Penicillium Toxins Research Articles

A Preliminary Study to Classify Corn Silage for High or Low Mycotoxin Contamination by Using near Infrared Spectroscopy

Mycotoxins should be monitored in order to properly evaluate corn silage safety quality. In the present study, corn silage samples (n = 115) were collected in a survey, characterized for concentrations of mycotoxins, and scanned by a NIR spectrometer. Random Forest classification models for NIR calibration were developed by applying different cut-offs to classify samples for concentration (i.e., μg/kg dry matter) or count (i.e., n) of (i) total detectable mycotoxins; (ii) regulated and emerging Fusarium toxins; (iii) emerging Fusarium toxins; (iv) Fumonisins and their metabolites; and (v) Penicillium toxins. An over- and under-sampling re-balancing technique was applied and performed 100 times. The best predictive model for total sum and count (i.e., accuracy mean ± standard deviation) was obtained by applying cut-offs of 10,000 µg/kg DM (i.e., 96.0 ± 2.7%) or 34 (i.e., 97.1 ± 1.8%), respectively. Regulated and emerging Fusarium mycotoxins achieved accuracies slightly less than 90%. For the Penicillium mycotoxin contamination category, an accuracy of 95.1 ± 2.8% was obtained by using a cut-off limit of 350 µg/kg DM as a total sum or 98.6 ± 1.3% for a cut-off limit of five as mycotoxin count. In conclusion, this work was a preliminary study to discriminate corn silage for high or low mycotoxin contamination by using NIR spectroscopy.

A novel chemical marker of tremorgenic mycotoxicosis detected by gas-chromatography/mass-spectrometry

Tremorgenic mycotoxicosis can arise from dietary exposure to secondary metabolite products of various fungal species, particularly those from thePenicillium genus. Although general toxin screens often rely on gas chromatography-mass spectrometry (GC/MS) and well-developed mass spectral library databases, two principal representativePenicillium mycotoxins, roquefortine and penitrem A, are unamenable to GC/MS owing to high molecular weights, low volatilities and/or high thermal instabilities. Reliance on GC/MS screens alone could therefore inadvertently result in failure to collect evidence of exposure to such tremorgenic mycotoxins. In this report we describe a newly discovered tremorgenic marker compound (TMC), the presence of which correlates highly with conclusive exposure toPenicillium toxins in explanation of clinical manifestations of tremorgenic mycotoxicosis. According to detailed mass spectral deconvolution, the compound is 210.0892 molecular weight, and amenable to GC/MS whether chemically underivatized or derivatized by methylation or trimethylsilylation. 1D and 2D NMR (nuclear magnetic resonance) studies on the isolated compound determined the TMC to be thePenicillium product terrestric acid, C11H14O4, which matches the molecular formula determined by high resolution mass spectrometry and thus provides an excellent target for assessment of mycotoxicosis by GC/MS.

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.

Mycotoxicoses in veterinary medicine: Aspergillosis and penicilliosis.

Molds and mycotoxins are contaminants of animal feed causing spoilage and clinical intoxication. Animal exposure to mycotoxins reflects diet composition with major differences occurring between animals kept predominantly of pastures, i.e. ruminants and horses, and those consuming formulated feed like pigs and poultry. Mixed feeds are composed of several ingredients, often sourced from different continents. Subsequently, practitioners may confront endemic diseases and signs of toxin exposure related to toxins imported accidentally with contaminated feed materials from other countries and continents. Mycotoxins comprise more than 300 to 400 different chemicals causing a variety of clinical symptoms. Mycotoxin exposure causes major economic losses due to reduced performance, impaired feed conversion and fertility, and increased susceptibility to environmental stress and infectious diseases. In acute cases, clinical symptoms following mycotoxin ingestion are often non-specific, hindering an immediate diagnosis. Furthermore, most mold species produce more than one toxin, and feed commodities are regularly contaminated with various mold species resulting in complex mixtures of toxins in formulated feeds. The effects of these different toxins may be additive, depending on the level and time of exposure, and the intensity of the clinical symptoms based on age, health, and nutritional status of the exposed animal(s). Threshold levels of toxicity are difficult to define and discrepancies between analytical data and clinical symptoms are common in daily practice. This review aims to provide an overview of Aspergillus and Penicillium toxins that are frequently found in feed commodities and discusses their effects on animal health and productivity.

