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An improved extraction and cleanup procedure for quantitative analysis of ergovaline in Neotyphodium-infected grass tissues by high-performance liquid chromatography was developed, utilizing aqueous 2-propanol-lactic acid as extraction solvent. Losses of sample material and time requirements were significantly reduced, handling procedures simplified, and ergovaline and internal standard ergotamine recovered with similar efficiency from extracts. Analyses can be carried out on very small amounts (2-5 mg of dry weight) of samples and another endophyte-alkaloid, peramine, determined in the same extracts. Calibration curves with 2-propanol-lactic acid were linear over the range 0.004-0.938 microM ergovaline (= 2-500 ng/mL) in extracts, corresponding to 0.04-10 microg/g in samples. The distribution of ergovaline in the plant was extremely heterogeneous, indicating low in-planta mobility and strong regulation of accumulation by the internal plant environment. In contrast, peramine was much more uniformly distributed. These results clearly demonstrate very large differences in the tissue specificities of ergovaline and peramine.

Grazing of Echinopogon spp. by livestock in Australia has caused symptoms similar to those of perennial ryegrass staggers. We observed an endophytic fungus in the intercellular spaces of the leaves and seeds of New Zealand and Australian specimens of Echinopogon ovatus. Culture of surface-sterilized seeds from New Zealand specimens yielded a slow-growing fungus. An examination in which immunoblotting and an enzyme-linked immunosorbent assay (ELISA) were used indicated that E. ovatus plants from Australia and New Zealand were infected with fungi serologically related to Neotyphodium lolii (the endophyte of perennial ryegrass) and other Epichloe and Neotyphodium spp. endophytic in pooid grasses. No lolitrems (the indole-diterpenoids implicated as the causative agents of perennial ryegrass staggers), peramine analogs, or ergot alkaloids were detected in the infected specimens by high-performance liquid chromatography or ELISA. However, in endophyte-infected E. ovatus plants from New Zealand, analogs of the indole-diterpenoid paxilline (thought to be a biosynthetic precursor of the lolitrems and related tremorgens) were detected by ELISA, and N-formylloline was detected by gas chromatography. Endophyte-free specimens of New Zealand E. ovatus did not contain detectable paxilline analogs or lolines and were more palatable than infected specimens to adults of the pasture pest Listronotus bonariensis (Argentine stem weevil). Hyphae similar to those of the E. ovatus endophyte were also found in herbarium specimens of Echinopogon nutans var. major, Echinopogon intermedius, Echinopogon caespitosus, and Echinopogon cheeli. This appears to be the first time that an endophytic Neotyphodium species has been identified in grasses endemic to New Zealand or Australia.

Five loline-type alkaloids, the known lolinine, N-formylloline, loline, N-methylloline and the new 5,6-dehydro-N-acetylloline, were characterized from the pooid-grass endophyte-infected Festuca argentina. These compounds are not responsible for the toxicity of the plant, as shown upon i.p. injection into mice. Their probable phytoalexine role and chemotaxonomical significance are discussed.

The objective of these experiments was to test the hypothesis that the major alkaloid classes found in endophyte-infected tall fescue could act as growth promoters for vascular smooth muscle. Bovine vascular smooth muscle cells (VSMC) from the dorsal metatarsal artery were grown in vitro and exposed to five concentrations (10(-6), 10(-8), 10(-9), 10(-11) and 0 M) of ergonovine, alpha-ergocryptine, ergovaline, and N-acetyl loline for 48 h. The mitogenic potential of the alkaloids was tested on both actively growing cells (serum fed, 10% fetal bovine serum) and quiescent cells (serum starved, 0.1% serum) to assess the need for serum constituents for alkaloid induced growth or toxicity. Ergonovine stimulated (P < .07) VSMC growth at 10(-6) M concentration in growing and quiescent culturs and at 10(-8) M concentration in quiescent cultures. alpha-Ergocryptine stimulated (P < .01) growth at 10(-6) M concentration in growing cultures and at 10(-8) and 10(-9) M concentrations in quiescent cultures. Ergovaline exhibited a dual activity on the growth of VSMC in culture, stimulating (P = .06) growth of quiescent cells at 10(-9) M concentration but inhibiting (P < .05) growth of growing cultures at concentrations of 10(-6) and 10(-9) M. This duality of activity was also noted for N-acetyl loline: N-acetyl loline stimulated (P < .05) growth of quiescent cultures at concentrations of 10(-8), 10(-9), and 10(-11) M but inhibited (P < .05) growth of growing cultures at concentrations of 10(-8) and 10(-9) M. The growth effects of the alkaloids in vitro on VSMC support in part the hypothesis that the alkaloids may contribute to the vascular complications noted in cattle grazing endophyte-infected tall fescue through hyperplasia of the intima. This would result in a decreased luminal diameter of the blood vessels and a resultant decrease in blood flow to the afflicted tissues. The diminished blood flow to tissues would result in tissue death and reduced ability to dissipate heat.

