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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

Service ~ Endophyte

Testing Laboratory

Lead by A. Morrie Craig

and Anita Holman

history of the oregon seed straw industries
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 industries3
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
history of the oregon seed straw industries4
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
crisis in japan
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
Tall fescue ~ Ergovaline HPLC Analysis

Perennial ryegrass ~Lolitrem B HPLC Analysis

threshold levels for clinical disease tall fescue
Threshold Levels for Clinical Disease (Tall fescue)

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

when the threshold is zero.

threshold levels for clinical disease perennial ryegrass
Threshold Levels for Clinical Disease (Perennial ryegrass)

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

when the threshold is zero.

sample analysis
Sample Analysis

Grind → Extract → Sample Prep

HPLC → Chromatogram → Analysis and Data Reduction

analysis of endophyte toxins
Analysis of Endophyte Toxins
  • Log in samples
    • Accession number and submission form
    • Enter into computer log and case records
analysis of endophyte toxins12
Analysis of Endophyte Toxins
  • Grind Samples
    • Use a grinding mill with 0.5 mm screen
    • Clean between each sample
    • Store in freezer until analysis
analysis of endophyte toxins13
Analysis of Endophyte Toxins
  • Schedule run
    • Priority
  • Label glassware
  • Weigh out run
    • Duplicate
      • Samples
      • Controls
      • Reference material
    • 1.00g ergovaline, 0.200g lolitrem B
analysis of endophyte toxins14
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
analysis of endophyte toxins15
Analysis of Endophyte Toxins
  • Turn in rotorack for 18-24 hours
analysis of endophyte toxins16
Analysis of Endophyte Toxins
  • Centrifuge samples
  • Pull out supernatant
analysis of endophyte toxins17
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
analysis of endophyte toxins18
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

analysis of endophyte toxins19
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
analysis of endophyte toxins20
Analysis of Endophyte Toxins
  • Evaluate chromatograms
    • Draw baselines
    • Look for ‘abnormal’ fingerprint
    • Quantify
analysis of endophyte toxins21
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


quality control program for the tall fescue and perennial ryegrass toxins ergovaline and lolitrem b
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



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

chn analysis of december sample
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

ergovaline crystalline standard osu method
Ergovaline Crystalline StandardOSU Method


5.6 µg/ml


Ergotamine epimer

Ergovaline epimer

2.8 µg/ml

1.12 µg/ml

0.28 µg/ml

new quality control reference material for interlaboratory analysis
New Quality Control Reference Material for Interlaboratory Analysis
  • Zero Seed
  • 300 ppb seed
  • 3300 ppb seed
chuck estill shanna ivey anita holman jennifer duringer a morrie craig

Dairy Case Studies

Winter 2003-2004

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

the problem
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
  • 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…
causes con t
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?
ms ms m z 593 of 5 minute peak

mw = 60

C=12, O=16, H=1


mw = 533.63

Molecular formula = C29H35N5O5

MS/MS (m/z 593) of 5 Minute Peak
where do we go from here
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