A preliminary study on mycotoxin contamination in red meat from registered abattoirs in South Africa.

The frequency of some major mycotoxins in marker tissues (liver and kidney) and in muscle tissue of slaughter pigs and cattle, obtained from registered abattoirs in South Africa, was studied. Samples of each three bovine carcasses were obtained from two abattoirs, and samples of three porcine carcasses were from a third abattoir. All samples originated from animals from subsistence farming. All samples were analysed for aflatoxins (AFB1, AFB2, AFG1, AFG2, deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEN) using immunoaffinity chromatography extract cleanup and high-performance liquid chromatography (HPLC). At a limit of quantification (LOQ) of 1 µg/kg (individual AFs, 100 µg/kg (DON), 1 µg/kg (OTA) and 20 µg/kg (ZEN)), no mycotoxins were detected in any of the samples.

Molecular analysis of Aspergillus section Nigri isolated from onion samples reveals the prevalence of A. welwitschiae.

The aim of this study was to isolate Aspergillus section Nigri from onion samples bought in supermarkets and to analyze the fungal isolates by means of molecular data in order to differentiate A. niger and A. welwitschiae species from the other non-toxigenic species of black aspergilli, and detect genes involved in the biosynthesis of ochratoxin A and fumonisin B2. Aspergillus section Nigri were found in 98% (94/96) of the onion samples. Based on the results of multiplex PCR (performed on 500 randomly selected strains), 97.4% of the Aspergillus section Nigri strains were recognized as A. niger/A. welwitschiae. Around half of them were subjected to partial sequencing of the CaM gene to distinguish one from the other. A total of 97.9% of the isolates were identified as A. welwitschiae and only 2.1% as A. niger. The fum8 gene, involved in fumonisin B2 biosynthesis, was found in 36% of A. welwitschiae isolates, but radH and pks genes, involved in ochratoxin A biosynthesis, were found in only 2.8%. The presence/absence of fum8 gene in the A. welwitschiae genome is closely associated with ability/inability of the isolates to produce fumonisin in vitro. Based on these results, we suggest that in-depth studies are conducted to investigate the presence of fumonisins in onion bulbs.

Distribution of fungi and their toxic metabolites in melon and sesame seeds marketed in two major producing states in Nigeria.

In this study, melon (n = 60) and sesame (n = 60) seeds purchased from markets within Benue and Nasarawa states, respectively, in Nigeria, during two seasons (dry and wet), were analysed for fungal and mycotoxin contamination in order to determine the safety of these foods for human consumption. Molecular analysis revealed the following seven fungal taxonomic groups in the foods: Aspergillus section Candidi, Aspergillus section Flavi, Aspergillus section Nigri, Cladosporium, Fusarium fujikuroi species group, Penicillium, and Pleosporales/Didymellaceae. A total of 78 microbial metabolites, including several mycotoxins, occurred in the foods. The most frequent mycotoxins in melon and sesame were aflatoxin B1 (occurrence: 76%) and alternariol monomethyl ether (occurrence: 59%), respectively. However, higher mean total aflatoxin levels occurred in sesame (17 µg kg-1) than in melon (11 µg kg-1). About 28 and 5% of melon and sesame, respectively, exceeded the 4 µg kg-1 total aflatoxin limit for oilseeds intended for direct human consumption in the European Union. Additionally, fumonisin B1 and moniliformin occurred only in sesame, whilst ochratoxins A and B occurred only in melon; ochratoxin B being reported for the first time in this food. Our data indicated seasonal variations in the fungal and mycotoxin contamination levels in both foods.

Mycobiota and mycotoxins in Portuguese pork, goat and sheep dry-cured hams.

