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.

Evaluation of Agronomic Characteristics, Disease Incidence, Yield Performance, and Aflatoxin Accumulation among Six Peanut Varieties (Arachis hypogea L.) Grown in Kenya.

Diseases contribute to attainment of less than 50% of the local groundnut potential yield in Kenya. This study aimed to evaluate the agronomic characteristics (flowering and germination), disease incidence, yield performance (biomass, harvest index, 100-pod, 100-seed, and total pod weight), and aflatoxin accumulation in six peanut varieties. A field experiment was conducted using four newly improved peanut varieties: CG9, CG7, CG12, and ICGV-SM 90704 (Nsinjiro), and two locally used varieties: Homabay local (control) and 12991, and in a randomized complete block design with three replications. The disease identification followed the International Crop Research Institute for the Semi-Arid Tropics (ICRISAT) rating scale and further isolation of fungal contaminants was conducted by a direct plating technique using potato dextrose agar. The aflatoxin levels in the peanuts were determined after harvesting using the ultrahigh performance liquid chromatography and fluorescence detection (UHPLC-FLD) technique. ICGV-SM 90704 showed the least average disease incidence of 1.31 ± 1.75%, (P < 0.05); the lowest total aflatoxin levels (1.82 ± 1.41 µg kg-1) with a range 0.00-0.85 µg kg-1 for total aflatoxins and a range 0.00-1.24 µg kg-1 for Aflatoxin B1. The locally used varieties (12991 and the control) revealed the highest disease incidence (5.41 ± 8.31% and 7.41 ± 1.88%), respectively. ICGV-SM 90704 was the best performing among all the six varieties with an average total pod weight (9.22 ± 1.19 kg), 100-pod weight (262.93 ± 10.8 g), and biomass of (27.21 ± 5.05 kg) per row. The 12991 variety and the control showed the least total pod weight (1.60 ± 0.28 and 1.50 ± 1.11 kg, respectively) (P = 0.0001). The newly improved varieties showed lower disease rates, low levels of aflatoxins, and higher yields than the locally used varieties.

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.

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.

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.

Seasonal Diversity and Occurrence of Filamentous Fungi in Smallholder Dairy Cattle Feeds and Feedstuffs in South Africa.

This study investigated 65 (35 in summer and 30 in winter) smallholder dairy cattle feeds from Free State and Limpopo provinces in South Africa from 2018 to 2019 for fungal contamination and assessed the impacts of seasonal variation on fungal contamination levels, isolation frequency, and diversity. Samples were examined for fungal contamination using macro- and microscopic approaches, and their identities were confirmed by molecular means. A total of 217 fungal isolates from 14 genera, including Aspergillus, Fusarium, and Penicillium, were recovered from feeds from both seasons. The most prevalent fungal species recovered were A. fumigatus and P. crustosum. Mycological analyses showed that 97% of samples were contaminated with one or more fungal isolates, with the summer fungal mean level (6.1 × 103 to 3.0 × 106 CFU/g) higher than that of feeds sampled during winter (mean level: 1.1 × 103 to 4.1 × 105 CFU/g). Independent sample t-test revealed that the isolation frequencies of the genera Aspergillus and Fusarium were significantly (p ≤ 0.05) higher in summer than winter, while Penicillium prevalence in both seasons was not statistically (p > 0.05) different. Furthermore, the Shannon−Weiner diversity index (H') revealed a higher fungal diversity in summer (H' = 2.8) than in winter (H' = 2.1). This study on fungal contamination could be used for future fungal control and mycotoxin risk management in South Africa.

Essential descriptors for mycotoxin contamination data in food and feed.

