mycotoxins overview and sampling to testingmycotoxins mycotoxins overview and sampling to testing...
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MycotoxinsMycotoxins Overview and Sampling to Overview and Sampling to
TestingTesting
John L. Richard, Ph.D.
Consultant to Romer Labs, Inc.
Webinar
August 3, 2012
Early History of Mycotoxins
Alsberg and Black, 1913
Described toxicity of mold metabolites
Resulted from studies of grain deterioration
Note: Agricultural Engineers were very much interested in this aspect of grain storageand were early experimenters with potentialproblems with fungal metabolites in grain
Chris Stackhouse Photography
Turkey “X” Disease-Blount, 1961
June 1960—large numbers turkeys dying:1. Short illness prior to death2. “Inappetent”3. Non-transmissible4. Somnolent, drooping wings5. Before death-neck arched backwards, would
“leap forward” with legs stretched backwards6. Kidneys enlarged, congested, small intestine
inflamed7. No known etiology by end of year8. 100,000 poults plus ducklings and pheasants died
Aspergillus flavus infestation and Aflatoxin Contamination of ECB Caterpillar Damaged Maize at Harvest in Iowa 1975
Aflatoxin as a Preharvest Issue
Lillehoj, et al, 1976
Biological Factors• Susceptible
Crop +• Compatible
Toxigenic Fungus
Environmental Factors• Temperature• Moisture• Mechanical Injury• Insect/Bird Damage• Fungus
Harvesting• Crop Maturity• Temperature• Moisture
Storage• Temperature• Moisture
Animals
Distribution &
ProcessingHumans
Factors Affecting Mycotoxin Occurrence in the Food ChainAnimal Products
(Adapted from Pestka & Casale 1989)
O
OO
O O
OCH3
Organisms - Aspergillus flavus, A. parasiticus, A. nomiusCommodities - Corn, Cottonseed, Peanuts, Tree NutsLD50 B1 Duckling (po), day-old 0.37 mg/kg
Rat (po), day-old 1.00 “Rat (po), 21 day-old 5.50 “
G1 Duckling (po), day-old 0.79 “M1 Ducking (po), day-old 16.60 “
Aflatoxins
Aflatoxins
Aflatoxins
Aflatoxins
Aflatoxins
Aflatoxins
Aflatoxin in Trout
Summary of FDA Levels for Aflatoxins (Total Aflatoxins or M1) in Animal Feed or Milk
Aflatoxins
COMMODITY FOR LEVEL
Corn, peanut products, cottonseed meal, and other feed and feed ingredients
Immature animals, dairy animals, animals not otherwise specified or when intended use
is not known.20 ppb
Corn and peanut products
Breeding beef cattle, breeding swine, mature poultry
100 ppb
Finishing swine of 100lbs or greater 200 ppb
Finishing (feedlot) beef cattle 300 ppb
Cottonseed meal Beef cattle, swine, poultry 300 ppb
Milk 0.5 ppb
Summary of EU Levels for Aflatoxins (B1, Total Aflatoxins or M1) in Animal Feed or Milk
Aflatoxins
COMMODITY FOR LEVEL
Cereals and processed products thereof, or cereals, with the exception of maize, to be
subjected to physical sorting or other physical treatment
Direct human consumption or as ingredient in feedstuffs
2 ppb B1; 4 ppb total
Maize to be subjected to sorting or other physical treatment
Direct human consumption or as ingredient in feedstuffs
5 ppb B1; 10 ppb total
Complete feedstuffs (with moisture content of 12%)
Dairy Cattle 5 ppb B1
Calves, lambs 10 ppb B1
Cattle, sheep, goats 50 ppb B1
Milk 0.05 ppb
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CH3
CH3O
OH
CH3
+NH3
O
OH
O
HO
O
OCH3
OH
OH
OHO
HO O
B1 : F1 = COCH2 CH(CO2 H)CH2 CO2 H; R2 = OH; R3 = HB2 : R1 = COCH2 CH(CO2 H)CH2 CO2 H; R2 = R3 = H
Organisms - Fusarium verticillioides, F. proliferatumCommodities - CornLD50 ?
