Mortality in broiler chicks on feed amended withFusarium proliferatum culture material or with purified fumonisin B1 and moniliformin

Two hundred twenty-eight male chicks (Columbia × New Hampshire) were given feed amended with autoclaved culture material (CM) ofFusarium proliferatum Containing fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and moniliformin in 3 separate feeding trials. Purified FB₁ and moniliformin were given separately and in combination in a fourth feeding trial. Birds were given amended rations at day 1 (Trial 1 and 4), day 7 (Trial 2), and day 21 (Trial 3) and their respective ration was given for 28 days (Trial 1), 21 days (Trial 2), 7 days (Trial 3), and 14 days (Trial 4). FB₁ concentrations were 546, 193, and 61 ppm; FB₂ were 98, 38 and 14 ppm; and moniliformin were 367, 193, and 66 ppm in the first 3 feeding trial regimens. Chicks in Trial 4 were given dietary concentrations of purified FB₁ at 274 and 125 ppm, and moniliformin at 154 and 27 ppm. FB₁ and moniliformin, both alone and in combination, produced dose-responsive clinical signs, reduced weight gains and mortality in chicks. Age of birds given amended feeds had little difference in the clinical response; however, those given the rations from days 7 or 21 were slightly less susceptible than those given rations beginning at 1 day of age. Additive effects were noted when the toxins were given in combination. When toxins were given separately, adverse effects took longer to occur. A system to monitor pattern and rate of defecation (RD) was developed for assessing the chicks' approach to feed, water and heat source as illness progressed. Our results indicate that chicks fed corn heavily infected withF. proliferatum under field conditions could suffer acute death similar to that described for spiking mortality syndrome during the first 3 weeks of age.     

Embryopathic and embryocidal effects of purified fumonisin B1 orFusarium 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.     

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. Disclaimer: The mention of firm names or trade products in this article does not imply that they are endorsed or recommended by the United States Department of Agriculture over other firms or similar products not mentioned.     

Fumonisins production by Fusarium proliferatum strains isolated from asparagus crown

The present work deals with the capability for producing fumonisin by Fusarium proliferatum strains isolated from asparagus in China. Fifty of F. proliferatum strains were randomly selected and incubated on cultures of maize grain and asparagus spear, respectively. Fumonisin levels (FB₁ and FB₂) were determined by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that all 50 strains produced fumonisins in maize culture within a wide range of concentrations, 10–11,499 μg/g and 2–6,598 μg/g for FB₁ and FB₂, respectively. On culture of asparagus spear,48 strains (96%) produced fumonisins in the range 0.2–781.6 μg/g and no detected to 40.3 μg/g for FB₁ and FB₂, respectively. All of F. proliferatum strains produced much higher levels of FB₁, FB₂ and total fumonisins (FB₁ + FB₂) in maize grain culture than in asparagus spear culture. Meanwhile, fumonisin B₃ (FB₃) was identified in all maize culture extracts and most of asparagus spear culture extracts. This is the first study carried out the fumonisin-producing ability of F. proliferatum strains isolated from asparagus in China. The information obtained is useful for assessing the risk of fumonisins contamination in asparagus spear. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Occurrence of fumonisin B1 and B2 in corn-based foodstuffs in Taiwan market

Corn-based human foodstuffs purchased in Taiwan were analyzed for fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) using high-performance liquid chromatography. Fifty-two (33.9%) and 32 (20.9%) of 153 samples were found to contain FB₁ (73–2395 ng/g) and FB2 (120–715 ng/g), respectively. The highest frequency of detection and also the highest FB1 concentrations were found in sweetcorn (50%, 1089 ng/g) and cornflour (50%, 608 ng/g), followed by corn snacks (33.3%, 2395 ng/g), miscellaneous corn products (33.3%, 73 ng/g), popcorn (31.8%, 1003 ng/g) and cornflakes (23.5%, 1281 ng/g). 16 corn snacks (= approximately 20.5% of the samples) had an average FB₁ and FB₂ content of 456 and 145 ng/g, respectively, while six sweetcorn (= 25%) samples were contaminated with an average of 400 ng/g of FB₁ and 65 ng/g of FB₂. Of the 22 pop-corn samples examined, 7 had an average of 347 ng/g and 116 ng/g of FB₁ and FB₂, respectively. During an analysis of the distribution pattern for the combined fumonisin levels of FB₁ and FB₂, it became apparent that more than 69% of tested samples had fumonisin concentrations below 100 ng/g, while 11.1% (or 17 samples) contained in excess of 600 ng toxins per g. These results clearly illustrated that commercially available corn-based foodstuffs for human consumption in Taiwan are frequently contaminated with FB₁ and FB₂.This revised version was published online in October 2005 with corrections to the Cover Date.     