Mycotoxin food contamination data is scattered, isolated, and poorly described. Reporting mycotoxin contamination data in a standardized manner is essential for collaborative research and integrated large-scale data analysis. The present study aimed to complement the existing European Food Safety Authority (EFSA) and Global Environment Monitoring System (GEMS) mycotoxin contamination data descriptors for application in low- and middle-income countries in particular. A three-round Delphi process was followed to establish a consensus on the missing descriptors. An invitation letter was first sent to 34 mycotoxin experts of an international collaboration of MYTOX-SOUTH®, of which 12 finally participated in the study. The response rate was 29.4% (10/34) in the Delphi I, 75% (9/12) in the Delphi II, and 83.3% (10/12) in the Delphi III rounds. The majority of the Delphi study participants were professors from 6 universities. Twenty-two descriptors (17 study level, 1 sample level, and 4 assay level) were proposed and were mainly related to pre and post-harvest periods of a food/feed sample. The pre-defined (>70% in the Delphi II and > 80% in the Delphi III) agreement among participants was achieved for all the proposed descriptors. The existing descriptors from EFSA (33) and GEMS (25) with the new proposed MYTOX-SOUTH® (22) descriptors, in total 80 descriptors, were arranged as study, sample, and assay categories and organized as a data submission template. Pre-testing of the template on three mycotoxin researchers indicated that the average time to fill out the form for a sample was 42 min. The current format helps mycotoxin contamination data to become more informative, reusable, and applicable especially to data from low- and middle-income countries. The above-proposed descriptors will help GEMS to provide technical cooperation with countries wishing to initiate and strengthen food contaminant monitoring programs. Similarly, the descriptors from the current study will be useful for EFSA as it regularly updates the Standard Sample Description. A standardized global reporting format for mycotoxin contamination data will enable national authorities to perform mycotoxins exposure and risk assessments and share data for international benchmarking. Standardized reporting and sharing of mycotoxin contamination data should be further advocated in ongoing research and become common practice in authorities, companies, academia, and other entities working on mycotoxin in food and feed.

Comprehensive analysis of multiple mycotoxins and Aspergillus flavus metabolites in maize from Kenyan households.

This study assessed the levels of mycotoxins in maize from Kenyan households. Further, local open pollinated maize varieties were compared with commercial hybrids to evaluate which variety is less susceptible to mycotoxin contamination. Four hundred and eighty (n = 480) maize samples were collected in the years 2018-2020 from households in Eastern, Western, Coastal and Lake Victoria regions of Kenya. Liquid chromatography coupled to tandem mass spectrometry was used to detect and quantify 22 mycotoxins, along with 31 Aspergillus flavus metabolites in the samples. Eastern Kenya had the highest aflatoxin (AF) contamination with 75% of samples having AF levels above the Kenyan regulatory limits (10 µg/kg), the highest concentration was 558.1 µg/kg. In Western Kenya, only 18% of samples had concentration levels above the Kenyan regulatory limits for AF with highest sample having 73.3 µg/kg. The Lake Victoria region had the most fumonisins (F) contamination, with 53% of the samples having fumonisin B1 (FB1) < 1000 µg/kg. However, only 20% of the samples surpassed the Kenyan regulatory limit for total fumonisins (2000 µg/kg) with the highest concentration being 13,022 µg/kg. In addition, 21.6% of samples from the Lake Victoria region had zearalenone (ZEN) and deoxynivalenol (DON) above regulatory limits for European countries (1000 µg/kg). Western region had the least A. flavus metabolites contamination (18%) while the Eastern region had the highest incidence of A. flavus metabolites (81%). Among the A. flavus metabolites, cyclopiazonic acid (CPA), beta-cyclopiazonic acid (ß CPA), flavacol (FLV) and methylcitreo-isocoumarin (MIC) positively correlated with each other but negatively correlated with the other metabolites. Significant positive co-occurrence was also noted among Fusarium mycotoxins: nivalenol (NIV) positively correlated with DON (r = 0.81), fusarenon-X (FX) (r = 0.81) and ZEN (r = 0.70). Negative correlations were observed between Aspergillus and Fusarium mycotoxins: aflatoxin B1 (AFB1) negatively correlated with FB1 (r = -0.11), FX (r = -0.17) and ZEN (r = -0.20). Local open-pollinated maize varieties (L-opv) were less susceptible to mycotoxin contamination compared to the commercial hybrids (C-hy). This study reveals that Kenyan maize is contaminated with multiple mycotoxins most of which are not regulated in Kenya despite being regulated in other parts of the world. A comprehensive legislation should therefore be put in place to protect the Kenyan public against chronic exposure to these mycotoxins. In addition to high yield, there is a need for commercial hybrid maize breeders to incorporate mycotoxin resistance as an important trait in germplasm improvement in seeds production.