Fumonisins
Fumonisin in Horse
Signs of Disease
Somnolence
Head pressing
Facial paralysis
Walking in circles
Difficulty walking
“Star gazing”
Fumonisins
OOH
O
OHCH2 OH
O
HHH
CH 3H
Organism - Fusarium graminearumCommodities - Wheat, Corn, Barley, OatsLD50 Mice (ip)=70-77 mg/kgNote: Minimum emetic dose in swine (po)=0.1 mg/kg
Deoxynivalenol
Deoxynivalenol
Deoxynivalenol
Summary of FDA Recommended Levels for Deoxynivalenol in Animal Feed
Animal or ClassIn the total ration
(ppm1)Human
ProductsFinished wheat 1
Swine & other Animals1
Grain & grain by- products
5
1excluding cattle and chickens, not to exceed 20% of diet of swine (40% for other species (this equates to 1.0 ppm in the diets of swine and 2ppm in the diets of other animals).
Deoxynivalenol
Summary of FDA Recommended Levels for Deoxynivalenol in Animal Feed
Animal or ClassIn the total ration
(ppm1)Products
Ruminating Beef, Feedlot Cattle older than 4 months & chickens; not to exceed 50% of diet
10
No Advisory level for raw wheat as the finished product is what is important!
Grain & grain by- products
Deoxynivalenol
EU Regulations for Deoxynivalenol
Deoxynivalenol
COMMODITY FOR LEVEL
Complementary and complete feedstuffs
Pigs0.9 ppm
Calves <4 months old, lambs, kids2 ppm
Animals not otherwise specified5 ppm
Cereals and cereal products intended for animal feed
Unspecified 8 ppm
Maize by-products intended for animal feed Unspecified 12 ppm
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Deoxynivalenol
Not considered a major problem in poultry but:
Is often found in poultry feed where there are problems
One of the most frequently found mycotoxins in
commodities
Is quite immunosuppressive in birds
May indicate other mycotoxins present
May be cause for reduced feed consumption
May be additive with other mycotoxins – look for them!
OH O CH3
OH
HH
O
H
H
O
Organism - Fusarium graminearum
Commodities - Corn, Barley, Wheat
LD50 ?
Zearalenone
Zearalenone
Zearalenone
ON
OOOH
H
OH O
CH3
HClOrganisms - Aspergillus ochraceus,
Penicillium verrucosumCommodities-Barley, Small Grains, Nuts, ETC.LD50 mg/kg
Rats (PO), weanling 22.00Trout (IP), 6 mo-old 3-5.53
Ochratoxin A
BarleyWheatOatsRyeGreen Coffee BeansPeasBeansPeanutsBreadSorghumOlives
BeerMixed FeedsMeatCheeseMilk powderHayRaisinsCurrentsSultanasWineGrape juice
Ochratoxin A - Occurrence
Ochratoxin
Ochratoxin
EU Regulations for Ochratoxin
Ochratoxin
Raw cereal grains 5 ug/kg
3 ug/kgAll products derived from cereals intended for direct human consumption
Dried vine fruit (currants, raisins and sultanas)
10 ug/kg
Pure Toxin
Calculated Dosages
Sufficient Numbers of Animals Given Similar Doses to Establish Significance
Administered Same Time Each Day
Usually Given As Separate Entity to Ensure Dose Administered
If Toxin Given With Food-Measure Food Intake
Characteristics:
Experimental Studies
Healthy Animals Used
Animals in Controlled Environment
Animals Fed and Watered Adequately
Ample Room-Animals Often Housed Individually
Light/Dark Cycle Controlled
Careful Measurement of Effects/Changes
Characteristics (cont.):
Experimental Studies
From U. Hofsteter, Biomin
Diagnosis of Mycotoxicoses
Mycotoxins Species Effect
Mycotoxin Combinations
AF + OA Chicks S, <A
Swine A, <A
AF + T-2 Chicks S
Swine <A
AF + DAS Chicks S
Swine A
Sheep S
AF + DON Chicks A
Swine <A
AF + FB1 Poults A
Swine S
AF + CPA Chicks <A
AF + KjA Chicks A
AF + Mon Chicks <A
OA + PCA Chicks OA
OA + CIT Chicks Antag.
