were calculated. Additionally, the serum sphinganine/sphingosine (Sa/So) ratio, a biomarker of effect, was determined using LC-MS/MS. A significantly higher Sa/So ratio was shown in the placebo group compared to both esterase treatments, demonstrating the efficacy of the esterase. Moreover, a significant decrease in serum FB1 area under the concentration-time curve (AUC) and an increase of faecal HFB1 AUC were observed after intraoral esterase administration. However, these effects were not observed with statistical significance after intragastric esterase administration with the current sample size.

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.

The efficacy and effect on gut microbiota of an aflatoxin binder and a fumonisin esterase using an in vitro simulator of the human intestinal microbial ecosystem (SHIME®).

Mycotoxin intoxication is in general an acknowledged and tackled issue in animals. However, in several parts of the world, mycotoxicoses in humans still remain a relevant issue. The efficacy of two mycotoxin detoxifying animal feed additives, an aflatoxin bentonite clay binder and a fumonisin esterase, was investigated in a human child gut model, i.e. the in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). Additionally, the effect of the detoxifiers on gut microbiota was examined in the SHIME. After an initial two weeks of system stabilisation, aflatoxin B1 (AFB1) and fumonisin B1 (FB1) were added to the SHIME diet during one week. Next, the two detoxifiers and mycotoxins were added to the system for an additional week. The AFB1, FB1, hydrolysed FB1 (HFB1), partially hydrolysed FB1a and FB1b concentrations were determined in SHIME samples using a validated ultra-performance liquid chromatography-tandem mass spectrometry method. The short-chain fatty acid (SCFA) concentrations were determined by a validated gas chromatography-mass spectrometry method. Colonic bacterial communities were analysed using metabarcoding, targeting the hypervariable V1-V3 regions of the 16S rRNA genes. The AFB1 and FB1 concentrations significantly decreased after the addition of the detoxifiers. Likewise, the concentration of HFB1 significantly increased. Concentrations of SCFAs remained generally stable throughout the experiment. No major changes in bacterial composition occurred during the experiment. The results demonstrate the promising effect of these detoxifiers in reducing AFB1 and FB1 concentrations in the human intestinal environment, without compromising the gastrointestinal microbiota.

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.

Co-Occurrence and Levels of Mycotoxins in Fish Feeds in Kenya.

This study determined the presence, levels and co-occurrence of mycotoxins in fish feeds in Kenya. Seventy-eight fish feeds and ingredients were sampled from fish farms and fish feed manufacturing plants and analysed for 40 mycotoxins using high-performance liquid chromatography-high resolution mass spectrometry. Twenty-nine (73%) mycotoxins were identified with 76 (97%) samples testing positive for mycotoxins presence. Mycotoxins with the highest prevalences were enniatin B (91%), deoxynivalenol (76%) and fumonisin B1 (54%) while those with the highest maximum levels were sterigmatocystin (<30.5-3517.1 µg/kg); moniliformin (<218.9-2583.4 µg/kg) and ergotamine (<29.3-1895.6 µg/kg). Mycotoxin co-occurrence was observed in 68 (87%) samples. Correlations were observed between the fumonisins; enniatins B and zearalenone and its metabolites. Fish dietary exposure estimates ranged between <0.16 and 43.38 µg/kg body weight per day. This study shows evidence of mycotoxin presence and co-occurrence in fish feeds and feed ingredients in Kenya. Fish exposure to these levels of mycotoxins over a long period of time may lead to adverse health effects due to their possible additive, synergistic or antagonist toxic effects. Measures to reduce fish feed mycotoxin contamination should be taken to avoid mycotoxicosis in fish and subsequently in humans and animals through residues.

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.

Occurrence and Levels of Aflatoxins in Fish Feeds and Their Potential Effects on Fish in Nyeri, Kenya.

Aflatoxins are fungal metabolites that contaminate foods and feeds, causing adverse health effects in humans and animals. This study determined the occurrence of aflatoxins in fish feeds and their potential effects on fish. Eighty-one fish feeds were sampled from 70 farms and 8 feed manufacturing plants in Nyeri, Kenya for aflatoxin analysis using competitive enzyme-linked immunosorbent assay. Fish were sampled from 12 farms for gross and microscopic pathological examination. Eighty-four percent of feeds sampled tested positive for aflatoxins, ranging from 1.8 to 39.7 µg/kg with a mean of 7.0 ± 8.3 µg/kg and the median of 3.6 µg/kg. Fifteen feeds (18.5%) had aflatoxins above the maximum allowable level in Kenya of 10 µg/kg. Homemade and tilapia feeds had significantly higher aflatoxin levels than commercial and trout feeds. Feeds containing maize bran and fish meal had significantly higher aflatoxin levels than those without these ingredients. Five trout farms (41.7%) had fish with swollen abdomens, and enlarged livers with white or yellow nodules, which microscopically had large dark basophilic hepatic cells with hyperchromatic nuclei in irregular cords. In conclusion, aflatoxin contamination of fish feeds is prevalent in Nyeri, and may be the cause of adverse health effects in fish in this region.