The objectives of the present work were to survey, for the first time, the contamination of Portuguese fresh and dry-cured meat products with ochratoxin A (OTA) and aflatoxin B1 (AFB1), and to determine the fungi potentially responsible for this contamination. A total of 128 samples including pork fresh legs, dry-cured legs and shoulders, as well as goat and sheep dry-cured legs were analysed. Mycological analysis of these samples yielded a total of 630 fungal isolates. Penicillium sp. was the dominant fungal genus in all products (66% of all isolates). Penicillium nordicum and Aspergillus westerdijkiae were only rarely isolated from pork ham samples. In fresh pork meat, 40% of the samples were contaminated with OTA at levels below 1 µg/kg. In pork dry-cured legs with 20 to 25 months of ripening, 43% of the samples showed detectable contamination, while 18% of the shoulder hams were contaminated. OTA was not detected in any of the goat and sheep samples. OTA contamination does not seem to be a risk in small-piece and short-ripe products like goat and sheep legs, but affects longer ripe products like pork legs and shoulders. Although aflatoxigenic fungi were identified, AFB1 was not detected in any sample, and it should not be considered a risk in dry-cured hams.

Enzymes for Detoxification of Various Mycotoxins: Origins and Mechanisms of Catalytic Action.

Mycotoxins are highly dangerous natural compounds produced by various fungi. Enzymatic transformation seems to be the most promising method for detoxification of mycotoxins. This review summarizes current information on enzymes of different classes to convert various mycotoxins. An in-depth analysis of 11 key enzyme mechanisms towards dozens of major mycotoxins was realized. Additionally, molecular docking of mycotoxins to enzymes' active centers was carried out to clarify some of these catalytic mechanisms. Analyzing protein homologues from various organisms (plants, animals, fungi, and bacteria), the prevalence and availability of natural sources of active biocatalysts with a high practical potential is discussed. The importance of multifunctional enzyme combinations for detoxification of mycotoxins is posed.

Prevalent Mycotoxins in Animal Feed: Occurrence and Analytical Methods.

Today, we have been witnessing a steady tendency in the increase of global demand for maize, wheat, soybeans, and their products due to the steady growth and strengthening of the livestock industry. Thus, animal feed safety has gradually become more important, with mycotoxins representing one of the most significant hazards. Mycotoxins comprise different classes of secondary metabolites of molds. With regard to animal feed, aflatoxins, fumonisins, ochratoxins, trichothecenes, and zearalenone are the more prevalent ones. In this review, several constraints posed by these contaminants at economical and commercial levels will be discussed, along with the legislation established in the European Union to restrict mycotoxins levels in animal feed. In addition, the occurrence of legislated mycotoxins in raw materials and their by-products for the feeds of interest, as well as in the feeds, will be reviewed. Finally, an overview of the different sample pretreatment and detection techniques reported for mycotoxin analysis will be presented, the main weaknesses of current methods will be highlighted.

First report of Fusarium foetens as a mycotoxin producer.

Fusarium foetens, a pathogen of Begonia plants, has been recently described as a new fungal species. This Fusarium species causes a destructive vascular wilt disease which leads to the death of the plant. Moreover, Fusarium species are known to produce a huge variety of secondary metabolites such as mycotoxins and phytotoxins. Here, we studied the toxicogenic profile of one F. foetens strain, isolated from maize, employing two methods based on the use of ultra-performance liquid chromatography coupled to mass spectrometry-ion trap-time of flight detection. The mycotoxins beauvericin and fusaric acid were detected in a pure culture of F. foetens. In addition, four fusaric acid analogs (10,11-dihidroxyfusaric acid, hydroxyfusaric acid, dehydrofusaric acid, and a hydroxylated unsaturated fusaric acid analog) were tentatively identified on the basis of their accurate mass and fragmentation patterns. Therefore, these preliminary data indicate that F. foetens isolated from maize is able to produce Fusarium mycotoxins including beauvericin and fusaric acid.

Variation of cassiicolin genes among Chinese isolates of Corynespora cassiicola.

Corynespora cassiicola is a species of fungus that is a plant pathogen of many agricultural crop plants, including severe target spot disease on cucumber. Cassiicolin is an important effector of pathogenicity of this fungus. In this study, we collected 141 Corynespora isolates from eighteen hosts, and the casscolin gene was detected in 82 C. cassiicola strains. The deduced protein sequences revealed that 72 isolates contained the Cas2 gene, two strains from Gynura bicolor harboured the Cas2.2 gene, and 59 isolates without a cassiicolin gene were classified as Cas0. Phylogenetic analyses was performed for the 141 isolates using four loci (ITS, ga4, caa5, and act1) and revealed two genetic clusters. Cluster A is composed of four subclades: subcluster A1 includes all Cas2 isolates plus 18 Cas0 strains, subcluster A2 includes the eight Cas5 isolates and one Cas0 isolate, and subclusters A3 and A4 contain Cas0 strains. Cluster B consists of 21 Cas0 isolates. Twenty-two C. cassiicola strains from different toxin classes showed varying degrees of virulence against cucumber. Cas0 or Cas2 strains induced diverse responses on cucumber, from no symptoms to symptoms of moderate or severe infection, but all Cas5 isolates exhibited avirulence on cucumber.

