Low Concentrations Of Deoxynivalenol Research Articles

Prevalence of mycotoxins from silage in a small beef cattle feedlot over a storage season: a case study

Challenges exist worldwide for the storage of animal feed, though year-long feeding of livestock is especially difficult in northern climates, including Canada. Usage of silage allows for crops to be harvested at the optimal nutritional value and stored for extended periods in customized storage systems. This case study investigated various feed management practices typical of small beef cattle feedlot farms in Southwestern Ontario, Canada. Crop samples were collected monthly and analyzed for mycotoxins and fungal secondary metabolites by liquid chromatography mass spectrometry (LC-MS/MS). Stored barley contained modest concentrations of deoxynivalenol and related compounds, as well as near trace amounts of fumonisin. Stored hay contained low concentrations of deoxynivalenol and beauvericin from the field. Hot spots of the Penicillium metabolite citrinin were detected after 7–8 months of storage in barley (39.2 ng g−1). Citrinin was also detected after 7–8 months in hay (16.3–17.5 ng g−1) along with the appearance of trace amounts of other storage toxins, including penitrem A (8.84 ng g−1), griseofulvin (34.8 ng g−1) and sterigmatocystin (14.1–261 ng g−1). This case study was a unique opportunity to use ‘citizen science’ to observe the onset of postharvest fungi and associated mycotoxins in various storage conditions allowing for the farmer to make necessary modifications before major problems begin. An improved understanding of the storage conditions that foster fungal growth and mycotoxin production in this working farm led to better agronomic practices, ultimately improving feed quality, livestock health, and profitability.

Response of Barley Genotypes to Fusarium Head Blight under Natural Infection and Artificial Inoculation Conditions.

Forty-eight spring barley genotypes were evaluated for deoxynivalenol (DON) concentration under natural infection across 5 years at Harrington, Prince Edward Island. These genotypes were also evaluated for Fusarium head blight (FHB) severity and DON concentration under field nurseries with artificial inoculation of Fusarium graminearum by the grain spawn method across 2 years at Ottawa, Ontario, and one year at Hangzhou, China. Additionally, these genotypes were also evaluated for FHB severity under greenhouse conditions with artificial inoculation of F. graminearum by conidial suspension spray method across 3 years at Ottawa, Ontario. The objective of the study was to investigate if reactions of barley genotypes to artificial FHB inoculation correlate with reactions to natural FHB infection. DON concentration under natural infection was positively correlated with DON concentration (r = 0.47, P < 0.01) and FHB incidence (r = 0.56, P < 0.01) in the artificially inoculated nursery with grain spawn method. Therefore, the grain spawn method can be used to effectively screen for low DON. FHB severity, generated from greenhouse spray, however, was not correlated with DON concentration (r = 0.12, P > 0.05) under natural infection and it was not correlated with DON concentration (r = −0.23, P > 0.05) and FHB incidence (r = 0.19, P > 0.05) in the artificially inoculated nursery with grain spawn method. FHB severity, DON concentration, and yield were affected by year, genotype, and the genotype × year interaction. The effectiveness of greenhouse spray inoculation for indirect selection for low DON concentration requires further studies. Nine of the 48 genotypes were found to contain low DON under natural infection. Island barley had low DON and also had high yield.

Examination of high-resolution feed intake data of growing-finishing pigs confronted with high deoxynivalenol contents present in their feed

Modern single space feeding systems for fattening pigs allow the detailed assessment of an individual animal’s feeding behaviour. In an experiment involving 96 fattening pigs, the influence of deoxynivalenol (DON) contaminated feed (&amp;gt; 4 500 µg/kg DON) on the zootechnical performance and feeding behaviour was compared with a feed with low DON concentration (&amp;lt; 900 µg/kg DON), this served as the control group. Additionally, in separate treatments, two commercial mycotoxin binders were added to the DON-contaminated feeds to assess if an expected DON effect could be attenuated. The high DON content significantly (P &amp;lt; 0.03) reduced daily feed intake (500–600 g/day). The DON group showed 240 g less daily gain compared to the control with 728 g/day. Both mycotoxin binders were seen to additionally depress weight gain by approximately 65 g/day (P &amp;lt; 0.01). The treatment did not affect the individual feeding behaviour as assessed by daily visits to the feeder, feed intake per visit and the highest feed intake per visit per day. These were influenced only by the pig and its pen, indicating that the animals developed a distinct behaviour within their respective groups. Behaviour analyses of persistency and day-to-day variation showed that the persistency was reduced and variation was increased when DON without or with binder was present. The DON contents therefore seemed to lead to a more erratic and less consistent behaviour that remained dependent on the animal group.

