Vomitoxin: Natural Occurrence on Cereal Grains and Significance as a Refusal and Emetic Factor to Swine

R. F. Vesonder B.S. and C. W. Hesseltine Ph.D.*

Fifty 12,13-epoxytrichothecenes have been reported in the literature; of these diacetoxyscirpenol, T-2 toxin, nivalenol and vomitoxin have been naturally occurring in cereal grains throughout the world. Vomitoxin is produced in ears of corn prior to harvest when wet, cool weather precedes which favours Gibberella zeae growth. This type of infected corn, which often contains vomitoxin, is associated with the refusal and emetic syndrome exhibited by swine.

Since 1916, emesis in humans resulting from grains is indicated by growth of the mould Fusaria in the field is meagre. The ability ingestion of food prepared from Fusarium­ G. zeae (perfect stage of F. graminearuml of G. zeae to render grains unwholesome for infected cereal grains has been recorded in on the kernels and flowering head. On corn, consumption by swine and other farm several areas around the world. In the Soviet G. zeae occurs as a reddish mould that animals may be dependent on the type of Union, .it was known as the drunken bread begins as observable growth on the ear tip. hybrid, but studies of whether G. zeae intoxication. Sporadic food poisoning featur­ This condition is referred to as Gibberella or infection can be controlled by breeding 8 ing vomiting, nausea, somnolence, headache pink ear rot • new plants have not been conducted. and convulsion occurred in Japan during Outbreaks of barley scabbed with Emetic substances were extracted from the 1950's and the late 1940's. These dis­ G. zeae in the Midwest region of the U.S. naturally moulded barley with water3,9,'3. orders were associated with consumption were reported in 1919, 19289 and in the A water extract obtained from barley of rice contaminated with Gibberella zeae 1930'sto. This barley was rejected by swine. infected with G. saubentii has been reported and Fusarium nivale1 and of foods made Barley from the 1928 U.S. crop exported to to cause vomiting when intubated into 2 from G. zeae infected wheat flour • In the Germany was also toxic to swine. Swine swine'4. Swine also refused feed made from southern part of Korea in 1963, barley force-fed this barley became sick and barley naturally infected with Fusaria and infected with F. graminearum was reported vomited, and eventually death ensued from barley cultured with G. saubenetti. Water to cause in humans the toxic disturbances preference not to eat. Equines also rejected extracts from F. graminearum'contaminated of nausea, vomiting, abdominal pains and feed containing mouldy barley, but ~ttle barley administered intraperitoneally (ipl diarrhoea!. Comparable sYmptoms 'were and chickens were not susceptible. Farm caused toxic disturbances in suckling mice observed in swine and a few cattle. In a animal disorders due to ingestion of scabbed and pigs but had no apparent effect on similar phenomenon, involving swine con­ barley in the U.S. subsided as production rabbits, rats, mature mice and day·old 3 suming corn naturally contaminated with moved from the humid Midwest environ­ chicks • F. graminearum in the U.S., a new tricho­ ment to the West. G. zeae·infected corn (12 to 100% of thecene, vomitoxin, was isolated and shown Wheat also is susceptible to Gibberella kernels were infectedl produced in northern to be the causative agent4. Whether or not infection, but its involvement in the swine Indiana in 1965 has been investigated for the factor causing emesis in humans is . sYndrome of refusal and emesis appears to emetic and refusal factors"; hog farmers identical to that in swine is unknown, be less frequent than that of barley and reported that this corn was refused by swine particularly since several trichothecenes corn. Fusaria-infected corn now seems to and caused a few pigs to vomit. In feeding have