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Fungal toxicosis in the mink (Mustela vison) McMillan, Kenneth Ronald 1965

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FUNGAL TOXICOSIS IN THE MINK (MUSTELA VISON) by KENNETH RONALD Mc MILLAN B . S c , U n i v e r s i t y of B r i t i s h C o l u m b i a , 1961 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e DIVISION OF ANIMAL SCIENCE We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA June, 1965 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of • Br i t i sh Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that per-mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that,copying or publi-cation of this thesis for financial gain shall not be allowed without my written permission*. Department of ANIMAL SCIENCE The University of Brit ish Columbia, Vancouver 8 5 Canada Date J u n e 18> 1 9 6 5 ABSTRACT The spontaneous occurrence of a l i v e r disease i n mink (Mustela vison) prompted an i n v e s t i g a t i o n of the animal's environment i n the search for a possible a e t i o l o g i c a l f a c t o r . The pathological findings suggested that the disease was diet-induced and po s s i b l y r e s u l t e d from the presence of a toxic factor i n the feed. A series of experiments were c a r r i e d out to asce r t a i n i f the con-d i t i o n arose through the action of a transmissible i n f e c t i v e agent or from an inadequate supply of some e s s e n t i a l n u t r i e n t . No evidence was obtained in support of e i t h e r of these p o s s i b i l i t i e s . Since the pathology noted was suggestive of a hepatotoxic factor or factors and was unlike any of the described i n t o x i c a t i o n s a r i s i n g from b a c t e r i a l metabolites, major attention was given to a study of the possible r o l e of the fungi as causative agents. Some fungi, when given the necessary conditions of temperature, moisture and substrate, elaborate toxins that are poisonous to a large number of species. The p o s s i b i l i t y that a fungal t o x i n was the cause of the disease i n mink was therefore investigated. A l i t e r a t u r e review covering the mycotoxicoses has been compiled. In order to test the s u s c e p t i b i l i t y of the mink to fungal t o x i c o s i s , a model was set up i n which mink were fed a r a t i o n containing a known fungal toxin, a f l a t o x i n . The mink proved hi g h l y susceptible to t h i s toxin. The pathology of a f l a t o x i c o s i s i n the mink has been documented. The fungal population of the mink r a t i o n and i t s cereal ingredients was examined f o r toxin-producing fungi. A series of t o x i c i t y assays were conducted to screen the large number of organisms i s o l a t e d from mink r a t i o n s . : Toxigenic f u n g i were i s o l a t e d from: wheat used i n mink r a t i o n i i formulations. The r o l e of cottonseed o i l as an agent contributing to t h i s l i v e r condition has been investigated. I t i s suggested that cottonseed o i l either contains a toxic component or contains a s p e c i f i c f a c t o r which can be used by a fungus i n some phase of i t s metabolism, and that the by-product of t h i s metabolic pathway may accumulate as a toxic compound. i i i TABLE OF CONTENTS PAGE i i i v i i v: 'ix :x x i i i INTRODUCTION 1 PART I AN HEPATO-HAEMANGIOMA-SYNDROME IN MINK I n t r o d u c t i o n 2 O c c u r r e n c e 2 A e t i o l o g y 4 C l i n i c a l Appearance 4 Gross P a t h o l o g y 5 H i s t o p a t h o l o g y 6 D i s c u s s i o n and Summary 9 PART I I LITERATURE REVIEW - THE MYCOTOXICOSES I n t r o d u c t i o n 13 A l i m e n t a r y T o x i c A l e u k i a 18 1. D i s e a s e 18 2. C l i n i c a l Symptoms 18 3. P a t h o l o g y 21 4. A e t i o l o g y 22 5. I s o l a t i o n and C h a r a c t e r i z a t i o n o f T o x i n 23 6. C h e m i c a l and B i o l o g i c a l C h a r a c t e r i s t i c s o f T o x i n 23 7. S i g n i f i c a n c e i n Human and A n i m a l N u t r i t i o n and D i s e a s e 25 8. Treatment and C o n t r o l . 26 S t a c h y b o t r y o t o x i c o s i s 27 1. D i s e a s e 27 2. C l i n i c a l Symptoms 28 3. P a t h o l o g y 30 4. A e t i o l o g y 30 5. I s o l a t i o n and C h a r a c t e r i z a t i o n o f T o x i n 31 6. S i g n i f i c a n c e i n Human and A n i m a l N u t r i t i o n and D i s e a s e 33 7. Treatment and C o n t r o l 35 ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF PLATES ACKNOWLEDGEMENTS PAGE • Yellowed Rice Disease 36 1. Disease 36 2. C l i n i c a l Symptoms 37 3. Pathology 38 4. Aetiology 39 5. I s o l a t i o n and Characterization of Toxin 40 6. Chemical and B i o l o g i c a l C h a r a c t e r i s t i c s of Toxin 40 7. Signific a n e i n Human and Animal N u t r i t i o n and Disease 44 Af l a t o x i c o s i s 45 1. Disease 45 2. C l i n i c a l Symptoms 46 3. Pathology 47 4. Aetiology 49 5. I s o l a t i o n and Characterization of Toxin 50 6. Chemical and B i o l o g i c a l C h a r a c t e r i s t i c s of Toxin 57 7. Experimental A f l a t o x i c o s i s i n Animals 62 8. Sign i f i c a n c e i n Human and Animal N u t r i t i o n and Disease 64 F a c i a l Eczema 66 1. Disease 66 2. C l i n i c a l Symptoms 67 3. Aetiology 68 4. Pathology 68 5. I s o l a t i o n and Characterization of Toxin 70 6. Chemical and B i o l o g i c a l C h a r a c t e r i s t i c s of Toxin 72 7. Experimental Sporidesmin Intoxication in Animals 74 8. Mode of Action of Toxin 75 9. Treatment and Control 76 Mycotoxicoses General 76 A s p e r g i l l u s t o x i c o s i s 76 Hyperkeratosis i n C a t t l e 80 Mouldy Corn Toxicosis 81 Poultry Haemorrhagic Syndrome 82 Mould Control i n Stored Grain 84 Bibliography 87 PAGE PART I I I EXPERIMENTAL AFLATOXICOSIS IN MINK Introduction Experimental A. Source of A f l a t o x i n B. Evaluation of Toxic Peanut Meal by Duckling Assay Duckling Assay No. 1 (1) Preparation of Extract (2) Animals and Treatment (3) Results Duckling Assay No. 2 (1) Rations (2) Animals and Treatment (3) Results Duckling Assay No. 3 (1) Rations (2) Animals and Treatment (3) Results C. A f l a t o x i c o s i s i n the Mink 105 Mink Assay No. 1 105 (1) Rations 105 (2) Animals and Treatment 106 (3) Results 106 Mink Assay No. 2 107 (1) Rations 107 (2) Animals and Treatment 108 (3) Results 108 D. Gross Pathology 110 E. Histopathology 115 Discussion and Summary 121 Bibliography 125 PART IV FUNGAL TOXICOSIS STUDIES I Introduction 126 Experimental 127 A. I s o l a t i o n of Fungi 127 B. Evaluation of T o x i c i t y of Fungi by Bio-Assay 127 98 98 98 99 99 99 99 99 99 99 101 101 101 101 101 105 Rabbit Dermal Assay 127 PAGE (1) Preparation of Extracts 127 (2) Animals and Treatment 128 (3) Results 128 Duckling Assay No. 4 130 (1) Preparation of Extracts 130 (2) Animals and Treatment 131 (3) Results 132 Duckling Assay No. 5 135 (1) Preparation of Extracts 136 (2) Animals and Treatment 136 (3) Results 138 Chick Embryo L e t h a l i t y Assays 139 Embryo L e t h a l i t y Assay No. 1 139 (1) Preparation of Extracts 139 (2) Animals and Treatment 139 (3) Results 139 Embryo L e t h a l i t y Assay No. 2 142 (1) Preparation of Extracts 142 (2) Animals and Treatment 142 (3) Results 142 Embryo L e t h a l i t y Assay No. 3 144 (1) Preparation of Extracts 144 (2) Animals and Treatment 144 (3) Results 144 IV 147 BibliographyPART V FUNGAL TOXICOSIS STUDIES II Introduction 148 Experimental 149 (1) Rations 149 (2) Animals and Treatment 150 (3) Results 150 Bibliography 157 GENERAL SUMMARY AND DISCUSSION 158 APPENDIX 161 vii TABLE LIST OF TABLES PAGE I . E x p e r i m e n t a l B a s a l R a t i o n f o r Mink 3 I I . E f f e c t o f I n c u b a t i o n P e r i o d of S t r a w I n o c u l a t e d w i t h S. a t r a and Dermal T o x i c i t y of E x t r a c t s 32 I I I . I n f l u e n c e o f Time of E x t r a c t i o n o f S. a t r a I n o c u l a t e d S t r a w on Removal o f D e r m a l l y T o x i c S u b s t a n c e s 32 IV. C h e m i c a l C h a r a c t e r i s t i c s o f Four Components o f A f l a t o x i n 53 V. P h y s i c o - C h e m i c a l C h a r a c t e r i s t i c s o f Four Components of A f l a t o x i n 54 V I . C h a r a c t e r i s t i c s o f L i v e r Damage i n D u c k l i n g s as r e p o r t e d by t h e I n t e r d e p a r t m e n t a l Working P a r t y on Groundnut T o x i c i t y R e s e a r c h (1962) 58 V I I . C o r r e l a t i o n o f R e s u l t s O b t a i n e d by Two Methods of A f l a t o x i n A s s a y 60 V I I I . D i s t i n g u i s h i n g C h a r a c t e r i s t i c s of F u m i g a t i n and F u m i g a c i n 78 IX. Dosage and M o r t a l i t y S c h e d u l e of D u c k l i n g s A s s a y No. 1 100 X. M o r t a l i t y S c h e d u l e o f D u c k l i n g s A s s a y No. 2 102 X I . Mean Body Weights o f S u r v i v i n g D u c k l i n g s . A s s a y No. 2 103 X I I . M o r t a l i t y S c h e d u l e o f D u c k l i n g s A s s a y No. 3 104 X I I I . Mink R a t i o n A s s a y No. 1 106 XIV. M o r t a l i t y P a t t e r n f o r Mink A s s a y No. 1 106 XV. Mink R a t i o n A s s a y No. 2 108 XVI. Mean Body Weights and S u r v i v a l P a t t e r n f o r Mink A s s a y No. 2 109 X V I I . Dosage S c h e d u l e o f D u c k l i n g s A s s a y No. 4 131 X V I I I . M o r t a l i t y S c h e d u l e o f D u c k l i n g s Assay No. 4 132 XIX. Mean Body Weight R e c o r d o f S u r v i v i n g D u c k l i n g s As s ay No. 4 133 XX. Gross P a t h o l o g y o f D u c k l i n g s A s s a y No. 4 134 XX I . M o r t a l i t y S c h e d u l e o f D u c k l i n g s A s s a y No. 5 137 v i i l TABLE PAGE X X I I . Embryo M o r t a l i t y D a t a A s s a y No. 1 140 X X I I I . Embryo M o r t a l i t y D a t a A s s a y No. 2 143 XXIV. Embryo M o r t a l i t y ' D a t a ;Assay|No. 3 145 XXV. Ranch-Type Mink R a t i o n 148 XXVI. E x p e r i m e n t a l Mink R a t i o n 149 X X V I I . Mean Body Weight of E x p e r i m e n t a l Mink 151 X X V I I I . D a i l y Feed I n t a k e (Grams) o f R e p r e s e n t a t i v e Mink on E x p e r i m e n t a l R a t i o n s 152 XXIX. Body Weight Data on E x p e r i m e n t a l Mink 154 XXX. D i g e s t i b i l i t y V a l u e s o f Mink R a t i o n F a t and P r o t e i n 155 XXXI. D e t e r m i n a t i o n of t h e E r y t h r o c y t e , L e u c o c y t e and H e m a t o c r i t V a l u e s of Mink 156 X X X I I . Mean D i f f e r e n t i a l Counts o f Mink 156 App end i x A. Mink C e r e a l G r a i n P e l l e t s 161 B. C o m p o s i t i o n on U.B.C. Mink C e r e a l R a t i o n 144-63 162 C. Composit i o n o f Czapek-Dox Agar Medium (Dehydrated) 162 D. C o m p o s i t i o n o f Duck Grower R a t i o n 163 i x : FIGURE LIST OF FIGURES PAGE 1. Chemical F r a c t i o n a t i o n from the Fungus Mat of P. islandicum Sopp A l 2. Chemical F r a c t i o n a t i o n from the F i l t r a t e of P. Islandicum Sopp 42 3. Procedure f o r the E x t r a c t i o n of A f l a t o x i n from Groundnut Meal 51 4. S t r u c t u r e s of A f l a t o x i n and B2 55 5. S t u r c t u r e s of A f l a t o x i n and G2 56 6. F r a c t i o n a t i o n of Sporidesmin 71 7. S t r u c t u r e of 'Luteoskyrin 73 PLATE NO. LIST OF PLATES Hepato-Haemangioma-Syndrome i n Mink 1. T y p i c a l HHS mink l i v e r 2. HHS mink l i v e r and m e s e n t e r y 3. Haemangiomatous mink l i v e r 4. HHS mink - p e r i t o n e u m w i t h haemorrhage 5. HHS mink - l i v e r , s p l e e n and m e s e n t e r i c f a t 6. G a s t r i c u l c e r s i n HHS mink 7. HHS mink showing a s c i t i c c o n d i t i o n 8. HHS mink l i v e r : G e n e r a l v i e w 9. HHS mink l i v e r : B l o o d v e s s e l ( v e i n ) w i t h b i l e d u c t s p i r a l l i n g 10. HHS mink l i v e r : Network o f b l o o d c y s t s 11. HHS mink l i v e r : S u b c a p s u l a r a r e a w i t h f i b r o u s - w a l l e d b l o o d c y s 12. HHS mink l i v e r : J u n c t i o n between c y s t s and e n d o t h e l i a l l i n i n g 13. HHS mink l i v e r : Edge o f b l o o d c y s t d i f f u s i n g i n t o s i n u s o i d s e n d o t h e l i a l l i n i n g 14a HHS mink l i v e r : P o r t a l t r i n i t y w i t h c e l l i n f i l t r a t i o n 14b HHS mink l i v e r : T r i n i t y w i t h c e l l i n f i l t r a t i o n 15. HHS mink l i v e r : Severe r e c e n t f i b r o s i s 16. HHS mink l i v e r : C i r r h o t i c a r e a w i t h n e o p l a s t i c c e l l mass 17. HHS mink l i v e r : C l o s e up of n e o p l a s t i c mass 18. HHS mink s p l e e n : Red p u l p w i t h i n c r e a s e d m e g a k a r y o c y t e s 19. HHS mink lymph node: Extreme oedema xi PLATE NO. LIST OF PLATES E x p e r i m e n t a l A f l a t o x i c o s i s i n Mink (FTS) 1. E u t h a n a s i a i n e x t r e m i s . Fed 2.5 p e r c e n t t o x i c m e a l . P e r i t o n e a l a s c i t e s . 2. Spontaneous d e a t h . Fed 10 p e r c e n t t o x i c m e a l . I n f l a m m a t i o n o f m e s e n t e r y . 3. Spontaneous d e a t h . Fed 5 p e r c e n t t o x i c m e a l . C a t a b o l i s m o r s o f t e n i n g o f m e s e n t e r i c f a t . 4. Spontaneous d e a t h . Fed 10 p e r c e n t t o x i c m e a l . C o n g e s t i o n and p e t e c h i a l haemorrhages of t h e p e r i c a r d i u m . 5. Spontaneous d e a t h . Fed 10 p e r c e n t t o x i c meal ( l e f t ) c l o s e up ( r i g h t ) and c o n t r o l mink ( c e n t r e ) . 6. Spontaneous d e a t h . Fed 10 p e r c e n t t o x i c m e a l . E c c h y m o t i c or s u f f u s i v e s u b c a p s u l a r haemorrhage i n t h e l i v e r . 7. Spontaneous d e a t h . Fed 2.5 p e r c e n t t o x i c meal.-Y e l l o w n o d u l a r l i v e r . 8. Spontaneous d e a t h . Fed 2.5 p e r c e n t t o x i c m e a l . L i v e r w i t h s u r f a c e n o d u l e s o f c o n g e s t i v e or ha e m o r r h a g i c t i s s u e . 9. Spontaneous d e a t h . Fed 1.25 p e r c e n t t o x i c m e a l . L i v e r w i t h s u r f a c e r e g e n e r a t i v e n o d u l e s . 10. E u t h a n a s i a . Fed 0.625 p e r c e n t t o x i c m e a l . L i v e r o f normal c o l o u r w i t h m i l i a r y f o c i o f n e c r o s i s . 11. Spontaneous d e a t h . Fed 2.5 p e r c e n t t o x i c m e a l . L i v e r p h o t o m i c r o g r a p h w i t h two haemorrhages. 12. Spontaneous d e a t h . Fed 1.25 p e r c e n t t o x i c m e a l . L i v e r w i t h b i l e d u c t p r o l i f e r a t i o n and r e g e n e r a t i v e n o d u l e s -13. Spontaneous d e a t h . Fed 5 p e r c e n t t o x i c m e a l . L i v e r w i t h b i l e d u c t c a s t s and i n f i l t r a t e d c e l l s . 14. E u t h a n a s i a . Fed 0.625 p e r c e n t t o x i c m e a l . L i v e r w i t h i n f i l t r a t e d c e l l s around b i l e d u c t s and p o r t a l v e i n s . PLATE NO. 15. E u t h a n a s i a . Fed 0.625 p e r c e n t t o x i c m e a l . I n f i l t r a t i o n o f c e l l s around b i l e d u c t s and p o r t a l v e i n . 16. Spontaneous d e a t h . Fed 5 p e r c e n t t o x i c m e a l . L i v e r w i t h f a t t y metamorphosis and n e c r o s i s . 17. E u t h a n a s i a . Fed 0.625 p e r c e n t t o x i c m e a l . L i v e r showing f o c a l n e c r o s i s . 18. Spontaneous d e a t h . Fed 2.5 p e r c e n t t o x i c m e a l . L i v e r w i t h b i l e d u c t p r o l i f e r a t i o n and v a c u o l a t i o n of parenchyma. 19. Spontaneous d e a t h . Fed 2.5 p e r c e n t t o x i c m e a l . L i v e r w i t h r e g e n e r a t i v e n o d u l e s . 20. Spontaneous d e a t h . Fed 5 p e r c e n t t o x i c m e a l . K i d n e y , g l o m e r u l u s w i t h r e d c e l l c a s t s i n Bowman's space. 21. E u t h a n a s i a . Fed 1.25 p e r c e n t t o x i c m e a l . K i d n e y w i t h c o r t i c a l c o n g e s t i o n . 22. E u t h a n a s i a . Fed 0.625 p e r c e n t t o x i c m e a l . K i d n e y c o r t e x , d i m i n u t i o n o f Bowman's space. 23. S u p e r f i c i a l s i m i l a r i t i e s i n l i v e r s o f HHS and FTS ( l e f t ) mink. x i i i ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o thank Dr. B.A. E a g l e s , Dean o f t h e F a c u l t y o f A g r i c u l t u r e and c h a i r m a n o f t h e D i v i s i o n o f A n i m a l S c i e n c e f o r h i s p e r m i s s i o n t o u n d e r t a k e t h i s s t u d y and f o r t h e use of d e p a r t m e n t a l f a c i l i t i e s . The a u t h o r i s g r a t e f u l t o Dr. A . J . Wood, P r o f e s s o r of A n i m a l S c i e n c e , f o r h i s g u i d a n c e , i n t e r e s t and u n f a i l i n g encouragement t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y . S i n c e r e t h a n k s a r e due t o Dr. P.L. S t o v e l l , Head of t h e A n i m a l P a t h o l o g y L a b o r a t o r y , F e d e r a l Department o f A g r i c u l t u r e , B r i t i s h C o l u m b i a D i v i s i o n , f o r h i s h e l p and i n t e r e s t and f o r t h e use of the l a b o r a t o r y f a c i l i t i e s w i t h o u t w h i c h t h i s work c o u l d not have been c o m p l e t e d . Thanks a r e due a l s o t o Dr. P. Simon and t h e s t a f f o f t h e A n i m a l P a t h o l o g y L a b o r a t o r y m e n t i o n e d above f o r t h e i r a s s i s t a n c e i n c e r t a i n phases of t h i s p r o j e c t . F i n a l l y t h e a u t h o r acknowledges most g r a t e f u l l y t h e t e c h n i c a l a s s i s t a n c e of M i s s K a r e n F o r g e r o n , Mr. C l i f f o r d H a r v ey, Mr. Edward Jung and Mr. D a v i d F a r r e l l . T h i s s t u d y has been s u p p o r t e d i n p a r t by a g r a n t from the Canada Department o f A g r i c u l t u r e . 1 INTRODUCTION The D i v i s i o n of Animal Science has, for some time, been operating an experimental mink colony i n order to study c e r t a i n aspects of the n u t r i t i o n of t h i s animal. During these i n v e s t i g a t i o n s , a l i v e r disease was en-countered and became a serious threat to the mink colony due to i t s untimely recurrences over the past s i x years. In order to prevent t h i s condition i n mink, i t was decided to pursue the matter i n i t s e n t i r e t y . Since the condition seemed to be caused by a toxin, presumably diet-induced, an i n v e s t i g a t i o n of the feed for toxin-producing organisms was c a r r i e d out. The r o l e of mycotoxins i n animal poisonings was at t h i s time gaining prominence i n the f i e l d of animal health. Since these toxins for the most part a f f e c t the l i v e r s of animals, i t was decided to examine the mink disease to see i f i t was due to a mycotoxin present i n the mink feed or formed i n the feed a f t e r i t has been mixed. The following discussions deal with the various aspects of the work that has been c a r r i e d out on the subject. They have been presented in the order i n which they were conducted. Each experiment has been discussed as an e n t i t y i n order to give a c o n t i n u i t y to events as they occurred. As a r e s u l t , a d e s c r i p t i o n of the o r i g i n a l disease syndrome i n mink i s treated f i r s t , and i s followed by an extensive review of the l i t e r a t u r e covering the f i e l d of the mycotoxicoses. Three papers are then presented dealing with various aspects of the work that was undertaken to elucidate the a e t i o -l o g i c a l conditions leading to the o r i g i n a l hepato-haemangioma-syndrome i n mink. PART I AN HEPATO-HAEMANGIOMA-SYNDROME (HHS) IN MINK INTRODUCTION 2 During the course of c e r t a i n investigations i n mink n u t r i t i o n , a severe degenerative change was noted i n the l i v e r s of some of the experi-mental subjects. These changes were of a d i s t i n c t i v e character and unlike any of the recognized pathological states that have been recorded f o r t h i s species. The condition can best be described as an apparent "hepatotoxi-cos i s " complicated i n i t s terminal stages by massive haemorrhages from the l i v e r . The condition was f i r s t noted when several animals, i n apparently excellent condition, died suddenly. Post mortem examination revealed that the deaths were due to a massive haemorrhage into the peritoneum a r i s i n g from the rupture of one or more s i t e s of damage at the l i v e r capsule. Se-vere degenerative changes were present i n a l l l i v e r s examined. OCCURRENCE During the l a t e summer and f a l l of 1959 a few deaths of adult and k i t mink occurred i n the u n i v e r s i t y experimental u n i t . The p r i n c i p a l autopsy f i n d i n g was the presence of a yellow, f a t t y l i v e r , or a l i v e r showing nodular c i r r h o s i s and often accompanied by a marked splenomegaly. These conditions arose in animals that had received the r a t i o n shown i n Table I. 3 TABLE I E x p e r i m e n t a l B a s a l R a t i o n I n g r e d i e n t P e r Cent H e r r i n g M e a l (72%) 32.0 Soya M e a l (501) 8.0 Meat S c r a p s (50%) 4.0 Whale M e a l (80%) 4.0 Ground Wheat 9.0 Oat G r o a t s 8.0 Skim M i l k Powder 4.0 De h y d r a t e d G r a s s 2.0 I o d i z e d S a l t 1.2 V i t a m i n P r e - m i x 1.6 R e f i n e d C o t t o n s e e d O i l 26.2 100.0 F i f t y pounds o f t h i s r a t i o n was b l e n d e d w i t h f i f t y pounds of t a p w a t e r t o y i e l d t h e "as f e d " mink f e e d . Over t h e c o u r s e of the s u c c e e d i n g n i n e months, twenty-one p e r c e n t o f a d u l t b r e e d i n g a n i m a l s succumbed t o t h e d i s e a s e . I n a d d i t i o n t h r e e k i t s from t h e 1960 c r o p d i e d and d i s p l a y e d s i m i l a r p a t h o l o g i c a l changes a t p o s t mortem. D u r i n g t h i s p e r i o d (1960) a number of r a t i o n changes were made on t h e a s s u m p t i o n t h a t t h e l i v e r damage m i g h t be due t o t h e absence of a n u t r i t i v e e n t i t y f r o m t h e r a t i o n . No e v i d e n c e was o b t a i n e d t o s u p p o r t t h i s c o n t e n t i o n . I n December o f 1960 t h e s u r v i v i n g a n i m a l s were p e l t e d out i n t h e hope t h a t c o m p l e t e d e p o p u l a t i o n o f t h e c o l o n y f o l l o w e d by r e s t o c k i n g would a r r e s t t h e i n c i d e n c e of t h e d i s e a s e . D e t a i l e d p o s t mortem e x a m i n a t i o n s were c a r r i e d out on t h e c a r c a s s e s r e c o v e r e d a f t e r p e l t i n g . The i n c i d e n c e of t h e l i v e r d i s e a s e was found t o be more t h a n n i n e t y - e i g h t p e r c e n t . I n J a n u a r y of 1961 t h e e x p e r i m e n t a l u n i t was r e s t o c k e d w i t h mink 4 fro m c o m m e r c i a l h e r d s where t h e r e had been no i n c i d e n c e o f h e p a t o p a t h y d u r i n g t h e p r e v i o u s t e n y e a r s . The d i s e a s e r e c u r r e d i n t h e ne w l y i n t r o -duced a n i m a l s i n September of 1962 and s p o r a d i c a l l y d u r i n g 1963, 1964 and 1965. AETIOLOGY The syndrome appears t o be a d i e t - i n d u c e d d i s e a s e r e s u l t i n g f r o m an exogenous o r endogenous f a c t o r o r f a c t o r s p r e s e n t i n t h e r a t i o n i n g r e d i e n t s o r p r o duced i n t h e f e e d subsequent t o m i x i n g . D u r i n g 1960 b o t h c e l l u l a r and c e l l - f r e e t i s s u e i n o c u l a , p r e p a r e d s h o r t l y a f t e r d e a t h f r o m a f f e c t e d mink, were i n t r o d u c e d p a r e n t e r a l l y i n t o n o r m a l mink. No e v i d e n c e was o b t a i n e d f o r t h e t r a n s m i s s i o n o f a d i s e a s e - p r o d u c i n g agent when t h e s e a n i m a l s were k i l l e d and examined e i g h t weeks p o s t i n j e c t i o n . P r e l i m i n a r y e x a m i n a t i o n o f t h e h i s t o p a t h o l o g i c a l f e a t u r e s o f b o t h a f f e c t e d a n i m a l s t h a t were k i l l e d and o t h e r s t h a t had d i e d as a r e s u l t o f m a s s i v e l i v e r haemorrhages, s u g g e s t e d t h a t t h e c o n d i t i o n a r o s e i n r e s p o n s e t o a t o x i c f a c t o r o r f a c t o r s . S u pport f o r t h i s c o n c l u s i o n was o b t a i n e d from t h e f a c t t h a t e x t e n s i v e b a c t e r i o l o g i c a l s t u d i e s o f a f f e c t e d l i v e r s , s p l e e n s , lymph nodes, i n t e s t i n e s , h e a r t s , l u n g s and o t h e r organs y i e l d e d no s u s p i c i o u s i s o l a t e s . The f a i l u r e t o passage any a c t i v e i n f e c t i o u s agent i n t h e mink t o mink t r a n s m i s s i o n t r i a l s m e n t i o n e d e a r l i e r l e n d s f u r t h e r s u p p o r t t o a n o n - i n f e c t i o u s a e t i o l o g y . CLINICAL APPEARANCE The a f f e c t e d mink t h a t had been r e a r e d , m a i n t a i n e d and a l l o w e d t o r e p r o d u c e on t h e e x p e r i m e n t a l r a t i o n s a t t a i n e d a c c e p t a b l e body w e i g h t , p e l t q u a l i t y and g e n e r a l a ppearance. A t a u t o p s y t h e g e n e r a l c o n d i t i o n o f t h e 5 body fat depots was excellent and c e r t a i n l y equivalent to those normally encountered i n ranch-reared mink. Affected mink that died in most cases did so following massive i n t r a p e r i t o n e a l haemorrhage from the l i v e r . In some cases death appeared to be the r e s u l t of peritoneal a s c i t e s , which was evident p r i o r to death by marked abdominal swelling. GROSS PATHOLOGY The feature most commonly present (70 per cent) i s that of a pale pink or yellow l i v e r of somewhat increased s i z e with a f i n e or coarsely nodular surface d i s p l a y i n g one or more large bulging c y s t i c , b l o o d - f i l l e d structures ( p r o v i s i o n a l l y termed haemangiomas) at the surface (Plates 1, 2 and 3). In most cases of spontaneous death, one of these structures had erupted and f i l l e d the peritoneal c a v i t y with blood (Plates 2 and 4). The l i v e r parenchyma usually contained numerous smaller b l o o d - f i l l e d cysts or haemangiomas and one or more addit i o n a l unerupted large structures (Plates 1, 2 and 3). In some cases where deaths occurred due to reasons other than l i v e r haemorrhage, for example, following the development of asc i t e s (15 per cent) (Plate 7), the l i v e r was yellow and nodular and did not contain the haemangiomas. In many cases there were various degrees of enlargement or congestion of the lymph nodes of the upper g a s t r o i n t e s t i n a l t r a c t , i n -cluding the hepato-splenic lymph node. Haematomas i n the spleen and omentum were also observed (Plate 5). The p a r t i c i p a t i o n of the omentum i n containing the haemorrhage from the l i v e r frequently led to an adhesive haematomatous mass involving the l i v e r and omentum (Plate 4). In twenty-five per cent of the cases the spleen was hyperplastic or hypertrophied (Plate 5). Gastric ulcers (8 per cent) (Plate 6), and surface plaque c a l c i f i c a t i o n of the l i v e r (4 per cent), were occa s i o n a l l y present. Kidney changes involving renal 6 i n f a r c t i o n s , p e t e c h i a l haemorrhages and f o c a l n e c r o s i s o c c u r r e d i n about t e n p e r c e n t o f t h e c a s e s a u t o p s i e d . T h o r a c i c s t r u c t u r e s were r a r e l y a f f e c t e d . There was one c a s e w i t h pulmonary haemorrhage o r haematoma and two w i t h s u b p l e u r a l d i a p h r a g m a t i c haematomas. I n t h i s d i s e a s e i n t r a l u m e n a l i n t e s t i n a l haemorrhages o r e n t e r i t i s were r a r e l y e n c o u n t e r e d . The subcu-t a n e o u s , m e s e n t e r i c and omental f a t d e p o t s were normal ( P l a t e 5 ) . I n c a s e s examined a f t e r e u t h a n a s i a w i t h c a r b o n d i o x i d e , t h e l i v e r was u s u a l l y a deep shade o f p i n k b u t w i t h t h e d i s t o r t e d v a s c u l a r s t r u c t u r e s no l e s s e v i d e n t (98 p e r c e n t ) . I n a few c a s e s r u p t u r e o f t h e haemangiomatous s t r u c t u r e s had j u s t o c c u r r e d , a p p a r e n t l y as a r e s u l t o f t h e s t r e s s a r i s i n g d u r i n g e u t h a n a s i a and p e l t i n g ( P l a t e 1 ) . HISTOPATHOLOGY The c a s e s examined f r o m r o u t i n e h a e m a t o x y l i n and e o s i n p a r a f f i n s e c t i o n s were found t o f a l l i n t o t h r e e m a j o r g r o u p s . The o b s e r v a t i o n s were l i m i t e d , f o r t h e most p a r t , t o t h e l i v e r , k i d n e y s , s p l e e n and lymph nodes. (a) Mink w h i c h d i e d due t o haemorrhage i n t o t h e p e r i t o n e u m f r o m t h e l i v e r . I n t h e s e c a s e s t h e l i v e r s were p i n k o r y e l l o w and n o d u l a r , w i t h m a r k e d l y d i s t o r t e d b l o o d v a s c u l a r s t r u c t u r e s ( c y s t s o r haemangiomas). (b) Mink w h i c h d i e d s p o n t a n e o u s l y f o l l o w i n g t h e development o f p e r i t o n e a l a s c i t e s , t h e h e p a t i c t i s s u e was y e l l o w and n o d u l a r ( p r o g r e s s i n g t o c i r r h o s i s ) b u t c o n t a i n e d no g r o s s l y d i s t o r t e d v a s c u l a r s t r u c t u r e s . ( c ) Mink w h i c h were s u b j e c t e d t o c a r b o n d i o x i d e e u t h a n a s i a a t p e l t i n g . These a n i m a l s appeared t o be h e a l t h y b u t on p o s t mortem 98 p e r c e n t o f them were found t o have g r o s s l y changed l i v e r s u s u a l l y w i t h d i s t o r t e d b l o o d v a s c u l a r s t r u c t u r e s . The mink i n c a t e g o r y (a) u s u a l l y d i s p l a y e d a g e n e r a l s e v e r e f a t t y metamorphosis of t h e l i v e r p r o g r e s s i n g t o a c e n t r o l o b u l a r n e c r o s i s t h r o u g h -out t h i s o r gan ( P l a t e 8 ) . The o n l y parenchymal c e l l s w h i c h appeared t o be a l i v e and p o s s i b l y r e g e n e r a t i v e , appeared as s m a l l i s l a n d s around the • p o r t a l t r i n i t i e s and c e n t r a l v e i n s ( P l a t e 9 ) . The r e m a i n i n g c e l l s were g r o s s l y d i s t e n d e d w i t h f a t and i n many ca s e s c o a l e s c e n c e had o c c u r r e d f o l l o w i n g r u p t u r e o f the c e l l w a l l s . I n a d d i t i o n , t h e r e were p r e s e n t a number of i n t e r m e d i a t e and l a r g e b l o o d - f i l l e d e n d o t h e l i u m - l i n e d s t r u c t u r e s ( P l a t e 1 0 ) , f r e q u e n t l y s u r r o u n d e d w i t h heavy f i b r o u s t i s s u e w a l l s . These w a l l s were p a r t i c u l a r l y e v i d e n t near t h e c a p s u l e ( P l a t e 1 1 ) . The b l o o d -f i l l e d spaces were, i n some c a s e s , l i n e d w i t h t i s s u e r e s e m b l i n g normal venous w a l l s , w h i l e i n o t h e r a r e a s t h e r e was o n l y a s i n g l e l a y e r o f e n d o t h e l i u m s e p a r a t i n g them from t h e l i v e r parenchyma ( P l a t e 1 2 ) . O c c a s i o n -a l l y t h i s endothelium c o u l d n o t be d e t e c t e d and t h e spaces l e d d i r e c t l y i n t o engorged s i n u s o i d s ( P l a t e 1 3 ) , e s p e c i a l l y i n t h e s u b c a p s u l a r a r e a s . Many of the b l o o d - f i l l e d spaces appeared t o have no e v i d e n t r e l a t i o n t o the p o r t a l v e i n . I n o t h e r c a s e s t h e spaces appeared t o l e a d i n or out o f ex-panded v e s s e l s . O f t e n t h e b l o o d had f l o w e d out from t h e s e spaces when the t i s s u e s were removed f o r f i x a t i o n . T h i c k a r e a s of f i b r o u s c o n n e c t i v e t i s s u e appeared t o have o c c u p i e d t h e p r e v i o u s l y b l o o d - f i l l e d c y s t s o r haemangiomas. I n a few specimens t h e r e was e v i d e n c e o f t h r o m b o s i s o f t h e s e s t r u c t u r e s . C e l l i n f i l t r a t i o n s o c c u r r e d i n some a r e a s ( P l a t e 14 a ) . The mink i n c a t e g o r y (b) showed s i m i l a r but s m a l l e r o r medium-sized abnormal b l o o d - c o n t a i n i n g spaces i n t h e l i v e r . O c c a s i o n a l l y p a r e n t e r a l haemorrhages were seen. These were b o t h s u b c a p s u l a r and deep i n t h e l i v e r 8 t i s s u e , and were accompanied by b l o o d pigment. The l i v e r s g e n e r a l l y showed d i f f u s e p a r e n t e r a l f a t t y metamorphosis o r n e c r o s i s w i t h many i r r e g u l a r t r a c t s o f s c a r t i s s u e , some o f w h i c h were l a r g e , dense and o c c a s i o n a l l y c a l c i f i e d . D i f f u s e o r f o c a l i n f i l t r a t i o n o f t h e p o r t a l a r e a s w i t h r e t i c u l o -h i s t i o c y t e s o r l y m p h o c y t e s o c c u r r e d ( P l a t e 14 b ) . A number of l i v e r s were m a r k e d l y engorged i n some a r e a s . I n some l i v e r s r e c e n t p a r enchymal c e l l d e g e n e r a t i o n was r a r e , a l t h o u g h many t r a c e s o f s c a r t i s s u e and t h e g e n e r a l derangement o f t h e c e l l u l a r a r c h i t e c t u r e s u g g e s t e d p r e v i o u s c i r r h o t i c i n c i d e n t s . I n t h e group ( c ) mink, t h e haemangiomatous s t r u c t u r e s and s u r r o u n d i n g o r i n t e r s p e r s e d bands o f f i b r o u s t i s s u e were a l s o p r e s e n t , as were t h e p o r t a l and i n t r a l o b u l a r i n f i l t r a t i o n s o f r e t i c u l o - h i s t i o c y t e s and l y m p h o c y t i c c e l l s . The p r i n c i p a l d i f f e r e n c e f r o m t h e group ( a ) mink r e s i d e d i n t h e g e n e r a l l i v e r parenchyma (between c y s t i c s t r u c t u r e s ) w h i c h appeared e s s e n t i a l l y n o r m a l a l t h o u g h t h e a r c h i t e c t u r e was s l i g h t l y d eranged. I n some c a s e s c e r t a i n a r e a s o f t h e parenchyma d i s p l a y e d c h r o n i c changes. There was damage t o t h e c o n n e c t i v e t i s s u e ground s u b s t a n c e and r e p l a c e m e n t o f t h e h e p a t i c c e l l s by f i b r o u s t i s s u e and by t h e b l o o d - f i l l e d s t r u c t u r e s ( P l a t e 1 5 ) . There was d i f f u s e i n f i l t r a t i o n o f e o s i n o p h i l s and m e g a k a r y o c y t e s , and a s u s p i c i o n o f e x t r a - m e d u l l a r y h a e m a t o p o i e s i s . I n some c a s e s t h e f i b r o s i s was t r a b e c u l a t e d and g e n e r a l i z e d c a u s i n g d i v i s i o n o f t h e l o b u l e s . S e v e r a l a n i m a l s i n t h i s group were found t o have masses o f m e t a p l a s t i c o r n e o p l a s t i c t i s s u e i n t h e l i v e r parenchyma i n a s s o c i a t i o n w i t h advanced f i b r o t i c changes ( P l a t e s 16 and 1 7 ) . I n many c a s e s , i n a l l t h r e e g r o u p s , t h e s p l e e n s showed l i t t l e o r no h i s t o l o g i c a l change. I n about t w e n t y - f i v e p e r c e n t , w h i c h d e m o n s t r a t e d g r o s s s p l e n o m e g a l y , t h e r e appeared t o be h y p e r p l a s i a o f t h e r e d p u l p w i t h