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.

Occurrence of Fungi and Mycotoxins in Fish Feeds and Their Impact on Fish Health.

The rapid population growth in developing countries has led to strong pressure on capture fisheries. However, capture fisheries have reached their maximal limits of fish production and are supplemented by farmed fish. The growth in aquaculture has led to high demand for fish feeds, which play a very important role in fish nutrition and health. Use of animal protein in fish feeds is expensive; hence, a majority of farmers from developing countries use local feed ingredients from plant origin as a source of dietary protein. However, these ingredients of plant origin provide the best natural substrates for fungi, which can be easily accompanied by mycotoxin development under suitable conditions. The locally made feed comprises ingredients such as soybeans, cottonseed cake, and wheat and maize bran which are mixed together and ground after which the compounded feed is pelleted and stored. Among the ingredients, maize and oilseeds are more susceptible for mycotoxigenic fungi compared to other ingredients. The outcomes of mycotoxin contamination in fish feeds are not different from other animal species intended for human consumption, and they are directly associated with production losses, particularly decreased weight gain and feed conversion, impaired immune system and reproductive performance, and increased fish mortality. Fish may also carry mycotoxin residues along the food chain, thus compromising human health. Hence, it is important to ensure the control of mycotoxin contamination in fish feeds, especially during the production and storage.

Effects of aflatoxin B1 on reproductive performance of farmed Nile tilapia.

This study evaluated the effect of dietary aflatoxin B1 (AFB1) on growth, milt and egg quality in matured Nile tilapia (Oreochromis niloticus). Triplicate groups of Nile tilapia (initial body weight 24.1 ± 2.6 g) were fed with either of four diets (Diets 1 to 4) designed to contain 0, 20, 200 and 2000 µg AFB1 kg-1 diets for 24 weeks. After 24 weeks of AFB1 exposure, growth was significantly (P <0.05) different between the control and the AFB1 exposed treatments in both sexes. No significant differences were observed in 17ß-oestradiol, absolute fecundity, oocytes volume and diameters between AFB1 exposure groups and the control group. However, we observed a significant reduction in relative fecundity and gonad somatic index (GSI) in females fed 2000 µg AFB1 kg-1 diet. On the other hand, we observed significant differences (P <0.05) in gonadosomatic index (GSI), testosterone, milt count and motility between males in the control group and AFB1 treatments. We conclude that rearing Nile tilapia with aflatoxin-contaminated diets for a prolonged period affects milt quality, fecundity (at higher doses) and growth performance. This implies that for optimal seed production, provision of aflatoxin free diets should be part of the management practices in Nile tilapia hatcheries.

Infestation mechanisms of two woodborer species in the mangrove Sonneratia alba J. Smith in Kenya and co-occurring endophytic fungi.