been capable of causing various lab­ be the major grain in the U.S. which has tests, swine rejected the naturally infected oratory animals to vomit2,5,6. Vomitoxin detrimental effects on swine performance. corn and the corn amended with water was also shown to be the principal toxin In 1972, corn contaminated with G. zeae extracts obtained from this infected corn. responsible for the phenomenon of feed caused extensive losses to swine operations, In addition, swine vomited when this water refusal exhibited by swine'. reaching epidemic proportions in the U.S. extract was administered by stomach tube, Sporadic Fusaria outbreaks in the corn intravenously or ip. The substances extrac­ History of refusal and emetic factors belt also occurred in Indiana in 1958 and ted with water from this infected corn in scabbed grains 1965" and in northwest Ohio in 1970, were separated into two fractions, based 12 Cereal grains that cause swine or farm 1975 and 1977 • Low temperature with on their solubility in methanol. Swine animals with simple stomachs to vomit on concomitant high moisture conditions, ingesting corn amended with the methanol­ ingestion and refuse feed usually connotes which usually resUlts in a delayed harvest, soluble fraction vomited initially but would a scab condition in grain. This disease in appears to favour G. zeae corn infection. return to eat without further symptoms of This was the situation in 1966 and 1972. nausea or other effects. Hence, it was In addition, the 1972 corn crop was not proposed that the water extract of G. zeae­ Ronald F. Vesonder is a resaarch chamist in the dried immediately because of energy shor­ infected corn contained a methanol·soluble Mycotoxin Microbiology end Biochemistry tages for operating dryers; this introduced emetic factor and a methanol·insoluble R8lIesrch. Fermentation Laboratory; andCliffordW. a storage variable. refusal factor. Hease/tine ia Chief olthe Fermenlstion Laboratory 81 the Northern Regional Research Canter. Unusually wet weather preceded harvest . Emetic substances associated with Fusaria Northern Regional Research center. Agricultural in northwest Ohio in 1977. Conditions were species isolated from cer~als have been Resaarch. Science and Education Administretion. conducive to G. zeae growth on corn reported, using pigeons as an assay tool'6. U.S. Department of Agriculture. Peoria. Illinois intended for swine feed, and much of this Emetic factors were produced on corn 61604. 12 corn was found to contain vomitoxin • inoculated in the field with OF. graminearum tThe mention of firm names or trade produClsdoes Even with these documented cases, informa­ and from liquid culture media fermented not imply that they are endorsed or recommended by tha US Department of Agriculture over other tion concerning environmental conditions with a strain of F. moniliforme, F. roseum, firms or similar products not mentioned. and factors governing production of refusal F. poae, F. culmorum and F. nivale. The and emetic factors on grains infected with emetic principle elaborated by F. monili· form" was lethal to the pigeon in 12 hours of nine corn samples collected by the Food 1.88 bs, 3H, CH 3-G=C; 2.16 m, 2H, CH 2; when injer.ted through the wing vein. In and Drug Administration from the Midwest 3.10 ABq, J=4 Hz, CH,; 3.60 d,1!::!, CH; Japan, at about the same time, nivalenol, in 1972, vomitoxin was found at levels 3.82 s, 2!:!, CH, -OH; 4.50 m, 1H, CH-OH; 8 trichothecene produced by F. nivale ranging from 15-28 ppm. These findings 4.80 m, 1H, CH-OH; O-CH-HC=C; 6.60 m, cultured on liquid media was reported',". show vomitoxin to be the main tricho­ CH=C. The mass spectrum of vomitoxin This Fu:;arium strain was isolated from thecene involved in the syndrome of refusal showed a molecular ion at 296.157, and mouldy rice that caused nausea and vomit­ and emesis exhibited by swine in the U.S. loss of