Isolation, identification, and characterization of Ustilaginoidea virens from rice false smut balls with high ustilotoxin production potential.

Ustilaginoidea (U.) virens grows on rice grains and leads to significant rice yield losses in most of the major rice producing areas. Meanwhile, ustiloxins produced by U. virens are a serious hazard to human health and ecological safety of farmlands. The other key point is that ustiloxins have been regarded as a novel resource with their potential in the treatment of cancers. There is no better way to extract ustiloxins than from pure culture of the high ustilotoxin-producing strains. U. virens has become a key research organism. However, due to the presence of some interference components, it is a certain difficulty in the successful isolation of the strain from the false smut balls. We present here a detailed study based on the separation, screening and identification of high ustiloxins-producing strains of U. virens. Through this study, we got a satisfactory success rate of separation and provided a good solution to the problem of separation. At the same time, this study provides quality resources for researchers interested in ustiloxins as anticancer agents.

Study of three interesting Amanita species from Thailand: Morphology, multiple-gene phylogeny and toxin analysis.

Amanita ballerina and A. brunneitoxicaria spp. nov. are introduced from Thailand. Amanita fuligineoides is also reported for the first time from Thailand, increasing the known distribution of this taxon. Together, those findings support our view that many taxa are yet to be discovered in the region. While both morphological characters and a multiple-gene phylogeny clearly place A. brunneitoxicaria and A. fuligineoides in sect. Phalloideae (Fr.) Quél., the placement of A. ballerina is problematic. On the one hand, the morphology of A. ballerina shows clear affinities with stirps Limbatula of sect. Lepidella. On the other hand, in a multiple-gene phylogeny including taxa of all sections in subg. Lepidella, A. ballerina and two other species, including A. zangii, form a well-supported clade sister to the Phalloideae sensu Bas 1969, which include the lethal "death caps" and "destroying angels". Together, the A. ballerina-A. zangii clade and the Phalloideae sensu Bas 1969 also form a well-supported clade. We therefore screened for two of the most notorious toxins by HPLC-MS analysis of methanolic extracts from the basidiomata. Interestingly, neither α-amanitin nor phalloidin was found in A. ballerina, whereas Amanita fuligineoides was confirmed to contain both α-amanitin and phalloidin, and A. brunneitoxicaria contained only α-amanitin. Together with unique morphological characteristics, the position in the phylogeny indicates that A. ballerina is either an important link in the evolution of the deadly Amanita sect. Phalloideae species, or a member of a new section also including A. zangii.

Fusarium Species and Their Associated Mycotoxins.

The genus Fusarium includes numerous toxigenic species that are pathogenic to plants or humans, and are able to colonize a wide range of environments on earth. The genus comprises around 70 well-known species, identified by using a polyphasic approach, and as many as 300 putative species, according to phylogenetic species concepts; many putative species do not yet have formal names. Fusarium is one of the most economically important fungal genera because of yield loss due to plant pathogenic activity; mycotoxin contamination of food and feed products which often render them unaccep for marketing; and health impacts to humans and livestock, due to consumption of mycotoxins. Among the most important mycotoxins produced by species of Fusarium are the trichothecenes and the fumonisins. Fumonisins cause fatal livestock diseases and are considered potentially carcinogenic mycotoxins for humans, while trichothecenes are potent inhibitors of protein synthesis. This chapter summarizes the main aspects of morphology, pathology, and toxigenicity of the main Fusarium species that colonize different agricultural crops and environments worldwide, and cause mycotoxin contamination of food and feed.

Penicillium Species and Their Associated Mycotoxins.

