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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.

J Vet Diagn Invest 2:352-354 (1990)

A mycological evaluation and in vivo toxicity evaluation of feed from 41 farms with equine leukoencephalomalacia Terrance M. Wilson, Paul E. Nelson, W. F. O. Marasas, Pieter G. Thiel, Gorden S. Shephard, Erick W. Sydenham, Hillman A. Nelson, P. Frank Ross Equine leukoencephalomalacia (ELEM) is of worldwide distribution and is caused by the mycotoxin fumonisin B1 (FB1), a metabolite of Fusarium moniliforme. 4,6 Studies also indicate that metabolites from various strains of F. moniliforme may be hepatocarcinogenic to rats.2,8 A recent review of the history, disease, seasonality, clinical syndromes, epidemiology, and differential diagnosis of equine leukoencephalomalacia has been published.7 The purpose of this study was to mycologically analyze commercial and noncommercial feeds associated with ELEM From the US Department of Agriculture, Animal and Plant Health Inspection Service, Science and Technology, National Veterinary Services Laboratories, PO Box 844, Ames, IA 50010 (Wilson, H. Nelson, Ross), the Fusarium Research Center, Department of Plant Pathology, Pennsylvania State University, University Park, PA 16802 (P. Nelson), and the Research Institute for Nutritional Disease, South African Medical Research Council, Tygerberg 7505, South Africa (Marasas, Thiel, Shephard, Sydenham). Received for publication April 3, 1990.

and to evaluate the toxigenicity of selected cultures of F. moniliforme. A mail and telephone survey was conducted with veterinary diagnostic laboratories in the East and Midwest between 1983 and 1986 to locate confirmed cases of ELEM. Telephone calls and correspondence were initiated to follow up all reported cases. Feed samples were collected on all farms, with emphasis on those feeds used l-3 weeks prior to the diagnosis of ELEM. Based on the feeding practices, the farms were divided into 5 groups (Table 1): 1) commercial nonpelleted feed, 2) commercial pelleted feed, 3) corn plus commercial pelleted feed, 4) mixed feeding—some on corn, some on corn plus commercial pelleted feed, and 5) corn or corn screenings. Clinical necropsy information, as well as macroscopic and microscopic material from most cases, was reviewed. In all cases, a necropsy and, in most cases, a histopathologic evaluation of brain tissue were conducted on at least 1 animal from each farm. Forty-one farms were surveyed with a wide range of breeds

Table 1. Number of farms and type of ration fed to horses exposed to Fusarium moniliforme and diagnosed as having ELEM.

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Brief communications Table 2. Cultures of Fusarium moniliforme recovered from individual components of 14 feed samples.

and ages represented in the 506 horses exposed. The attack rate and case fatality rates are shown in Table 1. In addition to the rations listed in Table 1, the horses were also being fed hay and/or were on pasture. The feed samples collected were cultured for Fusarium species by a previously described method,9 and 500 F. moniliforme isolates were obtained. Isolates were lyophilized and stored at the Fusarium Research Center, Pennsylvania State University, University Park, Pennsylvania. Fourteen of the feed samples were mixtures of corn, oats, and pellets. The components of these samples were separated and cultured individually. Cultures of F. moniliforme were recovered from each type of feed component. In 3 of the 14 feed samples composed of corn, oats, and pellets, F. moniliforme was recovered from every feed ingredient cultured and, in most cases, the fungus grew in pure culture (Table 2). Ten pellets from each of 3 randomly selected feed samples were dipped in alcohol, flamed, and cultured. Ten additional pellets from each of these 3 samples were also cultured with-