PLATE 1 HHS Mink. T y p i c a l l i v e r : p i n k i s h w i t h c o a r s e l y n o d u l a r s u r f a c e .and b u l g i n g c y s t i c b l o o d - f i l l e d s t r u c t u r e s , (haemangiomas). PLATE 2 HHS Mink. Y e l l o w l i v e r x ^ i t h c o a r s e l y n o d u l a r s u r f a c e and b u l g i n g c y s t i c b l o o d - f i l l e d s t r u c t u r e s (haemangiomas). L a r g e c y s t on r i g h t l o b e was t h e s i t e o f f a t a l haemorrhage. PLATE 3 HHS. Mink. L i v e r : p o s t e r i o r s u r f a c e showing c o a r s e n o d u l a r i t y and haemangiomas. PLATE 3 PLATE 4 HHS Mink. Death s p o n t a n e o u s : haemorrhage i n t o p e r i t o n e u m , omental a d h e s i o n . PLATE 5 HHS Mink. Haematomas i n s p l e e n . NOTE h e a l t h y c o n d i t i o n o f m e s e n t e r i c f a t . PLATE 6 .HHS Mink. Many s m a l l g a s t r i c u l c e r s . PLATE 7 HHS Mink. C l i n i c a l appearance f o l l o w i n g t h e development of p e r i t o n e a l a s c i t e s . PLATE 8 HHS Mink. L i v e r : s e v e r e f a t t y changes and c e n t r o l o b u l a r n e c r o s i s . Case o f spontaneous d e a t h . (33x) PLATE 9 HHS Mink. L i v e r : p o r t a l t r i n i t y showing v e i n and s p i r a l l i n g b i l e d u c t w i t h a few a d j a c e n t v i a b l e parenchymal c e l l s . Case o f spontaneous d e a t h . (168x) PLATE 9 PLATE 10 HHS Mink. L i v e r : network of b l o o d - f i l l e d haeman-giomas or c y s t s . A u t o p s y f o l l o w i n g e u t h a n a s i a . (43x) PLATE 11 HHS Mink. L i v e r : s u b c a p s u l a r a r e a w i t h l a r g e haemangioma and heavy w a l l o f . f i b r o u s c o n n e c t i v e t i s s u e . (168x) PLATE 12 HHS Mink. L i v e r : c l o s e up of l i v e r shown i n P l a t e 10. J u n c t i o n between haemangiomas showing e n d o t h e l i a l l i n i n g . (168x) PLATE 12 PLATE 13 HHS Mink. L i v e r : d i r e c t c o n n e c t i o n between expanded s i n u s o i d s and m a r g i n o f l a r g e haemangioma w i t h e n d o t h e l i a l l i n i n g . A u t o p s y f o l l o w i n g euthanasia.. (57x) PLATE 14 a HHS Mink. L i v e r : d i f f u s e d e g e n e r a t i o n o f parenchyma w i t h c e l l u l a r i n f i l t r a t i o n o f p o r t a l t r i n i t y and c e n t r a l v e i n a r e a . Case o f spontaneous d e a t h . (168x) PLATE 14 b HHS Mink. L i v e r : a degree of c e n t r o l o b u l a r p arenchymal d e g e n e r a t i o n and c e l l u l a r i n f i l t r a t i o n • o f p o r t a l a r e a . A utopsy f o l l o w i n g e u t h a n a s i a . (83x) PLATE 14 a PLATE 14 b PLATE 15 HHS Mink. L i v e r : s u b c a p s u l a r a r e a , s e v e r e r e c e n t f i b r o t i c t r a c t s and edge of a s m a l l haemangioma at l e f t m a r g i n . Case o f spontaneous d e a t h . (83x) PLATE 16 HHS Mink. L i v e r : n e o p l a s t i c t i s s u e a s s o c i a t e d w i t h a r e a o f advanced f i b r o t i c changes. Case o f spontaneous d e a t h . (117x) PLATE 17 HHS Mink. L i v e r : c e l l u l a r d e t a i l o f n e o p l a s t i c m a s s , ' p r o b a b l y n o n - m a l i g n a n t and p o s s i b l y a r i s i n g f rom e n d o t h e l i a l e l e m e n t s . Case o f spontaneous d e a t h . (368x) PLATE 17 PLATE 18 HHS Mink. S p l e e n : a r e a o f r e d p u l p w i t h i n c r e a s e o f m u l t i n u c l e a t e d c e l l s ( m e g a k a r y o c y t e s ) . Case o f spontaneous d e a t h . (528x) PLATE 19 HHS Mink. Lymph node: s i n u s h y p e r p l a s i a and extreme oedema. Case o f spontaneous d e a t h . (23x) PLATE 18 PLATE 19 9 larger than normal numbers of megakaryocytes or other multinucleated c e l l s (Plate 18). Occasional groups of red-staining c e l l s , p o s s i b l y eosinophils, were noted. Subcapsular haematomas were noted grossly i n a few spleens, however the microscopical p i c t u r e shed l i t t l e l i g h t on the pathogenesis of these structures. In approximately twenty-five per cent of the cases, as noted previous-l y , there was a gross enlargement of one or more lymph nodes, usually in the abdominal c a v i t y . In these, the h i s t o l o g i c a l picture was one of sinus hyperplasia progressing to extreme oedema (Plate 19). There was some damage to the connective ti s s u e ground substance around the blood vessels i n some lymph nodes. A degree of hyperplasia of lymph f o l l i c l e s and some erythro-phagia existed i n a few nodes. Although damage to kidney t i s s u e was minimal, some of the animals of group (c) showed a marked congestion of t h i s organ and t h i s was evident i n the vessels of the cortex and medulla. DISCUSSION AND SUMMARY With the exception of some animals that were noted to be a s c i t i c , v i r t u a l l y no c l i n i c a l signs preceded the f a t a l haemorrhages i n the experi-mental mink, despite the tremendous upset i n l i v e r a rchitecture which be-came apparent at autopsy. With few exceptions, appetite, weight gains, p e l t q u a l i t y , general body condition and reproductive c a p a b i l i t i e s were unimpaired. Despite t h i s , when the balance of the population was l i q u i -dated, ninety-eight per cent of the mink were affected with hepatopathy. The gross l i v e r changes were b a s i c a l l y i d e n t i c a l i n 70 per cent of the spontaneous deaths and i n 90 per cent of those pelted i n 1960. This probably indicated that the changes were a c h a r a c t e r i s t i c sequela to an 10 exposure t o some h e p a t o x t c i n f l u e n c e f o r a p e r i o d o f t h r e e t o s i x months or l o n g e r . I n perhaps 15 p e r c e n t o f spontaneous d e a t h s t h e r e d e v e l o p e d a c i r r h o t i c s e q u e l a c a u s i n g or accompanied by p e r i t o n e a l a s c i t e s . Some c a s e s o f a c u t e h e p a t i c n e c r o s i s or f a t t y metamorphosis, o c c u r r i n g i n l a t e 1959, p r e c e d e d the m a j o r i t y o f l o s s e s by a few months. There may have been an a e t i o l o g i c a l r e l a t i o n s h i p between t h e s e and the c h a r a c t e r i s t i c changes n o t e d the f o l l o w i n g March, i f , as i s p r o b a b l y the c a s e , t h e t o x i c i n f l u e n c e i s c a p a b l e o f p r o d u c i n g a c u t e d e a t h when p r e s e n t i n h i g h e r c o n c e n t r a t i o n s . The c a r d i n a l c h a r a c t e r i s t i c s o f the g r o s s p a t h o l o g i c a l changes i n the c l a s s i c a l c a s e s (86 p e r c e n t o v e r a l l ) can be b r i e f l y l i s t e d a s : (1) T o t a l r e p l a c e m e n t o f normal l i v e r a r c h i t e c t u r e w i t h p a l e or y e l l o w n o d u l a r parenchyma i n t e r s p e r s e d w i t h deep or S u b c a p s u l a r and o f t e n b u l g i n g , b l o o d - c o n t a i n i n g s t r u c t u r e s . (2) I n spontaneous d e a t h s , haemorrhage of an abnormal v a s c u l a r s t r u c t u r e e r u p t s t h r o u g h t h e l i v e r c a p s u l e i n t o t h e p e r i t o n e u m . Even when exposed t o the a i r a t a u t o p s y , t h e b l o o d does n o t c l o t . O f t e n t h e r e i s an i n v o l v e m e n t o f the omentum w h i c h p e r i o d -i c a l l y may have succeeded i n c o n t a i n i n g t h e haemorrhage. T h i s i s s u g g e s t e d by the p r e s e n c e o f o l d , o r g a n i s i n g n o d u l a r haematomas of v a r i o u s s i z e s a t t a c h e d t o and a s s o c i a t e d w i t h a d h e s i o n s o f the omentum o r t h e l i v e r . (3) I n c a s e s o f h e p a t o p a t h y n o t e d a f t e r e u t h a n a s i a , the l i v e r parenchyma by c o m p a r i s o n w i t h the l i v e r s i n t h o s e w h i c h s u f f e r e d spontaneous d e a t h , was l e s s changed i n terms o f c o l o u r and c o n s i s t e n c y . W h i l e t h i s d i f f e r e n c e m ight be e x p e c t e d i n a l i v e r n o t d r a i n e d o f b l o o d by i n t r a p e r i t o n e a l haemorrhage, the h i s t o l o g i c a l p i c t u r e tended t o c o n f i r m t h a t t h e r e was l e s s parenchymal change. (4) I n about t w e n t y - f i v e p e r c e n t of the c a s e s t h e r e was enlargement of t h e s p l e e n and some of t h e lymph nodes o f t h e g a s t r o i n t e s t i n a l c h a i n e s p e c i a l l y t h o s e d r a i n i n g t h e stomach, l i v e r , s p l e e n , p a n c r e a s , and s m a l l i n t e s t i n e . (5) I n a few c a s e s , haematomas were n o t e d t o have o c c u r r e d i n t h e s p l e e n and, i n one c a s e , t h e l u n g . (6) Other f i n d i n g s such as g a s t r i c u l c e r s , r e n a l changes and d i a -p h r a g m a t i c haemorrhages were not p r e s e n t i n h i g h i n c i d e n c e and p o s s i b l y s h o u l d not be c o n s i d e r e d i n the p a t h o g e n e s i s o f HHS. I t was found t h a t t h r e e somewhat a r b i t r a r y d i v i s i o n s c o u l d be used f o r d e s c r i b i n g t h e h i s t o l o g i c a l changes i n t h e l i v e r . The b a s i s f o r d i f f e r -e n t i a t i o n i n v o l v e d a c o m b i n a t i o n o f t h e g r o s s and h l s t o p a t h o l o g i c a l o b s e r -v a t i o n s . I t p l a c e d t h e m a j o r i t y (70 p e r c e n t ) of spontaneous d e a t h s i n t o group (a) w h i c h showed g r o s s haemangiomatous s t r u c t u r e s , and a m i n o r i t y (15 p e r c e n t ) o f such d e a t h s i n t o group (b) w h i c h were w i t h o u t g r o s s haemangio-mas. These d i e d from p e r i t o n e a l a s c i t e s r a t h e r t h a n f a t a l haemorrhages i n t o t h e abdominal c a v i t y . The t h i r d g roup, ( c ) , was c o m p r i s e d of the m a j o r i t y (90 p e r c e n t ) of mink g i v e n e u t h a n a s i a and found t o have t h e g r o s s l i v e r haemangiomas. The p r i n c i p a l d i f f e r e n c e s between the l i v e r s o f group (a) w h i c h d i e d , and t h o s e of group ( c ) w h i c h were k i l l e d , r e s i d e d i n t h e more a c u t e g e n e r a l parenchymal n e c r o s i s or d e g e n e r a t i o n i n t h e l i v e r s o f t h e mink t h a t d i e d . The more c h r o n i c f i b r o t i c and haemangiomatous changes were e q u a l l y e v i d e n t i n b o t h g r o u p s . The l i v e r s o f mink p l a c e d i n group ( b ) , i n w h i c h d e a t h f o l l o w e d t h e development o f a s c i t e s , showed c h r o n i c changes. S m a l l e r 12 d i s t o r t e d blood v a s c u l a r s t r u c t u r e s were present m i c r o s c o p i c a l l y and i n most cases there was extensive d i f f u s e and a c t i v e c i r r h o s i s i n v a r i o u s stages. C e l l i n f i l t r a t i o n s were noted i n a l l three groups, mostly i n the p o r t a l t r i n i t y areas, while metaplasia and n e o p l a s i a were noted i n conjunc-t i o n w i t h f i b r o t i c changes i n l i v e r s of group (c) which were given euthanasia. At l e a s t twenty-five per cent of a l l mink showed r e t i c u l o e n d o t h e l i a l stimulus by gross h y p e r p l a s i a of the spleen and lymph nodes. M i c r o s c o p i c a l l y these enlarged organs showed c e l l u l a r s timulus, mostly of non-lymphocytic elements, and oedema i n the case of the nodes. H i s t o l o g i c a l examination shed l i t t l e l i g h t on the pathogenesis of subcapsular haematomas noted on some spleens. PART I I LITERATURE REVIEW THE MYCOTOXICOSES 13 INTRODUCTION M o r t a l i t y i n d o m e s t i c a n i m a l s r e s u l t i n g from t h e i n g e s t i o n o f mouldy f e e d s t u f f s has r e c e i v e d a g r e a t d e a l o f a t t e n t i o n i n r e c e n t y e a r s . These s t u d i e s have shown t h a t t h e c a u s a t i v e agent has been i n many c a s e s a t o x i c s u b s t a n c e e l a b o r a t e d by one o r more of t h e common f u n g i . The t e r m m y c o t o x i c o s e s has been used t o d e s c r i b e t h e syndromes r e s u l t -i n g f r o m t h e i n g e s t i o n o f t h e s e t o x i c f u n g a l m e t a b o l i t e s . B o t h humans and a n i m a l s have been a f f e c t e d . A l t h o u g h humans a r e s u s c e p t i b l e t o t h e s e d i s e a s e s , t h e t r a c e a b l e i n c i d e n c e o f m o r t a l i t y has n o t been v e r y h i g h , e x c e p t i n R u s s i a where t h e d i s e a s e c a l l e d a l i m e n t a r y t o x i c a l e u k i a ( h e r e -a f t e r r e f e r r e d t o as ATA) has been p r e v a l e n t i n humans who have i n g e s t e d o v e r w i n t e r e d mouldy g r a i n i n f e c t e d w i t h t h e fungus F u s a r i u m s p o r o t r i c h o i d e s . The m y c o t o x i c o s e s a r e c o m p l i c a t e d d i s e a s e s . The symptoms d i s p l a y e d by a f f e c t e d a n i m a l s are b o t h v a r i e d and complex. The syndrome o f f u n g a l t o x e m i a i s i n many c a s e s s i m i l a r t o t h a t a r i s i n g f rom n u t r i t i o n a l d e f e c t s and f r o m c h e m i c a l p o i s o n i n g s . The d i a g n o s i s o f t h e d i s e a s e o f t e n r e q u i r e s t h a t a l l o t h e r p o s s i b l e agents o r f a c t o r s , f o r example b a c t e r i a l , v i r a l , c h e m i c a l and n u t r i t i o n a l , be e x c l u d e d b e f o r e t h e fungus c a n be i n c r i m i n a t e d as t h e c a u s a t i v e a g e n t . S i n c e f u n g i a r e u b i q u i t o u s and p r e s e n t t o some e x t e n t i n a l m o s t a l l f e e d s t u f f s , a d e t a i l e d s t u d y o f t h e f u n g a l p o p u l a t i o n o f a s u s p e c t f e e d i s e s s e n t i a l . The d i s e a s e must be r e p r o d u c e d i n h e a l t h y a n i m a l s w i t h t o x i n s e l a b o r a t e d i n t h e l a b o r a t o r y by p u r e i s o l a t e s o f t h e s u s p e c t e d f u n g a l s p e c i e s i n o r d e r t o p r o v i d e a d e f i n i t e c o n f i r m a t i o n o f t h e i n i t i a l d i a g n o s i s . M i c r o s c o p i c f u n g i d i s p l a y a g r e a t d i v e r s i t y i n m e t a b o l i s m a f f e c t i n g c o u n t l e s s b i o c h e m i c a l t r a n s f o r m a t i o n s of. a h o s t o f d i f f e r e n t s u b s t a n c e s . ; 14 Some f u n g a l m e t a b o l i t e s show a n t i b i o t i c p r o p e r t i e s w h i l e o t h e r s produce t o x i n s w h i c h a r e h a r m f u l t o man and a n i m a l s . F u n g a l m e t a b o l i t e s have been t h o u g h t t o be waste o r s t o r a g e p r o d u c t s of m e t a b o l i s m . I f some of t h e s e p r o d u c t s i n h i b i t c o m p e t i t o r s , t h e y f a c i l i t a t e s p e c i e s s u r v i v a l . D a l g l i e s h , as r e p o r t e d by M i l l e r ( 1 9 6 1 ) , s u g g e s t e d t h a t enzyme systems u n a b l e t o d e a l w i t h e x c e s s s u b s t r a t e m i g h t c o n v e r t i t t o m e t a b o l i c b y - p r o d u c t s w h i c h a r e e l i m i n a t e d by the o r g a n i s m s . The same r e f e r e n c e r e p o r t s on t h e work of Nord (1955) who s u g g e s t e d t h a t t h e s e f u n g a l m e t a b o l i t e s a r e p r o d u c e d i n y i e l d s e x c e e d i n g f u n c t i o n a l r e q u i r e m e n t s and t h e s e m e t a b o l i t e s a c c u m u l a t e because t h e enzyme systems i n v o l v e d i n t h e i r o x i d a t i v e sequences become s a t u r a t e d w i t h r e s p e c t t o t h e i r s u b s t r a t e s . R u s s i a n s c i e n t i s t s have been f a c e d w i t h t h e p r o b l e m o f human and a n i m a l m y c o t o x i c o s i s f o r a l o n g p e r i o d . They have r e p o r t e d numerous c a s e s o f ATA among humans who have e a t e n g r a i n o r c e r e a l i n v a d e d by F. s p o r o t r i c h o i d e s . The t o x i n e l a b o r a t e d by t h i s fungus has a d e g e n e r a t i v e e f f e c t on t h e b l o o d c e l l s o f t h e h o s t and t h e r e f o r e t h e c o n d i t i o n has been d e s c r i b e d as an a p l a s t i c anaemia. The b l o o d p i c t u r e p o r t r a y s a p r o g r e s s i v e l e u c o p e n i a and g r a n u l o p e n i a w i t h an accompanying d e p l e t i o n i n bone marrow a c t i v i t y . I t has been shown t h a t t h e fungus e l a b o r a t e s a t o x i n w h i c h i s t h e s p e c i f i c agent r e s p o n s i b l e f o r the d i s e a s e . The R u s s i a n l i t e r a t u r e c o n t a i n s many r e p o r t s o f a n i m a l i n t o x i c a t i o n s due t o t h e c o n s u m p t i o n o f mouldy f e e d s t u f f s . The fungus S t a c h y b o t r y s a t r a has been t h e c a use o f p o i s o n i n g s i n h o r s e s and c a t t l e t h a t have been f e d mouldy g r a i n . O t h e r f u n g i have been i s o l a t e d f r o m t o x i c g r a i n known t o have ca u s e d o u t b r e a k s of f u n g a l t o x i c o s i s . Members o f t h e g e n e r a Mucor, R h i z o p u s , A l t e r n a r i a , T r i c h o t h e e i u m , F u s a r i u m and P e n i c i l l i u m have been i s o l a t e d f r o m mouldy c o r n . ' Numerous s p e c i e s of t h e genus A s p e r g i l l u s have been i s o l a t e d from mouldy feedstuffs and have been incriminated in animal poisonings. It i s of in t e r e s t to note that the incidence of mycotoxicosis may depend on the conditions under which the grain i s stored following harvest. Although some fungi do invade the grain while i t i s s t i l l developing on the plant, none of these fungi are known to be t o x i c . The fungi responsible for the disease evidently invade the ri p e harvested grain during storage over the winter months, and with the onset of spring, elevated temperatures and a high moisture content in the grain provide a favourable environment for fungal p r o l i f e r a t i o n . In Southern England during 1960, a new aspect of fungal poisoning became apparent when a large number of turkey poults died from the ingestion of feed in which mouldy groundnut meal had been included as a supplementary protein source. Subsequently the disease was reported in ducklings and young pheasants. Examination of groundnut meals from c e r t a i n t r o p i c a l countries, v i z . B r a z i l , India, Kenya, showed that they contained a s t r a i n of the mould A s p e r g i l l u s flavus which had produced a toxin in the meals. This toxin, since named a f l a t o x i n (the Report of the Interdepartmental Working Party on Groundnut T o x i c i t y Research, 1962), i s extremely l e t h a l to turkeys, ducklings, r a b b i t s , calves, pigs, sheep and other animals. Methods of i s o l a t i o n and concentration of t h i s toxin have been perfected by research workers in England and the United States and attempts to characterize and chemically i d e n t i f y the toxin have met with considerable success. The causative agent i n groundnut poisoning i s one of the many fungal contaminants present in the meal. The t o x i n i s produced in extremely minute amounts, hence the technique for i t s i s o l a t i o n and p u r i f i c a t i o n have been d i f f i c u l t . For many years, New Zealand has been plagued by sheep losses which have now been shown to be due to fungal poisoning with a toxin elaborated by P i t h o m y c e s c h a r t a r u m . The d i s e a s e has been c a l l e d " f a c i a l eczema", a name d e s c r i p t i v e o f t h e l e s i o n s t h a t o c c u r on t h e f a c e and neck o f sheep t h a t have i n g e s t e d the f u n g a l t o x i n , s p o r i d e s m i n . T h i s t o x i n damages th e l i v e r and i n t e r f e r e s w i t h t h e c a t a b o l i s m o f c h l o r o p h y l l w i t h t h e r e s u l t t h a t p h y l l o e r y t h r i n , a breakdown p r o d u c t of c h l o r o p h y l l , c i r c u l a t e s i n t h e b l o o d s t r e a m . P h y l l o e r y t h r i n i s a photodynamic s u b s t a n c e and when sheep i n whose b l o o d t h e s u b s t a n c e i s c i r c u l a t i n g a r e exposed t o s u n l i g h t , l e s i o n s o f p h o t o s e n s i t i z a t i o n o c c u r on t h e f a c e s and n e c k s . New Z e a l a n d w o r k e r s have i n v e s t i g a t e d f a c i a l eczema e x t e n s i v e l y d u r i n g t h e p a s t f i f t e e n y e a r s e s -p e c i a l l y a t Ruakura (McMeekan, 1959) i n an attempt t o u n d e r s t a n d and c o n t r o l t h e s e a s o n a l o u t b r e a k s o f t h i s d i s e a s e i n c o m m e r c i a l f l o c k s . I n a l l t h e s e f u n g a l p o i s o n i n g s i t i s i m p o r t a n t t o r e c o g n i z e s t r a i n v a r i a t i o n i n t o x i n p r o d u c t i o n . I n any s p e c i e s o f mould t h e r e a r e a l a r g e number o f s u b - s t r a i n s o n l y one or two of w h i c h may produce t h e h a r m f u l agent. T h i s p r e s e n t s a v e r y d i f f i c u l t s c r e e n i n g p r o b l e m i n d i s c o v e r i n g t h e t o x i n -p r o d u c i n g v a r i a n t . A dermal t e s t e m p l o y i n g t h e e t h e r e x t r a c t of c o n t a m i n a t e d f o d d e r has been s u g g e s t e d by R u s s i a n w o r k e r s (Mayer 1953) f o r s c r e e n i n g t h e s u s p e c t e d f e e d f o r t o x i c i t y . A p o s i t i v e t e s t i s i n d i c a t e d by haemorrhage and n e c r o s i s on t h e shaved a r e a of a r a b b i t ' s back a f t e r s e r i a l a p p l i c a t i o n of t h e s u s p e c t e d t o x i n p r e p a r a t i o n . A n o t h e r i m p o r t a n t f a c t o r f o r t o x i n p r o d u c t i o n i s the i n f l u e n c e of e n v i r o n m e n t a l c o n d i t i o n s on b o t h t h e g r o w t h of t h e fungus and i t s a b i l i t y t o produce t o x i n . The f o l l o w i n g i s a r e v i e w of the s c i e n t i f i c and s e m i - t e c h n i c a l l i t e r a t u r e f r o m t h e v a r i o u s c o u n t r i e s of t h e w o r l d where the m y c o t o x i c o s e s a r e p r e v a l e n t . F o r c o n v e n i e n c e the r e v i e w has been p r e p a r e d a c c o r d i n g t o t h e c l i n i c a l symptoms and p a t h o l o g i c a l s t a t e s caused by s p e c i f i c t o x i n s t h a t have been i s o l a t e d . A s u r v e y o f t h e l i t e r a t u r e p e r t a i n i n g t o t h e s u b j e c t of the mycotoxicoses has shown that various authors have attempted to describe the diseases from the point of view of causative organism or organisms. This review i s treated by the method mentioned as i t i s f e l t that i n s u f f i * c i e n t information i s on hand to permit consideration of the mycotoxicoses under the generic or s p e c i f i c designations of the various offending fungi. The diseases for which no s p e c i f i c toxins have been i s o l a t e d and character-ized have been discussed under the heading MYCOTOXICOSES GENERAL. Although the d e s c r i p t i o n of these diseases does not follow the pattern used f o r the most part i n t h i s report, i t is thought that t h e i r importance cannot be ignored by a complete omission from the discussion. 18 ALIMENTARY TOXIC ALEUKIA 1. DISEASE A l i m e n t a r y t o x i c a l e u k i a (ATA) i s a d i s e a s e o f humans ca u s e d by t h e i n g e s t i o n o f mouldy g r a i n or g r a i n p r o d u c t s t h a t have been p r e p a r e d from c r o p s l e f t i n the f i e l d o v e r t h e w i n t e r . The d i s e a s e has been r e p o r t e d o n l y from R u s s i a where i t has been endemic and r e c o g n i z e d c l i n i c a l l y f o r t h e p a s t f i f t y y e a r s . ATA i s a n o n - c o n t a g i o u s , n o n - i n f e c t i v e , f r e q u e n t l y f a t a l d i s e a s e i n w h i c h t h e e s s e n t i a l c l i n i c a l symptoms have been v a r i o u s l y d e s c r i b e d as a p l a s t i c anaemia, h a e m o r r h a g i c a l e u k i a , a g r a n u l o c y t o s i s o r p a n m y e l o t o x i c o s i s . ATA i s most f r e q u e n t l y o b s e r v e d i n r u r a l a r eas a l t h o u g h u r b a n p o p u l a t i o n s have been a f f l i c t e d due t o the c o n s u m p t i o n o f mould g r a i n o r i t s f l o u r . The d i s e a s e may o c c u r i n one r e g i o n and be c o m p l e t e l y absent from a n o t h e r . The i n c i d e n c e o f t h e d i s e a s e i s g r e a t l y i n f l u e n c e d by c l i m a t i c c o n d i t i o n s a t h a r v e s t i n g . G r a i n i s n o r m a l l y h a r v e s t e d i n t h e f a l l , but i f weather i s i n c l e m e n t a t t h i s t i m e , i t may be l e f t l y i n g i n t h e f i e l d t h r o u g h -out t h e w i n t e r . Under t h e s e c o n d i t i o n s i t may be exposed t o a mean tempera-t u r e o f -15° C. W i t h t h e o n s e t o f s p r i n g , t h e t e m p e r a t u r e may r i s e t o 15° -20° C. A g r a d u a l t h a w i n g o c c u r s f a v o u r i n g t h e growth of v a r i o u s f u n g i . 2. CLINICAL SYMPTOMS Mayer (1953) s t a t e s t h a t t h e r e a r e f o u r s t a g e s i n t h e development o f t h e d i s e a s e . These may be c l a s s i f i e d a s : (1) l o c a l a c t i o n of t h e t o x i n (2) i n c u b a t i o n o r p e r i o d o f s e n s i t i z a t i o n (3) a n a p h y l a c t i c changes ( 4 ) reconvalescence. F i r s t Stage - This stage i s characterized by changes in the buccal c a v i t y and g a s t r o i n t e s t i n a l t r a c t . A f t e r consuming toxic food, the i n d i v i d u a l experiences a burning sensation in the mouth, tongue, throat, palate, oesophagus and stomach. This sensation i s a d i r e c t r e s u l t of the l o c a l e f f e c t of the toxin on the mucous membranes where, presumably, i t sets up a mild inflammation. G a s t r o i n t e s t i n a l lesions of a s i m i l a r nature may occur. Within a few days there develops an acute g a s t r o e n t e r i t i s with diarrhoea, nausea, vomiting and excessive p e r s p i r a t i o n . The body temperature usually remains normal but in severe cases may r i s e about 102° F. At t h i s stage there i s excessive s a l i v a t i o n , acute oesphagitis, g a s t r o e n t e r i t i s , abdominal pains, v e r t i g o , headache; and a f e e l i n g of coldness at the extremities. If the consumption of toxic food ceases these symptoms may disappear within 2-3 days, but i f consumption of the toxic food continues the symptoms may p e r s i s t for 5-9 days and then subside spontaneously. Mayer (1953) states that 92 per cent of a l l cases of an outbreak are recognized during the f i r s t stage of ATA. Second Stage - The disappearance of the l o c a l symptoms which characterize the f i r s t stage i s immediately followed by a symptomless period of 2 weeks :t 2 months when the'toxin exerts i t s destructive action on the bone marrow. This e f f e c t i s manifested by v i s i b l e haemorrhagic spots on the skin. Haematological examination of the peripheral blood reveals a state of progressive leucopenia, a granulopenia and a r e l a t i v e lymphocytosis. This stage i s often c a l l e d the leucopenic stage. There is also a decrease in erythrocytes, p l a t e l e t s and haemoglobin. Neutrophils show toxic granulation Weakness, v e r t i g o , headache, p a l p i t a t i o n and s l i g h t asthma may occur at t h i s stage. The skin and mucous membranes may be i c t e r i c , the pupil s of the eye 20 may be d i l a t e d , the pulse i s soft and l a b i l e , the blood pressure i s decreased, the sounds of the heartbeat appear muffled and the patient becomes f e b r i l e . I f consumption of toxic food i s discontinued, there i s usually a complete recovery or the disease may then proceed to the t h i r d stage. Third Stage - The onset of the t h i r d stage i s usually abrupt and may be e s p e c i a l l y v i o l e n t under conditions of stress such as physical exertion, fatigue, common cold and i n s o l a t i o n . In t h i s stage, four e s s e n t i a l c l i n i c o -p athological changes due to t o t a l bone marrow atrophy develop: (1) haemorrhagic d i a t h e s i s (2) necrosis, e s p e c i a l l y necrotic angina (3) septic state (4) t y p i c a l haematological changes. This stage l a s t s from 5-20 days. The f i r s t v i s i b l e sign of t h i s stage i s the appearance of petechial haemorrhages on the skin of the trunk, chest and abdomen, in the a x i l l a r y and inguinal regions, on the l a t e r a l surfaces of the arms and thighs, on the inner areas of the body which are exposed to f r i c t i o n , on the mucous membranes of the mouth, on the tongue, palate and t o n s i l s . Necrotic angina follows. These necrotic changes i n the mouth and throat begin i n the form of a c a t a r r h a l condition. The basis of these necrotic changes i n the mouth and throat i s the impairment of the leu c o c y t i c -phagocytic and the r e t i c u l o e n d o t h e l i a l functions of the body defenses. This impairment and the changed permeability of the blood vessels favour the penetration pf i n f e c t i v e agents into the tissues and into the blood stream (Mayer, 1953). The lesions i n the mouth extend to the palate, gums, mucosa of the cheeks and descend to the larynx, vocal cords, lungs, stomach, bowels etc. Necrosis may develop i n several i n t e r n a l organs. The lesions are round, 5-7 mm i n diameter, of greyish colour, situated on a hyperaemic and 21 b l e e d i n g m u cosal s u r f a c e w h i c h shows no p r o t e c t i v e i n f l a m m a t o r y r e a c t i o n . The l e s i o n s f u s e i n t o a b l a c k f u n g o i d mass i n s e v e r e c a s e s . By f u s i n g , t h e l e s i o n s d e v e l o p t o a c o n s i d e r a b l e s i z e and p e n e t r a t e t h e m u s c u l a r t i s s u e below. Secondary i n f e c t i o n by b a c t e r i a o c c u r s . The r e g i o n a l lymph g l a n d s become s w o l l e n . F r e q u e n t l y t h e s u b m a n d i b u l a r and c e r v i c a l lymph g l a n d s become so s w o l l e n t h a t t h e p a t i e n t e x p e r i e n c e s d i f f i c u l t y i n s w a l l o w i n g . I n some c a s e s d e a t h may be due t o s t r a n g u l a t i o n . F o u r t h S t age - The d i r e c t i o n and n a t u r e o f t h e f o u r t h s t a g e depends on the s e v e r i t y o f t h e t o x i c o s i s . Hence t h e d u r a t i o n o f t h e r e c o v e r y p e r i o d i s v a r i a b l e . The n e c r o t i c and h a e m o r r h a g i c phenomena d i s a p p e a r a f t e r about two weeks o f t r e a t m e n t . F o r the r e p a i r o f t h e damage t o t h e h a e m a t o p o i e t i c s ystem, a much l o n g e r p e r i o d i s r e q u i r e d . I t may t a k e two months b e f o r e a l l f u n c t i o n s o f t h e bone marrow a r e back t o n o r m a l : f i r s t t h e l e u c o c y t e s , t h e n t h e g r a n u l o c y t e s , t h e p l a t e l e t s and f i n a l l y t h e e r y t h r o c y t e s . 3. PATHOLOGY The changes i n t h e b l o o d p i c t u r e f o l l o w a p a t t e r n c o r r e s p o n d i n g t o th e c l i n i c a l s t a g e s o f t h e d i s e a s e . I n t h e f i r s t s t a g e , t h e r e i s a s l i g h t l e u c o c y t o s i s and a t t h e same t i m e a d i s a p p e a r a n c e o f e o s i n o p h i l s , a ly m p h o p e n i a and a n e u t r o p h i l i a . T h i s i s t h e r e s u l t o f t h e a c t i o n o f t h e t o x i n on t h e bone marrow. As more t o x i n i s i n g e s t e d , t h e a c t i o n on t h e bone marrow i s g r e a t e r and t h e r e i s an i n c r e a s e i n t h e p e r i p h e r a l b l o o d e l e m e n t s . There i s a moderate l e u c o p e n i a , a moderate h y p e r c h r o m i c anaemia and a drop i n t h e p l a t e l e t c o u n t . I n t h e t h i r d s t a g e a c r i t i c a l low i n t h e l e u c o c y t e c o u n t i s r e a c h e d . Three s t a g e s a r e d i s t i n g u i s h e d i n t h e development of f u n c t i o n a l changes i n t h e h a e m a t o p o i e t i c s y s t e m and a r e due t o : (1) stimulation by smaller doses of the entering poison (2) oppression by larger accumulated doses (3) regeneration of the impaired elements. The f i r s t of these is detected in the peripheral blood p i c t u r e . The peak of the oppression i s studied from both post mortem and c l i n i c a l f i n d i n g s . At t h i s stage, the bone marrow shows the t y p i c a l macroscopic and microscopic appearance of global myelotoxicosis of a l l the blood forming elements. In the necrotic stage of ATA the e n t i r e lymphatic and r e t i c u l o e n d o t h e l i a l systems are affected together with the spleen and l i v e r . 4. AETIOLOGY Russian workers have i s o l a t e d numerous fungi from the toxic cereals causing ATA. Included among these were representatives from the following genera: A l t e r n a r i a Fusarium Cladosporium P e n i c i l l i u m A s p e r g i l l u s The Fusarium species have been most frequently implicated in ATA. Fusarium  sporotrichoides was observed to be the chief cause of spoilage of over-wintered grain. Sarkisov found 222 samples of toxic g rain which contained F. sporotrichoides and feeding experiments with t h i s fungus produced diseases s i m i l a r to ATA. The Fusarium species are members, many having imperfect forms, of the c l a s s Ascomycetes. They have been included with the Fungi Imperfecti. The genus Fusarium includes a l l fungi with c o n i d i a of a v i v i d colour (white, 23 y e l l o w , orange, b l a c k ) ; i t has a s p r e a d i n g m y c e l i u m w i t h s p i n d l e - s h a p e d c o n i d i a ( w h i c h a t m a t u r i t y a r e m a n y - c e l l e d ) and b r a n c h i n g c o n i d i o p h o r e s . The fungus m i g h t a t t a c k any p a r t of the g r a i n k e r n e l b u t u s u a l l y t h e e n t i r e g r a i n i s c o v e r e d w i t h f i n e m y c e l i u m and t o x i n i s produced by some s t r a i n s under f a v o u r a b l e c l i m a t i c c o n d i t i o n s . A f t e r t h e tha w i n g o f the snow t h e f i n e m y c e l i u m w i l l d r y v e r y q u i c k l y and w i l l n o t be e a s i l y seen w i t h the naked eye (Mayer, 1953). 5- ISOLATION AND CHARACTERIZATION OF TOXIN S i n c e t h e R u s s i a n w o r k e r s i n d i c a t e t h a t t h e t o x i n i s c o n t a i n e d i n the l i p i d p o r t i o n o f the g r a i n , the t o x i n must be i s o l a t e d and i t s t o x i c i t y c h e c k e d . F a t s o l v e n t s ( e t h e r , a c e t o n e , c h l o r o f o r m , p e t r o l e u m e t h e r ) a re used f o r e x t r a c t i o n o f the t o x i n . The o f f i c i a l t e s t used i n R u s s i a f o r t h e d e t e c -t i o n o f t o x i c i t y i n g r a i n employs e t h e r f o r t h e e x t r a c t i o n o f t h e s u s p e c t e d t o x i n s . Two e t h e r e x t r a c t s a r e p r e p a r e d i n a S o x h l e t a p p a r a t u s , each from 50 grams of g r a i n . The f i r s t i n v o l v e s e x t r a c t i o n f o r s i x h o u r s and the second f o r t h r e e d a y s . 