L. G. Rice

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 μg/g to 126 μg/g with 75% of the cases having at least 1 sample above 10 μg/g. Feeds associated with PPE ranged from less than 1 μg/g to 330 μg/g with 71% of the cases having at least 1 sample greater than 10 μg/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).

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 B1 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.

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.

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 μg fumonisin B1 per gram. Screenings containing 92 μg 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 B, (17 μg/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.

Experimental Equine Leukoencephalomalacia, Toxic Hepatosis, and Encephalopathy Caused by Corn Naturally Contaminated with Fumonisins

A study to evaluate the effects of dietary fumonisin B, was conducted using 6 ponies (4 test and 2 control). A ration naturally contaminated with fumonisin B, was fed in 3 phases: 1) 44 ppm fumonisin B1, 2) less than 1 ppm fumonisin B1, and 3) 88 ppm fumonisin B,. 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 B, diet for 98 days. Phase 2 consisted of a diet with < 1 ppm fumonisin B, 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.

Fumonisin B2 in Cultured Fusarium Proliferatum, M-6104, Causes Equine Leukoencephalomalacia

with panretinal necrosis had evidence of retinal vascular disease. Vascular disease was limited to retinal vessels, unlike systemic hypertension in dogs in which similar changes can also be seen in choroidal vessels. In persons with acute and chronic glaucoma, the ganglion cell and nerve fiber layers are usually the only retinal layers significantly affected. 1,3 In 1 recent report, photoreceptor numbers were decreased in 24 glaucomatous human eyes with IOPs > 35 mm Hg. The globes in our study, however, routinely showed necrosis in the inner nuclear, outer nuclear, photoreceptor, and retinal epithelial layers in addition to marked changes in the ganglion cell layer. The functional significance of the retinal necrosis is potentially severe. Animals with retinal necrosis would not be expected to regain vision regardless of a decrease in IOP. The difference between the animals who regain sight and those who do not may be at least partially due to differences in the severity and distribution of retinal necrosis. The filtration apparatus was affected in all globes. Early changes were characterized by atrophy with trabecular cells showing hypertrophy. Later in the disease process, profound atrophy made the filtration apparatus undiscernible. Mitosis and plump mesenchymal cells in the plexuses were indicative of a cellular proliferative response that occurs even as the structure undergoes severe atrophy within 9 days of the initial rise in IOP. Regardless of whether these changes are primary or secondary, the consequences of a closed filtration apparatus would be a decrease in conventional aqueous outflow, which may contribute to a further rise in IOP. In summary, changes noted within the first 11 days of increased IOP are 1) the filtration apparatus initially exhibited a proliferative response with eventual progression to severe atrophy; 2) ganglion cell abnormalities and optic nerve malacia occurred early and at relatively low pressures; and 3) the nontapetal retina was consistently more severely affected than the tapetal retina in terms of panretinal necrosis. In these globes, a threshold level of >45 mm Hg appears to be necessary for some of the more severe changes.

Analysis of Corn and Cultured Corn for Fumonisin B1 by HPLC and GC/MS by Four Laboratories

Fusarium moniliforme, an important ear rot pathogen of corn, occurs on a variety of plant hosts. Corn worldwide is frequently contaminated by this fungus. A number of toxicoses are believed attributable to the consumption of moldy corn or corn screenings, including equine leukoencephalomalacia (ELEM) and pulmonary edema/hydrothorax in swine. 7,8 Recently, fumonisins were isolated and characterized from F. moniliforme cultures. Fumonisins are structurally similar to a host-specific phytotoxin of tomato isolated from the unrelated fungus Alternaria alternata f. sp. lycopersici. 3,4 Fumonisin B, has been identified in moldy corn from southern Africa and in samples of corn associated with ELEM cases. 11,12,17 Injection of purified fumonisin B1 produced ELEM in a horse and pulmonary edema/hydrothorax in swine. Analytical data concerning the levels of fumonisins in naturally contaminated corn and corn screenings, especially those associated with animal toxicoses, are just beginning to appear in the literature. Laboratory cultures of Fusarium mondiforme produce fumonisin B1 levels as high as several parts per thousand. In naturally contaminated corn and screenings, the levels are expected to be much lower. Earlier reports indicate that levels of l-150 μg/g of fumonisin B1 were measured in samples associated with ELEM, and levels of l330 μg/g were found in samples associated with toxicoses in swine. Nonproblem samples contained lower levels, generally < 6 μg/g. 12 Analytical methods of assaying fumonisin in corn or cultures include TLC;5,12 HPLC of derivatives (Ware GM: 1990, Determination of Fumonisins in corn by HPLC. Association of Food and Drug Officials of the Southern States Technical Program, April 1990, Paper #21); liquid-secondary ion mass spectrometry; 15 and hydrolysis followed by derivatization and detection of the esterified tricarballylic acid moiety, 14 or the TMS ether or TFA ester of the 22 carbon aminopentol backbone. 11,16 Validated analytical methods are needed for assessing the risk of consuming fumonisin contaminated grains and feed. The primary objective of this study was to obtain comparative analytical information regarding a fluorescamine procedure developed at the National Veterinary Services Lab-