Ion mobility-mass spectrometry to extend analytical performance in the determination of ergot alkaloids in cereal samples.

This work evaluates the potential of ion mobility spectrometry (IMS) to improve the analytical performance of current liquid chromatography-mass spectrometry (LC-MS) workflows applied to the determination of ergot alkaloids (EAs) in cereal samples. Collision cross section (CCS) values for EA epimers are reported for the first time to contribute to their unambiguous identification. Additionally, CCS values have been inter-laboratory cross-validated and compared with CCS values predicted by machine-learning models. Slight differences were observed in terms of CCS values for ergotamine, ergosine and ergocristine and their corresponding epimers (from 3.3 to 4%), being sufficient to achieve a satisfactory peak-to-peak resolution for their unequivocal identification. A LC-travelling wave ion mobility (TWIM)-MS method has been developed for the analysis of EAs in barley and wheat samples. Signal-to-noise ratio (S/N) was improved between 2.5 and 4-fold compared to the analog LC-TOF-MS method. The quality of the extracted ion chromatograms was also improved by using IMS.

Effects of Heating, Pelleting, and Feed Matrix on Apparent Concentrations of Cereal Ergot Alkaloids in Relation to Growth Performance and Welfare Parameters of Backgrounding Beef Steers.

As the contamination of cereal grains with ergot has been increasing in Western Canada, studies were undertaken to evaluate the impacts of heating (60, 80, 120, or 190 °C) alone or in combination with pelleting on concentrations of ergot alkaloids. Fifteen samples of ergot-contaminated grain from Alberta and Saskatchewan were assayed for R and S epimers of six alkaloids (ergocryptine, ergocristine, ergocornine, ergometrine, ergosine, and ergotamine) using HPLC MS/MS. Five samples with distinct alkaloid profiles were then selected for heating and pelleting studies. Heating resulted in a linear increase (p < 0.05) of total R and total S epimers with increasing temperature, although some individual R epimers were stable (ergometrine, ergosine, ergotamine). Pelleting also increased (p < 0.05) concentrations of total R and total S epimers detected, although ergometrine concentration decreased (p < 0.05) after pelleting. A feeding study arranged in a 2 × 2 factorial structure used 48 backgrounding Angus-cross steers fed four different diets: (1) Control Mash (CM, no added ergot), (2) Control Pellet (CP), (3) Ergot Mash (EM), or (4) Ergot Pellet (EP). Pelleting heated the ergot to 90−100 °C under 4 bars pressure, but the ergot used in the feeding study was not otherwise heated. Alkaloid concentrations of EM and EP varied by up to 1.1 mg/kg depending on the feed matrix assayed. No differences among treatments were noted for growth performance, feed intake, feed conversion, concentrations of serum prolactin and haptoglobin, hair cortisol, or in temperatures of extremities measured by infrared thermography. The only negative impacts of ergot alkaloids were on blood parameters indicative of reduced immune function or chronic inflammation. Pelleting did not heighten the negative clinical outcomes of ergot, although alkaloid concentrations of pelleted feed increased depending on the matrix assayed. It was hypothesized that the heat and pressure associated with pelleting may enhance the recovery of alkaloids from pelleted feed.

Using On-Farm Monitoring of Ergovaline and Tall Fescue Composition for Horse Pasture Management.

Central Kentucky horse pastures contain significant populations of tall fescue (Schedonorus arundinacea (Schreb.) Dumort) infected with an endophyte (Epichloë coenophialum (Morgan-Jones and Gams) Bacon and Schardl) known to produce several ergot alkaloids, with ergovaline in the highest concentration. While most classes of horses are not adversely affected by average levels of ergovaline in pastures, late term pregnant mares have a low tolerance to ergovaline and the related ergot alkaloids. Endophyte-infected tall fescue has been known to cause prolonged gestation, thickened placenta, dystocia, agalactia, and foal and mare mortality. The University of Kentucky Horse Pasture Evaluation Program utilizes ergovaline and endophyte testing, as well as pasture species composition, to calculate ergovaline in the total diet in broodmare pastures. This data is used to develop detailed management recommendations for individual pastures. Application of these recommendations has led to reduced tall fescue toxicity symptoms on these farms, as well as improved pasture management and improved forage quality and quantity.

