TOXIC and THERAPEUTIC EVALUATION OP STILBAMIDINE and HYDROXYSTILBAMIDINE in CANINE BLASTOMYCOSIS DISSERTATION Presented in Parti

TOXIC AND THERAPEUTIC EVALUATION OP STILBAMIDINE AND HYDROXYSTILBAMIDINE IN CANINE BLASTOMYCOSIS

DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy In the Graduate School of The Ohio State University

ANDRE""LAGACE', D.V.M.

The Ohio State University 1957

Approved by:

7?. Adviser Department of Veterinary Pathology ACKN OWLEDGMESTTS

To Dr* C.R. Cole, chairman of the Department of Veteri nary Pathology at Ohio State University, I express my most sincere gratitude. Without his guidance, encouragement and understanding, it would have been impossible to ac complish this work. To the Quebec Agricultural Research Council, I am very grateful for their generous scholarship. To Dr G. Labelle, dean of the Quebec School of Veteri nary Medicine, I extend my thanks. To the techrJ cal personnel of the department I am very grateful. To my wife, Jeannine, for her encouragement, comfort and understanding during those years of studies, I will never be able to put into word3 all my appreciation.

ii TABLE OP CONTENTS Page INTRODUCTION AND OBJECTIVES ...... 1 LITERATURE R E V I E W ...... 1|. Stllbamidine and hydroxystllbamidine Physical and chemical p r o p e r t i e s ...... k Pharmacologic action and toxicity ...... 7 Other pharmacologic effects ...... 2J+ Mechanism of action ...... 2ij. Fungistatic properties ...... 27 Treatment of blastomycosis...... 30 TOXICITY OF STILBAMIDINE AND HYDROXYSTILBAMIDINE FOR D O G S ...... 33 Objectives ...... 33 Materials and Methods ...... 33 Results ...... 35 A) Pilot injection of stllbamidine ..... 36 B) Effects of high doses of stllbamidine . . 1+3 C) Effects of low doses of stllbamidine . . . 53 D) Effects of high doses of hydroxystil- b a m i d i n e ...... 66 E) Effects of low doses of hydroxystil- b a m i d l n e ...... 71+ F) Schedule modification and therapeutic trial using dosage of dlamldlnes .... 79 G) Stllbamidine and hydroxystllbamidine for the treatment of canine blastomycosis • • 67 THE PATHOGENESIS OF STILBAMIDINE AND HYDROXYSTIL BAMIDINE TOXICITY ...... 91 Objectives ...... 91 Materialsj Methods and R e s u l t s ...... 91 H) Delay in toxicity after fatal dosage of diami di n e s ...... 91

iii iv

Page I) Effect of concurrent administration of corti sone (10 mg/kg) and diamidines...... 98 J) Effect of histamine phosphate ...•••• 109 K) Effect of concurrent administration of anti histamine and diamidines ..••••.. 110 L) Comparison of thiamine deficiency in cats and dogs with diamidine toxicity in dogs • llij. EFFECTS OF STILBAMIDINE AND 2-HYDR0XYSTILBAMIDINE ON EXPERIMENTAL CANINE BLASTOMYCOSIS...... 135 Objectives ...... 135 Materials and Methods 135 Results ..... Ikl M) Dogs inoculated intratracheally with 0.25 ml of packed cells ...... li+l N) Dogs inoculated intratracheally with 0.£0 ml of packed cells ...... lij.6 0) Dogs inoculated intravenously with 0.25 ml of packed cells ...... 168 P) Dogs inoculated intravenously with 0.1 ml of packed c e l l s ...... 181 Q) Dogs inoculated subcutaneously with 1.0 ml of packed yeast ...••••...... 187 DISCUSSION OF TREATMENT OF BLAST0MTC0SIS ...... 201 SUMMARY AND CONCLUSIONS ...... 211

BIBLIOGRAPHY 220 LIST OF TABLES

Table Page 1. RESUME OF ALL TEST ANIMALS FOR STILBAMIDINE TOXICITY ...... 1*1 2. RESUME' OF ALL TEST ANIMALS FOR HYDEOXYSTIL- BAMIDINE TOXICITY ...... 1|2 3. EFFECTS OF HIGH DOSES OF STILBAMIDINE ......

I4 . EFFECTS OF LOW DOSES OF STILBAMIDINE...... %. 5. EFFECTS OF HIGH AND LOW DOSES OF HYDROXYSTIL- BAMIDINE ...... 67

6 . SCHEDULE MODIFICATION OF A HIGH DOSAGE OF THE DIAMIDINES...... 80

7. STILBAMIDINE REGIMEN ...... 8 8

8 . HYDROXYSTILBAMIDINE R E G I M E N ...... 90 9. LIST OF ALL TEST ANIMALS FOR STUDY OF PATHOGENESIS ...... 92 10. DELAY IN TOXICITY AFTER FATAL DOSAGE OF DIAMIDINES 93

1 1 . CONCURRENT ADMINISTRATION OF CORTISONE (10 mg/kg) AND DIAMIDINES...... 100 12. CONCURRENT ADMINISTRATION OF ANTIHISTAMINE (10 mg/ kg) AND DIAMIDINES ...... 112 13. THIAMINE DEFICIENCY IN CATS AND DOGS ...... 120 llj.. SUMMARY OF ROUTE AND DOSE OF B^ PER MAT IT IDIS AND TREATMENT ...... 1 3 9 l^. CLINICAL SUMMARY OF DOGS INFECTED I.T. WITH 0.5 ML YEAST ...... II4.7 16. CLINICAL SUMMARY OF PRETREATED DOGS INFECTED I.T. WITH 0.5 ML Y E A S T ...... 156 17. COMPARISON OF UNTREATED, STILBAMIDINE AND HYDROXY- STILBAMIDINE TREATED DOGS INFECTED I.T. WITH 0.5 ML YEAST ...... 162+ vi

Table Page 18. CLINICAL SUMMARY OF DOGS INFECTED I.V. WITH 0.25 ML YEAST ...... 169 19. COMPARISON OF UNTREATED, STILBAMIDINE AND HYDROXY- STILBAMIDINE TREATED DOGS INFECTED I.V. WITH 0.25 ML Y E A S T ...... 175 20. DISSEMINATION OF B. DERMATITIDIS IN DOGS INFECTED INTRATRACHEALLY ...... 179 21. CLINICAL SUMMARY OF DOGS INFECTED I.V. WITH 0.10 ML Y E A S T ...... 182 22. COMPARISON OF TREATED AND UNTREATED DOGS INFECTED 1.V. WITH 0.10 ML Y E A S T ...... 181+ 23. DISSEMINATION OF B^ DERMATITIDIS IN DOGS INFECTED INTRAVENOUSLY OR SUBCUTANEOUSLY...... 192 21+. DAYS BErWEEN INFECTION AND D E A T H ...... 191+

2$, INFLUENCE OF ROUTE OF INOCULATION AND TREATMENT ON DISSEMINATION OF BLASTOMYCOSIS ...... 195 26. DISTRIBUTION OF B. DERMATITIDIS IN TREATED AND UNTREATED D O G S ...... 196 27. DISTRIBUTION OF B. DERMATIT IDIS IN 19 TREATED AND 10 UNTREATED D O G S ...... 197 28. DISTRIBUTION OF B. DERMATITIDIS IN 23 DOGS WITH DISSEMINATED FATAL INFECTION ...... 199 29. DISTRIBUTION OF B. DERMATITIDIS SHOWING FUNGIS TATIC ACTIVITY AND INCREASED OCULAR AND CENTRAL NERVOUS DISEASE IN DIAMIDINE TREATED DOGS . . . 200 LIST OP ILLUSTRATIONS Figure Pag© 1. PHOTOGRAPH- OPISTHOTONOS AND EXTENSOR RIGIDITY.... 40 2. PHOTOMICROGRAPH- PERIVASCULAR AND PARENCHYMAL HEMORRHAGE IN THE SUPERIOR COLLICULUS...... 40 3. PHOTOMICROGRAPH- PROTEINACEOUS MATERIAL DIFFUSING FROM VESSELS .. 55 4. PHOTOMICROGRAPH- NECROSIS, GITTER CELLS AND INCLUSION BODIES IN INFERIOR COLLICULUS...... 55 5. PHOTOGRAPH- EXTENSIVE HEMORRHAGIC UNCEPHALOMALACIA OF THE COLLICULI...... 59

6 . PHOTOGRAPH- HEMORRHAGIC UNCEPHALOMALACIA OF THE COLLICULI...... 69 7. PHOTOGRAPH- HEMORRHAGE IN CEREBELLUM AND PONS 85