6. CHEMICAL AND BIOLOGICAL CHARACTERISTICS OF THE TOXIN Sev e r a l , b i o l o g i c a l t e s t s have been p r o p o s e d f o r the d e t e c t i o n o f t o x i c g r a i n . (1) The a p p l i c a t i o n o f the e t h e r e x t r a c t o f t h e g r a i n t o s e v e r a l denuded s p o t s on the s k i n o f a w h i t e r a b b i t . I f t h e e x t r a c t i s n o n - t o x i c , the s k i n r e m a i n s normal o r t h e r e i s o n l y m i l d s c a l i n g . The t e s t i s d o u b t f u l i f t h e r e i s s l i g h t oedema, r e d n e s s and s c a l i n g o f t h e s k i n w i t h i n 7 d a y s . I f t h e e x t r a c t i s t o x i c , 24 there w i l l be oedema, redness and necrosis 2 hours a f t e r a p p l i c a t i o n of the extract to the skin. This i s the o f f i c i a l t e s t . (2) The addition of an ethanol extract to a culture of Saccharomyces  cer e v i s i a e to see i f the extract w i l l suppress the fermentative powers of the yeast (Mishoosten, 1946). (3) A pharmacological test in which a drop of the extract i s tested to ascertain i f i t w i l l cause the w i l t i n g of chrysanthemums (E l p i d i n a , 1945). (4) A test which involves a change in colour of a c i d i f i e d grain by the addition of potassium permanganate and methylene blue (Manilova, 1947). (5) Kosin and Yershov (1948) worked out a method for detection of t o x i c i t y in grain through changes in colouration of a s o l u t i o n of t i n i n hydrochloric acid., (6) A l i s o v a (1946) obtained a serum p r e c i p i t a t e through the immuniza-t i o n of rabbits with condensates of fungal medium of Fusarium  poae. The toxin r e s i s t s heating (100° C), acid and a l k a l i treatments. Heat-ing at 121° C in an autoclave did not destroy the t o x i c i t y of the grain. Darkened seeds proved to be most t o x i c . The t o x i c i t y of the grain i s said to be unchanged by cooking and storage. The percentage of t o t a l nitrogen in toxic grain i s decreased whereas there i s an increase in non-protein amino nitrogen. The infected seeds contain less starch, t h e i r oxidase and peroxidase i s decreased while t h e i r amylase i s increased. The toxin of proso i s a nitrogen containing phytotoxin which i s said 25 t o d e s t r o y complex n i t r o g e n s u b s t a n c e s i n t h e a n i m a l body e s p e c i a l l y t he immunoproteins o r a n t i b o d i e s (Mayer, 1953). The t o x i n may be c o n s i d e r e d an a n t i b i o t i c . I t i s a l s o t hought t h a t i t i s a l i p o p r o t e i n t h a t s p l i t s i n t h e i n t e s t i n e i n t o p r o t e i n and a n o t h e r s u b s t a n c e w h i c h behaves l i k e an o x y d e r i v a t i v e o f c o u r m a r i n . C h l o r a m p h e n i c o l has been r e s p o n s i b l e f o r a number o f d e a t h s due t o anaemia, g r a n u l o c y t o p e n i a , f a t a l a p l a s t i c anaemia and h e p a t i t i s i n t h e U n i t e d S t a t e s ( S t u r g e o n , 1952; C l a n d o n e t a l , 1952; S m i l e y e t a l , 1952). The bone marrow p i c t u r e i n t h e s e c a s e s was s i m i l a r t o t h a t caused by ATA i n R u s s i a . I t i s n o t known whether t h e t o x i n i s a m i x t u r e o f w h i c h p a r t i s an a n t i b i o t i c d e r i v e d from t h e f u s a r i u m and a p a r t i s a t o x i n from t h e g r a i n . There a r e two f r a c t i o n s o f the t o x i n , (1) an i n f l a m m a t o r y agent w h i c h causes h y p e r a e m i a , oedema and i n f l a m m a t i o n o f t h e s k i n , (2) a n e c r o t i c f a c t o r p r e s e n t i n t h e u n s a p o n i f i a b l e f r a c t i o n o f t h e e x t r a c t and w h i c h c a u s e s n e c r o s i s . 7. SIGNIFICANCE IN HUMAN AND ANIMAL NUTRITION AND DISEASE Anyone who has consumed a l a r g e q u a n t i t y o f g r a i n made t o x i c by F. s p o r o t r i c h o i d e s may d e v e l o p ATA. The d i s e a s e u s u a l l y a f f e c t s humans between the ages o f 8 and 50 y e a r s . O u t s i d e t h i s age range t h e d i s e a s e may assume u n u s u a l s e r i o u s n e s s . The t h e o r y t h a t t h e d i s e a s e was due t o a d e f i c i e n c y o f v i t a m i n s o f the B complex o r a s c o r b i c a c i d has been d i s p r o v e d , but a d e f i c i e n c y o f t h e s e v i t a m i n s d o e s ' a g g r a v a t e t h e c o n d i t i o n . I n c l u s i o n o f t h e s e v i t a m i n s i n t h e d i e t o f a p e r s o n a f f e c t e d w i t h t h e d i s e a s e r e s u l t s i n a r e d u c t i o n i n t h e s e v e r i t y o f t h e syndrome. The two chief methods for the diagnosis of ATA are: (1) The examination of the grain for a toxin or toxin-producing fungus. (2) An evaluation of the blood p i c t u r e . Complete recovery from the disease depends on the amount of toxic grain consumed, the extent of the i n t o x i c a t i o n , the complications that may have resulted and the type of treatment and care which the patient receives. The three chief causes of death are: (1) bleeding and sepsis (2) stenosis of the g l o t t i s (3) multiple embolism of the b r a i n and volvulus of the i n t e s t i n e . The fate of the toxin in the animal i s unknown and the exact method of involvement of the haematopoietic system i s yet to be ascertained. M o r t a l i t y from the disease i s extremely rare during the f i r s t and second stages and then i t i s the r e s u l t of massive haemorrhage in the prenecrotic phase. About 80 per cent of those who die reach the t h i r d stage and may show signs of oedema of the g l o t t i s with asphyxia (Mayer, 1953). Recurrences of. the diesease are possible and there is no s p e c i f i c immunity. Ingestion of toxic grain w i l l always cause a new attack, or a relapse may occur a f t e r recovery from the t h i r d stage i f the patient undergoes physical s t r a i n or develops i n f e c t i o n . 8. TREATMENT AND CONTROL There i s no s p e c i f i c treatment. Since the toxin i s non-antigenic, immunization i s impossible. The methods for preventing the disease include the elimination of the toxic food from the d i e t , stimulation of haemato-p o i e s i s , blood transfusions, preventing secondary in f e c t i o n s and 27 r e h a b i l i t a t i o n on a favourable d i e t . During the second stage of the disease, sulphonamides, a n t i b i o t i c s , vitamins and calcium preparations may be given. Mouthwash, purgatives and emetics are also used. 1. DISEASE Stachybotryotoxicosis i s one of the mycotoxicoses caused by the ingestion of toxic feedstuffs and straw in which the fungus Stachybotrys atra is present. The disease, l i k e the other mycotoxicoses, Is caused by a toxin produced by the fungus i n the feed. It a f f e c t s horses although other animals and humans are suceptible. Drobotko (1945), Vyshelesski (1948) and others have reported on t h i s disease in horses. Boichenko and Kalitenko (1959) have found t h i s disease i n c a t t l e which had ingested straw infected with the fungus. In the United States, Forgacs and co-workers (1958) have experi-mentally induced the disease i n horses, c a t t l e and other animals and have reported on the s i m i l a r i t y between t h i s disease and that described i n the Russian l i t e r a t u r e . kept i n stables and fed straw which has been stacked under conditions favouring mould growth. During the winter, the incidence of the disease increases reaching i t s peak from February to A p r i l . The m o r t a l i t y rate during t h i s period may be quite high. When the horses are turned out to pasture i n the springtime, the disease subsides. This t o x i c o s i s i s endemic; It recurs i n succeeding years on farms where i t has been present. STAGHBOTRYOTOXICOSIS The disease i s seasonal, beginning i n the f a l l when the animals are 28 2. CLINICAL SYMPTOMS The symptoms o f t h e d i s e a s e i n h o r s e s v a r y w i d e l y d e p e n d i n g on the c o n c e n t r a t i o n o f t h e f u n g a l t o x i n i n the mouldy f e e d . S t a c h y b o t r y o t o x i c o s i s may o c c u r i n e i t h e r o f two f o r m s , t h e t y p i c a l f orm w h i c h i s d i v i d e d i n t o t h r e e s t a g e s and t h e a t y p i c a l o r " s h o c k i n g " form. TYPICAL FORM F i r s t S t a g e - As a r e s u l t o f the i n g e s t i o n o f s u b l e t h a l amounts o f s t r a w o r hay c o n t a m i n a t e d w i t h S. a t r a , h o r s e s d e v e l o p c h a f f i n g o f t h e l i p s and c o r n e r s o f t h e mouth w i t h i n one t o t h r e e d a y s . The l i p s a r e u s u a l l y s w o l l e n . I n 2-10 days s t o m a t i t i s d e v e l o p s . The mucous membranes of t h e mouth c a v i t y a re h a e m o r r h a g i c , s w o l l e n and g e n e r a l l y d i s e a s e d . L a r g e a r e a s o f n e c r o s i s appear on t h e i n s i d e s o f the c h e e k s , on t h e gums, p a l a t e and on t h e tongue. E x c e s s i v e s a l i v a t i o n o c c u r s and i s g e n e r a l l y accompanied by an o f f e n s i v e odour. The s u b m a x i l l a r y lymph nodes become e n l a r g e d and p a i n f u l t o t h e t o u c h . There i s a marked l o s s o f a p p e t i t e . The body t e m p e r a t u r e i s normal a l t h o u g h i n c r e a s e s o f 0.5° - 1.0° C f o r a s h o r t d u r a t i o n o c c u r . I n some a n i m a l s t h e l e s i o n s a r e n o t i c e a b l e as o n l y a s l i g h t p e e l i n g o f t h e l i p s and o r a l mucosa and may v e r y e a s i l y be o v e r l o o k e d . The f i r s t s t a g e may l a s t f r om 8-12 d a y s , b u t may e x t e n d f r o m 15-30 days i n c a s e s where v e r y s m a l l amounts of t o x i c f o d d e r a r e consumed. Removal o f t h e t o x i c f o d d e r f r o m t h e d i e t d u r i n g t h i s s t a g e r e s u l t s i n c o m p l e t e r e c o v e r y from t h e d i s e a s e . .. .. Second Stage - I n c a s e s where t h e d i s e a s e i s p r o l o n g e d by t h e c o n t i n u o u s i n g e s t i o n o f the t o x i n , t he h e a l t h o f t h e a n i m a l s d e t e r i o r a t e s . There a r e changes i n t h e b l o o d p i c t u r e . There i s a marked t h r o m b o c y t o p e n i a and l e u k o p e n i a . The t h r o m b o c y t e s d e c r e a s e f r o m 40-60 p e r 3000 e r y t h r o c y t e s t o 8-15 p e r 3000 e r y t h r o c y t e s , w h i l e t h e l e u c o c y t e s d e c r e a s e t o about 200 o r 29 less per cubic m i l l i m e t e r of blood. Blood c o a g u l a b i l i t y i s lessened. During t h i s stage the symptoms of the f i r s t stage are either t o t a l l y absent or appear as an intense necrosis of the mucous membranes of the mouth and l i p s . This stage l a s t s from 15-20 days. Removal of the toxic fodder during t h i s stage w i l l not ne c e s s a r i l y lead to a cessation of the disease. Third Stage - This stage i s characterized by an elevation of the body temperature as high as 41.5° C. Heart a c t i v i t y diminishes and the blood p i c t u r e becomes aggravated. The pulse weakens, i n t e s t i n a l disorder i s frequently observed and most of the animals appear depressed. The mucosa of the o r a l c a v i t y becomes very oedematous, b a c t e r i a l i n f e c t i o n takes place and a f o u l odour emanates from the mouth. There i s a decrease i n thrombocytes to 3-1 per 3000 erythrocytes and the leucocytes to 400-100 per cubic millimeter of blood. This stage l a s t s from 1-6 days and the f a t a l i t y rate i s exceedingly high. Pregnant mares us u a l l y abort before death. ATYPICAL FORM The a t y p i c a l or "shock" form of stachybotryotoxicosis develops a f t e r the ingestion of large amounts of toxic fodder. It occurs with 5-10 hours a f t e r ingestion of the toxin. This type of t o x i c o s i s i s rare among farm animals. The c l i n i c a l p i c t u re of the "shock" form i s e n t i r e l y d i f f e r e n t from that of the t y p i c a l form. This condition i s characterized by the rapid onset of the disease and general d e t e r i o r a t i o n of the animal. The l o c a l necrosis of the t y p i c a l form i s absent i n the a t y p i c a l . The symptoms are those of increased body temperature, disorder of the cardiovascular sys-tem, oedema of the lungs and a general disorder of the nervous system. There i s a loss of r e f l e x action and marked h y p e r i r r i t a b i l i t y . Blindness and bleeding from the nose and anus may occur. The duration of the i l l n e s s i s from 24-36 hours and the r e s u l t i s generally f a t a l . 30 The horse becomes s l u g g i s h and avoids movement whenever p o s s i b l e . In c o n t r a s t w i t h the t y p i c a l form, there are no blood d y s c r a s i a s . Increased p e r i s t a l t i c a c t i o n and diarrhoea are sometimes observed. Death i s u s u a l l y due to r e s p i r a t o r y f a i l u r e . 3. PATHOLOGY Macroscopic - S t a c h y b o t r y o t o x i c o s i s i s c h a r a c t e r i z e d by profuse haemorrhage i n many t i s s u e s and i n some cases n e c r o s i s . The mucous and serous membranes of the body as w e l l as the subcutaneous t i s s u e and muscle, the spleen, l a r g e i n t e s t i n e , mesenteries, c o s t a l p l e u r a , diaphragm and other parenchymatous organs are p r i m a r i l y a f f e c t e d . The lungs are oedematous and haemorrhagic, and haemorrhages are a l s o observed i n the b r a i n , s p i n a l cord, meninges, suprarenals and lymphatic nodes. The heart and l i v e r are not markedly a f f e c t e d . As mentioned p r e v i o u s l y , the mucous membranes of the mouth and g a s t r o i n t e s t i n a l t r a c t become inflamed and develop areas of necro-s i s . In the buccal c a v i t y the n e c r o t i c l e s i o n s appear as ovoid, y e l l o w i s h -grey papules. The lymph nodes e s p e c i a l l y the submaxillary glands are swollen and haemorrhagic. Mic r o s c o p i c - In the haematopoietic bone marrow, c e l l u l a r elements are decreased e s p e c i a l l y the granulocytes. The haemorrhagic f o c i i n v a r i o u s t i s s u e s c o n t a i n very small pigmented bodies which were thought to be of fungal nature. However Forgacs and co-workers (1958) were unable to i s o l a t e S. a t r a from l e s i o n s present i n a c a l f which had died from t h i s t o x i c o s i s . 4. AETIOLOGY Causative agent - Stachybotrys a t r a i s an imperfect form of the c l a s s Ascomycetes, order M o n i l i a l e s , f a m i l y Dematiaceae. Corda (1937) founded the 31 genus Stachybotrys w i t h one species S. a t r a . This f a m i l y i s c h a r a c t e r i z e d by s i n g l e dark conidiophores a p i c a l l y bearing a c l u s t e r of t h i c k and short p h i a l i d e s ( b a s i d i a ) . C o n i d i a are borne at the apex of each p h i a l i d e , each conidium being s i n g l e , dark, subglobose to ovoid. The d i s t r i b u t i o n of S. a t r a i s worldwide and the fungus can grow at temperatures ranging from 2° - 25° C. The fungus favours a moisture content of 60-100 per cent, but a r e l a t i v e humidity of only 30 per cent i s r e q u i r e d f o r i n i t i a t i n g growth. The fungus does not grow p r o f u s e l y as compared w i t h the a s p e r g i l l i , p e n i c i l l i a , mucor and the l i k e . Thus i t i s d i f f i c u l t to i s o l a t e . The c o n i d i a survive temperatures of -40° C, but under c o n d i t i o n s of high humidity they d i e w i t h i n one hour at 60° - 65° C and w i t h i n 5 minutes at 100° C. The fungus i s destroyed at 80° C i n one hour. Spores are destroyed during the biothermal decomposition of manure yet survive passage through the gut. 5. ISOLATION AND CHARACTERIZATION OF TOXIN Forgacs and co-workers (1958) observed that the fungus f l o u r i s h e d and produced t o x i n f o r long periods on c e r t a i n s u b s t r a t e s . Using a s p e c i a l s t r a i n of S. a t r a under l a b o r a t o r y c o n d i t i o n s , they found that the t o x i n i s formed i n moist straw w i t h i n 10 days and a t t a i n s a high l e v e l i n 19 days which i s maintained f o r at l e a s t 414 days. The t o x i c substance can be e f f e c t i v e l y e x t r a c t e d from the straw w i t h i n three hours, the bulk of the m a t e r i a l being removed during the f i r s t hour. Anhydrous ether was the best solvent f o r e x t r a c t i o n . Toxic s t r a i n s of S. a t r a are determined by applying ether e x t r a c t s to shaved areas on a r a b b i t ' s s k i n and observing the dermal inflammatory r e a c t i o n produced. From experimental observations, i t was found that the t o x i n reacted 32 TABLE I I E f f e c t o f I n u c b a t i o n P e r i o d of S t r a w I n o c u l a t e d w i t h S. a t r a and Dermal  T o x i c i t y o f E x t r a c t s . * I n c u b a t i o n P e r i o d Wt (mg) E t h e r - E x t r a c t a b l e Dermal (days) M a t e r i a l /0.125 ml R e a c t i o n 10 0.6 SE 19 1.3 N 25 1.6 N 33 1.6 N 47 1.7 N 74 1.6 N 109 1.0 N 125 0.9 N 160 0.8 N 220 0.8 N 414 0.4 N SE = s l i g h t erythema N = n e c r o s i s * F o r g a c s et a l ( 1 9 5 8 ) . TABLE I I I I n f l u e n c e o f Time of E x t r a c t i o n o f S. a t r a I n o c u l a t e d S t r a w on Removal o f D e r m a l l y T o x i c S u b s t a n c e s . * E x t r a c t i o n Dermal Time ( h o u r s ) Per Cent Wt. (mg) /0.125 ml R e a c t i o n 1 1.28 1.750 PE 2 0.12 0.577 ME 3 0.08 0.117 ME 4 0.05 0.061 0 5 0.03 0.061 0 6 0.03 0.037 0 7 0.03 0.036 0 PE = pronounced erythema ME = moderate erythema 0 = n e g a t i v e r e a c t i o n * F o r g a c s e t a l (1958) 33 with the gastric juice In the stomach and was absorbed in a water-soluble form. This observation was confirmed by digestion of toxic straw with pepsin at a low pH which rendered the toxin water-soluble and left the extracted straw non-toxic. The toxin is extremely potent (Drobotko, 1945, Forgacs et al , 1958). It is resistant to sunlight, ultra violet light and X-rays. It is thermo-stable. It is not inactivated by a two per cent concentration of organic or inorganic acids, but is destroyed by alkalis. The toxin is non-antigenic. 6- SIGNIFICANCE IN HUMAN AND ANIMAL NUTRITION AND DISEASE For a number of years the horse was thought to be the only large animal susceptible to stachybotryotoxicosis, but it has recently been discovered that other farm animals, e.g., cattle (Boichenko and Kalitenko, 1959), sheep and swine (Forgacs et al , 1958) could also develop this form of toxicosis. Boichenko and Kalitenko (1959) report on cases of poisoning in cattle by S. atra toxin during 1958-59 in five provinces of the Ukraine where an estimated 5000 head of cattle died. The illness was more intense in adult cattle than in the young stock. The symptoms were the same as observed in horses where there was diarrhoea, elevation of body temperature, haemorrhages and necrosis of the mucous membranes etc. Milk production dropped 25-30 per cent in affected cows. Al l external symptoms were similar to those seen in horses. The animals died within 4-6 days. The blood picture was similar to that of horses affected by the toxin. The patho-logical conditions displayed by cattle were haemorrhage in various tissues, degeneration of the liver and disorder of the alimentary canal. Forgacs et al (1958) found that the calf was less susceptible than other animals. In experimentally induced toxicosis in calves, death was 34 p r o d uced w i t h i n 17 days. I n sheep and s w i n e , d e a t h o c c u r r e d w i t h i n 24 h o u r s . W i t h t h e l a b o r a t o r y a n i m a l s i t was found t h a t mice and g u i n e a p i g s were h i g h l y s u s c e p t i b l e , d y i n g w i t h i n 5 days a f t e r d a i l y f e e d i n g w i t h t o x i c o a t s . Dogs d i e d i n 4-6 days a f t e r d a i l y a d m i n i s t r a t i o n o f t h e t o x i n . W i l s o n and W i l s o n (1961) d e m o n s t r a t e d t h a t c e r t a i n moulds produce o x a l i c a c i d i n c e r e a l g r a i n s i n amounts w h i c h are t o x i c f o r c h i c k e n s . Schumaier et a l (1963) d e m o n s t r a t e d t h a t a l i p i d - s o l u b l e n e c r o t i c , agent produced by S. a t r a d e s t r o y s t h e e p i t h e l i u m o f t h e mouth and c r o p of t h e c h i c k . F o l l o w i n g t h i s , Greek (1964) t e s t e d w a t e r and l i p i d - s o l u b l e f r a c t i o n s o f S. a t r a i n r e l a t i o n t o o x a l i c a c i d t o x i c i t y f o r c h i c k s . They found t h a t the w a t e r - s o l u b l e f r a c t i o n d e p r e s s e d growth r a t e by about 30 p e r c e n t and damaged th e e p i t h e l i u m o f t h e mouth. At the 3 p e r c e n t l e v e l o x a l i c a c i d r e d u c e d g r o wth d r a s t i c a l l y b u t d i d n o t damage the e p i t h e l i u m of t h e mouth and c r o p . S. a t r a t o x i n and o x a l i c a c i d were t h e r e f o r e c o n c l u d e d t o be d i s t i n c t l y d i f f e r e n t . D r o b o t k o (1945) r e p o r t e d on an i l l n e s s i n humans who had been exposed t o hay or s t r a w i n f e c t e d w i t h S. a t r a . T h i s i l l n e s s m a n i f e s t e d i t s e l f as a r a s h on t h e s k i n e s p e c i a l l y on t h e p a r t s most s u b j e c t t o p e r s p i r a t i o n . The r a s h f r e q u e n t l y became a m o i s t d e r m a t i t i s and t h e a f f e c t e d a r e a was c o v e r e d w i t h d r i e d l a y e r s o f s e r o u s e x u d a t e s from t h e s k i n . S i m u l t a n e o u s l y t h e r e o c c u r r e d c a t a r r h a l o r a n g i n a l p h a r y n g i t i s and r h i n i t i s w i t h b l o o d exudate and cough. The p a t i e n t c o m p l a i n e d o f p a i n s i n the t h r o a t , b u r n i n g i n the nose and c o n g e s t i o n i n the c h e s t . The body t e m p e r a t u r e u s u a l l y r emained n o r m a l . By a p p l y i n g t h e mould i t s e l f t o human s k i n i t was p o s s i b l e t o p r o d u c e th e t y p e of d e r m a t i t i s as seen i n t h e n a t u r a l human i n f e c t i o n . I n some c a s e s t h e r e was l e u c o p e n i a , but n e v e r below 2000 p e r c u b i c m i l l i m e t e r of b l o o d . T h i s i n d i c a t e d t h a t t h e a b s o r p t i o n o f t h e t o x i c s u b s t a n c e t h r o u g h / t h e s k i n and i n h a l a t i o n o f t h e s p o r e s a r e s u f f i c i e n t t o cause p o i s o n i n g . There i s no immunity t o t h e d i e s e a s e and r e c u r r e n c e o f t h e t o x i c o s i s among t h e same a n i m a l s i s p o s s i b l e . The d i s e a s e i s n o t c o n t a g i o u s . I n e x p e r i m e n t a l c a s e s where h e a l t h y h o r s e s have been k e p t i n c l o s e c o n t a c t w i t h a f f e c t e d ones, but f e d a d i f f e r e n t b a t c h o f hay, t h e h e a l t h y ones d i d not d e v e l o p t h e d i s e a s e . S t a c h y b o t r y o t o x i c o s i s i n h o r s e s and c a t t l e d i s p l a y s a h i g h m o r t a l i t y r a t e . 7. TREATMENT AND CONTROL As soon as a n i m a l s become i n f e c t e d a l l s u s p e c t e d f o d d e r s h o u l d be o m i t t e d f r o m t h e r a t i o n s . Treatment o f a f f e c t e d a n i m a l s v a r i e s w i t h t h e ty p e and s t a g e o f t h e t o x i c o s i s . I n t h e f i r s t s t a g e t h e t o x i c f o d d e r s h o u l d be removed and t h e b e d d i n g changed. I n t h e second and t h i r d s t a g e s v a r i o u s t h e r a p e u t i c a g e n t s have been s u g g e s t e d . I n o r d e r t o d i s i n f e c t t h e gut and t o s t i m u l a t e h e a r t a c t i v i t y , 10 p e r c e n t s t e r i l i z e d s o l u t i o n o f CaCl2 i n t r a -v e n o u s l y i n q u a n t i t i e s o f 100-200 ml and a d r e n a l i n e s u b c u t a n e o u s l y s h o u l d be c o n s e c u t i v e l y i n j e c t e d . I n c a t t l e , B o i c h e n k o and K a l i t e n k o (1959) s u g g e s t e d t h e f o l l o w i n g : -(1) The i n t r o d u c t i o n o f a 10 p e r c e n t N a C l s o l u t i o n (200 ml) i n t o t h e stomach d a i l y f o r 2-3 days. (2) A d m i n i s t r a t i o n o f a s o l u t i o n o f 10-15 grams o f w h i t e h e l l e b o r e i n 500 ml o f w a t e r . (3) F o r t h e e l i m i n a t i o n of d e f e c t s i n t h e b l o o d c i r c u l a t o r y system, t h e i n t r o d u c t i o n i n t o t h e b l o o d o f a g l u c o s e s o l u t i o n w i t h an a d d i t i o n o f benzo-sodium s a l t o f c a f f e i n i n normal d o s e s . (4) For the removal of in f e c t i o u s material from the stomach, stomachs are washed out with a 1/1000 so l u t i o n of potassium permanganate or a 1-2 per cent s o l u t i o n of sodium bicarbonate. The most e f f e c t i v e r e s u l t s are obtained when treatment i s applied r i g h t at the s t a r t of the i l l n e s s . In t h i s way, the recovery i s usually 90-100 per cent. Horses that have recovered from the i l l n e s s should not be worked for a month. Thus i n an attempt to control stachybotryotoxicosis, the most e f f e c t i v e approach i s prophylaxis. (1) The prevention of the contamination of fodder by fungi. (2) The prevention of the spreading of the fungus beyond areas already infected. Dark so o t - l i k e straw indicates that S. atra i s already present i n dangerous q u a n t i t i e s . (3) The a n n i h i l a t i o n of e x i s t i n g hotbeds of the toxic fungus on the farms. YELLOWED RICE DISEASE 1. DISEASE Throughout the r i c e eating countries of the world, there has been a high Incidence of l i v e r carcinoma, l i v e r c i r r h o s i s and other l i v e r diseases. In recent years the Japanese have investigated t h i s condition and have traced i t s occurrence to the presence of a toxic agent(s) In "yellowed" r i c e . Various fungi were is o l a t e d from t h i s r i c e Including P e n i c i l l i u m islandicum  Sopp. The climate of the country which i s suitable for the growing of r i c e 37 also affords favourable conditions for the growth of fungi, not only i n farmyards and warehouses but also in the consumers' kitchens. It i s quite possible that the l i v e r injury and other l i v e r symptoms could be due to a n u t r i t i o n a l factor l i k e p r otein or choline deficiency, but the Japanese workers were unwilling to accept a n u t r i t i o n a l aetiology to explain the high incidence of hepatic carcinomas among the population. It was suspected that some unknown substance contributing to l i v e r injury and carcinogenic action was present in the food. Rice being the staple food was therefore the primary suspect. Thus the Japanese workers, suspecting yellowed r i c e to be the cause of the hepatic condition, aimed at the follow-ing targets to elucidate the nature and cause of these hepatomas. (1) A knowledge of the c h a r a c t e r i s t i c s of the poisonings by the so-c a l l e d yellowed r i c e , e s p e c i a l l y a l t e r a t i o n s of the l i v e r . (2) The discovery of the o r i g i n of the active agents causing these a l t e r a t i o n s . (3) The i s o l a t i o n and i d e n t i f i c a t i o n of the toxic compounds i n the metabolites of P. islandicum Sopp. (4) An Investigation into the r o l e played by toxic compounds in the development of yellowed r i c e t o x i c i t y and the hepatic a l t e r a t i o n s . 2. CLINICAL SYMPTOMS Mice and rats displayed the following symptoms when exposed to a d i e t of yellowed r i c e . Mice; At the beginning of the feeding period they became inactive with r u f f l e d fur and assumed a crouching p o s i t i o n . In the acute cases feed intake and body weight decreased markedly from the s t a r t whereas in the chronic cases they v a r i e d over a very long period and f i n a l l y decreased as death was 38 approached. The animals became depressed and l o s t t h e i r muscular and cutaneous tensions, r e s p i r a t i o n decreased and they lapsed into a coma. Death followed within a few hours. Rats: Rats proved to be more r e s i s t a n t to the t o x i c o s i s than mice. For the f i r s t two weeks the d a i l y feed intake was almost normal but decreased s l i g h t l y as time elapsed. The rats became inactive with r u f f l e d fur around the 70th day and then feed intake and body weight decreased u n t i l death intervened. About one month p r i o r to death these animals became worse with a general emaciated body condition. In another group of animals feed intake and body weights fluctuated over a 250 day period and then gradually de-creased as death was approached. 3. PATHOLOGY Mice: In the acute cases the l i v e r s of the dead mice were on macroscopic examination d i f f u s e l y atrophic though t h e i r surfaces were smooth and s l i g h t l y yellowish in colour. The c e n t r a l portion of the l i v e r lobules were d i f f u s e l y red and s l i g h t l y excavated. M i c r o s c o p i c a l l y there was centrolobular necrosis of varying degrees with d i f f u s e f a t t y metamorphosis of the l i v e r c e l l s . In the necrotic f o c i there were i n f i l t r a t i o n s of endothelial and lymphatic c e l l s together with debris of necrotic l i v e r c e l l s . Karyorrhexis and k a r y o l y s i s of l i v e r c e l l s were observed in the zones surrounding the necrotic f o c i . In some cases where the mice survived a longer time, there was l i v e r c i r r h o s i s with ascit e s on autopsy. In the more chronic form of the t o x i c o s i s the mice on autopsy displayed l i v e r c i r r h o s i s with d i f f u s e l y granulated surfaces and greyish white coarse nodules i n some cases. M i c r o s c o p i c a l l y there were c i r r h o t i c changes featured by complete reconstruction cf l i v e r lobules forming fibrous septa connecting the p e r i p o r t a l spaces and the centrolobular regions with each other. The coarse nodules were characterized by tumourous p r o l i f e r a t i o n of l i v e r c e l l s . In other cases there was f o c a l necrosis located outside the centrolobular region with i n f i l t r a t i o n of endothelial c e l l s and leucocytes. In some cases only mononuclear c e l l i n f i l t r a t i o n and increased f i b r o s i s i n the p e r i p o r t a l regions were observed. Rats: In the acute cases there were macroscopic changes denoted by d i f f u s e atrophy of the l i v e r . The l i v e r surface was smooth. H i s t o l o g i c a l examina-t i o n of the l i v e r s showed sporadic coagulation and vacuolation of the l i v e r c e l l s but no necrosis was observed in the l i v e r lobules. There was marked pleomorphism of the l i v e r c e l l s with destructive changes of n u c l e i ; i r r e g u l a r trabeculae were also observed i n the l i v e r c e l l s . In the chronic cases there was f i n e to coarse nodular l i v e r c i r r h o s i s . H i s t o l o g i c a l l y there was broad septal f i b r o s i s connecting p e r i p o r t a l spaces with centrolobular regions and occasionally there was p r o l i f e r a t i o n of the b i l e ducts i n these f i b r o t i c areas. Other organs were affected, including the pancreas which showed vacuolation and necrosis of the acinous c e l l s , the kidneys where degeneration of the renal tubules and deposition of calcium-like substance in the medulla were observed, the myocardium, the spleen, the thymus and the t e s t i c l e s . 4. AETIOLOGY The agent responsible for the condition known i n Japan as yellowed r i c e disease i s a fungus i d e n t i f i e d as P e n i c i l l i u m islandicum Sopp. P. i s -landicum Sopp i s described as having conidia which are .ovoid to e l l i p -t i c a l , thin-walled and smooth. The conidiophores are heavy-walled, 40 d u l l y e l l o w t o g r e e n ; c o l o n i e s a r e f i b r o u s t o f l o c c o s e o r f l o c c o s e f u n i -c u l o s e , m o s t l y i n b u f f t o orange shades. Other f u n g i have been i s o l a t e d f r o m y e l l o w e d r i c e known t o have caused t h e d i s e a s e . These a r e : P. c i t r i n u m T h o r n P. t o x i c a r l u m M l k a y e P. r u g u l o s u m Thorn P. tardum Thorn P. c i t r e o v i r i d e B i o u r g e . i j 5. ISOLATION AND CHARACTERIZATION OF TOXIN P. i s l a n d i c u m Sopp was e x t r a c t e d w i t h m e t h anol and s u b j e c t e d t o the f r a c t i o n a t i o n p r o c e d u r e as o u t l i n e d i n F i g . 1. From t h i s m e t h a n o l e x t r a c t a t o x i c compound l u t e o s k y r i n was i s o l a t e d . The f i l t r a t e o f P. i s l a n d i c u m  Sopp c u l t u r e medium was e x t r a c t e d as o u t l i n e d i n F i g . 2. T h i s e x t r a c t gave a t o x i c c h l o r i n e - c o n t a i n i n g p e p t i d e . The d i r e c t i o n o f t h e f r a c t i o n a t i o n was d e t e r m i n e d by t o x i c i t y t e s t i n g i n a n i m a l s and h i s t o p a t h o l o g i c a l e x a m i n a t i o n each t i m e one f r a c t i o n was o b t a i n e d . P. t o x i c a r l u m and P. c i t r i n u m were a l s o a l c o h o l e x t r a c t e d and p r o d u c e d compounds o f a t o x i c n a t u r e but l e s s t o x i c t h a n t h o s e o f P. i s l a n d i c u m Sopp. 6. CHEMICAL AND BIOLOGICAL CHARACTERISTICS OF THE TOXINS Working w i t h the fungus P. i s l a n d i c u m Sopp, t h e Japanese w o r k e r s were a b l e t o i s o l a t e two t o x i c components. One of t h e components was i s o l a t e d f r o m t h e m e t h a n o l e x t r a c t of t h e f u n g a l mat and was s l o w - a c t i n g and l i p o p h i l i c . The o t h e r component was i s o l a t e d f r o m t h e f u n g a l c u l t u r e medium and was f a s t - a c t i n g and h y d r o p h i l i c . Combining i s o l a t i o n p r o c e d u r e s w i t h b i o l o g i c a l t e s t s and p a t h o l o g i c a l e x a m i n a t i o n s , t h e two components were F i g . 