Mycotoxins Overview and Sampling to Testing

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1 Mycotoxins Overview and Sampling to Testing Webinar August 3, 2012 John L. Richard, Ph.D. Consultant to Romer Labs, Inc.

2 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

3 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 ,000 poults plus ducklings and pheasants died

4 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

5 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)

6 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

7 Aflatoxins

8 Aflatoxins

9 Aflatoxins

10 Aflatoxins

11

12 Aflatoxins

13 Aflatoxin in Trout

14 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

15 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 Milk 0.05 ppb

16 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?

17 Fumonisin in Horse Signs of Disease Somnolence Head pressing Facial paralysis Walking in circles Difficulty walking Star gazing

18 Fumonisins

19 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

20 Deoxynivalenol

21 Deoxynivalenol

22 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 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).

23 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!

24 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

25 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!

26 Zearalenone OH O CH 3 H HO H O H Organism - Fusarium graminearum Commodities - Corn, Barley, Wheat LD 50? H O

27 Zearalenone

28 Zearalenone

29 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 Trout (IP), 6 mo-old CH 3

30 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

31 Ochratoxin

32 Ochratoxin

33 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

34 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

35 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

36 From U. Hofsteter, Biomin

37 Diagnosis of Mycotoxicoses

38 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

39 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.

40 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).

41 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

42 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

43 Lot Test Procedure Sample Mill Sample Preparation Analysis Test Result

44 Sampling Error Total Error Sample Preparation Error Analytical Error Lot Sample Preparation Analysis ppb

45 SAMPLING ERROR

46

47

48 TRUCK SHIP OR BARGE

49 LOT (50,000 KG PPB=? SAMPLE 2 KG PPB LOT PPB = SAMPLE PPB? PPB < LIMIT?

50 LETS PAUSE FOR A SAMPLING COMMENT

51 REDUCING ERROR

52

53

54 DYNAMIC LOT

55 HIGH CONCENTRATIONS OF AFLATOXINS

56 MAXIMUM AFLATOXIN COTTONSEED 5,000,000 PPB PEANUT 1,000,000 PPB CORN 400,000 PPB

57 Lot ( 19 ppb) Uncertainty

58 SAMPLE PREPARATION ERROR

59 SAMPLE PREPARATION MILL TO GRIND SAMPLE REMOVE SUBSAMPLE

60 One seed 10,000 pieces Mill

61

62 ANALYTICAL ERROR

63 Rugged Sensitive Accurate Rapid

64 All Effects Shelled Corn at 20 ppb Test Variance Test Variance 2 lb Romer,50 g 56.3 TLC,1 al lb 53.3 Wiley, 50 g 5.0 HPLC,1 al. 4.6 Total Total 62.9 Range 20 +/- 37 Range 20 +/- 16

65 Cost / Benefits Cost: Increase sample size Benefits: Reduce uncertainty

66 Technology Overview - Rapid Testing ELISA Technology AgraQuant Lateral Flow Tests AgraStrip Fluorometric assays FluoroQuant

67 Analytical Service Don t want to analyze sample on your own? Union Singapore Tulln -> Short Turnaround, Customized, Test report

68 Mycotoxins Overview, Sampling and Testing Thanks Webinar for Listening August 3, 2012 John L. Richard, Ph.D. Consultant, Romer Labs, Inc.

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