Mary Ann Dombrink-Kurtzman

Lymphocyte cytotoxicity and erythrocytic abnormalities induced in broiler chicks by fumonisins B1 and B2 and moniliformin fromFusarium proliferatum

Peripheral blood lymphocytes were isolated from broiler chicks that had ingested feed amended with autoclavedFusarium proliferatum culture material containing fumonisin B1 (FB1), fumonisin B2 (FB2) and moniliformin. Lymphocyte viability was determined for birds that were placed on amended rations at day 1 or day 7 of age at three different levels of mycotoxins, ranging from 61–546 ppm FB1, 14–94 ppm FB2 and 66–367 ppm moniliformin. Reduction of the tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], to yield MTT formazan, based on mitochondrial metabolic activity, was used to assess cell viability. Lymphocyte cytotoxic effects were observed in all treatment groups on day 21; chicks that started on amended feed at day 1 of age were affected more than those that started at day 7. Abnormal erythrocytes resembling early stages of erythroblasts were observed in peripheral blood from test chicks. Abnormally shaped red cells (poikilocytes) having a spindle-shape with one or both ends pointed were present. Some red cells appeared to be undergoing mitosis. Both reduced lymphocyte viability and abnormal erythrogenesis occurred in chicks given feed amended withF. proliferatum culture material containing FB1, FB2 and moniliformin.

Fumonisin and beauvericin induce apoptosis in turkey peripheral blood lymphocytes.

Fumonisins, a family of mycotoxins produced by Fusarium verticillioides (synonym Fusarium moniliforme Sheldon) and F. proliferatum, have been associated with various deleterious effects in different animal species. Serological, hematological and pathological effects and mortality have previously been observed in broiler chicks fed F. proliferatum culture material containing known concentrations of fumonisin, moniliformin and beauvericin. Turkey peripheral blood lymphocytes were exposed in vitro for 72 hours to fumonisin B1(FB1), fumonisin B2(FB2), hydrolyzed fumonisin B1 (HFB1), moniliformin and tricarballylic acid (TCA) (0.01-25 μg/ml). A decrease in cell proliferation, as determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] bioassay, occurred in the order: FB2 > FB1 > HFB1, with IC50 = 0.6 μM, 1 μM and 10 μM, respectively. Internucleosomal DNA fragmentation and morphological features characteristic of apoptosis were observed following exposure to fumonisin B1 and beauvericin; cytoplasmic condensation and membrane blebbing were seen by light microscopy. Tricarballylic acid and moniliformin did not interfere with cell proliferation. Results suggested that fumonisin B1 and beauvericin may affect immune functions by suppressing proliferation and inducing apoptosis of lymphocytes.

Embryopathic and embryocidal effects of purified fumonisin B1 or Fusarium proliferatum culture material extract on chicken embryos

One hundred eight fertile eggs (Columbia × New Hampshire) were assigned to 10 groups of 10 eggs each (2 control groups had 14 eggs each). Five groups of eggs were inoculated on day 1 of incubation, while the other 5 groups were inoculated on day 10. The inoculum of the 4 treatment groups on both day 1 and 10 consisted of 1,10, or 100 µM purified fumonisin B1 (FB1) or a culture material extract (CME) ofFusarium proliferatum, having known amounts of FB1, FB2 and moniliformin (FB1 20 µM; FB2 4 µM and moniliformin 7 µM). Inoculum consisted of the respective toxin(s) dissolved in 100 µl double distilled, autoclaved water (diluent). Control eggs were inoculated with diluent only. Mortality was both dose- and time-responsive in all treatments. Eggs inoculated on day 1 with 1 µM FB1 had 50% mortality; 10 µM FB1 had 70% mortality; 100 µM FB1 had 100% mortality; and CME had 100% mortality. Eggs inoculated on day 10 with 1,10 or 100 µM FB1 or CME had 30, 60, 90 and 80% mortality, respectively. Normal chicks were hatched from all control eggs. The median death times (MDT50) were inversely dose-responsive in all treatments, ranging from 3.0 to 7.4 days in embryos exposed on day 1 and from 3.2 to 9.0 days in those exposed on day 10. Early embryonic changes in exposed embryos included hydrocephalus, enlarged beaks and elongated necks. Pathologic changes were noted in liver, kidneys, heart, lungs, musculoskeletal system, intestines, testes and brain toxin-exposed embryos.

