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17. TOXICOLOGY OF ERGOT ALKALOIDS IN AGRICULTURE

RICHARD A.SHELBY

Department of Plant Pathology, 209 Life Sciences,Auburn University, AL 36849, USA

Ergot is a two edged sword. The sclerotia of Claviceps may contain usefulpharmaceutical compounds, but as young plant pathologists we were all taughtin our introductory course about the impact of ergot on human health. Welearned about the misery of “St. Anthony’s Fire” and how these mycotoxinsfrom a phytopathogenic fungus can directly impact our society. Like the Irishpotato famine, and countless other epiphytotic disasters, this one has shapedthe course of human destiny. Early historic records are descriptive in theiraccounts of the effects of eating ergot bodies in grain, but recent accounts havegrown increasingly rare. This is due largely to our understanding of Clavicepsas a plant pathogen. We can control the fungus with fungicides (McLaren,1994; Schultzet et al., 1993; Mielke, 1993) or resistant varieties (Pageau et al.,1994) and remove sclerotia from grain with modern equipment. Ergot alkaloids,however, still find their way into our food supply, making the need for testingjust as important as ever. Small numbers of sclerotia can still evade the graincleaning process. In a 5-year survey of food products in Canada, Scott et al.(1992) found ergot alkaloids in 118/128 rye flour samples, with the highestamount being almost 4ppm total alkaloids. They found that other commoditieswere also contaminated: wheat flour was positive in 68/93 samples; bran 29/35; triticale flour 24/26; and baked rye products 52/114. The amounts foundwere generally insufficient to cause symptoms of ergotism in humans, but thepotential is present if the diet is heavy in contaminated grain products. Onedocumented case of human ergotism is described in Fuller (1968). Point-Espirit,France, was exposed to ergot in their bread, due to a local supply of flourwhich had not been thoroughly inspected. This factual account of the tragedyshould be required reading for anyone who doubts the importance of thoroughlytesting food products for traces of ergot alkaloids. Several people died, andmany more suffered irreversible injuries, some from psychotic episodes similarto those produced by LSD-25.

The ergot alkaloids produced by C. purpurea depend to some extent on thehost infected. Rye (Secale cereale) produces some of the highest amounts ofergot alkaloids, mainly ergocristine, ergotamine, and lesser amounts ofergosine, ergocornine, ergokryptine, and ergonovine (Scott et al., 1992).Wheat (Triticum aestivum) ergot contains a similar distribution of alkaloids asdoes the hybrid of the two (Triticale). Barley, on the other hand, seems toproduce more ergotamine than ergocristine (Porter et al., 1987), while other

Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under theHarwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group.

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grasses like fescue (infected with Claviceps only) produce more ergosine than isfound on other host grasses. Of course the story becomes infinitely morecomplex if the full range of Claviceps species and hosts are considered.

17.1. ERGOT ALKALOIDS IN ANIMAL FEED

Perhaps even more common are ergot alkaloids in animal feeds. We tend to takeless care in screening animal feeds for ergot alkaloids, perhaps believing thatanimals are more resistant to their toxic effects, but this is not the case. A recentsurvey of ergotism in American livestock (Burfening, 1994) found 655 publishedreports of ergot in the period from 1970–1992. These included case studies ofintoxication of mainly cattle, sheep, and swine. Reports of ergotism in livestockvaries from year-to-year in the United States, probably due to varying rainfalland temperature, but in the grain producing areas of the Northern plains, it canbe found in most years (Lamey et al., 1982). Several syndromes are associatedwith the ingestion of ergot alkaloids by livestock.

Nervous ergotism In this type of ergotism, the animal “staggers” as ifintoxicated. Later the animal may experience paralysis of the posterior limbs,followed by drowsiness, and/or convulsions. Extreme cases of this type ofergotism sometimes result in mortality. Horses, sheep, and even carnivores canexperience this type of ergotism, but rarely cattle.

Gangrenous Ergotism Because some of the ergot alkaloids causecontraction of smooth muscle (vasoconstriction), capillary beds can havereduced blood flow. In cases of chronic exposure, this leads to diminishedblood supply, degeneration of the tissues, and eventual gangrene and loss of theafflicted body part. Ears, tails, and often hooves are the target organs. Thesesymptoms are almost identical to those in humans having the worst cases ofexposure and in the throes of “St. Anthony’s Fire”. In animals, these symptomsare exaggerated by cold weather, when capillary circulation in the extremities isfurther restricted. This type of ergotism is most common in cattle. In severalclinical cases, a total alkaloid load amounting to about 0.2% of the diet,caused these symptoms (Burfening, 1994).

Agalactia In dairy animals lactation is severely curtailed by exposure toergot alkaloids. One documented endocrine effect is reduction in prolactine,which in turn reduces milk production. This malady can not only reduce milkproduction, but reduce the weaning weight of calves whose mothers wereintoxicated by ergot, and in swine, can reduce the birth weight andsurvivability of piglets in the litter. Schneider et al. (1986) reported that 1%ergot in the diet of sows caused a piglet birth weight reduction of about halfthat of control sows, and 100% mortality of piglets by three days afterfarrowing, compared to 3% mortality in the control group (Table 1).

