Mycoflora and Co-Occurrence of Fumonisins and Aflatoxins in Freshly Harvested Corn in Different Regions of Brazil

Natural mycoflora and co-occurrence of fumonisins (FB(1), FB(2)) and aflatoxins (AFB(1), AFB(2), AFG(1) and AFG(2)) in freshly harvested corn grain samples from four regions of Brazil were investigated. Fusarium verticillioides was predominant in all samples. Analysis of fumonisins showed that 98% of the samples were contaminated with FB(1) and 74.5% with FB(1) + FB(2), with toxin levels ranging from 0.015 to 9.67 mu g/g for FB(1) and from 0.015 to 3.16 mu g/g for FB(2). Twenty-one (10.5%) samples were contaminated with AFB(1), seven (3.5%) with AFB(2) and only one (0.5%) with AFG(1) and AFG(2). Co-contamination with aflatoxins and fumonisins was observed in 7% of the samples. The highest contamination of fumonisins and aflatoxins was observed in Nova Odessa (SP) and Varzea Grande (MT), respectively. The lowest contamination of these mycotoxins was found in Varzea Grande and Nova Odessa, respectively.

Equine leukoencephalomalacia (ELEM) due to fumonisins B1 and B2 in Argentina

In August 2007 an outbreak of neurological disease and sudden death in Arabian horses occurred in a farm located in Coronel Rosales County, Buenos Aires Province, Argentina. The animals were on a pasture of native grasses and supplemented ad libitum with corn kernels and wheat bran. Three horses were observed having acute neurologic signs including blindness, four leg ataxia, hyperexcitability, aimless walking and circling, followed by death in two of them. Four other horses were found dead overnight without a history of neurologic signs. The morbidity, mortality and lethality rates were 11.6%, 10% and 85.7%, respectively. Grossly, the brain showed focal areas of hemorrhage, brown-yellow discoloration and softening of the sub-cortical white matter. The microscopic brain lesions consisted of extensive areas of malacia within the white matter of the cerebral hemispheres, brainstem and cerebellum, characterized by rarefaction of the white matter with cavitations filled with proteinaceous edema, multifocal hemorrhages and mild infiltration by neutrophils, and rare eosinophils. Swollen glial cells with abundant eosinophilic cytoplasm, distinct cell borders, intracytoplasmic deeply eosinophilic globules and eccentric, hyperchromatic, occasionally pyknotic nucleus were present throughout the areas of rarefaction hemorrhage, edema and necrosis. The feed supplements contained 12,490µg/kg of fumonisin B1 and 5,251µg/ kg of fumonisin B2. This is the first reported outbreak of ELEM associated with consumption of feed supplements containing high concentrations of fumonisins in Argentina.

Fumonisins and Alternaria alternata lycopersici toxins: sphinganine analog mycotoxins induce apoptosis in monkey kidney cells.

Fusarium moniliforme toxins (fumonisins) and Alternaria alternata lycopersici (AAL) toxins are members of a new class of sphinganine analog mycotoxins that occur widely in the food chain. These mycotoxins represent a serious threat to human and animal health, inducing both cell death and neoplastic events in mammals. The mechanisms by which this family of chemical congeners induce changes in cell homeostasis were investigated in African green monkey kidney cells (CV-1) by assessing the appearance of apoptosis, cell cycle regulation, and putative components of signal transduction pathways involved in apoptosis. Structurally, these mycotoxins resemble the sphingoid bases, sphingosine and sphinganine, that are reported to play critical roles in cell communication and signal transduction. The addition of fumonisin B1 or AAL toxin, TA, to CV-1 cells induced the stereotypical hallmarks of apoptosis, including the formation of DNA ladders, compaction of nuclear DNA, and the subsequent appearance of apoptotic bodies. Neither mycotoxin induced cell death, DNA ladders, or apoptotic bodies in CV-1 cells expressing simian virus 40 large T antigen (COS-7) at toxin concentrations that readily killed CV-1 cells. Fumonisin B1 induced cell cycle arrest in the G1 phase in CV-1 cells but not in COS-7 cells. AAL toxin TA did not arrest cell cycle progression in either cell line. The induction of apoptosis combined with the widespread presence of these compounds in food crops and animal feed identifies a previously unrecognized health risk to humans and livestock. These molecules also represent a new class of natural toxicants that can be used as model compounds to further characterize the molecular and biochemical pathways leading to apoptosis.