A Concise History of Mycotoxin Research.

Toxigenic fungi and mycotoxins entered human food supplies about the time when mankind first began to cultivate crops and to store them from one season to the next, perhaps 10,000 years ago. The storage of cereals probably initiated the transition by mankind from hunter-gatherer to cultivator, at the same time providing a vast new ecological niche for fungi pathogenic on grain crops or saprophytic on harvested grain, many of which produced mycotoxins. Grains have always been the major source of mycotoxins in the diet of man and his domestic animals. In the historical context, ergotism from Claviceps purpurea in rye has been known probably for more than 2000 years and caused the deaths of many thousands of people in Europe in the last millennium. Known in Japan since the 17th century, acute cardiac beriberi associated with the consumption of moldy rice was found to be due to citreoviridin produced by Penicillium citreonigrum. This toxin was believed to be only of historic importance until its reemergence in Brazil a few years ago. Other Penicillium toxins, including ochratoxin A, once considered to be a possible cause of Balkan endemic nephropathy, are treated in a historical context. The role of Fusarium toxins in human and animal health, especially T-2 toxin in alimentary toxic aleukia in Russia in the 1940s and fumonisins in equine leucoencephalomalasia, is set out in some detail. Finally, this paper documents the story of the research that led to our current understanding of the formation of aflatoxins in grains and nuts, due to the growth of Aspergillus flavus and its role, in synergy with the hepatitis B virus, in human liver cancer. During a period of climate change and greatly reduced crop diversity on a global basis, researchers tasked with monitoring the food system need to be aware of fungal toxins that might have been rare in their working careers that can reappear.

Silage and livestock health

Silage making involves preserving forage crops by fermentation of water-soluble carbohydrates (sugars) to organic acids. The fermentation is relatively uncontrolled and inadequate acidification can allow the growth of undesirable microorganisms such as enterobacteria and clostridia. Ingress of oxygen to the outer layer of the silo or bale during the storage period encourages growth of yeasts and moulds. Mycobacterium bovis may survive in silages with pH above 5.0. Outgrowth of clostridia in aerobically deteriorating silage can lead to high counts of butyric acid spores in silage that can contaminate milk and cheese. Milk may be contaminated with Penicillium toxins and be rejected for containing antibiotics. Specific disease risks to livestock from poorly preserved silage include bacterial endotoxicosis, listeriosis, botulism, mycotoxicosis and nitric acid poisoning in addition to reduced feed value. Rapid acidification is essential once the crop is ensiled to restrict the growth of enterobacteria, listeria and clostridia. Oxygen barrier film, in addition to reducing loss of dry matter during the storage period, prevents clostridial outgrowth in the outer layer of the silo and is also likely to reduce the risk of listeriosis.

Aspergillus and Penicillium toxins in chestnuts and derived products produced in Italy

Occurrence of aflatoxins, ochratoxin A, sterigmatocystin, cyclopiazonic acid, citrinin, roquefortine C and mycophenolic acid in fresh chestnuts and dried chestnut products was surveyed. A total of eighty-two samples were collected from retail outlets located in northern Italy. After specific extraction and purification through immunoaffinity or prepacked columns, mycotoxins were analysed using HPLC-FLD or HPLC-MS/MS. In fresh chestnuts, mycotoxins were rarely detected, while a widespread contamination was found in dried products, particularly in chestnut flour. The incidence of aflatoxin B1 was 92.0 and 40.0% in chestnut flour (maximum value 58.6 μg kg−1) and dried chestnuts, respectively; in chestnut flour, the percentage of samples exceeding the value of 2.0 μg kg−1 for aflatoxin B1 (maximum limit fixed by EC Regulation 165/2010 in dried fruits) was 24.0%. Chestnut flour was also often contaminated with ochratoxin A, citrinin, roquefortine C and mycophenolic acid, showing sometimes high values (particularly for mycophenolic acid); in 80% of samples, more than four mycotoxins were detected. To the best of our knowledge, no data are available from previous studies about the occurrence of citrinin, roquefortine C and mycophenolic acid in chestnuts and derived products. These results showed that probably the contamination occurred in the post-harvest, during exsiccation, storage and sorting; the high incidence and concentration found in chestnut flour are probably related to the practice of producing it by grinding of the product coming from sieving of dried chestnuts.