CH 3, H,O, hydroxymethyl or ing in 25 young people. The emetic-produc­ in 1972. formaldehyde as characterized by fragment ing Fusaria strain6 described subsequently iOn peaks at m/e 281, 278, 265 and 286, received much attention by investigators respectively. The most prominent ion, throughout the world. In a screen of Fusaria Classification of Fusaria involved in at m/e 248, which corresponds to loss of species using chemical and bioassay methods, fusariotoxicoses both water and formaldehyde, is consistent it was found that the F. poae strain pro­ Since Link's description of the genus for keto at C-8, hydroxyl at C-7 and duced the trichothecenes T-2 toxin, HT-2 Fusarium in 1809, classification of this hydroxymethyl at C-B. toxin and neosolaniol on a peptone-supple­ species has followed many schemes: The Vomitoxin was converted into its tri­ mented Czapek-Dox media at 25 and thousand Fusaria that had been described acetate with acetic anhydridepyridine (m.p. I8 27°C . Trichothecenes were not detected by the 1930's was lumped into sixteen 155-156° C) with a molecular ion 422.155. in the culture liquor for F. moniliforme and groups, six subgroups and 142 species The NMR spectrum in CDCI 3: 8 0.96 S, F. nivale strains. The PD strains on moist varieties'3; About 50 species were recognised CH 3; 1.86 s, 3H, CH 3; 1.90 s, 3H, CH 3; corn at 5 and 25° C and on Richards solu­ in the Fusarium genus"; All Fusaria were 2.12 s, 3!:!, CH 3; 2.20 s, 3H, CH 3; 2.95 ABq, tion at 25° C have been studied'. Extracts reduced to nine species",'6; and there are J=4, Hz 2H, CH-CH,-O; 3.89 d, CH" obtained from the F. poae strain cultured several other classification systems""'. 5.2 m, 1H, CH-OAc; 4.30 s, 2H, CH 20Ac; on corn at 8° C and from the Richards The differences in opinion among myco­ 4.76 d, J=5.5, CH-CH=C; 6.50 d;-J=5.5, . solution induced emesis in pigeons either logists in classifying Fusaria species neces­ CH=C; 6.04 s, 1H-:G!:!-OAc. by intubation into the crop or intravenous sitate identification of the sY:item followed injection through the wing at a non-lethal to describe this species. In the mycotoxi­ concentration. The emetic was identified cological literature synonomous names of Natural occurrence of vomitoxin as T-2 toxin. Acetyl T-2 toxin was isolated Fusaria, dependent upon classification and trichothecenes from shaken liquid cultures of F. poae system, are: F. roseum = F. graminearum and shown to be a less potent emetic to = F. equiseti = F. scirpi = F. avenaceum Prior to the finding of vomitoxin occurring pigeons than T-2 toxinl9 . The role of = F. culmorum (Section: Roseum, Gibbo­ naturally in corn and its documentation as the agent responsible for emesis and refusal acf!tyl T-2 toxin in the syndrome of emesis sum, Fusarium, Discolor); F. tricinctum = 4 and refusal in swine remains obscure, since F. poae = F. sporotrichioides = F. sporo­ syndrome exhibited by swine ,', 2 ppm of T-2 toxin was reported in feed associated it has not been detected in feedstuffs, in trichie/la {Section: Sporotrichiellal. 29 cereal grains or on corn cultured with Fusarium species that have the ability with a lethal toxicoses in dairy cattle . Fusaria species in the laboratory. to parasitize plants are indigenous in soil. Su bsequently, these two tric!hothecenes, as The conjecture was made that, since fungi. Fusaria of the Sporotrichiella section well as diacetoxyscirpenol (DAS) and only the F. poae strain produced emetic and Roseum section have been isolated nivalenol (NIV), have been found in cereals factors, perhaps the other four PD strains from cereal grains; toxins produced by these throughout the world. Table 1 summarizes may no longer be identical with the original species, which have been associated with the the natural occurrence of vomitoxin, and strains and that T-2 toxin and acetyl T-2 disorders in farm animals, are: T-2 toxin Table 2 cites