Insect damage on trees can severely affect the quality of timber, reduce the fecundity of the host and render it susceptible to fungal infestation and disease. Such pathology weakens or eventually kills the host. Infestation by two insect woodborer species (a moth and a beetle) is causing mortality of Sonneratia alba, a wide-ranging pioneer mangrove species of the Indo-Pacific. Establishing the infestation mechanism of the two insect woodborer species is an initial and essential step towards understanding their ecological role in the mangroves and in determining sustainable management priorities and options. Our main objectives were to investigate the infestation mechanism employed by the two insect woodborers which infest S. alba trees, to establish the occurrence of secondary infestation by endophytic fungi in the infested S. alba branches, and to explore a control management option to the woodborer infestation. We conducted an external inspection of infested branches in two large embayments in Kenya, Gazi Bay and Mida Creek, and by splitting infested branches we determined the respective internal infestation mechanisms. Infested wood samples from Gazi Bay and Mida Creek were incubated at 28±1°C for 3-5 days to establish the presence of fungi. A survey was conducted in both Gazi Bay and Mida Creek to ascertain the presence of ants on S. alba. The infestation characteristics of the two insect woodborer species were different. It took 6-8 months for the beetle to kill a branch of 150 cm-200 cm long. For the moth to kill a branch, it depended upon several factors including the contribution by multiple species, other than the moth infestation alone. A total of 15 endophytic fungal species were identified. Two ant species Oecophylla longipoda and a Pheidole sp. inhabited 62% and 69% respectively of sampled S. alba trees in Gazi Bay whereas only Pheidole sp. inhabited 17% of the sampled S. alba trees in Mida Creek. In summary, we have documented the time it takes each woodborer species to kill a branch, the infestation mechanism of the two insect woodborers, and we hypothesized on the role of two ant species. The presence of several different fungal species was ascertained, and we discussed their possible role in the infested wood. Our results cannot unambiguously associate the woodborers and identified fungi. We recommend further studies to investigate the presence or absence, and if present, the nature of fungi in the gut of the woodborers.

Genetic Profiling of Aspergillus Isolates with Varying Aflatoxin Production Potential from Different Maize-Growing Regions of Kenya.

Highly toxigenic strains of Aspergillus flavus have been reported to frequently contaminate maize, causing fatal aflatoxin poisoning in Kenya. To gain insights into the environmental and genetic factors that influence toxigenicity, fungi (n = 218) that were culturally identified as A. flavus were isolated from maize grains samples (n = 120) from three regions of Kenya. The fungi were further characterized to confirm their identities using a PCR-sequence analysis of the internal transcribed spacer (ITS) region of rDNA which also revealed all of them to be A. flavus. A subset of 72 isolates representing ITS sequence-based phylogeny and the agroecological origin of maize samples was constituted for subsequent analysis. The analysis of partial calmodulin gene sequences showed that the subset consisted of A. flavus (87%) and Aspergillus minisclerotigenes (13%). No obvious association was detected between the presence of seven aflatoxin biosynthesis genes and fungal species or region. However, the presence of the aflD and aflS genes showed some association with aflatoxin production. The assessment of toxigenicity showed higher aflatoxin production potential in A. minisclerotigenes isolates. Given that A. minisclerotigenes were mainly observed in maize samples from Eastern Kenya, a known aflatoxin hotspot, we speculate that production of copious aflatoxin is an adaptative trait of this recently discovered species in the region.

Aflatoxin Binders in Foods for Human Consumption-Can This be Promoted Safely and Ethically?

Aflatoxins continue to be a food safety problem globally, especially in developing regions. A significant amount of effort and resources have been invested in an attempt to control aflatoxins. However, these efforts have not substantially decreased the prevalence nor the dietary exposure to aflatoxins in developing countries. One approach to aflatoxin control is the use of binding agents in foods, and lactic acid bacteria (LAB) have been studied extensively for this purpose. However, when assessing the results comprehensively and reviewing the practicality and ethics of use, risks are evident, and concerns arise. In conclusion, our review suggests that there are too many issues with using LAB for aflatoxin binding for it to be safely promoted. Arguably, using binders in human food might even worsen food safety in the longer term.

Correction: Mitema, A. et al. The Development of a qPCR Assay to Measure Aspergillus flavus Biomass in Maize and the Use of a Biocontrol Strategy to Limit Aflatoxin Production. Toxins 2019, 11, 179.

The authors wish to make the following correction to their paper [...].

Activation of the p38/MAPK pathway regulates autophagy in response to the CYPOR-dependent oxidative stress induced by zearalenone in porcine intestinal epithelial cells.