Neurotoxicity of Penicillium crustosum secondary metabolites: Tremorgenic activity of orally administered penitrem A and thomitrem A and E in mice

Several cases of neurological disease in dogs after poisoning by food- and feed-borne Penicillium toxins in Norway during the last years have uncovered a lack of knowledge regarding the toxicity and mechanism of action of neuroactive mycotoxins. In the present study, the lowest tremor-inducing dose after single oral administration of penitrem A to mice was 0.50 mg/kg bw. The estimated half maximal effective dose (ED50) in respect to the visual tremor scale was 2.74 mg/kg bw. Mice receiving the maximum penitrem A dose (8 mg/kg bw) suffered severe spontaneous tremors and even convulsions. Thomitrem A and E are penitrem analogues lacking the C-16–C-18 ether linkage and possessing an olefin at C-18–C-19. Compared with penitrem A, the lowest tremor-inducing dose of thomitrem A was 16-times higher (8 mg/kg bw) and thomitrem E was found to be non-tremorgenic at the highest dose tested (16 mg/kg bw). During a recovery phase of two weeks post administration animals appeared restored and no changes in feeding and other biological processes were observed. An initial dose-related weight reduction was observed 2 days after penitrem A administration. Penitrem A was absorbed and distributed to gastrointestinal tract, liver, kidneys and brain in the mice. Elimination of penitrem A appeared to be mainly hepatic and the highest concentration levels were found 1 h post administration for all investigated organs. The relationship between liver and gastrointestinal tract concentration levels showed time-dependent linear correlation and a doubling within 1.5 h.

Poisoning by penicillium toxins—Clinical and brain receptor effects

Poisoning by penicillium toxins—Clinical and brain receptor effects

Mycotoxins in small grains and maize: Old problems, new challenges

This paper reviews the challenges relating to chronic contamination of small grains and maize with deoxynivalenol and related compounds, fumonisin and the use of ensiled cereals in cool dairy areas. Uncertainties in the tolerable daily intakes for deoxynivalenol and fumonisin are discussed as they have the potential to affect current regulatory limits. In addition, climate change is resulting in more extreme rainfall and drought events which favour formation of deoxynivalenol and fumonisin, respectively. The development and refinement of models for predicting mycotoxin accumulation from weather data will become an essential tool for managing these events. Such models are also important for providing timely food aid to developing countries, which experience increased occurrence of acute toxicities, especially in children. Chronic contamination of silage in some areas with some Penicillium toxins deserves more attention in terms of their economic effects and possible implications for the purity of milk.

Influence of gamma-radiation on the occurrence of toxigenic Pennicium strains and mycotoxins production in different feedstuffs

One hundred and fifty random poultry and animal feed samples were collected and analyzed for Penicillium toxins. The effect of gamma irradiation on the production of mycotoxins in feeds was studied. The results revealed the isolation of 12 Penicillium species. Out of 179 pure cultures of Penicillium species only 104 isolates (58%) were mycotoxin-producers. The isolates have the ability to produce, citrinin, patulin, ochratoxin A, rubratoxin B and cyclopiazonic acid with variable levels. Of the 150 samples examined, 70 were positive for Penicillium toxins. Analysis of feed samples revealed the occurrence of citrinin, ochratoxin A, cyclopiazonic acid and rubratoxin B. Irradiation at a dose 4.0 kGy reduced the Penicillium growth greatly relative to unirradiated controls and there was no growth and mycotoxin production at dose 5.0 kGy. Treatment of feed samples with 10 kGy detoxified citrinin by 92.5 to 97.5% and ochratoxin A by 68.8 to 78.5%. A dose of 15 kGy was sufficient for complete destruction of citrinin and by 90 to 95% for ochratoxin A. Patulin, cyclopiazonic acid and rubratoxin B were not detect at 5.0 kGy. Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 130-144