Does low concentration mycotoxin exposure induce toxicity in HepG2 cells through oxidative stress?

The purpose of this study was to determine whether exposure to low concentrations of deoxynivalenol (DON), T-2 toxin (T-2) and patulin (PAT) in a human hepatocellular carcinoma cell line (HepG2) exerts toxic effects through mechanisms related to oxidative stress, and how cells deal with such exposure. Cell viability was determined by the MTT and protein content (PC) assays over 24, 48 and 72 h. The IC50 values detected ranged from >10 to 2.53 ± 0.21 μM (DON), 0.050 ± 0.025 to 0.034 ± 0.007 μM (T-2) and 2.66 ± 0.66 to 1.17 ± 0.21 µM (PAT). The key players in oxidative stress are the generation of reactive oxygen species (ROS), lipid peroxidation (LPO) and mitochondrial membrane potential (MMP) dysfunction. The results obtained showed that PAT, DON and T-2 did not significantly increase LPO or ROS production with respect to the controls. Moreover, PAT and DON did not alter MMP, though T-2 increased MMP at the higher concentrations tested (17 and 34 nM). In conclusion, the exposure of HepG2 cells to nontoxic concentrations of T-2 condition them against subsequent cellular oxidative conditions induced by even higher concentrations of mycotoxin.

The Food-Associated Ribotoxin Deoxynivalenol Modulates Inducible NO Synthase in Human Intestinal Cell Model.

The intestinal epithelium possesses active immune functions including the production of proinflammatory cytokines and antimicrobial molecules such as nitric oxide (NO). As observed with immune cells, the production of NO by the intestinal epithelium is mainly due to the expression of the inducible NO synthase (iNOS or NOS2). Epithelial immune functions could be affected by many factors including pathogenic microorganisms and food-associated toxins (bacterial and fungal). Among the various mycotoxins, deoxynivalenol (DON) is known to alter the systemic and intestinal immunity. However, little is known about the effect of DON on the production of NO by the intestinal epithelium. We studied the impact of DON on the intestinal expression of iNOS using the Caco-2 cell model. In line with its proinflammatory activity, we observed that DON dose-dependently up-regulates the expression of iNOS mRNA. Surprisingly, DON failed to increase the expression of iNOS protein. When testing the effects of DON on cytokine-mediated induction of iNOS, we found that very low concentrations of DON (ie, 1 µM) decrease the amount of iNOS protein but not of iNOS mRNA. We demonstrated that DON's effect on iNOS protein relies on its ability to activate signal pathways and to increase iNOS ubiquitinylation and degradation through the proteasome pathway. Taken together, our results demonstrate that although DON causes intestinal inflammation, it suppresses the ability of the gut epithelium to express iNOS and to produce NO, potentially explaining the increased susceptibility of animals to intestinal infection following exposure to low doses of DON.

Dual effects exerted in vitro by micromolar concentrations of deoxynivalenol on undifferentiated caco-2 cells.

