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Physiological effects of Astragalus miser var. serotinus, (timber milkvetch) on sheep, rats, and mice. Mosher, Gary Alfred 1970

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< THE PHYSIOLOGICAL EFFECTS OF ASTRAGALUS miser var. serotinus. (TIMBER MILKVETCH) ON SHEEP, RATS, AND MICE by GARY ALFRED MOSHER B.S.A., University of British Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS. FOR THE DEGREE OF MASTER OF SCIENCE IH AGRICULTURE in the Division of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree tha permission 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 publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver 8, Canada ABSTRACT Feeding trials were conducted during 1968 and 1969 to study the physiological effects of Astragalus miser var. serotinus, timber n&lkvetch, (TMV) in sfieep. In Trial I, TMV was collected from Kamloops and Clinton, pelleted and fed to U groups of Dorset Horn wethers at levels of 0, 35, 70 and 100$. The symptoms exhibited by the sheep fed 100$ TMV included backward flexion of the fetlock - joints, hind limb paralysis and inco—ordination of movements. The presence of longi-tudinal ulcers in the intestines was a characteristic lesion observed in the sheep fed 100$ TMV. Fatty infiltration and hemorrhages in the liver and kidney were also observed. After the vetch was cut, dried and pelleted, the miserotoxin content dropped from 2.3 - 3.4$ to 0.9$. Trial II was carried out under actual grazing conditions near Kamloops.' Yearling ewes fed freshly cut TMV showed symptoms similar to those observed in Trial I. However, the symptoms were less severe and could be observed only when the sheep were exerted. The intestinal ulcers were found to be confined to the jejunum. The activity of the serum glutamic - oxaloacetic transaminase was significantly (p <.05) elevated in the sheep fed TMV indicating extensive tissue destruction. The level of thiamine in blood in both the trials was found to be within the normal range indicating that the beneficial effects of thiamine as a therapeutic agent in TMV poisoning may be indirect. When rats and mice were fed TMV, i t was found that they developed a syndrome characterized by the manifestation of convulsive movements terminating in a state of inactivity and a significant decline in body temperature. Extensive hemorrhages were seen in the gastric mucosa. By chromatographic procedures i t has been shown that miserotoxin is hydrolyzed to glucose and 3 - nitropropionic acid in the stomach of monogastric animals. In ruminants, miserotoxin is hydrolyzed to glucose and 3 - nitro - 1 - propanol which appears to be responsible for the toxicity. TABLE OF CONTENTS PAGE I. Introduction 1 II. Review of Literature 2 A. History of Timber Milkvetch (TMV) Poisoning 2 B. In Vivo Catabolic Products of Miserotoxin 7 C. Control of TMV Poisoning 11 III. Trial I. - THE EFFECTS OF TIMBER MILKVETCH ON SHEEP MAINTAINED UNDER HOUSED CONDITIONS 12 A. Introduction 12 B. Material and Methods 12 (a) Feeding Trial with Sheep 12 (b) Clinical Biochemical Investigations 13 (c) TMV Analysis 14-C. Results and Discussion 14 IV. Trial II.- THE EFFECTS OF TMV ON SHEEP GRAZING UNDER RANGE CONDITIONS 36 A. Introduction 36 B. Material and Methods 36 (a) Feeding Trial with Sheep 36 (b) Clinical Biochemical Investigations 37 C. Results and Discussion 38 V. Trial III.- THE EFFECTS OF TMV ON MICE 45 A. Introduction 45 B. Materials and Methods 45 (a) Feeding Trial with Mice 45 (b) Feeding Trial with Rats 45 (c) Effects of TMV Derivations on Mice 4-6 C. Results and Discussion 46 VI. Trial IV.- METABOLISM OF MISEROTOXIN IN THE ANIMAL BODY 51 A. Introduction 51 B. Material and Methods 51 (a) Metabolism of Miserotoxin in the Rat 51 (b) Metabolism of Miserotoxin in Sheep 52 C. Results and Discussion 52 VII. Conclusions 55 VIII. Bibliography 58 IX. Appendices 61 LIST OF TABLES TABLE PAGE I. The occurrence of miserotoxin in the three toxic varieties of TMV" and the minimum dose required of each to produce symptoms in 1 week old chicks. 7 II. Average daily gains, feed efficiencies and daily feed intakes for the 0 , 35, 70 and 100$ TMV fed sheep. 15 III. Average blood hemoglobin percent levels in sheep fed 0 , 35, 70 and 100$ TMV. 23 IV. Average microhematocrit values for sheep fed 0, 35, 70 and 100$ TMV. 23 V. Average blood glucose concentration in sheep fed 0 , 35, 70 and 100$ TMV. 25 VI. Average serum glutamic oxaloacetic transaminase (SGOT) levels in sheep fed 0 , 35, 70 and 100$ TMV. 25 VII. Average serum glutamic pyruvic transaminase (SGPT) levels in sheep fed 0 , 35, 70 and 100$ TMV. 27 VIII. Average total blood thiamine levels in sheep fed 0 , 35, 70 and 100$ TMV. 27 IX. Average TPP effect with respect to hexose in blood thiamine pyrophosphate determination for the 0 , 35, 70 and 100$ TMV fed sheep. 30 X. Average TPP effect with respect to pentose in blood thiamine pyrophosphate determination for sheep fed 0 , 35, 70 and 100$ TMV. 30 XI. Average creatine phosphokinase (CPK) activities in sheep fed 0$ and 100$ TMV. 34 XII. Percent dry matters for TMV. 34 XIII. Proximate analysis of TMV. 34 XIV. Average microhematocrit values for the control (Groupl) and hand-fed (Group 3) TMV sheep. 40 XV. Average SGOT and SGPT activities for the control (Group i) and hand-fed (Group 3) TMV sheep. 40 XVI. Average creatine phosphokinase activities for the control (Group I) and hand-fed TMV (Group 3) sheep. 41 XVII. Average total blood thiamine levels in the control (Group I) and hand-fed TMV (Group 3) sheep. 4-3 XVIII. Average TPP effects in the control (Group I) and hand-fed TMV (Group 3) sheep. 4-3 XIX. Effects of TMV derivatives on mice. 49 XX. Gm$ hemoglobin at different absorbance values. 65 XXI. Nitrogen concentration at respective absorbances. 65 XXII. SGOT activity as derived from standard solutions. 66 XXIII. SGPT activity as derived from standard solutions. 67 XXIV. The effect of time on thiochrome destruction by UV light. 69 XXV. Details of incubation for the assay of TPP activity. 70 XXVI. Determination of hexose by the Anthrone method. 71 XXVII. Determination of pentose by the Qrcinol method. 72 XXVIII. Miserotoxin concentration (mg/50mg plant material) at various absorbance levels. 77 XXIX. Individual responses of the 100$ TMV group (sheep 51, 52, 65 and 73) in the 1968 feeding t r i a l . 78 LIST OF APPENDICES PAGE Appendices I. - XII 62-80 LIST OF FIGURES FIGURE PAGE I. Structure of miserotoxin 5 II. Fetlock joint flexion in poisoned sheep #52 17 III. Front leg inco-ordination in poisoned sheep #52 17 IV. Type 1 ulceration of sheep intestine 19 V. Type 2 ulceration of sheep intestine 19 VI. Hematoxylin-eosin intestine section from a TMV poisoned sheep exhibiting sloughing epithelium (X 400) 20 VII. Hematoxylin-eosin section of TMV poisoned sheep liver tissue exhibiting widespread necrosis (X 4-00) 22 VIII. Hematoxylin-eosin section of TMV poisoned sheep liver tissue exhibiting fatty infiltration (X .100) 22 IX. Average hematocrit and hemoglobin changes in sheep fed 0 and 100$ TMV 24-X. Average SGOT levels in the sheep fed 0 , 70 and 100$ TMV 28 XI. A TMV poisoned mouse 48 XII. Hematoxylin-eosin section of TMV poisoned sheep kidney exhibiting necrosis (X 400) 80 ACKNOWLEDGEMENTS I wish to express my thanks to Dr. W.D. Kitts, Professor and Chairman, Department of Animal Science for allowing the use of the departmental facilities. To Dr. C.R. Krishnamurti, Assistant Professor, I gratefully express my thanks for the encouragement, consultation, and assistance received throughout the course of this study. I also wish to thank Dr. V.C. Brink, Professor of Plant Science, for his assistance in harvesting timber milkvetch and for his proposing sites of timber milkvetch growth. Special thanks must be given to Dr. R.H. Handford and Dr. A. McLean of the Canada Department of Agriculture Research Station, Kamloops, B.C., for their valuable co-operation and assistance. 1 am also grateful to the many people who assisted me in this study especially Mr. Jim Campbell and Mr. Ron Charles. To Mrs. R.M. Tait, I am grateful for her preparing microscopic sections, used in this study. I would especially like to thank the B.C. Cattle (Beef) Grower's Association for their most generous financial assistance. Astragalus miser var. serotinus ( Timber milkvetch ) 1. I . INTRODUCTION O f t h e more t h a n 1 2 0 0 known s p e c i e s o f t h e g e n u s A s t r a g a l u s , 200 a r e n a t i v e t o N o r t h A m e r i c a . The s p e c i e s , m i s e r ( B a r n e b y I96I4.) h a s b e e n r e p o r t e d t o c o n t a i n two v a r i e t i e s o b l o n g i f o l i u s a n d s e r o t i n u s , b o t h com-m o n l y c a l l e d t i m b e r m i l k v e t c h a n d a c u t e l y p o i s o n o u s t o l i v e s t o c k . The n o r t h e r n v a r i e t y , s e r o t i n u s , i s f o u n d i n A l b e r t a a n d S a s k a t c h e w a n t o a l i m i t e d d e g r e e , b u t o c c u r s m a i n l y i n t h e i n t e r i o r d r y b e l t o f B r i t i s h C o l u m b i a , w h e r e i t h a s become a m a j o r e c o n o m i c t h r e a t t o t h e r a n c h i n g i n d u s t r y . A s u r v e y i n 1 9 6 7 i n d i c a t e d t h a t t h e r e i s a n a v e r a g e l o s s o f 15t) c a t t l e b y d e a t h e a c h y e a r i n B r i t i s h C o l u m b i a a s a r e s u l t o f s e r o t i n u s p o i s o n i n g . T h i s d i d n o t i n c l u d e l o s s e s i n s l a u g h t e r e d c a t t l e w h i c h h a d s u f f e r e d f r o m e m a c i a t i o n , l o s s e s o f p o t e n t i a l c a l f c r o p s , a d d i t i o n a l management a n d t r e a t m e n t c o s t s , o r l o s s e s t o o t h e r t y p e s o f l i v e s t o c k , s u c h a s s h e e p a n d h o r s e s . I n t h e p a s t , t i m b e r m i l k v e t c h t o x i c i t y i n a n i m a l s h a s b e e n r e f e r r e d t o b y a v a r i e t y o f n a m e s : C l i n t o n h o r s e d i s e a s e , m o u n t a i n f e v e r , j a c k p i n e f e v e r , t i m b e r t r o u b l e , t i m b e r g r a s s d i s e a s e , r o a r i n g d i s e a s e a s w e l l a s t h e K a m l o o p s c a t t l e d i s e a s e . R e c e n t l y h o w e v e r , i t h a s b e e n c a l l e d A s t r a g a l u s p o i s o n i n g o r t i m b e r m i l k v e t c h p o i s o n i n g . T i m b e r m i l k v e t c h p o i s o n i n g o c c u r s p r i m a r i l y i n c a t t l e a n d s h e e p , s i n c e t h e y c o m p r i s e t h e m a j o r i t y o f g r a z i n g a n i m a l s f e e d i n g on r a n g e s s u p p o r t i n g t i m b e r m i l k v e t c h . Due t o t h e s i g n i f i c a n t e c o n o m i c l o s s e s a s s o c i a t e d w i t h t i m b e r m i l k v e t c h p o i s o n i n g , t h i s s t u d y was u n d e r t a k e n t o i n v e s t i g a t e t h e p h y s i o l o g i c a l e f f e c t s p r o d u c e d i n t h e a n i m a l b y t i m b e r m i l k v e t c h a n d t o e v a l u a t e t h e n a t u r e o f t h e t o x i c p r i n c i p l e w i t h a v i e w t o f o r m u l a t i n g s u i t a b l e t h e r a p e u t i c m e a s u r e s . 2. I I . REVIEW OF LITERATURE A. History of Timber Milkvetch (TMV) Poisoning (1) E a r l y Findings (a) Occurrence of T o x i c i t y The f i r s t o f f i c i a l report on TMV i n B r i t i s h Columbia was presented by Bruce (1912) who reported an outbreak i n Similkameen d i s t r i c t where 1^ 00 sheep from a f l o c k of 900 died. In t h i s p a r t i c u l a r instance kk% of the f l o c k died even though p r a c t i c a l l y a l l of the animals were aff e c t e d . C a t t l e , sheep and horses are known to be susceptible to the plant. Among ruminants, l a c t a t i n g cows and ewes are poisoned very q u i c k l y . Although native stock are more r e s i s t a n t than those newly introduced, Bruce con-cluded that 50 - 75% of animals were affected by TMV poisoning generally, with m o r t a l i t y ranging anywhere from 2 - 7$%. (b) Symptoms and Observations Subsequently Bruce (192?) described the t o x i c i t y of Astragalus by feeding the plant to sheep, c a t t l e and horses, ( i ) Sheep The e a r l i e s t symptoms consist of frequent u r i n a t i o n and sudden attacks of heart trouble i n which the heart beats very r a p i d l y (paroxysmal tachycardia). These attacks are s h o r t - l i v e d but can cause death. Excitement or unusual exertion w i l l induce the attacks e s p e c i a l l y i f the surrounding environment i s hot or the d i g e s t i v e t r a c t i s f u l l of food. In addition, incoordination of hind and f r o n t legs as well as d i f f i c u l t r e s p i r a t i o n e s p e c i a l l y a f t e r exertion appear as secondary c h a r a c t e r i s t i c s . In severe cases, complete prostration with the head pulled over the shoulders i s common. Accompanying these symptoms, there 3. can be wheezing or roaring, coughing, nasal discharge, grinding of teeth and protrusion of a cyanotic tongue. Appetite remains good but emaciation i s common. Temperature can r i s e a few degrees a f t e r struggling f o r breath, more often expiratory, which would i n d i c a t e a b i l a t e r a l p a r a l y s i s of the larynx. ( i i ) C a t t l e Symptoms are very s i m i l a r to those observed i n sheep but incoordination i s f i r s t noted i n the hind quarters and knuckling of the hind limbs i s frequently observed. Since animals are s t i l l mobile, e x c i t e -ment or excessive exertion w i l l induce heart attack with consequent death. ( i i i ) Horses The cardiac and r e s p i r a t o r y symptoms f o r c a t t l e and sheep apply to the horse as we l l . However, a f t e r being excited, the animal breaks into a profuse p e r s p i r a t i o n and death from asphyxia can occur. The attack