8 . PHOTOMICROGRAPH- VACUOLATION AND HEMORRHAGE IN CEREBELLUM AND PONS...... 85 9. PHOTOGRAPH- TRYPAN BLUE STAINING OF CEREBELLUM AND COLLICULI...... 101 10. PHOTOMICROGRAPH- NECROSIS OF A VESSEL...... 101 11. PHOTOGRAPH- BILATERAL HEMORRHAGIC LESIONS IN COLLICULI, CEREBELLUM AND PONS...... 103 12. PHOTOGRAPH- ENCEPHALOMALACIA IN THALAMUS...... 103 13. PHOTOMICROGRAPH- VACUOLATION AND SWOLLEN VASCULAR ENDOTHELIUM IN A CAT...... 122 14. PHOTOGRAPH- HEMORRHAGIC LESIONS IN CEREBELLUM AND COLLICULI...... 128 15. PHOTOGRAPH- HEMORRHAGIC LESIONS IN MEDULLA...... 128 16. CHART- LOCATION OF LESIONS, HIGH DOSAGE OF STILBAMIDINE...... 133 17. CHART- LOCATION OF LESIONS, LOW DOSAGE OF STILBAMIDINE...... 135 vii VIII Figure Page 18. CHART- LOCATION OF LESIONS, HIGH DOSAGE OF HYDROXY ST ILB AMID IN E ...... 134 19. CHART- LOCATION OF LESIONS, LOW DOSAGE OF HYDROXYST ILBAMIDINE...... 134 20. PHOTOGRAPH- LUNGS OF AN INTRATRACHEALLY INFECTED DOG...... 154 21. PHOTOMICROGRAPH- BRAIN GRANULOMA, CONTAINING GIANT CELLS AND YEAST...... 154 22. PHOTOMICROGRAPH- GRANULOMA IN CHOROID...... 161 23. PHOTOMICROGRAPH- GRANULOMATOUS INFLAMMATION IN LATERAL VENTRICLE...... 161 24. PHOTOMICROGRAPH- SUBCUTANEOUS GRANULOMA, HIGH MAGNIFICATION ...... 172 25. PHOTOMICROGRAPH- SUBCUTANEOUS GRANULOMA...... 172 INTRODUCTION AND OBJECTIVES

Recently, North American blastomycosis has been rec ognized more frequently in the canine specie. In the midwest area, as a systemic fungus infection it is second in impor tance only to histoplasmosis. Previous work done at this Uni versity has provided new information concerning the clinicopathologic, radiologic, pathologic, and mycologic nature of canine North American bl&stomycoBls. The use of these diag nostic criteria will help the practicing veterinarian to rec ognize clinical cases. Now that fairly useful diagnostic procedures have been developed the urgent need is for a spe cific therapeutic agent. A search of the literature revealed that veterinarians have consistently failed in attempts to treat canine blasto mycosis. Sulfamides, penicillin, streptomycin, aureomycin, and other antibiotics, have been useless. Although stilbamidlne has been known to possess fungistatic properties since

In the literature, the toxicity of stllbamidine for dogs was controversial. We produced peculiar central nervous system signs and lesions in one dog after only a few injec tions of stilbamidine. We then attempted to characterize the clinicopathologic nature of this toxicity and pursued studies designed to determine the maximum safe dosage for dogs.

After determining the minimum lethal amount of Blastomyces dermatltldls and the maximum dose of diamidines which could be tolerated, we endeavored to evaluate the pro phylactic and therapeutic efficacy of stilbamidine and

2 -hydroxystilbamidine in experimental canine blastomycosis.

Our specific objectives were as follows:

1. To determine the maximum safe dose and regimen of

stilbamidine and 2 -hydroxystilbamidlne for dogs.

2. If the drugs prove to be toxic, an attempt will be

made to characterize the syndrome and determine

the pathogenesis of diamldlne toxicity.

3. To establish approximate minimum lethal dose of

B. dermatltldls for dogs, and characterize the 3

clinical and pathological aspects of blastomycosis.

Ij.. To evaluate the prophylactic and therapeutic effi cacy of stilbamidine and 2-hydroxystilbamidine in

experimental canine blastomycosis. LITERATURE REVIEW

A search of the literature revealed that veteri narians have consistently failed in attempts to treat canine 65, 98 95 blastomycosis* Sulfamides, penicillin, strepto- 90 8k 9k mycin, aureomycin, ^ and other antibotics, ^ have been useless. Although stilbamidine has been known to possess fungistatic properties since 19k5 and therapeutic value in human blastomycosis since 195l»10^ no report could be found concerning the effects of stllbamidine on canine blastomycosis. Pertinent information will be revealed concerning the physical and chemical properties, pharmacologic action, toxicity, therapeutic value in man and fungistatic proper ties of stllbamidine and related compounds*

Physical and chemical properties* —

Formulas:

H2N H H NH2 C - N - ( CH2 ) 10 - N - C NH NH Common name : SYNTHALIN Chemical name: Decamethylene diguanidine

k Common name : STILBAMIDINE Chemical name: i+jlj.1 stilbenedicarboxamidine

H2N^ /------\ HO OH I------\ ^ NH2 c HN - 0 = i - C D ^ N H Common name : HYDROXYS!ILBAMIDINE Chemical name: a-hydroxy-li,^1stilbenedlcarboxaml- dlne

Stllbamidine is a white crystalline powder. It cam be heated in solution to 70-80 C for a short time (fifteen minutes) without decomposition. A melting point cannot be determined as stllbamidine turns yellow at 200 C and de composes at 290 C. The compound is soluble in water up to i|.0 grams in 100 ml at 25 C. It Is fairly soluble In glucose solutions and moderately soluble in blood-citrate mixtures (0.08 mg/ml). Pulton and Goodwin^ mentioned that stllbami dine added to human or mouse sera in concentration greater than 0*5> per cent precipitates out as a base which is insolu ble in water but soluble in dilute hydrochloric acid. In the earlier years, stllbamidine has been used in the form of the free base and also, in the form of the di- hydrochlorlde. Now, the dlisethionate salt, containing only 60 per cent of the base present in the hydrochloride salt, 6 is used for clinical and experimental purposes because of Its greater solubility and better tolerance during Intraven ous injections. Maximum tolerated doses of the dllsethionate are from $0 per cent to 100 per cent greater than that of 99 the hydrochloride. The powder is stable in the dark, even if it may turn pale yellow, with no significant alteration in the absorption spectra, toxicity or therapeutic action.^ Even the solution remains unaltered for several months in total darkness. The solution is definitely affected by fifteen minutes of sun light with marked increase in toxicity and decrease in thera- Mi peutic activity in both man and animals.

This deterioration is due to the action of ultra violet light on the unsaturated stilbene linkage. Stllbami dine also has a marked adsorption on proteins and other com- 3 3 ,ii2 ,5 5 pounds of large molecular weight or surface area.

Another characteristic of stllbamidine is its bril liant blue fluorescence, even in extremely dilute solution, when exposed to ultraviolet light. On adsorption on tissue or decalso it emits a relatively strong white-greenish fluorescence. Stilbamidine also possesses a strong narrow 55,117 spectral absorption band with a maximum of 329 millimicrons.

Hydroxystilbamidine is a yellow crystalline powder. Like stllbamidine, it is stable in the dark, but unstable when exposed directly to heat, sunlight or ultraviolet light. 7

It has the same general characteristics as stllbamidine. In solution, It emits a faint reddish fluorescence much weaker than the blue fluorescence of stllbamidine. On adsorption, Its white-greenish fluorescence Is stronger than 117 that given by stllbamidine. In man It Is considered less toxic than stllbamidine and did not produce, to date, the late trigeminal neuropathy, which has been a sequella of the latter.

Determination: Several methods have been designed to determine qualitatively or quantitatively the level of stllbamidine or

2-hydroxystilbamidine In body fluids or tissues. The marked properties of adsorption, insolubility, fluorescence, and the specific absorption spectra have been used in those methods. However, they are not usable at the very low drug concentration which could still be at the therapeutic level. Furthermore, the accuracy of some methods was often variable.

Pharmacologic action and toxicity.— Absorption. distribution and excretion of these drugs in the body must be known before they can be employed intelligently as treatments.

High concentration of stilbamidine in the blood and urine could be detected in man and various experimental animals, within a few minutes after administration by either U2.S5.126 oral, subcutaneous, intramuscular or intravenous routes. 8

After a single intravenous or intramuscular injection of a maximal tolerated dose, Wien‘S and Pulton and Goodwin^ ob served peak blood levels of 30 to I4.O microgram/cc. These quickly fell within thirty minutes. After two hours, less than 2 microgram/cc were detectable. Even after large oral doses, peak levels were maintained for only a short period of time. In mice, the subcutaneous route afforded slight pro longation of the high blood levels. According to Pulton, levels less than 2 microgram/cc are capable of definite therapeutic and prophylactic activity in mice. 55 Henry and Grindley studied the rate of urinary ex cretion of stllbamidine in two patients receiving single in jections and found that approximately 10 per cent was ex creted over a 2£ day period. After the first two hours, there was a rapid fall in excretion, and at the end of the period, scarcely any stilbamidine could be detected by the method employed (the crude "spot technique."). 59 Kirk and Henry in 1944* noted a cumulative action of the drug. After nine intravenous injections of 50 mg of stilbamidine on alternate days in a man they noticed a rapid increase in rate of excretion of the drug. By the seventh or eighth injection, 75 to 85 per cent of the daily amount administered was excreted. About 53 per cent of the drug was excreted when measured by the chromatographic technique.

