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Lead by A. Morrie Craig and Anita Holman

Service ~ Endophyte Testing Laboratory. Lead by A. Morrie Craig and Anita Holman. History of the Oregon Seed & Straw Industries. 1987-1991: Field burning of grass straw phase-out completed 250,000 acres down to 40,000 acres. History of the Oregon Seed & Straw Industries.

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Lead by A. Morrie Craig and Anita Holman

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  1. Service ~ Endophyte Testing Laboratory Lead by A. Morrie Craig and Anita Holman

  2. History of the Oregon Seed & Straw Industries • 1987-1991: Field burning of grass straw phase-out completed • 250,000 acres down to 40,000 acres

  3. History of the Oregon Seed & Straw Industries • Exportation of straw begins to increase • Pollution control tax credit from OR legislature • Export of grass straw to Japan grows to 250,000 tons/year in 1991 • Seed companies intensify use of endophyte since it lessens the need for pesticides, fertilizers and water • Pushes up concentration of ergot alkaloids in straw, creating the need for clinical-related research and testing and research for a long term, proactive solution

  4. History of the Oregon Seed & Straw Industries • 1991-1999 • Grass seed export increases over 200% (250,000 to 500,000 tons/year) • Represents 250,000 acres/year that are no longer burned • 1996: Oregon Legislature votes on money to be directed towards endophyte research at OSU

  5. Crisis in Japan • 1998: shipments to Japan were shut down by Japanese gov’t d.t. fear of high level of endophyte toxins • Dr. Craig, seed industry and National Hay Association established the endophyte testing program and reaffirmed commitment to finding a long term solution • Reopened export to Japan • 1999-present: funding from the OR Ag Fiber Assn, state of Oregon and USDA was granted to study practical and clinical aspects of endophyte-infected forages and their effects on livestock • 2002: export increases to over 600,000 tons/year

  6. Number of Test Requests Increasing ↑ Crisis in Japan

  7. Tall fescue ~ Ergovaline HPLC Analysis Perennial ryegrass ~Lolitrem B HPLC Analysis

  8. Threshold Levels for Clinical Disease (Tall fescue) *Except for mares in the last 60 to 90 days of pregnancy, when the threshold is zero.

  9. Threshold Levels for Clinical Disease (Perennial ryegrass) *Except for mares in the last 60 to 90 days of pregnancy, when the threshold is zero.

  10. Sample Analysis Grind → Extract → Sample Prep HPLC → Chromatogram → Analysis and Data Reduction

  11. Analysis of Endophyte Toxins • Log in samples • Accession number and submission form • Enter into computer log and case records

  12. Analysis of Endophyte Toxins • Grind Samples • Use a grinding mill with 0.5 mm screen • Clean between each sample • Store in freezer until analysis

  13. Analysis of Endophyte Toxins • Schedule run • Priority • Label glassware • Weigh out run • Duplicate • Samples • Controls • Reference material • 1.00g ergovaline, 0.200g lolitrem B

  14. Analysis of Endophyte Toxins • Add mixing solutions • Ergovaline: 10 ml CHCl3, 1 ml internal standard (ergotamine tartrate) and 1 ml 0.001 N NaOH • Lolitrem B: CHCl3:MeOH (2:1 v/v) • Cap

  15. Analysis of Endophyte Toxins • Turn in rotorack for 18-24 hours

  16. Analysis of Endophyte Toxins • Centrifuge samples • Pull out supernatant

  17. Analysis of Endophyte Toxins • Extraction • Ergovaline: manifold with SPE (1.0g silica and 0.5g sodium sulfate) • Condition with CHCl3 • Add 5 ml supernatant • Wash twice with 1 ml 75% CHCl3/25% acetone • Wash with 1.5 ml MeOH • Elute with 2.5 ml MeOH

  18. Analysis of Endophyte Toxins • Concentrate • Ergovaline: dry eluent in N2 evaporator at 50°C • Lolitrem B: dry 1.8 ml supernatant in N2 evaporator • Reconstitute • Ergovaline: 0.5 ml MeOH • Lolitrem B: 0.5 ml CHCl3:ACN (4:1 v/v) • Vortex and sonicate • Final “filter” • Ergovaline: centrifuge • Lolitrem B: centrifuge in Nanosep microcentrifuge tubes • Add this solution to amber HPLC vials

  19. Analysis of Endophyte Toxins • Run samples on HPLC system • Program computer and auto sampler • Create sequence and load method • Make mobile phase • Warm up system and prime pump • Load vials into auto sampler • Start injections

  20. Analysis of Endophyte Toxins • Evaluate chromatograms • Draw baselines • Look for ‘abnormal’ fingerprint • Quantify

  21. Analysis of Endophyte Toxins • Enter results into log file • Generate client summary and certificates • Fax of phone results • Send results and billing information • File lab copy of results • Transfer completed samples to long term storage freezer Sample

  22. Quality Control Program for the Tall Fescue and Perennial Ryegrass Toxins, Ergovaline and Lolitrem B Oregon State University College of Veterinary Medicine Dr. Craig’s Laboratory

  23. GOAL Accurate and consistent results from the analysis of samples for ergovaline and lolitrem B between laboratories in Oregon, Missouri and Japan

  24. Make the Perfect Standard

  25. NMR of Ergovaline Synthesized by Dr. Forrest Smith in December DMSO H2O

  26. CHN Analysis of December Sample Expected Found C 57.22% 57.02% H 6.11% 6.33% N 10.11% 10.04% 2 Ergovaline + 2 Tartaric acid + H2O

  27. Ergovaline Crystalline StandardOSU Method Ergovaline 5.6 µg/ml Ergotamine Ergotamine epimer Ergovaline epimer 2.8 µg/ml 1.12 µg/ml 0.28 µg/ml

  28. New Quality Control Reference Material for Interlaboratory Analysis • Zero Seed • 300 ppb seed • 3300 ppb seed

  29. Dairy Case Studies Winter 2003-2004 Chuck Estill, Shanna Ivey, Anita Holman, Jennifer Duringer, A. Morrie Craig

  30. The Problem • Reproductive problems at four dairies in the Willamette Valley in fall-winter 2003 • cows not coming into heat as frequently • silent heats • decreased conception rates

  31. Causes • Viral or bacterial • Dr’s Estill and Holcheck involved → exams showed no sign of pathogen involvement • Mycotoxin • screened for two dairies → negative • Something else • same innoculum used for ensiling process by all four dairies • “new” toxin…

  32. Causes con’t • Endophyte • prolactin: no obvious trend • ergovaline and lolitrem B: low levels in hay and silage • new peak: RT = 5 minutes in EV assay • only in silage, not hay from same field • another ergot alkaloid?

  33. Chromatograms from Four Dairies

  34. HPLC of Collected Peak

  35. MS of 5 Minute Peak

  36. mw = 60 C=12, O=16, H=1 Ergovaline mw = 533.63 Molecular formula = C29H35N5O5 MS/MS (m/z 593) of 5 Minute Peak

  37. Taking it one step further…MS7 of 5-minute peak m/z 533 5-minute peak m/z 533

  38. Where do we go from here? • Identify the molecule • Nuclear Magnetic Resonance Spectroscopy • How many C and H? • C,H, & N Analysis • What percent is the molecule composed of C,H, & N? • Post-incident (2004…) • Reproduction problems abated • 5 minute peak has decreased in some cases, others it is still there

  39. Questions?

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