may subside a f t e r $ - 10 minutes and the animal w i l l regain i t s nor-mal state with evidence of exhaustion. The resistance of the animal i s lowered making i t more susceptible to i n f e c t i o u s agents culminating i n pneumonia. In general, poisoned sheep, c a t t l e and horses w i l l die within a few days or weeks from cardiac p a r a l y s i s . Others may die of pneumonia months l a t e r due to secondary i n f e c t i o n . I f the vetch i s removed ea r l y from t h e i r d i e t , some animals w i l l r e t a i n a s l i g h t weakness of the hind quarters and e x h i b i t mild interference with the hind f e t l o c k s provided they survive the f i r s t few months. Macroscopically the lesions are those of cardiac f a i l -ure. P e r i c a r d i t i s i s f a i r l y common as i s the congestion of br a i n and s p i n a l cord. Some evidence of n e p h r i t i s has also been reported. M i c r o s c o p i c a l l y , there i s evidence of degeneration of the vagus nerve and of the superior and l a t e r a l columns of the s p i n a l cord. ( i v ) Rabbits and Guinea-Pigs W i l l i s (19h9) conducted TMV feeding t r i a l s with both l a c t a t i n g rabbits and guinea-pigs. The rabbits reared normal young and there was a s l i g h t l o s s i n body weight only i n those which consumed the 50 or 100$ vetch r a t i o n . Even these d i d not e x h i b i t t y p i c a l symptoms. Guinea-pigs r e c e i v i n g vetch i n amounts up to 67% of t h e i r d i e t gained weight and r a i s e d normal young. However, those l a c t a t i n g females, r e c e i v i n g a 100$ vetch d i e t , died within 3 - h weeks showing only s l i g h t weight l o s s . W i l l i s suggested that death was a r e s u l t of starvation even though h i s proximate analysis of TMV indicated that the nutrient con-tent of the plant was adequate to maintain l i f e . (2) Recent Findings No f u r t h e r i n v e s t i g a t i o n on Astragalus poisoning was reported u n t i l 1°52 when MacDonald v e r i f i e d the symptoms of t o x i c i t y f o r c a t t l e and sheep previously described by Bruce. In addition, MacDonald studied the d i s t r i b u t i o n of Astragalus i n B r i t i s h Columbia and described i t s growth requirements and other c h a r a c t e r i s t i c s . (a) Newly Described Symptoms and Observations Nicholson ( I 9 6 3 ) conducted grazing and feeding t r i a l s to inv e s t i g a t e the t o x i c i t y of the plant, the quantity of plant material required to bring about the f i r s t symptoms of poisoning, and the stage of growth at which the plant i s most poisonous. He reported that TMV i s r e l a -t i v e l y unpalatable to animals and i s consumed only when grasses and legumes have been depleted by the pressure of grazing or when e a r l y spring grazing i s done before grass growth has commenced. Generally i t was found that l a c t a t i n g beef cows would develop symptoms of Astragalus poisoning within 10 - 20 days a f t e r consuming 3hl - 425 pounds of hand-cut green TMV. Under grazing conditions however, the development of symptoms vary, depending on the rate of ingestion of the vetch. A very important observation i n t h i s 5. study was that the manifestation of symptoms i n those animals that had con-sumed a c r i t i c a l amount of vetch may be induced by moving and exerting the animals. I t was also concluded that Astragalus miser var. serotinus i s poisonous to c a t t l e and sheep from e a r l y growth to the beginning of the seed-pod stage i f consumed i n s u f f i c i e n t amounts. ob l o n g i f o l i u s found on the mountain meadows i n Colorado, Wyoming, Utah, Nevada and Arizona. Williams and Binns (1967) reported that t h i s plant i s so poisonous during pre-bloom and bloom growth stages that 0.25 l b . w i l l u s u a l l y be l e t h a l . They concluded that sheep are l e s s susceptible to the poison than c a t t l e . Previously, Buckeridge (1965) indicated that 1.12 l b s . of the dried plant f a t a l l y poisoned a 225 l b . steer i n h hours. i n TMV was a nitrogen-containing compound. Williams and Binns (I967) devel-oped a procedure (Appendix X) to extract and p a r t i a l l y p u r i f y the t o x i c p r i n c i p l e of TMV. The f i n a l e x traction product i s a yellowish-brown extremely hygroscopic gum which emits a sweet medicinal odor. Very recently Stermitz et a l . (1969) i d e n t i f i e d the poisonous p r i n c i p l e of TMV as miserotoxin (Figure I) based on nuclear magnetic resonance, i n f r a - r e d and mass s p e c t r a l analyses as well as chemical t e s t s . The southern counterpart of the v a r i e t y , serotinus, i s (b) 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 P r i n c i p l e , Miserotoxin Buckeridge (1965) had indicated that the poisonous p r i n c i p l e Figure I. Structure of Miserotoxin (B glucoside of 3 - n i t r o -1-propanol) HO H H (c) E f f e c t s of Miserotoxin. on the Animal ( i ) Bioassay with Chicks Williams and Binns (1967) administered o r a l l y 5ml. of a semi-purified extract of TMV, equivalent to 1 gram of plant material, to week old chicks. Using the LD ^ of the chicks as an index of plant t o x i -c i t y , they observed a l o s s of t o x i c i t y as the plant matured, p a r t i c u l a r l y beyond the e a r l y seed stage. Flowers or seeds tended to d i l u t e the t o x i -c i t y of the other plant parts. Acutely poisoned birds exhibited depression, lowered heads, closed eyes, drooped wings and r u f f l e d feathers. I n i t i a l l y there was a gradual p a r a l y s i s of the legs followed by complete p a r a l y s i s and death. Body temperature dropped r a p i d l y from 106 - 107°F to 85°F before death. Death occurred 1 - 3 hours a f t e r the administration of a l e t h a l dose. C h r o n i c a l l y poisoned birds exhibited b a s i c a l l y the same syndrome extended over 1; - 5 days. ( i i ) Concentration of Miserotoxin i n Plants Cooke (1955) described a procedure for the determination of 3 - n i t r o p r o p i o n i c a c i d i n the legume, Indigofera endecaphylla. Using a modified version of t h i s procedure, Williams and Norris (1969) studied the concentration of miserotoxin i n 3 v a r i e t i e s of TMV. They found that the v a r i e t y , serotinus, contained the highest l e v e l of toxin and was comparable to the other v a r i e t i e s i n producing symptoms i n chicks (Table I ) . The study of Williams and Norris (1969) also established that the concentration of miserotoxin was associated with the maturity of the plant, a t t a i n i n g a maximum at the bloom and e a r l y pod stages. The r a t i o of l e a f l e t s , p e t i o l e s , flowers, seeds and pods to each other at any one time and the degree of drying and bleaching as influenced by the hours of sun-l i g h t were considered responsible f o r the v a r i a t i o n s observed. Williams (1969) implied that the s o i l and weather conditions are a d d i t i o n a l factors 7. TABLE I. The occurrence of miserotoxin in the three toxic varieties . - - of TMV and the minimum dose required o f each to produce symptoms in 1 week old chicks. (Williams and Norris (1969)-Collection Site Misero-toxin Present Percent Miserotoxin Minimum dose in grams to produce symp-toms in chicks Variety Pre-bloom Bloom Oblongifolius Sanpete County, Utah + 1.1* 1.5 2.0 Oblongifolius Cache County, Utah 2.5 3.2 1.0 Serotinus Kamloops, B.C. + 3.2 2.7 1.0 Hylophilus Gallatin County, Montana + 2.6 2.7 1.0 Hylophilus Teton County Wyoming + 2.6 2.7 1.0 which influence the concentration of miserotoxin in the plants. He also reported that plants collected locally in 1968 had up to 3.k% miserotoxin whereas plants in the same stage of growth 200 miles away from Logan, Utah contained only 1.5%. Plants collected at the same elevation within a few miles of each other varied by 1 - 1.5$. In 1969, a dry spring resulted in none of the local collections having levels in excess of 2.3$. B. In Vivo Catabolic Products of Miserotoxin Recently, Williams et al. (1969) studied the toxicity of Astragalus to different species of animals by administering the extracts of 3 poisonous varieties, hylophilus, serotinus, and oblongifolius, and compared these with the effects produced by feeding sodium nitrite, 3-nitro-l-propionic acid (3-NPA) and 3-nitro-l-propanol (3-NPOH). Chickens given 3-nitro-l-propionic acid or the extracts of the 3 vetch varieties developed signs of poisoning and had identical values for 50$ and 100$ lethal doses. On the other hand 8. chickens given sodium n i t r i t e d i d not develop signs of t o x i c i t y u n t i l the n i t r i t e l e v e l was doubled suggesting that the immediate cause of death was not n i t r i t e but a 3-carbon n i t r o compound resembling 3-nitropropionic a c i d . Rabbits poisoned with extracts of the v a r i e t i e s o b l o n g i f o l i u s , serotinus, or hylophilus containing 137mg. NO^ per Kg. body weight died of methemo-globinemia. C l i n i c a l signs d i d not develop when rabbits were given a l e t h a l dose of the plant extract followed immediately by intravenous i n j e c t i o n of methylene blue at the rate of 3nig./Kg. body weight. Rabbits given 3-nitro-propionic acid died even when treated with methylene blue. C a t t l e were reported to be f a t a l l y poisoned by feeding the v a r i e t y o b l o n g i f o l i u s at the rate of 25mg. NC^/Kg. body weight, but intravenous administration of methylene blue was not e f f e c t i v e as a treatment. Symptoms were not apparent i n ca t t l e when 3-nitropropionic acid was fed, but 3-nitro-1-propanol caused death with i n i t i a l symptoms t y p i c a l of Astragalus poison-ing. In both groups, the common lesions included microhemorrhages scattered through the brain and between the myocardial muscle bundles, ecchymotic hemorrhages over the epicardium of the l e f t v e n t r i c l e , p r i m a r i l y over the apex, petechiae underlying the tracheal mucosa, congested thyroid glands and parotid lymph nodes, as w e l l as some degree of pulmonary congestion. (1) 3-nitro-l-propanol (3-NPOH) Stermitz et a l . (1969) were the f i r s t to report that o r a l treatment of a IkOKg. c a l f with 8 grams of 3-NPOH, corresponding, to a toxi c dose of the o r i g i n a l plant material, caused death with symptoms i d e n t i c a l with those of Astragalus poisoning. Miserotoxin was also s a i d to be metabolized to 3-NPOH i n l i v e s t o c k rumen f l u i d . Upon treatment with 2 N HC1, miserotoxin hydro-lyzed i n v i t r o to form glucose and 3-NPOH (Williams e_t a l . 1969). The same workers suggested that a f t e r miserotoxin i s degraded, the 3-NPOH in the rumen i s r a p i d l y absorbed and transported v i a the blood to the brain where i t d i r e c t l y a f f e c t s the s i t e s c o n t r o l l i n g r e s p i r a t o r y and muscular responses. Using chromatographic techniques ¥illiams et a l . ( 1 9 7 0 ) confirmed that miserotoxin was metabolized to 3-NPOH in the rumen f l u i d of both cattle and sheep and that 3-NPOH was the major metabolite responsible for the toxicity. ( 2 ) 3-nitropropionic acid (3-NPA) 3-NPA, a dibasic acid, was f i r s t shown by Gorter ( 1 9 2 0 ) to be identical with hiptagenic acid, the aglycone of hiptagin, a toxic gluco-side isolated from Hiptage benghalensis. Morris et a l . ( 1 9 5 U ) reported that the ingestion of creeping indigo, Indigofera endecaphylla, resulted in loss of appetite, dizziness, abortion and death of dairy cattle. Cooke ( 1 9 5 5 ) stated that sheep and rabbits exhibited toxic symptoms similar to dairy cattle when fed creeping indigo from which they isolated B-nitro-propionic acid. Both Cooke ( 1 9 5 5 ) and Morris et a l . ( 1 9 5 U ) maintained that B-nitropropionic acid was identical with hiptagenic acid. Hutton et a l . ( 1 9 5 8 ) substantiated that the l i v e r i s the main organ affected by the toxic principle of creeping indigo specifically in rabbits, mice and sheep. How-ever, the failure to duplicate the symptoms by feeding B-nitropropionic acid, suggests that this acid i s not the toxic principle of creeping indigo, con-trary to a l l other findings. Both Cooke ( 1 9 5 5 ) and Morris et a l . ( 1 9 5 4 ) used 1 - 2 week old chicks to test the toxicity of the semi-purified extract of creeping indigo and a synthetic source of B-nitropropionic acid. Both of these compounds k i l l e d chicks within 0 . 