They believed in the hypothesis that the kidneys rapidly 9 excrete any excess of the drug circulating in the plasma after saturation has been reached. It seemed necessary to maintain a certain concentration of drug in the blood stream in order to exert a significant effect on organisms. Once saturation has been reached the dosage must be graduated to achieve an optimum constant plasma concentration and thus avoid excessive renal excretion of the drug. If Intravenous injections Eire used, it is likely that small and frequently repeated doses will be more satisfactory, after saturation has been reached, than larger doses at intervals of two or three days. They, then, suggested the possible superiority 59 of the subcutaneous or intramuscular routes. 126 Wien, in 191J.6# using the fluorescent technique, observed approximately 15 per cent excretion during the first twenty-four hours after a standard intravenous or intra muscular dose of 2 mg/kg. With successive dally injections, the percentage excreted increased progressively until 70 to 80 per cent of the daily dose was excreted by the seventh or eighth dose. When high dally doses (10 mg/kg) were administer ed, the proportionate amount excreted was diminished when compared to excretion on low daily doses (1 mg/kg). The daily amount eliminated was stated to be approximately the same regardless of dose. This suggested to Wien a limited ability of the body to excrete stllbamidine. He assumed that a large amount of stilbamidine was either metabolized or ex creted in the non-fluorescent form. 10 96 In 1950, Saltzman et al using the modified fluor escent technique, did not agree with the above findings* They did not notice any grossly significant change in the rate of excretion in any subject, although the excretion for as many as 16 injections was determined. After intravenous in jections of 150 mg of stilbamidine diisethionate the urinary excretion in a normal subject was I4..2 per cent in four hours and 10*1 per cent in twenty-four hours. They also mentioned that the daily average excretion of 2 -hydroxystilbamidine in a similar experiment varied between 6 and 7 per cent of the quantity injected. 53 Hawking, in 19l|l|., using the spot technique, showed that stllbamidine quickly disappears from the plasma of mice and rabbits even during the first half hour after adminis tration. In rabbits, from a theoretical 16 mg/100 cc of blood, it went down to 0.05 mg/100 cc of blood after two hours. and to 0.005 mg/100 cc of blood after six hours. 113 In 1951*' analysis made by Snapper et al showed that considerable amounts of stllbamidine and 2 -hydroxystil

bamidine are deposited in the parenchymatous organs, especially in the liver, kidneys and adrenals. Often about one-third of the total quantity injected could be recovered from these organs. The deposition seemed to vary in differ ent species. In mice more dlamldine is found in the kidneys

than in the liver; in man and rabbits, the opposite is true. 11

After deposition diamidines remain in the liver and adrenals for a long time. Twenty-three months after the last injec tion of 2 -hydroxystilbemidine appreciable amounts could be recovered from a myeloma patient. Traces of stilbamidine were found in one liver, thirty-nine months after the last 113 injection. Snapper et al also noted that liver and kidneys of normal mice contained less diamidines than the same organs of mice bearing a transplantable hepatoma subcutaneously. Considerable amounts are deposited in the hepatoma. Lymphosarcoma and mammary carcinoma of mice con tained only traces. ii6 In 1955t Geraci et aI used the method of Lieben and Snapper in a patient that had received 7 g® of 2 hydro xys tilbamidine in a thirty-two-day course. Two months after the end of the treatment analysis revealed 5$ in the liver and 2 mg in the brain, compared to 2 mg in the liver and none in the brain of a control patient. 56 In 195>2, Henry et al mentioned that even late in a series of injections, no detectable amount of stilbamidine was present in the blood plasma or blood corpuscles of a sheep. The liver of a sheep which died three months after the termination of a course of injections (presumably from the delayed toxic effects of the drug) contained a high pro portion of stilbamidine. There appeared to be no tendency for hydrolysis of the amidine group to the amide group to occur in the body. This is important since the first 12 hydrolysis product is more toxic than the parent compound. They also noted that rats appear to be considerably more sensitive to the toxic action of the drug than are mice or guinea pigs. According to their admittedly limited observa tions* stilbamidine is heavily adsorbed in the rat liver after Injection. Even when death ensues at a later stage* it has been largely eliminated from the liver before death occurs. In this respect the rat seemed to differ radically from the sheep. In the case of a rabbit which had received a course of Injections and was killed two months later* no organ* even the skin* was entirely free from stllbamidine. They added that more work will be necessary in order to determine whether or not storage of stilbamidine produces any pronounced Interference with fat or mineral metabolism* which might well explain the delayed toxic action of the drug.

Hepato-renal function: 32 In 1914.0 * Devine showed that in rabbits* a single Intravenous injection of half of the lethal dose (15 mg/kg) did not cause any alteration in blood sugar level but the blood urea rose from 15 nag per cent to over 100 mg per cent. Single injections of 25 mg/kg Intravenously were tolerated with difficulty. Within two hours the blood sugar rose almost 100 per cent and then declined rapidly to normal. The animals continued to reveAl evidence of prolonged 13

Impairment of renal function with either transient or fatal uremia and death after eight to ten days. Successive small doses (6 daily injections of 5 mg/kg) produced no alteration in the blood chemistry. 127 In 191+3* Wien et al observed hyperglycemia only vith doses near the toxic level. Adrenalin hyperglycemia was reduced by previous injection of stilbamidine and the adrenal glands seemed to play a part in the glycemlc response. Stllbamidine hyperglycemia was reduced in adren- alectomized animals. Ergotoxine sometimes abolished the hyperglycemic action of the diamidines. No depletion of liver glycogen could be demonstrated four hours after a single injection of propamidine in a large number of rats. Repeated injections produced marked depletion of liver glycogen which was associated with signs of toxemia, anorexia and starvation. They noticed an Increase in blood- urea and non-protein nitrogen at levels that were not influ encing blood sugar. In toxic doses, fatty degeneration of the liver and some cloudy swelling of the kidneys were found. Their observations have been supported by pathologic exami nation of the livers of rabbits, mice, guinea pigs and cows. 105 Seager and Castlenuovo reported that oral adminis tration of 100 mg/kg dally to mice for 120 days resulted in fatty metamorphosis of the liver and sometimes marked degeneration of the renal convoluted tubules. Rabbits could 11*. tolerate the same dosage subcutaneously for only a few days; 10 mg/kg dally killed all rabbits before 10 doses were ad ministered* The same lesions were found In the liver and kidneys. When 10 mg/kg was fed for one week to mice* dis tention of the central and Interlobular veins of the liver as well as cloudy swelling and degenerative changes In the renal tubules were noted. An evaluation of the experimental renal damage pro duced by the diamidines is hindered somewhat by the wide fluctuation in blood urea levels in animals suffering from the anorexia, syncope, and general toxemia immediately following even therapeutic doses of the diamidines injected 99 intr avenous ly •

Respiratory effects: Large doses of stllbamidine by parenteral routes produced in mice and rabbits an increase In the respiratory rate; later respirations became slow and forced. Dyspnea may have been due to spasm of the bronchioles, and the death from respiratory failure. In rats and guinea pigs, Wien has concluded that there is no evidence of any direct depression 125 of the respiratory center.

Blood and vascular effects: 125 59 Wien and Kirk and Henry reported a fall in blood pressure. Wien attributed this fall in blood pressure to vasodilation in the smaller arteries and arterioles. The IS dilatation was believed to be a direct action on the blood vessels and independent of any central or nervous mechanism. Wien showed that this depressor effect is reduced slightly in the intact animal by atropine and that the pressor action of adrenalin was counteracted markedly by the diamidines* Atropine and adrenalin have been used to overcome the immedi ate syncopal reactions in man, but neither agent gave good results* Kirk and Henry suggested the intramuscular route to obviate to the immediate toxic reaction, but added that it is a painful process. Most of the clinicians today use the slow-drip method* 73 In 19^9, Mackintosh found that diamidines produced a sudden fall of arterial pressure in the dog 20 to 25 seconds after injection. The blood or plasma obtained during this period was found to contain histamine* He believed at the time that the main site of histamine release was the liver. The vascular effects were not prevented by anti histamine drugs* Later, other workers (Riley and West in particular) found that most of the histamine of the body is located in 17 92 93 the mast cells. * * Until that time heparin was the only constituent known in those cells. After injection of diami dines, they noted an explosive bursting of the mast cells with dispersion of their basophilic granules. Bursting was 39 also noted by Fawcett in 195^ after intraperitoneal 16 injection of water which resulted in osmotic disruption of the subserous mast cells with their subsequent death. There was no damage to other cell types* A series of increasing doses of diamidines had a graded morphological effect upon mast cells and resulted in a graded increase in the amount of histamine that appeared in the peritoneal fluid. He con cluded that it is unlikely that this compound acts by simply lysing the plasma membrane. Ten days after mast cells were lysed with water he found that diamidines released little or no histamine* Lately, Arvy and Quivy enumerated the mast' cells in the intestine of the dog and found a very good correlation with the maximal amount of histamine contained in the differ ent portions. Esophagus was poor in mast cells, the stomach was the richest, and there was a gradual diminution from the stomach to the colon* Mast cells were most numerous in the muscularis mucosa. They mentioned that the pleura of cattle was the richest site of mast cells* 93 In 1955* Riley found differences between the effects on the same tissue of rats and mice* Tissue mast cells in the ears and subcutis of the mouse are much more resistant to the damaging effect of diamidines than are those of the rat. Less than $0 per cent of the histamine was released in the mouse compared to 98 per cent In the rat. Apparently the process of histamine liberation and disruption of mast cells 17 plays a small part in the mode of action of the liberator in the mouse; other toxic factors are more prominent. This may have a bearing on the higher susceptibility of the rat. Riley also noticed that a dose which on first injec tion produced pronounced symptoms of histamine release, no longer does so when injected a second or third time, unless the dose is highly increased. In this way chronic doses of normally lethal amounts of the liberator have been given without any observable effects. Once the mast cells are dis rupted the histamine release appears to be minimal. 50 Halpern et al in 1955» stated that the disorders produced by histamine liberators are more severe than those elicited by histamine Itself. They believed that histamine liberated at the cellular level must behave, in certain respects, differently from outwardly applied histamine. The local instantaneous concentration, the way of accosting the effectors and the gradient of destruction differ greatly. 68 In 19Sbt Linde 11 and Westling believed that the in crease in blood histamine, following stilbamidine injections, apparently is due, at least in part, to inhibition of hlsta- minase and concomitant accumulation of histamine. lii la 1955* Butler found that at low blood concentra tion of histamine in dogs the coagulation time was reduced 10 per cent without any change in total platelet count. Higher blood concentration of histamine resulted in 19 per 18 cent reduction In coagulation time, disintegration and clump ing of the platelets, trapping of platelets in capillaries with a reduction in the platelet count and hemoconcentration. This action of histamine does not occur in vitro and is apparently dependent upon the immaturity of the developing platelet. Heparin will not alter this manifestation. 66 Lately, Lecomte and Bounameaux, contrary to Butler, denied the reduction of platelets by histamine and attributed hemostatic activity to it. They ascribed the reduction of platelets to the histamine-liberator itself, which they demonstrated to be effective in vivo and in vitro. 50 Halpern et_ al showed that a considerable increase in capillary permeability in the dog was produced by an hista mine releaser, polyvinylpyrrolidone. They attributed this effect to the release of endogenous histamine. They saw evidence of rapidly developing changes in the appearance of the endothelial cells, edema and widespread hemostasis. Stasis and increased permeability are aggravated by the histamine-induced constriction of the efferent venules. 79 Mota and Beraldo, in 1953* noticed that stilbamidine and compound lj.8/8 0 , another histamine releaser, in creased the coagulation time in rats and counteracted the anti-coagulation action of heparin in vivo. They stated that this strongly suggests a protamin-like action of this compound, which results in the formation of insoluble 19 complexes that remove heparin from the system.