incidence of T-2 toxin, DAS toxin may be the emetics :PD described. causes a hemorrhagic syndrome in swine and and NIV. In a survey of corn from the 1972 Each PD strain on autoclaved corn at 28° C cattle'9; zearalenone causes a hyperestrQ­ crop for Fusarium toxins, it was found that was re-examined for abilitY to elab6rate genism condition in swine3.; vomitoxin is a extracts from 93 samples caused a dermal rejection and emetic factors. Each corn potent refusal and emetic in swine'; leuko­ reaction by the rabbit skin bioassay42. The sample was evaluated for toxic factors by encephalmalacia is associated with equine dermatitic effect was attributed to T-2 a pig bioassay based on the degree of ingestion of corn contaminated with toxin, although it was not detected. Nine acceptance of corn amended with extracts F. moniliforme (Section: Liseola), and the of these samples were re-examined, six of obtained from the cultured corn ','1. Corn factor is unidentified31 . Fusaria of the which produced a skin irritation to the cultured with each PD strain was refused Sporotrichiella section are found less rabbit". Vomitoxin levels of 15 to 28 ppm by swine. Analyses of the refused corn for frequently on corn at harvest in temperate were found in four samples, but T-2 toxin trichothecenes showed that the corn fer­ zones, but it is more of a problem on cob and DAS were not detected in the nine mented with F. culmorum contained corn stored in a crib and overwintered. samples by GC-MS. Vomitoxin was detected vomitoxin. The method of detection was a However, colonists of the Roseum, Gibbo­ in Austrian and Canadian corn refused by 33 thin-layer chromatographic procedure that sium or Fusarium (Discolor) section are swine and also has been reported in corn in France37 and South Africa38 and in was rather insensitive for the trichothecenes common on corn in the U.S. 42 T-2 toxin, HT-2 toxin, acetyl T-2 and barley in Japan . Vomitoxin levels ranging from 0.40 to fusarenone-X. These strains are being Physiochemical properties re-investigated for their ability to produce 1.8 ppm were reported in feedstuffs refused toxins by extensive isolation procedures of vomitoxin by swine and T-2 toxin at a lower level, as Vomitoxin characterized as 3,7,15-trihy­ well as DAS and zearalenone36 . Since and. the detection system of gas chromato­ 4 graphy-mass spectrometry (GC-MSl. droxy-12,13-epoxytrichothec-9-ene-8-one ,' zearalenone often occurs in corn naturally . The Fusarium outbreak in U.S. corn is also referred to as Rd toxin and 4-deoxy­ contaminated with G. zeae, it has been nivalenoI 3'. The latter trivial name implies suggested that zearalenone may enhance in 1972, which was associated with swine 38 refusal to eat this contaminated corn, that the vomitoxin structure is like nivalenol the refusal syndrome • In a mouse bio­ perplexed investigators around the world but lacks a C4 hydroxy group. Vomitoxin assay, based on their being able to reject as to whether toxic factors were involved. (CIS H2.06), a white solid crystallized from drinking water containing trichothecenes, Contaminated predominantly with F. grami­ ethyl acetate and hexane as fine needles mp zearalenone (100 mgfl) did not enhance 153-154°C; (o:)~ + 6.5 (C 0.07 ethanol), is the refusal response for low levels (2 mg/I) nearum, corn produced in northwest Ohio, 48 which caused swine to vomit on ingestion soluble in ethyl acetate, methanol, ethanol of either T-2 toxin, DAS or vomitoxin • followed by refusal to eat was shown to and chloroform; insoluble in hexane; and contain a new trichothecene, vomitoxin4. slightly soluble in water. The ultraviolet F. graminearum isolates from this corn absorption of vomitoxin in methanol showed elaborated vomitoxin on autoclaved grain, a maxima at 226, and its infrared spectrum Analysis of vomitoxin and vomitoxin was demonstrated as the showed absorption at 3340-3460 broad, Trichothecenes such as vomitoxin and major emetic and refusal factor to swine' . 2950 and 1680 cm-!. nivalenol, which contain three and four A level of 40 ppm of vomitoxin was found The nuclear magnetic resonance spec­ hydroxyl groups, respectively, and a

