A. Morrie Craig

Correlation of ergovaline and lolitrem B levels in endophyte- infected perennial ryegrass (Lolium perenne)

The varieties of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotiphodium lolii contain several classes of toxic alkaloids, including ergopeptide alkaloids and lolitrem alkaloids. Lolitrem B, a potent tremorgen, is generally considered to be the predominant alkaloid in endophyte-infected perennial ryegrass. Ergovaline, a vasoconstrictor normally associated with endophyte-infected tall fescue (Festuca arudinacea), is also present in endophyte infected perennial ryegrass. Clinical signs of animals ingesting endophyte-infected perennial ryegrass are consistent with the presence of lolitrem B. However, clinical signs normally associated with ergovaline poisoning are not usually observed in animals ingesting endophyte-infected perennial ryegrass. A survey was conducted to quantitate both lolitrem B and ergovaline in 459 perennial ryegrass straw samples received at the Oregon State University College of Veterinary Medicine. Samples were analyzed for each alkaloid using separate high-performance liquid chromatography analyses. A strong positive correlation between the 2 alkaloids (r 2 = 0.7335) was observed, especially in the samples containing <3,000 ppb (ng/g) lolitrem B. The threshold levels above which clinical signs typically occur are 2,000 ppb lolitrem B and 300–400 ppb ergovaline. All of the samples analyzed contained <425 ppb ergovaline.

Improved Extraction and HPLC Methods for Ergovaline from Plant Material and Rumen Fluid

Ergovaline, the main ergopeptine alkaloid produced in tall fescue infected with Acremonium coenophialum, is known to cause tall fescue toxicosis. Current methods in use for quantifying ergovaline in plant material have several disadvantages, including large solvent volumes and long analysis time. We report here improvements in extraction and cleanup and the high-pressure liquid chromatographic methods. Our improvements include a 24-hour extraction time, the use of smaller solvent volumes during sample preparation, and fast analysis on the polymeric reverse-phase column. In addition to allowing the analysis of large batches to assist practitioners in the accurate diagnosis of fescue toxicosis, our method is also easily modified for other matrices, such as rumen fluid.

Semiquantitative determination of ergot alkaloids in seed, straw, and digesta samples using a competitive enzyme-linked immunosorbent assay.

Ergot alkaloids present in endophyte-infected (E+) tall fescue cause fescue toxicosis and other toxic effects in livestock that consume infected plant tissue, leading to significant financial losses in livestock production each year. The predominant method currently in use for quantifying ergot alkaloid content in plant tissue is through high-performance liquid chromatography (HPLC), which quantifies the amount of ergovaline, one of many ergot alkaloids in E+ plant tissue. The enzyme-linked immunosorbent assay (ELISA) method used in this study detects quantities of nonspecific ergot alkaloids and therefore accounts for greater amounts of the total ergot alkaloid content in E+ tissue than does HPLC. The ELISA can also be used to more expediently analyze a larger number of forage samples without sophisticated and costly analytical equipment and therefore could be more desirable in a diagnostic setting. The purpose of this study was to evaluate the between-day and within-run variability of the ELISA and to determine the binding efficiency of 6 ergot alkaloids to the 15F3. E5 antibody used in the competitive ELISA to ascertain its feasibility as a quick analysis tool for ergot alkaloids. Straw samples had an average coefficient of variation (CV) for concentration of 10.2% within runs and 18.4% between runs, and the seed samples had an average CV for concentration of 13.3% within runs and 24.5% between runs. The grass tissue-based lysergic acid standard curve calculated from the ELISA had an average r 2 of 0.99, with a CV of 2.1%. Ergocryptine, ergocristine, ergocornine, and ergotamine tartrate did not bind strongly to the 15F3.E5 antibody because of the presence of large side groups on these molecules, which block their binding to the antibody, whereas ergonovine and ergonovine maleate were bound much more efficiently because of their structural similarity to lysergic acid. Clarified rumen fluid was tested as an additional matrix for use in the ergot alkaloid competitive ELISA to determine whether future livestock metabolism experiments on the postingestion fate of ergot alkaloids in ruminants could utilize this assay as a quick screening tool for the presence of nonspecific ergot alkaloids in rumen fluid. HPLC and ELISA procedures were compared for their ability in determining ergot alkaloid toxicity based on the repeatability of the procedures and on the specific compounds they measure. The ratio of ELISA concentration to HPLC concentration (ergovaline) varied from 2.00 to 2.81 in seed samples and from 0.62 to 8.66 in straw samples, showing no consistent pattern between the 2 methods. Based on the lack of data at present for the identity of the toxin causing endophyte toxicosis and the lack of agreement between the ergovaline HPLC and ELISA analyses for ergot alkaloids, each method is equally valid as an indicator of toxicity and is the best means for determining the quantity of the specific toxin(s) they measure.