Central nervous system effects: 125 Wien In 192+3# lh short-term experiments, did not notice any significant effect of stilbamidlne on the central nervous system of guinea pigs, rabbits and mice. 71 In 1939, Lourie and Yorke mentioned that a subcu taneous dose of 10 mg/kg of stilbamidlne in the dog is usual ly fatal, and that $ mg/kg is tolerated. Even after moderate size doses dogs often vomited and defecated, and sometimes there was swelling of the face and transient weakness and tremors which disappeared within an hour. With larger doses, bordering on the lethal, these signs were accompanied by collapse due presumably to respiratory and circulatory dis turbances. If the animal recovered from these immediate effects a peculiar spastic paresis developed three to four days later; when this condition appeared, the termination was almost invariably fatal. 60 Kirk and S&ti observed transient paresis in dogs after large doses of propamidine. In 19U&, Oastler and Pidler ^ observed selective central nervous system lesions of a constant type in dogs after intravenous injections of fresh 1 per cent aqueous solution of stilbamidlne autoclaved at 5 lbs. pressure for twenty minutes. In most animals these lesions were associ ated with definite and characteristic neurological signs. 20

Ten dogs were used; they received 1 mg/kg of stilbamidlne daily during the first week, 1.5 mg/kg during the second week, and 2 mg/kg during the third week. Five dogs had clinical manifestation of central nervous system damage; four of them died in a few days and showed macroscopic and microscopic lesions post-mortem. One animal recovered from early neuro logical signs and was killed three weeks after completion of the course of treatment; it showed a healing lesion in each caudate nucleus. The five other animals had no clinical signs that could be definitely attributed to the central nervous system; three showed nothing abnormal in the brain, but the two others showed microscopic lesions in the brain stem, cerebral cortex and spinal cord. The characteristic clinical manifestation was ex treme and generalized spasticity which resembled decerebrate rigidity. At necropsy they found cerebral softening, inflam mation of the meninges, together with hemorrhage and throm bosis within the cerebrum. Microscopically, cellular infil tration, thickening of the walls of blood vessels, and myelin degeneration was noted. The liver was essentially normal in all animals ex cept one, where small scattered foci of central lobular necrosis and a moderate degree of fatty changes were found. The kidneys were normal except for slight fatty degeneration

in 5 dogs. 21

There was no apparent relationship between the de gree or neurological damage and the amount of drug given* The authors emitted the hypothesis that the small vascular lesions found in some dogs could be the primary condition which is followed by intravascular thrombosis, myelin degen eration and infiltration with erythrocytes and leucocytes* They added that the sudden onset of ataxia and unconscious ness which was seen in some dogs immediately after injection, suggest that vascular spasm and local anoxia may be impor tant factors. They also mentioned that these cerebral lesions could be the result of a special tissue suscepti bility of dogs. 106 In 19*4-7» Sen Gupta published a letter in discus sion of the last article. He mentioned that 2 to 5 mg/kg of atilbamidine given six days a week for 1f? to 20 Injections in the rhesus monkey and the rabbit failed to produce any clinical signs or brain lesions bearing the slightest re semblance to those described by Oastler and Fidler. In rabbits given autoclaved stilbamidlne death was caused by great damage to the liver and the kidney, and widespread de generative changes and hemorrhages in different internal organs* No abnormality was seen in the nervous tissues of those rabbits. He believed that the autoclaving of stilbamidine solution used by Oastler and Fidler could have produced some chemical changes which lead to an Increased toxicity. 22

He also mentioned that the intense vascular changes and the infiltration with polymorphonuclear leucocytes resemble an acute infective inflammation of the brain and meninges* Furthermore, he finds it hard to realize how neuronic or myelin degeneration could be fairly studied without using Weigert-Fal or similar stains or special staining for Nissl's granules. 82 In 19i+2, Napier and Sen Gupta described a late chronic neuropathy confined largely to the distribution of the fifth cranial nerve. This neuropathy following the use of stilbamidlne is unlike any other drug-induced or naturally occurring syndrome, except possibly that following 99 trichloroethylene or streptomycin. From two to five months after termination of therapy there is a rapid onset of vary ing degree of hypesthesia and hypalgesia over the face and over the upper cervical sensory dermatomes. There is also some burning paraesthesia about the nose and the eyes which tend to subside slowly. On occasion, the same findings have been reported to extend to the neck and waist. Many investigators have reported this peculiar 26 neuropathy. Collard and Hargreaves reported almost 100 per cent incidence of this complication among British servicemen. 25 Collard and Nevln in 1946, believed that the nuclei affected were the chief pontine nucleus and the descending

nucleus. The mesencephalic root of the fifth nerve appeared 23 to be Intact by the preservation of the jaw-Jerk. Since the cells of the affected nuclei are different structurally from the latter, they believed in a selective action of stilbami** dine. They emitted the hypothesis that the three-months latent period may be due to the fixation of the drug by the cells and that a toxic substance is liberated later by meta bolic changes. They also added that it is perhaps possible that stilbamidlne is absorbed at the sensory nerve endings and produces the lesions when it reaches the cell body. 23 In 1952, Cohen et al reported the death of a 3-year- old infant apparently due to stilbamidlne toxicity. The patient received intravenous injections for thirty days, starting with 30 mg daily and working up to 1^0 mg daily dur ing the last eighteen days. The patient then developed con vulsions, coma, irregular respiration and a decerebrate type of rigidity. A spinal tap showed no cells. Autopsy revealed acute yellow atrophy of the liver, fatty infiltration of the brain and a fatty infiltration of kidney tissue. In contrast to stilbamidlne, the use of 2-hydroxystilbamidine has never caused a late neuropathy even when a much Il6 higher total dosage has been given. In 1955# CJeraci et al reported an unusual allergic reaction to 2-hydroxystilbamidine. The drug was given daily for thirty days without any reaction; after the 3 1 st, 32nd, and 33**<1 injection the patient developed an intense prurit&s. When the drug was 2U tried a few days later an alarming anaphylactic-like syndrome appeared.

Other pharmacologic effects: 125 In 191+3, Wien found that concentration as low as 1 /10000 to 1/25000 could stimulate smooth muscle of rabbits Intestine and uterus. These effects were reduced, but not abolished by atropine. Higher concentration (1/5000) pro duced spontaneous contraction of striated muscle. Guinea pigs smooth muscle did not respond. Wien postulated that the drug increased the sensitivity of nerve muscle prepara tions by reducing the concentration of calcium and lowering the calcium/phosphorus ration. In both dogs and rabbits, 127 Wien at al reported a slight fall in serum potassium with in the first five hours after Injection of stilbamidine. Wien also noticed the inhibition of adrenalin stimulation of tissues after they had been exposed to dilute solutions of stilbamidlne.