Risk assessment of exposure to multiple mycotoxins in food

Moulds may produce a diversity of toxins such as aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins and others. Although toxicological, environmental and epidemiological studies have addressed the problem of these toxins one by one, more than one mycotoxin are found usually in the same contaminated food. Risk assessment for humans potentially exposed to multimycotoxins suffers very much from the lack of adequate food consumption data. Furthermore, for a given mycotoxin, synergism and antagonism with other mycotoxins, found in the same food commodities, are not taken into account. Aflatoxin B1 and ochratoxin A belong to the most frequently occurring mycotoxins. This has repeatedly been demonstrated, however, normally, the risk resulting from their simultaneous occurrence is not considered. A descriptive study was developed to monitor air fungal contamination in one hospital food unit.

Mycotoxin contamination of Maize and Guinea corn from markets in Plateau State, Nigeria

Maize (Zea mays) and guinea corn (Sorghum bicolor) are major food items in Plateau state, Nigeria. A multistage sampling technique was used to select the markets and store/warehouses used for this study; sample collection employed a simple random sampling method from different sampling points within designated areas. A total of 18 representative samples were collected and analyzed for the following mycotoxins: aflatoxins (Aflatoxin B1 - AFB1, Aflatoxin B2 - AFB2, Aflatoxin G1 - AFG1 and Aflatoxin G2 - AFG2), fumonisins (Fumonisin B1 - FB1 and Fumonisin B2 - FB2 ) and cyclopiazonic acid (CPA). Out of 12 samples analyzed for Aflatoxins, AFB1 was detected in 5, AFB2 in 1, AFG1 in 1 and AFG2 in 6 samples respectively. The highest concentration of AFB1 and AFG2 were found in maize samples from Pankshin market. Only maize samples from Mangu market were contaminated with AFB2 and also harboured the lowest concentration of AFG2. AFG1 contamination occurred in only guinea corn from Shendam market. and FB1 was detected in all 18 samples analyzed. The mycotoxin CPA was not detected in any of the samples. Aflatoxins levels in analyzed samples were regarded as safe based on Nigerian and European Union maximum permissible levels of 4g/kg. With the exception of two samples, FB1 levels in analyzed maize samples were within European Union maximum permissible levels of 1,000 to 3000g/kg. The health and food safety implications of these results for the human and animal population are further discussed.

A review of mycotoxins in food and feed products in Portugal and estimation of probable daily intakes

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that occur naturally in agricultural commodities worldwide. Aflatoxins, ochratoxin A, patulin, fumonisins, zearalenone, trichothecenes and ergot alkaloids are presently the most important for food and feed safety. These compounds are produced by several species that belong to the Aspergillus, Penicillium, Fusarium and Claviceps genera and can be carcinogenic, mutagenic, teratogenic, cytotoxic, neurotoxic, nephrotoxic, estrogenic and immunosuppressant. Human and animal exposure to mycotoxins is generally assessed by taking into account data on the occurrence of mycotoxins in food and feed as well as data on the consumption patterns of the concerned population. This evaluation is crucial to support measures to reduce consumer exposure to mycotoxins. This work reviews the occurrence and levels of mycotoxins in Portuguese food and feed to provide a global overview of this issue in Portugal. With the information collected, the exposure of the Portuguese population to those mycotoxins is assessed, and the estimated dietary intakes are presented.