1 C h e m i c a l F r a c t i o n a t i o n from t h e Fungus Mat o f P. i s l a n d i c u m Sopp * Fungus mat powder o f P. i s l a n d i c u m M e t h a n o l e x t r a c t e x t r a c t e d w i t h p e t r o l e u m e t h e r P e t r o l e u m s o l u b l e f r a c t i o n R e s i d u e Pigment i n c r y s t a l O i l e x t r a c t e d w i t h e t h e r E t h e r s o l u b l e f r a c t i o n R e s i d u e Methanol s o l u b l e f r a c t i o n suspended i n w a t e r and ex-t r a c t e d w i t h e t h e r Res i d u e E t h e r s o l u b l e f r a c t i o n E t h e r i n s o l u b l e f r a c t i o n f i l t e r e d Pigments i n ether I extracted with 5% NaHCO-Gummy residue NaHCC-3 soluble f r a c t i o n Ether solution extracted with 10% N a 2 C 0 3 1 : N a 0 C O o Ether solution I extracted with 5% NaOH I NaOH soluble f r a c t i o n Luteoskyrin (Pigment X) * Kobavashi et a l , ( 1 9 5 8 ) Ether solution Water sol u t i o n concentrated, extracted with n-butanol n-butanol soluble Extract 42 Fi g - 2 Chemical F r a c t i o n a t i o n from the F i l t r a t e of  P. islandicum Sopp * F i l t r a t e of P. islandicum c u l t u r e media E t h y l acetate s o l u b l e f r a c t i o n concentrated under diminished pressure 1/10 Concentrated S o l u t i o n e x t r a c t e d w i t h e t h y l acetate Water s o l u t i o n e x t r a c t e d w i t h n-butanol n-butanol s o l u b l e f r a c t i o n Water s o l u t i o n C h l o r i n e - c o n t a i n i n g peptide i n c r y s t a l * Kobayashi et a l , (1958) 43 f i n a l l y described as:-(1) luteoskyrin, the l i p o p h i l i c , slow-acting component (2) chlorine-containing peptide, the h y d r o p h i l i c , f a s t - a c t i n g component. Luteoskyrin was i s o l a t e d as yellow hexagonal c r y s t a l s from acetone, melting point 278° C and a molecular formula Cgg 0^, w i t n a skeleton of dihydrobianthraquinone. The compound i s markedly photosensitive. The chlorine-containing peptide is a newly found compound with a v i o l e n t t o x i c i t y and with the following chemical properties: melting point 251° C and a molecular formula C 2 5 I^g N 5 6g G I 2 . I t appears as white needles on c r y s t a l l i z a t i o n from methanol. The compound i s composed fo two 1-serine, one d-serine, one d-amino butryic acid, and one beta-phenyl-beta-amino propionic acid molecules. These amino acids are presumably combined with each other in a r i n g form e i t h e r by the peptide linkage or by the ester linkage between the a l c o h o l i c hydroxl group in a serine and the carboxyl group i n an amino acid. Uraguchi et a l (1961) tested crude extracts of P. islandicum Sopp on mice and r a t s by dosing o r a l l y . The extracts proved toxic for both mice and rats within 24 hours and up to 7 days for doses of 1.5-5.0 grams per kilogram body weight. Extracts suspended in water proved more toxic than those suspended i n o i l . This supported the theory that the h y d r o p h i l i c agent, the chlorine-containing peptide was more potent than the l i p o p h i l i c agent, lut e o s k y r i n . Luteoskyrin, though not e a s i l y absorbable, i s poisonous to mice and r a t s . It causes acute l i v e r i n jury. The death pattern i n the rat i s quite v a r i a b l e but most m o r t a l i t i e s occur within 2-4 days. The LD^Q in mice on subcutaneous i n j e c t i o n i s 147 mg per kilogram body weight and 221 mg per 43A F i g . 7. HO 0 OH S t r u c t u r e of l i i i t e o s k y r i n k i l o g r a m body w e i g h t when g i v e n p e r orum. A c u t e p o i s o n i n g by t h e compound d e v e l o p s r a t h e r s l o w l y and t h e t o x i c symptoms appear about 1 0 h o u r s b e f o r e d e a t h . On a u t o p s y t h e p a t h o l o g i c a l p i c t u r e i s one of l i v e r c i r r h o s i s and l i v e r i n j u r y s i m i l a r t o t h a t d e s c r i b e d e a r l i e r . The c h l o r i n e - c o n t a i n i n g p e p t i d e i s e a s i l y a b s o r b a b l e and i s r a p i d l y i n j u r i o u s t o t h e l i v e r o f mice and r a t s . The l i f e s p a n o f the a n i m a l s when dosed w i t h t h i s compound i s v e r y s h o r t and t h e a n i m a l s succumb i n l e s s t h a n 2 4 h o u r s . Death i s p r o l o n g e d by d e c r e a s i n g dosage. The LD^Q f o r m i c e on subcutaneous i n j e c t i o n i s 0 . 4 7 4 mg p e r k i l o g r a m body w e i g h t , 0 . 3 3 5 mg p e r k i l o g r a m body w e i g h t when g i v e n o r a l l y . Acute p o i s o n i n g by t h e compound d e v e l o p s soon a f t e r a d m i n i s t r a t i o n . The p a t h o l o g i c a l c o n d i t i o n s a r e l i v e r i n j u r y d i s p l a y e d by v a c u o l a t i o n of the l i v e r c e l l s , d i l a t i o n of s i n u s o i d s and haemorrhage. I n l e s s a c u t e c a s e s t h e r e i s d i f f u s e f a t t y metamorphosis of l i v e r c e l l s . P. t o x i c a r i u m - t h e t o x i c agent c o n t a i n e d i n t h e a l c o h o l i c e x t r a c t of t h i s f u n g u s , when o r a l l y a d m i n i s t e r e d t o h i g h e r v e r t e b r a t e s , i n d u c e s an a s c e n d i n g p a r a l y s i s a f f e c t i n g s e l e c t i v e l y the motor neurons of t h e s p i n a l c o r d and t h e m e d u l l a o b l o n g a t a ( U r a g u c h i 1 9 6 1 ) . The t o x i c compound was i s o l a t e d as a y e l l o w pigment o f p o l y e n e . P. c i t r i n u m - c i t r i n i n was i s o l a t e d as a t o x i c pigment from t h e f i l t r a t e o f t h e f u n g a l medium. I t has an LD50 o f 6 0 mg p e r k i l o g r a m body w e i g h t f o r t h e mouse on subcutaneous i n j e c t i o n . 7 . SIGNIFICANCE IN HUMAN AND ANIMAL NUTRITION AND DISEASE I n Japan t h e a g r i c u l t u r e i s c e n t e r e d around t h e r i c e c r o p s and t h e d i e t a r y l i f e o f t h e p e o p l e i s based on r i c e , each i n d i v i d u a l consuming about 1 5 0 k i l o g r a m s of r i c e a n n u a l l y . 45 Mould r i c e grains are not always detectable on macroscopic observation and may very e a s i l y be overlooked. From the viewpoint of food s a n i t a t i o n , mouldy r i c e , as i n the case of other mouldy foods discussed, should be re-garded as an important hazard to health and attempts should be made to a l l e v i a t e the p o s s i b i l i t y of r i c e becoming mould infe s t e d . In the experimental yellowed r i c e t o x i c o s i s , i t was noticed that rabbits were more susceptible to the t o x i c o s i s than mice or r a t s . Rabbits developed l i v e r damage s i m i l a r i n feature but stronger i n degree than that of mice or r a t s . The s u s c e p t i b i l i t y to the poison v a r i e s with the age of the animal. In mouldy r i c e t o x i c o s i s i t i s revealed that the adult animals are more susceptible than the young; the animals at the intermediate stage are more susceptible than at an e a r l i e r stage. The intermediate stage i n mice age corresponds to the adult age i n man. The feature of the intermediate stage, according to Mikaye et a l (1960), can be considered to be a balanced status i n l i v i n g animal's response to the toxic invasion. AFLATOXICOSIS (TURKEY X DISEASE) 1. DISEASE In recent years, the high incidence of m o r t a l i t y i n domestic animals and poultry due to the ingestion of mould infested groundnut meal imported from t r o p i c a l countries has given impetus to serious i n v e s t i g a t i o n of the r o l e of fungi as a causative agent i n these animal i n t o x i c a t i o n s . 46 In 1960, a large consignment of B r a z i l i a n groundnut meal used i n turkey r a t i o n s i n B r i t a i n caused the death of thousands of turkeys aged be-tween 3 and 20 weeks. I t was estimated that more than 100,000 turkeys died in that outbreak (Carnaghan and Sargeant, 1961). Most of the dead birds had been fed a r a t i o n containing ten per cent B r a z i l i a n groundnut meal as a pro-t e i n supplement. This disease was c a l l e d Turkey X disease as no s p e c i f i c agent could be found to be d i r e c t l y responsible for the a f f l i c t i o n . The f i r s t reports of t h i s disease indicated that groundnut meal from B r a z i l was implicated, but l a t e r reports indicated that samples of meal from East A f r i c a , India and some of the meals commercially available i n the United States were also implicated. 2. CLINICAL SYMPTOMS In turkeys, affected birds died within a week. They l o s t t h e i r appetites, became markedly l e t h a r g i c and developed a weakness of the wings. A c h a r a c t e r i s t i c a t t i t u d e of head, neck and legs was adopted at the time of death. In ducklings, there has been an i n t e r v a l which may be as long as two weeks or more from the commencement of feeding toxic groundnut meal to the onset of m o r t a l i t y . This i n t e r v a l seems to be influenced by the t o x i c i t y of the sample, the amount consumed and the age of the ducklings. The f i r s t signs of the disease are inappetance and poor growth rate. Ducklings may s t a r t to eat and then lose i n t e r e s t i n the food. The crop i s not well f i l l e d . A cry i n d i c a t i n g discomfort may be heard. A conspicuous feature occasionally seen i n white-skinned older birds i s a purple d i s c o l o r a t i o n of the legs arid f e e t . Young ducklings develop ataxia followed by convulsions s h o r t l y before death and die i n opisthotonus. The legs and feet are markedly haemorrhagic. 47 In c a t t l e , a tendency to scour i s noticed. They are generally i n poor condition during t h i s stage and hold t h e i r t a i l s i n a s l i g h t l y r a i s e d p o s i t i o n as though i n discomfort. There i s inappetance followed by a reduction i n growth rate. A f t e r about 16-25 weeks on the toxic d i e t , terminal symptoms characterized by severe tenesmus occur 2-4 days before death. The symptoms In pigs are not very marked. Harding et a l (1963) reported that pigs became depressed and were i n a generally i n a c t i v e and poor condition. There was a reduction i n growth. Otherwise pigs did not show any other d e f i n i t e signs of i l l health. In general the young animals were more susceptible than the older ones. 3. PATHOLOGY Ducklings - Post mortem findings vary according to the t o x i c i t y of the sample and to the s u r v i v a l time of the ducklings. In young ducklings that die of the groundnut poisoning within a week af t e r hatching, the l i v e r i s putty-coloured and s l i g h t l y enlarged. The kidneys may be pale, s l i g h t l y swollen and contain a few d i f f u s e l y scattered haemorrhages. Petechial haemorrhages are frequently scattered i n the pancreas. In ducklings surviving up to three weeks of age, the l i v e r changes are much more pronounced. The l i v e r shrinks and there i s a firmness i n texture. In older ducklings, l i v e r c i r r h o s i s i s more marked and progresses to the stage of nodular hyperplasia. The gall-bladder i s usually distended. Petechial haemorrhages occur i n the pancreas and larger and more d i f f u s e haemorrhages i n the kidneys. H i s t o l o g i c a l l y the main changes occur i n the l i v e r . Diffuse 48 degenerative changes are found i n the parenchymal c e l l s . These c e l l s be-come swollen and many are vacuolated. The n u c l e i become enlarged and karyorrhexis and k a r y o l y s i s occur. Concurrently, rapid and extensive pro-l i f e r a t i o n of b i l e duct epithelium takes place. These changes are v i s i b l e within 6 days of feeding a r a t i o n containing 10 per cent toxic groundnut meal (Asplin and Carnaghan, 1 9 6 1 ) . In the pancreas, d i f f u s e areas of acinar degeneration are i n v a r i a b l y present. In the kidneys, multiple and d i f f u s e haemorrhages i n the i n t e r s t i t i a l tissues are present. C a t t l e - On post mortem, the carcasses were dark and congested and showed varying degrees of br u i s i n g over the r i b s . The most marked feature was the appearance of the l i v e r . It was pale i n colour with darker red mottling i n large patches, and was very hard and firm. The gall-bladder was gr e a t l y distended and contained thick dark b i l e . The peritoneal membrane of both l i v e r and gall-bladder had a s l i g h t g l i s t e n i n g appearance due to subperi-toneal oedema. The peritoneal c a v i t y contained a large quantity of straw-coloured f l u i d ( a s c ites) which produced a soft c l o t when allowed to stand. The heart tended to be small and f l a c c i d . The kidneys were pale and yellow-coloured surrounded by wet f a t t y t i s s u e . H i s t o l o g i c a l l y , the l i v e r s showed severe d i f f u s e f i b r o s i s with ex-tensive b i l e duct p r o l i f e r a t i o n and chronic endophlebitis of centrolobular and hepatic veins. Pigs - The l i v e r was deep yellowish-brown with f i n e wrinkles on i t s surface. The centres of the lobules were of a darker colour than the rest of the organ. The gall-bladder appeared s l i g h t l y spongy. The walls of the g a l l -bladder were thick and oedematous and the cut surface of the l i v e r showed a coarsely nodular pattern. The pe r i t o n e a l c a v i t y contained a s c i t e s . The lungs were extensively petechiated and tbe trachea contained blood-stained 49 f r o t h . The c o n t e n t s o f t h e caecum and t h e f i r s t p a r t of t h e c o l o n were d a r k r e d d i s h - b r o w n and t a r r y . H i s t o l o g i c a l l y , t h e f i r s t l e s i o n s appear t o be a r a p i d p r o g r e s s i v e s t e a t o s i s and p r o l i f e r a t i o n o f d u c t u l e c e l l s w h i c h a t f i r s t f o r m o n l y i m p e r f e c t d u c t u l e s . P r o l i f e r a t i o n o f r e t i c u l i n and c o l l a g e n q u i c k l y f o l l o w s i n a s s o c i a t i o n w i t h t h e p r o l i f e r a t i n g d u c t u l e s . These p r o l i f e r a t i n g d u c t u l e s become more numerous and w e l l - d i f f e r e n t i a t e d , l e a v i n g t h e p e r i p h e r y o f the n o d u l e and moving g r a d u a l l y i n towards t h e c e n t r e . Karyomegaly appears e v e n t u a l l y r e s u l t i n g i n l a r g e b i z z a r e n u c l e i . S t r a n d s of c o l l a g e n accompany the d u c t u l e s and a l s o b e g i n t o f o r m around t h e c e n t r a l v e i n s so t h a t t h e l o b u l e i s e v e n t u a l l y d i s s e c t e d and c o n t a i n s a p r o m i n e n t c e n t r o l o b u l a r a r e a o f f i b r o s i s . 4. AETIOLOGY The w i d e s p r e a d o c c u r r e n c e of t h i s t y p e of p o i s o n i n g s u g g e s t e d t h a t t h e c a u s a t i v e agent might be a m i c r o o r g a n i s m . A s t r a i n of t h e common mould A s p e r g i l l u s f l a v u s was f ound t o be r e s p o n s i b l e . T h i s s t r a i n was i s o l a t e d f r o m t o x i c g r o u n d n u t s and p r o duced the t o x i c f a c t o r when grown i n p u r e c u l t u r e on s t e r i l e g r o u n d n u t s . A s p e r g i l l u s f l a v u s i s a member o f t h e group o f f u n g i of t h e o r d e r Mucedineae i n t h e c l a s s F u n g i I m p e r f e c t i . F u n g i l a c k i n g a c o m p l e t e l i f e c y c l e a r e r e l e g a t e d t o t h i s c l a s s . However a number of known s p e c i e s of A s p e r g i l l u s p o s s e s s a s e x u a l s t a g e c h a r a c t e r i z e d by a s c o s p o r e s borne i n a s a c , t h e a s c u s , and t h e s e forms a r e a s s i g n e d t o t h e c l a s s A s c o m y c e t e s . The a s e x u a l s t a g e of A s p e r g i l l i w i t h w h i c h we a r e c o n c e r n e d i s c h a r a c t e r i z e d by a v e g e t a t i v e m y c e l i u m " c o n s i s t i n g o f s e p t a t e b r a n c h i n g hyphae f r o m w h i c h s p o r e - b e a r i n g c o n i d i a l s t r u c t u r e s a r i s e a e r i a l l y . The s p o r e s or c o n i d i a a r e p r o d u c e d on c o n i d i o p h o r e s g r o w i n g perpendicular to the surface of the substrate. The conidiophore terminates i n a swollen t i p ( v e s i c l e ) which may be globose, subglobose or hemispherical. The v e s i c l e bears f e r t i l e c e l l s or sterigmata on i t s surface. Conidia varying g r e a t l y i n colour, si z e and shape are borne i n chains on the t i p s of the sterigmata. The con i d i a of A. flavus vary from yellow-green to dark green and are pyriform. A. flavus i s a common mould which can be i s o l a t e d from many stored dried foodstuffs and from t r o p i c a l s o i l s . I t grows r a p i d l y but requires somewhat more moisture than other moulds. At t r o p i c a l temperatures (30° C), i t w i l l grow at 80-85 per cent r e l a t i v e humidity and above. This corres-ponds with groundnut meal and kernels having a moisture content of 9-16 per cent or more. 5. ISOLATION AND CHARACTERIZATION OF TOXIN The extraction procedure as outlined by Sargeant et a l (1961) has i n general been adopted by other workers with various modifications. This method b a s i c a l l y consists of extracting the meal with hot methanol and removing the solvent under vacuum. The methanol extract i s suspended i n water and continuously extracted with chloroform, the chloroform f r a c t i o n being separated and the solvent removed. The extract i s suspended i n a mixture of 50 ml methanol and 50 ml petroleum ether, 5 ml of water i s added and the mixture i s shaken. The methanolic layer i s separated o f f and concentrated. This contains the toxic extract. Using t h i s procedure, Sargeant et a l (1961) were able to concentrate the toxic material 250 times. A f l a t o x i n i s the name given to the toxic product of the mould A s p e r g i l l u s flavus Link ex F r i e s . A f l a t o x i n i s present i n very small amounts in toxic groundnuts. I t s concentration i s a few parts per m i l l i o n 51 Procedure for the Extraction of A f l a t o x i n from Groundnut Meal * Toxic B r a z i l i a n Groundnut Meal Extracted with hot methanol for 18 hours Soluble Methanol - brown o i l y residue Extracted Toxic (12%) Water and Chloroform added Chloroform Soluble Extract C -Toxic (1.5%) Petroleum ether Methanol Water Lower layer Methanol Soluble Extract E Toxic (0.4%) Non-toxic (88%) Water Soluble Extract D Non-toxic (10%) Upper layer Petroleum ether and Water-Soluble Extract F Non-toxic (1%) * Sargeant et a l (1961) 52 in highly t o x i c samples. Van der Zijden et a l (1962) i s o l a t e d i n c r y s t a l l i n e form a toxin from A. flavus which had caused Turkey X disease. Mould cultures were grown on s t e r i l e groundnut meal and on glucose-ammonium n i t r a t e medium. The fungus was extracted with methanol/chloroform, and the residue remaining a f t e r evaporation of the material was chromatographed on a column of s i l i c a gel in chloroform/1% ethanol; the zone fluorescent f r a c t i o n s i n u l t r a v i o l e t l i g h t were further p u r i f i e d by t h i n layer chromatography on k i e s e l gel G column chromatography over k i e s e l gel G / c e l i t e and f i n a l l y f i l t r a t i o n through a short column of alumina. The toxin was obtained i n gleaming white c r y s t a l s . Other workers including Nesbitt et a l (1962), Asao et a l (1963) and Chang et  al (1963) have i s o l a t e d and p u r i f i e d a f l a t o x i n f r a c t i o n s from various pre-parations of A. fla v u s. Of the twelve or more f r a c t i o n s that have been i s o l a t e d , two have been shown to be t o x i c . These have been labeled a f l a -toxin B and G. These two components with Rf values of 0.5 and 0.6 fluoresce i n u l t r a v i o l e t l i g h t . A f l a t o x i n B exhibits blue fluorescence and a f l a -toxin G exhibits green fluorescence. Both a f l a t o x i n B and G are composed of two separate f r a c t i o n s B-p B 2 and G^, G 2. Hodges et a l (1964) have reported on the i s o l a t i o n of a f l a t o x i n from a cul t u r e of P e n i c i l l i u r o puberulum which was found growing on a sample of mouldy peanuts. Four components with Rf values i d e n t i c a l to those of a f l a t o x i n B^, B 2, G^ and G 2 were obtained. The toxin, when administered to two or three-day-old Pekin white ducklings, produced the t y p i c a l b i l e duct p r o l i f e r a t i o n type of l i v e r damage that i s c h a r a c t e r i s t i c of the a f l a t o x i n e f f e c t . The L D ^ Q for these ducklings was about 2.3 mg per kilogram of body weight. A f l a t o x i n B^ has a molecular weight of 312, melting point 265-270° C. TABLE IV Chemical C h a r a c t e r i s t i c s of Four Components of A f l a t o x i n * A f l a t o x i n Fluorescence i n U.V. l i g h t at 365 raj* ( p r i n c i p a l wavelength) Melting Point Molecular Weight Chemical Formula Elementary Analysis C H % 0 B l Blue 265-270 312 C17 H12°6 65.3 3.6 31. 2 B 2 Blue 305-309 314 C17 H14°6 64.6 4.3 30. 9 Gl Green 247-250 328 C17 H12°7 62.3 3.9 33. 7 G 2 Green 237-240 330 C 1 7 H 1 4 0 7 61.7 4.5 33. 7 * Ref. Hartley et al (1963) TABLE V Physico - Chemical C h a r a c t e r i s t i c s of Four Components o f A f l a t o x i n * S p e c i f i c Rotation Molar Exti n c t i o n C o e f f i c i e n t s Fluorescence Spectrum A f l a t o x i n C ( i n chloroform) £ 2 6 5 ra/j £363 mp E x c i t a t i o n Wavelength (mji) Emission Wavelength (mjj) B l -550 0.101 12,000 22,000 365 425 B2 -492 0.100 11,700 23,400 365 425 G l -533 0.133 10,500 18,700 365 450 G2 -473 0.184 9,700 21,000 365 450 C «• Concentration * Ref. Hartley et a l (1963) Fig^_4 S t r u c t u r e s of A f l a t o x i n and B 2 OCH, A f l a t o x i n Bi 0 .0 \AAA OCH, A f l a t o x i n B r F i g - 5 S t r u c t u r e s of A f l a t o x i n and G2 Aflatoxin Elementary analysis gave C - 6 5 . 3 % ; H - 3 . 6 % ; 0 - 3 1 . 2 % . The chemical formula i s C^y H-^ 2 . A f l a t o x i n B 2 has a molecular weight of 3 1 4 , melting point 3 0 5 - 3 0 9 ° C . Elementary analysis gave C - 6 4 . 4 % ; H - 4 . 3 % ; 0 - 3 0 . 9 % . The chemical formula i s C^y Hj^ Og. A f l a t o x i n G-l has a moleular weight of 3 2 8 , melting point 2 4 7 - 2 5 0 ° C . Elementary analysis gave C - 6 2 . 3 % ; H - 3 . 9 % 0 - 3 3 . 7 % . The chemical formula i s C J J O7. A f l a t o x i n G 2 has a molecular weight of 3 3 0 , melting point 2 3 7 - 2 4 0 ° C. Elementary analysis gave C - 6 1 . 7 % ; H - 4 . 5 % ; O - 3 3 . 7 % . The chemical formula i s C^y H-^ O7. A f l a t o x i n B^ can be converted to B 2 by hydrogenation i n ethanol for 1 5 minutes using platinum oxide c a t a l y s t at room temperature and atmospheric pressure. S i m i l a r l y a f l a t o x i n G^ can be converted to G 2 by hydrogenation using 1 0 per cent palladium on charcoal as c a t a l y s t . The hydrogenation occurs at the double bond in the terminal dihydrofuran r i n g . Hydrogenation lowers t o x i c i t y . The formulae for a f l a t o x i n B and G have been suggested on the bases of microanalysis and mass spectrometric molecular weight determinations. Asao  et a l ( 1 9 6 3 ) have suggested the following structures for the compounds (Fi g . 4;and 5 ) . 6 . CHEMICAL AND BIOLOGICAL CHARACTERISTICS OF TOXIN The presence of a f l a t o x i n i n a sample can be detected by two methods: ( 1 ) The b i o l o g i c a l e f f e c t s of the toxin ( 2 ) The c h a r a c t e r i s t i c fluorescence i n u l t r a v i o l e t l i g h t . B i o l o g i c a l Assay - the b i o l o g i c a l assay involves the extraction of the toxin from the groundnut meal or substrate by the method of Sargeant et a l ( 1 9 6 1 ) or one of i t s modifications. Using the above method, the extract 58 w h i c h i s c o n c e n t r a t e d 250 t i m e s on a w e i g h t b a s i s i s a d m i n i s t e r e d t o d a y - o l d d u c k l i n g s o r a l l y and the t o x i c i t y o f t h e sample i s a s s e s s e d by t h e time t a k e n f o r i t t o k i l l t h e d u c k l i n g s . A more dependable c r i t e r i o n f o r t o x i c i t y c an be o b t a i n e d by u s i n g o l d e r d u c k l i n g s and m e a s u r i n g t o x i c i t y by the p r e s e n c e and e x t e n t o f l e s i o n s p r o d u c e d i n the l i v e r and t h e p r o l i f e r a t i o n o f b i l e d u c t e p i t h e l i u m t h a t o c c u r s . TABLE V I C h a r a c t e r i s t i c s o f L i v e r Damage i n D u c k l i n g s as r e p o r t e d by t h e  I n t e r d e p a r t m e n t a l Working P a r t y on Groundnut T o x i c i t y R e s e a r c h (1962) L e v e l s o f S e v e r i t y o f T o x i c i t y C h a r a c t e r i s t i c s Found L i v e r Damage Stong The d u c k l i n g s d i e w i t h t h e c h a r a c t e r i s t i c l i v e r l e s i o n s (+ + + +). Moderate The d u c k l i n g s s u r v i v e and a r e k i l l e d 7 days a f t e r t h e s t a r t o f d o s i n g . C h a r a c t e r i s t i c s o f h i s t o l o g i c a l l i v e r l e s i o n s a r e p r e s e n t t o a marked degree (+ + + o r + + + +) S l i g h t The d u c k l i n g s s u r v i v e and are k i l l e d 7 days a f t e r t he s t a r t of d o s i n g . H i s t o l o g i c a l l i v e r l e s i o n s a r e p r e s e n t but a r e l e s s s e v e r e t h a n i n t h e "moderate" group (+ o r + +). N i l D u c k l i n g s s u r v i v e and a r e k i l l e d a f t e r 7 days. No h i s t o l o g i c a l changes a r e o b s e r v e d i n t h e l i v e r s . S p e n s l e y (1963) has r e p o r t e d on b i o l o g i c a l t e s t s u s i n g 5-day i n c u b a t e d hen's embryos. The t o x i n i s i n o c u l a t e d i n t o t h e y o l k s a c , and i f d e a t h o c c u r s w i t h i n 48 h o u r s , the t o x i n i s c o n s i d e r e d h i g h l y f a t a l . De Iongh e t a l (1962) have m e n t i o n e d t h e use of d u c k l i n g embryos i n t e s t i n g a f l a t o x i n 59 samples. P i a t t et a l (1962) have mentioned the use of chick embryos for a f l a t o x i n assay. Verret et a l (1964) have used chick embryos with great success i n the assay of a f l a t o x i n t o x i c i t y . The route of inoculation was e i t h e r v i a the yolk sac or a i r c e l l . The development of the embryo was observed for the f u l l 21-day period. A dose-response r e l a t i o n s h i p was exhibited i n that t o x i c i t y of the samples was r e l a t e d to the m o r t a l i t y at the time of hatching. A f l a t o x i n samples from both toxic groundnut meal and mould culture extracts were l e t h a l to the embryos. Legator and Withrow (1964) have studied the e f f e c t of a f l a t o x i n on m i t o t i c d i v i s i o n i n lung tissue c u l t u r e c e l l s . They found that a f l a t o x i n suppressed m i t o t i c d i v i s i o n i n human embryonic lungs 4 to 12 hours a f t e r exposure of the c e l l s to the toxin. Juhasz and Greczi (1964) tested an extract of mould-infested groundnuts i n c a l f kidney tissue culture and found that i t destroyed both cytoplasm and nucleus. Smith (1963) demonstrated that low concentrations of p u r i f i e d aflatox-i n i n h i b i t the incorporation of leucine into protein by various l i v e r preparations. Four of these toxins, B^, B2, G^, G 2 (Smith and McKernan, 1962), show t h i s a c t i v i t y . 200Ug of toxin completely i n h i b i t e d incorpora-t i o n , whereas l O u g toxin gave 10-20 per cent reduction i n rate of incorpora-t i o n . Duckling l i v e r s l i c e s were more s e n s i t i v e to the toxin than rat l i v e r s l i c e s . He also observed d i s t i n c t vacuolation of monkey kidney c e l l s i n tissue c u l t u r e on addition of 0.3ng of a f l a t o x i n B^ to 1 ml of the tissue medium. With the addition of 3.0 Ug of the toxin, growth was completely i n h i b i t e d and c e l l d estruction was observed. U.V. fluorescence - In order to detect a f l a t o x i n by u l t r a v i o l e t fluorescent technique, the groundnut meal i s f i r s t defatted by extracting with petroleum ether for about two hours. The defatted sample i s then extracted with hot 60 methanol for about 6 hours. The methanol removes a l l the toxin together with many other substances which are separated by shaking the extract with chloroform. The chromatography of the chloroform extract on thi n layers of aluminium oxide and the examination of the chromatograms under u l t r a v i o l e t l i g h t permit the l e v e l of t o x i c i t y to be c l a s s i f i e d as: Very high - containing more than 2 p.p.m. High - containing 0.5 - 2.0 p.p.m. Medium - containing 0.1 - 0.5 p.p.m. Low or Negative - containing less than 0.1 p.p.m. TABLE VII Cor r e l a t i o n of Results Obtained by Two Methods of A f l a t o x i n Assay (Report of Interdepartmental Working Party on Groundnut T o x i c i t y Research, 1962) Method 1 B i o l o g i c a l Test (Ducklings) Method 2 Fluorescence Test Strong Very high > 2.0 P' .p .m. high 0. .5 - 2.0 P' .p .m. Moderate Upper range of medium 0. .1 - 0.5 P .p .m. Sl i g h t Lower range of medium ' 0, .1 - 0.5 P .p.m. N i l Lower or negative > 0.1 P .p .m. For p u r i f i e d a f l a t o x i n Bj_, i t has been reported that a lesser concen-t r a t i o n of toxin as observed by u l t r a v i o l e t fluorescence i s necessary. A "strong" b i o l o g i c a l test indicates an approximate a f l a t o x i n B^ concentration of 0.25 to 1 p.p.m. with a "moderate" b i o l o g i c a l test ranging from 0.05 to 0.25 p.p.m. A " s l i g h t " b i o l o g i c a l test indicates less than 0.05 p.p.m. Smith and McKernan (1962) report the i s o l a t i o n of 12 components of a f l a t o x i n which fluoresced under u l t r a v i o l e t l i g h t . Extracts of these 12 components were administered to day-old ducklings and 5 of these 12 extracts a f t e r 5 d a i l y applications gave l i v e r lesions s i m i l a r to those seen i n turkey X disease. The mould A. flavus Link ex F r i e s does not produce a single toxin but rather a mixture of c l o s e l y r e l a t e d toxins. The toxin-producing p o t e n t i a l of these s t r a i n s v a r i e s as regards to both t o t a l production and the propor-t i o n i n which the active components of toxin are synthesized. The composi-t i o n of a f l a t o x i n v a r i e s depending on the s t r a i n of fungus, substrate, microclimate and extraction procedure used for preparation of toxin (Hartley  et a l . 1963). A f l a t o x i n B]_ has an LD50 of 18.2jig for day-old ducklings. A f l a t o x i n Gi i s less toxic and has an L D ^ Q of about 39.2^£g for day-old ducklings. A f l a t o x i n B 2 has an L D ^ Q of 84.8^<g and a f l a t o x i n G 2 has an L D ^ Q of 172.5^ug for day-old ducklings (Hartley et a l , 1963). Fischbach et a l (1965) re-ported on the d e t o x i f i c a t i o n of a f l a t o x i n with a 5 per cent sodium hypo-c h l o r i t e s o l u t i o n . These same workers also exposed a highly contaminated peanut meal sample to a 10 per cent chlorine gas atmosphere overnight and found that i t had l o s t i t s t o x i c i t y as indicated by i t s reduced fluorescence i n u l t r a v i o l e t l i g h t and i t s non - t o x i c i t y to chick embryos. The T r o p i c a l Products I n s t i t u t e Report (1962) stated that toxic peanut meal was not made free of toxin by heat, exposure to acid, a l k a l i , c h l o r i n e , sulphur dioxide, gamma r a d i a t i o n and u l t r a v i o l e t l i g h t without s e r i o u s l y impairing the n u t r i t i v e and other properties of the meal. 62 7. EXPERIMENTAL AFLATOXICOSIS IN ANIMALS Following the outbreak of groundnut t o x i c i t y i n England, i t became necessary to f i n d a suitable experimental animal for t e s t i n g the t o x i c i t y of samples. A s p l i n and Carnaghan (1961) tested the t o x i c i t y of c e r t a i n ground-nut meals on poultry and found that ducklings were the most hig h l y suscept-i b l e . Young ducklings were more susceptible than older ones. Various workers administered a f l a t o x i n o r a l l y to ducklings ranging i n age from 1-7 days and induced deaths at i n t e r v a l s of from 1-5 days, with the day-old ducklings succumbing to the toxic e f f e c t s as early as the f i r s t day. In the older ducklings the c h a r a c t e r i s t i c l i v e r lesions were noticed. Thus the t e s t i n g of the extracts i n ducklings became a s u i t a b l e c r i t e r i o n f or t o x i c i t y . The r e s u l t s of the ducklings tests as compared with feeding t r i a l s done on turkeys and chicks indicated that ducklings were the most susceptible to the toxic factor and that they succumbed to the e f f e c t s more r a p i d l y and on rations containing lower l e v e l s of toxic groundnuts than do turkey poults (Asplin and Carnaghan, 1961). Experimental feeding t r i a l s were also done on rats and guinea pigs. Rats were fed samples of B r a z i l i a n groundnut meal at the 20 per cent l e v e l f or 6 months. The weights of rats fed the B r a z i l i a n groundnut meal were lower than the contols. Males showed a diff e r e n c e of about 70 grams whereas the weights of the females were not s i g n i f i c a n t l y d i f f e r e n t . The food intake and conversion of the rats on the toxic meal were also lower than those of the c o n t r o l r a t s . A f t e r 30 weeks of experimentation, post mortem examina-t i o n of the rats showed macroscopic lesions of the l i v e r and lungs. The l i v e r s were grossly abnormal, twice the weight of the l i v e r s of the c o n t r o l s , with a brownish-yellow i r r e g u l a r nodular surface, red and greenish cysts and 63 numerous y e l l o w i s h f o c a l l e s i o n s . The k i d n e y s were a l l m a c r o s c o p i c a l l y n o r m a l , but h i s t o l o g i c a l l y showed d e g e n e r a t i v e changes. The f e e d i n g of t h e t o x i c groundnut meal t o r a t s , t h e r e f o r e , f a i l e d t o r e p r o d u c e t h e a c u t e l i v e r damage as i t appeared i n t u r k e y s and d u c k l i n g s . However, t h e p r e s e n c e of t h e l e s i o n s on t h e l i v e r a f t e r p r o l o n g e d f e e d i n g i n d i c a t e s t h a t t h e groundnut meal was c a r c i n o g e n i c ( L a n c a s t e r et a l , 1961). P a t e r s o n e t a l (1962) f e d d i e t s c o n t a i n i n g t o x i c groundnut meal a t t h e 15 p e r c e n t l e v e l t o g u i n e a p i g s . A l l a n i m a l s f e d t h e t o x i c meal showed po o r w e i g h t g a i n s , d e v e l o p e d g r o s s oedema o f t h e subcutaneous t i s s u e s and d i e d i n f r o m 17-55 d a y s . B u t l e r and Barnes (1963) p r o d u c e d h e p a t i c c a r c i n o m a s i n r a t s f e d d i e t s c o n t a i n i n g 4 - 0 . 8 p.p.m. a f l a t o x i n . These same w o r k e r s a l s o o b s e r v e d l i v e r l e s i o n s i n g u i n e a p i g s dosed w i t h a f l a t o x i n . The g u i n e a p i g p r o v e d much more s e n s i t i v e t o t h e a c u t e e f f e c t s o f a f l a t o x i n t h a n t h e r a t . D i c k e n s and Jones (1963) were a b l e t o i n d u c e tumours i n r a t s a t t h e s i t e o f i n j e c t i o n by subcutaneous a d m i n i s t r a t i o n o f a f l a t o x i n . These tumours were o f the sarcomatous t y p e s and were s u c c e s s f u l l y t r a n s p l a n t e d t o o t h e r r a t s . H a r d i n g e t a l (1963) r e p o r t on f e e d i n g r a t i o n s c o n t a i n i n g 17.5 - 20 p e r c e n t t o x i c groundnut meal t o p i g s . Changes i n t h e l i v e r s p r o g r e s s e d t h r o u g h s t e a t o s i s , d u c t u l e p r o l i f e r a t i o n and p e r i c e l l u l a r f i b r o s i s t o p a r e n -chymal k a r y o m e g a l y , d i s s e c t i n g f i b r o s i s and f i n a l l y n o d u l a r h y p e r p l a s i a . The l e s i o n s were sometimes accompanied by e l e v a t e d serum g l u t a m i c - o x a l a c e t i c t r a n s a m i n a s e and a l k a l i n e p h o s p h a t a s e l e v e l s , i n c r e a s e d h e p a t i c c o n c e n t r a -t i o n s of f a t , r e d u c e d h e p a t i c l e v e l s o f v i t a m i n A and low t h y r o i d t o body w e i g h t r a t i o ' s . P i g s a r e t h e o n l y s p e c i e s t o show g e n e r a l i z e d j a u n d i c e . A l l c r o f t and L e w i s (1963) r e p o r t on f e e d i n g r a t i o n s c o n t a i n i n g 20 p e r cent toxic groundnut meal to calves, f i r s t - c a l v i n g h e i f e r s and older cows. There was a reduction i n growth rate of calves accompanied by u n t h r i f t i n e s s and loss of appetite. Death followed i n 16-25 weeks. The post mortem findings include f i b r o s i s of the l i v e r , a s c i t e s and v i s c e r a l oedema. The most noticeable biochemical symptoms were: (1) an increase i n serum a l k a l i n e phosphatase l e v e l s up to the 12th week followed by a decline to normal values during the terminal period. (2) almost complete absence of vitamin A i n the l i v e r s at death. H i s t o l o g i c a l examination of the l i v e r showed centrolobular necrosis of the l i v e r , ductal c e l l hyperplasia and veno-occlusive disease. Loss of condi-t i o n occurred i n the h e i f e r s a f t e r 7 months. In older animals no c l i n i c a l abnormality was noted. In milking cows on a d i e t containing 15 per cent toxic groundnut meal, there was a s i g n i f i c a n t f a l l i n milk y i e l d . 