Penicillium are very diverse and cosmopolite fungi, about 350 species are recognized within this genus. It is subdivided in four subgenera Aspergilloides, Penicillium, Furcatum, and Biverticillium; recently the first three has been included in Penicillium genus, and Biverticillium under Talaromyces. They occur worldwide and play important roles as decomposers of organic materials, cause destructive rots in the food industry where produces a wide range of mycotoxins; they are considered enzyme factories, and common indoor air irritants. In terms of human health are rarely associated as human pathogen because they hardly growth at 37°, while the main risk is related to ingestion of food contaminated by mycotoxins produced by several species of Penicillium. Various mycotoxins can occur in foods and feeds contaminated by Penicillium species, the most important are ochratoxin A and patulin; for which regulation are imposed in a number of countries, and at a less extent cyclopiazonic acid. In this chapter we summarize the main aspect of the morphology, ecology and toxigenicity of Penicillium foodborne mycotoxigenic species which belong mainly in subgenus Penicillium sections Brevicompacta, Chrysogena, Fasciculata, Penicillium, and Roquefortorum.

A Rapid Assay to Detect Toxigenic Penicillium spp. Contamination in Wine and Musts.

Wine and fermenting musts are grape products widely consumed worldwide. Since the presence of mycotoxin-producing fungi may greatly compromise their quality characteristics and safety, there is an increasing need for relatively rapid "user friendly" quantitative assays to detect fungal contamination both in grapes delivered to wineries and in final products. Although other fungi are most frequently involved in grape deterioration, secondary infections by Penicillium spp. are quite common, especially in cool areas with high humidity and in wines obtained by partially dried grapes. In this work, a single-tube nested real-time PCR approach-successfully applied to hazelnut and peanut allergen detection-was tested for the first time to trace Penicillium spp. in musts and wines. The method consisted of two sets of primers specifically designed to target the ß-tubulin gene, to be simultaneously applied with the aim of lowering the detection limit of conventional real-time PCR. The assay was able to detect up to 1 fg of Penicillium DNA. As confirmation, patulin content of representative samples was determined. Most of analyzed wines/musts returned contaminated results at >50 ppb and a 76% accordance with molecular assay was observed. Although further large-scale trials are needed, these results encourage the use of the newly developed method in the pre-screening of fresh and processed grapes for the presence of Penicillium DNA before the evaluation of related toxins.

[Forensic medical diagnostics of intoxication with certain poisonous mushrooms in the case of the lethal outcome in a hospital].

The present study was undertaken with a view to improving forensic medical diagnostics of intoxication with poisonous mushrooms in the cases of patients' death in a hospital. A total of 15 protocols of forensic medical examination of the corpses of the people who had died from acute poisoning were available for the analysis. The deathly toxins were amanitin and muscarine contained in various combinations in the death cap (Amanita phalloides) and the early false morels (Gyromitra esculenta and G. gigas). The main poisoning season in the former case was May and in the latter case August and September (93.4%). The mortality rate in the case of group intoxication (such cases accounted for 40% of the total) amounted to 28.6%. 40% of the deceased subjects consumed mushrooms together with alcohol. The poisoning caused the development of either phalloidin- or gyromitrin-intoxication syndromes (after consumption of Amanita phalloides and Gyromitra esculenta respectively). It is emphasized that the forensic medical experts must substantiate the diagnosis of poisoning with mushroom toxins based on the results of the chemical-toxicological and/or forensic chemical investigations. The relevant materials taken from the victim or the corpse should be dispatched for analysis not only within the first day but also on days 2-4 after intoxication. The mycological and genetic analysis must include the detection and identification of mushroom microparticles and spores in the smears from the oral cavity, vomiting matter, wash water, gastric and intestinal contents. In addition, the macro- and microscopic morphological signs, clinical data (major syndromes, results of laboratory studies, methods of treatment) should be taken into consideration as well as the time (season) of mushroom gathering, simultaneous poisoning in a group of people, and other pertinent information.

The mycotoxin definition reconsidered towards fungal cyclic depsipeptides.

