Glenn A. Bennett

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.

Distribution of Fumonisins in Food and Feed Products Prepared from Contaminated Corn

The fate and distribution of the fumonisins B1 (FB1) and B2 (FB2) were determined in products obtained from naturally contaminated corn used for ethanol fermentation and wet milling operations. Fumonisins are stable to the conditions used in ethanol fermentations and tend to concentrate in the distillers dried grain, a fraction generally used for animal feed. No toxin was found in the ethanol. Starch from wet milling of corn, naturally contaminated at 13.9 micrograms fumonisin B1/g, was free of detectable toxin. The other fractions contained fumonisins at the following levels: gluten (5.1-5.8 micrograms FB1/g, 4.7-4.9 micrograms FB2/g); fiber (2.7-5.7 micrograms FB1/g, 2.1-3.1 micrograms FB2/g); and germ (1.3-3.1 micrograms FB1/g, 0.7-1.6 micrograms FB2/g). The steep water and process water contained 22% of the recoverable fumonisins. A combination of analytical methodologies was required to determine fumonisins in the different products from the wet milling process.

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.

Fertility ofFusarium moniliforme from maize and sorghum related to fumonisin production in Italy

Forty-three strains ofFusarium moniliforme isolated from infected maize and sorghum plants in Italy were assayed for their ability to produce fertile crosses with “A” and “F” mating population tester strains, in relation to their ability to produce fumonisins on maize substrate. Most of the strains isolated from maize (ear and stalk rot and maize-based feed), producing fumonisin B1 (FB1) and B2 (FB2) (up to 4,100 and 855 mg/kg, respectively), belonged to the “A” mating population. All of the strains isolated from sorghum belonged to the “F” mating population and produced little or no FB1 and FB2. This is the first report of the occurrence of mating population “F” in Europe. Our data on strains from Italy are consistent with previous studies from the United States that found significant differences in sexual fertility and fumonisin production between strains from maize and sorghum.

Analysis of fumonisin B1 in corn by capillary electrophoresis.

Intact fumonisins contain two tricarballylic acid groups and can therefore acquire a net negative charge. The anionic nature of the fumonisins is the basis behind the widely used method for cleanup of corn with strong anion exchange (SAX) columns. This property also enables the fumonisins to be separated by electrophoretic techniques which, until now, have not been applied to the analysis of fumonisins in corn. Fumonisin B1, extracted from corn with 80/20 (v/v) methanol/water and isolated with a commercially available affinity column, was derivatized with fluorescein isothiocyanate for analysis by capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). Recoveries from corn fortified with 0.25 to 5.0 ppm FB1 averaged 89% (range 71 to 102%). As little as 0.05 ppm FB1 could be detected in corn. For corn naturally contaminated with FB1, the CZE-LIF method compared favorably to established SAX/HPLC and C18/HPLC methods. Capillary electrophoresis can be used for quantitation of FB1 in corn, with minimal use of organic solvents and provides an additional tool for confirming fumonisin contamination.

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.

Affinity column clean-up for the analysis of fumonisins and their hydrolysis products in corn.

Fumonisins, toxic metabolites of certain Fusarium molds, can be found in corn and corn‐based foods at levels sufficient to cause disease in livestock. Widely used methods of analysis involve organic extraction followed by isolation with either C18 or strong anion exchange columns. An affinity column procedure that simultaneously isolated both intact and hydrolyzed fumonisins from corn is reported. The columns were prepared using two monoclonal antibodies with differing specificities, the first (P2A5–3‐F3) bound intact fumonisins, while the second (P2F11–3‐H7) bound their hydrolysis products. Corn samples were extracted with methanol/phosphate buffer (80:20, v:v) and the extract was diluted and applied to the affinity column. The recoveries of FB1 from corn spiked with 0.5–8.0 ppm averaged 82%. The recoveries of HFB1 over the range 0.25–1.25 ppm averaged 102%. Affinity columns capable of binding both intact and hydrolyzed fumonisins allow for the analysis of both types of toxins simultaneously from a singl...