Ergovaline, found in Acremonium coenophialum-infected tall fescue, is considered to be responsible for many symptoms associated with fescue toxicosis. Rats were tested to determine time-related thermoregulatory responses to acute treatment with ergovaline during specific thermal challenges. Isolated ergovaline was administered to rats (15 micrograms/kg body mass, i.p.) at controlled ambient temperatures (Ta) of 7 to 9 degrees C (cold) and 31 to 33 degrees C (hot). Treatment at cold Ta resulted in a decrease in rectal temperature (Tre) from 38.0 to 37.0 degrees C at 50 min after injection (P < .05) without complete return to preinjection value at 100 min. Tail temperature (Ttail) exhibited a concomitant 1C degree decrease (P < .05) after ergovaline injection at cold Ta. Also, metabolic heat production decreased from 15.7 to 11.7 W/kg at 20 min after injection of ergovaline (P < .05), with return to normal value at 40 min after injection. Injection of ergovaline at hot Ta increased Tre from 39.0 to 40.6 degrees C at 80 min after injection (P < .05), with no return to preinjection value at 100 min. This was attributed to a reduction in heat transfer across the tail as indicated by the decrease in Ttail from 37.1 to 36.4 degrees C at 40 min after injection (P < .05) and an initial increase in metabolic heat production from 8.4 to 9.4 W/kg at 3 min after injection (P < .05). Ambient temperature was found to be a major determinant of ergovaline response, which included alterations in both thermogenic and thermolytic mechanisms that control thermal balance.

Research on livestock toxicoses caused by Acremonium (endophyte)-infected grasses strongly implicate the ergopeptine alkaloids with A. coenophialum-infected fescue and paxilline and the lolitrem alkaloids with A. lolii-infected perennial ryegrass as the causative agents. Isolation, identification, and detection of these toxins involves extraction with appropriate solvents, clean-up procedures, and chromatographic methods with known standards. Thin-layer, high-performance liquid and gas chromatography along with ultraviolet and mass spectrometric (i.e., electron impact, chemical ionization, tandem mass) characterizations have been reported. These methods have varying degrees of success depending on the matrix from which the alkaloids have been extracted. Ergovaline is the primary ergopeptine alkaloid isolated from cultures of A. coenophialum and also from infected fescue grass and seeds toxic to livestock. Other compounds isolated from the endophyte-infected fescue include: lysergic acid amide (ergine), the clavine class of ergot alkaloids (chanoclavine I, agroclavine, elymoclavine, penniclavine), the pyrrolizidine alkaloids (N-formylloline, N-acetylloline, N-methyloline, N-acetylnorloline), and the unique pyrrolopyrazine alkaloid peramine. The loline alkaloids and peramine have been more associated with the insect-deterrent properties of the endophyte-infected fescue than with livestock toxicoses. Also, both peramine and the ergopeptine alkaloids (ergovaline, ergotamine) have been isolated from A. lolii-infected perennial ryegrass. More recently, paxilline and lolitrem B have been detected in laboratory cultures of A. coenophialum isolated from tall fescue. The ergot alkaloids in endophyte-infected perennial ryegrass may be more related to decreased animal productivity (weight gains, reproduction problems), whereas the lolitrems cause the staggers syndrome. The detection, isolation, identification, and analyses of these compounds from Acremonium-infected grasses is presented.