Contamination of crops used for food and feed production with Fusarium mycotoxins, such as deoxynivalenol (DON), raise important health and economic issues all along the food chain. Acute exposure to high DON concentrations can alter the intestinal barrier, while chronic exposure to lower doses may exert more subtle effects on signal transduction pathways, leading to disturbances in cellular homeostasis. Using real-time cellular impedance measurements, we studied the effects exerted in vitro by low concentrations of DON (0.37–1.50 μM), relevant for mycotoxin-contaminated food, on the proliferation of undifferentiated Caco-2 cells presenting a tumorigenic phenotype. A 1.5 μM concentration of DON maintained cell adherence of non-proliferating Caco-2 cells, whilst arresting the growth of actively proliferating cells compared with control Caco-2 cells in vitro. At 0.37 μM, DON enhanced Caco-2 cell metabolism, thereby triggering a moderate increase in cell proliferation. The results of the current study suggested that low concentrations of DON commonly detected in food may either limit or sustain the proliferation of colon cancer cells, depending on their proliferation status and on DON concentration. Soluble factors released by Lactobacillus strains can partially counteract the inhibitory action of DON on actively proliferating colon cancer cells. The study also emphasized that real-time cellular impedance measurements were a valuable tool for investigating the dynamics of cellular responses to xenobiotics.

Effect of deoxynivalenol (DON) mycotoxin on in vivo and in vitro porcine circovirus type 2 infections

Deoxynivalenol (DON) is a mycotoxin produced by Fusarium spp and is a common contaminant of grains in North America. Among farm animals, swine are the most susceptible to DON because it markedly reduces feed intake and decreases weight gain. Porcine circovirus type 2 (PCV2) is the main causative agent of several syndromes in weaning piglets collectively known as porcine circovirus-associated disease (PCVAD). The objectives of this study were to investigate the impact of DON on PCV2 replication in NPTr permissive cell line, and to determine eventual potentiating effects of DON on PCV2 infection in pigs. Noninfected and infected cells with PCV2 were treated with increasing concentrations of DON (0, 70, 140, 280, 560, 1200ng/mL) and cell survival and virus titer were evaluated 72h postinfection. Thirty commercial piglets were randomly divided into 3 experimental groups of 10 animals based on DON content of served diets (0, 2.5 and 3.5mg/kg DON). All groups were further divided into subgroups of 6 pigs and were inoculated with PCV2b virus. The remaining pigs (control) were sham-inoculated with PBS. In vitro results showed that low concentrations of DON could potentially increase PCV2 replication depending on virus genotype. In vivo results showed that even though viremia and lung viral load tend to be higher in animal ingesting DON contaminated diet at 2.5mg/kg, DON had no significant effect on clinical manifestation of PCVAD in PCV2b infected animals. DON has neither in vitro nor in vivo clear potentiating effects in the development of porcine circovirus infection despite slight increases in viral replication.

Deoxynivalenol impairs the immune functions of neutrophils

Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., is toxic to many animal species, with pigs being the most sensitive species to the toxin. The aim of the present study was to determine the effects of DON on pig polymorphonuclear cells (PMNs), the first line of defense against infection. PMNs isolated from pig blood samples were stimulated with LPS to mimic infection. DON (0.5-10 μM) altered three main functions of pig PMNs: LPS-induced secretion of IL-8, chemotaxis, and phagocytosis capability. This alteration of PMN properties was due to apoptotis induced by DON exposure. Using Western blot and flow cytometry, we demonstrated that this process included the permeabilization of the mitochondrial outer membrane and the activation of caspase-3. The effect of DON was mediated by the phosphorylation of the p38 mitogen-activated protein kinase within the first 30 min of exposure. This study provides evidence that low concentrations of DON can alter the immune functions of porcine PMNs and suggests the involvement of p38 mitogen-activated protein kinase in the signal transduction pathway. These immunosuppressive effects of DON may have implications for humans and/or animals when eating contaminated food/feed.