Mechanism of action: Synthalin, the drug from which stilbamidine originated, was once used in diabetes. For this reason, its therapeutic effects in trypanosomiasis were thought to be due to hypogly- 70 cemia. However, In 1937* Lourie and Yorke proved that hypo glycemia was not produced by synthalin at therapeutically active doses. Since that time, many hypotheses have appeared regarding the mechanism of action of the diamldines in their 25 different uses. Apparently, no single hypothesis is satis factory for all diseases. Since Trypanosoma rhodesiense is stilbamidine- sensitive compared to the other species (T. lewis and T . cruzi, which are stilbamidine-resistant) the Investigators have looked for basic differences that could account for this fact. The stilbamidine-sensitive species did not have their 20 oxygen uptake inhibited by cyanide whereas stilbamidine- resistant species were found to be very sensitive to such 123 inhibition. Species of Leishmania sensitive to diamidine are also very sensitive to inhibition of their oxygen uptake 20,123 by cyanide. Since the stilbamidine-sensitive species of Trypano- somes have glucose and oxygen requirements which are tenfold 20 greater than that of the stilbamidine-lnsensitive group, 71 Lourie and Yorke in 1939* suggested a ’’sugar blockade” pro duced by inhibition of the intrinsic aerobic glucose metabol ism of the trypanosomas. 62 7 Kohn and Berhelm stated that propamidine Inhibits 9 the catabolic metabolism of bacteria. Bichowsky inferred that in bacteria and protozoa, the diamidines tend to con centrate primarily within bodies rich in nucleic acid. There, as basic substances, they may combine with nucleic acids or the nucleoproteins and interfere with the utilization of an essential metabolite or enzymatic pathway. Berheim also 26 noted that the oxygen uptake of suspensions of rat kidney and liver was not inhibited by U/QOOO propamidine, but rat brain oxidation of glucose was reduced 50 per cent by concen tration of nucleic acid and nucleoprotein present in the rat liver or kidney* 63, 6k Kopac showed that stilbamidine disassociated protamine nucleate complexes, and that it was not so with the other diamidines. Stilbamidine trapped the nucleate by the formation of insoluble stilbamidine-nucleate complexes* Kopac also showed in tissue culture that stilbamidine selectively destroyed certain types of mammary carcinomas and transplantable lymphosarcomas of the rat* Those findings have been used to explain the degeneration damages in the 5 th nerve nuclei, specific for stilbamidine* 110,111*,115 _ , Snapper, after using stilbamidine to re lieve pain in multiple myeloma, noticed the appearance of basophilic Inclusion bodies in myeloma cells of his patients. These inclusions were present only in patients manifesting globinemia or Bence-Jones proteinuria. The cytoplasmic granules proved to contain rlbose nucleic acid and stilbami dine or 2-hydroxystilbamidine. They disappeared when the enzyme rlbosenuclease was applied to the cell* Snapper con cludes that stilbamidlne and 2 -hydroxystilbamidine interferes with the metabolism of the myeloma cells. 27

The variation In diamldine action on related species of trypanosomas, and the effectiveness of all diamldines In protozoal infection (despite the fact that only stilbamidine produces protein denaturatlon) point to the fact that at least two mechanisms are probably involved: first, the inhi bition of certain specific enzyme systems and secondly, the alteration of nucleoproteins which may have a greater or lesser role in the enzyme systems of different species of 99 bacterial, protozoal, and tumor cells.

Fungistatic properties.— Stilbamidine has proven 11, 69 useful in the treatment of human trypanosomiasis but, the therapeutic index was too narrow for use on large animals. Similar compounds, pentamidine andid l|.ii^V-dlamldl- sl|.1 16, 30 no dlmethylstilbsne, were not more successful. Stilbamidine was also useful in the treatment of leishmaniasis,^*2,3,21,31*,128 caniae piroplasmos^s,^,^ ,^ and human "tic douloureux."1^0 It has also been used to re- lleve pain In cases of human multiple myeloma.109'115'116 Today the drug is known for its fungistatic properties.

In 19l+5>, there was a report by Elson on the in vitro fungistatic properties of propamidine. Blastomyces dermatltldis. Sporotrichum schenckll and Achorion schoenlel- nli were inhibited with concentrations of 0.018 mg/20 cc 10k agar. Se&bury and Artis, in 191*6* found stilbamidine to 28 to© fungistatic in vitro against Histoplasma canaulaturn at a concentration of 10 mg per 100 cc of medium. However, in clinical trials, the course of the disease was not modified 103 to a significant degree. In 1950, the first case of cutaneous blastomycosis 21+ treated with propamidine showed very good results. In 100-101 19^1 and 1952, Schoentoach et al treated four cases of systemic blastomycosis with stilbamidine and noticed good results; three of the patients suffered trigeminal neuropa thy. Since then stilbamidine and 2-hydroxystilbamidine, the latter following a clinical trial by Snapper and McVay111 in 1953* have been used extensively against blastomycosis. It has also been tried on some other fungi, follow- * 4 11,19,22,118,122 ing in vitro studies that were encouraging. lil In 1950# Fisher found that propamidine did not alter 78 the course of cryptococcosis in mice. In 1952, Miller et al concluded after treating three cases, that the Intravenous administration of stilbamidine does not affect the clinical course of cryptococcosis in man. 88 As early as 191+5* Parsons treated two cases of human histoplasmosis with stilbamidine and neostibosan and reported some improvement in one patient. Again, in 191+9, Seabury^^ noted some temporary improvement in two patients but stilbamidine did not modify the course of the disease to

a significant degree. Ellis et al in 1952 noticed visible amelioration of a lesion of the epiglottis due to Histoplasma 29 capsulatum after topical use of varidase and propamidine. This lesion had been resistant to a previous treatment with ethyl vanillate. In 1955# Nejedly and Baker J reported the successful treatment of a localized case of histoplasmosis of the hard palate and gums. No histoplasma were seen after 20 mg of hydroxystilbamidine, U8 In 195i|.* Grebe reported a rapid and complete heal ing of a bone lesion due to Goccidloides lmmitls after treat ment with 2-hydroxystilbamidine. However, Gephart and HanlonkS noticed the persistence of the meningeal invasion by coccidloides after a course of twenty-seven days of 112 stilbamidine. In 1955* Snapper .et al reported on seven cases of coccidioidomycosis treated with hydroxystilbamidine. As much as 23 gm was given to a patient but no evidence of curative effect was noticed. In five patients there was evi dence of a suppressive effect on the progression of the disease two to six months after the end of treatment. In 23 1952, Cohen at al reported the death of a 3-ye&**-old child treated with stilbamidine and suffering from coccidioido mycosis and tuberculosis. After an intensive treatment of thirty days, the child developed convulsions, irregular respiration, coma, and a decerebrate type of rigidity. Death was attributed to the toxic effects of stilbamidlne, which according to the authors are not mentioned in any other reports in the literature. 30

77 In 19^2, Miller et al treated successfully with stilbamidine, a case of actinomycosis In man that was re sistant to penicillin and sulfadiazine. 51 Harrell et al treated successfully with stilbami dine a case of sporotrichosis that was resistant to iodide therapy. In 1955* a case of systemic sporotrichosis was 2 t 6 -• treated by Geraci et al with hydroxystilbamidine, with great amelioration but after the thirtieth injection the patient showed a sudden allergic reaction. In the subse quent injection of hydroxystilbamidine he presented alarming anaphylactic-like symptoms and the treatment was replaced by iodides which did not control the infection. Thrush oesophagitis in three infants was also treated 129 successfully with hydroxystilbamidine. Hydroxystilbamidine was found ineffective in the 91 treatment of two cases of tinea capitis.

Treatment of blastomycosis.— ’Potassium iodide, one of the oldest treatments for North American blastomycosis, hi was first employed empirically by G-ilchrlst in 1902. Until recent years it has been used extensively. In the majority of cutaneous cases marked Improvement has been ob tained with this drug. Some cutaneous cases failed to respond and when used in an allergic patient the drug often 75*76,107 increased the symptoms. In systemic blastomycosis the oral use of potassium iodide has usually been followed 31 18,75 by dismal results; there may be temporary Improvement 102 but the course of the disease Is usually not altered* The use of iodine in a strongly allergic patient may bring a recrudescence of the disease, with sometimes a fatal 7k outcome. To obviate to this Martin recommended the de sensitisation of all allergic patients* before starting treatment, using increasing doses of vaccine as a skin test ing agent* The poor results obtained with potassium iodine in systemic blastomycosis has prompted the clinicians to use a large number of therapeutic agents, usually with very in consistent results* Intravenous sodium iodine, intravenous arsenicals, 75 colloidal copper sulfate, vaccine therapy, penicillin, 29 81 67 sulfamides, diethylstilbestrol, aureomycin, and surgery* 12,57,58,12k including lobectomy, have been used against the systemic form of the disease. Most of the agents already mentioned plus arspena- mine, silver nitrate, tincture of iodide, bichloride of mercury, 1 per cent copper sulfate, phenol, cauterization, freezing with carbon dioxide, radium, and several others 75 have been used on the cutaneous lesions* 102 According to Schwarz and Baum, potassium iodide x-ray therapy had no effect on the ultimate course of the disease; they considered surgery the best procedure if the 32 lesions are localized* 36 In 19k5* Elson's report on the In vivo fungistatic properties of propamidine opened the way to a new era in the treatment of blastomycosis* In 1950, the first case of cutaneous blastomycosis treated with propamidine showed very 2k 100,101 good results* In 1951 and 1952, Schoenbach et al treated successfully four systemic cases. Heilman used it 5U successfully in experimental blastomycosis of mice. Since then hydroxystilbamidine has been Introduced to obviate to the annoying fifth nerve neuropathy produced by stilbamidine. These two drugs have been used by most clinicians, often in conjunction with surgical procedures, and the results have been encouraging. It is interesting to note that the im provement has usually been noted several months after the end of treatment. Most of the clinicians used daily doses of 150 mg of stilbamidine or 225 ®g of hydroxystilbamidine. The drugs are diluted with 5 per cent glucose and given very slowly by vein, usually by the drip method. However, when it was Impossible to give it intravenously, the more painful intramuscular route has been used. Totals 2 to 20 grams have been given in one or several courses. l8 Cherniss and Waisbren in 1956 mentioned that their results did not coincide with those of other;workers in re gard to the efficacy ; of stilbamidlne when used in recommend ed dosage. However, they also noted from the current 33 literature that stilbamidine or hydroxystilbamidine are the drugs of choice for systemic blastomycosis, the former being more potent, the latter less toxic.1 3 ,2 7 ,2 8 ,2 9 ,lj.0 ,5 2 ,6 l,8 0 , 86,87,97,119,120