Gravid mares grazing endophyte-infested (E+) tall fescue exhibit increased gestation lengths, agalactia, foal and mare mortality, tough and thickened placentas, weak and dysmature foals, increased sweating during warm weather, reduced serum prolactin and progesterone, and increased serum estradiol-17 beta levels. Also, E+ tall fescue hay is less digestible than endophyte-free (E-) hay. Unlike many other species, horses consuming E+ tall fescue do not exhibit increased body temperature. Young horses consuming only E+ pasture do not gain as well as those consuming E- pasture. There is little difference in gain when the pasture is supplemented with enough concentrate to meet NRC requirements for growth. Neither selenium injections nor supplementing with corn at 50% of the NRC requirements for energy reduces the effects of toxic tall fescue on reproduction and lactation in gravid mares. It seems that the alkaloids of E+ tall fescue are serving as D2 dopamine receptor agonists. This activity would explain their prolactin-lowering effect. Domperidone, a dopamine receptor antagonist, is effective in preventing the signs of tall fescue toxicosis in horses without neuroleptic side effects.

A historic profile of endophyte-induced tall fescue toxicosis is presented. A chronology of events is presented, beginning with the importance of finding Balansia-infected grasses in a tall fescue pasture in north central Georgia. This initial finding was followed by the discovery that another related endophyte was present in tall fescue and other major forage grasses. This species of endophyte was identified as Acremonium coenophialum. After this report was the important discovery that cattle performance was poor on Acremonium-infected tall fescue. Thus, this endophyte and its presence in tall fescue was implicated as the cause of tall fescue toxicosis and fescue foot. It was later established that this endophyte also produced ergot alkaloids. The related endophyte of perennial ryegrass, A. lolii, was subsequently shown to be responsible for the ryegrass staggers syndrome. Several other species of Acremonium have been associated with other important forage and turf grasses. Finally, important papers leading to the revelations that endophytic fungi and their grass hosts are ecologically significant and that most should be considered mutualistic symbioses are reviewed. Symbiotic grasses have enhanced physiological and morphological characteristics that offer biotechnological exploitations on one hand, but on the other solutions to the toxicity of tall fescue are difficult because grasses free of their fungal partner are generally ecological failures.

Plant toxins are the chemical defenses of plants against herbivory. Grasses have relatively few intrinsic toxins, relying more on growth habit to survive defoliation and endophytic fungal toxins as chemical defenses. Forage grasses that contain intrinsic toxins include Phalaris spp. (tryptamine and carboline alkaloids), sorghums (cyanogenic glycosides), and tropical grasses containing oxalates and saponins. Toxic effects of these grasses include neurological damage (Phalaris staggers), hypoxia (sudangrass), saponin-induced photosensitization (Brachiaria and Panicum spp.), and bone demineralization (oxalate-containing grasses). Endophytic toxins in grasses include ergot alkaloids in tall fescue and tremorgens (e.g., lolitrem B) in perennial ryegrass. Lolitrems cause neurological effects, producing the ryegrass staggers syndrome. Annual ryegrass toxicosis is caused by corynetoxins, which are chemically similar to tunicamycin antibiotics. Corynetoxins are produced by Clavibacter bacteria that parasitize a nematode, Anguina agrostis, that may infect annual ryegrass. Corynetoxins inhibit glycoprotein synthesis, causing defective formation of various blood components of the reticulo-endothelial system. Another mycotoxin in ryegrass is sporidesmin, which causes liver damage and secondary photosensitization (facial eczema). Fusarium toxins such as zearalenone and trichothecenes also occur in forage grasses. Kikuyugrass poisoning results in severe damage to the ruminal epithelium and omasal mucosa, and neurological signs. The causative agent, which may be associated with army worm predation of the grass, has not been identified. The properties and significance of these toxins are reviewed.