The Mycotoxin Deoxynivalenol Potentiates Intestinal Inflammation by Salmonella Typhimurium in Porcine Ileal Loops

Background and AimsBoth deoxynivalenol (DON) and nontyphoidal salmonellosis are emerging threats with possible hazardous effects on both human and animal health. The objective of this study was to examine whether DON at low but relevant concentrations interacts with the intestinal inflammation induced by Salmonella Typhimurium.MethodologyBy using a porcine intestinal ileal loop model, we investigated whether intake of low concentrations of DON interacts with the early intestinal inflammatory response induced by Salmonella Typhimurium.ResultsA significant higher expression of IL-12 and TNFα and a clear potentiation of the expression of IL-1β, IL-8, MCP-1 and IL-6 was seen in loops co-exposed to 1 µg/mL of DON and Salmonella Typhimurium compared to loops exposed to Salmonella Typhimurium alone. This potentiation coincided with a significantly enhanced Salmonella invasion in and translocation over the intestinal epithelial IPEC-J2 cells, exposed to non-cytotoxic concentrations of DON for 24 h. Exposure of Salmonella Typhimurium to 0.250 µg/mL of DON affected the bacterial gene expression level of a limited number of genes, however none of these expression changes seemed to give an explanation for the increased invasion and translocation of Salmonella Typhimurium and the potentiated inflammatory response in combination with DON.ConclusionThese data imply that the intake of low and relevant concentrations of DON renders the intestinal epithelium more susceptible to Salmonella Typhimurium with a subsequent potentiation of the inflammatory response in the gut.

The mycotoxin Deoxynivalenol inhibits the cell surface expression of activation markers in human macrophages

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. It exhibits several toxic effects including impaired growth and immune dysregulation. Macrophages play pivotal role in the host defense; upon activation, they express several specific cell surface receptors that are important in adhesion and cell signaling. Several studies have demonstrated that DON can affect macrophages, however, very few data are available concerning the effect of DON on human macrophages, and the effect on macrophage cell surface receptors is unknown. In the present study, human blood monocytes, differentiated in vitro into macrophages, were activated with IFN-γ, in the presence or absence of low concentrations of DON. The expression of CD11c, CD13, CD14, CD18, CD33, CD35, CD54, CD119 and HLA-DP/DQ/DR was analyzed by flow cytometry. As expected, macrophage activation by IFN-γ upregulated the expression of CD54, CD14, CD119 and HLA-DP/DQ/DR. Incubation with DON decrease the cell surface expression of these activation markers in a dose-dependent manner. When cells were treated with 5 μM DON, the mean fluorescence intensity measured for the expression of these receptors was the same as that observed in non-activated macrophages. This inhibitory effect of DON was only observed when the mycotoxin was applied before the activation signal. Taken together, our results suggest that low concentration of DON alter macrophage activation as measured by the expression of cell surface markers. This may have implications for human health when consuming DON contaminated feed.

Immunotoxicology of mycotoxins produced by Fusarium fungi—Low concentrations of deoxynivalenol interfere with nucleotide metabolism

Immunotoxicology of mycotoxins produced by Fusarium fungi—Low concentrations of deoxynivalenol interfere with nucleotide metabolism

Analysis of the chromosome 2(2H) region of barley associated with the correlated traits Fusarium head blight resistance and heading date

Fusarium head blight (FHB) is a major disease of barley (Hordeum vulgare L.) that results in reduced grain yield and quality through the accumulation of the mycotoxin deoxynivalenol (DON). Coincident QTL for FHB severity, DON concentration, and heading date (HD) map to a region of chromosome 2(2H) designated Qrgz-2H-8. It is unclear whether disease resistance at this locus is due to a pleiotropic effect of late HD by delaying the host exposure to the pathogen or a tightly linked resistance gene. The objectives of this study were to develop a set of near isogenic lines (NILs) for the Qrgz-2H-8 region and to genetically dissect the QTL region containing the coincident traits. Two NIL populations were developed consisting of F(2)- and F(4)-derived recombinants from a cross between a BC(5) line carrying the donor parent (Chevron) alleles in the Qrgz-2H-8 region and the recurrent parent M69. Analysis of field and marker data from these NILs revealed that the Chevron alleles conditioning FHB resistance, late HD, and low DON concentration were successfully introgressed into the BC(5) parent line and were segregating among NILs. QTL analysis of the F(4)-derived population showed that the HD QTL is adjacent to the FHB QTL. Furthermore, a single NIL was identified that was similar to the resistant BC(5) parent for FHB severity and the early flowering parent M69 for HD. These results indicate that the relationship between FHB and HD at the Qrgz-2H-8 region is likely due to tight linkage rather than pleiotropy.