5 - U.O hours manifesting the same syndrome. The two toxic agents were shown to be similar by numerous identification techniques. Matsumoto et a l . ( 1 9 6 l ) reported that intra-peritoneal injections of rats with 3-NPA produced methemoglobinemia reach-i n g a maximum of 2 5 - 3 0 $ in 1 9 - 1 2 0 minutes and slowly decreasing there-after. Soon after injection a syndrome developed, including inactivity, d i f f i c u l t breathing, slow side-to-side head movement, convulsive r o l l i n g , followed by flaccid paralysis with death occurring generally within 2 U 0 1 0 . minutes a f t e r i n j e c t i o n . A f t e r intravenous i n j e c t i o n of 3-NPA, only 1% of the r a t s survived when the methemoglobin l e v e l exceeded 25%. Injected 3-NPA was found i n the blood, l i v e r and brain , but mostly i n the l a t t e r . The ra t s excreted a l l the i n j e c t e d 3-NPA within 36 hours a f t e r i n j e c t i o n . The f a c t that no methemoglobin was produced i n v i t r o by incubating 3-NPA with blood indicates that 3-NPA i s metabolized at a s i t e other than the blood. They suggested that the 3-NPA t o x i c i t y syndrome i s probably due to an i n h i b i -t i o n of an enzyme system at a s i t e that controls muscular movement especi-a l l y i n b r a i n ti s s u e where the 3-NPA concentration was the highest. Williams et a l . ( 1 9 6 9 ) concluded that i n c a t t l e , e i t h e r 3-NPA was not r a p i d l y absorbed, or a l e s s t o x i c compound was formed, since a l e t h a l dose on the basis of n i t r a t e content could be given without any t o x i c symptoms. I t appears that monogastric animals are d e f i n i t e l y affected by 3-NPA u s u a l l y r e s u l t i n g i n death. However, the controversy concerning 3-NPA poisoning of ruminants would tend to indicate that these species are not f a t a l l y a ffected by t h i s a c i d . ( 3 ) N i t r i t e Poisoning An elevated blood methemoglobin, c h a r a c t e r i s t i c of n i t r i t e poison-ing has been suggested to indicate the involvement of the n i t r o group of miserotoxin i n producing the c h a r a c t e r i s t i c symptoms of TMV t o x i c i t y . How-ever, r a b b i t s were the only species that died from methemoglobinemia as a r e s u l t of consuming TMV. Williams et a l . ( 1 9 6 9 ) concluded that i n rabbits miserotoxin was not absorbed as a 3 -carbon n i t r o sidechain and the methemo-globinemia observed was due to n i t r i t e poisoning. Other monogastric animals, such as r a t s and chicks, had elevated blood methemoglobin l e v e l s i n the studies described, but the poisoning has been p o s i t i v e l y a t t r i b u t e d to the ef f e c t s of the carbon sidechain moiety. The absence of increased methemo-globin l e v e l s i n the: ruminants unlike i n the monogastric animals can be substantiated by the i n d i v i d u a l findings of Brooks ( 1 9 3 U ) and Case ( 1 9 5 ? ) • 11. They showed that the inclusion of readily fermentable carbohydrates, such as glucose, in the diet of ruminants either limited the formation of methemoglobin or increased i t s reduction. In the case of Astragalus poisoning, a miserotoxin degradation product is glucose, which could impart this carbohydrate effect being present at a moderately high concen-tration. C. Control of TMV Poisoning (1) Herbicide Treatment Williams and Binns (196?) reported that cattle had to consume from 2 - 3 times as much TMV treated with silvex (alkyl amine salts of 2-(2,U,5-trichlorophenoxy)-propionic acid), as those consuming untreated vetch, in order to develop symptoms of Astragalus poisoning. This treat-ment follows the principle of natural senescence in which the disappear-ance of green color in the plant accompanied a decrease in poison concen-tration. Hence at a specific stage of bleaching, the plant can be ingested by animals without any harmful after-effects. (2) Limitation of Grazing If poisoned animals are removed from access to TMV and offered good feed and water, slow recovery w i l l occur generally to a point where affected animals can be sold for slaughter and most of their economic value returned to the owner (Willis 19U9)• He also suggested that since dry females are not so susceptible to poisoning as lactating cows, less risk is involved i f they are allowed to graze vetch infested areas provided over-grazing is not permitted. Williams et a l . (1969) suggested that non-lactating sheep are much more tolerant to vetch than are cattle. In those areas where TMV grows extensively and grazing must be allowed, the above suggestions may be applied. 1 2 . I I I . TRIAL 1 . THE EFFECTS OF TIMBER MILKVETCH ON SHEEP MAINTAINED UNDER HOUSED CONDITIONS A. Introduction Since the animals most frequently poisoned by timber milkvetch (TMV) under range grazing conditions are ruminants, sheep were selected as the experimental animals. The f i r s t t r i a l was designed to study the p h y s i o l o g i c a l e f f e c t s of timber milkvetch on sheep maintained i n the sheep research u n i t on the U.B.C. campus. I t was thought that t h i s would f a c i l i -tate subsequent t r i a l s to be conducted under a c t u a l range conditions. B. M a t e r i a l and Methods (a) Feeding T r i a l with Sheep ( 1 ) C o l l e c t i o n of TMV: During the month of June ( 1 9 6 8 ) , two separate samples of TMV were c o l l e c t e d from ranges i n the v i c i n i t y of Kamloops and C l i n t o n , B r i t i s h Columbia. Approximately two-hundred pounds (dry weight) of TMV were hand cut, placed on fin e mesh screens, and dri e d indoors where the temperature range was between 2 0 ° C and i | 0 ° C . A f t e r drying, the vetch was baled and wrapped i n p l a s t i c bags to minimize l e a f l o s s during transport from Kamloops to the U n i v e r s i t y of B.C. campus. In addition, 8 0 0 pounds (dry weight) of a mixture containing 10% TMV (by weight) and 30% Kentucky bluegrass (Poa  p r a t e n s i s ) , Junegrass (Koeleria c r i s t a t a ) , Yarrow ( A c h i l l e a lanulosa) and Canada bluegrass (Poa compressa) were c o l l e c t e d from the same area using a t r a c t o r mounted three foot c u t t i n g bar. This l o t was barn dri e d i n the same way as the previous one with i t s TMV composition being approximated by measuring small random samples. ( 2 ) Rations: Four d i f f e r e n t r a t i o n s , containing 0 , 3 5 , 7 0 , and 1 0 0 $ vetch were 13. prepared. An alfalfa (Medicago sativa) and smooth brome (Bromu's ihermis) 10-mesh ground hay mixture comprised the 0$ TMV ration and also served as the control ration. The 100$ and 70$ vetch samples were also ground through a 10-mesh grinder. One portion of the 70$ vetch mixture was diluted with two parts of the alfalfa-brome grass mixture to formulate the 35$ vetch ration. In order to prevent possible selective elimination of TMV by the sheep a l l the four rations were pelleted (l/8 inch size). Iodized cobalt salt, commercial mineral mix and water were supplied free choice through-out the duration of the experiment. (3) Experimental Animals: Sixteen Dorset Horn wethers ranging from 20Kg. to 25Kg. were ran-domly divided into four groups. Prior to the experiment the sheep were dewormed by drenching them with 2 grams of Thibenzole per head. (4) Housing: Each group of four sheep was kept in a 10 ft . x 8 ft. pen in the north end of the U.B.C. sheep unit. (5) Feeding: The sheep were fed ad libitum twice daily in order to maintain the animals at maximal intake. Daily records of feed consumption were kept. (6) Weighing and Sampling: The animals were weighed at one week intervals. Blood serum and heparinized blood were also collected weekly, (b) Clinical Biochemical Investigations As a guide line to the state of health of the sheep consuming TMV, the concentration of certain metabolites, hemoglobin, and the activity of selected enzymes were determined in the blood at weekly intervals. Blood glucose was determined using the Worthington glucose oxidase method. Hematocrits were determined for a l l blood samples using the standard microhematocrit method. Hemoglobin was measured by the method ih. of Lucas ( 1 9 6 1 ) (Appendix I . ) . Blood protein free f i l t r a t e s were prepared by p r e c i p i t a t i o n with zinc s u l f a t e and sodium hydroxide as described by the Somogyi - Shaffer - Hartman method (Oser, 1 9 6 5 ) (Appendix I I . ) . Serum n i t r a t e and n i t r i t e were determined by the method of Diven et a l . ( 1 9 6 2 ) (Appendix I I I . ) . Methemoglobins were determined by the procedure of Evelyn and Malloy (1938) (Appendix IV.). The a c t i v i t y of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase was determined using the procedure described by Bergmeyer ( 1 9 6 5 ) ( A p p e n d i x V.) and (Appendix VI.) r e s p e c t i v e l y . Creatine phosphokinase was determined i n blood serum with the procedure provided i n the CaLBiochem creatine phosphokinase t e s t pack # 8 6 9 2 l U . Blood thiamine was determined by a method of Gyorgy and Pearson (I967). Thiamine pyrophosphate e f f e c t was measured by the procedure of Albanese (I967) (Appendix V I I I . ) . The t r i a l was run f o r a period of 3 0 days at which time a l l the prepared rations were consumed. At the end of t h i s period the animals were s a c r i f i c e d , and samples of l i v e r , kidney, and other t i s s u e s were c o l l e c t e d and f i x e d i n 10$ Formalin. The tissues were embedded i n wax, sectioned and stained with a hematoxylin-eosin s t a i n , (c) TMV Analysis From samples of the barn dr i e d , U 0 - m e s h ground TMV and f r e s h cut TMV, dry matter determinations were made. The proximate analysis was per-formed as outlined i n the A.O.A.C. Methods of Analysis (I960). C. Results and Discussion (a) Feeding T r i a l Table I I . shows the body weight response and feed intake f o r the four groups of sheep. The average d a i l y gain and feed intake were much lower i n the 100$ group than i n the other three groups. The feed e f f i c i -ency on the other hand, was low among the control sheep but improved when TMV was added at l e v e l s of 3 5 and 70$. D a i l y feed intake was lower i n the TABLE II. Average d a i l y gains, feed e f f i c i e n c i e s and d a i l y feed intakes f o r the 0 , 3 5 , 7 0 and 1 0 0 $ TMV fed sheep. Level of TMV 0 $ 3 5 $ 7 0 $ 1 0 0 $ Animal Control 5 1 5 2 6 5 7 3 Average Average d a i l y gain (lb./day/head) 0 . 3 3 0 . 1 * 3 0 . 3 3 - 1 . 0 - 1 . 0 0 . 6 3 0 . 3 7 - 0 . 3 5 Feed e f f i c i e n c y ( l b . feed/lb. gain) 9 . 1 7 . 2 6 . 0 5 . 0 7 . 0 — D a i l y feed intake ( l b . feed/day/head) 3 . 0 1 3 . 1 2 2 . 0 0 . 8 0 . - U 3 3 . 1 7 2 . 5 7 1 . 7 4 Average t o t a l gain (lb./head) 9 . 9 1 2 . 9 9 . 9 - 1 0 . 0 - 2 1 . 5 1 2 . 9 1 1 . 1 - 7 . 5 1 6 . 70% than i n the 0% and 35$ groups. The t o t a l gain i n body weight i s d e f i n -i t e l y lower i n the sheep fed 1 0 0 $ TMV. Generally, there i s an extensive setback i n feed intake and body weight gain i n those sheep consuming a 100$ TMV r a t i o n . Hence TMV consumption at high rates can be associated with a r e s u l t a n t d e t e r i o r a t i o n i n general body condition, (b) Symptoms Ind i v i d u a l responses of each sheep i n the 1 0 0 $ TMV group (sheep no. 5-Lj 5 2 , 65 and 73) are l i s t e d i n Appendix XII. The symptoms observed i n t h i s t r i a l are backward f l e x i o n of f e t l o c k j o i n t s and r e s u l t a n t walking on these j o i n t s ; i n addition a backward extension of the f r o n t legs was obvious so that the animal appeared to kneel on i t s forearms. Figures I I I I I show a poisoned sheep manifesting these symptoms. Other symptoms observed i n the 100$ TMV group were incoordination of the limbs, d i f f i c u l t r e s p i r a t i o n with wheezing or roari n g , grinding of teeth, frequent u r i n a t i o n and r a p i d pulse rate (160/minute as opposed to a normal of 75/minute). These symptoms of TMV poisoning were manifested by sheep # 5 2 . A reduced intake of feed by some sheep i n the 1 0 0 $ group was a t t r i b u t a b l e to t h e i r f i n d i n g the p e l l e t e d vetch unpalatable. Only the l i m i t e d feed intake r e s u l t e d i n these animals becoming emaciated. Many workers ( W i l l i s , l Q k Q ; Nicholson, 1963) have reported that TMV contains adequate nutrients to maintain l i f e . The proximate a n a l y s i s given i n Table XV f o r TMV c l o s e l y corresponds to the analyses of other workers and substantiates the,view that the n u t r i t i v e content w i l l maintain l i f e . During the l a s t ten days of the experiment blood sampled from the 100$ group s p e c i f i c a l l y , appeared very dark red i n c o l o r as compared to the b r i g h t red color of normal sheep blood. In ad d i t i o n , t h i s blood tended to c l o t immediately upon withdrawal from the animal rather than to remain unclotted f o r a few minutes l i k e normal sheep blood does. Figure I I . Fetlock j o i n t f l e x i o n i n TMV poisoned sheep #£2. Figure I I I . Front leg incoordination i n TMV poisoned sheep #52. 18. These two modifications in blood therefore appear to be associated with a TMV poisoned state. (c) Lesions At autopsy, one apparant difference between the control sheep and those fed vetch was the presence of extensive ulceration of the intestine in a l l three vetch groups. The ulceration was more prominent in the small intestine of most affected animals. Basically, two distinct types of ulcer-ation were observed microscopically. One form could be described as a raised ridge-like projection extending along the intestinal mucosa. These projections varied in width from 0.3 to 1.5 cm. and in length from 6 to 20 cm. (Figure IV). The second type of ulcer was seen as a series of band-like circles on the intestinal mucosa. This is more clearly shown in Figure V. Both forms, especially the latter, contained many petechial hemorrhages interspersed among more massive hemorrhages. (d) Histopathology Microscopically, hematoxylin-eosin stained sections of control and treated tissues only showed differences in the intestinal preparations. In those intestinal sections from sheep consuming vetch there was a sloughing off of the intestinal epithelium. (Figure VI.). The extensive degree of damage, particularly to the inner lining of the small intestine of vetch consuming sheep only, indicates that this region must be the major site of digestion of this plant. Furthermore, tissue damage must be induced by some plant constituent, miserotoxin for example, or by some digestion product of the plant, such as 3-nitropropanol or 3-nitropropionic acid. It also seems applicable that this region of the gut would be the main site of absorption of the chemical agent, which there-by initiates its toxic effect on the other susceptible body tissues. Hence:, the blood must serve as the transport medium for this toxic principle to reach these other target tissues, such as the heart. Figure IV. Type 1 Ulceration of sheep intestine Figure V. Type 2 Ulceration of sheep intestine 20. Figure V I . Hematoxylin-eosin in tes t ine sect ion from a TMV poisoned sheep exh ib i t ing sloughing of the epithelium (x/,.00). 