Natural Occurrence of Fumonisins in Rice with Fusarium Sheath Rot Disease.

Twenty samples of rough rice (Oryza sativa) (unpolished kernels) collected during the 1995 harvest season from Arkansas (seven samples) and Texas (13 samples) were obtained from rice fields known to include plants with symptoms of Fusarium sheath rot putatively caused by Fusarium proliferatum. Samples were analyzed for fumonisin B1 (FB1) at three laboratories using three different extracting solvents by high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA) methods. Forty percent of the samples were positive for FB1 at levels ≤4.3 µg/g by HPLC. The same samples contained FB1 at ≤3.6 µg/g when measured by an ELISA method. Most samples that were positive for FB1 were positive for fumonisin B2 (FB2) and fumonisin B3 (FB3) by HPLC at levels ≤1.2 µg/g. Very good agreement was obtained among the two laboratories using HPLC methods and the third using ELISA. Shelling of the unpolished rice results in hull and brown rice fractions. In a sample that contained 4.3 µg/g in whole kernels, the fumonisin level was very high in hulls (≤16.8 µg/g) and low in brown rice (≤0.9 µg/g). Milling of brown rice results in bran and white rice fractions. Fumonisins were found in bran at a level of ≤3.7 µg/g but were below the level of detection by HPLC in white rice. The presence of fumonisins (FB1, FB2, and FB3) was confirmed by fast atom bombardment/mass spectrometry. This is the first report of fumonisins in naturally contaminated rice in the United States.

Importance of sphingolipids and inhibitors of sphingolipid metabolism as components of animal diets.

Sphingolipids are highly bioactive compounds that participate in the regulation of cell growth, differentiation, diverse cell functions, and apoptosis. They are present in both plant and animal foods in appreciable amounts, but little is known about their nutritional significance. Recent studies have shown that feeding sphingomyelin to female CF1 mice treated with a colon carcinogen (1,2-dimethylhydrazine) reduced the number of aberrant colonic crypt foci; longer-term feeding also affected the appearance of colonic adenocarcinomas. Therefore, dietary sphingolipids should be considered in studies of the relationships between diet and cancer. Sphingolipids have also surfaced as important factors in understanding the mechanism of action of a recently discovered family of mycotoxins, termed fumonisins. Fumonisins are produced by fungi commonly found on maize and a few related foods, and their consumption can result in equine leukoencephalomalacia, porcine pulmonary edema and a number of other diseases of veterinary animals and, perhaps, humans. A cellular target of fumonisins is the enzyme ceramide synthase, and disruption of sphingolipid metabolism by fumonisins has been established by studies with both cells in culture and animals that have consumed these toxic mycotoxins. These findings underscore the ways in which sphingolipids and agents that affect sphingolipid utilization should be given consideration in selecting animal diets for nutritional and toxicological studies.

Absence of detectable fumonisins in the milk of cows fed Fusarium proliferatum (Matsushima) Nirenberg culture material.

Fumonisins, a group of mycotoxins produced by the ubiquitous fungi Fusarium moniliforme and F. proliferatum, were first identified about eight years ago. They have been shown to cause a variety of health effects in animals, including epidemiological evidence of esophageal cancer in humans. Cattle are less sensitive to ill effects than horses and swine. Fumonisins are common contaminants of low quality grain fed to cattle. Culture material containing fumonisins (FB1, FB2, and FB3) was mixed into the total diet and fed for 14 days to two midlactation Jersey cows to determine if fumonisins are excreted in milk. The dietary equivalent of fumonisin was approximately 75 ppm and the two cows consumed an average of 3 mg fumonisin B1/kg body weight (bwt)/day. Fumonisins were not detected in any of the milk samples by two analytical laboratories using methods with a sensitivity of 5 ng/ml. Except for transient diarrhea at the beginning of the contaminant feeding period and an increase in serum cholesterol, clinical and hematologic changes were not observed in the animals. The appearance or carry over of fumonisins from feed to milk in dairy cows does not appear to be significant and likely not a hazard or food safety concern for humans.

Evaluation of a liquid chromatographic method for the determination of fumonisins in corn, poultry feed, and Fusarium culture material.