Sodium cyanate alters glutathione homeostasis in rodent brain: relationship to neurodegenerative diseases in protein-deficient malnourished populations in Africa

Sodium cyanate, a neurotoxic chemical in rodents, primates and humans, is implicated in neurodegenerative disorders in protein-deficient populations subsisting in parts of Africa on the cyanogenic plant cassava. The molecular and cellular mechanisms of cyanate neurotoxicity are not understood. This study investigates the effect of sodium cyanate on glutathione (GSH) homeostasis in rodent brain and liver in vitro and in vivo. GSH levels in mouse brain were rapidly, time- and dose-dependently decreased following intraperitoneal administration of 100, 200 or 300 mg/kg sodium cyanate. By contrast, GSH disulfide (GSSG) levels were increased and GSH/GSSG ratios were decreased in a dose-dependent manner in rat brain. Sodium cyanate depleted GSH levels in all regions of mouse brain. Brain glutathione reductase activity was dose-dependently inhibited, while glutathione peroxidase activity was not affected by sodium cyanate. The disruption of GSH homeotasis, as evidenced by reduced tissue GSH/GSSG ratios, likely results from cyanate-induced inhibition of glutathione reductase activity. The results of this study suggest that cyanate neurotoxicity, and perhaps cassava-associated neurodegenerative diseases, are mediated in part by disruption of glutathione homeostasis in neural tissue.

Vitamin E and exertional rhabdomyolysis during endurance sled dog racing

Exertional rhabdomyolysis (ER) is common in sled dogs, animals with high energy expenditures that consume high fat (60% of ingested calories) diets. Associations between pre-race plasma [vitamin E] and total antioxidant status (TAS) and risk of developing ER were examined in dogs competing in the 1998 Iditarod race. Pre-race blood samples were collected from 750 dogs and a second sample was collected from 158 dogs withdrawn from the race at various times. Plasma creatine kinase activity was used to identify withdrawn dogs with ER. There was no association between pre-race plasma [vitamin E] and risk of development of ER. Dogs that developed ER started the race with higher TAS, but when withdrawn, had lower TAS than unaffected dogs and had similar pre-race [vitamin E] but higher [vitamin E] at time of withdrawal. Hence, the risk of ER in sled dogs is not affected by plasma [vitamin E] before the race.

Cases of ergotism in livestock and associated ergot alkaloid concentrations in feed

Ergot-induced disease in humans was known long before Biblical times and has been the root cause for countless human epidemics spanning from the early fourteenth century to the late sixteenth century. In contrast, many of these same ergot alkaloids have been utilized for their medicinal properties to mitigate migraine headaches and have had indications as anti-carcinogens. Although ergot alkaloids have been used for centuries by humans, basic pharmacokinetic data has not been documented for clinical disease in livestock. Consequently, a threshold dose and accurate dose-response data have yet to be established. Throughout the past several years, new detection techniques have emerged to detect these alkaloids at the parts per billion (ppb) level which has allowed for new efforts to be made with respect to determining threshold levels and making accurate clinical diagnoses in affected animals. This perspectives article provides a critical initial step for establishing a uniform interpretation of ergot toxicosis from limited existing data.

Molecular Characterization of Sheep Ruminal Enrichments that Detoxify Pyrrolizidine Alkaloids by Denaturing Gradient Gel Electrophoresis and Cloning

An enrichment of strictly anaerobic bacteria from ovine rumen fluid, which has previously been named L4M2, is known to detoxify animal hepatotoxins from the pyrrolizidine alkaloid family. These toxins are present in the tansy ragwort plant (Senecio jacobaea). These plants have been described in livestock animals’ range forages in regions of the world such as the Northwest United States and South Africa. The bacterial enrichment was characterized by molecular cloning techniques and by the molecular fingerprinting technique of denaturing gradient gel electrophoresis (DGGE). Phylogenetic analysis of the enrichment revealed that the consortium is composed of no more than five putative bacterial species which associated to the Anaerovibrio, Desulfovibrio, Megasphaera, Prevotella, and Synergistes generas. These are all known to exist in the upper gastrointestinal tract of ruminant animals. This work improved upon previous attempts to characterize the consortium by obtaining nearly full-length ribosomal 16S rDNA sequences through cloning. The DGGE results were directly compared to the cloning data by polymerase chain reaction (PCR) amplifying eight phylogenetically representative clones and analyzing them by DGGE. Direct DGGE analysis of the enrichment displayed greater 16S diversity than the clone library used in this study, suggesting that at least one of the organisms present in the enrichment comprises less than 1% of the total cell population. These data will be used to further refine the enrichment in hopes of future use as a probiotic, which could be administered to animals challenged by the presence of tansy ragwort in their forage.

