Project Number: 6227-32630-001-32
Project Type: Specific Cooperative Agreement

Start Date: Feb 15, 2008
End Date: Feb 14, 2013

Objective:
The overall goal is to develop an additive containing microbes that will degrade ergopeptide alkaloids in the rumen and thus render them non-toxic. A secondary goal is to define the role of the various alkaloids produced by endophyte-infected forages that adversely affect animal health.

Approach:
Preliminary studies show that common earthworms (Lumbricus terrestris) can survive on compost made from high ergoalkaloid tall fescue. Further investigations will examine the ability of earthworms and their anaerobic microbes to degrade ergopeptides and metabolize lysergic acid. If such are identified, total DNA will be extracted and 16s rRNA genes will be amplified by Polymerase Chain Reaction (PCR) to create a clonal library as a means of identifying bacterial species/strains. To further understand the degradation of ergovaline to lysergic acid in the rumen, the fate of radiolabeled 14C ergovaline (being prepared by Dr. Forest Smith, Auburn University) in artificial rumen experiments will be determined. Unknown metabolites will be identified by LC/MS/MS. Studies will be undertaken to evaluate changes in the rumen population (i.e., numbers of bacteria, archae, protozoa, and fungi) while animals adapt to the presence of ergovaline in the diet. Rumen microbes in sheep will be monitored during adaptation to ergovaline over two 28-day trial periods in a crossover experimental design. Ergovaline and lysergic acid levels will be measured and correlated to microbial populations. More than likely, not all of the ergot alkaloids involved in fescue toxicosis have been identified. A feeding trial will be conducted using a 4 X 4 Latin square statistical design. Fistulated steers will be fed endophyte-containing straw at four concentrations of ergovaline for 28 days. Concentrations of ergovaline, ergovalinine, ergocornine, loline, and lysergic acid will be measured in the ruminal digesta, urine, and, where possible, serum. Associations between unknown peaks in LC analysis and toxicosis will be constructed to identify possible toxins. Individual alkaloids are currently quantitated by a HPLC and fluorometric assay. A number of peaks representing possible toxins are not well defined. New LC/MS/MS chromatographic parameters will build on earlier work and negative ion chemical ionization of thermally desorbed and partially fragmented compounds will be used to separate and characterize these compounds. New molecules will be tested for vasoconstrictive properties as an indicator of involvement in toxicosis. Vasoconstriction is associated with fescue toxicosis and vasoconstriction assays have been developed. These assays will be used to compare the physiological response of veins to ergotomine, ergovaline, ergonovine, ergocyptine, lysergic acid alkaloids, and other alkaloids. Feed samples associated with outbreaks of toxicosis/staggers will be investigated to identify other alkaloids possibly associated with poor animal health. Threshold levels of ergovaline and lolines in fescue and ryegrass that adversely affect beef and dairy cattle, and sheep were initially established from feeding trial results and in clinical outbreaks of toxicosis/staggers. Concerns have arisen regarding the effects of cold environmental conditions on previously established threshold levels. New studies will be conducted in conjunction with Animal Science units that will include large numbers of animals under varying environmental condition.