Currently, next to the major classes, cyclic depsipeptides beauvericin and enniatins are also positioned as mycotoxins. However, as there are hundreds more fungal cyclic depsipeptides already identified, should these not be considered as mycotoxins as well? The current status of the mycotoxin definition revealed a lack of consistency, leading to confusion about what compounds should be called mycotoxins. Because this is of pivotal importance in risk assessment prioritization, a clear and quantitatively expressed mycotoxin definition is proposed, based on data of widely accepted mycotoxins. Finally, this definition is applied to a set of fungal cyclic depsipeptides, revealing that some of these should indeed be considered as mycotoxins.

[Metabolites of Toxigenic Fungi in Lichens of Genera Alectoria, Bryoria, Evernia, Pseudevernia, and Usnea].

Complexes of mycotoxins in fruticose lichens of 14 species belonging to five genera of the family Parmeliaceae were characterized by size, composition, and content of individual components. It was shown that species of the genus Bryoria always contain five mycotoxins (sterigmatocystin, mycophenolic acid, citrinin, emodin, and alternariol). In Evernia and Pseudevernia, this list is supplemented with zearalenone, diacetoxyscirpenol, and cyclopiazonic acid or fumonisins. It was noted that Alectoria and Usnea are distinguished by a peculiar set of toxic metabolites and occupy an intermediate position according to their number. The similarities and distinctions of the mycotoxin profile in species belonging to the same genus and in specimens from different habitats are discussed.

Development of a risk management tool for prioritizing chemical hazard-food pairs and demonstration for selected mycotoxins.

We developed a simple tool for ranking chemical hazard-food pairs to assist policy makers and risk managers selecting the hazard-food pairs that deserve more attention and need to be monitored during food safety inspections. The tool is based on the derivation of a "Priority Index" (PI) that results from the ratio of the potency of the hazard and the consumer exposure. The potency corresponds to a toxicity reference value of the hazard, whereas the exposure results from the combination of the concentration of the hazard in the food, and the food consumption. Tool's assumptions and limitations are demonstrated and discussed by ranking a dataset of 13 mycotoxins in 26 food items routinely analyzed in Switzerland. The presented ranking of mycotoxin-food pairs has to be considered as relative due to scarce exposure data availability, and uncertainties in toxicity reference values. However, this representative example allows demonstrating the simplicity and the ability of the PI tool to prioritize chemical hazard-food pairs.

[Metabolites of Toxigenic Fungi in Lichens of the Genera Nephroma, Peltigera, Umbilicaria, and Xanthoria].

The component composition of mycotoxin complexes is characterized in foliose lichens of the genera Nephroma, Peltigera, Umbilicaria, and Xanthoria. The interspecies differences in the genus Peltigera are expressed by the number of metabolites detected, from seven in P. aphthosa to three in P. canina, P. didactyla, P. praetextata, and P. rufescens. In Nephroma arcticum eight mycotoxins occurred regularly, with mycophenolic acid in especially high quantities in comparison with other lichens. In Umbilicaria, of six permanent components the content of alternariole is the highest, and in Xanthoria the content of emodine is the highest. Variation of the quantitative content of mycotoxins in general and of species of lichens is discussed, as is expansion of the background spectrum of these metabolites in collections from different territories.

[Secondary fungal metabolites (mycotoxins) in lichens of different taxonomic groups].

Secondary fungal metabolites (mycotoxins) in 22 lichen species of the families Parmeliaceae, Nephromataceae, Umbilicariaceae, Ramalinaceae, Cladoniaceae, Peltigeraceae, and Teloschistaceae were identified determined by enzyme immunoassay enzyme-linked immunosorbent assay. The following mycotoxins were identified found in these lichens in a broad concentration range with a frequency of 70-100%: sterigmatocystin (7-2090 ng/g), alternariol (20-6460 ng/g), and emodin (45-94500 ng/g). Mycophenolic acid frequently occurred in 19 lichen species; citrinin, in 17 species; diacetoxyscirpenol, in 11 species; cyclopiazonic acid, in 10 species; and zearalenone, in 9 species. PR toxin was regularly detected in three lichen species; deoxynivalenol, fumonisins, and ochratoxin A, in two species; and T-2 toxin and ergot alkaloids, in one species. Aflatoxin B1 was detected in only six species with a frequency of 2-42%, whereas roridin A was identified present in 10% of Hypogymnia physodes samples.