Polyphasic approach including MALDI-TOF MS/MS analysis for identification and characterisation of Fusarium verticillioides in Brazilian corn kernels

Fusarium verticillioides is considered one of the most important global sources of fumonisin contamination in food and feed. Corn is one of the main commodities produced in the Northeastern Region of Brazil. The present study investigated potential mycotoxigenic fungal strains belonging to the F. verticillioides species isolated from corn kernels in 3 different Regions of the Brazilian State of Pernambuco. A polyphasic approach including classical taxonomy, molecular biology, MALDI-TOF MS and MALDI-TOF MS/MS for the identification and characterisation of the F. verticillioides strains was used. Sixty F. verticillioides strains were isolated and successfully identified by classical morphology, proteomic profiles of MALDI-TOF MS, and by molecular biology using the species-specific primers VERT-1 and VERT-2. FUM1 gene was further detected for all the 60 F. verticillioides by using the primers VERTF-1 and VERTF-2 and through the amplification profiles of the ISSR regions using the primers (GTG)5 and (GACA)4. Results obtained from molecular analysis shown a low genetic variability among these isolates from the different geographical regions. All of the 60 F. verticillioides isolates assessed by MALDI-TOF MS/MS presented ion peaks with the molecular mass of the fumonisin B1 (721.83 g/mol) and B2 (705.83 g/mol)

The naturally occurring food mycotoxin fumonisin B1 impairs myelin formation in aggregating brain cell culture.

The effects of subchronical applications of the mycotoxin Fumonisin B1 (FB1) were analyzed in vitro, using aggregating cell cultures of fetal rat telencephalon as a model. As cells in the aggregates developed from an immature state to a highly differentiated state, with synapse and compact myelin formation, it was possible to study the effects of FB1 at different developmental stages. The results showed that FB1 did not cause cell loss and it had no effects on neurons. However it decreased strongly the total content of myelin basic protein, the main constituent of the myelin sheath, during the myelination period (DIV 18-28). The loss of myelin was not accompanied by a loss of oligodendrocytes, the myelinating cells. However FB1 had effects on the maturation of oligodendrocytes, as revealed by a decrease in the expression of galactocerebroside, and on the compaction of myelin, as shown by a reduction of the expression of the mnyelin/oligodendrocyte glycoprotein MOG. The content of the cytoskeletal component glial fibrillary acidic protein (GFAP) was decreased in differentiated astrocytes, exclusively, while neurons were not affected by 40 microM of FB1 applied continuously for 10 days. In summary, FB1 selectively affected glial cells. In particular, FB1 delayed oligodendrocyte development and impaired myelin formation and deposition.

Metodologias analíticas para determinação das fumonisinas em milho e alimentos à base de milho

Fumonisins are mycotoxins occurring worldwide, mainly in maize and maize-based food products, which could affect animal and human health. This paper reviews analytical methodologies for the determination of these fungal toxins in foods. It includes extraction, cleanup, derivatization procedures, detection, quantification, and confirmation procedures. Initial attempts at gas chromatographic methods and thin layer chromatography were supplanted by liquid chromatographic methods, mainly performed with fluorometric detection, or mass spectrometry detection, enabling the analysis of polar and thermolabile chemicals without chemical derivatization, which results in lower limits of detection. Alternative methods, such as enzyme linked immunosorbent assay or zone capillary zone electrophoresis, are also described.

Relative populations and toxin production by Aspergillus flavus and Fusarium verticillioides in artificially inoculated corn at various stages of development under field conditions

The presence, development and production of mycotoxins by Aspergillus flavus and Fusarium verticillioides were studied in corn ears under field conditions after artificial contamination of corn silks. The planted area was divided into five treatments: T1, inoculated with A.flavus solution containing 1 x 10(8) spores, ears covered; T2, inoculated with F. verticillioides solution containing 1 X 10(8) spores, ears covered; T3, inoculated with E verticillioides plus A. flavus solution containing 1 x 10(8) spores of each, ears covered; T4, sprayed with sterile phosphate-buffered saline, ears covered; TS, non-sprayed silks, uncovered ears. Soil and air samples were also collected and analysed for the occurrence of fungi. Water activity, relative air humidity, rainfall and temperature were determined to assess the correlation between abiotic factors and the presence of fungi in the samples. Contamination with the inoculated fungus predominated in T1 and T2. In the other treatments, F. verticillioides was the most frequently isolated contaminant irrespective of treatment. Considering the production of mycotoxins, a positive relation between the production of fumonisins B-1 and B-2 and the frequency of F. verticillioides was statistically verified in all treatments. (C) 2007 Society of Chemical Industry.