The possibility of supplementing livestock diets with an aluminosilicate to protect them from fescue toxicosis was investigated. An in vitro study showed that hydrated sodium calcium aluminosilicate (HSCAS) removed greater than 90% of the ergotamine from aqueous solutions at pH 7.8 or lower, indicating a high affinity of ergotamine for HSCAS in vitro. Rats fed diets containing tall fescue seed infested (E+) with the endophytic fungus Acremonium coenophialum had lower (P less than .05) feed intakes and weight gains than did rats fed diets containing uninfested (E-) tall fescue seed. When feed intake by rats fed the E- seed diet was limited to that of rats fed the E+ seed diet, weight gains did not differ, but testes weights and serum prolactin (PRL) concentrations were lower (P less than .05 and .10, respectively) in rats receiving E+ seed. Supplementing E+ seed diets with HSCAS did not eliminate effects of E+ seed on intake, PRL, or testes weights. Sheep fed E+ tall fescue hay had higher (P less than .05) rectal temperatures than did sheep fed an equal amount of E- tall fescue hay, but OM and N digestion coefficients did not differ between the two hays. Supplementing E+ hay diets with HSCAS did not eliminate the effect of E+ hay on rectal temperatures. Addition of 2% HSCAS to tall fescue hay diets did not affect apparent absorption by sheep of OM, N, Ca, P, Na, K, or Cu, but it reduced (P less than .05) the apparent absorption of Mg, Mn, and Zn.(ABSTRACT TRUNCATED AT 250 WORDS)

Grasses infected with clavicipitaceous fungi have been associated with a variety of diseases including classical ergotism in humans and animals, fescue foot and summer syndrome in cattle, and rye-grass staggers in sheep. During the last decade it has been recognized that many of these fungal infections are endophytic; a fungal endophyte is a fungus that grows entirely within the host plant. Inspection of field collections and herbarium specimens has revealed that such infections are widespread in grasses. The chemistry associated with these grass-fungal interactions has proved to be interesting and complex, as each grass-fungal pair results in a unique "fingerprint" of various alkaloids, of which some are highly toxic to herbivores. In many cases the presence of an endophyte appears to benefit the plant by increasing drought resistance, or by increasing resistance to attack by insects, thus improving the overall survivability of the grass. This review will focus on alkaloids that have been reported in endophyte-infected grasses.

Stipa robusta (= Stipa vaseyi) is a perennial grass found in certain areas of the southwestern United States. It is commonly known as sleepygrass, as horses that ingest this grass may become profoundly somnolent or stuporous for periods of time lasting up to several days. In an attempt to determine the active principle(s), fractionation of a methanolic extract of sleepygrass infected with an Acremonium endophyte has yielded lysergic acid amide (20 micrograms/g dry wt), isolysergic amide (8), 8-hydroxylsergic acid amide (0.3), ergonovine (7), chanoclavine-I (15), and N-formylloline (18). Related alkaloids have been found in many endophyte-infected grasses. The dominant alkaloid constituent in sleepygrass, lysergic acid amide, has not previously been identified in a grass in such high concentration. Lysergic acid amide is likely to be the basis for the extreme sedative effects on animals, given past pharmacological work on the compound from the ergot fungus Claviceps paspali.

A group of 48 crossbred steers (approx 250 kg each) were used to determine the effects of various diets and treatments on serum prolactin concentrations and rectal temperatures. The steers were randomly assigned to groups fed the following: (1) endophyte fungus-infested fescue hay; (2) ammoniated endophyte fungus-infested fescue hay; (3) endophyte fungus-infested fescue hay plus 1 g of thiabendazole (TBZ)/9.1 kg of body weight at 7-day intervals; (4) ammoniated endophyte fungus-infested fescue hay plus 1 g of TBZ/9.1 kg at 7-day intervals; (5) ground Bermuda grass hay; and (6) endophyte-fungus-free fescue hay. Blood samples for prolactin determinations and rectal temperatures were obtained biweekly beginning on week 3 and continuing through week 9. A significant correlation (P less than 0.05) between low prolactin concentrations and high rectal temperatures in cattle eating endophyte-infested fescue hay was determined; however, as the prolactin concentrations approached normal (control animal) concentrations, this relationship between serum prolactin and rectal temperatures was not observed. Two antifungal agents used in this experiment (thiabendazole and ammonia) appeared to have different effects on the variables measured. Thiabendazole had no significant effect on prolactin concentrations or rectal temperatures of cattle ingesting endophyte-infested fescue hay, whereas ammoniation of this hay induced significantly higher (P less than 0.05) prolactin concentrations and lower rectal temperatures than in steers receiving only endophyte-infested fescue hay. Therefore, ammonia may be valuable not only as an aid in determining the cause of the fescue problem but also as a practical solution to some of the fescue-related economic problems in the cattle industry. Also, prolactin concentrations may be valuable in diagnosing fescue-related problems.