Toxic interaction of fumonisin B1 and fusaric acid measured by injection into fertile chicken egg

Toxic interactions of fusaric acid and fumonisin B₁, two mycotoxins produced byFusarium moniliforme, were studied in the chicken embryo. The yolk sacs of fertile White Leghorn eggs were injected before incubation with separate and combined solutions of either fusaric acid and or fumonisin B₁. The toxins were administered in either a sterile 10 mM buffered phosphate solution, pH 6.90, which produced a final pH of 6.6 ± 0.2, or sterile distilled water. Toxicity was based on absence of egg pip at the end of the 21-day incubation period. Toxins administered in the phosphate buffer solution were more toxic than those administered in distilled water. When both toxins were combined in equal concentrations and injected into eggs, increased toxicity resulted. Fusaric acid was shown to be a mild toxin to the eggs and when a relatively nontoxic concentration of it was combined with graded doses of fumonisin B₁, a synergistic toxic response was obtained. Fusaric acid is only moderately toxic to the chicken egg, however its co-occurrence with other fusaria toxins found on corn and other cereals might present possible antagonisms or synergisms. The results of this egg model suggest that fusaric acid might play a role in enhanced and unpredicted toxicity in mammalian systems if it is consumed with other mycotoxins.     

Natural occurrence of fumonisins and their correlation to Fusarium contamination in commercial corn hybrids growth in Argentina

Fifty commercial corn hybrids with different endosperm characteristics, vegetative cycle length and cross class grown in the same geographical area (Cordoba Province, Argentina) were analysed for fumonisin accumulation. All hybrids analysed showed fumonisin B₁ and B₂ contamination ranging from 185 to 27,050 ng/g for FB₁ and from 40 to 9950 ng/g for FB₂. Although most of the hybrids analysed had flint-type endosperm, two hybrids with dent-type endosperm (e.g. Prozea 10 and AX 746) showed the highest level of fumonisin (37,000 ng/g) and more FB₂ than FB₁ (FB₂/FB₁ ratio 2.42), respectively. There was no correlation between fumonisin concentration and length of the vegetative cycle. Among 18 hybrids examined for Fusarium species contamination there was also no correlation between fumonisin contamination and the level of infection with Fusarium species (Section Liseola). Eighteen hybrids showed fumonisin levels lower than 1000 ng/g. This result suggests that there is some possibility of selecting hybrids resistant or less susceptible to fumonisin and Fusarium contamination.     

A review and update of animal toxicoses associated with fumonisin-contaminated feeds and production of fumonisins by Fusarium isolates

During the 1989 corn harvest season, numerous reports of equine leukoencephalomalacia (ELEM) outbreaks and a pulmonary edema (PPE) syndrome in swine from several regions of the United States were received by the National Veterinary Services Laboratories (NVSL), Ames, Iowa. Previous and concurrent research linked Fusarium moniliforme and fumonisin-contaminated feeds to both diseases. Chemical and mycological investigations revealed fumonisin B₁ (FB₁) concentrations of 20 to 360 ppm in suspect swine feeds and 8 to 117 ppm in suspect equine feeds. Nonproblem feeds contained concentrations below 8 ppm. Fusarium moniliforme and Fusarium proliferatum were isolated from both problem and nonproblem equine and swine feeds. When cultured on autoclaved corn, the F. moniliforme and F. proliferatum isolates produced respective FB₁ and fumonisin B₂ (FB₂) that range from less than 5 to more than 2450 ppm and less than 5 to more than 1000 ppm, respectively. Isolates from both problem and nonproblem feeds produced high levels (greater than 500 ppm) in culture. Reported here is a review of chemical and mycological data resulting from the study of several cases of PPE and ELEM.     

Biosynthesis of labeled fumonisins in liquid cultures of Fusarium moniliforme

Fumonisins were readily produced in cultures of Fusarium moniliforme using a defined liquid medium. Addition of 200 mg of d₃-methyl L-methionine to 100-ml cultures of F. moniliforme gave increased overall yields and high levels of deuterium (²H) incorporation into fumonisin B₁. Approximately 90% of the resulting fumonisin B₁ contained 6 deuterium atoms, while 9% of the product contained 3 deuterium atoms. Deuterium was shown to be incorporated exclusively in the methyl groups of the fumonisin backbone. The addition of as little as 5 mg of labeled methionine stimulated fumonisin production, but only about 5% of the fumonisin produced contained 3 deuterium atoms.Abbreviations ELEM equine leukoencephalomalacia Mention of companies or products by name does not imply their endorsement by the US Department of Agriculture over others not cited.     