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out surface flaming. All unflamed pellets yielded cultures of F. moniliforme. The organism was not cultured from any of

the 10 pellets from 1 flamed sample; however, 1 of 10 and 5 of 10 pellets from the other 2 flamed samples yielded cultures of F. moniliforme. Obviously, the organism survives the pelleting process. Autoclaved corn colonized with 100 selected isolates of F. moniliforme representing various geographic locations were fed to 1-day-old ducklings as previously reported5 After incubation at 25 C for 3 weeks, the cultures were ground and mixed with commercial chicken mash (1:1 w/w) and fed ad libitum for 14 days to groups of 41-day-old Peking ducklings. Control diets consisted of autoclaved noninoculated yellow corn mixed with chicken mash (1:1 w/w). Mortality was recorded daily, and the mean day of death was calculated from the number of ducklings that died on given days. An isolate was considered toxic if the death rate was greater than 50%. Ninety of the culture isolates were toxic (Table 3). The mean day of death for toxic strains ranged from 4.0 to 8.5 days, and the amount of culture material consumed ranged from 8 to 498 g/day. Culture material consumption ranged from 642 to 1,200 g for nontoxic isolates. There was no correlation between mean day of death and feed/ingredient type. Additionally, there was no correlation between mean day of death and geographic location where the feeds were collected. The high rate of toxic isolates and the ability of the organism to survive the pelleting process suggests the potential for toxicity in most feeds. The clinical data obtained were similar to those in previous reports on ELEM. 1,3 Mycologic examination of feed collected from areas of incidents of ELEM included in the survey resulted in 500 isolations of F. moniliforme. Culture material from 100 selected isolates was fed to 1-day-old ducklings with toxicity demonstrated in 90% of the cultures. Additional work is needed to determine if FB1 is the component of the culture material toxic to ducklings.

Acknowledgements We appreciate the assistance of Dr. Mark C. Thurmond and Mrs. Lois Klotz.

Table 3. Geographic location and number of Fusarium moniliforme isolates toxic to ducklings.

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References 1. Buck WB, Haliburton JC, Thilsted JP, et al.: 1979, Equine leukoencephalomalacia. In: Comparative pathology of naturally occurring and experimental cases. Proc Annu Meet Am Assoc Vet Lab Diagn 22:239-258. 2. Gelderblom WCA, Jaskiewic K, Marasas WFO, et al.: 1988, Fumonisins — novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme. Appl Environ Microbiol 54: 1806-1811. 3. Haliburton JC, Buck WB: 1986, Equine leukoencephalomalacia. Curr Top Vet Med Anim Sci 33:75-79. 4. Kriek NPJ, Kellerman TS, Marasas WFO: 1981, a comparative study of the toxicity of Fusarium verticillioides (=F. moniliforme) to horses, primates, pigs, sheep, and rats. Onderstepoort J Vet Res 48:129-131.

5. Kriek NPJ, Marasas WFO, Thiel PG: 1981, Hepato- and cardiotoxicity of Fusarium verticillioides (F. moniliforme) isolates from South African maize. Food Cosmet Toxicol 19:447-456. 6. Marasas WFO, Kellerman TS, Gelderblom WCA, et al.: 1988, Leukoencephalomalacia in a horse induced by Fusarium moniliforme B1. Onderstepoort J Vet Res 55:197-203. 7. McCue PM: 1989, Equine leukoencephalomalacia. The Compendium 11:646-651. 8. Wilson TM, Nelson PE, Knepp CR: 1985, Hepatic neoplastic nodules, adenofibrosis, and cholangiocarcinomas in male Fisher 344 rats fed corn naturally contaminated with Fusarium moniliforme. Carcinogenesis 6: 1155-1160. 9. Wilson TM, Nelson PE, Ryan TB, et al.: 1985, Linking leukoencephalomalacia to commercial horse rations. Vet Med 80: 63-68.

Announcement Beginning October 1, 1990, all manuscripts should be submitted to the new Editor, Dr. Lenn R. Harrison, Veterinary Diagnostic and Investigational Laboratory, P.O. Box 1389, Tifton, Ga. 31794. (Area code 912/386-3340)

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A mycological evaluation and in vivo toxicity evaluation of feed from 41 farms with equine leukoencephalomalacia.

Brief Communications 352 nosis of pseudorabies (Aujeszky’s disease). Proc Annu Meet Am Assoc Vet Lab Diagn 20:375-390. 5. Hill HT, Seymore CL, Egan...
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