TOXICITY OP STILBAMIDINE AND HYDROXYSTILBAMIDINE FOR DOGS The objectives of this study were as follows: 1 ) to characterize the clinical and pathological aspects of stilbamidine toxicity in dogs, 2 ) to investigate the toxicity of the related 2 -hydroxystilbamidine, and 3 ) to establish a relatively safe treatment schedule at an acceptable therapeutic level.

MATERIALS AND METHODS Thirty-four healthy dogs of mixed breeding, varying in age from six months to three years were selected (Tables 1 and 2). During a baseline observation period of at least three weeks, they were immunized against canine distemper and infectious canine hepatitis with a commercial killed vaccine given in three doses at weekly intervals. After treatment for external and internal parasites, the following baseline studies were made: urinalysis, blood cell counts, hemoglobin, bromsulfaleln liver function tests, phenolsulph- talein kidney function tests, and physical (including neuro logic and ophthalmoscopic) examinations. These studies were % continued during the experimental period when indicated* The mean arterial hlood pressure (femoral artery) was recorded on selected dogs before and after treatment. The isethionate salt of two diamidine drugs, stilbamidine ibth*stilbenedicarboxamidine) and hydroxystilbamidine (2 -hydroxy-i}.,i^.,stilbenedicarboxamidine) were manufactured and supplied by the Wm. S. Merrell Company, Cincinnati, Ohio* Stilbamidlne was supplied in 20 cc ampules, each containing 150 mg of stil bamidlne isethionate sterile powder. Hydroxystilbamidine was supplied in 20 cc ampules, each containing 225 of hydroxystilbamidine isethionate sterile powder. The drugs were weighed as rapidly as possible under feeble artificial light, and put in individual amber vials* Each dose was diluted with 2 cc of 2 per cent procaine solu tion and administered within one hour after preparation into the thigh muscles using the right and left sides for alter nate doses* Care was taken not to expose the drug to heat or sunlight at any time* The dosage given each animal is pre sented in tables under results. The animals were caged individually in a room with lighting of low intensity and were not exposed to the direct rays of the sun* The dogs were fed a commercial kibble ration (23 per cent protein) and received fresh horse meat twice a week. A few dogs received a diet relatively low in proteins (11|*5 per 35 cent) made according to the following formula: corn meal 73 lbs*, wheat shorts 20 lbs., meat said bone scrapings $ lbs*, lard 2 lbs., cod liver oil 100 cc (1000 units of vitamin A and 1000 units of vitamin D/cc) and salt 1 lb* Necropsy was performed on all dogs as soon as possi ble after natural death or immediately after euthanasia* Tissues showing lesions as well as representative sections of all organs were fixed in 10 per cent isotonic neutral formalin, embedded in paraffin and sectioned at 6 microns; eyes were Infiltrated in celloldin and sectioned at 10 microns* Where indicated the following histopathologic methods were employed: Well-Weigert for myelin sheaths; oil red-0 and Nile Blue sulfate for lipids; Bauer, Grldley and Grocott methenamine silver for fungi; hematoxylin and eosin for routine cellular differentiation; Masson* s trichrome for collagen; and periodic acid Schiff reaction for aldehyde groups* Evidence of functional pathology attributed to drug toxicity was recorded by motion pictures; morphologic pathology was recorded by still photographs.

RESULTS Each experiment will be described separately, and will Include the objectives, particulars of the materials and methods, signs, necropsy findings, hlstopathology and a discussion. We emphasize that only positive data were recorded* 36

A) Pilot injection of stilbamidine Since the literature reviewed disclosed only contro versial information about the toxicity of stilbamidine for dogs we attempted to give a course of the drug, with the maximum doses used in man. Dog 5392 was a 26-pound male, approximately 8 -yearA3 old. A subcutaneous injection of 0*02 mg of atropine diluted in 2 cc of saline was followed 10 minutes later by intra muscular administration of 36 mg of stilbamidine (3 mg/kg) diluted in 2 cc of 2 per cent procaine. No abnormal reactions were noticed. The next day, moderate ataxia of a few minutes duration occurred 20 minutes after the same dose of stilbami dine was given. He was in good health on the third day when treatment was omitted. On the fourth day, the dog was found on its right side, in extreme opisthotonos with the front legs extended and severe extensor rigidity. The hindlegs were also extended but making continual paddling movements. The tail was rather rigid and curved upward. The eyes were bulging; the pupils contracted but responded to light. The corneal reflex was also present. The dog had some breath ing difficulty, and the tongue was rather cyanotic. For humane reasons the dog was destroyed, although he presented no evidence of pain. (Fig. 1). 37

Necropsy findings: Hemorrhages and edema were present at the site of in jection, between the muscle bundles, and in the popliteal lymph nodes. The lungs showed some congestion and pneumonia but the most significant findings were in the central nervous system. Striking lesions were noted in the area of the dendate nuclei in the cerebellum and in both inferior colllcull and superior colllculi in the midbrain. These lesions con sisted of bilateral, hemorrhagic and softened foci, measur ing from 1 to 5 millimeters in diameter.

His topathology: A section through the medulla oblongata at the level of the middle of the inferior olive disclosed only a few bilateral hemorrhages in the area of the lateral cuneate nuclei and of the inferior olive. Congested vessels were seen along the medial lemniscus. A transversal section through the cerebellum, at the level of the dendate and fastigial nuclei, showed large areas of congestion, perivascular hemorrhages, often extensive, vacuolatlon and edema. Those lesions were distributed in the grey matter, in the area of the nuclei, and did not ex tend into the convolutions of the cerebellum. The majority of hemorrhagic foci could be associated with a vessel. Some blood vessel walls showed proliferation of endothelial cells; others showed necrotic changes, pyknosls and karyorrhexis of 38 the endothelial cells. Pale eosinophilic material around some vessels was interpreted as edema and appeared to be dif fusing from the vessel into the adjacent parenchyma. Vacuo- lation was extensive throughout the gray substance. A few gitter cells were seen in areas of demyellnation. Peculiar homogeneous basophilic bodies were found immediately outside the walls of vessels, or less frequently in the brain paren chyma or inside capillaries. They were round (but a few were elongated) and measured 20 microns or less in diameter. There was no cuffing of the blood vessels and no neutrophils were seen in the parenchyma. A section through the pons did not show lesions. A transversal section through the inferior colliculi presented extensive hemorrhagic lesions (like those in the cerebellum) in both colliculi, extending down wards in the area of the teeto-olivary tracts• Numerous basophilic bodies, vacuolation and edema were also present. Sections through the thalamus, cerebrum, hippocampus and striatum showed only a few congested vessels.

Discussion: The signs and lesions of toxicity presented by this dog were similar to those described by Oastler and Fidler, after intravenous injections of autoclaved solutions of stil bamidine. Some of the objections of Sen Gupta,especially about the autoclaving of stilbamidine, are then refuted. Pig. 1- Dog 55-392 showing opisthotonos and extensor rigi dity.

Pig. 2- Superior colliculus of dog 55-69 showing perivas cular and parenchymal hemorrhage.