The performance of a liquid chromatographic method for determining fumonisins in corn, animal feeds, and culture material was evaluated. Efficiencies of extractions with the following solvent systems were determined: acetonitrile-water (50 + 50, v/v), methanol-water (75 + 25, v/v), and 100% water. The acetonitrile solvent gave both higher extraction efficiencies and faster extraction times than the other 2 solvents. Extraction was followed by C18 solid-phase extraction column cleanup. Fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) were measured by precolumn derivatization with o-phthalaldehyde followed by isocratic separation on a C18 reversed-phase column with a mobile phase of 50 mM potassium dihydrogen phosphate (pH 3.3)-acetonitrile (60 + 40). Commercially prepared poultry feed, corn, and Fusarium spp. corn cultures were analyzed at the following levels: FB1, 1.5 to 15,000 micrograms/g; FB2, 0.5 to 4000 micrograms/g; FB3, and 0.17 to 1,500 micrograms/g. Recoveries were 91-94%, 90-100%, and 81-93% for FB1, FB2, and FB3, respectively. Precision (coefficient of variation) was determined with pooled field samples and ranged from 2% at 19 micrograms/g for FB1 to 9% at 0.17 microgram/g for FB3. Time and pH studies of the formation of the fluorescent derivative and its stability were conducted. Complete reaction occurred at pHs above 7.9, with optimal pH for chromatography between 8.0 and 8.5. No statistically significant response differences were detected for reaction times ranging from 4 to 40 min; however, the detector signal was significantly reduced when reaction times were shorter than 4 min. Chromatograms of samples were free of interferences for all feeds, corn, and culture material tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental equine leukoencephalomalacia, toxic hepatosis, and encephalopathy caused by corn naturally contaminated with fumonisins.

A study to evaluate the effects of dietary fumonisin B1 was conducted using 6 ponies (4 test and 2 control). A ration naturally contaminated with fumonisin B1 was fed in 3 phases: 1) 44 ppm fumonisin B1, 2) less than 1 ppm fumonisin B1, and 3) 88 ppm fumonisin B1. All ponies were monitored daily, weighed weekly, and limit fed at a rate of 0.8% body weight plus hay. Feed intake was measured daily, and a serum chemistry panel was completed once or twice weekly. Four to 7 days after initiation of the trial (Phase 1), all 4 test ponies had decreased feed consumption, and selected serum chemistry parameters were markedly elevated. On day 9, 1 pony died acutely with mild encephalopathy and hepatic necrosis. Another pony, euthanized on day 45, also had mild encephalopathy and hepatic necrosis. The remaining 2 test ponies continued the 44 ppm fumonisin B1 diet for 98 days. Phase 2 consisted of a diet with < 1 ppm fumonisin B1 for 120 days. During this phase, the serum chemistry values of the 2 ponies returned to normal. Following Phase 2, the 2 ponies were fed a diet containing 88 ppm fumonisin B1. After 75 days, 1 animal died of equine leukoencephalomalacia with mild hepatic necrosis. On day 78, the remaining pony was euthanized after showing distress; it also had leukoencephalomalacia and hepatic lesions.

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 isolated 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 produces 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.

Experimental reproduction of ELEM. A study to determine the minimum toxic dose in ponies.

An experiment to gain insight into the minimum toxic dose of fumonisins was conducted by feeding ponies rations with known fumonisin concentrations. Naturally contaminated corn screenings (CS) were blended with pellets, corn, and molasses to formulate individual daily diets. One group of 4 ponies was fed a ration with fumonisin B1 (FB1) varying from less than 1 ppm to 22 ppm. A second group of 5 ponies was fed a ration at varying rates containing 8 ppm FB1 for 180 days. A panel of clinical chemistry parameters was evaluated twice weekly for both groups. One pony in the first group died of equine leukoencephalomalacia (ELEM) after 225 days of which the final 55 days' diet contained 22 ppm FB1. Approximately 9 days prior to death, this animal experienced elevated liver chemistry values. All 5 ponies in the second group experienced mild, transient, clinical signs; were euthanized at 180 days; and had mild, histopathological brain lesions.

Characterization of an epizootic of pulmonary edema in swine associated with fumonisin in corn screenings.

In 1989, corn screenings were associated with acute interstitial pulmonary edema, hydrothorax, and death in swine. Attack rate was 5-50%, case fatality rate was 50-90%, and clinical course was 1-2 days. Screenings from farms with pigs affected with pulmonary edema contained 20-330 micrograms fumonisin B1 per gram. Screenings containing 92 micrograms fumonisin B1 per gram fed to weanling pigs caused pulmonary edema and death. Sterilized corn inoculated with Fusarium moniliforme and diluted 1:1 with clean corn contained fumonisin B1 (17 micrograms/g) and caused acute pulmonary edema when fed for 5 days. Survivors developed subacute hepatotoxicosis with individual hepatocellular necrosis, hepatomegalocytosis, and increased numbers of mitotic figures. Similar liver lesions occurred in pigs given fumonisin B1 intravenously at 0.8 mg/kg body weight for 14 days.