21. Examination of microscopic sections of liver tissue from the 100$ TMV sheep, showed extensive tissue necrosis with widespread accumulation of red blood cells in the extracellular spaces (Figure VII.). Also seen in the microscopic section of TMV sheep liver was fatty infiltration of the tissue. The presence of fat is indicated by the clear spaces dispersed among the liver cells as seen in Figure VIII. (e) Blood Analysis Blood hemoglobin and hematocrit values of sheep fed different levels of TMV are given in Tables III and IV. Over the duration of the experiment apparent but no significant (p<0.05) increases in both the hemoglobin and hematocrit values for the 100$ group were evident as com-pared to the other groups. Guyton (1966) has stated that tissue anoxia resulting from decreased blood flow through the tissues increases the rate of red cell production, with resultant increase in the hematocrit, as would be experienced in cardiac failure. Similarily an interference with respir-ation would decrease blood oxygenation and affect hematocrit values. There-fore i t follows that the cardiac and respiratory involvements, observed in Astragalus poisoned animals, would probably result in increased hematocrit values, especially during the latter stages of poisoning. A further explana-tion for elevated hematocrits could be attributed to a hemoconcentration effect commonly found in many pathological states in which erythrocyte numbers remain constant but blood fluid volume decreases. Figure IX shows graphically, hematocrit and hemoglobin changes in both control and 100$ vetch sheep as discussed. Blood glucose levels are presented in Table V. Generally, a l l four groups experienced a marked depression in blood glucose which reached its lowest point by the third week. Since the control group exhibited the same effect as the other three groups, i t would be justified to say that Figure VII. Hematoxylin-eosin section of TMV poisoned sheep liver tissue exhibiting widespread necrosis (x^OO). Figure VIII. Hematoxylin-eosin section of TMV poisoned sheep liver tissue exhibiting fatty infiltration (x100). TABLE I I I . Average blood hemoglobin percent levels i n sheep fed 0 , 35, 70 and 100$ TMV. Blood Hemoglobin $ (gm./lOOml.) Level of TMV Day 1 Day 9 Day 18 Day 29 0$ Hi. 9 12.6 11.8 12.3 35$ 12.9 11*. 3 12.1 12.1* 70$ 14.5 12.6 11.2 10.6 100$ l l . u 11.3 13.5 13-7 TABLE IV. Average microhematocrit values for sheep fed 0 , 35, 70 and 100$ TMV Microhematocrit Percent Level of TMV Day 1 Day 9 Day 18 Day 29 0$ 31.9 29.5 30.5 30.9 35$ 31.1 29.8 31.3 32.8 70$ 32.3 33.8 28.5 31.0 100$ 32.5 33.3 31*.7 36.1 24. Figure IX. Average hematocrit and hemoglobin changes in sheep fed 0 and 100$ TMV. t t .21 '18 M 3 0 Time ( days) TABLE V. Average blood glucose concentration i n sheep fed 0 , 3 5 , 7 0 and 1 0 0 $ TMV. Blood Glucose (mg. $ ) Level of TMV Day 1 Day 9 Day 1 8 Day 2 8 Day 3 0 0 $ 5 9 5 0 . 5 3 8 1*5 5 9 3 5 $ 6 2 . 5 1*5 3 7 1 * 0 . 5 7 0 $ 5 7 5 6 1*3 6 5 5 0 . 5 1 0 0 $ 5 7 . 5 1*6 1*0 6 1 — TABLE VI. Average serum glutamic oxaloacetic transaminase (SGOT) l e v e l s i n sheep fed 0 , 3 5 , 7 0 and 1 0 0 $ TMV. *SG0T A c t i v i t y (GOT units/ml. serum) Level of TMV Day 1 Day 9 Day 18 Day 28 Day 30 0$ 3 5 5 0 . 3 1*2 5 6 . 3 7 7 3 5 $ 3 6 . 3 1*1*.3 5 0 . 3 7 3 70$ 5 6 . 3 70 81.8 1 2 5 100$ 1*5.3 5 1 6 8 . 3 1 1 5 . 7 *A SGOT un i t i s defined as: one unit of enzyme that w i l l catalyze 1 uraole of substrate per minute under the conditions of the r e a t i o n procedure. 26. TMV did not account for this alteration in blood glucose levels. The decrease could be attributed to the fact that the sheep required an adjustment period to their pelleted rations. After this interval, the animals became more adapted to their surroundings and feed and thus were able to re-establish glucose levels comparable to those existent before the experiment commenced. Serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) levels are presented in Tables VI and VII respectively. Buck et al. (I96l) have reported that SGPT levels in normal adult sheep range from 0.5 to 19 GPT units per ml. of serum with a mean of 8.4.. Also given were SGOT values for sheep which were 54 - 128 GOT units per ml. of serum for adults and 97 - 121 GOT units per ml. of serum for lambs. Experimental values obtained in this study for both the transaminases f a l l well within the above ranges. If on the other hand, a comparison is made between the control SGOT activities and those of the 70$ and 100$ groups, a significant difference (p<0.05) existed during the last two weeks of the experiment between the control and 2 TMV groups. On this basis, there is an indication of elevated and abnormal SGOT activity in sheep fed 70$ or more TMV. This effect is presented in Figure X. Elevations in SGOT and SGPT activities, have been consistently associated with diseases involving tissue necrosis, such as myocardial infarctions, infections and toxic hepatitis, as well as muscular dystrophy. These two enzymes have been shown to be most concentrated in skeletal and heart, muscle, brain, liver and kidneys in decreasing content respectively, as well as in normal serum (Wroblewski and LaDue, 1955). When tissue undergoes necrosis the damaged cells release trans-aminases into the surrounding tissue fluid and thence to the circulating blood where serum transaminase activities will be elevated. Buck et al. (1961).has shown that serum SGOT and SGPT activities are directly TABLE VII . Average serum glutamic pyruvic transaminase (SGPT) levels i n sheep fed 0 , 35, 70 and 100$ TMV. *SGPT A c t i v i t y (SGPT units/ml. serum) Level of TMV Day 1 Day 9 Day 18 Day 28 Day 30 0$ 5 5 8 11 .5 11 8 12 10 14 — 70$ 5 1* H i 16 — . 100$ 6 . 5 5 10 12 Wroblewski units TABLE V I I I . Average t o t a l thiamine levels i n Sheep fed 0 , 35, 70 and 100$ TMV. Total Blood Thiamine (jig./ml. blood) Level of TMV Day 1 Day 9 Day 18 Day 28 0$ 0.079 0.081* 0.071 0.103 35$ 0.099 0.117 0.073 0.101* 70$ 0.097 0.116 0.081* 0.108 100$ 0.088 0.092 0.077 0.107 2 8 . Figure X. Average SGOT levels in the sheep fed 0, 70 and 100$ TMV. I$0-| S o -ls' I • I • 1 1—I Time (days) 29. proportional to the degree of tissue necrosis and that necrotic tissue con-tains considerably less transaminase activity than does normal tissue. The elevated SGOT and SGPT levels observed in this study are an indication of tissue necrosis as seen previously in the histopathological sections of liver and kidney. The blood thiamine levels are presented in Table VIII. Generally there appears to be very l i t t l e difference in the blood thiamine level between the four treatment groups at any time throughout the experimental period. Within these groups, thiamine levels are consistent throughout the first three weeks specifically. Gyorgy and Pearson (1967) have stated that most of the blood thiamine is contained in the red blood cells. Since hemato-crit determinations indicate red blood cell volume, any change in the hematocrit value will result in a corresponding change in the blood thiamine level. In the last week of the experiment, a l l four groups exhibited a slight increase in blood thiamine levels. This increase could be related to the higher hematocrit values (Table IV) also evident during this period. In the present study the determination of blood thiamine was undertaken to check the rationality of the suggestion by Nicholson (1963) that thiamine hydrochloride administration had a therapeutic effect on Astragalus poisoned cattle and sheep. He indicated that the most recently poisoned animals showed the greatest response to thiamine treatment, but only after the vetch was removed from their diet. Untreated animals required three times longer to recover, after removal of vetch from their diet, than did thiamine injected animals. An immediate response was shown by poisoned animals receiving intramuscular injections of thiamine hydro-chloride at the rate of 4.00 mgm. per adult cow and 100 mgm. per sheep or lamb. The mechanism by which thiamine exerts its action on poisoned animals is not known. Blood thiamine levels given in Table X eliminate the possibility TABLE I X . A v e r a g e TPP e f f e c t w i t h r e s p e c t t o h e x o s e i n b l o o d t h i a m i n e p y r o p h o s p h a t e d e t e r m i n a t i o n f o r t h e s h e e p f e d 0 , 3 5 , 70 a n d 100$ TMV. T P P e f f e c t ( $ ) L e v e l o f TMV D a y 1 D a y 9 D a y 18 D a y 28 0$ 2 7 . 0 1 0 2 4 . 6 3 1 8 . 0 9 35$ 3 6 . 7 7 0 4 9 . 4 0 4 7 . 8 6 70$ 3 5 . 3 8 0 5 6 . 4 8 1 7 . 8 7 100$ 2 3 . 8 3 4 . 8 1 1 . 2 3 5 0 . 8 3 TABLE X . A v e r a g e TPP e f f e c t w i t h r e s p e c t t o p e n t o s e i n b l o o d t h i a m i n e p y r o p h o s p h a t e d e t e r m i n a t i o n f o r s h e e p f e d 0 , 3 5 3 70 a n d 100$ TMV. T P P e f f e c t ( $ ) L e v e l o f TMV D a y 1 D a y 9 D a y 18 D a y 28 0$ 4 . 8 4 2 0 . 9 5 2 2 . 3 9 1 .45 35$ 7 .15 3 .22 1 7 . 0 5 2 2 . 3 1 70$ 0 2 1 . 2 5 6 .98 9 . 9 0 100$ 1 7 . 0 6 9 .81 4 . 7 6 9 . 7 7 31. of there being a thiamine deficiency per se in the TMV poisoned ruminant. Another approach was made to measure the thiamine status of the sheep fed TMV by the determination of the transketolase activity in the erythrocytes. The activity of this enzyme is measured as the percent thiamine pyrophosphate (TPP) effect which is the response of transketolase to the in vitro addition of TPP (Brin et al., 1960 and Bruns, 1959). TPP supplementation has l i t t l e effect on the transketolase activity of normal animal blood in vitro (Albanese, 1967). However, addition of TPP in vitro to a thiamine deficient blood sample results in increased hexose formation due to the enhancement of transketolase activity. Albanese suggested for hexose that a TPP effect within a range of 15 - 25$ is suggestive of marginal thiamine deficiency, while a severe deficiency would exist in excess of 25$ with clinical signs. These values apply for humans. Brin et al. (1960) has previously utilized the transketolase assay in rat studies. It was found that TPP effect rose to 40$ during manifestation of clinical thiamine deficiency. Use of supplemental TPP in vitro and thiamine hydrochloride in vitro returned values to near normal in the deficient hemolysates and animals respectively but no change was apparent in treated control rats. Hence, supplementation markedly increased both pentose disappearance and hexose formation. Dreylus (1962) found that severe symptoms and signs of deficiency became evident in rats when enzymatic activity had droppped to 10$ of normal and TPP effect had risen to 64$. It was noted that in early phases of deficiency in vitro addition of TPP is capable of restoring the system to a normal level of activity. However, with progressive depletion, restoration declines progressively. Thiamine administration into the animal will restore.activity to normal even after a lengthy deprivation time. The TPP effect with respect to hexose and pentose are given in Tables IX and X respectively. TPP effect in the TMV groups is equally 32. comparable with the control group on the basis of pentose utilization. There-fore the thiamine status of sheep fed TMV is comparable to those fed the basal ration. However on the basis of hexose formation by this same reaction, TPP effect was very apparent in a l l three TMV groups especially during the third and fourth weeks of the t r i a l , whereas the control group manifested a consis-tently lower TPP effect throughout the t r i a l . The pentose TPP effect does not indicate a thiamine deficient state in any of the groups. When these TPP effect levels are compared to values in the literature, they do not f a l l within the range which would suggest thiamine deficiency. Albanese (1967) suggested that a severe thiamine deficiency occurred in rats when pentose TPP effect exceeded 3 0 $ TPP effect. No group experienced a change in excess of 2 2 . 