Toxicity of the mycotoxins fumonisins B1 and B2 and Alternaria alternata f. sp. lycopersici toxin (AAL) in cultured mammalian cells

Fumonisins B₁ and B₂ and AAL toxin are a series of structurally related mycotoxins. Fumonisins B₁ and B₂, produced by Fusarium moniliforme Sheldon induce toxic hepatitis and hepatomas in rats and leukoencephalomalacia in horses. The cancer-promotion assay which has been used to guide their purification is slow and consumes large amounts of sample. We have examined a series of cultured mammalian cell lines in order to develop a more rapid and sensitive bioassay system, which may be useful for examining structure-activity relationships and the mechanism(s) of action of these toxins. Of 9 rat hepatoma cell lines tested, all except the two most de-differentiated lines were sensitive to the three toxins, with a toxic response visible by 48 h. Approximate IC₅₀ values for the most sensitive hepatoma line, H4TG, were 4, 2 and 10 g/ml for fumonisins B₁, B₂ and AAL toxin, respectively in 100 l cultures. Among 15 cell lines from other sources, only MDCK dog kidney epithelial cells were sensitive (IC₅₀ = 2.5, 2 and 5 g/ml, respectively). Studies in co-cultures of sensitive and insensitive cell lines and in cultures of a sensitive cell line over a range of cell densities indicated that cytotoxicity of fumonisins B₁ and B₂ does not involve metabolite activation to a derivative stable enough to diffuse to adjacent cells.Abbreviations AAL toxin Alternaria alternata f. sp. lycopersici toxin - IC₅₀ concentration giving 50% inhibition of cell proliferation     

Developmental effects of fumonisin B1-containingFusarium moniliforme culture extract in CD1 mice

Pregnant Charles River CD1 mice were treated with a semipurified extract ofFusarium moniliforme culture containing 0, 12.5, 25, 50 or 100 mg FB₁/kg each day orally (diluted in distilled water) between gestational days (GD) 7 and 15 to evaluate the developmental toxicity of FB₁. Following sacrifice of dams on GD 18, litters were examined for gross abnormalities and divided equally for skeletal or visceral examination by routine techniques. Significant maternal mortality was observed at doses of 50 and 100 mg FB₁/kg. Dose-dependant decreases in maternal body weight gains, number of live offsprings per litter, and mean body weight of the offspring were produced at FB₁ doses of 25 mg/kg or higher. The percentage of implants resorbed increased at all doses in a dose-dependant manner. A dose-dependant increase, except at the lowest dose tested, in the incidence of ossification deficits involving digits and sternum, short and wavy ribs, and hydrocephalus of lateral and third ventricles was also evident. Cleft palate was seen only at the highest FB₁ dose. Maternal intoxication manifested as a dose-dependant increase in the severity of ascites associated mainly with increased histopathologic scores reflecting hepatocellular damage at day 18. Concommittant increases in serum alanine amino transferase (ALT) on GD 12, reflecting parenchymal liver cell damage, was also observed at all doses above 12.5 mg of FB₁/kg. These results suggest that FB₁-containingF. moniliforme culture extract is developmentally toxic in mice, and that this toxicity may be mediated by maternal hepatotoxicity.     

Occurrence and toxicity ofFusarium subglutinans from Peruvian maize

Twenty-five samples of maize kernels collected at harvest time from geographically different corn fields in Peru, were examined for the occurrence of toxigenicFusarium species. The most frequently recovered species wereF. subglutinans (48%),F. moniliforme (46%), andF. equiseti (5%). OtherFusarium species isolated (up to 1%) includedF. graminearum, F. acuminatum, F. solani, F. oxysporum, andF. culmorum. Assays ofFusarium culture extracts usingArtemia salina larvae, showedF. subglutinans as one of the most toxigenic species, and its toxicity was mostly correlated to the capability to produce beauvericin (BEA). All eight tested isolates ofF. subglutinans grown on autoclaved corn kernels produced BEA (from 50 to 250 mg/Kg) as well as moniliformin (M) (from 70 to 270 mg/Kg). This is the first report on BEA and M production by maize isolates ofF. subglutinans from South America.     

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C₁₈ chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.     

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (11) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/ methanol extraction. Fumonisins B₁ and B₂ were extracted from the CM by water, but not chloroform/ methanol, and were present in the toxic diets at concentrations of 93–139 and 82–147 ppm, respectively. Nontoxic diets contained 22 ppm fumonisin B₁ and 65 ppm fumonisin B₂.Abbreviations CM culture material - ELEM equine leukoencephalomalacia Mention of a trademark, proprietory name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

Spargelstangenuntersuchungen zur Haupterntezeit auf Infektionen mitFusarium spp. und Kontaminationen mit Fumonisin B1