Occurrence of Ergot Alkaloids in Barley and Wheat from Algeria.

The natural occurrence of six major ergot alkaloids, ergometrine, ergosine, ergotamine, ergocornine, ergokryptine and ergocristine, as well as their corresponding epimers, were investigated in 60 cereal samples (barley and wheat) from Algeria. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and a QuEChERS extraction method were used for sample analysis. The results revealed that 12 out of 60 samples (20%) were contaminated with ergot alkaloids. Wheat was the most contaminated matrix, with an incidence of 26.7% (8 out of 30 samples). The concentration of total ergot alkaloids ranged from 17.8 to 53.9 µg/kg for barley and from 3.66 to 76.0 µg/kg for wheat samples. Ergosine, ergokryptine and ergocristine showed the highest incidences in wheat, while ergometrine was the most common ergot in barley.

A critical review of analytical methods for ergot alkaloids in cereals and feed and in particular suitability of method performance for regulatory monitoring and epimer-specific quantification.

Cereals and feed contaminated with ergot alkaloids (EAs) have been of concern for several decades. Nowadays, analysis of EAs is focused on ergometrine, ergotamine, ergosine, ergocristine, ergocryptine (a mixture of α- and ß-isomers) and ergocornine and their related -inine epimers as listed in the European Commission Recommendation 2012/154/EU. Liquid chromatography with fluorescence detection has been used for quantification of EAs for decades whilst LC-MS has become the work-horse for quantification of EAs in the last decade. However, in LC-MS analysis matrix effects of different magnitudes exist for each EA epimer, especially ergometrine/ergometrinine, even after different clean-up procedures. This leads to an underestimation or overestimation of EAs levels. Moreover, isotopic labelled standards for EAs are still not available in the market. This review aims to provide background information on different analytical methods, discuss their advantages and disadvantages and possible advancement. Moreover, the method performance requirements to support forthcoming regulations are also discussed.

A Simple LC-MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass.

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography-mass spectrometry (LC-MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC-MS (for peramine). This work provides the first highly sensitive quantitative LC-MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Co-exposure of the Mycotoxins Lolitrem B and Ergovaline in Steers Fed Perennial Ryegrass ( Lolium perenne) Straw: Metabolic Characterization of Excreta.

Past research showed a strong linear correlation between levels of the mycotoxins lolitrem B (LB, a tremorgen) and ergovaline (EV, an ergot alkaloid and potent vasoconstrictor) in perennial ryegrass (PRG) forage. The purpose of this study was to characterize the excretion of these two compounds in beef cattle consuming PRG straw and to utilize liquid chromatography-tandem mass spectrometry to investigate the metabolism of LB and EV in excreta. Four groups of steers ( n = 6/group) were fed endophyte-infected PRG for 64 days (2256/638, 1554/373, 1012/259, or 247/<100 µg/kg LB/EV). Concentrations of LB and EV in both PRG straw and feces showed a linear relationship to each other. Feces reflected a dose-response for both mycotoxins, with values increasing most rapidly through 21 days then plateauing. Urine contained no detectable level of either compound or the ergoline lysergic acid. Screening for metabolites showed oxidation and reduction biotransformations for both toxins, with additional conjugation products detected for ergovaline.

Effects of grazing different ergovaline concentrations on vasoactivity of bovine lateral saphenous vein.

