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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 l.jpg

Service ~ Endophyte

Testing Laboratory

Lead by A. Morrie Craig

and Anita Holman


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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


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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


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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


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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



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Tall fescue ~ Ergovaline HPLC Analysis

Perennial ryegrass ~Lolitrem B HPLC Analysis


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Threshold Levels for Clinical Disease (Tall fescue)

*Except for mares in the last 60 to 90 days of pregnancy,

when the threshold is zero.


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Threshold Levels for Clinical Disease (Perennial ryegrass)

*Except for mares in the last 60 to 90 days of pregnancy,

when the threshold is zero.


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Sample Analysis

Grind → Extract → Sample Prep

HPLC → Chromatogram → Analysis and Data Reduction


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Analysis of Endophyte Toxins

  • Log in samples

    • Accession number and submission form

    • Enter into computer log and case records


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Analysis of Endophyte Toxins

  • Grind Samples

    • Use a grinding mill with 0.5 mm screen

    • Clean between each sample

    • Store in freezer until analysis


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Analysis of Endophyte Toxins

  • Schedule run

    • Priority

  • Label glassware

  • Weigh out run

    • Duplicate

      • Samples

      • Controls

      • Reference material

    • 1.00g ergovaline, 0.200g lolitrem B


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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


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Analysis of Endophyte Toxins

  • Turn in rotorack for 18-24 hours


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Analysis of Endophyte Toxins

  • Centrifuge samples

  • Pull out supernatant


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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


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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


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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


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Analysis of Endophyte Toxins

  • Evaluate chromatograms

    • Draw baselines

    • Look for ‘abnormal’ fingerprint

    • Quantify


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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


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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


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GOAL Ryegrass Toxins, Ergovaline and Lolitrem B

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


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Make the Perfect Standard Ryegrass Toxins, Ergovaline and Lolitrem B


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NMR of Ergovaline Synthesized by Ryegrass Toxins, Ergovaline and Lolitrem B Dr. Forrest Smith in December

DMSO

H2O


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CHN Analysis of December Sample Ryegrass Toxins, Ergovaline and Lolitrem B

Expected Found

C 57.22% 57.02%

H 6.11% 6.33%

N 10.11% 10.04%

2 Ergovaline + 2 Tartaric acid + H2O


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Ergovaline Crystalline Standard Ryegrass Toxins, Ergovaline and Lolitrem B OSU Method

Ergovaline

5.6 µg/ml

Ergotamine

Ergotamine epimer

Ergovaline epimer

2.8 µg/ml

1.12 µg/ml

0.28 µg/ml


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New Quality Control Reference Material for Interlaboratory Analysis

  • Zero Seed

  • 300 ppb seed

  • 3300 ppb seed


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Dairy Case Studies Analysis

Winter 2003-2004

Chuck Estill, Shanna Ivey, Anita Holman, Jennifer Duringer, A. Morrie Craig


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The Problem Analysis

  • 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


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Causes Analysis

  • 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…


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Causes con’t Analysis

  • 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?





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mw = 60 Analysis

C=12, O=16, H=1

Ergovaline

mw = 533.63

Molecular formula = C29H35N5O5

MS/MS (m/z 593) of 5 Minute Peak


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Taking it one step further…MS Analysis7 of 5-minute peak m/z 533

5-minute peak m/z 533


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Where do we go from here? Analysis

  • 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


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Questions? Analysis


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