DNA damage by mycotoxins

Mycotoxins are toxic fungal metabolites which are structurally diverse, common contaminants of the ingredients of animal feed and human food. To date, mycotoxins with carcinogenic potency in experimental animal models include aflatoxins, sterigmatocystin, ochratoxin, fumonisins, zearalenone, and some Penicillium toxins. Most of these carcinogenic mycotoxins are genotoxic agents with the exception of fumonisins, which is currently believed to act by disrupting the signal transduction pathways of the target cells. Aflatoxin B 1 (AFB 1), a category I known human carcinogen and the most potent genotoxic agent, is mutagenic in many model systems and produces chromosomal aberrations, micronuclei, sister chromatid exchange, unscheduled DNA synthesis, and chromosomal strand breaks, as well as forms adducts in rodent and human cells. The predominant AFB 1–DNA adduct was identified as 8,9-dihydro-8-( N 7-guanyl)-9-hydroxy–AFB 1 (AFB 1– N 7-Gua), which derives from covalent bond formation between C8 of AFB 1–8,9-epoxides and N 7 of guanine bases in DNA. Initial AFB 1– N 7-guanine adduct can convert to a ring-opened formamidopyrimidine derivative, AFB 1–FAPY. The formation of AFB 1– N 7-guanine adduct was linear over the low-dose range in all species examined, and liver, the primary target organ, had the highest level of the adduct. Formation of initial AFB 1– N 7-guanine adduct was correlated with the incidence of hepatic tumor in trout and rats. The AFB 1– N 7-guanine adduct was removed from DNA rapidly and was excreted exclusively in urine of exposed rats. Several human studies have validated the similar correlation between dietary exposure to AFB 1 and excretion of AFB 1– N 7-guanine in urine. Replication of DNA containing AFB 1– N 7-guanine adduct-induced G→T mutations in an experimental model. Activation of ras protooncogene has been found in AFB 1-induced tumors in mouse, rat, and fish. More strikingly, the relationship between aflatoxin exposure and development of human hepatocellular carcinoma (HHC) was demonstrated by the studies on the p53 tumor suppressor gene. High frequency of p53 mutations (G→T transversion at codon 249) was found to occur in HHC collected from populations exposed to high levels of dietary aflatoxin in China and Southern Africa. Furthermore, AFB 1-induced DNA damage and hepatocarcinogenesis in experimental models can be modulated by a variety of factors including nutrients, chemopreventive agents, and other factors such as food restriction and viral infection, as well as genetic polymorphisms.

EFFECTS OF MYCOTOXINS ON CYTOKINE PRODUCTION AND PROLIFERATION IN EL-4 THYMOMA CELLS

The thymoma cell line EL4.IL-2 (EL-4) was used as a T-cell model to assess the immunotoxic effects of several mycotoxins produced by the Aspergillus-Penicillium and the Fusarium groups. EL-4 cells were stimulated with phorbol 12-myristate 12-acetate (PMA) in the presence of mycotoxins at various concentrations for 5 d and culture supernatants were analyzed for interleukins (IL) IL-2 and IL-5 by enzyme-linked immunosorbent assay (ELISA). The cytokine effects were further related to proliferation and cell viability using the MTT [3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay with absorbance at 570 nm (A570) as the endpoint indicator. IL-2 and IL-5 levels were dramatically increased by cyclopiazonic acid at 50-1000 ng/ml, whereas IL-2 was significantly decreased at 10 microgram/ml. Proliferation was slightly increased at 100-1000 ng/ml cyclopiazonic acid but markedly depressed at 5 and 10 microgram/ml. When EL-4 cells were exposed to 5 and 10 microgram/ml of ochratoxin A, IL-2 production was markedly increased while IL-5 production was significantly decreased. The A570 was significantly decreased by ochratoxin A at 10 microgram/ml. IL-2 and Il-5 production was almost totally suppressed by patulin at concentrations > or = 500 ng/ml and by T-2 toxin at > or = 5 ng/ml. These effects occurred concurrently with marked depression of A570 in the MTT assay. Although A570 was unaffected by either zearalenone or alpha-zearalenol exposure, both IL-2 and IL-5 levels were significantly elevated by these toxins at 5 or 10 microgram/ml. IL-2 and IL-5 production were not affected in EL-4 cells cultured with either the Aspergillus-Penicillium toxins aflatoxin B1 and secalonic acid or the Fusarium toxins wortmannin, fumonisin B1, or fusaric acid at concentrations up to 10 microgram/ml. In total, the EL-4 culture studies indicated that cyclopiazonic acid, ochratoxin A, zearalenone, and alpha-zearalenol could stimulate cytokine production whereas patulin and T-2 toxin were inhibitory. Cytokine dysregulation was not always related directly to perturbations in proliferation. The results suggest that the EL-4 thymoma cell line could be a simple and effective in vitro model for evaluating immunotoxicity of various classes of environmental chemicals.