Fumonisin B1 concentrations in feeds from 45 confirmed equine leukoencephalomalacia cases.

During the fall of 1989 and winter of 1990, numerous reports of equine leukoencephalomalacia (ELEM) occurred from many regions of the United States. Typically, horses were consuming feed partially or entirely composed of corn and/or corn screenings. From October 1989 through May 1990, samples from 55 confirmed or suspected ELEM cases were received at the National Veterinary Services Laboratories, Ames, Iowa, for fumonisin B1 analysis. Samples from 9 cases in 1984-1985 were also obtained. Fumonisin B1, a mycotoxin produced by Fusarium moniliforme, causes ELEM, but little is known of naturally occurring toxic or safe levels in feeds. To determine what levels of fumonisin B1 in feeds are associated with ELEM, 45 selected cases were studied. The fumonisin B1 concentrations ranged from less than 1 ppm to 126 ppm, with the majority of the samples above 10 ppm. All types of feeds were included: corn, screenings, sweet feeds, and commercially pelleted rations. The length of exposure varied from 7 to greater than 35 days. Horse feed samples not associated with ELEM were also collected and analyzed. None of the nonproblem feed samples contained fumonisin B1 levels greater than 8 ppm.

Concentrations of fumonisin B1 in feeds associated with animal health problems.

Ninety-eight samples of feeds associated with 44 cases of equine leukoencephalomalacia (ELEM) and 83 samples of feed associated with 42 cases of a porcine pulmonary edema syndrome (PPE) were analyzed for fumonisin B1 (FB1). For comparison purposes, 51 feed samples not associated with PPE or ELEM were also analyzed. Feed associated with ELEM contained FB1 ranging from less than 1 microgram/g to 126 micrograms/g with 75% of the cases having at least 1 sample above 10 micrograms/g. Feeds associated with PPE ranged from less than 1 microgram/g to 330 micrograms/g with 71% of the cases having at least 1 sample greater than 10 micrograms/g. Quantitation was by high performance liquid chromatography (HPLC)/fluorescence using the fluorescamine derivative with confirmation by thin layer chromatography (TLC) and/or gas chromatography/mass spectroscopy (GC/MS).

Novel occurrence of cyclopiazonic acid in sunflower seeds.

Cyclopiazonic acid (CPA), a secondary metabolite produced by several species of Aspergillus and Penicillium, was detected in moldy sunflower seed screenings fed to sows who experienced conception problems and feed refusal. Tandem mass spectrometry confirmed the CPA which was estimated by thin-layer chromatography (TLC) to be present at 10 ppm. A Penicillium species was isolated from the moldy seeds, but in culture it did not produce CPA.

Production of fumonisins by Fusarium moniliforme and Fusarium proliferatum isolates associated with equine leukoencephalomalacia and a pulmonary edema syndrome in swine.

Fumonisin B1 (FB1) and FB2 were isolated from corn cultures of both Fusarium moniliforme and Fusarium proliferatum. Respective concentrations in culture materials of FB1 and FB2 ranged from 960 to 2,350 and 120 to 320 micrograms/g for F. moniliforme and from 1,670 to 2,790 and 150 to 320 micrograms/g for F. proliferatum. Thin-layer chromatography, gas chromatography-mass spectroscopy, high-performance liquid chromatography, and liquid secondary ion mass spectroscopy were used for detection. Fumonisins from F. proliferatum have not previously been reported.

Fumonisin B1 levels associated with an epizootic of equine leukoencephalomalacia.

During the fall of 1989, an episode of equine leukoencephalomalacia involved 18 of 66 purebred Arabian horses at a breeding/training stable in Arizona. Of the 18 horses affected, the condition was fatal in 14. These horses, as well as 48 unaffected horses, had been fed a diet containing a substantial amount of white corn screenings. Gross pathologic findings included liquefactive necrosis in parts of the cerebral white matter and hemorrhagic foci of various sizes in the brain stem. Histopathologic findings included rarefied white matter with pyknotic nuclei and eosinophilic cytoplasm. Thin-layer chromatography, high-performance liquid chromatography, and gas chromatography/mass spectroscopy were utilized to identify and quantitate fumonisin B1 in 3 samples of corn from the farm. Concentrations of fumonisin B1 range from 37 to 122 ppm. Fumonisin B2 was also detected. Using information on diet, animal weights, and feeding practices, estimates of total fumonisin B1 dosage were determined. This is the first definitive report on equine leukoencephalomalacia and associated fumonisin B1 concentrations.