Serohematologic Alterations in Broiler Chicks on Feed Amended with Fusarium Proliferatum Culture Material or Fumonisin B1 and Moniliformin

Two hundred twenty-eight male broiler chicks (Columbia x New Hampshire) were given feed amended with autoclaved culture material of Fusarium proliferatum containing fumonisin B1 (FB1) at 61, 193, and 546 ppm, fumonisin B2 (FB2) at 14, 38, and 98 ppm, and moniliformin at 66, 193, and 367 ppm in 3 separate feeding trials (amounts of toxin in each trial, respectively). Birds were started on amended rations at days 1, 7, and 21 and continued on their respective ration until they were 28 days old. Purified FB1 (125, 274 ppm) and moniliformin (27, 154 ppm) were given separately and in combination (137 and 77 ppm, respectively), starting on day 1 and continuing for 14 days. Of serum chemistry parameters, only glucose was significantly decreased. Significant increases were noted in serum cholesterol, sodium, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and γ-glutamyl transferase. Of the hematologic parameters, significant decreases were noted in red blood cell counts, hemoglobin, packed cell volume, and white blood cell counts. Immunologic changes included impaired anti-Newcastle disease antibody hemagglutination inhibition titers associated with relative decreases in total serum globulins and increases in albumin/globulin ratios. The changes were noted in all treatment groups when compared to controls.

Activation of rat splenic macrophage and lymphocyte functions by fumonisin B1

Fumonisins represent a family of toxic, structurally related metabolites produced by fungi that are found in corn worldwide. We investigated the effects of the mycotoxin, fumonisin B(1), on rat splenic macrophage and lymphocyte functions. Pretreatment (24 h) of resident macrophages with fumonisin B(1) (1, 10, and 100 microg/ml) significantly (p<0.01) stimulated nitric oxide production (0.48, 2. 60, and 4.40 nmol nitrite/well, respectively), compared with the response of untreated macrophages (no nitrite detected), after 72 h of culture. Fumonisin B(1) (1 and 10 microg/ml) and IFN-gamma acted in an additive manner to activate nitric oxide production. The response of IFN-gamma (50 U/ml)-activated macrophages (1.68 nmol nitrite/well) was potentiated (3.52, 4.96, and 4.44 nmol nitrite/well) by fumonisin B(1) (1, 10, and 100 microg/ml, respectively). In addition, fumonisin B(1) significantly (p<0.05) potentiated Con A (1.25 to 5 microg/ml) (1.46- to 2.62-fold increases)- and antiTCR, IL-2 or antiTCR+IL-2 (1.72- to 2.60-fold increases)-induced proliferation of splenic cells in the presence of the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (NMA). These results show two distinct and separate effects of fumonisin B(1): it induces nitric oxide production by macrophages and it stimulates T cell proliferation.

The sequence of the isoepoxydon dehydrogenase gene of the patulin biosynthetic pathway in Penicillium species.

Interest in species of the genus Penicillium is related to their ability to produce the mycotoxin patulin and to cause spoilage of fruit products worldwide. The sequence of the isoepoxydon dehydrogenase (idh) gene, a gene in the patulin biosynthetic pathway, was determined for 28 strains representing 12 different Penicillium species known to produce the mycotoxin patulin. Isolates of Penicillium carneum, Penicillium clavigerum, Penicillium concentricum, Penicillium coprobium, Penicillium dipodomyicola, Penicillium expansum, Penicillium gladioli, Penicillium glandicola, Penicillium griseofulvum, Penicillium paneum, Penicillium sclerotigenum and Penicillium vulpinum were compared. Primer pairs for DNA amplification and sequencing were designed from the P. griseofulvum idh gene (GenBank AF006680). The two introns present were removed from the nucleotide sequences, which were translated to produce the IDH sequences of the 12 species for comparison. Phylogenetic relationships among the species were determined from rDNA (ITS1, 5.8 S, ITS2 and partial sequence of 28S rDNA) and from the idh nucleotide sequences minus the two introns. Maximum parsimony analysis showed trees based on rDNA and idh sequences to be congruent. It is anticipated that the genetic information obtained in the present study will aid in the design of probes, specific for patulin biosynthetic pathway genes, to identify the presence of these mycotoxigenic fungi.

The isoepoxydon dehydrogenase gene of the patulin metabolic pathway differs for Penicillium griseofulvum and Penicillium expansum

Purified DNA from isolates of Penicillium griseofulvum and P. expansum was used as a template to amplify a 600-bp fragment of the isoepoxydon dehydrogenase (idh) gene of the patulin biosynthetic pathway. Primer pairs designed from the P. griseofulvum gene (GenBank accession AF006680) to amplify specific regions of the idh gene yielded similar-sized bands for all strains. Asymmetrical amplification produced DNA products for sequencing and DNA sequences were translated to produce the corresponding amino acid sequences. After removal of two introns present in the region sequenced, amino acid sequences were compared. There were 12 amino acid differences between P. expansum and P. griseofulvum in the coding region. The differences correlated with the amount of patulin previously produced in culture, with strains of P. griseofulvum producing the greatest amounts of patulin.