This effect can also be demonstrated on sheep reproduction (Table 2). In thisexperiment, 152 sheep were fed ergot during gestation.


ERGOT TOXICOLOGY 471

Reduced Gain Some livestock species, such as beef cattle, have reducedaverage daily gains (ADG) when ergot alkaloids are present in the diet. Thishas been demonstrated with both Claviceps (Table 3) and with endophyticfungi (Table 4).

This relatively new type of ergot toxicosis was first described as “fescuetoxicosis”, but other pasture grasses can be affected, too. It is caused byendophytic fungi in the genus Neotyphodium (formerly Acremonium) which aresystemic symbionts in several important genera of pasture grasses, includingFestuca and Lolium. White et al. (1993) listed 47 species of grasses in 12 genera

Table 1 Effect of rye ergot on swine reproduction(from Schneider et al., 1986)

Table 2 Effect of ergot on sheep reproduction (from Burfenicng, 1994)

Table 3 Effect of barley ergot on ADG of heifers (from Burfening, 1994)

Table 4 Effect of endc ophytic fungi on ADG i in steers (from Sch midt et al., 1982)


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in which they had observed endophytic fungi in herbarium specimens. Thisunusual group of fungi may share a common phylogeny with Claviceps, (Glennet al., 1996) and indeed produce some of the ergot alkaloids, but in differentcombinations. Ergovaline is most often the ergot alkaloid observed in plantsinfected by endophytic fungi (TePaske et al., 1993; Porter, 1995). Ergine is alsoproduced, and lesser amounts of ergosine. The endophytic fungi produce muchlower concentrations of these alkaloids than are found in sclerotia of Claviceps.It is also common for seed lots to have a simultaneous infection of bothNeotyphodium and Claviceps, resulting in an even more toxic mixture.

Unlike Claviceps, which is a phytopathogen, these fungi confer enhancedsurvivability upon their hosts by mechanisms which are poorly understood atthe present time. Certainly involved in this process is reduced feeding byherbivores of all classes, including insects which normally feed upon the grasshost. The endophytic fungi generally reproduce vegetatively by spread of fungalmycelium in the seed of host. Like Claviceps, these fungi inhabit the floralportion of the host and invade the seed, but do not form sclerotia, or conidia invitro. As the seed germinates, the endophyte resumes growth, colonizes theyoung host plant, and eventually moves up the bolting culm into the paniclewhere the life cycle is complete (Figure 1). They are spread exclusively in theviable seed of their host.

17.2. ROUTES OF CONTAMINATION

When one considers the number of fungal species capable of producing ergotalkaloids and also the broad host range of Claviceps and the endophyticNeotyphodium species (Flieger et al., 1997), it is hardly surprising that foodand feed can become contaminated. The USA National Pest Information Service(NAPIS) database lists 457 records of C. purpurea in grain from the period1984–1995. Almost all of the major grain crops are susceptible to a species ofClaviceps (Table 5). Some of these diseases are rare, or limited in geographicaldistribution. C. gigantea, for example, is limited in distribution to a small areaof Mexico (Fuentes et al., 1964). This may be due to strict habitat requirementsof the pathogen, but there have been no attempts to spread the pathogen (nor

Table 5 Species of Claviceps found on important grain crops



should there be). C. purpurea, on the other hand, is cosmopolitan in distribution,and we may not have discovered its complete host and geographic range. It is byfar the most common and troublesome member of the genus. C. africana is aspecies that appears to be expanding into Japan, Thailand, South America,Australia, and the United States (Reis, et al., 1996). Worldwide, at least 40species of Claviceps have been described, but most are parasites of noncultivatedgrasses. (See also chapter 3 on Claviceps taxonomy in this book.)

Contamination of feed and food occurs by two routes: the grain crop itself isinfected, or infected grasses growing as weeds in the crop are harvested withthe grain and sclerotia become mixed with the grain. Barley, for example, canbe contaminated by sclerotia from annual rye grass (Lolium multiflorum)which was a weed in the barley field. Similarly, grazing animals can ingestsclerotia from Claviceps infected grasses which are harvested with hay, or areingested from the field in direct grazing common in the USA. Contamination of

Figure 1 Life cycle of Neotyphodium coenophialum, the endophyte of tall fescue. AViable seed of the grass host contains viable mycelium of the fungal endophyte. At thisstage it may be quite toxic to herbivores. B The seed germinates and the fungus begins togrow intercellularly into the stem of the plant. C Stems of the mature plant are completelycolonized. When the plant forms an inflorescence, mycelium of the endophyte moves upthe culm, into the panicle, and eventually into maturing ovaries, where the cycle is complete


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feeds can also result when infected grains are processed into pelleted feeds,under the mistaken impression that heating will denature the ergot alkaloids.As proven by Scott et al. (1992), heat denaturation by baking is not 100%, andsome of the active compound may be present in processed feed. Fajardo et al.(1995) also found that ergot alkaloids survived not only the process offormation into pasta, but the cooking process as well.

