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

www.knowmycotoxins.com

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®

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MycotoxinsMycotoxins Overview, Sampling and Overview, Sampling and

TestingTesting

John L. Richard, Ph.D.

Consultant, Romer Labs, Inc.

Webinar

August 3, 2012Thanks for Listening