39 1*0

I f p* t* ii , . f * » TABLE 1. RESUME OP ALL TEST ANIMALS FOR STILBAMIDINE TOXICITY

Total Total Blastomyces

Dog. So. Age/mo. Sex . Wt/k£ .. injections drug/mg inoculation Toxicity 55392 36 M 12,0 2 72.0 No Fatal 55316 18 M 10*0 5 55.0 No Fatal 55387 8 P 8.2 47 311.6 No None 541252 24 M 12.0 5 66.0 No Fatal 5569 6 P 8.2 5 73.8 No Fatal 55386 12 P 10.0 5 90.0 No Fatal 55314 18 M 10.0 3 75.0 No Fatal 551862 24 P 7.3 33 760.2 * None * 551863 24 M 6 *l 33 656.2 None 5580 24 F 11.8 2 47.2 Yes Fatal 551861 12 M 9 .5 3 57.0 Yes Fatal 551031 24 M 8.2 4 32.8 Yes None 551032 12 M 8.2 3 28.7 Yes None 551041 12 P 8.2 4 32.8 Yes None 551034 6 F 6,4 10 1*4.8 Yes None 551035 12 M 7.7 9 49.7 Yes None 551036 24 M 7*7 77 308.1 Yes None

inoculated with Blastomyces dermatitidis after the stilbamidine course. TABLE 2. RESUME OP ALL TEST ANIMALS FOR HYDROXYSTILBAMIDINE TOXICITY

Total Total Blastomyces Dor No, Age/mo• Sex Wt/kg injections drug/mg inoculation Toxicity

55313 12 M 11.0 1*6 808.5 No None 5568 8 P 8.2 59 819.6 * None 55391 2k M 11.0 k 82.5 No Fatal 55390 12 M 8.2 k 114.5 No Fatal 55388 12 P 7.3 k 102.2 No Fatal 55393 18 M 12.0 3 lii4.0 No Fatal 5590 8 M 10.0 117 4065.0 *** None 551037 12 M 5.0 5 37.5 Yes None 551038 12 M 8.2 k 49.2 Yes None 551039 12 P 6.8 k 40.8 Yes None 551070 12 M 12.0 10 177.0 Yes None 551071; 2k M 12.0 6 110.7 Yes None 551033 12 F 6.8 11 112.2 Yes None 551042 12 M 8.6 7 90.3 Yes None 551072 6 P 5.0 6 k5.° Yes None 551067 36 P 6.0 77 6 8 4 .O Yes None

^inoculated with Blastomyces dermatitidis after end of drug course, "^inoculated with Blastomyces dermatitidis after 1020 mg of hydroxystilbamidine.

£ B) Effects of high doses of stilbamldine

Objectives: This experiment was conducted concurrently with ex periment C, using doses about twice as large. The objective was to determine the effects on dogs of stllbamidine at a level equivalent to the usual therapeutic doses of man. 9 Since Bichowsky noticed that ribose and desoxyribose nucleic acids inhibited the in vitro bacteriostasia of stllbamidine, it has generally been recommended to use a diet low in animal proteins. Such a diet has been used in dog 55-311+ to study its possible effects on toxicity. Stllbamidine was always administered intramuscularly according to the schedule listed in Table 3. Dog 55-69 was a 6-month old female weighing 8,2 kg. Ten minutes after receiving 0,02 mg of atropine, the dog was given 2 mg/kg of stllbamidine isethionatej on succeeding days the dose was respectively 3»0> 1*5> 1»5» and 1»0 mg/kg (total dose 73*8

Clinical findings: No unusual signs were noticed during the first five days. On the sixth day at 9:00 a.m., the dog was found dead, lying on the left side, the head in opisthotonos, legs ex tended laterally, and the mouth open. Evidence in the cage suggested that the dog had been in convulsions before death. TABLE 3. EFFECTS OF HIGH DOSES OF STILBAMIDINE

Dav 1 2 3 ii 5 6 7 8 9 10 Body T otal dose/mg _ _ Dog No# wt/kg Dose mg/kg 72.0 55392 12.0 3 3 NX 73.3 5569 8.2 2 3 1.5 1.5 1 D NX 90.0 55386 10.0 2 3 1.5 1.5 IN N N N N 75.0 553ik* 10.0 2 3 2 .5 ND

D - died from drug to x ic ity . N - nervous signs. X - euthanasia because of irreversible CNS disease. - received a low protein diet.

4=~ 4=- h$

Necropsy findings: The lungs, spleen, tonsils and kidneys were congested. The stomach contained some black material, positive when tested for the presence of blood. The liver showed a few yellow streaks, extending deep Into the parenchyma. No inflammation was found at the site of the drug injections in the thighs. Plre-red blood covered the meninges. A mid- sagittal section through the brain, midbrain, cerebellum and medulla, disclosed a large hemorrhagic and necrotic area, 1 by 0.75 cm, formed by the left inferior and superior eolliculi. An identical lesion was found on the right side, but it was located slightly more externally. Some pin point hemorrhages could be seen in the medulla of the cere bellum, the brain stem, in the area of the inferior olive, the hypoglossal nerve and the central gray matter of the medulla oblongata.

Histopathology: Lesions in the inferior and superior eolliculi were extensive (Pig. 2). They consisted of focal hemorrhages, some coalescent, sometimes with a visible necrotic vessel in the center. Neutrophils were scattered throughout the lesions but more numerous in the perivascular spaces. Those lesions extended downward into the tegmentum; the area of the red nuclei showed no lesions other than some proteinaceous material around the blood vessels. Neurons k*> throughout the lesions were undergoing degeneration; demyelination was extensive. In addition to the quadrigeminal bodies, the brachium conjunctivum, the central tegmental fasciculus and the spinotectal tract were involved. The cerebellum showed the same type of extensive lesions, but only in its medulla. Both dendate and fastlgial nuclei were involved. Sections through the pons and brain stem showed small perivascular hemorrhages along the nucleus gracilis. Some neuronal degeneration was seen in the hypoglossal nucleus, and extensive degeneration of neurons, demyelination, and congestion of capillaries were found in the area of the area of the cuneate nucleus. The area of the inferior olive also showed some petechial hemorrhages. Other sections of the brain were essentially normal except for some satellltosis in the striatum and spinal cord, and some neuronophagla in a lumbar segment of the cord. Dog 55-386 (1 year-old female weighing 10 kg) received daily injections of 2.0, 3*0, 1*5* 1*5* and 1.0 mg/ kg of stllbamidine Isethionate, reaching a total dose of 90 mg.

Clinical findings: During the first four days, the dog showed neither nervous signs nor change in habitus and appetite. After the injection of atropine, the pulse rate jumped from a normal 1+7

of 90/min. to 120/min., end after the injection of stll bamidine the rate was 220/min. the second day, and 160/min. the third, fourth and fifth day. The dog received its last injection at 3:30 p.m. on the fifth day and at 3*1+5 she ate and vomited immediate ly. At lj.:00 p.m., she was in a frenzy, repeatedly tunned to one side or the other, made running movements, tried to get up and then fell backward hitting the head on the floor and walls. She continually snapped her jaws, bit the iron bars of the cage and salivated profusely; the lips were bleeding. The head and legs were kept extended. The signs so closely mimicked those of rabies that a veterinarian unfamiliar with stilbamidine toxicity would surely consider a diagnosis of rabies. At 6:00 p.m., the same day, the dog was calm, re mained in a standing position, but leanadc against the wall and swayed laterally. The pupils responded well to light and placing, hopping, and righting reactions were present. The next day, at 9:00 a.m., the animal was lying on the side, with neck and legs extended but not rigid. When propped in a standing position the dog stood unsteadily and when we crossed h6n front legs she was unable to correct their position. If we supported the dog and helped her to walk, the right front leg would not flex and was thrown up very high; the left front leg would not flex and dragged the 14.8 ground. Placing reactions were nil, hopping reactions were slow in the front legs, and nil in the hindlegs. Righting reactions were slow; corneal reflex was present. Head was kept well extended at all time. At 8:00 p.m., the dog was lying on its left side in opisthotonos (head and tail pulled dorsally) and extensor rigidity. She did not appear to be in pain and occasionally made bicycling movements. On the eighth day, the dog was in the same state, but weaker; it was euthanized on the ninth day.

Necropsy findings: Besides some enteritis, apparently due to hookworms, contusions of the muscles of the head, and some emphysema throughout the lungs, the only other gross significant find ings were in the brain. A mid-sagittal section of the brain showed a necrotic and hemorrhagic focus, about 6 by £ mm., in the inferior eolliculi, and a similar lesion about 1+ x ij. mm., in the medulla of the cerebellum. A sagittal section slightly to the right of midline disclosed lesions in both eolliculi, and in the cerebellum.

Hi s topathology: Lesions in the medulla of the cerebellum, including the dendate and fastigial nuclei, consisted of necrotic vessels, sludging of blood, perivascular and diffuse 1+9 hemorrhages, demyelination, neutrophils, a few gitter cells, cuffing of the larger vessels with neutrophils, plasma cells, lymphocytes and glia cells, and neuronal degeneration. Several degenerating Purkinge cells were also seen. Similar bilateral lesions were seen in the superior colliculus and tegmentum; round areas of demyelination was demonstrated in the tegmentum. The red and oculomotor nuclei were not affected, but the optic stratum and spino tectal tracts were extensively involved. The lesions were of the same type, extensive, but well limited in the inferior eolliculi. A section of the pons, at the level of the trapezoid body, showed some necrotic capillaries containing sludged blood in the area of the spinal tracts and nuclei of the fifth nerve, accompanied by neutrophils, demyelination, and neuronal degeneration. Similar lesions were seen in the superior vestibular nuclei, but the nuclei of the abducens nerve were apparently free of injury. We saw satellitosis and congestion in several other locations, including the thalamus, caudate and lenticular nuclei, the cerebrum and cord at several levels, but no lesion like those described above.