8. SIGNIFICANCE IN HUMAN AND ANIMAL DISEASE AND NUTRITION In the United States, B r i t a i n and Canada, a f l a t o x i n has not been found i n the groundnut products used f o r human consumption as the nuts are u s u a l l y of the hand-picked grade. Hodges et a l (1964) report on the i s o -l a t i o n of A s p e r g i l l u s flavus from a sample of peanuts sold f or human con-sumption. Extraction of the peanuts and subsequent assay of the extract i n ducklings showed that the sample contained the toxic factor of A s p e r g i l l u s  f l a v u s . Peanut butter has been examined by Genest and Smith (1963) and has been found to be free of a f l a t o x i n . Spensley (1963) reports that groundnut o i l used for margarine i s toxin free even though i t has been produced from contaminated peanuts. This i s the r e s u l t of the action of a l k a l i s which are used i n the r e f i n i n g process. 65 A f l a t o x i n i s found to a f f e c t a wide range of animals. Poultry, i n -cluding turkeys, ducklings, pheasants and to a lesser extent chickens are susceptible to the t o x i c o s i s . Pigs, Calves, sheep, rabbi t s , guinea pigs, r a t s , etc. are also h i g h l y susceptible. The young of animals are usually more susceptible than the adults. Thus i n human n u t r i t i o n , the use of groundnut products for infant n u t r i t i o n should be very c a r e f u l l y c o n t r o l l e d e s p e c i a l l y in t r o p i c a l countries where groundnut meal i s r e a d i l y a v a i l a b l e and thus i s a very economical and handy pr o t e i n source. A f l a t o x i n has been found i n milk of cows fed a d i e t containing toxic groundnut meal. De Iongh et a l (1964) have i d e n t i f i e d a f l a t o x i n in the milk of cows and rats fed rations containing toxic groundnut meal. The toxins secreted were not i d e n t i c a l to the known' toxins but were just as t o x i c . They suggested that there was a s l i g h t a l t e r a t i o n i n the toxin during i t s passage through the animal. De Iongh (1964) also reported on the detection of the t o x i n i n the muscles and l i v e r s of c a t t l e and hogs fed toxic groundnut meal. A l l c r o f t and Carnaghan (1963) have found a f l a t o x i n i n milk of cows fed a toxic d i e t . However they were unable to detect any a f l a t o x i n in the l i v e r s of these same cows nor i n the eggs of hens fed a toxic d i e t . Low l e v e l s of a f l a t o x i n i n cottonseed meal has been the cause of hepatomas i n trout r e s u l t i n g in a high m o r t a l i t y rate. A f l a t o x i n has also been found i n corn, coconut and cocoabean. 66 FACIAL ECZEMA 1. DISEASE F a c i a l eczema i s endemic i n New Z e a l a n d where i t has been t h e sco u r g e o f t h e sheep i n d u s t r y s i n c e 1882 ( C l a r e , 1955). T h i s i s a hepatogeneous d i s e a s e and owes i t s name t o t h e e x t e r n a l l e s i o n s w h i c h appear on t h e f a c e s o f t he a n i m a l s t h a t have t h e p o i s o n o u s s u b s t a n c e i n t h e i r b l o o d s t r e a m s . These l e s i o n s o f p h o t o s e n s i t i z a t i o n t h a t appear on t h e f a c e and neck a r e cau s e d by t h e p r e s e n c e i n t h e b l o o d s t r e a m o f a breakdown p r o d u c t o f c h l o r o p h y l l c a l l e d p h y l l o e r y t h r i n . P h y l l o e r y t h r i n s e n s i t i z e s t h e a n i m a l , and on e x p o s u r e t o s u n l i g h t , t h e e x t e r n a l l e s i o n s appear on t h e f a c e , e a r s and neck o f t h e a n i m a l . The p r i m a r y e f f e c t o f f a c i a l eczema t o x i c i t y i s n o t however t h e s e e x t e r n a l l e s i o n s . F i r s t l y , l e s i o n s appear i n t h e l i v e r and as a r e s u l t o f l i v e r m a l f u n c t i o n , p h y l l o e r y t h r i n i s formed. L i v e r I n j u r y may be p r e s e n t w i t h o u t t h e e x t e r n a l l e s i o n s of p h o t o s e n s i t i z a t i o n . F a c i a l eczema v a r i e s f r o m y e a r t o y e a r , i s s e a s o n a l , and i s a s s o c i a t e d w i t h u n u s u a l c l i m a t i c c o n d i t i o n s . The m a j o r i t y o f c a s e s o c c u r i n t h e f a l l and a r e g e n e r a l l y p r e c e d e d by an u n u s u a l l y d r y summer f o l l o w e d by warm, humid weather w i t h an abundance o f r a i n f a l l and a h i g h t e m p e r a t u r e a t t h e s o i l s u r f a c e . The t o x i c o s i s i s n e i t h e r c o n t a g i o u s n o r i n f e c t i o u s , and tend s t o be f i x e d i n l o c a t i o n though i t may v a r y w i t h i n a g i v e n a r e a . Thus one p a r t i c u -l a r p a s t u r e may be t o x i c w h i l e an a d j o i n i n g s i m i l a r p a s t u r e may be n o n - t o x i c ( C l a r e , 1955). McMeekan ( 1 9 5 9 ) , i n r e p o r t i n g on t h e p r o g r e s s i n f a c i a l eczema r e -s e a r c h t h a t had been made up t o t h a t t i m e , o u t l i n e d s i x s t e p s t h a t l e d t o th e s u c c e s s o f New Z e a l a n d ' s s c i e n t i s t s i n the u n d e r s t a n d i n g o f t h i s 67 t o x i c o s i s : (1) Recognition of the features of the pathology of the disease. (2) The development of techniques f o r the c o l l e c t i o n of t o x i c grass. (3) The d i s c o v e r y of a small animal assay f o r t o x i c i t y . (4) Chemical f r a c t i o n a t i o n and p u r i f i c a t i o n of the t o x i n and the I d e n t i f i c a t i o n of an associated chemical. (5) The development of the "beaker" t e s t f o r t o x i c i t y . (6) The i n c r i m i n a t i o n of a s p e c i f i c fungus. 2. CLINICAL SYMPTOMS I Sheep - The f i r s t disturbance i s u s u a l l y the onset of diarrhoea i n some animals on the t h i r d and f o u r t h day a f t e r consumption of the t o x i c grass. This was a s s o c i a t e d w i t h inappetance and d u l l n e s s at about the s i x t h day, but the sheep soon improved both i n demeanour and a p p e t i t e . This p e r i o d of apparent recovery was a b r u p t l y terminated as the sheep became p h o t o s e n s i t i z e d from the tenth day onwards. In severe cases, d i a r r h o e a and dehydration increased u n t i l death, and the animals d i d not d i s p l a y p h o t o s e n s i t i z a t i o n . The p h o t o s e n s i t i z e d animals avoided the s u n l i g h t . The inflamed oedematous l e s i o n s which r a p i d l y develop on the l i p s , face, e y e l i d s and v u l v a caused marked i r r i t a t i o n . This was demonstrated by frequent head shaking, rubbing and r e s t l e s s n e s s . These signs together w i t h the c h a r a c t e r -i s t i c drooping ears made p o s s i b l e the r e c o g n i t i o n of p h o t o s e n s i t i z e d animals. Slow seepage of the inflammatory exudate through the s k i n covering the ears and face began a f t e r 24-48 hours. Scab formation followed and then the exudation was no longer apparent. The p e r i o d of dermal t o x i c i t y may l a s t f o r s e v e r a l weeks, but tends to disappear w i t h the advent of the c o l d weather. In the t e r m i n a l stages of the t o x i c o s i s , i c t e r u s may develop; i t i s 68 pronounced and e a s i l y d e t e c t e d i n some a n i m a l s w h i l e i n o t h e r s the c o n d i t i o n i s s l i g h t and not e a s i l y d i s c e r n e d . C a c h e x i a i s f r e q u e n t l y o b s e r v e d f o l l o w e d by d e a t h i n a few weeks. E x a m i n a t i o n o f the b l o o d r e v e a l s b i l i r u b i n e m i a and l e u c o c y t o s i s . 3. AETIOLOGY F a c i a l eczema i s caused by t h e consumption o f p e r e n n i a l r y e g r a s s t h a t i s i n f e c t e d w i t h t h e fungus P i t h o m y c e s c h a r t a r u m . T h i s fungus was f i r s t i d e n t i f i e d as Sporodesmium b a k e r i , b u t l a t e r was r e c l a s s i f i e d as P i t h o m y c e s  c h a r t a r u m . The genus Sporodesmium i s d e s c r i b e d as h a v i n g c o n i d i o p h o r e s w h i c h a re c l u s t e r e d , d a r k , s h o r t and s i m p l e , each b e a r i n g a t e r m i n a l c o n i d i u m ; c o n i d i a are d a r k and q u i t e l a r g e , and c o n t a i n many c e l l s w h i c h a re i r r e g u l a r l y a r r a n g e d . These c o n i d i a a r e o b l o n g t o o v o i d . The fungus i s s a p h r o p h y t i c . I t i s an i m p e r f e c t form o f t h e c l a s s Ascomycetes, o r d e r M o n i l i a l e s , f a m i l y Dematiaceae. 4. PATHOLOGY The g e n e r a l p a t h o l o g i c a l f i n d i n g s i n f a c i a l eczema c o n s i s t e d p r i m a r i l y o f g r o s s changes i n t h e l i v e r . There were g r e a t v a r i a t i o n s i n c o l o u r , s i z e , t e x t u r e and s i t e s o f l e s i o n s f o u n d . I n t h e most a c u t e form o f t h e d i s e a s e , t h e o r g a n i s m a r k e d l y e n l a r g e d w i t h rounded edges and a y e l l o w i s h - g r e e n d i s c o l o r a t i o n . The s u p e r f i c i a l l o b a r , h e p a t i c , c y s t i c and common b i l e d u c t s were v e r y p r o m i n e n t and t h i c k e n e d w i t h oedema i n v o l v i n g t h e i r w a l l s and t h e p e r i - d u c t a l t i s s u e s . D i s t e n s i o n o f the g a l l - b l a d d e r was common i n t h e s e a c u t e c a s e s and t h e r e was oedema and c o n g e s t i o n i n many i n s t a n c e s . I n t h e l e s s a c u t e c a s e s , n e i t h e r d i s c o l o r a t i o n n or enlargement o f t h e l i v e r was 69 noted. Patchy variegations were seen on either or both of the r i g h t and l e f t lobes. The l i v e r s were always firm and cut with resistance revealing a connective tissue network, the mesh of which consisted of discolored lobules of parenchymatous t i s s u e . In some of the chronic cases there was complete occlusion of the b i l e duct. A constant finding was the enlargement of the lymph node in the porta hepatis. The peritoneal c a v i t y usually contained an excess of f l u i d b i l e . The spleen was dark and enlarged or normal. The kidneys were coloured with b i l e . H i s t o p a t h o l o g i c a l l y three degrees of the l i v e r damage are noticed. (1) There i s l i t t l e damage i n the parenchyma. A marked leucocytosis i s observed i n the l i v e r c a p i l l a r i e s and i n the p o r t a l system. The walls of the b i l e duct are very thick. (2) In the second condition there i s much greater damage. The b i l e c a p i l l a r i e s increase extending almost around the l i v e r lobules. There are fibrous thickenings i n the wall of the b i l e ducts together with necrotic areas i n the parenchyma. (3) Marked necrosis of l i v e r tissue i s present e s p e c i a l l y i n the l e f t lobe which atrophies u n t i l no hepatic c e l l s remain. The large b i l e ducts are blocked with c e l l debris and the walls are thickened with evidence of necrosis. The intermediate b i l e ducts are o b l i t e r a t e d . Serum from animals with l i v e r lesions contained increased amounts of b i l i r u b i n , transaminases and c h o l e s t e r o l . The seve r i t y of l i v e r damage was in general re l a t e d to the extent of the increase of the serum constituents. There i s a marked increase i n serum glutamicroxalacetlc transaminase and ch o l e s t e r o l i n sheep with l i v e r damage and in some cases b i l i r u b i n e m i a can 70 be detected. 5. ISOLATION AND CHARACTERIZATION OF TOXIN Many attempts at extraction p u r i f i c a t i o n and f r a c t i o n a t i o n of the toxin of f a c i a l eczema were made. This was done by using the toxic rye grass as a source of the poison. It was possible to concentrate the poison to 1/200,000 to 1/400,000 of the weight of the grass by the use of adsorption and p a r t i t i o n chromatography. A chemical procedure for extracting the toxin was outlined by E.P. White (1959) which consisted of ether extraction of the culture and concen-t r a t i n g the extracts to at least 1/20 of i t s o r i g i n a l weight with l i t t l e loss of t o x i c i t y . The procedure may be outlined as follows: (1) The homogenized culture i s shaken with about four times i t s volume of ether i n separatory funnels. (2) Evaporation of the ether i n a cold a i r b l a s t . (3) The extract i s dissolved in a small amount of ether and the insoluble beaker test compound i s f i l t e r e d o f f . (4) Evaporation.of ether. (5) The residue i s taken up with methanol. (6) The methanol soluble part i s d i l u t e d to 80 per cent with petroleum ether. (7) Rotary evaporation of methanol to dryness. (8) Passage through acetic acid deactivated alumina columns using ether. (9) Cold evaporation of ether. F r a c t i o n a t i o n o f S p o r i d e s m i n * T o x i c G rass (900 grams) e x t r a c t e d w i t h w a t e r R e s i d u e ( n o n - t o x i c ) e v a p o r a t e d R e s i d u e ( c o n c e n t r a t e d 25x) d i s s o l v e d i n methanol R e s i d u e S o l u t i o n ( c o n c e n t r a t e d 50x) : h a r c o a l i z e d , d i l u t e d tfith w a t e r , e x t r a c t e d tfith p e t r o l e u m e t h e r I^O-methanol phase P e t r o l e u m e t h e r phase e v a p o r a t e d R e s i d u e ( c o n c e n t r a t e d 450x) d i s s o l v e d i n e t h e r , t r a n s f e r r e d t o a l u m i n a column, d e a c t i v a t e d w i t h a c e t i c a c i d , 200 ml e t h e r e l u a t e c o l l e c t e d , e v a p o r a t e d r E x t r a c t Residue (concentrated 3,000x) transferred to alumina V column i n benzene eluted with benzene Up to and including part of f i r s t orange zone (non-toxic) Later part of orange zone, dark zone, and small gap Later zones (non-toxic) evaporated Residue (concentrated 20,000x) transferred to Mg carbonate : e l i t e column i n CS2, eluted vith CS 9, then ether Dark Zone from CS2 (non-toxic) Ether eluate evaporated 13 mg Residue (toxic) (concentrated 70,000x) * White. E.P. (1959) 72 6. CHEMICAL AND BIOLOGICAL CHARACTERISTICS OF TOXIN The toxic compound that causes f a c i a l eczema i s c a l l e d sporidesmin. Ronaldson et a l (1963) have i s o l a t e d and i d e n t i f i e d two toxic metabolites of the fungus Pithomyces chartarum, sporidesmin (C^g 1 * 2 1 ( 2 2 ) ^ % °6 S 2 ^ and sporidesmin-B (C^g H 2 0 C l N 3 O 5 S 2 ) . Sporidesmin i s a hydroxy sporidesmin-B. The substance i s c o l o u r l e s s . The extracted toxin when fed to guinea pigs in amounts of 0.5 mg over a 3 week period causes severe l i v e r damage. Several bioassay techniques and an empirical test for determining the t o x i c i t y of pasture grass have been reported. These include: (1) Guinea pig test outlined by Evans and P e r r i n (1957) i n which the t o x i c i t y of the pasture sample i s r e l a t e d to the extent of l i v e r damage a f t e r 3 weeks of feeding. ( 2 ) Rabbit test (Dodd, 1960) - t h i s test i s much more rapid than the guinea pig. t e s t . (3) Murphy and Worker (1960), F a s t i e r (1961) outlined a test using c e l l s i n a r t i f i c i a l c u l t u r e . The toxic substance when incubated with hela c e l l s , human amniotic c e l l s , guinea p i g kidney c e l l s showed s l i g h t t o x i c o s i s . Unsuccessful attempts to demonstrate n e u t r a l i z i n g antibodies i n the sera of guinea pigs dosed with P. chartarum have been made (F a s t i e r , 1961). An i n d i r e c t chemical test f or t o x i c i t y c a l l e d the "beaker t e s t " was devised by P e r r i n (1959) and was further improved by Sandos (1959). This substance i s obtained as an insoluble residue when the to x i c p r i n c i p l e i s dissolved i n ether. This insoluble material appears as a white substance on the walls of the beaker in which the toxin i s mixed with ether. The test can 74 be c a r r i e d out in about 4 hours and i s rapid and r e l i a b l e f or the evaluation of t o x i c i t y of grass samples. The substance of the "beaker t e s t " i s i n i t s e l f non-toxic. 7. EXPERIMENTAL SPORIDESMIN INTOXICATION IN ANIMALS Experimental investigations on the f a c i a l eczema toxin have been done using guinea pigs and rabbits as well as sheep. Sheep developed the features of the t o x i c o s i s as was noticed i n the natural disease condition. L i v e r damage was observed and lesions of photosensitization appeared on the face on exposure to sunlight. In guinea pigs, P e r r i n (1957) distinguishes between four d i f f e r e n t stages of l i v e r damage: (1) Mild f a c i a l eczema - the l i v e r weight and colour are normal. On further examination of the l i v e r surface, there appear small l i g h t patches g i v i n g a flecked appearance. H i s t o l o g i c a l l y there i s an o b l i t e r a t i v e c h o l a n g i t i s and some p r o l i f e r a t i o n of b i l e duct epithelium. There may be some necrotic f o c i on the l i v e r . (2) Moderate f a c i a l eczema - the l i v e r weight i s increased and the l i v e r surface shows considerable f l e c k i n g . H i s t o l o g i c a l l y there i s an extensive o b l i t e r a t i v e c h o l a n g i t i s together with p r o l i f e r a -t i o n of b i l e duct epithelium. The necrotic l e s i o n s are more pronounced. (3) Severe f a c i a l eczema - the l i v e r i s considerably increased in siz e and weight and i s generally l i g h t i n colour. The surface i s conspicuously flecked, i s of a rough texture and shows a greenish-grey network probably due to degraded b i l e pigment. Large necrotic areas are present. The l i v e r f e e l s g r i t t y and rubbery when cut and exposed surfaces have a fibrous appearance. H i s t o l o g i c a l l y , the p r o l i f e r a t i n g b i l e duct epithelium i s the most s t r i k i n g feature with masses of tubules and und i f f e r e n t i a t e d epithelium occupying more than h a l f of the l i v e r section. Fibrous tissue and necrotic areas are increased. B i l e ducts are usu a l l y o b l i t e r a t e d and have been replaced by fibrous tissue and masses of smaller ductules. (4) Very severe f a c i a l eczema - the amount of l i v e r damage i s such that the animal does not survive the test period. At death the animal i s jaundiced and the body tissues are b i l e stained. Damage to the smaller b i l e ducts i s considerable but i f the s u r v i v a l time i s short, weight increase, loss of colour of the l i v e r and the amount of b i l e duct p r o l i f e r a t i o n may be small. S l a t e r et a l (1964) tested the e f f e c t of sporidesmin i n r a t s . They found that the rat i s more r e s i s t a n t to the toxin than the other species tested (sheep, guinea pigs, rabbits) and that the rat responds to the toxin i n a d i f f e r e n t way. There was l i t t l e evidence of l i v e r damage i n rats and t h i s was confirmed by l i v e r function tests v i z . serum b i l i r u b i n and a l k a l i n e phosphatase. Rats however developed a s c i t e s and p l e u r a l effusions. • Sporidesmin was shown to increase c a p i l l a r y permeability. 8. MODE OF ACTION OF TOXIN Worker (1960) reported that the transport of the toxin into the l i v e r i s by way of the p o r t a l route. The p o r t a l c i r c u l a t i o n i s the main route by which the toxin enters the l i v e r following absorption into the i n t e s t i n e . The toxin i s unstable i n the systemic c i r c u l a t i o n . Thus the uptake of the toxin by the lymphatics i s of no importance so f a r as l i v e r lesions are 76 concerned. The fact that the toxin i s inactivated i n the general c i r c u l a t i o n suggests that the uptake of the toxin by the l i v e r i s instantaneous and the lesions produced must be due to the f r a c t i o n of toxin removed from the por t a l i blood the f i r s t time i t passes through the l i v e r . 9. TREATMENT AND CONTROL The prevention of f a c i a l eczema i s lim i t e d to good management. Animals should not be allowed to graze on toxic pastures. The "beaker t e s t " should be employed as a screening method f or t o x i c i t y of pastures. Also since f a c i a l eczema i s a seasonal disease, the occurrence of a large spore load i n a pasture can be predicted by observing the weather conditions. MYCOTOXICOSES GENERAL ASPERGILLUSTOXICOSIS As a r e s u l t of the investigations c a r r i e d on i n Russia concerning ATA in humans and stachybotryotoxicosis i n animals, i t was then found necessary to test various other species of common moulds f or t o x i c i t y to animals. A large assortment of fungi were tested f o r dermal re a c t i o n on rabbits and the r e s u l t s of these tests revealed that the genus A s p e r g i l l u s was a prime causative agent i n fungal t o x i c o s i s . A s p e r g i l l u s fumigatus Waksman et a l (1942) obtained a n t i b i o t i c substances from A. fumigatus by growing the fungus under s p e c i a l conditions. By growing the fungus i n Czapek-Dox medium containing 40 gm glucose; 3 gm NaN03; 1 gm K H 2 P O 4 ; 0.5 gm MgS0^ . 7 H 20; 0.01 gm F e S 0 4 . 7 H 2 0 ; and 100 ml H 20, the a n t i b i o t i c substance fumigacin was produced i n three days at 28° C., reached i t s maximum i n 7-8 days and then gradually disappeared. The reduction i n a c t i v i t y was due to either the destruction of the active substance or to i t s transformation intO| an in a c t i v e or at least a less active compound. Oxford and R a i s t r i c k (1942) from t h i s fungus produced a maroon-coloured pigment, fumigatin, which has a n t i b a c t e r i a l properties. This was formed in Raulin-Thom medium a f t e r 30-35 days incubation. The method of i s o l a t i o n of these two substances were d i f f e r e n t , fumigacin being adsorbed onto n o r i t , then treated with ether and f i n a l l y extracted with chloroform. Fumigacin and fumigatin are two d i s t i n c t l y d i f f e r e n t compounds varying i n mode of formation, chemical nature and a n t i b a c t e r i a l a c t i v i t y . The chemical formula for fumigacin i s C 3 2 H^ 2 Og. Fumigatin has the formula Cg Hg O 4 . A n t i b a c t e r i a l tests have shown that fumigacin has a high a n t i b i o t i c a c t i v i t y and may be considered a true a n t i b i o t i c agent whereas fumigatin possesses l i t t l e a n t i b i o t i c a c t i v i t y and thus appears to be s i m i l a r to that c h a r a c t e r i s t i c of quinones. Fumigacin i s active against gram p o s i t i v e b a c t e r i a e s p e c i a l l y c o c c i and spore formers and has l i t t l e a c t i v i t y against gram negative b a c t e r i a ; fumigatin does not exhibit such marked dif f e r e n c e s . However fumigatin deteriorates on standing. Fumigacin appears to be f a i r l y toxic to animals, 1 mg of the c r y s t a l l i n e preparation k i l l i n g a 20 gram mouse i n 24 hours. A s p e r g i l l u s clavatus A s p e r g i l l u s clavatus i s morphologically d i s t i n c t from A. fumigatus, and i t s a n t i b i o t i c substance i s also markedly d i f f e r e n t . The a n t i b i o t i c 78 TABLE VIII Distinguishing C h a r a c t e r i s t i c s of Fumigatin and Fumigacin * C h a r a c t e r i s t i c Properties Fumigatin Fumigacin Medium produced in Stage of growth at which substance is l a r g e l y formed Method of extraction from medium C r y s t a l l i z a t i o n of substance Appearance of c r y s t a l s Carbon content, % Presence of nitrogen Melting point Chemical nature .Units of a n t i b i o t i c a c t i v i t y S p e c i f i c nature of a n t i b i o t i c a c t i v i t y Has been i s o l a t e d from a culture of A. fumigatus in Raulin-Thom medium Has been i s o l a t e d from A. fumigatus culture i n Czapek-Dox medium. Late stage (30-55 days) Early stage (3-10 days) Direct extraction of medium with chloroform From petroleum Maroon-coloured needles 57.1 Absent 116° C Quinone 12,000 to 100,000 L i t t l e difference i n a c t i v i t y upon gram negative (E. c o l i ) b a c t e r i a Adsorption on n o r i t , ex-t r a c t i o n with chloroform, following ether treatment From alcohol on cooling Pure white needles 62.7 Present (3.77.) 185-187° C Unknown 10,000 to 6,000,000 Gram negative b a c t e r i a (E. c o l i ) are far less s e n s i t i v e (10,000 units) than gram p o s i t i v e b a c t e r i a (B. mycoides -2,000,000 units S. lutea - 6,000,000 u n i t s ) . * Waksman, Horning and Spencer (1942) c l a v a c i n i s produced i n glucose - n i t r a t e medium aft e r 3 days incubation at 28° C , and diminishes on further incubation. The highest a c t i v i t y was usually obtained i n 6 days and the culture was usually active as long as 3 weeks. Clavacin i s active against gram negative b a c t e r i a (E. c o l i ) and possesses greater a c t i v i t y against B. s u b t i l i s than against B. mycoides. Thus c l a v a c i n i s highly active against both gram negative and gram p o s i t i v e b a c t e r i a , the degree of a c t i v i t y being s i m i l a r . Clavacin was i s o l a t e d from the culture either by extraction with ether or chloroform or by adsorption onto n o r i t followed by ether and chloroform e l u t i o n . Clavacin i s f a i r l y toxic to animals. Its chemical formula i s C7 Hg O4. A s p e r g i l l u s flavus White and H i l l (1942) obtained a s p e r g i l l i c acid from the mould A. flavus growing i n Czapek-Dox l i q u i d medium. The presence of the active substance does not depend on the a c t i v i t y of the mould i t s e l f since luxuriant mould growth may occur without the formation' of a s p e r g i l l i c acid. A s p e r g i l -l i c acid exhibits a n t i b a c t e r i a l a c t i v i t y against c e r t a i n gram negative as well as gram p o s i t i v e b a c t e r i a . It i s an amphoteric substance of the chemical composition ^20 N2 ^2* * s highly toxic to mice. Jones et a l (1943) produced a s p e r g i l l i c acid from a s t r a i n of A. flavus growing on media containing brown sugar (2 per cent) in addition to 2 per cent tryptone and 0.5 per cent NaCl. The addition of 2 per cent brown sugar increased the y i e l d of a s p e r g i l l i c acid from 250 mg per l i t r e to 400 mg per l i t r e . They found that a s p e r g i l l i c acid was more active against gram p o s i t i v e c o c c i than against the anaerobes which produce gas gangrene and the gram negative b a c i l l i . McKee et a l (1943) produced an a n t i b i o t i c substance, f l a v i c i d i n , 80 from a s t r a i n of A. flavus by submerged growth i n a modified Czapek-Dox medium. This substance i s s i m i l a r to p e n i c i l l i n and has been c a l l e d p e n i c i l l i n F. I t s chemical formula i s H 2 0 O 4 N 2 S. The b i o l o g i c a l c h a r a c t e r i s t i c s of f l a v i c i d i n and p e n i c i l l i n are s i m i l a r : (1) They are highly active against gram p o s i t i v e organisms and r e l a t i v e l y Inactive against gram negative b a c i l l i . ( 2 ) They are both highly soluble. (3) They protect mice i n equal degrees against pneumococcus i n f e c t i o n . (4) Organisms r e s i s t a n t to p e n i c i l l i n are also r e s i s t a n t to f l a v i -c i d i n but not to other a n t i b i o t i c substances. (5) An enzyme active against p e n i c i l l i n i s also active against f l a v i c i d i n but not against other a n t i b i o t i c substances. HYPERKERATOSIS IN CATTLE Afte r the success of the Russian workers in discovering various fungi to be the cause of a number of diseases in domestic animals, attempts were made i n the United States to trace the cause of many animal poisonings to the fungi. C a r l l et a l (1953) i s o l a t e d a toxic fungus from a food concen-trate which was the cause of hyperkeratosis i n c a t t l e . A. clavatus was the fungus and an ether extract of t h i s fungus proved dermally toxic to r a b b i t s . An ether extract of the fungus grown on corn, when fed to calves, produced the symptoms of f i e l d cases of bovine hyperkeratosis. In 1954 and 1955, the same workers Isolated A. clavatus and A. fumigatus from toxic corn and produced hyperkeratosis-like symptoms i n calves and horses. Forgacs et a l (1954) reported on the i s o l a t i o n of fungi from a suspected hyperkeratosis food sample which consisted of p a r t i c l e s of mouldy bread. A s p e r g i l l u s tamarii, A. flavus, A. c h e v a l e r i were is o l a t e d and the 81 e x t r a c t s , when d e r m a l l y t e s t e d on r a b b i t s and c a l v e s , p r oduced h y p e r a e m i a w i t h i n 24 h o u r s w i t h A. c h e v a l e r i showing a more pronounced r e a c t i o n . A. c h e v a l e r i i n o c u l a t e d b r e a d , when f e d t o c a l v e s , p r oduced t h e g e n e r a l symptoms of h y p e r k e r a t o s i s . Z i n c h e n k o (1959) r e p o r t e d on a d i s e a s e i n swine i n R u s s i a c a u s e d by t h e e a t i n g o f f e e d w h i c h c o n s i s t e d o f meat and bone meal found t o c o n t a i n A. f l a v u s and S c o p u l a r i o p s i s b r e v i c a u l i s . MOULDY CORN TOXICOSIS S i p p e l e t a l (1953) r e p o r t e d on t h e p o i s o n i n g o f swine and c a t t l e by the i n g e s t i o n o f mouldy c o r n . E a r l y o u t b r e a k s o f t h i s t o x i c o s i s were a s s o c i a t e d w i t h t h e c o n s u m p t i o n o f mouldy s o f t c o r n l y i n g on t h e ground. Subsequent o b s e r v a t i o n s r e v e a l e d t h a t b o t h h a r d and u n h a r v e s t e d c o r n were i n f e c t e d w i t h f u n g i . B u r n s i d e , S i p p e l et a l (1957) r e p r o d u c e d t h e d i s e a s e by f e e d i n g a ground s u s p e n s i o n o f mouldy c o r n t o swine and c a t t l e and by f e e d i n g the mouldy c o r n i t s e l f . They a l s o i s o l a t e d 13 f u n g i r e p r e s e n t i n g v a r i o u s g e n e r a and s p e c i e s f r o m samples o f mouldy c o r n and two o f t h e s e f u n g i were t o x i c . Of the 13 f u n g i i s o l a t e d , 9 were o f t h e s p e c i e s A s p e r g i l l u s f l a v u s L i n k , 3 o f t h e s p e c i e s F u s a r i u m m o n i l i f o r m e and one was P e n i c i l l i u m rubrum S t o l l . Two of t h e s e were t o x i c t o swine when f e d i n pur e c u l t u r e , one b e i n g P. rubrum S t o l l and t h e o t h e r , one of the A. f l a v u s L i n k s p e c i e s . Mouldy c o r n t o x i c o s i s i s s e a s o n a l , a p p e a r i n g d u r i n g t h e l a t e summer and e a r l y f a l l when the a n i m a l s a r e f o r a g i n g c o r n i n the f i e l d . The d i s e a s e m a n i f e s t s i t s e l f i n two f o r m s , t h e a c u t e and the c h r o n i c , w i t h symptoms and p a t h o l o g y s i m i l a r t o t h a t r e p o r t e d f o r o t h e r f u n g a l d i s e a s e s i n t h e l i t e r a -t u r e e s p e c i a l l y t h o s e e n c o u n t e r e d i n R u s s i a . E x t r a c t s o f P. rubrum and A. f l a v u s were t o x i c t o m i c e . 82 As w i t h t he o t h e r m y c o t o x i c o s e s , mouldy c o r n t o x i c o s i s does not depend on t h e amount o f f u n g a l c o n t a m i n a t i o n p r e s e n t on t h e c o r n , b u t on t h e typ e o f f u n g i p r e s e n t . O f t e n t h e d e l e t e r i o u s f u n g i a r e p r e s e n t i n a s m a l l amount compared w i t h t he e x t e n s i v e f u n g a l i n f e s t a t i o n t h a t i s p r e s e n t . The t o x i c p r i n c i p l e I s not t h e fungus i t s e l f , b u t a s u b s t a n c e s e c r e t e d by t h e fungus w h i c h c a n be e x t r a c t e d w i t h f a t s o l v e n t s . Thus the de r m a l t e s t on r a b b i t s w i t h e t h e r e x t r a c t s o f t h e f e e d i s of p r i m e i m p o r t a n c e i n s c r e e n i n g mouldy f e e d samples f o r t o x i c i t y . POULTRY HAEMORRHAGIC SYNDROME Around 1950, t h e r e appeared a new d i s e a s e o f p o u l t r y w h i c h was c l i n i c a l l y e x p r e s s e d by h a e m o r r h a g i c t i s s u e s . Baker and J a q u e t t e (1953) named i t "haemorrhagic syndrome". The d i s e a s e i s p r e v a l e n t i n b r o i l e r s f r o m 4-7 weeks o l d , b u t i t c a n o c c u r i n b i r d s o f any age. I t has been o b s e r v e d t h a t t he d i s e a s e v a r i e s from y e a r t o y e a r and most a r e a s r e p o r t t h a t o u t -b r e a k s o f the d i s e a s e o c c u r f r o m l a t e f a l l t h r o u g h t h e s p r i n g when t h e t e m p e r a t u r e and m o i s t u r e c o n d i t i o n s a r e i d e a l f o r f u n g a l g r o w t h and t o x i n format i o n . V a r i o u s t h e o r i e s have been advanced c o n c e r n i n g t he p o s s i b l e c a u s a t i v e a g e n t . These i n c l u d e : (1) A n t i b i o t i c s w h i c h d e p r e s s t h e growth o f i n t e s t i n a l b a c t e r i a c o n s i d e r e d t o be n e c e s s a r y f o r s y n t h e s i s o f v i t a m i n K. T h i s w o u l d i n c l u d e t he a d d i t i o n o f c o c c i d i o s t a t s , e.g. s u l f a q u i n o x a -l i n e , t o t h e d i e t t h u s c a u s i n g d r u g t o x i c i t y o r an a n t i v i t a m i n K e f f e c t . (2) Use of a d i e t l a c k i n g v i t a m i n K r e q u i r e m e n t s . (3) C a u s i n g a v i t a m i n o s i s . K a f t e r u n u s u a l s t r e s s . 