Influence of macro- and micronutrient fertilization on fungal contamination and fumonisin production in corn grains

The aim of this study was to investigate the effects of nutrients (nitrogen, zinc and boron) on fungal growth and fumonisins production in corn samples obtained at the beginning of grain formation and at harvest. Three nitrogen doses were applied to the corn plants through soil in combination with three zinc doses and two boron doses during sowing. Mycological analysis of grains, using Dichloran Rose-Bengal Chloramphenicol Agar, collected at the beginning of formation demonstrated a fungal population predominantly of yeasts. Analysis of freshly harvested corn revealed a higher frequency of Penicillium spp. (72%) and F verticillioides (27%). High Performance Liquid Chromatography analysis revealed that 100% of grains were contaminated with fumonisins B, at levels ranging from 0.3 to 24.3 mg/kg and 93% contaminated with fumonisin B(2) at levels ranging from 0.05 to 5.42 mg/kg. Nitrogen (50 kg/ha) in combination with boron (0.5 kg/ha) resulted in an increased fumonisin B2 production. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1 alpha partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B(1) and B(2) production levels. Among the isolated strains, 96 were identified as Fusarium verricillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1 alpha sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verricillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r=0.95 and r=0.79 (correlation of FUM1 with FB(1) and FB(2), respectively, P < 0.0001): r=0.93 and r =0.78 (correlation of FUM19 with FB(1) and FB(2). respectively, P < 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity. (c) 2010 Elsevier B.V. All rights reserved.

Effects of prolonged oral administration of fumonisin B(1) and aflatoxin B(1) in rats

The effects of prolonged oral administration (21 days) of fumonisin B(1) (FB(1)) and aflatoxin B(1) (AFB(1)) were evaluated on male Wistar rats. The animals were housed in individual metabolic cages and submitted to the following treatments: 1-0 mug AFB(1) + 0 mg FB(1)/100g bw.; 2-72 mug AFB(1)+ 0 mg FB(1)/100 g bw; 3-0 mug AFB(1) + 0.5 mg FB(1) g bw; 4-0 mug AFB(1) + 1.5 mg FB(1)/100 g bw; 5-72 mug AFB(1) + 0.5 mg FB(1)/100g bw; 6-72 mu gAFB(1) + 1.5 mg FB(1)/100g bw. on day 21, the rats were sacrificed for evaluation. The results showed that treated animals presented differences in body weight and absolute/relative weights of liver and kidney as well as altered hepatic function and cholesterol blood levels. Rats fed with the greatest doses of AFB(1) and FB(1) gained less weight (2.79 g/day) at the end of the experimental period; their blood concentrations of liver enzymes aspartate aminotransferase (AST) and alkaline phosphatase (AP) were above control levels (130.35 mu /l and 471.00 mu /l, respectively). Blood cholesterol increased in the groups treated with the highest dose of FB(1) or FB(1) associated with AFB(1). Histopathology revealed the occurrence of apoptosis in the liver of rats exposed to FB(1). The association of aflatoxin B(1) with fumonisin B(1) at higher dose probably potentiated the effects of the higher dose of fumonisin B(1)acting singly.

