Mycotoxins Overview and Sampling to Testing Webinar August 3, 2012 John L. Richard, Ph.D. Consultant to Romer Labs, Inc.
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 storage and were early experimenters with potential problems with fungal metabolites in grain Chris Stackhouse Photography
Turkey X Disease-Blount, 1961 June 1960 large numbers turkeys dying: 1. Short illness prior to death 2. Inappetent 3. Non-transmissible 4. Somnolent, drooping wings 5. Before death-neck arched backwards, would leap forward with legs stretched backwards 6. Kidneys enlarged, congested, small intestine inflamed 7. No known etiology by end of year 8. 100,000 poults plus ducklings and pheasants died
Aflatoxin as a Preharvest Issue Aspergillus flavus infestation and Aflatoxin Contamination of ECB Caterpillar Damaged Maize at Harvest in Iowa 1975 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 Humans Distribution & Processing Animal Products Animals Factors Affecting Mycotoxin Occurrence in the Food Chain (Adapted from Pestka & Casale 1989)
Aflatoxins O O O Organisms - Aspergillus flavus, A. parasiticus, A. nomius Commodities - Corn, Cottonseed, Peanuts, Tree Nuts LD 50 B 1 Duckling (po), day-old 0.37 mg/kg O O OCH 3 Rat (po), day-old 1.00 Rat (po), 21 day-old 5.50 G 1 Duckling (po), day-old 0.79 M 1 Ducking (po), day-old 16.60
Aflatoxins
Aflatoxins
Aflatoxins
Aflatoxins
Aflatoxins
Aflatoxin in Trout
Aflatoxins Summary of FDA Levels for Aflatoxins (Total Aflatoxins or M1) in Animal Feed or Milk 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 Breeding beef cattle, breeding swine, mature poultry 100 ppb Corn and peanut products 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
Aflatoxins Summary of EU Levels for Aflatoxins (B1, Total Aflatoxins or M1) in Animal Feed or Milk 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 Dairy Cattle 5 ppb B1 Complete feedstuffs (with moisture content of 12%) Calves, lambs 10 ppb B1 Cattle, sheep, goats 50 ppb B1 www.knowmycotoxins.com Milk 0.05 ppb
Fumonisins OH O OH O O OH OH CH 3 CH 3 HO CH 3 O HO O O O CH 3 OH +NH 3 B 1 : F 1 = COCH 2 CH(CO 2 H)CH 2 CO 2 H; R 2 = OH; R 3 = H B 2 : R 1 = COCH 2 CH(CO 2 H)CH 2 CO 2 H; R 2 = R 3 = H Organisms - Fusarium verticillioides, F. proliferatum Commodities - Corn LD 50?
Fumonisin in Horse Signs of Disease Somnolence Head pressing Facial paralysis Walking in circles Difficulty walking Star gazing
Fumonisins
Deoxynivalenol H O H H OH O O OH CH 2 OH CH 3 H Organism - Fusarium graminearum Commodities - Wheat, Corn, Barley, Oats LD 50 Mice (ip)=70-77 mg/kg Note: Minimum emetic dose in swine (po)=0.1 mg/kg
Deoxynivalenol
Deoxynivalenol
Deoxynivalenol Summary of FDA Recommended Levels for Deoxynivalenol in Animal Feed In the total ration Products Animal or Class (ppm 1 ) Finished wheat Human 1 Grain & grain byproducts Swine & other Animals 1 5 1 excluding 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 In the total ration Products Animal or Class (ppm 1 ) Grain & grain byproducts 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!
Deoxynivalenol EU Regulations for Deoxynivalenol COMMODITY FOR LEVEL Pigs 0.9 ppm Complementary and complete feedstuffs Calves <4 months old, lambs, kids 2 ppm Animals not otherwise specified 5 ppm Cereals and cereal products intended for animal feed Unspecified 8 ppm Maize by-products intended for animal feed Unspecified 12 ppm www.knowmycotoxins.com
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!
Zearalenone OH O CH 3 H HO H O H Organism - Fusarium graminearum Commodities - Corn, Barley, Wheat LD 50? H O
Zearalenone
Zearalenone
Ochratoxin A H O O O OH O N O H Organisms - Aspergillus ochraceus, Penicillium verrucosum Commodities-Barley, Small Grains, Nuts, ETC. LD 50 Cl H mg/kg Rats (PO), weanling 22.00 Trout (IP), 6 mo-old 3-5.53 CH 3
Ochratoxin A - Occurrence Barley Wheat Oats Rye Green Coffee Beans Peas Beans Peanuts Bread Sorghum Olives Beer Mixed Feeds Meat Cheese Milk powder Hay Raisins Currents Sultanas Wine Grape juice
Ochratoxin
Ochratoxin
Ochratoxin EU Regulations for Ochratoxin Raw cereal grains 5 ug/kg All products derived from cereals intended for direct human consumption 3 ug/kg Dried vine fruit (currants, raisins and sultanas) 10 ug/kg
Experimental Studies Characteristics: 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
Experimental Studies Characteristics (cont.): 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
From U. Hofsteter, Biomin
Diagnosis of Mycotoxicoses
Mycotoxin Combinations Mycotoxins Species Effect 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 Effect OA + 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).
Mycotoxicosis Diagnosis Criteria: 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
Mycotoxicosis Diagnosis Criteria (cont.): 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
Lot Test Procedure Sample Mill Sample Preparation Analysis Test Result
Sampling Error Total Error Sample Preparation Error Analytical Error Lot Sample Preparation Analysis ppb
SAMPLING ERROR
TRUCK SHIP OR BARGE
LOT (50,000 KG PPB=? SAMPLE 2 KG PPB 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
Rugged Sensitive Accurate Rapid
All Effects Shelled Corn at 20 ppb Test Variance Test Variance 2 lb 266.5 Romer,50 g 56.3 TLC,1 al. 27.9 10 lb 53.3 Wiley, 50 g 5.0 HPLC,1 al. 4.6 Total 350.7 Total 62.9 Range 20 +/- 37 Range 20 +/- 16
Cost / Benefits Cost: Increase sample size Benefits: Reduce uncertainty
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Mycotoxins Overview, Sampling and Testing Thanks Webinar for Listening August 3, 2012 John L. Richard, Ph.D. Consultant, Romer Labs, Inc.