83 (4) Reduced absorption of vitamin K owing to i t s low content in the d i e t or to i n t e s t i n a l l e s i o n s . (5) L i v e r damage from i n d e f i n i t e causes r e s u l t i n g i n an increased flow of b i l e necessary for optimum absorption of vitamin K. (6) I n h i b i t i o n of prothrombin production required for proper functioning of the b l o o d - c l o t t i n g mechanism. Anderson and co-workers (1954) fed chicks a r a t i o n that did not contain c o c c i d i o s t a t s nor a n t i b i o t i c s and got a 10 per cent m o r t a l i t y . They suspected that the corn or soybean meal was the toxic f r a c t i o n of the d i e t . Forgacs and C a r l l (1958) observed the s i m i l a r i t y between the c l i n i c a l and pathological p i cture of poultry haemorrhagic syndrome, ATA and stachybotryo-t o x i c o s i s . Using fungal i s o l a t e s from feed scattered i n the l i t t e r of b r o i l e r houses where the disease was enzootic,' they reproduced acute toxic signs in chickens s i m i l a r to those reported i n f i e l d conditions. Later on, they confirmed these findings by inducing a chronic t o x i c o s i s . Forgacs et  a l (1961) induced the t o x i c o s i s by addition of fungi to the l i t t e r and feed of chickens and were able to prevent the disease by an addition of a n t i -fungal compounds to the feed. Among the fungi i s o l a t e d by Forgacs et a l (1955, 1961) from feed and l i t t e r i n outbreaks of poultry haemorrhagic syndrome were A s p e r g i l l u s clavatus, A. flavus, A. fumigatus, A. glaucus, Paecilomyces v a r i o t i , P e n i c i l l i u m citrinum, P. purpurogenum, P. rubrum, a species of A l t e r n a r i a and several other u n i d e n t i f i e d p e n i c i l l i a , a l l of which were shown to induce a mycotoxic condition i n chickens. Chickens eating mouldy feed develop diarrhoea and an extensive haemorrhagic condition. The extent of the condition depends on the potency of the toxin and the amount of feed ingested. Since the p r o l i f e r a t i o n of fungi depends p a r t l y on the moisture content of the feed and the dampness of 84 the l i t t e r , the maintenance of dry conditions i n the l i t t e r and feed and the continuous replacement of damp feed and l i t t e r i s necessary f o r the control of t h i s t o x i c o s i s i n poultry. Forgacs et a l (1961) noted that Scopulariopsis species was present i n feed Involved i n t h i s poultry disease. However t h i s fungus was present p r i o r to the decline of the t o x i c o s i s . Ether extracts of the toxic feed were tested on rabbits for the dermal reacti o n . At the 5th week of fungal growth the test was p o s i t i v e , whereas at the 6th week i t was negative although fungi were s t i l l present i n the feed. The r o l e of Scopulariopsis i n mouldy feed t o x i c o s i s of chickens i s s t i l l obscure. Forgacs et a l (1961) suggested that the fungus s u c c e s s f u l l y competed with toxic fungi o r i g i n a l l y present i n the feed f o r some common nutr i e n t , or produced an antimetabolite to the toxic fungi. Schumaier et a l (1963) reported on stachybotryotoxicosis of chickens experimentally dosed with ether extracts of the fungus. This l i p i d - s o l u b l e toxin reduced growth rate and produced lesions i n the epithelium of the mouth and crop. The toxin was r e s i s t a n t to autoclaving f o r one hour at 121°C and 15 pounds pressure. MOULD CONTROL IN STORED GRAIN Contamination by and p r o l i f e r a t i o n of fungi i n feedstuffs which give r i s e to the problem of fungal t o x i c o s i s i n animals i s d i r e c t l y due to the conditions under which feedstuffs are stored a f t e r harvesting. Fungi may eit h e r invade the grain while i t i s s t i l l developing on the plant or during storage. Fungi belonging to the genera A l t e r n a r i a , 85 Helminthosporium, Fusarium, Cladosporium, Diplodia, Rhizopus and a number of others are thought to invade the seed while i t i s developing on the plant, whereas A s p e r g i l l u s and P e n i c i l l i u m species usually contaminate the grain during storage. It has been proved that storage fungi do not invade the seeds p r i o r to harvest even when the r i p e seeds are l e f t standing i n the f i e l d under moist conditions. However within a week a f t e r harvesting, rapid and extensive invasion by storage fungi i s encountered. Various phy s i c a l conditions influence the invasion of stored grain by fungi, v i z . moisture content, temperature, time, amount of previous mould i n f e c t i o n , damage due to insects and mites. Features responsible for mould spoilage i n cereal grain (1) Storage - I t has been suggested that the grain i s too moist (over 16 per cent moisture) when harvested and the high moisture content favours mould p r o l i f e r a t i o n . I t i s also possible that the storage of moist grain i n granaries for long periods r e s u l t s i n the oxidation of l i p i d s and free f a t t y acids i n the grain by moulds, thus giving r i s e to the toxic f a c t o r . The l i p i d content of proso-millet i s very high and the grain also contains the enzyme l i p a s e which, under su i t a b l e conditions, causes the decomposition of the seed l i p i d s . Thus spoilage of proso-millet i s very rapid. Wheat, rye, barley, corn, oats and buckwheat are also involved i n outbreaks of fungal t o x i c o s i s . (2) Weather - Experiments have shown that grain l y i n g i n the f i e l d during the winter i s non-toxic. I t i s only a f t e r the thawing of the snow that the grain becomes t o x i c . Thus i t i s the rapid change i n temperature together with the high moisture i n the 86 grain that bring about t h i s spoilage. (3) Insects and mites - Previous damage done to grain by insects and mites makes i t more susceptible to fungal contamination. (4) E f f e c t of moulds - Physical agents alone are not responsible for the toxin production i n cereal grain. The grain i t s e l f must be mould infested. 1 None of these conditions i s s o l e l y responsible for the extensive p r o l i f e r a -t i o n of the moulds, but rather some or a l l of these conditions act together to bring about the mould i n f e s t a t i o n . Moulds are normally present on or i n a l l grain or feed ingredients and there i s no p r a c t i c a l way of removing them. In order to c o n t r o l mould growth i n stored f e e d s t u f f s , the most important factors to be considered are temperature, moisture and the supply of oxygen and n u t r i e n t s . P e l l e t i n g i s a p r a c t i c a l way of making safe feedstuffs that would otherwise be highly susceptible to mould growth and heating. Christensen (1957) suggests three p o s s i b i l i t i e s f or c o n t r o l l i n g the growth of storage fungi in grains. (1) The use of fungicides (2) Storage under toxic or inert gases (3) Drying the grain to reduce the moisture content to a safe l e v e l . Certain fungicides (chlorinated phenols, 3 per cent ethyl mercury phosphate) have been tested with l i m i t e d success in c o n t r o l l i n g mould spoilage i n g r a i n . However, as yet no e f f e c t i v e non-toxic fungicide has been developed for use i n human and animal f e e d s t u f f s . 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PART I I I EXPERIMENTAL AFLATOXICOSIS IN THE MINK 98 INTRODUCTION A f l a t o x i n , elaborated by A s p e r g i l l u s flavus Link ex F r i e s , has been shown to be l e t h a l to many domestic and laboratory mammals and birds (Asplin and Carnaghan, 1961; A l l c r o f t and Lewis, 1963; Butler-et a l , 1963). Intoxicated animals displayed marked l i v e r damage characterized by necrosis and haemorrhage. Ducklings fed aflatoxin-containing peanut meal showed destruction of the parenchymal c e l l s of the l i v e r and extensive p r o l i f e r a t i o n of the e p i t h e l i a l c e l l s of the b i l e duct (Asplin and Carnaghan, 1961). The s i m i l a r i t y i n the l i v e r pathology of aflatoxin-poisoned animals and that displayed by mink dying from an hepato-haemangioma-syndrome (McMillan, 1965) suggested that the mink disease might be due to a s i m i l a r fungal toxin. To test t h i s postulate, a series of experiments were c a r r i e d out to determine the e f f e c t of the i n c l u s i o n of various l e v e l s of toxic peanut meal i n normal ranch mink r a t i o n s . Preliminary t r i a l s with ducklings were c a r r i e d out to evaluate a commercial peanut meal that was suspected to contain a f l a t o x i n . EXPERIMENTAL A. Source of A f l a t o x i n Several bags of B r a z i l i a n peanut meal that were suspected of contain-ing a f l a t o x i n were obtained from a commercial source. M i c r o b i o l o g i c a l examination of representative samples of t h i s meal revealed that the b a c t e r i a l and fungal counts were many f o l d higher than those normally encountered i n Canadian f e e d s t u f f s . The presence of the genus A s p e r g i l l u s as the dominant fungal contaminant was noted. The moisture content of the meal was twelve per cent. 99 B. Evaluation of Toxic Peanut Meal by Duckling Assay Duckling Assay No. 1 (1) Preparation of Extract: As a preliminary to assays with mink, representative two kilogram samples from one bag were subjected to the methanol extraction and concentration procedure suggested by Sargeant et a l (1961). Af t e r evaporation of the methanol, the extracted residue from two kilograms of the meal was made up to 50 ml with water and used for assay in ducklings. (2) Animals and Treatment: Twenty, three-day-old ducklings were divided into f i v e groups of four and were housed i n wire brooder cages. Four of the groups received s i n g l e per orum graded doses of the meal extract, the f i f t h group received an equivalent volume of d i s t i l l e d water and served as a c o n t r o l . They were offered a commercial duck grower r a t i o n which was known to be adequate to support rapid growth. A l l ducklings that died or that survived to the end of the experimental period were submitted to post mortem examination. Representative tissues were selected f or h i s t o p a t h o l o g i c a l study. (3) Results: The r e s u l t s of t h i s assay are provided i n Table IX. Duckling Assay No. 2 (1) Rations: Having established the presence of toxin i n the commercial peanut meal, a second duckling assay was c a r r i e d out by incorporating samples from three d i f f e r e n t bags of peanut meal into a commercial T A B L E I X Dosage and M o r t a l i t y S c h e d u l e o f D u c k l i n g s  A s s a y No. 1 Days on A s s a y Dosage Volume of Meal E x t r a c t (ml) E q u i v a l e n t (grams) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16-21 M o r t a l i t i e s 2.00 80 0 0 0 1 2 4 - - - -- - - - - - 4/4 1.00 40 0 0 0 0 0 4 4/4 0.50 20 0 0 0 0 0 1 0 0 0 0 0 0 2 0 3 0 3/4 0.25 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/4 C o n t r o l _ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/4 101 duck grower r a t i o n at the t w e l v e p er c e n t l e v e l by w e i g h t . T h i s a s s a y was i n t e n d e d t o a s c e r t a i n t h a t bag of peanut meal w h i c h would p r o v i d e t h e h i g h e s t c o n c e n t r a t i o n o f t o x i n f o r mink a s s a y . (2) A n i m a l s and Tr e a t m e n t : Three groups of f o u r , t h r e e - d a y - o l d d u c k l i n g s were housed i n w i r e b r o o d e r cages and o f f e r e d one o f the peanut meal supplemented r a t i o n s . A f o u r t h group was o f f e r e d the same b a s a l r a t i o n s u p p l e -mented w i t h a peanut meal sample t h a t was known t o be f r e e o f t o x i n . (3) R e s u l t s : The r e s u l t s o f t h i s a s s a y a r e p r e s e n t e d i n T a b l e X. P o s t mortem and h i s t o p a t h o l o g i c a l f i n d i n g s a g a i n i n d i c a t e d t h a t the d u c k l i n g d e a t h s were due t o a f l a t o x i n p o i s o n i n g . By u s i n g the time r e q u i r e d t o i n d u c e d e a t h as a c r i t e r i o n o f a f l a t o x i n c o n t e n t , i t c a n be c o n c l u d e d t h a t t h e peanut meal i n Bag No. 1 p r o b a b l y c o n t a i n e d the h i g h e s t l e v e l o f t o x i n . The body w e i g h t changes i n the f o u r groups of d u c k l i n g s g i v e n i n T a b l e X I p r o v i d e d c o n f i r m a t i o n o f t h i s a s s u m p t i o n . D u c k l i n g A s s a y No. 3 (1) R a t i o n s : A t h i r d a s s a y u s i n g d a y - o l d d u c k l i n g s was c a r r i e d out on t o x i c peanut meal from Bag No. 1 and on e x t r a c t s p r e p a r e d from meals i n Bags Nos. 1 and 3 (used i n A s s a y No. 2 ) . These same meals were f e d t o d u c k l i n g s a f t e r e x t r a c t i o n by the p r o c e d u r e o f S a r g e a n t e t a l (1 9 6 1 ) . (2) A n i m a l s and T r e a t m e n t : T h i r t y , d a y - o l d d u c k l i n g s were d i v i d e d i n t o s i x groups of f i v e . TABLE X M o r t a l i t y S c h e d u l e o f D u c k l i n g s A s s a y No. 2 Days on A s s a y Group Bag 0 3 5 7 10 12 14 17 M o r t a l i t i e s 1 1 0 0 0 4 4/4 2 2 0 0 0 0 1 2 3 4 4/4 3 3 0 0 0 1 4 4/4 4 ( C o n t r o l ) 0 0 0 0 0 0 0 0 0/5 o ho TABLE X I Mean Body Weights of S u r v i v i n g D u c k l i n g s  A s s a y No. 2 Days on A s s a y Group Bag 0 3 5 7 10 12 14 17 1 1 30.7/4 99.0/4 101.9/4 2 2 85.5/4 126.5/4 146.6/4 153.2/4 174.5/3 193.0/2 211.0/1 3 3 88.0/4 105.5/4 109.5/4 122.0/3 4 ( C o n t r o l ) 7 9 . 8 / 5 111.5/5 156.5/5 205.0/5 287.5/5 400.5/5 506.0/5 709.0/5 TABLE X I I M o r t a l i t y S c h e d u l e o f D u c k l i n g s A s s a y No. 3 Days on A s s a y Group Treatment Meal o r Meal E q u i v a l e n t (grams) 0 1 2 3 4 5 6 7 8 9 10 M o r t a l i t i e s 1 10% c r u d e peanut meal (Bag No. 1) D a i l y T o t a l 0.8 x days s u r v i v a l 0 0 0 0 0 1 0 2 5 5/5 2 10% e x t r a c t e d peanut meal (Bags Nos. 1 & 3) 0.8 x days s u r v i v a l 0 0 0 0 0 0 0 0 0 0 0 0/5 2 x 0.25 ml ex-t r a c t (Bag No. 1) 20 40 0 5 5/5 2 x 0.50 m l ex-t r a c t 40 80 0 4 5 5/5 5 2 x 0.25 ml ex-t r a c t (Bag No. 3) 20 40 0 2 0 0 3 0 0 0 0 0 0 3/5 6 2 x 0.50 ml ex-t r a c t (Bag No. 3) 40 80 0 1 2 4 4 5/5 105 They were g i v e n the t r e a t m e n t s o u t l i n e d i n T a b l e X I I . (3) R e s u l t s : The r e s u l t s o f t h i s a s s a y , as shown i n T a b l e X I I , c o n f i r m t h o s e o b t a i n e d i n A s s a y No. 2 i n d i c a t i n g t h a t the meal i n Bag No. 1 c o n t a i n e d the h i g h e r l e v e l of t o x i n . The d u c k l i n g s t h a t r e c e i v e d t h e e x t r a c t e d meal a l l s u r v i v e d t h e a s s a y p e r i o d and grew at a n ormal r a t e s u g g e s t i n g t h a t the e x t r a c t i o n p r o c e d u r e had been adequate. Thus the meal e q u i v a l e n t w e i g h t s shown i n T a b l e s I X and X I I , s h o u l d p r o v i d e a r e a s o n a b l e i n d i c a t i o n on a w e i g h t b a s i s of the l e t h a l i t y of t h e s e t o x i n - c o n t a i n i n g meals f o r d a y - d i d and t h r e e - d a y - o l d d u c k l i n g s . A f l a t o x i c o s i s i n t h e Mink Mink A s s a y No. 1 The peanut meal sample, Bag No. 1, shown t o c o n t a i n a f l a t o x i n by d u c k l i n g a s s a y , was used as a s o u r c e of t o x i n f o r t h e mink a s s a y s . B o t h s p e c t r o p h o t o m e t r y and c h r o m a t o p l a t e a s s a y s and c h i c k embryo l e t h a l i t y t r i a l s c o n f i r m e d t h a t the t o x i n r e s p o n s i b l e f o r the d u c k l i n g m o r t a l i t y was a f l a t o x i n . (1) R a t i o n : A p r e l i m i n a r y a s s a y was c a r r i e d out i n w h i c h t h e known t o x i c peanut meal was i n c o r p o r a t e d i n a n ormal mink r a t i o n a t t h e f i v e p e r c e n t l e v e l (wet f e e d b a s i s ) . The c o n s t i t u e n t c o m p o s i t i o n o f t h e r a t i o n i s g i v e n i n T a b l e X I I I . 106 TABLE XIII Mink Ration Assay No. 1 Ingredients Per Cent 20.0 Ground Barley 300 Ground Wheat 900 Oat Groats 600 Wheat Bran 200 Vitamin Supplement 10.0 Peanut Meal 5.0 Ground F i s h Frames 17.5 Ground Poultry Waste 7.5 Beef L i v e r 5.0 Cottonseed O i l 5.0 Water 30.0 100.0 (2) Animals and Treatment: Twenty female mink k i t s (age 100 days) were f u l l fed on the r a t i o n given i n Table XIII. Fresh feed and water were provided d a i l y . The animals were maintained on the r a t i o n u n t i l death occurred. (3) Results: TABLE XIV  M o r t a l i t y Pattern for Mink  Assay No. 1 Days to Death No. of Animals 7 1 10 12 12 7 The mean d a i l y wet feed intake of the mink over the period of su r v i v a l amounted to approximately 100 grams. For animals that 107 survived for ten days, the intake of toxic peanut meal was therefore 5 grams per day or a t o t a l of 50 grams for the period. The mean body weight of the mink over the ten-day s u r v i v a l period was approximately 750 grams. The peanut meal intake per gram of p h y s i o l o g i c a l body mass (W 0 , 7 5) was therefore in the order of 0.035 gram. The ducklings that survived for an average of eight days on a r a t i o n containing 10 per cent of the same peanut meal weighed 56 grams and consumed on the average 8 grams of feed per day. Their d a i l y intake of peanut meal was approximately 0 . 8 gram or again 0.035 gram per unit of p h y s i o l o g i c a l mass on the basis of these assays i t appeared that the female mink k i t and the one to three-day-old ducklings are both equally susceptible to poisoning with a f l a t o x i n . This f i n d i n g allowed the design of a further mink assay, based on the use of toxic meal, to demonstrate the pathology of a f l a t o x i n i n t h i s species. The d e s c r i p t i o n of the pathological features noted are included with those recorded for Mink Assay No. 2. Mink Assay No. 2 The r e s u l t s from Mink Assay No. 1 suggested that much lower l e v e l s of the toxic peanut meal would have to be incorporated i n a normal mink r a t i o n to induce the chronic pathological l i v e r changes that have come to be associated with fungal t o x i c o s i s . To t h i s end a second mink assay was c a r r i e d out. (1) Rations: The toxic peanut meal was incorporated into a normal mink r a t i o n at a s e r i e s of graded l e v e l s as shown i n Table XV. These rations were a l l mixed at one time and i n s u f f i c i e n t q uantities to 108 permit the experiment to continue for 300 days. Each r a t i o n was then subdivided into five-pound aliquots and frozen i n p l a s t i c bags. The feed was stored at 0° F. The amount required f or each day's feeding was thawed at room temperature overnight.prlorrtb'feeding. TABLE XV Mink Ration Assay No. 2 Ingredients A B C D E Ground Barley 300 Ground Wheat 900 Oat Groats 600 22, ,50 23.75 25.00 24.38 24, ,69 Wheat Bran 200 Vitamin Supplement 10, .00 10.00 10.00 10.00 10, ,00 F i s h Frames 17 .50 17.50 17.50 17.50 17, ,50 Poultry Wastes 7, .50 7.50 7.50 7.50 7, .50 Cottonseed O i l 5, .00 5.00 5.00 5.00 5, .00 L i v e r 5, .00 5.00 5.00 5.00 5. .00 Water 30, .00 30.00 30.00 30.00 30, .00 Toxic Peanut Meal 2, .50 1.25 0.00 0.62 0. .31 100, .00 100.00 100.00 100.00 100. .00 (2) Animals and Treatment: F i f t y male k i t s (mean weight 1100 grams), 150 * 5 days of age, were subdivided into f i v e representative groups of ten. The animals were housed i n standard wire mink cages. Feed and water were provided to appetite. (3) Results: The record of the s u r v i v a l pattern f o r the mink i s given i n Table XVI. From a knowledge of the feed intake of the male mink at the body weights shown i n Table XVI, i t can be estimated that the TABLE XVI Mean Body Weights and Survival Pattern for Mink Assay Ho. 2 Ration No. Peanut Meal Level Per Cent Mean Initial Weight (grams) Mean Final Weight (grams) Mean Time to Death (days) Dry Basis Wet Basis A 5.00 2.50 1049 826 28.4 B 2.50 1.25 1079 856 55.4 C 0.00 0.00 1029 1227 286 D 1.25 0.625 1143 1020 286 * E 0.625 0.312 1174 1104 286 * One mink died and one was killed at 160 days from this group. The other eight survived and were killed at 286 days. 110 animals i n r a t i o n groups A and B ingested approximately 100 grams of the toxic meal i n 28.4 and 55.4 days r e s p e c t i v e l y . The animals i n groups D and E that were k i l l e d a f t e r 286 days consumed s l i g h t l y i n excess of 200 grams of the toxic meal. D. Gross Pathology In general, mink which were fed 10 per cent, 5 per cent, and 2.5 per cent (dry weight basis) of aflatoxin-containing meal i n t h e i r rations showed at autopsy quite obvious gross pathological changes of a d i s t i n c t i v e nature (Assay Nos. 1 and 2). With the lower l e v e l s of toxic meal (1.25 per cent and 0.625 per cent) the changes noted a f t e r euthanasia, with one exception, were for the most part r e s t r i c t e d to the l i v e r . Skin and Subcutaneous Tissues There were no external signs of bleeding i n any mink fed aflatoxin-containing meal. Three mink at the 5 per cent l e v e l showed some f a t t y changes in the subcutaneous areas and i n three other cases from t h i s group, as well as i n f i v e animals on the 10 per cent l e v e l , there was oedema of the subcutaneous f a t or of the loose connective tis s u e i n the flank areas. Blood Vascular System The p r i n c i p a l abnormality was the i n a b i l i t y of the blood to c l o t when the vessels were transected at autopsy. This occurred i n ten (50 per cent) of the mink on the 10 per cent l e v e l and i n a l l ten animals on the 5 per cent l e v e l , as well as i n four of those at the 2.5 per cent l e v e l . This feature was absent from the control and lower dosage groups, that i s , the single death case and those that were s a c r i f i c e d . I l l Peritoneal Cavity The p r i n c i p a l feature noted was that of marked peritoneal a s c i t e s which occurred i n ten mink (50 per cent) at the 10 per cent l e v e l . At the 2.5 per cent l e v e l , there were three cases of marked and two cases of s l i g h t a s c i t e s out of the ten animals on the experiment (Plate 1). No obvious c l i n i c a l cases of as c i t e s occurred i n the groups fed lower l e v e l s of the meal. Other changes were inflammation of the mesentery (two mink at the 10 per cent l e v e l and one at 5 per cent) (Plate 2); haemorrhages i n the omentum (one mink at 5 per cent); and catabolism or softening of the f a t depots (three mink at 5 per cent and two at 2.5 per cent) (Plate 3). The only death at the 1.25 per cent l e v e l demonstrated t h i s change. Two animals at the 5 per cent and one at the 2.5 per cent l e v e l showed the presence of paint-brush haemorrhages beneath the per i t o n e a l surface of the diaphragm. One mink at the 10 per cent l e v e l showed a general congestion or haemorrhage i n the serous membranes of the peritoneal c a v i t y . Thoracic Cavity Sixteen'mink fed at the 10 per cent l e v e l (80 per cent), four mink at the 5 per cent l e v e l (40 per cent), and two mink at the 2.5 per cent l e v e l (20 per cent) displayed marked congestion and petech i a l haemorrhages of the pericardium (Plate 4). Three mink at the 5 per cent l e v e l and one at the 2.5 per cent l e v e l had enlarged rounded hearts or r i g h t v e n t r i c u l a r hypertrophy. One mink of t h i s group developed a hydropericardium. Four mink fed at the 10 per cent l e v e l displayed marked pulmonary congestion; otherwise the lungs were normal. 112 L i v e r The l i v e r s of the mink receiving 10 per cent meal were either yellow and necrotic or f a t t y i n texture (50 per cent) (Plate 5) or were merely extremely pale but with a shallow, congested subcapsular area on the anterior surface (30 per cent). In two cases there were ecchymotic haemorrhages i n the subcapsular area (Plate 6). In the other two mink there was merely a passive hyperaemia of the l i v e r . At the 5 per cent l e v e l , four of ten mink showed a mottled, orange-coloured l i v e r ; i n three cases the l i v e r was pi n k i s h with p e c u l i a r t h i n red cross s t r i a t i o n s ; two had a pinkish l i v e r without s t r i a t i o n s ; and one showed a l i v e r which was pale yellow and nodular. This was the f i r s t l i v e r to show nodules and i t was noted that s l i c e s of the organ would f l o a t i n water, suggestive of a high f a t content. At the 2.5 per cent l e v e l , one l i v e r was uniformly yellow with marked surface nodulation and several regenerative nodules (1-3 mm) (Plate 7). The remaining eight a l l showed haemorrhages of one form or another against a pink, yellow, or orange-coloured parenchyma; one only of these showed surface nodulation. This was a pale, though s u p e r f i c i a l l y , congested l i v e r with surface nodules of highly congested or haemorrhagic tissue (Plate 8). Of the mink fed at the 1.25 per cent l e v e l , the one that died was found to have a l i v e r of yellow-orange hue but with nodular patches of subcapsular regenerative t i s s u e . These patches resembled haemorrhagic areas (Plate 9). The mink that was s a c r i f i c e d on the following day had a normal l i v e r . The remaining eight mink, together with the ten mink at the 0.625 per cent l e v e l , when s a c r i f i c e d a f t e r nine and one-half months, were found to have l i v e r s of apparently 113 normal c o l o u r and t e x t u r e . However, t h e s e l i v e r s showed t h e p r e s e n c e o f a f i n e u n i f o r m n o d u l a t i o n o v e r t h e e n t i r e s u r f a c e and were s l i g h t l y p a l e r i n c o l o u r t h a n t h e l i v e r s o f t h e c o n t r o l group w h i c h were k i l l e d and examined a t t h e same t i m e . Some o f t h i s p a l e n e s s was due t o a d i s t i n c t p a t t e r n o f m i l i a r y i r r e g u l a r - s h a p e d y e l l o w i s h f o c i ( a p p a r e n t l y n e c r o t i c ) w h i c h showed t h r o u g h the c a p s u l e ( P l a t e 1 0 ) . S p l e e n No changes were n o t e d i n t h e s p l e e n s of mink f e d t o x i c meal a t the 10 p e r c e n t l e v e l . A t t h e 5 p e r c e n t l e v e l , 50 p e r c e n t o f t h e mink, and a t t h e 2.5 p e r c e n t l e v e l , 20 p e r c e n t o f the mink d i s -p l a y e d c o n g e s t i o n and m o t t l i n g o f the s p l e e n . Three a d d i t i o n a l mink of t he l a t t e r group showed a pronounced d a r k e n i n g o f t h i s o r g a n . Lymph Nodes A g e n e r a l enlargement o f b o t h body and v i s c e r a l lymph nodes was n o t e d i n s i x mink a t t h e 10 p e r c e n t l e v e l , i n two mink a t t h e 5 p e r c e n t l e v e l and i n one mink at t h e 2.5 p e r c e n t l e v e l . One o t h e r mink at t h e 10 p e r c e n t l e v e l showed haemorrhages of v a r i o u s lymph nodes. F o u r o t h e r s a t t h e 5 p e r c e n t l e v e l d i s p l a y e d e n l a r g e -ment, and i n some c a s e s c o n g e s t i o n , of v a r i o u s nodes of the g a s t r o -i n t e s t i n a l t r a c t . T h i s f i n d i n g was d u p l i c a t e d i n two mink a t t h e 2.5 p e r c e n t l e v e l . K i d n e y s No g r o s s changes were n o t e d i n t h e k i d n e y s o f mink f e d t o x i c peanut meal a t the 10 p e r c e n t l e v e l , b u t i n t h o s e f e d a t the 5 p e r c e n t and 2.5 p e r c e n t l e v e l s , one a n i m a l i n each group d e m o n s t r a t e d extreme p a l e n e s s of the c o r t e x o f t h e k i d n e y . One mink f e d a t the 2.5 p e r c e n t l e v e l showed s m a l l r e n a l i n f a r c t s . 114 Stomach A number of the mink fed at the 10 per cent l e v e l had almost black, b i l e - s t a i n e d and possibly blood-containing mucus in both stomach and i n t e s t i n e s ; however, there were no changes recorded f o r the g a s t r i c t i s s u e s . In the 5 per cent group, three mink displayed mucosal and sub-mucosal g a s t r i c haemorrhages and one d i s t i n c t g a s t r i c bleeding. A f i f t h mink had d i s c r e t e g a s t r i c u l c e r s . In the 2.5 per cent l e v e l , two had haemorrhages of the walls of the stomach and one a large number of small d i s c r e t e u l c e r s . Intestine Except f o r the black gelatinous heavy exudate, no other changes were noted i n mink at the 10 per cent l e v e l . In those fed 5 per cent peanut meal, s i x cases of generalized haemorrhagic e n t e r i t i s were recorded; one was severe. Two mink at the 2.5 per cent l e v e l also had e n t e r i t i s , another mink had duodenal haemorrhages and three others had haemorrhages throughout the length of the i n t e s t i n e . Myelopoietic Tissues One mink fed at the 5 per cent and three mink at the 2.5 per cent l e v e l s were noted to have a bright red and watery consistency of the bone marrow. General Observations Some of the weight loss and fat catabolism noted was undoubted-l y due, i n part, to the mink becoming anorexic p r i o r to death. In general, i t was f e l t that both the acute assay (No. 1), and the t i t r a t i o n assay (No. 2) were successful i n i l l u s t r a t i n g the suscepti-b i l i t y to and response of mink to crude a f l a t o x i n contained i n peanut meal. 115 E. H i s t o p a t h o l o g y I n a s s e s s i n g t h e h i s t o p a t h o l o g i c a l changes t h a t were o b s e r v e d i n mink, a number o f r e f e r e n c e s t h a t d e s c r i b e d the changes n o t e d i n v a r i o u s s p e c i e s d i s p l a y i n g a f l a t o x i c o s i s , were c o n s u l t e d . A few changes were n o t e d i n t h e mink t i s s u e s w h i c h have n o t been r e p o r t e d e l s e w h e r e . Many o f t h e r a d i c a l h i s t o p a t h o l o g i c a l changes w h i c h have been o b s e r v e d i n o t h e r s p e c i e s were absent f r o m t h e mink t i s s u e s . The organs and systems w i l l be c o v e r e d i n the a p p r o x i m a t e o r d e r o f t h e i r i m p o r t a n c e i n l i g h t o f the changes n o t e d . The s t u d i e s were n o t e x h a u s t i v e . L i v e r (a) The b l o o d v a s c u l a r s t r u c t u r e s True haemorrhages i n t o the l i v e r parenchyma o c c u r r e d i n two mink f e d t o x i c peanut meal a t the 10 p e r c e n t l e v e l ( P l a t e 6 ) , and i n e i g h t o f t h o s e f e d a t the 2.5 p e r c e n t l e v e l . These haemorrhages seemed t o be c o n f i n e d t o the immediate s u b c a p s u l a r a r e a s . Recent and r e s o l v i n g haemorrhages w h i c h d i s p l a y e d abnormal a r c h i t e c t u r e were n o t e d . I n a d d i t i o n , b l o o d pigment, some of w h i c h was i n g e s t e d by macrophages, was o b s e r v e d i n t h e s e - h a e m o r r h a g i c a r e a s ( P l a t e 1 1 ) . One mink f e d a t the 0.625 p e r c e n t l e v e l showed an a r e a o f p e r i -v a s c u l a r oedema and f i b r o s i s around the c e n t r a l v e i n . The l a s t m e n t i o n e d change i n v o l v i n g c e n t r a l v e i n s was n o t n o t e d i n the l i t e r a -t u r e r e f e r e n c e s f o r o t h e r s p e c i e s . Haemorrhages i n t o the l i v e r parenchyma have been o b s e r v e d i n t u r k e y s . There was no e v i d e n c e i n the mink of the v e n o - o c c l u s i v e d i s e a s e i n v o l v i n g the c e n t r a l and h e p a t i c v e i n s w h i c h have been r e p o r t e d i n c a t t l e . The p h l e b o s i s o f p o r t a l v e i n s w i t h o b l i t e r a t i o n , r e p o r t e d i n the d u c k l i n g , was absent 116 i n the mink. (b) The B i l i a r y System , B i l e d u c t p r o l i f e r a t i o n was n e i t h e r a d i s t i n c t n o r r e c o g n i z -a b l e f e a t u r e i n the l i v e r s o f mink w h i c h were f e d the h i g h e r l e v e l s of t o x i c peanut m e a l . I t was n o t e d i n mink f e d a t the 2.5 p e r c e n t l e v e l . I n the l o w e s t l e v e l g r o u p s , w h i c h f o r the most p a r t d i d not d i e n a t u r a l l y , b i l e d u c t p r o l i f e r a t i o n was a r e l a t i v e l y marked f e a -t u r e ( P l a t e 1 2 ) . Some b i l e d u c t c a s t s were n o t e d i n the l i v e r s o f mink f e d the meal at the 5 p e r c e n t l e v e l ( P l a t e 1 3 ) . The m a t e r i a l was o f a h y a l i n e n a t u r e and u s u a l l y was not a t t a c h e d t o the b i l i a r y e p i t h e l i u m . These f e a t u r e s o b s e r v e d i n the mink have been n o t e d i n o t h e r s p e c i e s . The b i l e d uct p r o l i f e r a t i o n has been r e c o r d e d f o r c a t t l e , i n c a l v e s a f t e r t h r e e months on t o x i c f e e d , i n p i g s ( s l i g h t t o moderate o n l y ) , i n t u r k e y s , i n d u c k l i n g s (by the s e v e n t h d a y ) , and i n c h i c k s . C a s t s i n t h e h e p a t i c b i l e d u c t s have a l s o been r e p o r t e d f o r d u c k l i n g s . C e r t a i n o t h e r f i n d i n g s w h i c h were not a p p a r e n t i n the mink t i s s u e s have been n o t e d i n c h i c k s . These i n c l u d e p r o l i f e r a t i o n o f b i l e d u c t u l e c e l l s w h i c h change t o b a s o p h i l i c s t a i n i n g p a r e n c h y m a l - t y p e c e l l s i n a t u b u l a r arrangement w i t h i n two weeks. G e n e r a l i z e d j a u n d i c e has been n o t e d i n p i g s ; oedema o f the w a l l o f the g a l l - b l a d d e r and s u r r o u n d i n g t i s s u e s has been n o t e d i n p i g s a f t e r 86 days on t o x i c f e e d ; i s l a n d s o f a p p a r e n t l y n ormal parenchymal c e l l s s u r r o u n d e d by masses o f b i l e d u c t u l e s and s u p p o r t i n g f i b r e s have been n o t e d i n d u c k l i n g s . ( c ) R e t i c u l o e n d o t h e l i a l elements A l y m p h o i d h y p e r p l a s i a , or a h y p e r p l a s i a w i t h l y m p o c y t e s and h i s t i o c y t e s was n o t e d i n t h e form of masses of i n f i l t r a t i n g c e l l s around t h e p o r t a l t r i n i t i e s and o c c a s i o n a l l y i n p e r i c e l l u l a r a r e a s . T h i s was seen o n l y i n t h e mink f e d t o x i c meal a t the l e v e l s o f 1.25 and 0.625 p e r c e n t ( P l a t e s 14, 15, 1 5 a ) . The l y m p h o i d and h i s t i o c y t e h y p e r p l a s i a i n cows, p i g s , t u r k e y s , d u c k l i n g s , and c h i c k e n s has been r e p o r t e d by o t h e r w o r k e r s . (d) C o n n e c t i v e T i s s u e and R e t i c u l i n There was a degree of p e r i h e p a t i t i s n o t e d i n one o r two mink f e d t o x i c meal a t the 5 p e r c e n t l e v e l . There may have been o t h e r c a s e s where t h e l i v e r c a p s u l e was not s u f f i c i e n t l y r e p r e s e n t e d i n the s e c t i o n s t o d e t e r m i n e t h i s . I t i s n o t known at p r e s e n t i f t h e r e was a degree o f p e r i c e l l u l a r f i b r o s i s i n some o f the l i v e r s . The a r c h i t e c t u r a l derangement s u g g e s t e d t h i s f i n d i n g . R e p o r t s of p e r i h e p a t i t i s i n o t h e r s p e c i e s b e i n g f e d a f l a t o x i n have n o t been n o t e d i n t h e l i t e r a t u r e . R e g e n e r a t i v e f i b r o s i s d i d not appear i n the mink, and t h i s was one