Association between vitamin E and enhanced athletic performance in sled dogs

PURPOSE To determine the association between prerace plasma vitamin E concentration and performance in sled dogs competing in the 1998 Iditarod Race. METHODS Prerace blood samples were collected from 670 dogs. Samples were analyzed for plasma vitamin E concentration while controlling for selected hematological and biochemical variables and signalment. Starting in teams of 16, exercise consisted of running up to 1159 miles pulling a laden sled and musher via checkpoints. The records of dogs that were withdrawn from the race for health reasons, fatigue, or strategic or technical reasons, and those of dogs that finished the race were analyzed. Multiple logistic regression and Cox proportional hazards analysis were used to determine factors associated with endurance. Multiple linear regression analysis was used to determine factors associated with team speed. RESULTS A total of 323 dogs (48%) were withdrawn from racing at various distances from the start. Median time to finish for 39 teams was 11.5 d and the winning time was 9.2 d. Dogs with prerace plasma vitamin E concentrations > 40.7 microg.mL-1 were 1.9 times more likely to finish (P = 0.0006) and had 1.8 times less of a risk of being withdrawn for every mile ran (P = 0.03) than were dogs with plasma vitamin E concentrations between 16.3 and 40.7 microg.mL-1. Neither a teams mean prerace vitamin E concentration, nor the proportion of dogs within a team with high (> 40.7 microg.mL-1) vitamin E concentration was associated with team speed. CONCLUSIONS Dogs with higher plasma vitamin E concentrations have enhanced endurance compared with dogs with lower plasma vitamin E concentrations, but the plasma vitamin E status of a team is not associated with team speed.

Uptake and transformation of soil [14C]-trinitrotoluene by cool-season grasses.

This study investigated the fate and uptake of [(14)C]-TNT from soil into orchardgrass (Dactylis glomerata), perennial ryegrass (Lolium perenne), and tall fescue (Festuca arundinacea) over a one year period in a greenhouse-controlled environment. Pots (n = 4 for each grass, containing 10 mg cold TNT/kg soil + 1.2 mg [(14)C]-TNT/kg soil and controls with no TNT) were exposed to light and temperature conditions typical of June at 45 degrees N for 369 days. Three plant harvests were made (63, 181, and 369 days), and soil and plant materials were monitored for [(14)C]-TNT and metabolite concentrations. The 11.2 mg/kg TNT dose was not phytotoxic to the plant species tested. Continual uptake of TNT into grass blades was observed over the one-year period, with a total accumulation of 1.3%, 0.9%, and 0.8% of the initial soil [(14)C]-TNT dose for orchard grass, perennial ryegrass, and tall fescue, respectively. All [(14)C]-TNT residue in plant material was incorporated as bound residue. At final harvest, radioactivity was concentrated most highly in the root > crown > blade for all species. Soil TNT was gradually reduced to aminodinitro-toluenes and then further to an unidentified metabolite(s). Overall, orchardgrass appeared to be the most efficient species at taking up TNT.

Design and in vitro evaluation of new rpoB‐DGGE primers for ruminants

Two new primer sets based on the rpoB gene were designed and evaluated with bovine and ovine rumen samples. The newly developed rpoB-DGGE primer set was used along with the 16S rRNA gene-V3, and another (old) rpoB-DGGE-based primer set from a previous study to in vitro compare the bovine and ovine rumen ecosystems. The results indicate a significant (P<0.001) difference in the microbial population between the two ruminants irrespective of the primers used in the analysis. Qualitative comparison of the data provides evidence for the presence of similar phyla profiles between the 16S rRNA gene and the newly developed rpoB primers. A comparison between the two rpoB-based primer sets (old and new) showed that the old rpoB-based primers failed to amplify phylum Bacteroidetes (a common phylum in the rumen) in both bovine and ovine rumen samples. The old and new rpoB-DGGE-based primers amplified a large number of clones belonging to phylum Proteobacteria, providing a useful insight into the microbial structure of the rumen. ChaoI, ACE, Simpson, and Shannon-Weaver index analysis estimated the bovine rumen to be more diverse than the ovine rumen for all three primer sets. These results provide a new insight into the community structure among ruminants using the newly developed primers in this study.