Previous research has demonstrated that exposure to ergot alkaloids reduces vasoactivity of serotonin (5HT) receptors. Chemical suppression of tall fescue seedhead production is a tool to reduce the level of exposure to ergot alkaloids by a grazing animal. Therefore, the objective was to evaluate contractility of lateral saphenous veins biopsied from mixed breed steers following a 87- to 101-d grazing period on 3-ha pastures of bermudagrass (Cynodon dactylon; n = 5 steers; BW = 340 ± 9 kg), or toxic endophyte-infected tall fescue (Lolium arundinaceum) that was not treated (n = 5 steers; BW = 300 ± 6; 0.56 ppm ergovaline) or was treated (n = 5 steers; BW = 294 ± 9 kg; 0.24 ppm ergovaline) with herbicide containing aminopyralid and metsulfuron-methyl. To evaluate contractility, biopsied veins were mounted in a multimyograph and exposed to increasing concentrations of a tall fescue seed extract (EXT; ergovaline source) and 5HT1B (CP 93129), 5HT1D (L-694,247), and 5HT2A (TCB2) agonists. All contractility data were normalized to a maximal response of 1 × 10-4 M norepinephrine and were analyzed as a split plot treatment design using SAS for effects of pasture treatment, agonist concentration, and the interaction. There was no contractile response to any concentration of 5HT1B agonist in any of the pasture treatments. There were pasture × concentration interactions for contractile responses to 5HT2A agonist (P < 0.01) and EXT (P < 0.01). For both EXT and TCB2, veins from bermudagrass steers were more vasoactive to the higher concentrations of these compounds (P < 0.05), and there were no differences between veins collected from the unsuppressed or seedhead-suppressed treatments (P = 0.66). There was also a pasture × concentration interaction for the contractile responses to 5HT1D agonist (P < 0.01). However, these responses were not sigmoidal and reached a zenith at 5 × 10-7 and 1 × 10-6 M. At these concentrations, the response was greatest for veins from the unsuppressed treatment (P < 0.05) and did not differ between veins from suppressed and bermudagrass treatments (P = 0.41). Although reduced levels of ergovaline in seedhead-suppressed pastures did not alter vasoactivity of 5HT2A or 5HT1B receptors in the lateral saphenous vein, elevated vasoactivity of 5HT1D in veins from unsuppressed tall fescue pasture treatment suggests that lower ergovaline levels in seedhead-suppressed pastures can influence the vascular effects of ergot alkaloids.

Meta-analysis of endophyte-infected tall fescue effects on cattle growth rates.

The objective of this study was to quantitatively summarize literature reporting endophyte-infected (Neotyphodium coenophialum) tall fescue (Festuca arundinacea) effects on cattle ADG. This meta-analysis evaluated endophyte infection level, climate, and forage yield using a literature dataset of 138 treatments from 20 articles. Three infection level measurements were tested: endophyte infection as a percentage of infected tillers (E%); ergovaline concentration in ppb ([E]); and total ergot alkaloid concentration ([TEA]). Three types of climate variables were used: base values (temperature, humidity, and relative humidity), climate indices (heat index and temperature-heat index [THI]), and novel climate variables accounting for duration of climate effects. Mixed effect models, weighted by 1/SEM, including a random effect of study were built for each factorial combination of measurement method and climate variable group. Because many studies were missing SEM, two datasets were used: one containing only data with SEM reported and one that also included missing-SEM data. For the complete-SEM dataset (CSD), models were weighted by 1/SEM. In the missing-SEM dataset (MSD) the mean reported 1/SEM was assigned as the weight for all missing SEM treatments. Although 18 initial models were created (2 × 3 × 3 factorial approach), the backward stepwise derivation resulted in models that included only endophyte infection level, suggesting a negative relationship between infection level and ADG. The CSD models predicted ADG to decrease 39 and 33 g/d with each increase of 100 ppb of [TEA] and [E], and by 39 g/d for each increase of 10% E%. In the MSD dataset, predicted ADG decreased by 39 and 33 g/d with each increase of 100 ppb of [TEA] and [E], and by 47 g/d for each increase of 10% E%. All relationships reported had P < 0.05. After visual inspection of the data, piecewise regression was used to identify an infection threshold (IT) of 60 ppb [E] and 11 E%, where the effect of infection level was constant on either side of the IT. The ADG was 40% and 49% greater for infection levels below the IT for [E] and E%, respectively. Across THI values in the analysis, ADG decreases ranged from 11.2% to 45.0% for cattle grazing endophyte-infected tall fescue compared to non-ergot alkaloid endophyte infected tall fescue. Pasture E%, [E], and [TEA] have a negative relationship with ADG in growing cattle, and increasing temperature decreases ADG when infection level is greater than the IT.