Removal of patulin from aqueous solutions by propylthiol functionalized SBA-15

Propylthiol functionalized SBA-15 silica was investigated to detoxify aqueous solutions contaminated with the regulated mycotoxin patulin. Micelle templated silicas with a specific pore size were synthetically modified to possess propylthiol groups, a functional group known to form Michael reaction products with the conjugated double bond system of patulin. BET surface area analysis indicated the propylthiol functionalized SBA-15 possesses channels with the pore size of 5.4 nm and a surface area of 345 m(2)g(-1). Elemental analysis indicates the silicon/sulfur ratio to be 10:1, inferring one propylthiol substituent for every ten silica residues. The propylthiol modified SBA-15 was effective at significantly reducing high levels of patulin from aqueous solutions (pH 7.0) in batch sorption assays at room temperature. The material was less effective at lower pH; however heating low pH solutions and apple juice to 60 °C in the presence of propylthiol functionalized SBA-15 significantly reduced the levels of patulin in contaminated samples. Composite molecular models developed by semi-empirical PM3 and empirical force field methods support patulin permeation through the mesoporous channels of propylthiol functionalized SBA-15. Density functional study at the B3LYP/6-31G(d,p) level predicts the proposed patulin adducts formed by reaction with the thiol residues exhibit less electrophilic properties than patulin. It is demonstrated the use of propylthiol functionalized SBA-15 is a viable approach to reduce patulin levels in aqueous solutions, including contaminated apple juice.

Comparative pathologic changes in broiler chicks on feed amendedwith Fusarium proliferatum culture material or purified fumonisinB1 and moniliformin

Feed amended with autoclaved culture material (CM) of Fusarium proliferatum containing fumonisin B1 (FB1) (61–546 ppm), fumonisin B2 (FB2) (14–98 ppm) and moniliformin (66–367 ppm) was given to 228 male chicks in three separate feeding trials. In a fourth feeding trial, purified FB1 (125 and 274 ppm) and moniliformin (27 and 154 ppm) were given separately and in combination (137 and 77 ppm, respectively). Chicks that died during the trial periods, survivors and controls were subjected to postmortem examination. Specimens (liver, kidney, pancreas, lung, brain, intestine, testis, bursa of Fabricius, heart and skeletal muscle) were examined grossly and preserved for subsequent histopathologic and ultrastructural examination. Prominent gross lesions in affected birds fed diets amended with CM or purified FB1 and moniliformin included ascites, hydropericardium, hepatopathy, nephropathy, cardiomyopathy, pneumonitis, gizzard ulceration, and enlarged bursa of Fabricius filled with caseous material. The various concentrations of FB1 and moniliformin in the amended rations produced well-defined dose–response lesions in all groups in all four trials. Histopathologic changes included hemorrhage, leucocytic infiltration, fatty change or infiltration, individual cell necrosis and fibrosis in liver, kidneys, lungs, heart, intestines, gizzard, bursa of Fabricius and pancreas. Edema and hemorrhage were prominent in brains of treated birds. Ultrastructural changes included cytoplasmic and nuclear enlargement of cells in affected liver, lungs, kidneys, heart and pancreas. There were thickened membranes of the smooth endoplasmic reticulum, dilation of the rough endoplasmic reticulum with loss of ribosomes and vacuolated or deformed mitochondria.

Determination of Deoxynivalenol in Infant Cereal by Immunoaffinity Column Cleanup and High-Pressure Liquid Chromatography–UV Detection†

The presence of deoxynivalenol (DON) in cereal-based baby food, a primary source of the first solid food for infants, was studied in order to develop a method to detect its presence at low concentrations. DON, produced primarily by Fusarium graminearum, is commonly isolated from grains and feed around the world and affects both animal and human health, producing diarrhea, vomiting, gastrointestinal inflammation, and immunomodulation. An aqueous extract of infant cereal was cleaned by means of an immunoaffinity chromatography column. After the eluate was evaporated and redissolved, DON was determined by high-pressure liquid chromatography-UV. The level of quantification for DON was 10 ppb for three types of infant cereal (mixed, barley, and oatmeal); the level of detection was 5 ppb. The protocol we have developed can measure DON between 10 to 500 ppb. An advisory level of 1 ppm for wheat products has been established by the U.S. Food and Drug Administration; however, the European Communities (EC) regulations have been set at 200 ppb for cereal-based foods for infants. Only 1 of 52 samples of barley-, mixed-, or oat-based infant cereal purchased in 2008 and 2009 in the United States exceeded the European standard.