in the northwest Ohio field corn. The natural trum of vomitoxin in CDCI 3 (tetramethyl­ carbonyl at the C. position, have been occurrence of vomitoxin was subsequently silane as internal standard) gave the follow­ extracted from grains with combinations of reported in the same area in 1977'2. In four ing chemical shifts: 8 1;13 s, 3 H, CH3-C; polar solvents. Vomitoxin was separated Table 1. Natural occurrence of vomitoxin on cereal grains and feed of its simplicity, rei iability and sensitivity. Vomitoxin applied in etf,ylacetate to shaved Country Commodity Concentration Reference backs of rabbits produced a visible vesica­ /lg/g tion at 1 /lg, but no irritation appeared on USA Corn 40 7,22 the shaved backs of mice (Vesonder, u,1pub­ lished results)' The animal SKin test may be USA Corn 15,18,20,28 22 expedient to eliminate from further study USA Corn 28 33 USA Corp 0.7 34 samples that test nevative; howeve" a posi­ tive test can result from compounds other USA Corn 12 35 USA Corn a 12 than trichothecenes. USA Corn 1,1.8 36 USA Commercial, mixed feed 0.04-0.06 36 USA Mixed feed 004,0.6,1 36 Vomitoxin - - Economics Japan Barley 7.3 32 The methodology for extraction of tricho­ France Corn 0.1-0.6 37 thecenes such as vomitoxin and nivalenol Canada Corn 7.9 33 from cereal grains is complex, tedious, Canada Feed 1.2 33 laborious and time consuming. The present Austria Corn 1.3, 7.9 33 state of the art for isolation of trichothe­ Austria Feed + 33 cenes from grains poses many questions Austria Corn b 47 concerning quantitative determinations. South Africa Corn 2.5 38 Further, this class of compounds has Zambia Corn 7.4 38 exacerbated precise chemical analytical a Vomitoxin was detected in a range of 0.5 to 10 /lg/g in 24 of 52 corn samples from methods due to their nonfluorescent pro­ Northwest Ohio. perties and their lack of intensity in the b Vomitoxin found in range of 1 to 20/lg/g in 56 maize samples obtained from Styria, ultraviolet and infrared region of the spec­ Austria. trum. Hence, quantitation measurements += Not quantified. have been limited to sophisticated equip­ ment (e.g., GC:MS and GLCl. Nevertheless, even though individual Table 2. Natural occurrence of T-2 toxin, nivalenol (NIV) and diacetoxyscirpenol (DAS) assessment of vomitoxin occurrence in on cereal grains cereal grains is meagre, circumstantial Country Commodity Concentration Reference evidence indicates that economic losses /lg/g have occurred to swine farmers because of this toxin. Farmers in northwest Ohio USA Corn T-2:2 29 in 1975 reported a major portion of their USA Mixed feed T-2:0.076, DAS:0.5 36 corn was refused by swine; this corn was Canada Barley T-2:25 39 heavily infected with G. zeae and had to France Corn T-2:0.02, NIV:4.8 37 be replaced with corn the swine would eat. Germany Corn DAS:31.5 40 The corn supply became short from grain India Sweet corn T-2:4, DAS:14 41 elevators in this area and had to be obtained Japan Barley NIV:detected 32 from suppliers as far as Peoria, Illinois, at an additional cost of SO.50/bushel. The from corn with 40% aqueous methanol4 Quantitation of the TMS derivative of losses sustained by the swine farmers in and vomitoxin and nivalenol from barley vomitoxin by GC:MS by the calibration northwest Ohio were staggering, and many with 50% aqueous ethanoI3'. Quantitation method in theSIM mode has been reported". faced financial disaster. As a result of this of trichothecenes by gas·liquid chromato­ Using a computerized GC: MS in the SI M severe G. zeae outbreak in 1975, northwest graphy (GLC) and GC-MS used in the mode vomitoxin, DAS, T-2 toxin, and Ohio farmers now