Ergovaline does not alter the severity of ryegrass staggers induced by lolitrem B.

AIM: To investigate a possible interaction between lolitrem B and ergovaline by comparing the incidence and severity of ryegrass staggers in sheep grazing ryegrass (Lolium perenne) containing lolitrem B or ryegrass containing both lolitrem B and ergovaline. METHODS: Ninety lambs, aged approximately 6 months, were grazed on plots of perennial ryegrass infected with either AR98 endophyte (containing lolitrem B), standard endophyte (containing lolitrem B and ergovaline) or no endophyte, for up to 42 days from 2 February 2010. Ten lambs were grazed on three replicate plots per cultivar. Herbage samples were collected for alkaloid analysis and lambs were scored for ryegrass staggers (scores from 0-5) weekly during the study. Any animal which was scored ≥4 was removed from the study. RESULTS: Concentrations of lolitrem B did not differ between AR98 and standard endophyte-infected pastures during the study period (p=0.26), and ergovaline was present only in standard endophyte pastures. Ryegrass staggers was observed in sheep grazing both the AR98 and standard endophyte plots, with median scores increasing in the third week of the study. Prior to the end of the 42-day grazing period, 22 and 17 animals were removed from the standard endophyte and AR98 plots, respectively, because their staggers scores were ≥4. The cumulative probability of lambs having scores ≥4 did not differ between animals grazing the two pasture types (p=0.41). CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence for ergovaline increasing the severity of ryegrass staggers induced by lolitrem B. In situations where the severity of ryegrass staggers appears to be greater than that predicted on the basis of concentrations of lolitrem B, the presence of other tremorgenic alkaloids should be investigated.

Links Between Genetic Groups, Host Specificity, and Ergot-Alkaloid Profiles within Claviceps purpurea (Fr.) Tul. on Slovenian Grasses.

In the present study, the genetic relationships and ergot-alkaloid production of the fungus Claviceps purpurea on grasses were investigated, to determine any associations between grass host specificity, ergot-alkaloid production, and geographic origin. C. purpurea sclerotia were obtained from wild and cultivated grasses along a 300-km climatic gradient, from sub-Mediterranean to continental climates. Twenty-one infected grass samples provided 39 sclerotia for analysis of the ergot alkaloids ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, and ergocristine, and their "-inine" epimers, using liquid chromatography-tandem mass spectrometry. C. purpurea ribosomal DNA underwent molecular classification to determine any grass host or geographic specificity of ergot-alkaloid composition for the different operational taxonomic units. Molecular analysis of sclerotia ribosomal DNA showed three genetic groups, with some associations with specific grass host taxonomic groups. The ergot-alkaloid composition data were in agreement with the data obtained by molecular methods. The most frequent ergot-alkaloid epimers were ergocristine, and ergosine. The total ergot-alkaloid concentrations in sclerotia varied from 59 to 4,200 mg kg-1, which corresponds to 0.059 to 4.2 mg kg-1 in animal feed (assuming ergot alkaloids at 1,000 mg kg-1 sclerotia). Therefore, grasses can be associated with significant levels of ergot alkaloids. In addition, the ergot-alkaloid compositions of C. purpurea sclerotia can be different for infections with different C. purpurea genetic groups, because these show different ergot-alkaloid compositions.