3 8 $ , TPP effect (pentose), which is well below Albanese's value of 3 0 $ . With respect to the transketolase assay the TPP effect as deter-mined from hexose formation indicates that a thiamine deficiency is present in a l l 3 TMV groups. Here the TPP effect is not only noticeably different from the average of the control, but is also within the severe deficiency range previously given for rats and humans (Albanese, 1967). It has been reported (Brin et al., i 9 6 0 ) that hexose TPP effect also gives an accurate indication of thiamine status. The TPP effect for the TMV groups suggests that a thiamine deficiency exists in these sheep. The hexose TPP effect like the pentose TPP effect indicates a thiamine deficiency i f a greater than 3 0 $ response in TPP effect is evident. On this basis, a thiamine deficiency exists in those sheep that consumed TMV. If a thiamine deficiency existed in these TMV sheep, a marked TPP effect with respect to both hexose and pentose would have occurred. In addi-tion blood thiamine levels would have shown an apparent decrease to some degree or other. The response of TMV poisoned sheep to thiamine injection 33. (Nicholson, 1963) would have to be explained on the basis that the animals were not suffering from a thiamine deficiency. However, thiamine s t i l l acts by some unknown mechanism to significantly improve the condition of TMV poisoned sheep. The serum creatine phosphokinases (CPK) activities are given in Table XI. There was no difference in the serum CPK activity between the control and the 100$ TMV group. The values obtained were comparable to the activity of 58 m /ml. reported for normal sheep (Buffey, 1969). Smith and Healy (1968) reported that CPK is found in considerable quantities in central nervous tissue and skeletal muscle. They found that CPK activity was considerably elevated in both central nervous and muscular disease while SGOT activity was only slightly increased in central nervous disease, but elevated considerably in muscular disease. By utilizing both enzyme determinations, an elevated CPK activity can be used to estimate any possible central nervous tissue involvement in a disease. The absence of any drastic changes in CPK activity indicate no apparent damage of central nervous tissue, in the sheep fed TMV. This coupled with the high SGOT activity observed earlier suggest probably muscle tissue breakdown. There was no evidence of either nitrite or nitrate in the blood, even though the procedure used had a sensitivity for nitrogen, as nitrate or nitrite of 0.05 parts per million, (f) TMV Analysis Analysis of the TMV fed to the experimental sheep by the method of Williams and Norris (1969) indicated that the misenioxin content was 1.65$ before pelleting and 0.9$ after pelleting. Hence, i t was concluded that the pelleting process lowered the TMV toxicity by action of excessive heat treat-ment on the plant toxin. The former miseatoxin level of 1.65$ is for that TABLE XI. Average creatine phosphokinase (CPK) activities in sheep fed 0$ and 100$. CPK Activity (m /ml.) Level of TMV Day 1 Day 9 Day 18 Day 28 0$ 53 60 4-9 47 100$ 52 49 54 55 TABLE XII. Percent dry matter levels for TMV, TMV $ Dry Matter $ Water Fresh Cut 10 90 Barn Dried 90 10 TABLE XIII. Proximate analysis of TMV. Constituent Average $ of the dry matter Ash 5.97 Crude Fiber 31.71 Ether Extract 2.37 Protein (N x 6.25) U.43 35. TMV which was sun and barn dried. For a similar TMV sample dried at a tempera-ture not exceeding 25°C, the toxin level is comparable with the 2.3$ - 3.4$ range suggested by Williams (1969). It is conceivable then, that the toxin level is subject to change by temperature thereby altering the potential toxi-city of the TMV plant. The TMV varied from early to late bloom in maturity while the grasses were in early florescence. Percent dry matter levels for TMV are given. (Table XII). In Table XIII, the proximate analysis of TMV is presented. 3 6 . IV. TRIAL II. THE EFFECTS OF TMV ON SHEEP GRAZING UNDER RANGE CONDITIONS A. Introduction The failure to produce toxicity in a l l the sheep fed 1 0 0 $ TMV during the f i r s t t r i a l suggested the possibility that the toxic factor(s) might have been destroyed extensively during the drying and/or pelleting process. To check this possibility, another feeding t r i a l was conducted under actual field conditions where the sheep were allowed to graze fresh TMV. B. Material and Methods (a) Feeding Trial with Sheep ( 1 ) Location: The experimental site was located on the Pemberton range, 3 0 miles east of Kamloops, B.C., at an altitude of 2 , 9 0 0 feet. On a westerly sloping hillside of approximately 20 acres, an area of 3 acres, where the density of TMV appeared to be maximal, was chosen. The other species of plants found grow-ing on this site were Junegrass (Koeleria cristata). Yarrow (Achillea lanulosa). Kentucky bluegrass (Poa pratensis) and Canada bluegrass (Poa compressa). The Yarrow was in f u l l flower while the grasses were in the early to late head stage. The duration of the t r i a l was 10 days (June 5 - June 1 5 ) , during which time the TMV matured from early flower to a mid-pod stage. On an adjacent one acre site, about 0 . 5 miles northwest of the first, a control plot was established. The vegetation was relatively free from TMV and was primarily covered with Junegrass (Koeleria cristata) which was only in a pre-head stage since this area was shaded from long hours of direct sunlight. Fresh TMV was handcut daily from the Pemberton range as well as from the Lac Dubois range and either fed immediately or frozen for use in the next 1 or 2 days. 37. (2) . Experimental Animals: Twenty-four Romneymarsh-Lincoln crossbred yearling ewes weighing in the range of 100-125 pounds were purchased locally from a range flock. At the end of the experiment a l l the sheep were slaughtered in Vancouver where blood and tissue samples were taken and carcasses examined internally for lesions. (3) Experimental Design: Three separate groups of 8 sheep each were restricted to areas enclosed by page wire hung on U foot high steel posts with a single top strand of barbwire. The control group was allowed to graze freely on the one acre site previously described. A second group grazed freely on the three acre vetch covered enclosure. In this same enclosure, a 15' x 15' pen was built to contain the third group which was fed only TMV, ad libitum, cut fresh daily. TMV density on this range was estimated at approximately 25 plants per square yard. Approximately 200 lbs. of TMV was frozen and stored for chemical determination of the toxic content and for in vitro fermentation tests as described below. (4-) Sampling and Treatment: Before the t r i a l was begun the animals were drenched with 1 fluid ounce of Piperanzine in 10ml. of water. Cobalt-iodized salt and water were provided free choice as was access to shade. (b) Clinical Biochemical Investigations (1) Blood: Blood was collected from the sheep on alternate days. The micro-hematocrit values, activities of SGOT, SGPT and CPK, as well as thiamine levels and TPP effect were determined using the methods described in Trial I. (2) TMV Analysis: In addition to the proximate analysis in dry matter determination previously described, three further tests were employed to assess the toxic 38. content of the plant material and i t s effect on cellulose digestion. TMV was run through an i n v i t r o fermentation technique as given i n Appendix IX. Rumen inoculum was obtained from a f i s t u l a t e d steer maintained on a die t of a l f a l f a hay. In addition to the disappearance of dry matter, cellulose digestion was determined by the method of Crampton and Maynard (1938). Miserotoxin from the TMV was extracted by procedure proposed by Williams and Binns (1967) as described i n Appendix X. The actual miserotoxin content of the vetch was estimated by a method given by Williams and Norris (1969) (Appendix XI). C. Results and Discussion ( 1 ) General: A l l hand-fed sheep (Group 3) ate TMV r e a d i l y with no apparent rejec-t i o n . The sheep foraging on t h e i r own (Group 2) grazed TMV with preference over grasses and other forbs. On the whole the two groups of sheep appeared to f i n d the TMV very palatable. (2) Symptoms; Only four of the eight sheep i n the hand-fed group (Group 3) showed symptoms of t o x i c i t y i n various forms, v i z . paralysis of hind quarters, s t i f f gait and f l e x i o n of the legs. These symptoms appeared r e a d i l y after the a n i -mals were excited, especially after blood was sampled. The f i r s t signs of t o x i c i t y were shown 6 days af t e r consuming TMV. When these sheep were shipped to Vancouver for slaughter, i t was observed that after a r r i v i n g at the packing plant, those i n Group 3 exhibited marked symptoms of t o x i c i t y i n the form of par a l y s i s , and le g fle x i o n s . Apparently the stress of shipping precipitated the t o x i c i t y syndrome. (3) Lesions: I n t e r n a l l y c h a r a c t e r i s t i c longitudinal ulcers i n the intestines were present i n a l l the sheep hand-fed TMV (Group 3) and only i n two of the 39. group grazing TMV (Group 2). These lesions were identical with those described in Trial I. In addition, i t was observed that most of the ulceration was con-fined to the jejunal region of the small intestine. (A) Histopathology: Microscopic appearance of the ulceration was the same as described and shown previously in Trail I. In this t r i a l , the severe fatty infiltration and necrosis of liver were not observed. No degenerative changes could be found in heart, brain, nervous and muscle tissues. (5) Blood Analysis: Microhematocrit levels are given in Table XIV. There appears to be no difference in the microhematocrit levels of the control (Group 1) and TMV groups. The activities of both SGOT and SGPT are presented in Table XV. The SGPT activity of the sheep in Group 3 (TMV) is within normal limits when the values are compared either to those of the control sheep (Group 1) or to the range (0.5 to 19 SGPT units/ml.) given by Buck et al. (1961). On the other hand, SGOT levels in the TMV sheep (Group 3) are significantly (p<.05) higher than those of the control sheep. The high SGOT values coupled with the absence of severe histopatho-logical lesions in the liver and kidney in this t r i a l suggests that the metabolic disturbance sets in earlier than extensive tissue distinction and that the duration of the t r i a l was too short for the latter to occur. There is no appreciable difference in the activity of CPK between the control and TMV groups (Table XVI) and compares well with the results obtained in Trial I. The normal level of CPK and the high activity of SGOT in the sheep fed TMV (Group 3) suggest that in the case of TMV poisoning the hepatic injury precedes any involvement of the central nervous tissue. TABLE XIV. Average microhematocrit values for the control (Group 1) and hand-fed TMV (Group 3) sheep. Microhematocrit Percent Group Day 0 1 2 3 4 5 6 7 8 9 10 Control TMV 28.9 31.6 30.1 31.3 30.6 31.5 29.8 30.5 30.3 30.9 32.1 32.5 30.3 29.9 32.9 32.6 29.5 32.5 29.3 32.0 31.0 32.7 TABLE XV. Average SGOT and SGPT activities for the control (Group 1) and hand-fed TMV (Group 3) sheep. SGOT Activity SGOT Activity SGOT units/ml. SGPT units/ml. Day Control TMV Control TMV 0 60 80 1 2 2 45 105 3 6 4 55 290 5 11 5 80 240 8 7 7: 85 235 4 5 8 65 240 3 U 9 75 250 6 8 10 72 210 8 11 41. TABLE XVI. Average creatine phosphokinase activities for the control (Group 1) and hand-fed TMV (Group 3) sheep. CPK Activity (ml|/ml.) Day Control TMV 0 51 46 1 53 31 3 47 34 4 46 38 5 49 70 7 46 ' 71 9 45 56 10 50 51 42. As shown in Table XVII, the total blood thiamine levels in both the control sheep (Group 1) and those fed TMV (Group 3), are within the nor-mal range of 0.05 - 0.08 pg/ml. of serum reported for dairy calves by Smith and Allen (1954). It therefore does not appear valid to state that a thia-mine deficiency per se develops in TMV toxicity. The TPP effects with respect to both hexose and pentose are presented in Table XVIII. There is no indication of a thiamine deficiency upon examin-ing both the hexose and pentose TPP effects for the TMV sheep. Some TPP$ values, calculated for hexose, indicate a mild state of thiamine deficiency in the sheep fed TMV (Group 3), but were not consistent enough to suggest a severe thiamine deficiency. Nitrite or nitrate could not be detected in the blood of either control or the TMV group. Methemoglobin concentration varied from a low of 0.27$ at the begin-ing to a high of 5.2$ at the end of the feeding t r i a l in the hand-fed TMV sheep-(Group 3). Methemoglobin could not be detected in the blood of the control sheep (Group 1) at any time. The slight methemoglobin increase in the TMV sheep was probably responsible for the darker colour imparted to their blood observed towards the end of the t r i a l . (6) Analysis of TMV for Toxic constituents: The miserotoxin levels of TMV collected during 1968 and 1969 were 2.57 and 3.14$ respectively. The slightly higher level for 1969 could be attributed to the wet spring in that year as compared to a drier spring in 1968. That i s , Williams (1969) has suggested that miserotoxin levels were highest in TMV, growing during wet seasons, since there would be less detoxi-fication by sun bleaching. TMV was harvested from locations both in the shade and direct sun-light. It was found that the pale green, narrow leaved, low growing TMV on TABLE XVII. Average total blood thiamine levels in the control (Group 1) and hand-fed TMV (Group 3) sheep. Day Total Blood Thiamine (ug/ml.) Control TMV 0 0 .042 0.033 1 0.04.7 0.041 2 0.045 0.045 3 0.051 0.046 4 0.053 0.050 5 0.049 0.037 6 0.057 0.041 7 0.055 0.034 8 0.051 0.034 9 0.048 0.031 10 0.052 0.030 TABLE XVIII. Average TPP effects in the control (Group 1) and hand-fed TMV (Group 3) sheep. TPP Effect (%) Hexose Pentose Day Control TMV Control TMV 0 16.62 5.42 10.36 9.98 1 13.71 21.27 7.65 6.78 2 14.07 18.25 9.23 25.00 3 18.23 14.35 5.04 3.17 4 18.75 25.42 8.49 14.16 5 10.26 31.16 3.84 3.23 6 11.69 19.15 7.97 7.71 7 13.43 31.13 4.25 5.88 8 18.27 24.14 8.39 7.86 9 10.34 19.12 11.57 12.06 10 11.94 21.34 10.32 9.72 44. the exposed open range is equally as toxic as the dark green, t a l l growing, broad leaved TMV found in shaded areas. A similar variation in the appear-ance of TMV under different environmental conditions has been reported by Davidson (1940). The miserotoxion level in the latter type of TMV was found to be 3.21$. Miserotoxin appeared to be confined mainly in the leaves and petioles with only small miserotoxin quantities being found in the pods, roots and flowers. Since a number of species of the genus Astragalus, are selenium accumulators, i t was necessary to establish whether the selenium content of Astragalus miser var. serotinus was high enough to induce selenium toxicity in animals. Wood (1949) has previously reported the selenium level to be 0.5 p.p.m. in serotinus. More recently, Brink and Fletcher found the selenium level to be less than 1.0 p.p.m. in serotinus sampled from various locations in B.C. These findings would classify serotinus as a non-seleniferous Astragalus, suggesting that i t is not toxic to animals by virtue of its high selenium content. 