OA + DON Chicks A
Mycotoxins Species EffectOA + T-2 Chicks A
Swine <A
OA + DAS Chicks <A
FB1 + DON Chicks FB1
Swine S
DON + MON Chicks <A
Poults MON
DON + T-2 Chicks A
FB1 + MON Poults MON
Swine A
Hens FB1
FB1 + T-2 Chicks <A
Poults A
FB1 + DAS Poults A
FB1 + OA Chicks A
T-2 + CPA Chicks <A
Mycotoxin Combinations
Summary
1. Interactions indicate that additive or less
than additive were predominant.
2. Synergism observed in 6/33 studies. Five
of those six involved aflatoxin.
3. Is synergism a major concern?
4. More studies are warranted.
Mycotoxicosis Diagnosis
Most of the mycotoxins cause disease in animals that
are nonpathognomonic--- can’t diagnose based on signs
or characteristics of the disease. So we look for the
mycotoxin(s).
A. Disease Manifestation
1. Observed changes in animal condition and behavior
2. Measured changes in animal condition and behavior
3. Intervention with measurement Urine, Feces, Blood - Chemistry, Microbiology, Immune Status
Criteria:
Mycotoxicosis Diagnosis
B. Postmortem Examination
1. Gross visible observations
2. Histopathologic observations
3. Chemistry of tissues and fluids
Urine, Blood, Rumen or Stomach Content, Specific Tissues
C. Examination of Feed – Testing –including Analysis
Criteria (cont.):
Mycotoxicosis Diagnosis
Mill
SampleSample
Analysis
Test Procedure
Lot
Test Result
Preparation
Lot Sample Preparation Analysis
Total Error
SamplingError
Sample Preparation
ErrorAnalytical
Error
ppb
SAMPLING ERROR
TRUCK
SHIP OR BARGE
LOT(50,000 KG
PPB=? PPB
SAMPLE2 KG
•LOT PPB = SAMPLE PPB?•PPB < LIMIT?
LETS PAUSE FOR A
SAMPLING COMMENT
REDUCING ERROR
DYNAMIC LOT
HIGH CONCENTRATIONS OF AFLATOXINS
MAXIMUM AFLATOXIN
COTTONSEED 5,000,000 PPB
PEANUT 1,000,000 PPB
CORN 400,000 PPB
Lot ( 19 ppb)
0 0 0 0 3 13 19 41 43 69
Uncertainty
SAMPLE PREPARATION ERROR
SAMPLE PREPARATION
• MILL TO GRIND SAMPLE
• REMOVE SUBSAMPLE
One seed
10,000 pieces
Mill
ANALYTICAL ERROR
Rapid
Accurate
Sensitive Rugged
Test Variance Test Variance
2 lb 266.5
Romer,50 g 56.3
TLC,1 al. 27.9
Total 350.7
HPLC,1 al. 4.6
Total 62.9Range 20 +/- 37 Range 20 +/- 16
Wiley, 50 g 5.0
10 lb 53.3
All Effects – Shelled Corn at 20 ppb
Cost / Benefits
Cost: Increase sample size
Benefits: Reduce uncertainty
• ELISA Technology–AgraQuant®
• Lateral Flow Tests–AgraStrip®
• Fluorometric assays–FluoroQuant®
Technology Overview -Rapid Testing
Analytical Service
Don´t want to analyze sample on your own?
• Union• Singapore• Tulln
-> Short Turnaround, Customized, Test report
MycotoxinsMycotoxins Overview, Sampling and Overview, Sampling and
TestingTesting
John L. Richard, Ph.D.
Consultant, Romer Labs, Inc.
Webinar
August 3, 2012Thanks for Listening