inspect ears of corn in selected ion mode is most attractive due zearalenone in feedstuffs was found36 . the field for visible signs of this mould. to increased sensitivity, precision and Ethyl acetate extracted T-2 toxin, DAS If the corn appears to have th is type of reliability. and zearalenone, and the polar solvent mould infection, it is withheld from feeding to swine, but can be a part of the feed given The authors have used a quantitative CH 30H:H.0 extracted vomitoxin from GLC method for vomitoxin with internal feedstuffs. to cattle or chickens which are not affected l standard '. The following procedure was Visualization of trichothecenes on TLC by this type corn. In 1977, when preharvest used to obtain sample extract: The corn plates has been made after spraying with corn in a four-county area in northwest sample (50-300 g) was dried 4 hr at 78° C. p-anisaldehyde reagent43 ; colour changes Ohio was surveyed, the farmers reported Each dried sample was blended with butanol of trichothecenes with a carbonyl at the that much of it was contaminated with in a blender (1 g/2 mil. This extraction step C. position were described as nonfluor­ G. zeae. Infected kernels ranged from 2 to removes corn oil, lipids and pigments in escent brown spots after spraying with 50% for 44 samples, and eight showed none. addition to other butanol soluble sub­ H. SO4.44 These spray reagents may be An incidence of 46% vomitoxin contamina­ stances. The corn freed of butanol solubles useful during the isolation of a particular tion was found in these samples collected was blended with 40% aqueous methanol trichothecene and may provide information from 26 farms in northwest Ohio. This (1 g/2 mil. Each sample was extracted on the degree of purity. Vomitoxin turns survey established that vomitoxin is pro­ twice by this procedure. The combined yellow to p-anisaldehyde spray reagent after duced in the field when wet and cool weather persists before harvest. Even higher CH3-OH: H. 0 extracts was evaporated to heating at 110° for a few minutes, and with dryness, and the remaining residue was H. S04 it turns a yellow brown colour. levels of vomitoxin might have occurred if chromatographed on a silica gel column The Rf values of vomitoxin are 0.7 in a rainy period had delayed harvest. Such a situation did occur in 1972 in the corn belt (10-30 g) presaturated with CHCI 3. The CHCI 3:CH3OH (8.2 v/v) and 0.1 in toluene: column was eluted consecutively with ethyl acetate:90% formic acid (6:3:1 v/v/v), of the United States, with a high level of Fusarium infection. Since corn rejected by CHCI 3, CHCI 3:CH30H (90:10), The latter Recently, it has been reported that column eluate was analyzed for vomitoxin 4-p-nitrobenzyl-pyridine4S produces a blue swine is often fed to animals that can by G LC on a column packed with 3% spot on a white background on TLC plates tolerate it, the actual econom ic losses from OV-101 on Gas Chrom after addition of for a number of trichothecenes (vomitoxin vomitoxin in swine rations or the presence trimethyl silating (TMS) reagent and the 0.1 /lg, nivalenol 0.05 /lg, DAS 0.2 Jlg and of other trichothecenes may not be internal standard DAS. Column temperature T-2 toxin 0.1 /lg), DAS turns blue purple accurately estimated. was programmed from 160-250°C at with p-dimethylaminobenzaldehyde46 in 5° C/minute. Vomitoxin retention time hydrochloric acid in ethanol. relative to the TMS derivative of DAS was The skin reaction response of small 1.3 minutes. A weight ratio range of vomi­ laboratory animals in response to tricho­ toxin/DAS of 0;5/1 to 1.5/1 was used. thecenes is appealing as a bioassay because REFERENCES

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This article is reprinted from the journal Process Biochemistry, Vol. 16 No.1 December/January 1980/81 Published by Wheatland Journals Ltd., Penn House, Penn Place, Rickmansworth, Herts.