Lipid metabolism of commercial layers fed diets containing aflatoxin, fumonisin, and a binder

Aflatoxins (AF) and fumonisins (FU) are a major problem faced by poultry farmers, leading to huge economic losses. This experiment was conducted to determine the effects of AF (1 mg/kg of feed) and FU (25 mg/kg of feed), singly or in combination, on the lipid metabolism in commercial layers and investigate the efficacy of a commercial binder (2 kg/t of feed) on reducing the toxic effects of these mycotoxins. A total of 168 Hisex Brown layer hens, 37 wk of age, were randomized into a 3 × 2 + 1 factorial arrangement (3 diets with no binder containing AF, FU, and AF+FU; 3 diets with binder containing AF, FU, and AF+FU; and a control diet with no mycotoxins and binders), totaling 7 treatments. The hens contaminated with AF showed the characteristic effects of aflatoxicosis, such as a yellow liver, resulting from the accumulation of liver fat, lower values of plasma very low-density lipoprotein and triglycerides, and higher relative weight of the kidneys and liver. Hepatotoxic and nephrotoxic effects of FU were not observed in this study. On the other hand, the FU caused a reduction in small intestine length and an increase in abdominal fat deposition. The glucan-based binder prevented some of the deleterious effects of these mycotoxins, particularly the effects of AF on hepatic lipid metabolism, kidney relative weight, and FU in the small intestine. © 2013 Poultry Science Association Inc.

Leucoencefalomalácia em equídeos da região Leste de Mato Grosso

Background: Fumonisins produced by Fusarium verticillioides are among the most important medical mycotoxins known. The intake of concentrate based on corn and corn by-products contaminated with fumonisins can cause severe poisoning in horses. The injuries are observed mainly in the white matter of the brain, and the disease is known as Equine Leukoencephalomalacia (ELEM). This study aims to describe and discuss the epidemiological, clinical and diagnostic aspects of an outbreak of ELEM occurred in three farms in the municipalities of Canarana and Agua Boa, in the eastern region of Mato Grosso, Brazil.Materials, Methods & Results: The outbreak occurred between May and August 2010. The disease affected six horses and four mules of different ages and sex. Clinical examination was only possible in animals with chronic evolution of the disease. All the affected animals showed neurological clinical signs such as ataxia and recumbency, which progressed to death or sudden death. Histopathological analysis showed foci of necrosis that predominantly affected the white matter, and the presence of gitter cells. Degenerative lesions were observed in the liver of the animals. Mortality rate ranged from 12.5 to 71%, and lethality reached 100%. The cases were preceded by sudden drops in the weather temperature. Fumonisins levels of 6.6 ppm were detected in the feed of the animals.Discussion: The presumptive diagnosis of leukoencephalomalacia was consistent and based on clinical and epidemiological studies. However, the definitive diagnosis was based upon the histological features of the brain including the presence of extensive areas of malacia. Moreover, the animals were being fed with corn or corn by-products contaminated with fumonisins levels considered to be toxic to equids. The mortality and lethality rates are in agreement with outbreaks described in previous studies. The animals showed neurological signs as the predominant clinical manifestation, with gait ataxia followed by recumbency, prostration and death between 24 h and 29 days. Similarly to other reports, the disease was more frequent in adult animals, which succumbed in 24-48 h. Conversely, the evolution of the disease in young animals was of 10 to 29 days. Sudden death was more prevalent in the mules. Previous studies have shown a predominance of cerebral and brainstem lesions in horses, whereas in mules the clinical signs are related to brainstem lesions. Corn and corn by-products are commonly used as energy supplementation to horses in the southern of Mato Grosso state, but outbreaks of the disease are uncommon. This may be influenced by the prevalence of hot climate conditions, which does not favor the production of toxin by the fungus. The atypical low weather temperatures (9-11 degrees C) observed prior to the outbreak could have contributed to the mycotoxin production by F. verticillioides, which requires temperatures between 8-12 degrees C to produce toxins. However, the disease in the region may be underestimated, considering that the practice of necropsies is not common among field technicians, mainly in the occurrence of sporadic deaths among horses intended for work. Preventive measures include avoiding the use of corn and corn by-products for horses after periods of sudden drops in temperature in the region. Furthermore, clinical and epidemiological surveys and post-mortem and histopathological analyses are undoubtedly important for appropriate differential diagnosis, especially in equids with neurological signs.