Newly discovered ergot alkaloids in Sorghum ergot Claviceps africana occurring for the first time in Israel.

Sorghum ergot is a disease caused commonly by C. africana. In 2015, ergot was identified for the first time in sorghum fields in Israel, leading to measures of eradication and quarantine. The aims of the study were to identify the ergot species by molecular and ergot alkaloid profile analysis, to determine the ergot alkaloid profile in pure honeydew and in infected sorghum silages and to estimate the safety of sorghum silages as a feed source. C. africana was rapidly and reliably identified by microscopical and molecular analysis. Dihydroergosine was identified as the major ergot alkaloid. Dihydrolysergol and dihydroergotamine were identified for the first time as significant ergot alkaloid components within the C. africana sclerotia, thereby providing for the first time a proof for the natural occurrence of dihydroergotamine. The sorghum silages were found to be safe for feed consumption, since the ergot alkaloids and the regulated mycotoxins were below their regulated limits.

Rapid Screening of Ergot Alkaloids in Sclerotia by MALDI-TOF Mass Spectrometry.

Ergot is a common disease of wheat and other cereal grains that is predominantly caused by Claviceps purpurea in the field, often affecting crop yield in addition to the environment. Infected grain can be contaminated with dark sclerotia, which contain fungal metabolites such as ergot alkaloids. The occurrence of ergot alkaloids in cereal grain is a major health concern for humans and livestock. Effective and rapid screening of these mycotoxins is crucial for producers, processors, and consumers of cereal-based food and feed grain. Established methods of ergot alkaloid screening based on LC-MS or GC-MS require laborious processes. A novel method using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS was developed to identify four ergot alkaloids. Using dihydroxybenzoic acid as the matrix, ergosine, ergocornine, ergocryptine, and ergocristine were readily detected in individual sclerotia of C. purpurea. The accuracy of the identified ergot alkaloids was further confirmed by tandem MS analysis. MALDI-TOF MS is suitable for high-throughput screening of ergot alkaloids because it permits rapid and accurate identification, simple sample preparation, and no derivatization or chromatographic separation.

Determination of the Ergot Alkaloid Ergovaline in Tall Fescue Seed and Straw Using a QuEChERS Extraction Method with High-Performance Liquid Chromatography-Fluorescence Detection.

Ergovaline is an ergot alkaloid produced by the symbiotic endophyte Epichloë coenophiala, which can colonize varieties of the cool-season grass tall fescue (Festuca arundinacea). It is the principle toxicant responsible for the vasoconstrictive and reproductive sequelae seen in "fescue toxicosis" in livestock which consume forage exceeding the threshold of toxicity established for this compound. A new method for extraction of ergovaline from tall fescue seed and straw was optimized and validated, on the basis of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, with high-performance liquid chromatography-fluorescence detection. Fourteen extraction solvents were tested; 2.1 mM ammonium carbonate/acetonitrile (50/50, v/v) had the highest and most consistent recovery (91-101%). Linearity, limit of detection, limit of quantitation, accuracy,and intra- and interday precisions for tall fescue seed and straw were 100-3500 µg/kg, 37 and 30 µg/kg, 100 µg/kg, 98%, 3.0 and 1.6%, and 3.8 and 1.0%, respectively. When the currently used solid-phase extraction (SPE) and QuEChERS methods were applied to 17 tall fescue straw samples, there was good agreement (correlation coefficient 0.9978). The QuEChERS method achieved the goals of eliminating chlorinated solvents and developing a fast, efficient, reliable method for quantitating ergovaline in tall fescue forage that can be applied in a high-throughput food safety laboratory.

Preference by sheep for endophyte-infected tall fescue grown adjacent to or at a distance from alfalfa.