4 5 . V. TRIAL III. THE EFFECTS OF TMV ON MICE A. Introduction In order to study the metabolic fate and distribution of the toxic conditions of the TMV, extracts of the plant as well as deproteinized body fluids from rats or sheep were administered to mice as described below. B. Materials and Methods (a) Feeding Trial with Mice (1) Experimental Animals: Twenty-five female mice weighing from 25 to 30gm. each were used. (2) Rations and Feeding: Dried TMV was ground through a 40-mesh screen and offered ad libitum to the experimental mice. Control mice were given a commercial rat feed (Purina). All mice were provided free access to water. (3) Experimental Design: Four groups of 5 mice each were fed TMV while the fifth group of 5 mice were used as the control. (4) Weighing and Sampling: A record was kept of the TMV consumed by each TMV group. Blood was obtained from the heart just prior to death in one the TMV groups and stored for subsequent analysis. (b) Feeding Trial with Rats (1) Experimental Animals: Twenty-four female Wistar rats weighing from 110 to I25gm. were used. (2) Rations and Feeding: The rations were the same as those fed to the mice. Both feed and and water were provided free choice. 46. (3) Experimental Design: Eight groups of 2 each were given TMV while 4 groups of 2 rats each served as the control. (4) Weighing and Sampling: The voluntary intake of a l l twelve groups was recorded. For analysis purposes, blood was sampled from the heart of one TMV group just prior to death for comparison with the control. (c) Effects of TMV Derivations on Mice (1) Experimental Design: Six groups of 4 mice were used in this experiment. All mice were given water and a commercial rat ration free choice. (2) TMV Derivatives Used: The mice were subjected to six different treatments. Five of these groups were injected intraperitoneally with either physiological saline (control), neutralized TMV sheep rumen fluid protein free filtrate, neutralized 3-nitro-propionic acid (4mg./ml.) or neutralized protein free filtrate of blood and intestinal fluid of rats poisoned by TMV. The sixth group was orally drenched with a semi-purified water extract of TMV containing miserotoxin in which 1ml. of the solution was equivalent to 1.0gm. of dried plant material. C. Results and Discussion (1) Mouse Feeding Trial: The extent to which the mice consumed the TMV over a specific period of time was responsible for determining the poisoning capability of the TMV. An average total intake of 2.15gm. by a mouse resulted in the appearance of symjfcoms within 36 hrs. with death following within 60 hrs. from the begin-ing of the feeding. Some mice refused the TMV for as long as 8 to 12 days. However, these mice did manifest symptoms of TMV poisoning and eventually died as a result of eating approximately 2gm. of TMV. Therefore i t appears 47. that the consumption of approximately 7gm. of TMV per 100gm. body weight over a time interval of between 2 and 12 days is adequate to k i l l mice. The appearance of a mouse after the onset of TMV poisoning symptoms is shown in Figure XT.". The most apparent change was the arching of the back while the animal remained lying on its ventral side. Previous to the mani-festation of this appearance, some of the mice exhibited a loss of equilibrium whereby they rolled over completely, staggered and were generally unsteady on their feet. Body temperature dropped significantly, being as low as 15° below the normal during the latter stages of toxicity. Heart rate slowed down markedly, especially when the mice assumed their inactive state exhibiting an arched back. Internally, there were massive hemorrhages on the mucosa of the entire stomach wall. There was no apparent damage to the intestinal lining as was seen in sheep. (2) Rat Feeding Trial: Most of the rats found TMV to be very palatable and consumed i t more readily than mice. Rats showed signs of poisoning within 36 hours and died within another 24 hours. Poisoned rats exhibited the same symptoms as was described for the mice but tended to be less erratic in convulsive movements. An average intake of 1.13gm. proved to be the lethal dose of dried TMV. It appears that 1gm. of TMV per 100gm. of body weight is a lethal dose for a rat. On this basis, mice would seem to be 7-fold more resistent to TMV. Analysis of the rat blood showed that SGOT activity in TMV rats was 551.3 SGOT units per ml. of serum as compared to 94.6 SGOT units per ml. of serum in the control rats. This is definitely a significant (p<0.5) indi-cation of extensive tissue damage in the TMV rats. In addition blood serum isocritate dehydrogenase (ICD) levels were found to be 645.16 ICD units in control rats and 1,774 ICD units in the TMV rats. The isocitrate dehydrogenase activity was determined by the procedure 4-3. Figure XI. A TMV poisoned mouse. given in Sigma bulletin #175. A significant (p<Q05) increase in ICD activity as was apparent in the TMV rats is indicative of acute hepatic injury. (3) TMV Derivative Effect on Mice: The result of the six treatments on the mice is presented in Table XIX. It would appear that an agent, found in rat blood and in the rat intestine, poisons mice producing symptoms similar to those produced by either 3-NPA, extract of TMV or dried TMV. However, this agent does not appear to be found in the rumen fluid of sheep that have developed TMV poisoning. Since the mice manifested similar symptoms with the latter four treatments, i t i s possible that 3-NPA or a closely related compound is the toxic principle involved. As was suggested previously in the literature, miserotoxin could be hydrolyzed into 3-NPA under the proper conditions. The absence of toxicity in the TMV rumen fluid could be explained by the fact that miserotoxin is hydrolyzed into 3-nitropropanol (3-NPOH) in the rumen. The toxicity symptoms of 3-NPA in rats, as previously described by Matsumoto TABLE XIX, Effects of TMV derivatives on mice. Treatment Dose (ml.) Times Given Results Physiological saline (control) 0,5 TMV sheep rumen fluid protein free filtrate pH 7. 0.5 3-nitropropionic acid (4mg./ml.) (neutralized) (3-NPA) 0.5 TMV rat intestinal contents protein free filtrate pH 7 0.5 TMV rat blood protein free filtrate 0.5 3 1/day 3 1/day No effect No effect Died within 3 hours; exhibited symptoms similar to those pre-viously described for TMV poisoned rats and mice. Died within 2 hours; exhibited symptoms similar to those for 3-NPA Died within 4 hours; exhibited symptoms similar to those of 3-NPA and rat intestine protein free filtrate TMV water extract (miserotoxin) (drench) 1.0 Typical symptoms of TMV poi-5 soning developed after 3 1/2 1/day days with death resulting after 5 days of treatment 50. et al. (1961), are exactly identical with those produced in both rats and mice by TMV. Isolation of 3-NPA in the blood and intestinal contents of TMV poisoned raonogastrics would confirm the mechanism of miserotoxin hydrolysis as suggested in this experiment. 5 1 . VI. TRIAL IV. METABOLISM OF MISEROTOXIN IN THE ANIMAL BODY A. Introduction The higher susceptibility of monogastric animals to the lethal action of TMV as compared to ruminants suggested that there might be a differ-ence in the metabolic fate of the invested toxin in these two groups. This t r i a l was undertaken to investigate this difference and identify the meta-bolites of miserotoxin in the blood of poisoned animals. B. Materials and Methods (a) Metabolism of Miserotoxin in the Rat ("0 Ilk vitro Acid Hydrolysis Concentrated hydrochloric acid was used to hydrolyze a sample of a water extract of TMV prepared by the procedure of Williams and Norris (1967). Along with this solution, a glucose solution and an untreated water extract of TMV were spotted on Whatman #1 chromatography paper and run for 12 hours in a solvent system consisting of isopropanol, water and ethyl acetate (2:1:2 v/v). Spots were developed with a spray containing anisaldehyde, acetic acid and sulfuric acid (5:50:1 v/v) and examined under ultraviolet light. (2) Phenylhydrazine Derivative Formation: In order to identify the metabolites of miserotoxin in the blood and intestinal contents of rats fed TMV, phenylhydrazine derivatives were prepared from these fluids by heating them for one half hour with phenylhydra-zine as outlined by Wild (1962). Similar derivatives were formed also from the acid hydrolyzed TMV water extract, the TMV water extract, and 3-NPA. These solutions were spotted on Whatman #1 chromatography paper and run for 12 hours in a solvent system consisting of n-butanol, ethanol and 0 . 5 N 52. ammonium hydroxide (7:1:2 v/V). Spots were yellow and readily examined under ultraviolet light. (b) Metabolism of Miserotoxin in Sheep (1) In vitro Metabolism of Miserotoxin: (i) Test for the presence of primary alcoholic groups in the in vitro fermentation fluid After incubating rumen liquor for 4-8 hours with a TMV substrate as outlined in Appendix IX, a protein free filtrate was prepared with a dilute aqueous solution of potassium permanganate for ten minutes, or until the solution turned brown in color so as to detect the formation of primary alcoholic groups. (ii) Phenylhydrazine Derivative Formation After testing the protein-free filtrate of rumen liquor for the presence of primary alcohols, the solution was treated with phenylhydrazine and the derivative formed and chromatographed as previously outlined. (2) In vivo-. Metabolism: Similar tests were done for the presence of primary alcoholic groups in the blood and rumen liquor of sheep poisoned by TMV in the 1969 feeding t r i a l . Phenylhydrazine derivatives were also formed from deproteinized blood and rumen liquor of sheep fed TMV as well as from 3-NPOH, and chroma-tographed on paper as described previously. 3-NPOH was synthesized from 3-bromo-1-propanol and sodium nitrite by the procedure of Norris (1970) for use as a standard solution. C. Results and Discussion (a) Metabolism of Miserotoxin in the Rat Glucose was found to have an Rf value of 0.49 in the solvent sys-tem employed. The acid hydrolized TMV extract produced a spot having the 53. same Rf value as glucose. A similar spot was not present in the chroma-togram of the unhydrolyzed TMV extract. This suggests that under the acid conditions of the monogastric stomach glucose may be split off from the 3 carbon-1-nitro side chain of miserotoxin. Since phenylhydrazine reacts with carboxylic acid to form derivatives, 3-NPA was used as a standard in this experiment to detect its presence in the other solutions. The phenylhydrazine derivatives of 3-NPA, the acid hydrolyzed TMV extract, the protein free filtrates of intestinal contents and blood from rats fed TMV, yielded visible yellow spots having an Rf value of 0.87. The unhydrolyzed TMV extract did not yield a spot with an equivalent Rf value. The acid hydrolysis of the TMV extract yields glucose and 3-NPA from miserotoxin as shown above. It appears that a similar breakdown of miserotoxin takes place in the acidic environment of the stomach of mono-gastric animals. The demonstration of the presence of 3-NPA in the blood and intestinal contents of rats fed TMV by the chromatographic behaviour of its phenylhydrazine derivatives provides evidence that 3-NPA is absorbed from the intestine into the blood. (b) Metabolism of Miserotoxin in Sheep A primary alcohol, when heated with a dilute aqueous solution of potassium permanganate, will produce manganese dioxide which imparts ar characteristic brown color to the solution 3-nitro-1-propanol used as the standard alcohol gave a positive reaction to this test. Similar positive tests were given by protein free filtrates of blood from sheep fed TMV and rumen fluid from in vivo and in vitro tests suggesting that a primary alcohol, probably 3-NPOH, was present in these fluids. The presence of 3-NPOH in the blood and rumen fluid of sheep was further confirmed by adding acid to the manganese dioxide solutions whereby the primary alcohol would be converted to the corresponding carboxylic acid. The acid could then be identified by the formation of derivatives of 5 4 . phenylhydrazines and by paper chromatography as outlined previously. Phenylhydrazines thus prepared from the blood and rumen f l u i d of sheep fed TMV produced spots on paper chromatograms with an Rf value of 0 . 8 3 . This corresponded to the Rf values of phenylhydrazine d e r i v a t i v e s prepared e i t h e r d i r e c t l y from 3-NPA or i n d i r e c t l y from 3-NPOH. The Rf values were a l s o s i m i l a r to those found from the blood and i n t e s t i n a l contents of monogastric animals. On the basis of these chromatographic fin d i n g s , i t appears that miserotoxin i s a c i d hydrolyzed i n t o glucose and 3-NPA i n the stomach of the monogastric animals. The 3-NPA i s absorbed i n t o the blood stream producing TMV t o x i c i t y symptoms. On the other hand, i n ruminants miserotoxin i s hydro-lyzed by the ac t i o n of the rumen m i c r o f l o r a to glucose and 3-NPOH (Williams, Van Kampen and Norri s , 1 9 6 9 ) . The 3-NPOH which i s absorbed i n t o the blood appears to be responsible f o r causing t o x i c i t y . 55 VII. CONCLUSIONS In the 1968 TMV feeding t r i a l (Trial I) conducted in the U.B.C. sheep unit, the symptoms observed in the sheep fed 100$ TMV were similar to those described by Bruce, 1912. However, a l l the sheep fed 100$ TMV did not suffer from the toxicity. This may be ascribed to the fact that the misentoxin content dropped from 2.3 -3.4-$ in fresh cut TMV to 1.65$ in the sun dried vetch and 0.9$ in the pelleted samples, which were actually fed to the sheep. The characteristic symptoms of TMV poisoning in sheep were backward flexion of the fetlock - joints of the hind limbs, occasional kneeling on the front legs and inco-ordination of movements. Grinding of teeth and breathing with a roaring noise were other symptoms observed. The presence of extensive intestinal ulceration has been found to be characteristic of TMV toxicity in sheep. A severe fatty infiltration and hemorrhage observed in the liver and kidney suggested that the toxic compound i s excreted by these organs. There was no evidence of a thiamine deficiency per .