P. Frank Ross

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.

A sensitive bioassay for detection of dietary estrogens in animal feeds.

Estrogen-responsive proliferation in the MCF-7 cell line was used as a bioassay for detection of dietary estrogens. The bioassay procedure was adapted to screen for estrogenic activity in feedstuffs that have been associated with hyperestrogenism in livestock. Methanolic feed extracts were added to the cell culture medium at μ1/ml concentrations for 4 days, after which the cell proliferation response was measured as DNA content. The half-maximal response for estradiol occurred at 2 pM, or 0.54 pg/ml. For zearalenone, a weaker estrogen, the half-maximal response occurred at approximately 200 pM, or 64 pg/ml. The bioassay was calibrated against a number of known estrogens (estradiol, diethylstilbestrol, zearalenone, zearalanol [cattle implant], β-zearalenol, zearalane), including the naturally occurring phytoestrogens (formononetin, genistein, daidzein, biochanin A, and coumestrol). The estrogenic activity of feed samples was expressed as equivalents of zearalenone (ppm zearalenone) that would have to be present to equally stimulate proliferation of the MCF-7 cells. The sensitivity of the bioassay was 0.05–0.1 ppm equivalents of zearalenone in feed, well below the threshold level associated with reproductive problems. The feed additive melengestrol acetate (MGA) showed no estrogenic activity in this assay. Estrogenic activity of feed extracts was confirmed by competitive inhibition with the antiestrogens tamoxifen or LY156758 (keoxifene) to show that stimulation of growth by feed extracts was through an estrogenic mechanism. Confirmation of known estrogens was by tandem mass spectroscopy. The assay is a sensitive and reliable screening procedure for detecting estrogenic activity in feedstuffs.

An HPLC Method for the Detection of Ergot in Ground and Pelleted Feeds

A high performance liquid chromatography (HPLC) method is described for detecting ergot in ground or pelleted forages and grains. Samples were extracted with alkaline chloroform, filtered, and applied to silica gel/organic binder cleanup columns. Following elution of pigments with acetone: chloroform, ergopeptine alkaloids were eluted with methanol and analyzed by HPLC with fluorescence detection. Average recovery of ergotamine, the major ergopeptine alkaloid produced by Claviceps, was 93%, with a relative standard deviation of 4.9%. The detection limit of ergotamine was approximately 50 ppb in all feedstuffs. Confirmation of ergopeptine alkaloids was accomplished by treating the parent ergopeptine alkaloids with 0.2% acetic acid to produce their -inine isomers and reexamining by HPLC with fluorescence detection or silica gel/organic binder column cleanup in combination with tandem mass spectroscopy. The method described is a valid alternative to microscopic inspection for detecting ergot contamination in ground or pelleted feedstuffs.

A Mycological Evaluation and in Vivo Toxicity Evaluation of Feed from 41 Farms with Equine Leukoencephalomalacia

nosis of pseudorabies (Aujeszky’s disease). Proc Annu Meet Am Assoc Vet Lab Diagn 20:375-390. 5. Hill HT, Seymore CL, Egan IT, Harris DL: 1983, Evaluation of the enzyme-linked immunosorbent assay and the microtitration serum-virus-neutralization tests as used in an epidemiological survey of Iowa, Illinois, and Missouri swine. Proc 3rd Int Symp World Assn Vet Lab Diagn 1:235-240. 6. Joo HS, Molitor TW, Leman AD: 1984, Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum. Am J Vet Res 45:2096-2098. 7. Kelling CL, Standinger WL, Rhodes MB: 1978, Indirect solidphase microradioimmunoassay for detection of pseudorabies virus antibody in swine sera. Am J Vet Res 39: 1955-1957. 8. Scherba G, Gustafson DP, Kanitz CL, et al.: 1980, Delayed hypersensitivity reaction to pseudorabies virus as a field diagnostic test in swine. J Am Vet Med Assoc 173: 1490-1493. 9. Synder ML, Erickson GA: 1981, Recommended minimum standards for an enzyme-linked immunosorbent assay (ELISA) in pseudorabies serodiagnosis. National Veterinary Services Lab, US Department of Agriculture, Ames, IA. 32 pp. 10. Wade TW, Brees J, Goyal SM: 1986, Comparison of latex agglutination and serum neutralization tests for the detection of pseudorabies antibodies in swine sera. Proc Annu Meet Am Assoc Vet Lab Diagn 29:401-408.

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-

Identification of Brucella abortus strain 19 by decreased ability to utilize erythritol as determined by gas liquid chromatography.

A method to identify Brucella abortus strain 19 by erythritol utilization using gas liquid chromatography (GLC) was developed. A total of 69 strains of B. abortus (41 virulent field strain isolates and 28 strain 19 isolates) were tested. Following incubation of the isolate with a standard amount of erythritol, the erythritol present in the cell suspension was acetylated and measured by GLC. Field strains of B. abortus utilized an average of 90.9% of the erythritol, whereas vaccine strains utilized an average of 42.4%. This difference in erythritol utilization will allow a more rapid identification of B. abortus strain 19.

Mass spectral confirmation of oosporein in poultry rations

9has provided a means to determine whether oosporein is present in grains and determine if it is a causative factor in renal disease in poultry. In this paper, we report on chemical ionization (CI) mass spectral investigations of oosporein for the purpose of confirming TLC tests for the presence of oosporein in samples of poultry rations. Mass spectrometry (MS) has recently become a routine tool for confirmation and identification of various toxicants encountered by Veterinary Diagnostic Laboratories (F. Ross, personal communication). Most reports 2,4