Asparagus spears collected from a total of six commercial plantings in Austria during the main harvest periods in May and June of 2003 and 2004 were examined for endophytic colonization byFusarium spp., particularlyF. proliferatum. Potentially toxigenic fungi such asF. proliferatum were isolated and identified by morphological characteristics using light microscopy. Fumonisin B₁ inF. proliferatum-infected asparagus spears was detected with IAS-HPLC-FLD or HPLC-MS/MS. The identity of endophytic fungi colonizing of a total of 816 individual spears was determined. The incidence of infection byF. proliferatum and otherFusarium spp. was highly dependent on location and sampling date. The dominantFusarium species among the endophytic microflora wasF. oxysporum. Other frequently isolated species includedF. proliferatum, F. sambucinum, F. culmorum, F. avenaceum andF. equiseti. The incidence ofF. proliferatum-infected asparagus spears was less than 10% at four of the six sampling locations. At the two remaining locations, 20–47% of the spears examined were infected withF. proliferatum. Further exploration of FB₁ generation in asparagus is required because the low levels of FB₁ (10–50 (μg/kg) detected in harvested spears in 2003 and 2004 cannot be explained by the results of this study.

     

Fumonisin B1 alters sphingolipid metabolism and immune function in BALB/c mice: Immunological responses to fumonisin B1

Fumonisins have been reported to have diverse effects on animals, including immunosuppression in chickens and feeder calves; therefore, the effects of fumonisin B₁ (FB₁) on immune function in BALB/c mice was investigated. When administered i.p. with sheep red blood cells (SRBC), 5 to 100 µg of FB₁ reduced the number of plaque-forming cells (PFC) produced against SRBC; however, when administered daily, 1 to 50 µg of FB₁ caused a 4 to 12-fold increase in the number of PFC after SRBC injection. Therefore, FB₁ is not only immunosuppressive; but also, immunostimulatory. To test the possibility that there may have been an immune response to FB₁ as an antigen, FB₁ was injected into mice and the number of splenic cells forming rosettes on FB₁-treated SRBC was determined. There were dose-dependent increases in the antigen-binding cells, with up to 4.9- and 4.6-fold increases, respectively, upon primary and secondary immunization. FB₁-binding immunoglobulins could be detected in sera from some treated mice, but this response was not obtained in every experiment. In summary, these results show that FB₁ has diverse effects on the immune system, causing both stimulation and suppression of the response to foreign antigens, and apparently inducing an antigenic response to FB₁.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - FB₁ fumonisin B₁ - FCS fetal calf serum - PBS phosphate buffered saline - PFC plaque forming cells - RFC rosette forming cells - SRBC sheep red blood cells     

Toxicity and moniliformin production by four recently described species of Fusarium and two uncertain taxa

Four recently described species, Fusarium nygamai, F. dlamini, F. beomiforme and F. napiforme and two uncertain taxa, F. nygamai from millet in Africa and Fusarium species from rice with Bakanae disease, were tested for toxicity and moniliformin production. Cultures grown on autoclaved corn were fed to groups of four one-day-old ducklings for 14 days. Isolates that caused the death of 3 or 4 out of 4 ducklings were considered to be toxic and analyzed for moniliformin. All 15 isolates of F. dlamini tested were nontoxic. The other taxa contained some isolates that were toxic to ducklings and produced moniliformin in corn cultures. This is the first report of moniliformin production by F. beomiforme (200–890 g/g), and F. napiforme (16–388 g/g), and by F. nygamai not obtained from millet in Africa (15–874 g/g). The highest production of moniliformin was obtained from the 19 isolates of F. nygamai from millet in Africa (4300–18200g/g) and the 15 isolates from rice with Bakanae disease (2300–19300 g/g). The taxonomic position of these two uncertain taxa should be re-evaluated.     

The implications of naturally occurring levels of fumonisins in corn for human and animal health

Contamination of corn with the fungus Fusarium moniliforme and its secondary metabolites, the fumonisins, has been associated with several human and animal diseases. This paper summarizes present knowledge and presents new data on the levels of fumonisins present in foods and feeds associated with these diseases as well as in commercial corn and corn-based products. The doses of fumonisins to which humans and animals consuming these products would be exposed are compared with those doses known to produce LEM in horses and hepatocarcinogenesis in rats. It is concluded that the known naturally occurring levels of fumonisins present a potential threat to human and animal health and realistic tolerance levels need to be set.     

Developmental toxicity of fumonisin in Syrian hamsters

The effects of fumonisin on development of Syrian hamster fetuses were studied using fumonisin B₁ and B₂ extracted fromFusarium moniliforme corn-culture and purified fumonisin B₁. A significant increase in litters with fetal deaths occurred with the high doses of purified (18 mg FB₁/kg) and culture-extracted (18 mg FB₁ plus 4.5 mg FB₂) fumonisin. It is concluded that prenatal exposure to fumonisin on days 8 and 9 of gestation is detrimental to fetal hamster survivability but does not induce clinical maternal intoxication at these doses. Equivalent doses of fumonisin B₁, whether from culture-extract or pure solution produced similar results.