Two experiments were conducted to assess preference by sheep for endophyte-infected tall fescue growing in monoculture at least 5 m away from alfalfa (fescue-middle (FM)) over endophyte-infected tall fescue growing adjacent (0.2 to 1 m; fescue-alfalfa (FA)) to alfalfa (FA), and the effect of legume scent on preference for endophyte-infected tall fescue. In Experiment 1, 10 six-month-old lambs were offered for 12 days a choice of freshly harvested FA and FM. On days 13 and 14, lambs were offered the same choice, except cages (to allow access only to scent) containing freshly harvested alfalfa were put in the feeders containing FA, whereas cages containing freshly harvested FM were included with the feeders containing FM. Forage intake was measured 1 h after feeding and at three consecutive 2-h intervals thereafter. FA contained greater (P<0.002) concentrations of the alkaloid ergovaline (360 ± 27 ppm) and CP (8 ± 0.4%) than FM (219 ± 27 ppm and 6 ± 0.4%, respectively). Lambs preferred (P<0.05) FA to FM during the 1st hour of feeding, but the differences became smaller and disappeared in later feeding periods (P<0.005). Lambs offered FA with alfalfa scent or FM with FM scent preferred (P<0.05) FA but only on the 2nd day. In Experiment 2, 10 six-month-old lambs were offered a choice of FM with cages (to allow access only to scent) containing freshly harvested alfalfa or FM for 8 days. During the following 4 days, FM in the cages was replaced with freshly harvested sainfoin. Preference was greater (P<0.05) for FM offered with alfalfa scent than for FM offered with FM scent only on days 4 and 8. When lambs were offered FM with alfalfa or sainfoin in cages, they preferred (P<0.05) tall fescue with sainfoin scent over fescue with alfalfa scent, but intake was variable across hours and days (P<0.001). It is concluded that (1) lambs adjusted their intake of and preference for FA and FM over successive feeding bouts within each day, likely owing to an attempt to balance intakes of nutrients and alkaloids and (2) olfactory cues influenced preference, but to a lesser extent than nutrients and alkaloids in endophyte-infected tall fescue.

Ergovaline and lolitrem B concentrations in perennial ryegrass in field culture in southern France: distribution in the plant and impact of climatic factors.

Perennial ryegrass (Lolium perenne) infected by Epichloë festucae var. lolii contains alkaloids that are responsible for toxicosis in several countries, but few cases are reported in Europe. Lolitrem B is generally the most abundant alkaloid and is recognized to be responsible for livestock staggers, whereas ergovaline is less frequently documented in perennial ryegrass. Lolitrem B and ergovaline were monitored over a three-year period in endophyte-infected perennial ryegrass 'Samson' sown in southern France. Alkaloid concentrations were strongly influenced by the stage of maturity of the plant; maximum concentrations were always measured at the fully ripe stage. Over the three years of analysis, variations in lolitrem B in the whole plant at the fully ripe stage were low (from 1296 to maximum 1871 µg/kg dry matter), whereas ergovaline varied considerably (from 526 to 2322 µg/kg dry matter), suggesting that abiotic factors play a key role in determining ergovaline levels in endophyte-infected perennial ryegrass.

Endophyte infection of tall fescue and the impact of climatic factors on ergovaline concentrations in field crops cultivated in southern France.

Tall fescue (Lolium arundinaceum) infected by Epichloe coenophiala contains ergot alkaloids responsible for fescue toxicosis in Australia, New Zealand, and the United States, with only a few cases occurring in Europe. The detection of Epichloe in 166 L. arundinaceum collected in southern France revealed that 60% were infected, 51% being high ergovaline producers. The ergovaline level in endophyte-infected tall fescue Kentucky 31 was monitored during 3 years in various parts of the plant. Maturation of plants, recorded according to the BBCH scale, appeared to be the main factor for estimating the risk of toxicity. Ergovaline levels of ≥300 µg/kg dry matter were obtained at the end of spring, the beginning of autumn, and mid-winter. Positive correlation between ergovaline level and cumulative degree-d was observed, whereas rainfall had no effect. These results suggest that the lack of fescue toxicosis observed in France cannot be explained by the lack of ergovaline in tall fescue.