Evaluation of environmental and management effects on Fusarium head blight infection and deoxynivalenol concentration in the grain of winter wheat

During the past decade European wheat ( Triticum aestivum L.) crops were increasingly affected by Fusarium head blight (FHB) infection and high deoxynivalenol (DON) concentration in the grain. This is discussed to be primarily caused by the application of conservation tillage practices and, simultaneously, enhanced cultivation of host crops such as maize and wheat. This study investigated the effect of several environmental and management factors on DON formation and, particularly, interactions between factors. Moreover, a methodology was developed to quantify the relative effect of single factors, and combinations of two or more management factors on the DON concentration in order to evaluate management strategies with regard to their potential effect on DON formation. In 2001 and 2002, FHB infection of wheat and DON concentration in the grain was determined on eight sites of a long-term on-farm tillage experiment located in southern and eastern Germany. In addition, three-factorial plot trials varying tillage, cultivar, and fungicide application were conducted at Goettingen, Lower Saxony, in 2001 and 2002. Based on these data, the severity of the effect of year, preceding crop, tillage, wheat cultivar and fungicide application was calculated as follows: mean DON value of the treatment with the highest DON concentration obtained in the study divided by the lowest treatment mean value. For each influencing factor the calculation was done on average of the other factors included. The following ranking order was obtained: annual variation of rainfall during wheat anthesis (4.4) = Fusarium infection/susceptibility of the preceding crop (wheat versus sugar beet, 4.3) = Fusarium susceptibility of the wheat cultivar grown (4.3–5.6) > soil tillage applied to cultivate wheat (1.3–3.4) ≥ fungicide application at anthesis of wheat (2.1). The relative effect of combinations of two or more management factors on the DON concentration can be calculated by multiplying severity factors. Limitations of this method are due to strong interactions between some of the factors (e.g. cultivar × fungicide application, cultivar × tillage). Within tillage treatments shallow mixing conservation tillage increased the DON concentration only slightly compared to conventional mouldboard ploughing, if previous wheat straw soil cover was reduced to about 30%. In this case, the severity of the effect of tillage was much smaller compared to year, preceding crop, and cultivar. Data also reveal that FHB infection values counted in the field do not give a reliable prediction of the DON concentration. It is concluded, that choosing a resistant cultivar is a powerful tool to ensure a low DON concentration in wheat grain even under highly infectious conditions. This strategy enables farmers to make use of the benefits of conservation tillage and, simultaneously, produce high quality wheat grain.

In vitro effect of deoxynivalenol on the differentiation of human colonic cell lines Caco-2 and T84.

The effects of low concentrations of deoxynivalenol (DON) on structural and functional characteristics of human colonic adenocarcinoma cell lines Caco-2 and T84 were examined. Scanning electron microscopic (SEM) analysis of the apical surfaces of Caco-2 cells revealed reduction or abnormal formation of brush borders in the presence of 50, 100 and 200 ng/ml of DON. Monolayer integrity of Caco-2 and T84 cells was studied using cells which were cultured on permeable membranes. The transepithelial electrical resistance (TEER) of Caco-2 cells was significantly reduced at 50, 100 and 200 ng/ml of DON, significant increase in lucifer yellow (LY) permeability was also observed in these cells at 100 ng/ml of DON. The TEER of T84 cells was significantly reduced at 100 and 200 ng/ml of DON. LY permeability significantly increased at 200 ng/ml of DON in T84 cells. Enzyme activities in Caco-2 cells were also examined. Alkaline phosphatase activity was reduced from the 6th to 15th day of culture in the presence of 100 or 200 ng/ml of DON, whereas sucrase-isomaltase activity was significantly decreased by adding 50 or 100 ng/ml of DON for 15 or 20 days. Protein content was attenuated only by treatment with 200 ng/ml of DON throughout the experimental period. The results indicate that DON interferes with structural and functional characteristics of differentiation in enterocytes at low doses.