§§, indicating that the therapeutic value of thiamine for TMV.poisoned sheep (Nicholson, 1963) may be indirect, probably through a general improvement in the metabolism of the animal. The 1969 TMV feeding t r i a l in the Kamloops region (Trial II) resulted in sheep developing TMV poisoning symptoms after consuming fresh TMV with a miserotoxin content of 3.14$. Paralysis of front and hind limbs was found to be the most common symptom which was very noticeable when the 56 sheep were excited and exerted. A complete manifestation of symptoms, as was seen in the 1968 t r i a l , was not evident in the 1969 t r i a l . This is partly because the duration of the feeding was not long enough and the t r i a l had to be terminated, the TMV having already matured to the pod stage with a low miserotoxin content. The intestinal ulcers in the sheep poisoned with TMV were confined mainly to the jejunal region. A significant elevation (p<.05) in SGOT activity was observed, suggesting tissue damage in the TMV poisoned sheep. However, the creatine phosphokinase activity did not show abnormal values which indicated the absence of destruction of central neaous tissue. The TMV poisoned sheep were found to have a normal thiamine status with no evidence of deficiency. Upon feeding TMV to rats and mice, massive hemorrages developed on the mucosa of the entire stomach. There was also a loss of body equilibrium with convulsive movements finally ending in an inactive state in which the animals exhibited an arched - back appearance. A lethal dose of TMV appeared to be 1 gm per 100 gm of body weight for mice. Both SGOT and ICD activity were elevated indicating the existence of muscle and hepatic tissue damage respectively. By comparing the chromatographic behaviour of phenylhydrazine derivatives of 3 - NPA with that of similar derivatives prepared from the blood and intestinal contents of rats and mice fed TMV, i t was concluded that miserotoxin was hydrolyzed under the acid conditions of the stomach to 3 - NPA. The 3 - NPA so produced appears to be responsible for the toxicity in the monogastric animals fed TMV. 57. However, in sheep, miserotoxin was found to be hydrolyzed by microbial action in the rumen into glucose and 3 - NPOH. This was confirmed by demonstrating the presence of primary alcoholic groups in blood in rumen fluid of sheep fed TMV and also in fluids obtained from in vitro fermentation tests. The 3 - NPOH formed in the rumen contents and absorbed into the blood of sheep fed TMV appears to be responsible for the toxicity. 58. VIII. BIBLIOGRAPHY 1. Albanese, A.A. (1967). "Newer Methods of Nutritional Biochemistry" Volume III p.p. 436 - 44-3 Academic Press (New York, London). 2. Barneby, R.C. (1964). Atlas of North American Astragalus. Memoirs of the New York Bot. Garden Vol. 13_ p. 1188. 3. Bergmeyer, H.U. (1965). "Methods of Enzymatic Analysis". 2nd Edition p.p. 842 - 853 Academic Press (New York, London). 4. Brin, M.j Tai, M.j Ostashever, A.S. and Kalinsky, H. (i960). The Effect of Thiamine Deficiency on the Activity of Erythrocyte Hemolysate Transketolase. J. Nut. 71 273 - 81. 5. Brink, V.C. and Fletcher, K. (1968). Note on the content of certain trace elements in range forages from south central B.C. Personal communications. 6. Brooks, M.M. (1934). Inhibition by Glucose of Methemoglobin Formation. Proc. Soc. Exp. Biol. Med. 32 63 - 68. 7. Bruce, E.A. (1927). Astragalus campestris and Other Stock Poisoning Plants of British Columbia. Canada Dept. of Agriculture Bulletin No. 88. 8. Bruns, F.H. (1959). Uber den Stoffwecheel von Ribose - 5 - Phosphat in Hamolysaten. Folia haemat. 76 317 - 330. 9. Buck, W.B.; James, L.F. and Binns, W. (l96l). Changes in Serum Transaminase Activities 1 Associated With Plant and Mineral Toxicity in Sheep and Cattle. Cornell Vet. J51 568 - 85. 10. Buckeridge, F.A. (1965). Toxicology and Structure of a Poisonous Principle from Astragalus miser. Ph.D. Dissertation Utah State University. 11. Buffey, V. (1969). Personal communications. 12. Case, A.A. (1957). Some Aspects of Nitrate Intoxication in Livestock. J. Am. Vet. Med. Assn. 13.0 323 - 327. 13. Cooke, A.R. (1955). The Toxic Constituent of Indigofera endecaphylla. Arch. Biochem. j>5_ 114 -120. 14. Crampton, E.W. and Maynard, L.A. (1938). The Relation of Cellulose and Lignin Content to the Nutritive Value of Animal Feeds. J. Nutr. 15_:383 - 386. 15. Davidson, J.F. (1940). A revision of the Genus Astragalus in B.C. Master of Arts Thesis, Dept. of Botany U.B.C. (April). 59. 16. Diven, E.H.; Pistor, W.J.; Reed, R.E.; Trautman, E.J. and Watts, R.E. (1962). The Determination of Serum or Plasma Nitrate and Nitrite. Am. J. Vet. Res. 23_:497 - 499. 17. Dreyfus, P.M. (1962). Clinical Application of Blood Transketolase Determinations*New Eng. J. Med. .267 596 - 598. 18. Evelyn, K.A. and Malloy, H.T. (1938). Microdetermination of Oxyhemoglobin, Methemoglobin, and Sulfhemoglobin in a Single. Sample of Blood. J..Biol. Chem. 126:655-662. 19. Gorter, K. (1920). Hiptagenic acid, the Toxic Principle of Hiptage benghalensis. Chem. Abstr. 1^ :1299 - 1305 (l92l). 20. Guyton, A.C. (1966). "Textbook of Medical Physiology" p.p. 109 - 113. W.B. Saunders Co. Philadelphia and London. 21. Gyorgy, P. and Pearson, H. (1967). "The Vitamins" 2nd Edition Academic Press (New York) Volume VII p.p. 69 - 82. 22. Hutton, E.M.; Windrum, G.M. and Kratzing, CC. (1958). Studies on the Toxicity of Indigofera endecaphylla. I. Toxicity For Rabbits. J. Nut. 6^ :321. - 38. II. Toxicity For Mice. J. Nut. 6J5:429 - 440. 23. Lucas, A.M. (l96l ) i "Atlas of Avian Hematology". Agriculture Monograph 25. p.p. 231 - 232. U.S.D.A. Washington, D.C. 24. MacDonald, M.A. (1952). Timber Milkvetch Poisoning on British Columbia Ranges. J. of Range Management 5.:l6 - 21. 25. Matsumoto, H.; Hylin, J.W. and Miyahara, A. (l96l). Methemoglobinemia in Rats Injected with 3 - Nitropropionic Acid, Sodium Nitrite, and Nitroethane. Toxicology and Applied Pharm. 3_:493 - 499. 26. Morris, M.P.j Pagan, C. and Warmke, H.E. (1954). Hiptagenic Acid, A Toxic Component of Indigofera endecaphylla. Science 119:322 - 323. 27. Nicholson, H.H. (1963). The Treatment of Timber -Milkvetch Poisoning Among Cattle and Sheep. Can. J. of An. Sc. 4J: p. 237 - 240. 28. Norris, F.A. (1970). Personal communication. 29. Official Methods of Analysis of the Association of Official Agricultural Chemists. 9th Edition (William Horwitz Ed.) Washington, D.C. (i960). 30. Oser, B.L. (1965). "Hawk's Physiological Chemistry". 14th Edition p. 1031. McGraw - Hi l l Book Co. (Toronto). 60. 31. Smith, J.B. and Healy, P.J. (1968). Elevated Serum Creatine Phosphokinase Activity in Diseases of the Central Nervous System in Sheep. Clin. Chim. Acta 21:295 - 296. 32. Smith, Q.T. and Allen, R.S. (1954). B - Vitamin Levels in the Blood of Young Dairy Calves Fed a Milk Replacement Diet with Aureomycin and Without. J. Dairy Sc. 27:1190 - 1197. 33. Stermitz, F.R.; Norris, F.A. and Williams, M.C. (1969). Misertoxin, a New Naturally Occurring Nitro Compound. J. Am. Chem. Soc. 9JL:4599 - 4560. 34. Wild, F. (1962). Characterisation of Organic Compounds. Second Ed. Cambridge University Press, p. 152. 35. Williams, M.C. (1969). Personal Communication. 36. Williams, M.C. and Binns, W. (1967). Toxicity of Astragalus miser Dougl., var. oblongifolius (Rydb.) Cronq. Weeds 15_:359 - 362. 37. Williams, M.C. and Norris, F.A. (1969). Distribution of Miserotoxin in varieties of Astragalus miser Dougl. ex Hook. Weed Sci. 17:236 - 238. 38. Williams, M.C; Norris, F.A. and Van Kampen (1970). Metabolism of Miserotoxin to 3 - Nitro - 1 - Propanol in Bovine and Ovine Ruminal Fluids. Am. J. Vet. Res. 3JL:259 - 262. 39. Williams, M.C.j Van Kampen, K.R. and Norris, F.A. (1969). Timber Milkvetch Poisoning in Chickens, Rabbits, and Cattle. Am. J. Vet. Res. 3J>:2185 - 2190. 40. Willis, T.G. (1949). "Timber Milkvetch Poisoning", p.p. 40 - 42. G.D.A. Range Station, Kamloops, B.C. Progress Report (1947 - 1953). 41. Wood, A.J. (1949). Timber Milkvetch Report. Dept. of Animal Science. University of B.C., Canada. 4.2. Wroblewski, F. and LaDuc, J.S. (1955). Serum Glutamic Oxalacetic Transaminase as an Index of Liver Cell Injury. J. Clin. Invest. 3^ :973-976. 61. IX. APPENDICES 62. APPENDIX I. DETERMINATION OF HEMOGLOBIN (LUCAS, 196l) To 10ml of 0.1$ ammonium hydroxide in a test tube, 0.02ml of heparinized whole blood was added. After adding 0.33ml of hydro-chloric acid (concentrated) the solution was mixed on a Vortex mixer. The absorbance of the solutions were read in a Bauch and Lomb Spectronic 20 at 410 up against a distilled water blank. All absorbance readings were converted into gm hemoglobin / 100ml of blood, (Table XX). APPENDIX II. PREPARATION OF PROTEIN FREE FILTRATES OF WHOLE BLOOD AND SERUM (OSER, 1965) One volume of blood was laked with 7 volumes of distilled water. To this was added 1 volume of 10$ Zn SO,; . 7 H 2 O . After mixing, 1 volume of 0.5N NaOH was added with continuous shaking. The mixture was then centri-fuged and the supernatant decanted for further use. In the same way, 1 volume of serum of plasma was laked with 8 volumes of H2O and 0.5ml each of 10$ ZnSO^  . 7H20 and 0.5N NaOH. APPENDIX I H . DETERMINATION OF NITRITES AND NITRATES IN SERUM (DIVEN et al., 1962) Procedure (a) Nitrite One ml of protein-free filtrate of serum was mixed with 1ml of 63. 0.2$ sulfanilamide in 20$ HC1 in a test tube. After mixing, 1ml of 0.02$ N - ( l - naphthyl) - ethylene diamine dihydrochloride was added and the solution was made up to 10ml with HoO and mixed. After 10 minutes the solution was read spectrophotometrically at 520 mu against a blank using HgO instead of 1ml of protein - free filtrate. (b) Nitrate The procedure is the same as used for nitrite except that copper (1.5 p.p.m.) and powdered zinc were added to the protein - free filtrate. Zinc was added to the protein - free filtrate with intermittent shaking until the solution turned gray. Standard curves were prepared using sodium nitrate (5 - 100 p.p.m.) as substitutes for the 1ml of protein - free filtrate. The standard curves for nitrate and nitrite are taken from Table XXI which relates nitrogen concentration to absorbance. APPENDIX IV. MICR0DETERMINATI0N OF METHEMOGLOBIN (EVELYN et al.,' 1938) Procedure Heparinized blood (O.lml) is mixed with 10ml of M/60 phosphate buffer, pH 6 .6 in a cuvette. After 5 minutes, the solution is read spectrophotometrically at 635 mu against a blank tube containing water. The absorbance is recorded as L]_. A drop of neutralized 10$ aqueous sodium cyanide in 12$ acetic acid is mixed with the contents in the cuvette and after 2 minutes a second reading L2 is taken. 64 Calculations The difference (Lj - L 2) is proportional to the methemoglobin concentration. Applying the difference,' the following equation describes methemoglobin determination: M = 100 (L r - L 2) 2.77 where M is equivalent to grams of methemoglobin per 100ml of blood. APPENDIX V. DETERMINATION OF SERUM GLUTAMATE - OXALOACETATE TRANSAMINASE (SGOT) (BERGMEYER, 1965) To 0.2ml of serum, lml of a substrate - buffer solution containing 0.1 M phosphate buffer, pH 7.4? 0.1 M L - aspartate; 2 X 10-3 M o t ' - oxogluterate was added. The solution was mixed by inversion and incubated at 37 degrees C for 60 minutes. One ml of ketone reagent (20mg 2,4 dinitrophenylhydrazine dissolved in 100ml of IN HCl) was added to the tube to terminate incubation. After 20 minutes at room temperature, 10ml of 0.4N NaOH were added. The solution was read spectrophotometrically at 530 mu against a blank In which ketone was added before the serum to denature transaminase activity. A standard curve was prepared by using difference dilutions of 0.002 M sodium pyruvate in the above method in place of the serum. Glutamate - oxaloacetate transaminase activity (SGOT units per ml of serum) is shown in Table XXII. 65. TABLE X X . Gn$ hemoglobin at different absorbance values. Absorbance Gm% Hemoglobin Absorbance Csa% Hemoglobin .323 7.84 .500 12.28 .364 8.89 .550 13.53 .368 9.00 .600 14.78 .400 9.78 .650 16.03 .450 11.03 TABLE X X I . Nitrogen concentration,at respective absorbances. Absorbance Nitrogen Concentration (~~m. nitrogen) Nitrite Nitrate (HBPI ) ( B E P O 0 0 0 0 0.080 0.3188 10 15 0.245 0.9564 30 40 0.420 1.5940 50 75 0.575 2.2316 70 105 0.740 2.8692 : 90 135 66. TABLE XXII. SGOT activity as derived from standard solutions. Test Tube Sodium Pyruvate (0.002M)(ml) Buffer-Substrate Solution (ml) *SG0T units Absorbance per ml of serum 1 0 1.0 0 0 2 0.05 0.95 21 0.052 3 0.10 0.90 42 0.105 4 0.15 0.85 64 0.152 5 0.20 0.80 97 0.197 6 0.25 0.75 140 0.240 * One unit of SGOT i s defined as that amount of the enzyme that will catalyze 1 p. mole of substrate per minute under the conditions of the assay. APPENDIX VI. DETERMINATION OF SERUM GLUTAMATE - PYRUVATE TRANSAMINASE DETERMINATION (BERGMEYER,1965) The substrate - buffer solution consisted of the following ingredients: 1.5gm KgH PO^ , 0.2gm KH2 PO^ , 0.03gm o( - oxoglutaric acid and 1.78gm DL - alanine in 100ml of distilled water. The pH was adjusted to 7.4 before making up the final volume. The procedure for individual serum determinations was the same as for the SGOT test except that the incubation time was 30 minutes for SGPT. A standard curve was prepared using different dilutions of the 0.002 sodium pyruvate solution with the SGPT buffer - substrate solution. SGPT activity (GPT units per ml of serum) is presented in Table XXIII. 