Holstein steers (112 to 258 kg BW) were fed diets high in endophyte-infected fescue (80 to 100%) in 15 latin square experiments to determine the relationships among BW, forage composition, voluntary consumption and digestion. Fescue DM intake (kg/d) increased linearly (P less than .05) with increasing BW (-.513 + .0275 BW; R2 = .69). Regressing log fescue intake (DM) on log BW resulted in a BW exponent of 1.1034 (R2 = .66) but did not account for any more variation in intake than the simple regression did. The percentage NDF in fescue was not related (P greater than .10) to fescue intake (kg/d) and was correlated only slightly with fescue intake as a percentage of BW (r = -.13; P less than .10). Adding the percentage of fescue in the diet or NDF in the fescue to the regression of DM intake on BW did not increase explained variation. Organic matter intake (kg) and digestibility (%) were not related (P greater than .10). Based on standardized partial regression coefficients, differences in voluntary intake accounted for 60% of the variation in the total quantity of OM digested, whereas differences in digestibility accounted for 40%. In conclusion, ingestibility of endophyte-infected fescue hay in relation to changing BW did not vary greatly. Concentrations of chemical constituents such as NDF and ADL in fescue were poorly correlated with feed consumption and digestibility.

A procedure was developed to isolate and determine ergot alkaloid production by Acremonium coenophialum, the endophytic fungus of tall fescue. The procedure established that macerated leaf sheath or pith from inflorescence stem placed either in a liquid medium or on a corn meal-malt extract agar medium produced isolated mycelium and characteristic conidia within a 3- to 3.5-week period. Once isolated, each fungus was placed in another liquid medium, M104T, where competent strains produced total ergot alkaloids ranging from 38 to 797 mg/liter. Several isolates were negative for ergot alkaloid synthesis. The production of ergot alkaloids by individual isolates was unstable; isolates rapidly degenerated in their ability to produce ergot alkaloids during subculture. However, the procedure as presented allows the assessment of an isolate for ergot alkaloid synthesis during its initial isolation.

The influence of supplemental phenothiazine (P) on growth and physiological criteria was studied in parasite-controlled calves consuming endophyte (Acremonium coenophialum)-infected tall fescue (TF). In Exp. 1, nine Angus heifer calves (312 kg) were supplemented with 227 g corn-mineral (CM) mix twice daily and allowed ad libitum access to either high-endophyte (HE) G1-307 (greater than 90% infected) or low-endophyte (LE) Kenhy (less than 1% infected) tall fescue hay, or HE G1-307 plus 2 g/d P in the daily supplement. Calves were kept in temperature-controlled rooms for 12 d at 21 degrees C followed by 7 d at 34 degrees C. In Exp. 2, 48 Angus steer calves (312 kg) were assigned to treatment groups consisting of calves grazing HE Kentucky-31 (57% infected) or LE Johnstone (less than 1% infected) TF, and supplemented daily with either .91 kg of a control CM mix or .91 kg of the CM mix containing 2 g P. The 112-d experiment was initiated on May 4 with BW and rectal temperature (RT) measurements and blood collected at 28-d intervals. In both experiments, calves receiving HE TF had lower (P less than .01) serum prolactin concentrations (PRL) at elevated ambient temperature and lower (P less than .01) serum alkaline phosphatase activities (AP) but higher (P less than .01) RT than calves consuming LE TF regardless of ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)