Zearalenone (ZEA) can widely contaminate crops and agricultural products. The ingestion of ZEA-contaminated food or feed affects the integrity and functions of the intestines. In this study, we aimed to find the potential protective mechanism against ZEA ingestion. We found that ZEA induced cell death in IPEC-J2 cells. Meanwhile, the cytoprotective autophagy was activated in ZEA-treated cells. Further studies demonstrated that a p38/MAPK inhibitor down-regulated autophagy and increased cell death compared to those of the controls. Furthermore, ZEA could induce the accumulation of ROS, and eliminating ROS with NAC resulted in a decline in cell death, p38/MAPK phosphorylation, and the expression of LC3-II compared to those of ZEA-group. In addition, cytochrome P450 reductase (CYPOR) was significantly increased in ZEA-treated cells compared to that in the controls, and an inhibitor of CYPOR decreased ROS levels and mitigated cell death compared to those of the ZEA-group. More importantly, we found that blocking both p38/MAPK signalling and autophagy could enhance CYPOR expression and elevate ROS levels. Overall, our study indicated that the p38/MAPK pathway could activate protective autophagy in response to the CYPOR-dependent oxidative stress that was induced by ZEA in IPEC-J2 cells.

The Development of a qPCR Assay to Measure Aspergillus flavus Biomass in Maize and the Use of a Biocontrol Strategy to Limit Aflatoxin Production.

Aspergillus flavus colonisation of maize can produce mycotoxins that are detrimental to both human and animal health. Screening of maize lines, resistant to A. flavus infection, together with a biocontrol strategy, could help minimize subsequent aflatoxin contamination. We developed a qPCR assay to measure A. flavus biomass and showed that two African maize lines, GAF4 and KDV1, had different fungal loads for the aflatoxigenic isolate (KSM014), fourteen days after infection. The qPCR assay revealed no significant variation in A. flavus biomass between diseased and non-diseased maize tissues for GAF4, while KDV1 had a significantly higher A. flavus biomass (p < 0.05) in infected shoots and roots compared to the control. The biocontrol strategy using an atoxigenic isolate (KSM012) against the toxigenic isolate (KSM014), showed aflatoxin production inhibition at the co-infection ratio, 50:50 for both maize lines (KDV1 > 99.7% and GAF ≥ 69.4%), as confirmed by bioanalytical techniques. As far as we are aware, this is the first report in Kenya where the biomass of A. flavus from maize tissue was detected and quantified using a qPCR assay. Our results suggest that maize lines, which have adequate resistance to A. flavus, together with the appropriate biocontrol strategy, could limit outbreaks of aflatoxicoses.

Curcumin-based photosensitization inactivates Aspergillus flavus and reduces aflatoxin B1 in maize kernels.

Different methods have been applied in controlling contamination of foods and feeds by the carcinogenic fungal toxin, aflatoxin, but nevertheless the problem remains pervasive in developing countries. Curcumin is a natural polyphenolic compound from the spice turmeric (Curcuma longa L.) that has been identified as an efficient photosensitiser for inactivation of Aspergillus flavus conidia. Curcumin mediated photoinactivation of A. flavus has revealed the potential of this technology to be an effective method for reducing population density of the aflatoxin-producing fungus in foods. This study demonstrates the influence of pH and temperature on efficiency of photoinactivation of the fungus and how treating spore-contaminated maize kernels affects aflatoxin production. The results show the efficiency of curcumin mediated photoinactivation of fungal conidia and hyphae were not affected by temperatures between 15 and 35 °C or pH range of 1.5-9.0. The production of aflatoxin B1 was significantly lower (p < 0.05), with an average of 82.4 µg/kg as compared to up to 305.9 µg/kg observed in untreated maize kept under similar conditions. The results of this study indicate that curcumin mediated photosensitization can potentially be applied under simple environmental conditions to achieve significant reduction of post-harvest contamination of aflatoxin B1 in maize.