67. TABLE XXEII. SGPT activity as derived from standard solutions. Test Tube Sodium Pyruvate (0.002M)(ml) Buffer-Substrate Solution (ml) *SGPT units Absorbance per ml of serum 1 0 1.0 0 0 2 0.1 0.9 27 0.105 3 0.2 0.8 57 0.193 4 0.3 0.7 95 0.275 5 0.4 0.6 137 0.344 6 0.5 0.5 205 0.420 ^Wroblewski units - One unit of SGPT is defined as that amount of the enzyme that will catalyze 1 u mole of substrate per minute under the conditions of the assay. APPENDIX VII. DETERMINATION OF BLOOD THIAMINE IN MOLE BLOOD (GYORGY AND PEARSON, 1967) Procedure One ml of fresh heparinized blood was placed in a 25ml centrifuge tube containing 4ml of distilled water, shaken and left standing for 10 minutes. This mixture was centrifuged (l600g) for 10 minutes after which the supernatant was decanted and the centrifugation repeated. The super-natant was drawn off and retained. The remaining centrifugal residue was suspended in 2.5ml of 2% trichloroacetic acid, centrifuged and the supernatant 68. added to the other supernatants previously extracted. To the combined supernatant, contained in a test tube, was added 1ml of 2 . 5 Ml sodium acetate followed by shaking, and the pH adjusted to 5.1 - 5.2. After the addition of 0.5ml of aqueous human prostate phosphatase (Sigma Chemical Co.) (2mg/ml) to the solution, i t was mixed and incubated for 12 hours at 37 degrees C. in a water bath. After incubation, 0.25ml of the enzyme - treated extract was added to 0.4.8ml of 20$ alkaline potassium ferricyanide solution to oxidize the solution. After 15 seconds of immediate and vigorous mixing, 0.48ml of 5 . 5 M sodium dihydrogen phosphate in 30$ hydrogen peroxide was introduced to reduce the solution. The thiochrome formed was immediately extracted into 4-.8ml of n - hexyl alcohol by vigorous mixing. The solution was centrifuged for 5 minutes, and the top alcohol layer (at least 4ml) was transferred quickly to a fluorometer and the fluorescence read in a Turner fluorometer. The fluorescence measured was recorded as % from the fluorometer set at range 3 with 47B and 2A secondary filters and a 7-60 primary f i l t e r . A f o i l covered cork was then used to seal the fluorometer tube before placing i t on a rack in a cabinet equipped with a mercury light source. The tube was left for 2 hours at a distance of three inches from 2 ultraviolet lamps of wavelength 2537 A degrees and 3650 A degrees. This treatment destroyed the thiochrome in the solution so that a second reading R2 was taken for the solution in the fluorometer. Standard solutions of thiamine ( . 0 5 and 1.0 ng/ml) were concurrently subjected to the same treatment as the unknown samples. 6 9 . Calculations With the particular ultraviolet lamp source, i t was necessary to determine the time required to destroy a l l the thiochrome in the test and standard solutions. Therefore a preliminary test was conducted using lug/ml thiamine solutions in order to find a time value when (R^ - R2) would be maximal. The results of this test are shown in Table XXIV. TABLE XXIV. The effect of time on thiochrome destruction by UV light. Sample Fluorometer Reading - (Range 3) - - - ' 0.05ug Time 0 R9 , D thiamine * ' ' • • • • • • • 1% _ R 2 ) per ml (R3.) 3 hours 2 hours 1 hour 0.5 hour 1 76 21 — — 55 2 81 — 2 6 — — 55 3 78 — — 33 — 45 4 81 — — — 48 33 Therefore, the minimal length of time required for destroying thiochrome by UV light treatment was 2 hours, which was used in this procedure. The thiamine in whole blood was calcuated as follows: ug thiamine/ml in standard A . ug thiamine/lOOml = X M X 100 u l i blood in sample B Where: Change of fluorescence of sample after irradiation. Change of fluorescence of thiamine standard after irradiation. of blood (% - R2) = A = (Bx - S2) = B = 70. APPENDIX VIII. DETERMINATION OF THIAMINE PYROPHOSPHATE (TPP) ACTIVITY IN THE ERYTHROCYTES (ALBANESE,*1967) Procedure The hemolysate was prepared by adding one volume of water to one volume of red blood cells previously separated by centrifuging heparinized blood and removing the plasma and buffy coat. (a) Incubation procedure: The protocol for the assay is outlined in Table XXV. TABLE XXV. Details of incubation for the assay of TPP activity. Tube Hemolysate B-Buffer TPP soln. Incubation Ribose Incubation 7.5$ (ml) (ml) (ml) time (min.) -5- time (min.) TCA at 38«C. Phos- at 38°C. (ml) phate (ml) A 0.5 0.45 30 0.2 60 6 B 0.5 T - 0.45 30 0.2 60 6 D 0.5 0.65 — — 6 R 0.5 (saline) (0.09$) 0.45 — 0.2 — 6 Tube A i s the hemolysate without the addition of TPP; Tube B contains hemolysate with added TPP while tube D i s a blank which i s used to determine the amount of hexose and/or pentose endogenous to the sample. 71 Tube R contains the substrate and i s used as a basis for determining the ribose utilized. After addition of 7.5$ trichloroacetic acid (TCA) and mixing, a l l four tube contents were centrifuged and the protein - free filtrates retained. These protein - free filtrates were used to determine the hexose formed (Anthrone method) and the pentose utilized (Orcinol method), (b) Determination of hexose: Hexose was determined by the Anthrone method using the protocol outlined in Table XXVI. TABLE XXVI. Determination of hexose by the Anthrone method. Tube Protein-free Hexose 7.5$ TCA Anthrone (cold) filtrate (ml) (lOOuglral) (ml) (ml) All reaction 1.0 10 tubes D - glucose 0.5 0.5 10 50 ug D - glucose 1.0 10 100 jig Blank 1 10 All.tubes were mixed and placed in a boiling water bath. At the end of 10 minutes, the tubes were cooled and read spectrophoto-metrically at 620mu.. (c) Pentose determination of pentose: Pentose was determined by the Orcinol method (Table XXVII). 72. TABLE XXVII. Determination of Pentose by the Orcinol method. Tube Protein-free filtrate(ml) Pentose (lOuglml) Water (ml) Orcinol Solution (ml) A,B and D 0.2 — 1.3 4.5 R 0.1 — 1.4 4.5 D - ribose (5 ug) 0.5 1.0 4.5 D - ribose (10 ug) 1.0 0.5 4.5 Blank 1.5 ...... 4.5 . . . . . All the tubes were mixed, and left in a boiling water bath for 20 minutes. After cooling, the tubes were read spectrophotometrically at 670mu. (d) Calculation: (i) Hexose ml of hexose formed = (ml hemolysate used X (ml of solution per incubation tube) X (ml of filtrate used) or (1/0.5 X (7.15/1) X l/l.O) = 14.3 Average absorbance per ug of hexose = (Absorbance of 50ug std.+Absorbance of lOOug std.) (SH) = 2 Since the dilution factor and absorbance /ug of hexose are constant for each tube. 11,3 X SH = KH (constant) Absorbance for tubes A," B and D respectively used in the calculation of the TPP effect as follows: 73. TH-L = ng hexose/ml hemolysate/hour without TPP = (A-D) X KH TH2 = ug hexose/ml hemolysate/hour with TPP = (B-D) X KH TPP effect = TH2 , - THi X 100 TH]_ (fO Pentose ml of pentose utilized = (ml of hemolysate used) X (ml of solution per incubation tube) X (ml of filtrate used) or (1/0.5) X (7.15/1) X (1/0.2) = 71.5 Average absorbance per = (Absorbance of 5ug std. + Absorbance ug of Pentose = (SP) of lQnk std.) Since the dilution factor and absorbance/ng of pentose are constant for each tube: 71.5 = KP (constant) SP Using this constant the TPP effect i s calculated as follows: (2R + B) = amount of pentose in the tubes before incubation TPn = ug pentose used/ml hemolysate/hour without TPP 1 = X & + D) - A X KP TP 2 = ug pentose used/ml hemolysate/hour with TPP = (2R + D) - B X KP TPP effect = TP 2 . - . T P 1 ; TPT X 100 Reagents: (i) B - buffer 40ml 0.9$ NaCl + 1030ml 1.15$ KC1 200ml 1.75$ KH^O^ + 10ml MgSO^  (pH7,A) 74. (a) TPP = thiamine pyrophosphate 1 volume (img TPP/ml solution) + 8 volumes B-buffer (iii) Substrate (Ribose - 5 - phosphate) Contains 7mg of ribose equivalent/ml. (iv) Anthrone Reagent 0.5gm anthrone + lOgm thiourea + 1000ml 66$ HgSO^  (v) Orcinol Reagent 4gm orcinol + 0.2gm ferric chloride + 100ml H20 + 1900ml 30$ HC1 APPENDIX IX. IN VITRO RUMEN FERMENTATION TECHNIQUE (a) Fermentation vessels and conditions: Erlenmeyer flasks (125ml) with rubber stoppers, fitted with bunsen valves were used as fermentation vessels. The incubation was carried out in a 39.5 degree C. drying oven for 48 hours. (b) Medium: To 1000ml of phosphate buffer, pH 6.5 were added: 3.5gm + Ka HCO3 + 0.75gm KC1 + 0.75gm NaCl + 0.15gm MgSO^  + 2gm urea (c) Procedure: To the fermentation flask, 25ml of mineralized buffers were added along with lgm of substrate, being either alfacel or TMV. Rumen fluid was obtained from a fistulated steer maintained on alfalfa hay and 75 strained through 8 layers of cheesecloth. The fluid was added at the rate of 20ml per flask and the flask contents were then gassed with CO2 to maintain anaerobic conditions. The flaScs were then tightly stoppered and fermentation allowed to continue for AS hours. The fermentation was terminated with the Somogyi - Shaffer - Hartman precipitating agent. The fermentation mixture was then centrifuged for 20 minutes at 3000 igp.and the residue retained for cellulose determination. APPENDIX X. EXTRACTION OF MLTEEROTOXIN FROM TMV (WILLIAMS AND NORRIS, 1967) Approximately 40gm of ground (40 - mesh) TMV were weighed into 1 a round - bottom medium porosity, A5 X 127mm Alundum extraction thimble. This was extracted with 200ml of 95$ ethanol on a Soxhlet extractor for 24. hours. The extract was cooled for 1 hour, centrifuged and the residue discarded. The supernatant was chilled for 8 hours at A.5 degrees C. and then filtered, rinsing several times with cold 95$ ethanol. This filtrate was reduced to near dryness in a rotary evaporator and then brought to 150ml with distilled water. After filtration, the residue was discarded while the aqueous fraction was extracted with several volumes of chloroform in a 500ml separatory funnel. The chloroform fractions were discarded and the aqueous phase again concentrated in a rotary evaporator to expel traces of ethanol and chloroform. The final volume of the aqueous solution was adjusted so that 1ml of solution equals lgm of the dried plant material. 76 APPENDIX XI. QUANTITATIVE DETERMINATION OF MISEROTOXIN IN TMV (WILLIAMS AND NORRIS, 1969) 0.5gm of dried ground 4-0 - mesh TMV were mixed with 10ml of IN HCl in a test tube. This mixture was allowed to extract at room temperature with frequent stirring for 2 hours. The solution was centrifuged and 1ml aliquots of the supernatant were pipetted into each of the 3 test tubes. One ml of 20$ KOH was added to each tube and the stoppered tubes were allowed to set overnight at room temperature. Five ml of distilled water were added to each test tube. Two ml of glacial acetic acid was added to one of the tubes which served as the blank. One ml of glacial acid, followed immediately by 1ml of Griess - Hosvay's reagent (0.5gm sulfanilic acid in 150ml 2N acetic acid and O.lg of 1 - naphthalamine HCl in 20ml distilled H20 and 150ml 2N acetic acid prior to using) were added to the other two test tubes. The color was allowed to develop for 20 minutes and then the solution was centrifuged for A minutes at 1500 R.P.M. Solutions were read spectr©photometrically at 530mu and the concentration of miserotoxin was computed from the standard curve for pure miserotoxin, (Table XXVIII) (Williams, 1969). 77. TABLE XXVIII. Miserotoxin concentration (mg/50mg plant material at various absorbance levels. Absorbance Miserotoxin (mg) Absorbance Miserotoxin (mg) 0.010 0.10 0.288 1.05 0.019 0.15 0.311 1.10 0.028 0.20 0.334 1.15 0.051 0.30 0.358 1.20 0.074 0.40 0.384 1.25 0.099 0.50 0.410 1.30 0.111 0.55 0.435 1.35 0.124 0.60 0.460 1.40 0.150 0.70 0.485 1.45 0.163 0.75 0.510 1.50 0.176 0.80 0.535 1.55 0.198 0.85 0.560 1.60 0.220 0.90 0.585 1.65 0.242 0.95 0.610 1.70 0.265 1.00 0.635 1.75 7 8 . APPENDIX XII. TABLE XHX. Individual responses of the 100$ TMV group (sheep 51, 52, 65 and 73) in the 1968 feeding t r i a l . Sheep 51 52 65 73 When feeding began day 1 day 1 day 1 day 1 When feeding ended day 11 day 22 day 30 day 30 Duration of feeding (days) 10 22 30 30 Total feed intake (lb) 8 11.25 95 76.5 Average daily gain (lb/day) -1.0 -1.0 0.63 0.37 Daily feed intake (lb) 0.8 0.A3 3.17 2.57 Feed efficiency (lb feed/lb gain) 5.0 7.0 When animals died or were killed died killed killed killed Sheep #51 This sheep did not readily consume i t s feed which was supplied ad libitum and exhibited extensive intestinal ulceration. Otherwise, this animal died without showing any grossly apparent TMV poisoning symptoms. Sheep #52 There was also extensive intestinal ulceration in this animal. Total feed consumption occurred during the first 10 days, after which the animal ate nothing. This animal exhibited the typical external symptoms of TMV poisoning as described by Bruce (1927). That i s , i ts actions were inco-ordinated; hind legs spread; foot below fetlock-joint knuckled and bent backwards on both legs so that the joints directly contacted the ground; 79. standing on backward extended forearms; gritting of teeth regularly with extensive frothing and foaming at the mouth; emaciated condition; respira-tion at L4/minute; pulse at 84/minute. All external symptoms were fi r s t apparent on Day 15. On Day 23, blood appeared very dark in color and clotted immediately in oxalate. Sheen #65 Feed was eaten constantly over the feeding period with no desire to refuse the feed. Feces appeared to be always soft ( not liquid) and to be excreted as a solid mass rather than as the typical marble form. Upon dissection, the liver appeared to be slightly enlarged and the internal organs, especially the heart, was infiltrated with fat, while the kidneys and intestines were surrounded by extensive fat depositions. Heart rate was l68/minute with respiration at l^/minute. There was some ulceration in the rmial 1 intestine. Sheep $73 Feed was consumed regularly over the experimental period. This sheep appeared healthy and thrifty throughout the duration of feeding. Pulse rate was l60/minute and respiration was lVminute. A l l of the sheep fed the 100$ TMV ration exhibited necrosis of the kidney (Figure XII). 80. FIGURE XII. Hematoxylin—eosin section of TMV poisoned sheep kidney t i s s u e e x h i b i t i n g necrosis (X400). 

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