o f the most s u r p r i s i n g f e a t u r e s of t h e p r e s e n t e x p e r i m e n t . T h i s change has been r e c o r d e d i n t h e l i t e r a t u r e f o r c a l v e s , cows, t u r k e y s , and d u c k l i n g s . I n a f l a t o x i -c o s i s o f t h e p i g a t y p e o f f i b r o s i s has been d e s c r i b e d where columns o f f i b r o u s c o n n e c t i v e t i s s u e l e a v e the normal p r e - e x i s t i n g s e p t a and d i s s e c t t h e l o b u l e s t o j o i n d l a r g e c e n t r a l f i b r o t i c a r e a s i n w h i c h t h e r e a r e s m a l l b l o o d v e s s e l s . I n the c a l f a s u b p e r i t o n e a l oedema over the s u r f a c e s o f t h e l i v e r and g a l l - b l a d d e r has been d e s c r i b e d . (e) H e p a t i c C e l l Parenchyma H e p a t i c c e l l n e c r o s i s , m o s t l y c e n t r o l o b u l a r and p o s s i b l y o f l y t i c n a t u r e , was w i d e l y p r e s e n t i n t h e l i v e r s o f a l l the mink f e d th e t o x i c peanut m e a l . T h i s n e c r o s i s was d i f f u s e and w i d e - s p r e a d i n 118 mink w h i c h d i e d on t h e f e e d i n g a s s a y s ( P l a t e 1 6 ) . S m a l l f o c i o f h e p a t i c n e c r o s i s were n o t e d i n mink w h i c h s u r v i v e d f o r n i n e and one-h a l f months on t o x i c meal f e e d i n g l e v e l s of 1.25 and 0 625 p e r c e n t ( P l a t e 1 7 ) . C e n t r o l o b u l a r o r parenchymal c e l l d e g e n e r a t i o n was a common f i n d i n g i n l e s s damaged p a r t s o f t h e l i v e r s a t a l l l e v e l s o f t o x i c f e e d i n g . Medium o r c o a r s e v a c u o l a t i o n of parenchymal c e l l a r e a s was a l s o n o t e d at a l l l e v e l s ( P l a t e 1 8 ) . I n o t h e r c a s e s where changes were not so a p p a r e n t , t h e r e was d i s t i n c t d i s o r g a n i z a t i o n o f the c o r d p a t t e r n s o f h e p a t i c c e l l s . L o b u l a r r e g e n e r a t i o n was n o t e d . Regener-a t i o n i n n o d u l a r form was one of t h e c h i e f f e a t u r e s o f mink f e d l e v e l s of t o x i c meal o f 2.5 p e r c e n t or l o w e r ( P l a t e s 12 and 1 9 ) . I n some of t h e mink f e d t h e l o w e s t l e v e l of meal t h e r e were some regene-r a t e d n o d u l e s w h i c h appeared t o be u n d e r g o i n g a c o a g u l a t i v e n e c r o s i s ( P l a t e 1 7 ) . ' H e p a t i c c e l l n e c r o s i s of a d i f f u s e c y s t i c n a t u r e has been n o t e d i n d u c k l i n g s . S m a l l f o c i o f h e p a t i c n e c r o s i s were a l s o ob-s e r v e d i n the l i v e r o f d u c k l i n g s . C e n t r o l o b u l a r or parenchymal c e l l d e g e n e r a t i o n was n o t e d i n c a l v e s and d i f f u s e d e g e n e r a t i o n was n o t e d i n t u r k e y s , and c h i c k s . Medium and c o a r s e v a c u o l a t i o n o f parenchymal c e l l s was n o t e d i n d u c k l i n g s and p i g s r e s p e c t i v e l y . D i s o r g a n i z a t i o n of t h e c o r d p a t t e r n was a f e a t u r e o f cows, p i g s , and d u c k l i n g s i n t h e l i t e r a t u r e . R e g e n e r a t i o n of l o b u l e s f r e e o f d u c t u l e , r e t i c u l i n , o r c o l l a g e n p r o l i f e r a t i o n has been r e p o r t e d f o r p i g s . N o d u l a r r e g e n e r a t i o n has a l s o been r e p o r t e d f o r s w i ne, and a f t e r 182 days o f f e e d i n g an a f l a t o x i n - c o n t a i n i n g m e a l , a c o a g u l a t i v e n e c r o s i s apparent-l y o c c u r r e d i n t h e r e g e n e r a t e d n o d u l e s . F e a t u r e s r e p o r t e d i n t h e l i t e r a t u r e w h i c h were not d e t e c t e d i n t h e mink were r e a c t i v e o r r e p a r a t i v e d u c t u l e p r o l i f e r a t i o n ( a p p a r e n t l y ) , r a d i a t i n g out from the p o r t a l t r i a d s as seen i n the d u c k l i n g ; n o d u l a r h y p e r p l a s i a ( g r o s s l y e n l a r g e d l i v e r ) has been r e p o r t e d f o r the d u c k l i n g ( t h i s i s c o n t r a r y t o our f i n d i n g i n duck-l i n g a s s a y s , where t h e l i v e r s were s i g n i f i c a n t l y s m a l l e r t h a n t h o s e o f the c o n t r o l s i n r e l a t i o n t o body w e i g h t s ) ; m e g a l o c y t o s i s has been n o t e d i n the l i v e r s o f cows and t u r k e y s ; c a r c i n o g e n e s i s has been o b s e r v e d i n the r a t and. i n monkeys; macrophage a c t i v i t y i n l i v e r s has been n o t e d f o r d u c k l i n g s ; k a r y o m e g a l y has been r e p o r t e d i n l i v e r s o f cows and p i g s . Other f e a t u r e s r e c o r d e d i n t h e p i g , b u t absent i n t h e mink a r e a n i s o c y t o s i s ; l a r g e , p a l e , e o s i n o p h i l i c and d i s o r g a n i z e d h e p a t i c c e l l s ; a s e r i e s o f d i l a t e d b l o o d v e s s e l s s u r r o u n d i n g t h e c e n t r a l v e i n ( a f t e r 86 d a y s ) ; and d i f f u s e s t e a t o s i s , ( a f t e r 60 d a y s ) . A c o n d i t i o n known as e x u d a t i v e h e p a t i s o r hepa-t o s i s has been d e s c r i b e d f o r g u i n e a p i g s ( P a t e r s o n e t a l , 1962). T h i s i s a p p a r e n t l y an emb r a c i n g term f o r many changes i n the l i v e r a n d . i t has n o t been used h e r e . K i d n e y s The p r i n c i p a l changes were n o t e d i n t h e mink a t t h e h i g h e s t l e v e l s o f t o x i c meal i n t a k e . Mink a t t h e 10 p e r c e n t l e v e l were s u f f e r i n g f r o m marked h y d r o p t i c c e l l e n l argement or v a c u o l a t i o n o f t h e t u b u l a r e p i t h e l i u m i n t h e c o r t e x . Mink f e d a t t h e 5 p e r c e n t l e v e l showed h y a l i n e c a s t s i n t h e c o r t i c a l t u b u l e s and e o s i n o p h i l i c c a s t s o r r e d c e l l c a s t s i n Bowman's spaces ( P l a t e 2 0 ) . There was g e n e r a l and marked c o n g e s t i o n o f t h e c o r t e x and m e d u l l a o f the k i d n e y s i n t h e mink f e d a t a l l l e v e l s ( P l a t e 2 1 ) . ^ Some o f t h i s may have been due t o t h e c a r b o n d i o x i d e e u t h a n a s i a p e r f o r m e d on the mink 120 at the two lowest l e v e l s of feeding. The casts in c o r t i c a l tubules were not noted i n l i t e r a t u r e reports i n other species; however, the hydroptic changes i n tubular epithelium, as well as an eo s i n o p h i l i c deposit i n the glomerular membranes, were noted i n r a t s . Glomerular n e p h r i t i s , which has been noted i n the turkey, was not c l e a r l y present in the mink although there were some areas where there was diminution of Bowman's space (Plate 22). The d i f f u s e i n t e r s t i t i a l haemorrhages noted in ducklings and the enlargement of n u c l e i of the proximal convoluted tubules noted i n the p i g were both absent i n the mink t i s s u e . Lymph Nodes As noted i n the gross d e s c r i p t i o n s , the lymph nodes of mink fed at the highest l e v e l were often enlarged and soft or oedematous throughout the body. At the 5 and 2.5 per cent l e v e l s , only the g a s t r o i n t e s t i n a l nodes were affected. On section, these enlarged lymph nodes were found to be oedematous, p a r t i c u l a r l y i n the hyaloid areas. There was some stimulation of macrophages in the sinusoidal areas. In the l i t e r a t u r e , v i r t u a l l y no observations on lymph nodes were noted, except i n the cow, where the mesenteric nodes were described as being oedematous. Spleen H i s t o l o g i c a l l y i t was noted that the red pulp contained numerous groups of dark s t a i n i n g c e l l s , suggesting a lymphocytic hyperplasia. I f th i s was so, i t i s contrary to one report for the pig which describes a depletion of lymphocytes in the spleen Harding  et a l , (1963). PLATE 1 FTS Mink. E u t h a n s i a i n e x t r e m i s . Mink f e d 2.5 p e r c e n t t o x i c m e a l . Marked p e r i t o n e a l a s c i t e s w i t h c l e a r f l u i d . Subcutaneous a r e a . shows i n a b i l i t y of b l o o d t o c l o t . PLATE 2 FTS. Spontaneous d e a t h s i n mink f e d 10 p e r c e n t t o x i c m e a l . L i v e r s p a l e x ^ i t h s u b c a p s u l a r c o n g e s t i o n . I n f l a m m a t i o n o f m e s e n t e r y shown by ar r o w s . PLATE 3 FTS. Spontaneous d e a t h i n mink f e d 5 per cent t o x i c m e a l . C a t a b o l i s m or s o f t e n i n g o f t h e m e s e n t e r i c f a t . PLATE 4 FTS. Spontaneous d e a t h i n mink f e d 10 p e r c e n t t o x i c m e a l . Arrow i n d i c a t e s p e r i c a r d i u m w h i c h shows c o n g e s t i o n and p e t e c h i a l haemorrhages. PLATE 1 PLATE 2 PLATE 3 PLATE 4 PLATE 5 FTS. Spontaneous- d e a t h s i n mink f e d 10 p e r c e n t t o x i c m e a l . G e n e r a l v i e w ( l e f t ) and c l o s e up ( r i g h t ) - showing p a l e y e l l o w f r i a b l e l i v e r w i t h l o b u l a r m a r k i n g s and oedematous m e s e n t e r i c lymph node. PLATE 6 FTS. Spontaneous d e a t h i n mink .fed 10 per c e n t t o x i c m e a l . L i v e r w i t h s u f f u s i v e sub-c a p s u l a r haemorrhage o f r i g h t l o b e . PLATE 6 PLATE 7 FTS. Spontaneous d e a t h i n mink f e d 2.5 p e r c e n t t o x i c m e a l ; Y e l l o w l i v e r , i r r e g u l a r s u r f a c e w i t h r e g e n e r a t i v e n o d u l e s . PLATE 8 FTS. .Spontaneous d e a t h i n mink f e d 2.5 p e r c e n t t o x i c m e a l . L i v e r w i t h s u r f a c e n o d u l e s o f c o n g e s t i v e o r h a e m o r r h a g i c t i s s u e . PLATE 9 FTS. Spontaneous d e a t h i n mink f e d 1.25 p e r c e n t t o x i c m e a l . L i v e r w i t h i r r e g u l a r p a t c h e s of s u b c a p s u l a r n o d u l a r r e g e n e r a t i o n . PLATE 8 PLATE 10 FTS. Mink g i v e n e u t h a n a s i a a f t e r n i n e and o n e - h a l f months f e e d i n g at t h e 0.625 per c e n t l e v e l of t o x i c meal. L i v e r o f normal c o l o u r w i t h m i l i a r y n e c r o t i c f o c i . PLATE 11 FTS. Spontaneous d e a t h i n mink f e d 2.5 p e r c e n t t o x i c m e a l . L i v e r p h o t o m i c r o g r a p h w i t h s u b c a p s u l a r r e s o l v i n g haemorrhages. (82-5x) PLATE 10 PLATE 11 PLATE 12 FTS. Spontaneous d e a t h i n mink f e d 1.25 p e r c e n t t o x i c m e a l . L i v e r p h o t o m i c r o g r a p h showing b i l e d u c t p r o l i f e r a t i o n and r e g e n e r a -t i v e n o d u l e s . (82.5x) PLATE 13 FTS. Spontaneous d e a t h i n mink f e d 5 per c e n t t o x i c m e a l . L i v e r p h o t o m i c r o g r a p h showing b i l e d u c t c a s t s and i n f i l t r a t e d c e l l s . (270x) PLATE 14 FTS. Mink g i v e n e u t h a n a s i a a f t e r n i n e and o n e - h a l f months f e e d i n g a t 0.625-per c e n t l e v e l o f t o x i c meal.- L i v e r p h o t o m i c r o g r a p h showing i n f i l t r a t e d c e l l s around b i l e d u c t s and p o r t a l v e i n s . (168x) PLATE 13 PLATE 14 PLATE 15 FTS. Mink g i v e n e u t h a n a s i a a f t e r n i n e and o n e - h a l f months f e e d i n g a t 0.625 p e r c e n t l e v e l o f t o x i c m e a l . L i v e r p h o t o m i c r o g r a p h showing i n f i l t r a t i o n o f c e l l s around b i l e d u c t s and p o r t a l v e i n . (528x) PLATE 15 a FTS. Spontaneous d e a t h on 8 1 s t ' d a y i n mink f e d 1.25 p e r ' c e n t t o x i c m e a l . L i v e r p hoto-m i c r o g r a p h showing c e l l d e g e n e r a t i o n and c e l l i n f i l t r a t i o n o r m e t a p l a s i a . (528x) PLATE 16 FTS. Spontaneous d e a t h i n mink f e d 5 p e r c e n t t o x i c m e a l . L i v e r p h o t o m i c r o g r a p h showing metamorphosis, and d i f f u s e n e c r o s i s . .(168k) PLATE 17 FTS. Mink g i v e n e u t h a n a s i a a f t e r n i n e and o n e - h a l f months f e e d i n g at 0.625 p e r c e n t l e v e l of t o x i c meal. L i v e r p h o t o m i c r o g r a p h showing s u b c a p s u l a r m i l i a r y f o c a l n e c r o s i s . (106x) (see P l a t e 1 0 ) . PLATE 18 FTS. Spontaneous d e a t h i n mink f e d 2.5 p e r c e n t t o x i c meal. L i v e r p h o t o m i c r o g r a p h showing b i l e d u c t p r o l i f e r a t i o n ( l e f t ) and v a c u o l a t i o n of parenchyma. (168x) PLATE 19 FTS. Spontaneous d e a t h i n mink f e d 2.5 p e r c e n t t o x i c m e a l . L i v e r p h o t o m i c r o g r a p h showing l a r g e r e g e n e r a t i v e n o d u l e and some b i l e d u c t p r o l i f e r a t i o n . (82.5x) PLATE 19 PLATE 20. FTS. Spontaneous d e a t h i n mink f e d 5 p e r c e n t t o x i c meal. K i d n e y p h o t o m i c r o g r a p h showing g l o m e r u l u s w i t h c o l l a p s e d c a p i l l a r y t u f t and r e d c e l l c a s t i n Bowman's space. . (528x) PLATE 21 FTS. Mink g i v e n e u t h a n a s i a a f t e r n i n e and o n e - h a l f months f e e d i n g at 1.25 p e r c e n t l e v e l o f t o x i c m e a l . K i d n e y p h o t o m i c r o g r a p h showing c o r t i c a l c o n g e s t i o n . (82.5x) PLATE 21 PLATE 22 FTS. Mink g i v e n e u t h a n a s i a a f t e r n i n e and o n e - h a l f months f e e d i n g at 0.625 p e r c e n t l e v e l o f t o x i c meal. K i d n e y p h o t o m i c r o g r a p h showing c o r t e x and d i m i n u t i o n o f Bowman's space i n r e n a l g l o m e r u l i . (168x) PLATE 23 S u p e r f i c i a l c o m p a r i s o n of. l i v e r s from.spon-taneous d e a t h s i n mink s u f f e r i n g t h e HHS d i s e a s e ( r i g h t ) and e x p e r i m e n t a l a f l a t o x i c o s i s due t o f e e d i n g w i t h t o x i c groundnut meal a t t h e 1.25 p e r c e n t l e v e l f o r S I days ( l e f t ) (see a l s o P l a t e 9 ) . The HHS l i v e r showed l a r g e haemangiomas o r b l o o d c y s t s . The d a r k p a t c h e s on t h e FTS l i v e r f rom a mink d y i n g t h e same day, r e s e m b l e d haemorrhages but were found h i s t o l o g i c a l l y t o be c o n g e s t e d areas o f s u b c a p s u l a r r e g e n e r a t i o n . 121 DISCUSSION AND SUMMARY Gross pathological changes involving tissues other than the liver-were e s s e n t i a l l y r e s t r i c t e d to the mink which died, i . e . those fed the toxic meal at the 2.5 per cent l e v e l (dry weight) and greater. The p r i n c i -pal changes can be l i s t e d i n r e l a t i o n to t h e i r appearance i n the three groups of mink which, being fed d i f f e r e n t l e v e l s , showed a consequent v a r i a t i o n i n mean death time. (a) Changes which appeared at a l l three l e v e l s (10, 5 and 2.5 per cent) 1. Haemophilia, i n a b i l i t y of the blood to c l o t or for bleeding to be co n t r o l l e d when mink were toe-clipped for blood samples. 2. Peritoneal a s c i t e s . 3. Congestion and petechial haemorrhages of the pericardium. 4. General enlargement of body and v i s c e r a l lymph nodes. In t h i s category i t i s f e l t that the changes might represent the presence of toxin at either dietary l e v e l regardless of time of exposure. However, they are equally l i k e l y to be a function of t o t a l intake of toxic meal, since the mean death times are roughly r e l a t e d to the le v e l s fed, thus: Per cent meal fed (Dry Wt.) Mean death time (Days) 10.0 10.5 5.0 28.4 2.5 55.4 to give a st r a i g h t l i n e r e l a t i o n s h i p . (b) Changes appearing only at the 10 per cent l e v e l 1. Marked pulmonary congestion. 2. Haemorrhages in various lymph nodes (one mink only). 122 3. P r e s e n c e o f masses o f b l a c k , b i l e - s t a i n e d (and p o s s i b l y b l o o d -c o n t a i n i n g ) mucus i n stomach and i n t e s t i n e s . These changes a r e pr e s u m a b l y a f u n c t i o n o f t h e h i g h dosage l e v e l and e i t h e r were n o t pr o d u c e d w i t h an e q u i v a l e n t t o t a l i n t a k e , (5 and 2.5 p e r c e n t ) o r , at l e a s t were not e v i d e n t when d e a t h o c c u r r e d . ( c ) Changes o c c u r r i n g at t h e 10 and 5 p e r c e n t l e v e l s b u t not at 2.5 p e r  c e n t 1. Oedema o f t h e subcutaneous f a t . 2. I n f l a m m a t i o n o f the mes e n t e r y . These changes a l s o s h o u l d be m a i n l y a f u n c t i o n o f dosage l e v e l s r a t h e r t h a n o f t o t a l i n t a k e o r t i m e o f e x p o s u r e . (d) Changes o c c u r r i n g o n l y at t h e 5 p e r c e n t l e v e l 1. D e g e n e r a t i o n o f subcutaneous f a t d e p o t s . 2. D i a p h r a g m a t i c haemorrhages. 3. E n l a r g e d rounded h e a r t o r r i g h t v e n t r i c u l a r h y p e r t r o p h y . 4. C o n g e s t i o n o r m o t t l i n g o f t h e s p l e e n . 5. Enlargement and c o n g e s t i o n o f g a s t r o i n t e s t i n a l lymph nodes. 6. Extreme p a l e n e s s o f t h e r e n a l c o r t e x . By t h e same s t a n d a r d s t h e s e g r o s s f e a t u r e s can be r e l a t e d t o t h e p e r i o d of t i m e exposed r a t h e r t h a n t o t h e l e v e l o r t o t a l i n t a k e o f t o x i n . ( f ) Changes i n organs o t h e r t h a n t h e l i v e r w h i c h o c c u r r e d o n l y a t the 2.5  p e r c e n t l e v e l These were n o n - e x i s t e n t e x c e p t f o r one c a s e o f m u l t i p l e r e n a l i n f a r c t i o n s . I f t h i s c a s e i s r e p r e s e n t a t i v e o f t h e f e e d i n g o f a f l a t o x i n , 123 i t would be a f u n c t i o n o f t i m e o f exposure r a t h e r t h a n o f t h e t o t a l i n t a k e of t o x i n w h i c h would n o t have g r e a t l y exceeded t h a t of mink d y i n g w h i l e on the 10 p e r c e n t l e v e l o f t o x i c m e a l . The g r o s s p a t h o l o g i c a l changes i n t h e l i v e r have been d e s c r i b e d i n d e t a i l . Mink d y i n g r a p i d l y as a r e s u l t o f h i g h - l e v e l f e e d i n g showed u n i f o r m l y p a l e o r y e l l o w f a t t y l i v e r s w i t h l i t t l e s i g n s of n o d u l a t i o n o r i r r e g u l a r i t y o f s u r f a c e . W i t h i n c r e a s i n g e x p o s u r e time a t lo w e r l e v e l s , t h e l i v e r s appeared p r o g r e s s i v e l y more p i n k i s h i n c o l o u r but w i t h s u r f a c e i r r e g u l a r i t i e s becoming marked. Some c a s e s showed n o d u l a r p r o t u b e r a n c e s o f p a l e or c o n g e s t e d t i s s u e . Two l i v e r s at the 10 per c e n t l e v e l and e i g h t at t h e 2.5 p e r c e n t l e v e l showed p i n p o i n t haemorrhages at t h e l i v e r c a p s u l e . At t h e s u b - l e t h a l l e v e l s o f 1.25 and 0.625 p e r c e n t meal ( d r y w e i g h t ) , t h e l i v e r s were normal i n c o l o u r and c o n s i s t e n c y e x c e p t f o r the p r e s e n c e o f numerous p a l e o r y e l l o w - c o l o u r e d s m a l l i r r e g u l a r f o c i w h i c h appeared t h r o u g h t h e c a p s u l e . D e t a i l s o f t h e h i s t o p a t h o l o g i c a l changes n o t e d i n t h e l i v e r , k i d n e y s , l y m p h a t i c g l a n d s and s p l e e n have been r e c o r d e d . They have been compared f o r t h e i r p r e s e n c e or o t h e r w i s e w i t h f i n d i n g s a v a i l a b l e i n t h e l i t e r a t u r e w h i c h r e f e r t o n a t u r a l or. e x p e r i m e n t a l a f l a t o x i c o s i s i n s p e c i e s o t h e r t h a n t h e mink. F i n d i n g s w h i c h were not d u p l i c a t e d i n our mink have a l s o been n o t e d f o r t h e s e o r g a n s . Of t h e organs examined h i s t o l o g i c a l l y f rom a f l a t o x i c mink, the l i v e r s , as expected, have been of g r e a t e s t i n t e r e s t . A l t h o u g h some haemorrhages o c c u r r e d i n mink at the 10 and t h e 2.5 p e r c e n t l e v e l s , t h e p r i n c i p a l changes were r e l a t e d t o t h e h e p a t i c c e l l s and t h e b i l e d u c t c e l l s . G e n e r a l d e g e n e r a t i o n l e a d i n g t o n e c r o s i s was o b s e r v e d i n the l e v e l s o f h i g h e r exposure t o t o x i c m e a l , (2.5 p e r c e n t , 5 per c e n t and 124 10 p e r c e n t ) w i t h mink t h a t d i e d . F o c a l n e c r o s i s , b i l e d u c t or d u c t u l e p r o l i f e r a t i o n and i n f i l t r a t i n g c e l l masses were seen more i n mink g i v e n e u t h a n a s i a a f t e r b e i n g f e d l o w e r l e v e l s o f t o x i n . N o d u l a r r e g e n e r a t i o n o c c u r r e d b o t h i n mink w h i c h d i e d on a meal l e v e l o f 2.5 p e r c e n t and ; t h o s e w h i c h s u r v i v e d and were examined l a t e r . I n the l a t t e r , some of t h e r e -g e n e r a t e d l o b u l e s or n o d u l e s were i n t u r n found t o be s u f f e r i n g f o c a l n e c r o s i s . N o t a b l y absent from a l l mink was c l e a r e v i d e n c e ( w i t h H and E s t a i n i n g ) o f any degree o f f i b r o u s c o n n e c t i v e t i s s u e i n c r e a s e e x c e p t i n v o l v i n g the c a p s u l e i n one or two c a s e s . T h i s i s of c o n s i d e r a b l e i n t e r e s t s i n c e i t i s known t h a t t h e mink l i v e r i s c a p a b l e of d e v e l o p i n g marked and e a s i l y v i s i b l e f i b r o s i s under c e r t a i n c o n d i t i o n s (Mc M i l l a n , 1965). The h i g h e r l e v e l s (10 and 5 p e r c e n t ) of t o x i c meal a r e c a p a b l e o f c a u s i n g a c u t e t u b u l a r parenchymal d e g e n e r a t i o n i n t h e r e n a l c o r t e x . I n some c a s e s t h e r e was e v i d e n c e o f venous t h r o m b o s i s a f f e c t i n g t h e k i d n e y as e v i d e n c e d by r e d c e l l c a s t s i n Bowman's space and s h r i n k i n g o f the g l o m e r u l a r c a p i l l a r y t u f t s . The l o c a t i o n o f t h e c o n j e c t u r a l thromboses i n r e n a l v e i n o r p o s t e r i o r v e n a c a v a c o u l d have been m i s s e d at. a u t o p s y . H i s t o l o g i c a l l y t h e s p l e e n was not m a r k e d l y changed. The lymph nodes showed s t i m u l a t i o n o f macrophages and a degree o f oedema i n some c a s e s w h i c h c o u l d have a c c o u n t e d f o r the g r o s s e n l a r g e m e n t . 125 BIBLIOGRAPHY 1. A l l c r o f t , R. and R.B.A. Carnaghan. B i o l o g i c a l e f f e c t s . Chem. and I n d . 50-53, 1963. 2. A l l c r o f t , R. and G. L e w i s . Groundnut t o x i c i t y i n c a t t l e : E x p e r i m e n t a l p o i s o n i n g o f cows and a r e p o r t on c l i n i c a l e f f e c t i n o l d e r c a t t l e . V e t . Rec. 75; 487-492, 1963. 3. A s p l i n , F.D. and R.B.A. Carnaghan. The t o x i c i t y o f c e r t a i n groundnut meals i n p o u l t r y w i t h s p e c i a l r e f e r e n c e t o . t h e e f f e c t on d u c k l i n g s and c h i c k e n s . V e t . Sec. _73: 1215-1218, 1961. 4. B u t l e r , W.H. and J.A. B a r n e s . T o x i c e f f e c t s o f peanut meal c o n t a i n i n g a f l a t o x i n t o r a t s and g u i n e a p i g s . B r i t . J . Cancer 17: 699-710, 1963. 5. C l e g g , F.G. and H. Bryson., An o u t b r e a k o f p o i s o n i n g i n s t o r e c a t t l e a t t r i b u t e d t o B r a z i l i a n groundnut m e a l . V e t . Rec. 74: 992-994, 1962. 6. Dougherty, E. and C G . P i c k a r d . Sub-acute and c h r o n i c l i v e r d i s e a s e s of t h e White P e k i n Duck. A v i a n D i s e a s e s l_i 217-234, 1963. 7. H a r d i n g , J . D . J , , J.T. Done, G. Le w i s and R. A l l c r o f t . E x p e r i m e n t a l groundnut p o i s o n i n g i n p i g s . Res. V e t . S c i . 4: 217-229, 1963. 8. Mc M i l l a n , K.R. Hepato-haemangioma-syndrome i n mink ( i n c l u d e d i n t h i s t h e s i s ) , 1965. 9. M o r t i m e r , P.H. The e x p e r i m e n t a l i n t o x i c a t i o n o f sheep w i t h s p o r i d e s m i n , a m e t a b o l i c product, o f P i t h o m y c e s c h a r t a r u m . IV. H i s t o l o g i c a l and h i s t o c h e m i c a l e x a m i n a t i o n s o f o r a l l y - d o s e d sheep. Res. V e t . S c i . 4: 166-185, 1963. 10. S a r g e a n t , K. , J . O ' K e l l y , R.B.A. Carnaghan and R. A l l c r o f t . The a s s a y of a t o x i c p r i n c i p l e i n c e r t a i n groundnut m e a l s . V e t . Rec. 73: 1219-1223, 1961. 11. Warnop, C C Groundnut t o x i c i t y i n p o u l t r y . V e t . J o u r . 119: 174-177, 1963,, 12. Zwart, D. and H.P. H a r d i n g . D i e t e t i c l i v e r c i r r h o s i s o f p i g s i n Ghana. Res. V e t . S c i . 5: 257-267, 1964. PART IV FUNGAL TOXICOSIS STUDIES I INTRODUCTION The o c c u r r e n c e o f an h e p a t i c d i s e a s e i n mink and the subsequent d e m o n s t r a t i o n t h a t mink are h i g h l y s u s c e p t i b l e t o a f u n g a l t o x i n s u ggest t h a t the mink environment s h o u l d be examined f o r a p o s s i b l e s o u r c e o f f u n g a l t o x i n . The n a t u r e o f t h e d i e t f e d t o mink c o n s t i t u t e s a v e r y f a v o u r a b l e menstruum f o r f u n g a l g r o wth. The main i n g r e d i e n t o f t h e c e r e a l p o r t i o n o f the mink's d i e t i s wheat. I t i s known t h a t C a n a d i a n wheat used i n a n i m a l r a t i o n s c a r r i e s a l a r g e burden of f u n g a l s p o r e s . The m o i s t u r e c o n t e n t o f normal mink r a n c h r a t i o n s i s h i g h and hence f a v o u r a b l e t o t h e growth of s p o i l a g e f u n g i . The p o s s i b i l i t y t h a t a fungus o r f u n g i o r i g i n a t i n g i n Ca n a d i a n wheat used i n a n i m a l r a t i o n s was t h e s o u r c e o f t o x i n prompted an i n v e s t i g a t i o n of C a n a d i a n wheat and t h e mink c e r e a l m ix f o r p o s s i b l e t o x i n -p r o d u c i n g f u n g i . A s u r v e y o f t h e w o r l d ' s l i t e r a t u r e p e r t a i n i n g t o the m y c o t o x i c o s e s s u g g e s t s t h a t f u n g i a r e f a s t i d i o u s m i c r o o r g a n i s m s w i t h r e g a r d t o t o x i n e l a b o r a t i o n and t h a t v e r y d e f i n e d c u l t u r a l c o n d i t i o n s must be met i n o r d e r t h a t t h e y may produce t o x i n . The f u n g a l t o x i n s a r e pr o d u c e d i n v e r y m i n u t e q u a n t i t i e s and r e q u i r e v e r y s e n s i t i v e a s s a y t e c h n i q u e s f o r t h e d e t e c t i o n o f t h e i r p r e s e n c e . W i t h t h i s i n f o r m a t i o n f o r g u i d a n c e , a s e r i e s o f b i o l o g i c a l a s s a y s were p e r f o r m e d t o see i f any of the f u n g a l i s o l a t e s from v a r i o u s g r a i n s o u r c e s were o f a t o x i c n a t u r e . These a s s a y s i n v o l v e d t h e use of r a b b i t s a f t e r t h e method of J o f f e ( 1 9 6 3 ) ; d u c k l i n g s , s i n c e t h i s a n i m a l has been shown t o be h i g h l y s u s c e p t i b l e t o a f l a t o x i c o s i s ; and c h i c k embryos, w h i c h have been used t o some e x t e n t i n a f l a t o x i n a s s a y ( R e p o r t o f t h e I n t e r -d e p a r t m e n t a l Working P a r t y on Groundnut T o x i c i t y R e s e a r c h , 1962). 127 EXPERIMENTAL A. I s o l a t i o n of F u n g i S i n c e the o r i g i n a l HHS syndrome i n mink seemed t o r e s e m b l e a p o i s o n i n g of a f u n g a l n a t u r e , C a n a d i a n Wheat, a m a j o r component o f t h e mink c e r e a l r a t i o n , has r e c e i v e d a g r e a t d e a l of a t t e n t i o n as a p o s s i b l e s o u r c e of t h e s e f u n g a l t o x i n s . Samples of U.B.C. mink c e r e a l r a t i o n , C a n a d i a n wheat and mouldy mink f e e d were i n o c u l a t e d onto Czapek-Dox agar medium and f u n g a l c o l o n i e s were i s o l a t e d by s e r i a l t r a n s f e r . Over one hundred s t r a i n s o f d i f f e r e n t f u n g a l s p e c i e s were i s o l a t e d . These were examined m i c r o s c o p i -c a l l y and t e n t a t i v e l y i d e n t i f i e d a c c o r d i n g t o genus. Among t h e s e f u n g i were members o f t h e f o l l o w i n g g e n e r a : A s p e r g i l l u s P e n i c i l l i u m A l t e r n a r i a F u s a r i u m P a e c i l o m y c e s P u l l a r i a Mucor. B. E v a l u a t i o n o f T o x i c i t y of F u n g i by B i o - a s s a y R a b b i t Dermal A s s a y (1) P r e p a r a t i o n o f E x t r a c t s : Pure c u l t u r e s of v a r i o u s f u n g i grown on a mink c e r e a l r a t i o n s u b s t r a t e were t r a n s f e r r e d t o S o x h l e t t h i m b l e s and e x t r a c t e d f o r s i x h o u r s w i t h anhydrous e t h y l e t h e r . A f t e r r e m o v a l of t h e e t h e r by r o t a r y e v a p o r a t i o n , t h e t o x i c i t y o f t h e e x t r a c t s was d e t e r m i n e d 128 u s i n g the r a b b i t dermal a s s a y t e c h n i q u e . (2) A n i m a l s and Tr e a t m e n t : White r a b b i t s o f about 1.5 k i l o g r a m body w e i g h t were used. A r e a s o f 1 square i n c h were shaved on the back of each a n i m a l . The f u n g a l e x t r a c t s were a p p l i e d t o t h e s e shaved a r e a s d a i l y and f o r p e r i o d s up t o 10 days. (3) R e s u l t s : From the l i t e r a t u r e o f t h e R u s s i a n w o r k e r s , i t would appear t h a t a r a p i d f u n g a l t o x i n a s s a y can be o b t a i n e d by a p p l y i n g e t h e r e x t r a c t s o f f u n g i t o t h e d e p i l a t e d s k i n of r a b b i t s . Mayer (1953) r e p o r t e d on t h e o f f i c i a l t e s t used i n R u s s i a n l a b o r a t o r i e s f o r t h e d e t e c t i o n of t o x i c i t y i n g r a i n . T h i s t e s t employed an e t h e r e x t r a c t of t h e t o x i c g r a i n . The e x t r a c t was a p p l i e d t o t h e shaved a r e a on a r a b b i t ' s back. I f t h e e x t r a c t was not t o x i c , t h e d e p i l a t e d a r e a r emained normal o r o n l y m i l d s c a l i n g was seen. The t e s t was doubt-f u l i f t h e r e was s l i g h t oedema, r e d n e s s and s c a l i n g o f t h e s k i n w i t h i n 7 days. I f the e x t r a c t was t o x i c , t h e r e was r a p i d oedema, r e d n e s s and n e c r o s i s o f t h e s k i n two hours a f t e r i t s a p p l i c a t i o n , f o l l o w e d l a t e r by the f o r m a t i o n o f a n e c r o t i c c r u s t . J o f f e (1963) used h i s v e r s i o n o f the o f f i c i a l R u s s i a n t e s t i n d e t e r m i n i n g the t o x i c i t y of pu r e f u n g a l c u l t u r e s . He used e t h e r or a l c o h o l e x t r a c t s o f t h e fungus grown on v a r i o u s media v i z . m i l l e t , wheat, b a r l e y . Two a p p l i c a t i o n s were made on t h e s k i n o f t h e r a b b i t w i t h an i n t e r v a l o f 24 h o u r s between each" a p p l i c a t i o n . The r e a c t i o n was r e c o r d e d f o r 48 h o u r s , but t h e r a b b i t s were o b s e r v e d f o r at l e a s t 6-8 days a f t e r the f i r s t a p p l i c a t i o n . A c c o r d i n g t o J o f f e , t h e r e a r e two types of skin reactions: (a) a leucocytorrhoeic r e a c t i o n which i s characterized by the formation on the skin surface of a whitish e a s i l y de-tachable f i l m which consists of a mass of leucocytes accumulating in the horny layer of the epithelium. (b) an oedemo-haemorrhagic reaction which involves an acute oedema and the appearance of haemorrhagic and necrotic symptoms. The i n t e n s i t y of the reaction was determined on the eight day and the other components were recorded on the t h i r d day. Forgacs and C a r l l (1962) stated that the type of substrate used for c u l t u r i n g a given fungus for toxin production should c l o s e l y approximate the composition of that from which the fungus was i s o l a t e d . In t h i s t e s t , the ether extract was d i l u t e d with o l i v e o i l before a p p l i c a t i o n . Necrosis was observed 48 hours a f t e r the second successive a p p l i c a t i o n of the extract. In the present study, a good rabbit dermal assay was not obtained. Several tests using pure cultures of various fungi were performed for durations of up to two weeks. In some cases there was s l i g h t oedema which could have been s i m i l a r to stage (a) of Joffe's t e s t , but there was no n e c r o t i c formation. None of the fungi tested gave a marked reactio n . J o f f e (1959) tested about 2000 d i f f e r e n t fungi belonging to 34 genera using the rabbit dermal assay as an index of t o x i c i t y . He found that Fusarium and Claddsporium species were the most highly toxic s t r a i n s , with A l t e r n a r i a , P e n i c i l l i u m and Mucor being less t o x i c . A s p e r g i l l u s species displayed l i t t l e or no t o x i c i t y . 130 Asp e r g i l l u s and P e n i c i l l i u m species made up the majority of the fungi i s o l a t e d from the mink environment and grain sources. Duckling Assay No. 4 An attempt was made to r e - e s t a b l i s h the condition whereby the fungi p r o l i f e r a t e i n the remains'of feed while i t i s on the mink cage and to assay an extract of t h i s fungal-contaminated feed i n ducklings. (1) Preparation of Extracts: A medium of the following composition was prepared: U.B.C. Mink Cereal Ration 144-63 3000 grams Cottonseed O i l 300 grams Water 2700 ml This contains approximately 45 per cent moisture. One unused and one used mink cage were obtained. The unused mink cage was auto-claved. 3 kilograms of the medium was spread over the top of each cage which was then placed i n a large p l a s t i c bag and sealed. These were incubated at atmospheric temperature of 19° C - 2 0 ° C for 5 days. At the end of t h i s incubation period, the medium was com'' p l e t e l y overgrown with an assortment of fungi. The bags were then opened and the medium was p a r t i a l l y d r i e d by blowing heated a i r onto the medium. The fungal mat and medium were removed from the cages and extracted according to the procedure of Sargeant et a l (1961). As a c o n t r o l , 3 kilograms of medium of the above composition were a i r - d r i e d and extracted i n the same manner. This medium was not incubated f o r the 5-day period. A l l three extracts were made up to 100 ml with cottonseed o i l . 