Special Issue "Aflatoxins"

Abstract: The safety characteristics of feed used in fish and crustacean aquaculture systems are an essential tool to assure the productivity of those animal exploitations. Safety of feed may be affected by different hazards, including biological and chemical groups. The aim of this preliminary study was to evaluate fungi contamination and the presence of aflatoxins in 87 samples of feed for sea bass, collected in Portugal. Molds were found in 35 samples (40.2%) in levels ranging from 1 to 3.3 log10 CFU∙g⁻¹. Six genera of molds were found. Aspergillus flavus was the most frequent, found in all positive samples, with a range from 2 to 3.2 log₁₀ CFU∙g⁻¹. Aspergillus niger was found in 34 samples (39.1%), ranging from 1 to 2.7 log₁₀ CFU∙g⁻¹. Aspergillus glaucus was found in 26 samples (29.9%) with levels between 1 and 2.4 log₁₀ CFU∙g⁻¹. Penicillium spp. and Cladosporium spp. were both found in 25 samples (28.7%). Fusarium spp. was found in 22 samples (25.3%), ranging from 1 to 2.3 log₁₀ CFU∙g⁻¹. All feed samples were screened for aflatoxins using a HPLC technique, with a detection limit of 1.0 μg∙kg⁻¹. All samples were aflatoxin negative.

Abstract: This research examined the expression patterns of 94 stress-related genes in seven maize inbred lines with differential expressions of resistance to aflatoxin contamination. The objective was to develop a set of genes/probes associated with resistance to A. flavus and/or aflatoxin contamination. Ninety four genes were selected from previous gene expression studies with abiotic stress to test the differential expression in maize lines, A638, B73, Lo964, Lo1016, Mo17, Mp313E, and Tex6, using real-time RT-PCR. Based on the relative-expression levels, the seven maize inbred lines clustered into two different groups. One group included B73, Lo1016 and Mo17, which had higher levels of aflatoxin contamination and lower levels of overall gene expression. The second group which included Tex6, Mp313E, Lo964 and A638 had lower levels of aflatoxin contamination and higher overall levels of gene expressions. A total of six “cross-talking” genes were identified between the two groups, which are highly expressed in the resistant Group 2 but down-regulated in susceptible Group 1. When further subjected to drought stress, Tex6 expressed more genes up-regulated and B73 has fewer genes up-regulated. The transcript patterns and interactions measured in these experiments indicate that the resistant mechanism is an interconnected process involving many gene products and transcriptional regulators, as well as various host interactions with environmental factors, particularly, drought and high temperature.

Abstract: Animal feeding studies have demonstrated that clay additives, such as bentonites, can bind aflatoxins in ingested feed and reduce or eliminate the toxicity. Bentonite deposits are found throughout the world and mostly consist of expandable smectite minerals, such as montmorillonite. The surfaces of smectite minerals can be treated with organic compounds to create surface-modified clays that more readily bind some contaminants than the untreated clay. Montmorillonites treated with organic cations, such as hexadecyltrimethylammonium (HDTMA) and phenyltrimethylammonium (PTMA), more effectively remove organic contaminants, such as benzene and toluene, from water than untreated clay. Similarly, montmorillonite treated with PTMA (K_d = 24,100) retained more aflatoxin B1 (AfB1) from aqueous corn flour than untreated montmorillonite (K_d = 944). Feed additives that reduced aflatoxin toxicity in animal feeding studies adsorbed more AfB1 from aqueous corn flour than feed additives that were less effective. The organic cations HDTMA and PTMA are considered toxic and would not be suitable for clay additives used in feed or food, but other non-toxic or nutrient compounds can be used to prepare surface-modified clays. Montmorillonite (SWy) treated with choline (K_d = 13,800) and carnitine (K_d = 3960) adsorbed much more AfB1 from aqueous corn flour than the untreated clay (K_d = 944). A choline-treated clay prepared from a reduced-charge, high-charge montmorillonite (K_d = 20,100) adsorbed more AfB1 than the choline-treated high-charge montmorillonite (K_d = 1340) or the untreated montmorillonite (K_d = 293). Surface-modified clay additives prepared using low-charge smectites and nutrient or non-toxic organic compounds might be used to more effectively bind aflatoxins in contaminated feed or food and prevent toxicity.

Abstract: Aflatoxin B₁ is a common contaminant of poultry feeds in tropical and subtropical climates. Research during the last five decades has well established the negative effects of the mycotoxin on health of poultry. However, the last ten years of relevant data have accentuated the potential of low levels of aflatoxin B₁ to deteriorate broiler performance. In this regard, any attempt to establish a dose-effect relationship between aflatoxin B₁ level and broiler performance is also complicated due to differences in types of broilers and length of exposure to the mycotoxin in different studies. Contrary to the prevalent notion regarding literature saturation with respect to aflatoxicosis of chicken, many areas of aflatoxicosis still need to be explored. Literature regarding effects of the mycotoxin on the gastrointestinal tract in this regard is particular scanty and non-conclusive. In addition to these issues, the metabolism of aflatoxin B₁ and recently proposed hypotheses regarding biphasic effects of the mycotoxin in broilers are briefly discussed.