17.3. TESTING OF FOOD AND FEED

The obvious solution to preventing human and animal intoxication by ergotalkaloids is a thorough regimen of testing agricultural products before they areconsumed. Fortunately, there are quite a few published methods designed todetect ergot alkaloids in agricultural commodities (see also chapter 10 Ergotalkaloid analytics by Jegorov in this book).

Thin-Layer Chromatography (TLC) This method has been used for manyyears to detect ergot alkaloids, and is still used due to its speed, simplicity, andrelatively low cost. To use TLC for agricultural commodities, one need only toextract the ergot alkaloids with a suitable solvent, concentrate by evaporation,or solvent partition, spot the sample on a thin-layer plate, and develop in asuitable solvent. Normal phase ergot alkaloid TLC is spotted on silica platesand developed in chloroform: methanol solutions (Svendsen and Verpoorte,1984). Plates can be viewed in UV light to visualize the fluorescent ergotalkaloids, or they can be sprayed with Ehrlich’s reagent which is a positive testfor the indole ring, common to most ergot alkaloids. While this method issimple, it does suffer from the problem of low sensitivity. Typically, this methodis accurate in the microgram range of detection, and quantitation is bycomparison with intensity of spots of control samples run on each plate. TLCworks well for Claviceps broth cultures, or pure sclerotia, but in cases of low-level contamination of agricultural commodities, the interference ofbackground materials may prevent a good chromatographic analysis.

High-Performance Liquid Chromatography (HPLC) This method takesadvantage of the same basic chemistry as TLC: ergot alkaloids have acharacteristic affinity for a solid, stationary phase relative to a mobile, liquidphase. The big advantages of HPLC are the rigidly controlled flow ratesgenerated by mechanical pumps as opposed to capillary flow in TLC, and theimproved accuracy and sensitivity of UV and fluorescence detectors. A recentreview of HPLC methods can be found in Flieger et al. (1997). Most HPLCmethods for ergot alkaloid analysis in food or feed samples utilize apreliminary cleanup by solvent partitioning (Scott and Lawrence, 1980) orsilica columns (Rottinghaus et al., 1993). With fluorescence detection, thismethod is sensitive in the nanogram range. Of the methods used in routinediagnostic work, HPLC provides the most accurate quantitative andqualitative analysis for ergot alkaloids in agricultural commodities.



Enzyme-Linked Immunoassay (ELISA) Immunological methods to detectergot alkaloids are based on the ability of an antibody or immunoglobulin tobind with a specific target alkaloid. These antibodies are produced by injectingmice, rabbits, or other animals with the alkaloid which has been conjugated toa strongly antigenic carrier molecule such as bovine serum albumin (Schran etal., 1979; Arens and Zenk, 1980; Shelby and Kelley, 1992). The immunoassaycan be in several formats, but the competitive ELISA has largely replaced theradioimmunoassay (RIA). The use of an enzymemediated color change in achromogenic substrate eliminates the need for radioisotopes. The specificity ofthe antibody can be manipulated somewhat by utilizing different conjugationchemistries, and thereby different binding sites on the alkaloid molecule. Thatportion of the alkaloid molecule distal to the point of conjugation to the carrieris the site recognized by the antibody. If that region is C-8 of the ergoline ring,then many more ergot alkaloids can be detected, because this region is commonto most ergot alkaloids. If, on the other hand, N-l is the point of conjugation,the antibody will be more specific, since the distal C-8 is the more variableregion of ergot alkaloids. The ELISA methods have the advantage of beingquicker and less expensive than other methods, with a level of sensitivity whichis at least equal to the HPLC methods. However, they suffer from the problemof quantitative inaccuracy due to cross-reactivity of the antibody with non-target compounds in the matrix. These method are more suited to rapid, largescale screening of agricultural samples for the presence of ergot alkaloids.Detailed accurate quantitation could then be conducted using a more accuratemethod, such as HPLC.

17.4. THE FUTURE OF ERGOT

Claviceps will always be around to make life interesting for plant pathologists.This pathogen has shown an ability to adapt to a variety of hosts, many ofwhich form the staple crops essential for human survival. It is unlikely that inthe foreseeable future this pathogen will be completely controlled byfungicides, resistant varieties, or phytosanitation. For this reason, it is essentialfor individuals in the food and feed industries to be kept aware of the potentialproblem posed by ergot in agricultural commodities. This is made moredifficult by the rarity of outbreaks of ergot intoxication, and the lack of publicawareness of the problem which could eventually lead to a dangeroussituation. From a purely academic standpoint, the phylogenetic relationshipsbetween Claviceps species and the endophytic fungi are beginning to beunderstood by modern molecular techniques. Even after more than 100 yearsof research in ergot alkaloid chemistry we are still finding new compoundsfrom ergot (Cvak et al., 1995). Certainly the future of ergot and the ergotalkaloids will be brighter than the past.


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