131 (2) Animals and Treatment: Twenty-five, day-old ducklings were divided into three groups of s i x and one group of seven. These were housed in groups in wire brooder cages and given the following extracts: Group 1 Used mink cage extract - 6 ducklings Group 2 New mink cage extract - 6 ducklings Group 3 Control extract - 6 ducklings Group 4 Control (cottonseed o i l ) - 7 ducklings Extracts were administered per orum using a syringe to which a th i n polythene tube was attached. A standard commercial duck r a t i o n and water were available ad libitum. Feed and water were kept away from the ducklings one hour p r i o r to and after dosing i n order to prevent r e g u r g i t a t i o n of extracts. Daily group weights were taken. Duck-lin g s that died or that survived to the end of the experimental period were submitted to post mortem examination. TABLE XVII Dosage Schedule of Ducklings Assay No. 4 Day Volume of Extract Equivalent Weight (ml) of Meal (grams) 1 0.1 3 2 0.2 6 3 - -4 0.2 6 5 0.4 12 6 - -7 0.4 12 8 0.8 24 9 - -132 TABLE XVIII  Mortality Schedule of Ducklings  Assay No. 4 Days on Assay Groups 1 2 3 4 5 6 7 8 9 Mortalities 1 - - - 1 3 4 6 6/6 2 - - 1 2 3 6 - 6/6 3 - - - - 0/6 4 * * 0/6 * Killed TABLE XIX Mean Body Weight Record of Surviving Ducklings  Assay No. 4 Days on Assay Groups 1 2 3 4 5 6 7 8 9 1 52.3 47.3 43.3 46.0 56.8 54.2 61.6 72.5 67.0 2 51.0 48.6 50.0 63.7 65.0 90.0 96.0 112.0 96.0 3 51.6 49.6 55.6 63.0 79.1 87.0 99.0 108.8 130.0 4 51.4 54.0 67.0 77.6 90.6 107.0 121.7 147.5 177.5 134 TABLE XX  Gross Pathology of Ducklings  Assay No. 4 Day of Group Death Pathological Findings 1 6 Pale yellow l i v e r 7 Haemorrhagic beak, pale l i v e r 7 Pale l i v e r 8 Haemorrhagic beak; yellow-coloured l i v e r , i n t e s t i n a l contents very f l u i d 9 Pale yellow l i v e r 9 Pale yellow l i v e r 2 3 Haemorrhagic beak, pale l i v e r 4 Haemorrhagic beak, pale l i v e r with minute pe t e c h i a l , some lung congestion with oedema 5 Haemorrhagic beak, pale l i v e r 6 Haemorrhagic beak, pale l i v e r 6 Haemorrhagic beak, pale l i v e r 9 Liver apparently normal 3 - Normal, s l i g h t l y stunted growth 4 - Normal. (3) R e s u l t s : The m o r t a l i t y and body weight data of Duckling Assay No. 4 are presented i n Tables X V I I I and XIX r e s p e c t i v e l y . The dose-response r e l a t i o n s h i p , i f body weight can be used as a c r i t e r i o n f o r expressing the response of the ducklings to the e f f e c t of dosing w i t h the e x t r a c t , shows that the e x t r a c t given to Group 1 ducklings caused a marked depression i n growth r a t e . The retarded growth of these ducklings and the m o r t a l i t y experienced by t h i s group, when compared with the Group 3 d u c k l i n g s , suggests that the e x t r a c t was of a t o x i c nature. The Group 2 ducklings d i s p l a y e d a high mor-t a l i t y , but t h e i r growth r a t e was not as reduced as that of the Group 1 d u c k l i n g s . The Group 3 ducklings survived but d i d not grow as r a p i d l y as the c o n t r o l d u c k l i n g s . The dosing of an e x t r a c t of the non-incubated medium had a depressing e f f e c t on the growth r a t e of the ducklings (Group 3 ) . This might have been due to a s t r e s s f a c t o r . One c o n s i d e r a t i o n that should not be ignored i s the f a c t that the f u n g i present i n the c e r e a l mix could be producing t o x i n at a very low l e v e l and t h e i r a b i l i t y to produce t o x i n might be enhanced by the presence of moisture and added energy i n the form of f a t . Wood (1963) has shown the b e n e f i c i a l e f f e c t of a d d i t i o n a l f a t to the t o x i n producing p o t e n t i a l of f u n g i . Duckling Assay No. 5 The r e s u l t s of Duckling Assay No. 4 suggested that e x t r a c t s of pure c u l t u r e s of moulds should be assayed f o r t o x i c i t y . To t h i s end, a number of known and unknown toxin-producing f u n g i were submitted to bio-assay. 136 (1) Preparation of Extracts: A medium of the following composition was prepared: U.B.C. Mink Cereal Mix 144-63 95 grams Cottonseed O i l 5 grams Water 400 ml. It was autoclaved at 121° C. and 15 pounds pressure for 15 minutes. This medium contained approximately 80 per cent moisture. The following fungal cultures were grown for 14 days on samples of th i s medium: 1. 2. 3. As p e r g i l l u s flavus 44 Asp e r g i l l u s flavus 45 P e n i c i l l i u m rubrum 4. Fusarium sp. (PE 78) 5. P e n i c i l l i u m sp. (Mink 101) 6. As p e r g i l l u s sp. (Mink 107) 7. As p e r g i l l u s sp. (Mink 109) 8. Asp e r g i l l u s sp. (GW 10) 9. Control At the end of t h i s time, the mycelial mat was separated from the medium and r e f r i g e r a t e d . The medium was centrifuged and the l i q u i d f r a c t i o n used f or assay. (2) Animals and Treatment: Sixty, two-day-old ducklings were divided into ten groups of s i x . Each group was further subdivided into two groups of three. Three ducklings i n each group were given 1.0. ml and the other three ducklings received 2.0 ml of extract per orum d a i l y . The ninth group served as a "treated" control and were given equivalent doses TABLE XXI M o r t a l i t y S c h e d u l e of D u c k l i n g s  A s s a y No. 5 Group C u l t u r e D e s i g n a t i o n Dosage T o t a l (ml) Deaths 1 1 A. f l a v u s 44 1.0 1/3 2.0 1/3 2 2 A. f l a v u s 45 1.0 1/3 2.0 0/3 3 3 P\ rubrum 1.0 2/3 2.0 1/3 4 4 P.E. 78 1.0 1/3 2.0 2/3 5 5 Mink 101 1.0 0/3 2.0 0/3 6 6 Mink 107 1.0 0/3 2.0 2/3 7 7 Mink 108 1.0 0/3 2.0 1/3 8 8 G.W. 10 1.0 0/3 2.0 2/3 9 9 C o n t r o l 1.0 0/3 2.0 0/3 10 10 0/3 0/3 138 o f c o t t o n s e e d o i l . The t e n t h group s e r v e d as an " u n t r e a t e d " c o n t r o l and were not dosed. A s t a n d a r d c o m m e r c i a l duck grower r a t i o n and w a t e r were a v a i l a b l e ad l i b i t u m . A l l d u c k l i n g s t h a t d i e d o r t h a t s u r v i v e d the e x p e r i m e n t a l p e r i o d were s u b m i t t e d t o p o s t mortem examinat i o n . (3) R e s u l t s : F o u r known t o x i n - p r o d u c i n g f u n g i and f o u r f u n g i i s o l a t e d f r o m mink c e r e a l mix and wheat were used i n t h i s a s s a y . A. f l a v u s 44 and 45 were known a f l a t o x i n - p r o d u c i n g s t r a i n s . P. rubrum and a f u s a r i u m s p e c i e s (PE 78) were known t o be t o x i c . These f o u r f u n g i were sup-p l i e d t h r o u g h t h e c o u r t e s y of the F e d e r a l Department o f A g r i c u l t u r e , D i v i s i o n o f A n i m a l P a t h o l o g y , B r i t i s h C o l u m b i a . . The w a t e r e x t r a c t of b o t h s t r a i n s o f A. f l a v u s d i s p l a y e d a low t o x i c i t y f o r d u c k l i n g s . T h i s r e s u l t was not s u r p r i s i n g as a f l a t o x i n was u s u a l l y o b t a i n e d from a m e t h a n o l e x t r a c t o f t h e f u n gus. The aqueous e x t r a c t s o f t h e medium of P• rubrum and t h e f u s a r i u m s p e c i e s d i s p l a y e d a h i g h e r t o x i c i t y l e v e l t h a n t h o s e of A. f l a v u s . The c u l t u r e s d e s i g n a t e d Mink 107, i s o l a t e d from mink c e r e a l mix and GW 10, i s o l a t e d from ground wheat, each caused d e a t h i n two o f t h r e e d u c k l i n g s a t t h e 2.0 ml dose l e v e l . On p a t h o l o g i c a l e x a m i n a t i o n o n l y t h e d u c k l i n g s dosed w i t h e x t r a c t s of GW 10 d i s p l a y e d o b s e r v a b l e s i g n s of l i v e r damage. The l i v e r s o f t h e s e d u c k l i n g s were y e l l o w - c o l o u r e d and m a r k e d l y h a e m o r r h a g i c . Subcutaneous haemorrhages were o b s e r v e d i n the webs and b e a k s . I n t h e o t h e r d u c k l i n g s t h a t d i e d as a r e s u l t of d o s i n g w i t h t h e v a r i o u s e x t r a c t s , l i v e r s r anged from a p p a r e n t l y normal t o p a l e and s l i g h t l y f l e c k e d . I n some c a s e s , s i g n s o f e n t e r i t i s were 139 o b s e r v e d . C h i c k Embryo L e t h a l i t y A s s a y s The use o f c h i c k embryos i n m y c o t o x i n a s s a y has been i n v e s t i g a t e d by P i a t t e t a l (1962) and more r e c e n t l y by V e r r e t e t a l (1964) . They have s u g g e s t e d t h a t t h e c h i c k embryo can p r o v i d e a r a p i d b i o - a s s a y f o r the d e t e c t i o n o f a f l a t o x i n . As a r e s u l t , c h i c k embryos were t e s t e d as a r a p i d s c r e e n i n g method f o r f u n g a l t o x i n s . Embryo L e t h a l i t y A s s a y No. 1 (1) P r e p a r a t i o n o f E x t r a c t s : 34 f u n g a l c u l t u r e s were grown i n a medium c o m p r i s i n g t h e aqueous e x t r a c t o f mink c e r e a l r a t i o n . The f u n g i and medium were e x t r a c t e d w i t h c h l o r o f o r m ; t h e c h l o r o f o r m l a y e r s were d r i e d w i t h anhydrous sodium s u l p h a t e and were r e d u c e d t o d r y n e s s i n a vacuum oven. 2 ml o f p r o p y l e n e g l y c o l was added t o each d r i e d e x t r a c t . (2) A n i m a l s and T r e a t m e n t : F i v e - d a y - o l d c h i c k embryos were used i n t h i s a s s a y . Each e x t r a c t was i n o c u l a t e d i n t o t e n embryos v i a t h e y o l k sac r o u t e , . f i v e a t t h e 0.1 ml l e v e l and f i v e a t the 0.2 ml l e v e l . Twenty embryos were each i n o c u l a t e d w i t h e q u i v a l e n t doses of p r o p y l e n e g l y c o l and s e r v e d as c o n t r o l s . A l l embryos were r e i n c u b a t e d . Samples f r o m a l l dead embryos were c u l t u r e d on b l o o d agar p l a t e s . (3) R e s u l t s : The r e s u l t s o f t h i s a s s a y a r e p r e s e n t e d i n T a b l e X X I I . I n t h i s a s s a y , p r o p y l e n e g l y c o l was used as t h e v e h i c l e f o r i n o c u l a t i o n . 140 TABLE X X I I Embryo M o r t a l i t y Data A s s a y No. 1 I n o c u l a t i o n V e h i c l e - P r o p y l e n e G l y c o l No. C u l t u r e Dose Embryos Day t o Death T o t a l B a c t e r i a l D e s i g n a t i o n (ml) I n o c u l a t e d 1 2 3 4 5 Deaths M o r t a l i t i e s Contaminat, i o n 101 0.1 5 4 0 0 0 1 5/5 100 • 5/5 0.2 5 4 1 0 0 0 5/5 100 5/5 103 0.1 5 1 1 2 1 0 5/5 100 5/5 0.2 5 0 0 1 3 0 4/5 80 4/4 104 0.1 5 1 0 0 0 0 1/5 20 -0.2 5 1 0 0 0 0 1/5 20 -105 0.1 5 1 0 0 0 0 1/5 20 -0.2 5 - _ ... _ _ -106 0.1 5 - - - - „ -0.2 5 1 0 0 0 0 1/5 20 -108 0.1 5 - - .., „ - - -0.2 5 0 1 0 1 0 2/5 40 2/2 109 0.1 5 - _ - - - - - -0.2 5 2 0 0 0 0 2/5 40 -110 0.1 5 - - _ - -0.2 5 •- - - - - - -111 0.1 5 - - _ - - - - -0.2 5 - - - - - - - -112 0.1 5 1 1 0 0 0 . 2/5 40 1/2 0.2 5 1 1 0 0 0 2/5 40 1/2 113 0.1 5 - - - - - -0.2 5 1 0 0 0 1 .2/5 40 1/2 114 0.1 5 - - _ - -0.2 5 1 0 0 0 0 1/5 20 -115 0.1 5 J. 0 0 0 0 1/5 20 -0.2 5 _ , „ _ - - -116 0.1 5 - - - - - - -0.2 5 - - - - - -117 0.1 5 1 0 0 0 0 1/5 20 -0.2 5 - _ -118 0.1 5 - _ - _ - - - _ 0.2 5 1 1 0 0 0 2/5 40 -119 0.1 5 1 0 0 0 0 1/5 20 -0.2 5 _ - - » „ _ ~ -120 0.1 5 1 0 0 0 1 2/5 40 2/2 0.2 5 0 1 0 0 0 1/5 20 -121 0.1 5 - „ . - - - - - -0.2 5 1 0 0 0 0 1/5 20 -TABLE X X I I ( C o n t i n u e d ) 141 No. C u l t u r e Dose Embroys Day t o Death T o t a l % B a c t e r i a l D e s i g n a t i o n (ml) I n o c u l a t e d 1 2 3 4 5 Deaths M o r t a l i t i e s C o n t a m i n a t i o n 122 0.1 5 2 3 0 0 0 5/5 100 5/5 0.2 5 1 4 0 0 0 5/5 100 3/5. 124 0.1 5 - - -0.2 5 1 0 0 0 0 2/5 40 2/2 125 0.1 5 1 3 0 0 0 4/5 80 4/4 0.2 5 1 4 0 0 0 5/5 100 5/5 126 0.1 5 - - - - - - - -0.2 5 1 0 0 0 0 1/5 20 1/1 127 0.1 5 0 2 0 3 2 5/5 100 5/5 0.2 5 2 1 1 1 0 5/5 100 5/5 128 0.1 5 - - -0.2 5 0 1 0 0 0 1/5 20 1/2 130 0.1 5 1 2 0 1 0 4/5 80 4/4 0.2 5 2 0 0 1 0 3/5 60 3/3 131 0.1 5 1 1 1 2 0 5/5 100 5/5 0.2 5 0 1 3 0 1 5/5 100 5/5 132 0.1 5 1 0 0 0 0 1/5 20 -0.2 5 1 0 0 0 0 1/5 20 -133 0.1 5 0 2 0 2 0 4/5 80 4/4 0.2 5 0 4 1 0 0 5/5 100 5/5 134 0.1 5 1 0 0 0 0 1/5 20 1/1 0.2 5 2 2 0 0 0 4/5 80 3/4 138 0.1 5 1 0 0 0 0 1/5 20 1/1 0.2 5 1 0 0 0 0 1/5 20 1/1 A. f l a v u s 0.1 5 2 0 0 0 0 2/5 40 2/2 44 0.2 5 1 1 0 0 1 3/5 60 2/3 A. f l a v u s 0.1 5 3 1 0 0 0 4/5 80 4/4 45 0.2 5 2 1 0 1 1 5/5 100 5/5 C o n t r o l 0.1 0.2 20 20 - - -142 T h i s compound was used s u c c e s s f u l l y by V e r r e t et a l ( 1 9 6 4 ) . Repeated a s s a y s , u s i n g t h i s v e h i c l e and o b s e r v i n g a s e p t i c p r e c a u t i o n s t o p r e v e n t u n n e c e s s a r y c o n t a m i n a t i o n o f m a t e r i a l , f a i l e d t o p r e v e n t b a c t e r i a l c o n t a m i n a t i o n Embryo L e t h a l i t y A s s a y No. 2 As a r e s u l t o f t h e f a i l u r e of p r o p y l e n e g l y c o l t o i n h i b i t b a c t e r i a l g r o w t h , a t r i a l a s s a y was p e r f o r m e d u s i n g 30 p e r c e n t m e t hanol as t h e i n o c u l a t i o n v e h i c l e . (1) P r e p a r a t i o n o f E x t r a c t s : The m y c e l i a l mat f r a c t i o n s from D u c k l i n g A s s a y No. 5 were l y o p h i l i z e d and e x t r a c t e d w i t h m e t h a n o l . The methanol was r e -moved under vacuum and t h e e x t r a c t s were r e s u s p e n d e d i n 30 p e r c e n t m e t h a n o l . (2) A n i m a l s and T r e a t m e n t : Doses of 0.1 ml and 0.2 ml p e r sample were i n o c u l a t e d i n t o f i v e embryos v i a t h e y o l k sac r o u t e . One group o f embryos was i n o c u l a t e d w i t h an e q u i v a l e n t volume o f 30 p e r c e n t methanol and s e r v e d as a c o n t r o l . A l l embryos were r e i n c u b a t e d and checked d a i l y . Samples from a l l dead embryos were c u l t u r e d on b l o o d agar p l a t e s . (3) R e s u l t s : The r e s u l t s of t h i s a s s a y are p r e s e n t e d i n T a b l e X X I I I . U s i n g known and unknown t o x i c f u n g i , i t was p o s s i b l e t o k i l l embryos w i t h o u t b a c t e r i a l i n t e r f e r e n c e . The e x t r a c t s of known t o x i n - p r o d u c i n g f u n g i e.g. A. f l a v u s 44, P. rubrum and the f u s a r i u m s p e c i e s , d i s p l a y e d a marked t o x i c i t y f o r embryos w i t h i n 143 TABLE X X I I I  Embryo M o r t a l i t y Data  A s s a y No. 2 I n o c u l a t i o n V e h i c l e - 30% M e t h a n o l No. C u l t u r e Dose Embryos Day t o Death T o t a l % B a c t e r i a l D e s i g n a t i o n (ml) I n o c u l a t e d 1 2 3 4 5 Deaths M o r t a l i t i e s C o n t a m i n a t i o n 101 0, .1 5 0 1 1 0 0 2/5 40 0. .2 5 1 0 1 0 1 3/5 60 107 0. .1 5 0 1 0 0 0 1/5 20 0. .2 5 1 2 0 0 0 3/5 60 109 0. .1 5 1 0 0 0 0 1/5 20 0, .2 5 1 0 0 1 0 2/5 40 F u s a r i u m 0. .1 5 5 0 0 0 0 5/5 100 0, .2 5 4 0 1 0 0 5/5 100 P. rubrum 0. .1 5 5 0 0 0 0 5/5 100 0, .2 5 5 0 0 0 0 5/5 100 A. f l a v u s 0. .1 5 5 0 0 0 0 5/5 100 44 0. .2 5 5 0 0 0 0 5/5 100 A. f l a v u s 0. .1 5 1 0 1 0 1 3/5 60 45 0. .2 5 2 0 3 0 0 5/5 100 C o n t r o l 0. 0. .1 .2 5 5 - - - •- - - -144 24 h o u r s . A. f l a v u s 45 was l e s s t o x i c . C u l t u r e 101 and 107 d i s -p l a y e d a moderate t o x i c i t y . Embryo L e t h a l i t y A s s a y No. 3 (1) P r e p a r a t i o n of E x t r a c t s : A f i v e p e r c e n t s u s p e n s i o n of mink c e r e a l c r u m b l e s i n w a t e r was used as the s u b s t r a t e . F i f t y - t w o f u n g a l c u l t u r e s were grown i n t h i s medium f o r 7 d a y s . The c u l t u r e s were i n c u b a t e d on a s h a k e r . At the end of t h i s p e r i o d , 100 ml of c h l o r o f o r m was added t o each f l a s k w h i c h was shaken f o r 24 h o u r s . The c h l o r o f o r m l a y e r was d r i e d w i t h anhydrous sodium s u l p h a t e and t h e s o l v e n t removed by r o t a r y e v a p o r a t i o n . The r e s i d u e s were c o l l e c t e d i n t e s t tubes and d r i e d i n a vacuum oven. 3 ml methanol was added t o each t u b e . The volume o f each sample was made up t o 10 ml w i t h d i s t i l l e d water. (2) A n i m a l s and T r e a t m e n t : 0.2 ml o f each e x t r a c t was i n o c u l a t e d i n t o each o f f i v e , s i x - d a y - o l d c h i c k embryos v i a t h e y o l k sac r o u t e . A l l embryos were r e i n c u b a t e d and checked d a i l y . Samples from a l l dead embryos were c u l t u r e d on b l o o d agar p l a t e s . (3) R e s u l t s : The r e s u l t s o f t h i s a s s a y are p r e s e n t e d i n T a b l e XXIV. Low t o x i c i t y f o r c h i c k embryos d i s p l a y e d by t h e s e e x t r a c t s s u g g e s t e d t h e i n e f f i c i e n c y of c h l o r o f o r m as a s o l v e n t f o r e x t r a c t i o n as compared w i t h methanol ( A s s a y No. 2 ) . 145 TABLE XXIV Embryo M o r t a l i t y Data  A s s a y No. 3 I n o c u l a t i o n V e h i c l e - 30% M e t h a n o l No. C u l t u r e Dose Embryos Day t o Death T o t a l % B a c t e r i a l D e s i g n a t i o n (ml) I n o c u l a t e d 1 2 3 4 5 Deaths M o r t a l i t i e s C o n t a m i n a t i o n 101 0 .2 5 1 0 0 0 0 1/5 20 102 0 .2 5 - -103 0 .2 5 - -104 0 .2 5 - -105 0 .2 5 - -106 0 .2 5 2 0 0 0 0 2/5 40 107 0 .2 5 - -108 0 .2 5 1 0 0 0 0 1/5 20 109 0 .2 5 0 0 0 1 0 1/5 20 110 0 .2 5 - -111 0 .2 5 1 0 0 0 0 1/5 20 112 0 .2 5 0 1 0 0 0 1/5 20 113 0 .2 5 0 1 0 0 0 1/5 20 114 0 .2 5 - -115 0 .2 5 1 0 0 0 0 1/5 20 116 0 .2 5 - -117 0 .2 5 - -118 0 .2 5 1 0 0 0 0 1/5 20 119 0 .2 5 1 0 0 0 0 1/5 20 120 0 .2 5 0 1 0 0 0 1/5 20 121 0 .2 5 0 1 0 0 0 1/5 20 122 0 .2 5 1 0 0 0 0 1/5 20 124 0 .2 5 - -125 0 .2 5 2 0 0 0 0 2/5 40 126 0 .2 5 1 0 0 1 0 2/5 40 127 0 .2 5 1 0 0 0 0 1/5 20 128 0 .2 5 2 0 0 0 0 2/5 40 129. 0 .2 5 1 0 0 0 0 1/5 20 130 0 .2 5 - -131 0 .2 5 1 0 0 0 0 1/5 20 132 0 .2 5 - -133 0 .2 5 - -134 0. .2 5 1 0 0 0 0 1/5 20 138 0 .2 5 - -146 TABLE XXIV (Continued) No. Culture Dose Embryos Day to Death Total % B a c t e r i a l Designation (ml) Inoculated 1 2 3 4 5 Deaths M o r t a l i t y Contamination AWW 1 0.2 5 -AWW 2 0.2 5 - -AWW 3 0.2 5 - -AWW 4 0.2 5 1 0 0 0 0 1/5 20 -AWW 5 0.2 5 1 1 0 0 0 1/5 20 -AWW 6 0.2 5 - -GW 1 0.2 5 1 0 0 0 0 1/5 20 -GW 2 0.2 5 0 1 0 0 0 1/5 20 -GW 3 0.2 5 0 1 0 1 0 2/5 40 -GW 10 0.2 5 - -A. flavus 0.2 5 0 0 0 1 0 1/5 20 -P- rubrum 0.2 5 0 0 1 0 0 1/5 20 0 Control (30% methanol) 0.2 20 - -147 BIBLIOGRAPHY Anonymous. T o x i c i t y associated with c e r t a i n batches of groundnuts. Report of Interdepartmental Working Party on Groundnut T o x i c i t y Research. June, 1962. Forgacs, J. and W.T. C a r l l . Mycotoxicoses. J . Vet. S c i . ]_'. 273-382, 1962. Jo f f e , A.Z. The mycoflora of overwintered cereals arid i t s t o x i c i t y . B u l l . Res. Gounc. I s r a e l 9D: 101-126, 1960. Jo f f e , A.Z. T o x i c i t y of overwintered cereals. Plant and S o i l s . XVII; 31-44, 1963. Mayer, G.F. Endemic panmyelotoxicosis i n the Russian grain b e l t . Part I I . The botany, phytopathology and toxicology of Russian cereal food. M i l i t a r y Surgeon 113: 295-315, 1953. P i a t t , B.S., R.J.C. Stewart and S.R. Gupta. The chick embryo as a test organism for toxic substances i n food. Proc. Nutr. Soc. 30: 21, 1962. Sargeant, K., J . O'Kelly, R.B.A. Carnaghan and R. A l l c r o f t . The assay of a toxic p r i n c i p l e i n c e r t a i n groundnut meals. Vet. Rec. 73: 1219-1223, 1961. Raper, K.B. and C. Thom. A Manual of the P e n i c i l l i a . Williams and Wilkins, Baltimore, Maryland, 1949. Thom, C. and K.B. Raper. Manual of the A s p e r g i l l i . Williams and Wilkins Co., Baltimore, Maryland, 1945. Verr e t t , M.J., J.P. Marliac and J . McLaughlin J r . Use of chicken embryo i n the assay of a f l a t o x i n t o x i c i t y . J . Assoc. O f f i c . Agr. Chem. 47: 1003-1006, 1964. Wood, A.J. Personal communication, 1963. PART V FUNGAL TOXICOSIS STUDIES 148 INTRODUCTION During l a t e 1964, a few cases of the hepato-haemangioma-syndrome occurred i n mink maintained on a ranch-type mink r a t i o n . The composition of the r a t i o n was as shown i n Table XXV. TABLE XXV Ranch-Type Mink Ration Ingredients Per Cent Cereal Mix 20.0 Horsemeat 10.0 The only constituent of the r a t i o n which had been unchanged since the o r i g i n a l disease had occurred was the cottonseed o i l f r a c t i o n . Up to that time, the problem had been approached from the point of view that a toxin had been elaborated by a fungus present in the mink feed. Since cottonseed meal containing a f l a t o x i n has been shown to have caused hepatomas in trout (Wolf and Jackson, 1963), i t was decided to re-examine the problem by feeding high l e v e l s of cottonseed o i l to mink to see i f the mink disease was due to a f l a t o x i n or an aflatoxin-type compound present in the cottonseed L i v e r Fishscraps Cottonseed O i l Water 5.0 50.0 7.0 8.0 100.0 149 EXPERIMENTAL To test t h i s postulate, s i x experimental rations were designed. These rations were adequate i n a l l the n u t r i t i o n a l requirements for the growth and maintenance of the young adult mink. (1) Rations: The composition of the basic experimental r a t i o n i s given i n Table XXVI. TABLE XXVI  Experimental Mink Ration Ingredients Per Cent Cereal Mix * 12.0 Fishmeal 9.6 Brewer's Yeast 0.7 D i s t i l l e r ' s Dried Solubles 0.7 Fishscraps 28.6 Horsemeat 5.8 Beef L i v e r 3.0 Fat Source 9.6 Water 30.0 100.0 * See Table A (Appendix) The following f a t sources were used in the s i x experimental r a t i o n s : (1) Cottonseed O i l (4) Lard (2) Rapeseed O i l (5) Beef Tallow (3) Safflower O i l (6) Lard and A f l a t o x i n The f a t content of these rations a f t e r mixing was 30 per cent. Crude a f l a -toxin was added to r a t i o n No. 6 at the 0.01 per cent l e v e l . The s i x experimental rations were pre-mixed into 350 pound l o t s . Each 150 r a t i o n was divided into ten-pound aliquots and frozen. The feed was stored at 0° F. The amount required for each day's feeding was thawed at room temperature overnight p r i o r to feeding. (2) Animals and Treatment: Seventy-two female and twelve male (84 t o t a l ) young adult mink were used in t h i s experiment. They were divided into s i x groups of fourteen, twelve females and two males to a group. They were maintained on these aforementioned rations for twelve weeks. Twelve a d d i t i o n a l mink were divided into s i x groups of two, one male and one female to a group. These mink were fed the same rations as the other seventy-two mink. Daily feed intakes were measured on these mink over a four-week period. At the end of twelve weeks, haematological indices were determined for ten mink from each group as well as for ten control mink. (3) Results: The mean body weight data of the surviving mink are presented in Table XXVII. Three deaths occurred i n the a f l a t o x i n - t r e a t e d group of mink during the experimental period. On autopsy, these animals displayed the gross pathology that was described previously for a f l a t o x i c o s i s i n the mink. Two mink died from the group that was given cottonseed o i l in the r a t i o n . On gross pathological examination, a f l a t o x i c o s i s was not indicated. In the other groups combined, three non-specific deaths occurred. The d a i l y feed intake data on the twelve mink are presented in Table XXVIII. The body weight data on these same mink are presented in Table XXIX. The d i g e s t i b i l i t y values for the r a t i o n , f a t and protein are presented i n Table XXX. TABLE XXVII  Mean Body Weight of Experimental Mink Mean Body Weights (Grams) Rations Sex Dec. 15 Jan. 15 Feb. 15 Mar. 15 Cottonseed O i l o* 1835 2075 2075 1870 Cottonseed O i l 9 945 890 1020 760 Rapeseed O i l d* 1625 1770 1745 1745 Rapeseed O i l 9 950 1070 920 850 Safflower O i l cr 1700 1650 1700 1690 Safflower O i l 9 980 990 980 890 Lard o* 1990 2035 2030 1970 Lard 9 920 910 870 800 Beef Tallow o* 1710 1670 1590 1520 Beef Tallow 9 940 920 880 835 Lard and A f l a t o x i n d* 1650 1675 1690 1670 Lard and A f l a t o x i n 9 1070 1015 970 950 TABLE XXVIII Daily Feed Intake (Grams) of Representative Mink on Experimental Rations Mink Days No. Sex Treatment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 o* Cottonseed O i l 141 143 138 147 138 165 86 102 127 117 118 61 114' 199 2 ? Cottonseed O i l 134 119 84 129 61 145 150 98 134 112 100 94 124 144 3 o* Rapeseed O i l 193 188 235 72 176 195 190 206 265 154 170 82 147 119 4 9 Rapeseed O i l 94 104 20 56 77 52 76 193 120 0 0 61 110 118 5 o* Safflower O i l 163 183 195 230 237 172 289 102 127 117 118 61 114 199 6 9 Safflower O i l 68 109 72 98 87 46 139 98 134 112 100 94 124 144 7 o* Lard 159 142 189 256 285 174 222 0 260 158 244 241 236 236 8 9 Lard 71 82 94 87 111 76 122 60 81 71 64 65 83 104 9 o* Beef Tallow 180 241 210 267 194 154 194 143 130 162 251 230 2 7 10 9 Beef Tallow 47 119 158 159 177 144 163 158 146 118 142 120 78 129 11 o* Lard and 173 195 239 200 184 138 162 173 159 126 187 169 156 150 12 9 A f l a t o x i n 0 38 2 102 92 90 76 60 95 68 137 92 106 105 TABLE XXVIII (Continued) Mink Mean No. Sex Treatment 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Intake 1 o* Cottonseed O i l 113 113 2 9 Cottonseed O i l 117 74 63 190 113 83 197 49 116 113 27 110 134 109 133 174 72 122 176 128 64 107 114 22 113 98 125 118 3 o* Rapeseed O i l 4 9 Rapeseed O i l 115 105 68 151 107 152 91 110 92 75 164 64 129 60 138 0 17 83 140 112 40 105 96 69 93 116 94 95 87 83 5 o* Safflower O i l 223 170 171 152 160 195 231 6 9 Safflower O i l 3 122 119 75 95 103 107 137 185 212 258 192 243 285 183 71 156 38 113 75 90 119 97 7 8 d* 9 Lard Lard 256 242 124 237 84 34 73 69 84 183 180 61 171 0 102 168 129 211 106 182 144 184 115 115 100 59 72 68 78 84 9 o* Beef Tallow 10 9 Beef Tallow 235 259 233 259 201 193 .234 119 162 144 152 196 171 111 199 112 112 213 146 105 176 189 83 184 217 194 154 167 169 146 11 o* Lard and 171 157 144 180 151 128 57 12 9 A f l a t o x i n 183 115 74 74 75 168 154 11.7 127 108 164 127 141 132 88 50 89 135 93 102 87 155 90 TABLE XXIX Body Weight Data on Experimental Mink Body Weight (Grams) Mink Day Day Day Day Day No. Treatment Sex 0 7 14 21 28 1 Cottonseed O i l cr 1180 1200 1200 1220 1230 2 Cottonseed O i l 9 960 1010 1000 990 990 3 Rapeseed O i l d* 1580 1700 1720 1730 1680 4 Rapeseed O i l ? 850 890 1030 1060 1050 5 Safflower O i l cr 1660 1860 2150 2240 2400 6 Safflower O i l 9 820 820 790 840 800 7 Lard cr 1440 1630 1760 1860 1890 8 Lard 9 900 930 870 840 920 9 Beef Tallow cr 1610 1740 1960 2030 2220 10 Beef Tallow 9 1190 1306 1360 1390 1540 11 Lard and cr 1300 1440 1570 1550 1700 12 A f l a t o x i n 9 1060 1060 1040 1030 1020 155 The haematological values for the mink blood are presented in Tables XXXI and XXXII. The r e s u l t s of the t o t a l white c e l l and d i f f e r e n t i a l c e l l counts suggest that a f l a t o x i c o s i s causes a marked leucopenia with a r e l a t i v e lymphocytosis. Cottonseed oil-treated-mink followed a s i m i l a r pattern. This hints at a suggestion that there i s a factor in cottonseed o i l that i s equal to or s i m i l a r to a f l a t o x i n . The duration of the experiment was not s u f f i c i e n t to allow for any noticeable pathological changes in the mink on cottonseed o i l . I f a f l a t o x i n i s present i n cottonseed o i l , i t would pre-sumably be at a very low l e v e l . TABLE XXX D i g e s t i b i l i t y Values of Mink Ration, Fat and Protein Rations Per cent D i g e s t i b i l i t y Ration Fat Protein Cottonseed O i l Rapeseed O i l Beef Tallow 87 .3 8 5 . 8 8 2 . 4 94 .5 95 .0 98 .2 93 .2 8 5 . 5 91 .3 156 TABLE XXXI Determination of the Erythrocyte, Leucocyte and Heamtocrit Values of Mink Mink Group Erythrocytes i n M i l l i o n s / c u . mm Leucocytes/cu. mm Hematocrit % Lard and A f l a t o x i n Cottonseed O i l Rapeseed O i l Safflower O i l Tallow Lard Control 8.9 9.9 9.7 9.9 9.9 9.7 9.9 4300 5400 7000 6200 8900 8500 8500 49.0 52.5 51.5 52.0 50.5 52.0 52.5 TABLE XXXII Mean D i f f e r e n t i a l Counts of Mink Mink Group Leucocytes as % B M * Lard and A f l a t o x i n 61 Cottonseed O i l 62 Rapeseed O i l 69 Safflower O i l 70 Tallow 71 Lard 72 Control 75 8 6 8 8 8 8 7 2 2 3 2 2 3 2 26 26 18 19 17 16 14 * P-polymorphonuclear neutrophil, S-staff c e l l , E-eosinophil, B-basophil, L-lymphocyte, M-monocyte. 157 BIBLIOGRAPHY A l l e n , M.R.P., E.V. Evans and I.R. Sibbald. Energy: Protein r e l a t i o n -ships i n the di e t s of growing mink. Can. J . Physiol, and Pharmocol. 42: 733-744, 1964. Anonymous. Hepatomas in trout. Nut. Res. 22: 208-210, 1964. Hartsough, G.R. and J . Gorham. Sanitation and Health. The Blue Book of Fur Farming, 120, 1965. Kubin, R. and M.M. Mason. Normal blood and urine values f o r mink. Cornell Vet. 38: 79-85, 1948. S i n c l a i r , D.G., E.V. Evans and I.R. Sibbald. The influence of apparent d i g e s t i b l e energy and apparent d i g e s t i b l e nitrogen i n the diet on weight gain, feed consumption, and nitrogen retention of growing mink. Can. J . Biochem. and Physiol. 40: 1375-1389, 1962. Wintrobe, M.M. C l i n i c a l Haemotology. Lea and Febiger, Philadelphia, 310-316, 1951. 158 GENERAL SUMMARY AND DISCUSSION An hepato-haemangioma syndrome (HHS) observed i n mink, appeared to be diet-induced and was possibly unique i n i t s manifestations. The target organ for the pathological changes was the l i v e r which showed severe gross a r c h i t e c t u r a l d i s r u p t i o n . The general body condition of the mink remained c h a r a c t e r i s t i c a l l y excellent, with death i n most cases being due to a massive i n t r a p e r i t o n e a l haemorrhage. The absence, both of d e b i l i t a t i v e changes and of favourable influence on the disease incidence when dietary adjustments were c a r r i e d out, appeared to eliminate any n u t r i t i o n a l disorder as a cause of the disease. The c a r d i n a l aspects of gross and histopathology i n HHS have been described i n d e t a i l with i l l u s t r a t i o n s . Most of the pathological changes suggested that the causative agent could be c l a s s i f i e d as an hepatoxin, possibly of a s p e c i f i c nature. A comprehensive examination of the l i t e r a -ture suggested that the toxin involved might be of fungal o r i g i n . An assay model was set up in mink by feeding groundnut meal con-taini n g crude a f l a t o x i n . This chemical i s a mycometabolite elaborated p r i n c i p a l l y by c e r t a i n s t r a i n s of As p e r g i l l u s flavus Link ex F r i e s , a common mould contaminant of animal and human foods. The p r i n c i p a l objec-t i v e s of t h i s assay were: 1. To determine the s u s c e p t i b i l i t y of mink to a c l a s s i c a l hepatopathic mycotoxin. 2. To compare the gross h i s t o p a t h o l o g i c a l changes i n HHS and experimental a f l a t o x i c o s i s in mink. As a r e s u l t of th i s fungal t o x i c o s i s study (FTS) the cardin a l comparative aspects of pathological changes i n HHS and FTS mink can be 159 l i s t e d : (a) The HHS (unknown) toxic factor and crude a f l a t o x i n are both p r i m a r i l y hepatotoxic i n mink. Both, when present i n s u f f i c i e n t l e v e l s , are apparently capable of causing acute hepatic damage and probably hepatic f a i l u r e . (b) The HHS toxin causes marked f i b r o t i c changes i n mink l i v e r s i n the natural disease. The r e s u l t i n g vascular d i s r u p t i o n may be the cause of the formation of haemangiomas or endothelium-lined blood cysts. Crude a f l a t o x i n , when fed experimentally at d i f f e r -ent l e v e l s does not cause the marked f i b r o t i c changes. While some true haemorrhages occur, there i s an absence of s i m i l a r haemangiomatous b l o o d - f i l l e d structures as seen i n the c l a s s i c a l HHS disease. (c) Except for a r e t i c u l o e n d o t h e l i a l stimulus noted in the spleens and lymph nodes of at least 25 per cent of HHS cases, and peritoneal a s c i t e s i n 15 per cent, there was a general absence of changes i n organs other than the l i v e r even i n cases of spontaneous death. By contrast, the FTS assay disease led to many changes outside the l i v e r and peritoneum i n mink which died spontaneously. The p r i n c i p a l s i t e s of change were the kidneys, myelopoietic t i s s u e s , and g a s t r o i n t e s t i n a l t r a c t . Haemophilia was present even i n mink fed the lowest experimental l e v e l of toxic meal. At l e v e l s of meal which led to death there was evidence of haemorrhage in many organs and s i t e s including the l i v e r . The r e t i c u l o e n d o t h e l i a l stimulus noted in about a quarter of the spontaneous HHS deaths was far more marked than s i m i l a r e f f e c t s i n FTS mink. Metaplasia and neoplasia were 160 were absent from the l i v e r s of mink fed s p e c i f i e d l e v e l s of aflatoxin-containing meal. These included sub-lethal l e v e l s involving nine and one-half months of exposure. Thus i n conclusion, the HHS toxin i s more s p e c i f i c for the mink l i v e r than i s a f l a t o x i n . It induces marked f i b r o t i c changes, haemangiomas or blood cysts, and perhaps neoplasia i n that organ. It can also be the cause d i r e c t l y or i n d i r e c t l y of marked r e t i c u l o e n d o t h e l i a l stimulus. These three c h a r a c t e r i s t i c s are in contrast to the changes in FTS mink, although there were many s i m i l a r i t i e s i n l i v e r s more acutely and severely affected. Plate 23 demonstrates s u p e r f i c i a l s i m i l a r i t i e s . The HHS syndrome probably takes longer to induce and may well be the r e s u l t of a f l u c t u a t i n g l e v e l of the toxic agent in the mink d i e t . Such a feature was not reproduced experimentally i n the mink fed crude a f l a t o x i n in groundnut meal. A mould source of hepato-toxin was sought extensively although not exhaustively i n the mink feed by c u l t u r a l and bio-assay methods. A preliminary experiment has been c a r r i e d out in mink feeding with high l e v e l s of various fats to shed l i g h t on the p o s s i b i l i t y of an HHS-inducing (fat-floluble) toxin i n cottonseed o i l . The l a t t e r was an important and consistent component of the rations of mink affected with the hepato-haemangioma syndrome. APPENDIX 161 TABLE A Mink Cereal Grain P e l l e t s Ingredients Amounts (lbs) Tomato Pomace 100 Ground Wheat 5 9 9 Wheat Bran IQQ Wheat Germ Meal 65 Oat Groats 150 F i s h Meal (70%) 200 Soya Meal (50%) 200 Linseed O i l Meal IQQ A l f a l f a Leaf Meal 100 Skim Milk Powder 100 Brewer's Yeast 20 Steamed Bone Meal 20 Iodized Salt 5 Molasses - Cane 100 S t a b i l i z e d Fat 150 Dry Vitamin A - 1,000,000 I.U. Dry Vitamin D - 150,000 I.U. 2,000 162 TABLE B Composition on U.B.C. Mink Cereal Ration 144-63 Ingredients lbs Oat Groats 400 Ground Wheat 400 Alfalfa Leaf Meal 75 Brewer's Yeast 25 Skim Milk 50 Herring Meal , 50 1,000 TABLE C Composition of Czapek-Dox Agar Medium (Dehydrated) Ingredients Grams Sucrose : 3Q.00 Sodium Nitrate 2.00 Dipotassium Phosphate 1.00 Magnesium Sulphate 0.50 Potassium Chloride 0.50 Ferrous Sulphate 0.01 Agar 15.00 49.01 TABLE D Composition of Duck Grower Ration Ingredients Corn Meal Ground Wheat Ground Oats Ground Barley Wheat Bran Wheat Shorts Meat Meal Fishmeal Soy Bean O i l Meal D i s t i l l e r ' s Dried Solubles Dehydrated Grass Meal ( V i t a Grass) Iodized Salt (1%) Ground Limestone Steamed Bone Meal Manganese Sulphate Potassium Iodide Vitamin Dry D 3 Chlorine Chloride R i b o f l a v i n Calcium Pantothenate Niacin A n t i b i o t i c Feed Supplement Vitamin Tested A Feeding O i l 1000 I.U. Vitamin Tested D Feeding O i l 150 I.U. Iron Oxide Copper Sulphate Cobalt Sulphate Guaranteed Analysis Protein - (min.) 16.0% Fat - (min.) 3.0% Fibre - (max.) 8.5% 

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