The role of the Oregon State University Endophyte Service Laboratory in diagnosing clinical cases of endophyte toxicoses.

The Oregon State University Colleges of Veterinary Medicine and Agricultural Sciences instituted the Endophyte Service Laboratory to aid in diagnosing toxicity problems associated with cool-season grasses in livestock. The endophyte (Neotyphodium coenophalum) present in tall fescue (Festuca arundinacea) produces ergopeptine alkaloids, of which ergovaline is the molecule used to determine exposure and toxicity thresholds for the vasoconstrictive conditions "fescue foot" and "summer slump". Another vasoconstrictive syndrome, "ergotism," is caused by a parasitic fungus, Claviceps purpurea, and its primary toxin, ergotamine. "Ryegrass staggers" is a neurological condition that affects livestock consuming endophyte (Neotyphodium lolii)-infected perennial ryegrass (Lolium perenne) with high levels of lolitrem B. HPLC-fluorescent analytical methods for these mycotoxins are described and were used to determine threshold levels of toxicity for ergovaline and lolitrem B in cattle, sheep, horses, and camels. In addition, six clinical cases in cattle are presented to illustrate diagnosis of these three diseases.

Clinical expression of lolitrem B (perennial ryegrass) intoxication in horses.

REASONS FOR PERFORMING STUDY: Perennial ryegrass staggers is purported to be a common neurological mycotoxicosis of horses but the case description lacks detail and evidence. OBJECTIVE: To describe the clinical syndrome of lolitrem B intoxication in horses, limiting tests to those that are applicable to clinical practice, and to assess the potential value of enzyme-linked immunosorbent assay (ELISA) tests for lolitrem B in horse body fluids. METHODS: Seven horses in 2 separate groups were fed perennial ryegrass seed and hay containing 2 ppm lolitrem B. Paired data were collected prior to and after 2 weeks exposure to lolitrem B, including video-documented neurological examination and clinical examination. RESULTS: All horses developed a variable degree of tremor and ataxia when exposed to lolitrem B. Tremor depended on the level of activity and included a subtle, rapid tremor of the eyeball. Ataxia was exaggerated by blindfolding and primarily involved a truncal sway and irregular, but predictable, limb placements. No change was detected in urine lolitrem B levels and, although plasma lolitrem B increased during the treatment period, levels did not correlate with the severity of clinical signs displayed. Limb swelling, heel lesions and serous nasal discharge were also observed in horses most severely intoxicated. CONCLUSIONS: The clinical effects of lolitrem B intoxication in horses primarily involve action-related tremors and symmetrical vestibular ataxia. Ergovaline may have caused the limb swelling, heel lesions and serous nasal discharge. Plasma ELISA for lolitrem B may be of diagnostic use in the future. POTENTIAL RELEVANCE: This study provides a clearer appreciation of the clinical signs and variability of perennial ryegrass intoxication in horses.

The role of solvents in the signal separation for quantitative (1)H NMR spectroscopy.

Compared to conventional means of quantitative drug analysis, NMR spectroscopy only provides a limited set of adjustable parameters to enhance the quality of the analysis such as pH value, temperature, auxiliary reagents, magnetic field strength or properties of the solvent. In this work we investigate the influence of the kind of solvent on the signal separation of decisive resonances in the NMR spectra of codergocrine mesilate and flupentixol dihydrochloride. Polarity and aromaticity of the solvent play a crucial role in the optimization of signal separation. However, the set of applicable solvents is usually limited due to certain boundary conditions like solubility of the agent. Therefore the effects of solubilizers as well as mixtures solvents on the chemical shift also have to be taken into account. The quantitative results obtained by means of (1)H NMR spectroscopy were found to be in good agreement with the results carried out using official HPLC methods of International Pharmacopoeias.