Abstract: Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B₁ (AFB₁), and aflatoxin G₁ (AFG₁) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB₁ production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB₁ production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB₁ production by A. flavus EGP9 and AFG₁ production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG₁ production by A. parasiticus SSWT 2999. Changes in AFB₁ production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

Abstract: At this time, no “magic bullet” for solving the aflatoxin contamination problem in maize and cottonseed has been identified, so several strategies must be utilized simultaneously to ensure a healthy crop, free of aflatoxins. The most widely explored strategy for the control of aflatoxin contamination is the development of preharvest host resistance. This is because A. flavus infects and produces aflatoxins in susceptible crops prior to harvest. In maize production, the host resistance strategy has gained prominence because of advances in the identification of natural resistance traits. However, native resistance in maize to aflatoxin contamination is polygenic and complex and, therefore, markers need to be identified to facilitate the transfer of resistance traits into agronomically viable genetic backgrounds while limiting the transfer of undesirable traits. Unlike maize, there are no known cotton varieties that demonstrate enhanced resistance to A. flavus infection and aflatoxin contamination. For this reason, transgenic approaches are being undertaken in cotton that utilize genes encoding antifungal/anti-aflatoxin factors from maize and other sources to counter fungal infection and toxin production. This review will present information on preharvest control strategies that utilize both breeding and native resistance identification approaches in maize as well as transgenic approaches in cotton.

Abstract: Aspergillus flavus and A. parasiticus infect peanut seeds and produce aflatoxins, which are associated with various diseases in domestic animals and humans throughout the world. The most cost-effective strategy to minimize aflatoxin contamination involves the development of peanut cultivars that are resistant to fungal infection and/or aflatoxin production. To identify peanut Aspergillus-interactive and peanut Aspergillus-resistance genes, we carried out a large scale peanut Expressed Sequence Tag (EST) project which we used to construct a peanut glass slide oligonucleotide microarray. The fabricated microarray represents over 40% of the protein coding genes in the peanut genome. For expression profiling, resistant and susceptible peanut cultivars were infected with a mixture of Aspergillus flavus and parasiticus spores. The subsequent microarray analysis identified 62 genes in resistant cultivars that were up-expressed in response to Aspergillus infection. In addition, we identified 22 putative Aspergillus-resistance genes that were constitutively up-expressed in the resistant cultivar in comparison to the susceptible cultivar. Some of these genes were homologous to peanut, corn, and soybean genes that were previously shown to confer resistance to fungal infection. This study is a first step towards a comprehensive genome-scale platform for developing Aspergillus-resistant peanut cultivars through targeted marker-assisted breeding and genetic engineering.

Abstract: A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies; eight diverse inbred maize lines for polymorphism identification within any maize gene sequence; four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes; and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline.

Abstract: Aflatoxin contamination caused by the opportunistic pathogen A. flavus is a major concern in maize production prior to harvest and through storage. Previous studies have highlighted the constitutive production of proteins involved in maize kernel resistance against A. flavus’ infection. However, little is known about induced resistance nor about defense gene expression and regulation in kernels. In this study, maize oligonucleotide arrays and a pair of closely-related maize lines varying in aflatoxin accumulation were used to reveal the gene expression network in imbibed mature kernels in response to A. flavus’ challenge. Inoculated kernels were incubated 72 h via the laboratory-based Kernel Screening Assay (KSA), which highlights kernel responses to fungal challenge. Gene expression profiling detected 6955 genes in resistant and 6565 genes in susceptible controls; 214 genes induced in resistant and 2159 genes induced in susceptible inoculated kernels. Defense related and regulation related genes were identified in both treatments. Comparisons between the resistant and susceptible lines indicate differences in the gene expression network which may enhance our understanding of the maize-A. flavus interaction.

Abstract: Key impediments to increased corn yield and quality in the southeastern US coastal plain region are damage by ear-feeding insects and aflatoxin contamination caused by infection of Aspergillus flavus. Key ear-feeding insects are corn earworm, Helicoverpa zea, fall armyworm, Spodoptera frugiperda, maize weevil, Sitophilus zeamais, and brown stink bug, Euschistus servus. In 2006 and 2007, aflatoxin contamination and insect damage were sampled before harvest in three 0.4-hectare corn fields using a grid sampling method. The feeding damage by each of ear/kernel-feeding insects (i.e., corn earworm/fall armyworm damage on the silk/cob, and discoloration of corn kernels by stink bugs), and maize weevil population were assessed at each grid point with five ears. The spatial distribution pattern of aflatoxin contamination was also assessed using the corn samples collected at each sampling point. Aflatoxin level was correlated to the number of maize weevils and stink bug-discolored kernels, but not closely correlated to either husk coverage or corn earworm damage. Contour maps of the maize weevil populations, stink bug-damaged kernels, and aflatoxin levels exhibited an aggregated distribution pattern with a strong edge effect on all three parameters. The separation of silk- and cob-feeding insects from kernel-feeding insects, as well as chewing (i.e., the corn earworm and maize weevil) and piercing-sucking insects (i.e., the stink bugs) and their damage in relation to aflatoxin accumulation is economically important. Both theoretic and applied ramifications of this study were discussed by proposing a hypothesis on the underlying mechanisms of the aggregated distribution patterns and strong edge effect of insect damage and aflatoxin contamination, and by discussing possible management tactics for aflatoxin reduction by proper management of kernel-feeding insects. Future directions on basic and applied research related to aflatoxin contamination are also discussed.

Abstract: Aflatoxins are carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Previous studies found that repeated serial mycelial transfer or treatment of A. parasiticus with 5-azacytidine produced colonies with a fluffy phenotype and inability to produce aflatoxins. To understand how these treatments affect expression of genes involved in aflatoxin production and development, we carried out expressed sequence tag (EST)-based microarray assays to identify genes in treated clones that are differentially expressed compared to the wild-type. Expression of 183 genes was significantly dysregulated. Of these, 38 had at least two-fold or lower expression compared to the untreated control and only two had two-fold or higher expression. The most frequent change was downregulation of genes predicted to encode membrane-bound proteins. Based on this result we hypothesize that the treatments cause changes in the structure of cellular and organelle membranes that prevent normal development and aflatoxin biosynthesis.