Dog 55-311l (1^-year old male weighing 10 kg) re ceived on three consecutive days, respectively, 2.0, 3*0» and 2.5 mg of stllbamidine isethionate. It received a total of 75 mg of the drug, and was on the same low-protein diet as dog 51+-1252. 50

Clinical findings: The dog remained healthy during the three days of treatment. After atropine, the pulse went from 80/mln to 90/min and up to 110/min after stilbamidine. Treatment was discontinued on the fourth day when at 12:00 p.m., the dog was depressed, but his reflexes and reactions were normal. At 1:1*5 p.m.j he was salivating profusely, walked incoordi nated and deviated to the right. He was able to walk up an 8-inch step, but fell when going down. A sudden noise caused violent maniacal excitement or frenzy with barking, biting or chewing, and rolling over and over toward the right for several minutes. He had periods of depression interrupted by convulsions. His legs and neck were not ex tended, and showed no extensor rigidity like other dogs described previously. The extensor postural thrust was deficient. At 3:00 p.m., we took a movie of the dog and by i*:00 p.m. he was unable to stand even when assisted. He rolled to the right, bicycling and lunging forward simul taneously. He died quietly at 5*00 p.m. the same day.

Necropsy findings: Pulmonary emphysema and hemorrhage in the jejunum and ileum seemed trivial in comparison with the extensive gross lesions in the brain. The medullary portion of the cerebellum was not hemorrhagic like in the preceding dogs, but instead it had a softened appearance and yellowish 51 discoloration. The brain stem also showed similar gross lesions. The pons and inferior and superior eolliculi showed bilateral hemorrhagic foci which were much more extensive on the left than the right side. An hemorrhagic focus in the right isthmic region of the pons, just back of the inferior colliculus, measured 5 by 4 mm; the similar lesion on the left side measured 2 by 2 mm.

Histopathology: The brain stem, at the level of the inferior olive, and the medulla of the cerebellum showed some congestion, a few perivascular hemorrhages, swollen myelin sheaths, ex tensive vacuolation and demyelation. Sections through the pons showed extensive, perivascular hemorrhages of recent origin, which were bilateral but more extensive on the left side. Some vessels were necrotic, others showed only sludged blood and were surrounded by eosinophilic material. There was slight endothelial proliferation in a few vessels. Neuronal degeneration was evident, but infiltration of neutrophils (seen in previous cases) was not demonstrable. The most severely affected structures were the left pontal reticular nucleus, medial reticular spinal tracts, central tegmental fasciculus, superior eolliculi, the in ferior eolliculi and tegmentum; lesions were bilateral, but more extensive on the left side. Several structures 52 included, in the affected areas were the oolliculi, optic, spinotectal, tecto-olivary, tectospinal, thalamo-olivary, rubrospinal and crossed vestlbulo-mesencephalic tracts, the oculo-motor and the lateral and deep tegmental nuclei. We saw some congestion, interstitial hemorrhages and emphysema throughout the lungs, extensive congestion and some hemorrhages in the cortex and medulla of both adrenal glands. The site of injection was characterized by frag mentation of muscular bundles with hyalin degeneration, edema and extensive infiltration of lymphocytes and macrophages.

Discussion: No dog of this group proved to be resistant to the drug toxicity; two dogs died about five hours after the first clinical signs, and it was necessary to euthanize the third dog after four days. It is significant to note that dog 55-386 of this experiment (B) and 55-316 of the following experiment (C), which lived four days after the apparition of clinical signs, presented diffuse infiltration of neutfcophils throughout the brain parenchyma; the latter dog also showing gitter cells. The dogs that died soon after the first clinical

signs like 55-69 and 5 5 -3 1 4 > showed only slight perivascular infiltration of neutrophils without invasion of the parenchyma. 53

Dog 55-314 of this experiment (B) and dog 54"1252 of experiment (C) were on a low protein diet. They differed from the other animals in having no extensor rigidity, neck rigidity or opisthotonos at any time before death. This difference in behavior could only be explained by the exten sive lesions found in the lateral vestibular nuclei of dog 54"1252 and the slight lesions in the same area in dog 55-314» since this nucleus has a function of maintaining or increasing extensor tonus of the muscles.

C ) Effects of low doses of stllbamidine

Objectives: Our immediate objective was to evaluate the toxic effects, if any, of doses of stllbamidine equivalent to the lowest therapeutic doses used in man. This information was necessary before a treatment schedule could be established for dogs. The dosage schedule for each dog is listed in Table 4» As in experiment (B), one animal (dog 54"1252) re ceived a diet low in animal protein.

Dog 55-316 weighed 10 kg (male, 18 months) and after a subcutaneous injection of 0.02 mg of atropine was given 10 mg of stllbamidine intramuscularly. No immediate reaction was noted. He received 20 mg of the drug on the second day, and 10 mg on the third and fourth day. Immediately after re ceiving only 5 mg of stllbamidine on the fifth day, he showed TABLE If.. EFFECTS OF LOW DOSES OF STILBAMIDINE

Day 1 2 3 k 5 6 7 8 ? 10 Body T otal Dog No. wt/kg Dose mg/kg ...... dose/mg 55316 10.0 1 2 l l o .5 NNN NX 55.0 55387 6.2 1 2 l l o .5 3.5 0.5 On alternate 311.6 day to day 26, daily to day 38, 1 mg.kg daily to day 56, 1.5 mg/kg d a ily to day 59, E 5ij.l2£2tf 12.0 1 2 l l o .5 NX 66.0

D - died from drug toxicity. E - euthanasia, no signs of toxicity. N - nervous signs. X - euthanasia because of irreversible CNS disease, -if - received a low protein diet.

vn 4=- Pig.3- Inferior colliculus of dog 55-316 (stllbamidine toxicity). Note the proteinaceous material diffusing from the capillaries.

Fig. 4- Inferior colliculus of dog 55-316 (stllbamidine toxicity). Note the necrosis, the gitter cells and the inclusion: bodies in some cells. 56 generalized tremors Tor a few minutes then laid down and re mained unusually quiet for a few hours. At the time of this reaction a total dose of 55 nig stllbamidine had been given and treatment was discontinued. No neurologic dis turbances could be demonstrated. At 9:00 a.m. on the seventh day, the dog was quieter than usual, but after examination all reflexes were judged normal. At 3*15 P«m« the same day he was swaying in the cage and his head was continually moving from side to side or up and down. The neck stayed well extended and so rigid that when we pressed downward on his head the hind feet raised from the floor. He was sway ing from side to side and the front legs were abducted to maintain a standing position. At times equilibrium was lost and he fell to his side; his head often hit the floor. When sitting, his head had a tendency to extend upward and some times, this made him lose his equilibrium and fall backward. The front left foot was the only one with good placing re actions. Hopping reactions were slow, late and hyper metric. Righting reactions were overactive. Corneal re flexes were present but slow and he seemed unable to recog nize objects as he looked straight ahead in a glassy stare. The next day, (third day after discontinuing treat ment) he laid on his side and was unable to stand. Occasionally he could stand on his front feet and then turn over on his back. When disturbed in the cage, he started 57 wild bicycling movements and propelled himself backward with the help of the walls and bars. When we held him in a standing position, he struggled as if to escape; when freed he reared upon his hind legs and fell backward and hit the floor very hard; holding the dog so his feet did not touch the floor incited continual gyration of his head and neck; the eyes turned upward and backward* No placing or hopping reaction could be elicited* His back was much more arched so the extended hind legs would touch the front legs. The front legs were more rigid than the hind legs and the elbows could be flexed only with much manual effort; the metacarpal and phalangeal articulation moved with ease. When we tried to hold him in a standing position, the hind legs slipped forward under him and the front legs became abducted. The front toes were knuckling. The dog was apparently blind, but not deaf. Corneal response (wink) could be elicited* A movie of the dog was taken the same day. On the tenth day, the dog was in the same state, but much weaker and breathing with the mouth open. No placing, hopping, or righting reaction could be demonstrated. For humane reasons he was destroyed and necropsied on the same day.

Necropsy findings: The lungs showed areas of emphysema and congestion.

All other significant findings were in the central nervous 58

system. The right inferior colliculus disclosed a large hemorrhagic focus, I4. mm in diameter. The cerebellum showed some punctate hemorrhages in the nuclei zona, accompanied by a yellowish discoloration and an apparent softening of the area.

Histopathology: The kidneys contained proteinaceous material in the glomeruli which was thought to be indicative of impaired glomerular filtration. An eye presented some congestion around the optic nerve, and some proliferation of the external cellular layer of the retina. All other significant lesions were in the brain. A transverse section through the cerebel lum revealed irregular staining in the medullary portion, dark patches of gliosis, foci of hemorrhage and a somewhat cribiform ground substance. At a higher magnification micro glia were seen infiltrating the arteriolar perivascular spaces and the adjacent parenchyma. The lumina of some arterioles and capillaries were occluded by purplish red bodies. Surrounding a few of these vessels, and in some others not occluded at this level, was a protein-like, acidophilic material (Pig. 3 ), which proved to be slightly positive to the periodic acid-Schiff reaction. Several of the blood vessels contained thrombus-like material and there was karyorrhexis of the endothelial nuclei indicating necrosis. 59

The nucleus and granular cytoplasm of some large neurons were faintly stained. Several empty spaces the size of neurons were seen, thus indicating complete loss of some nerve cells. Round purplish-red bodies, about 15 microns in diameter, and postive to the periodic acid-Schiff reaction were found in the medulla and In the granular layer of the folia of the cerebellum. Gitter cells with granular cytoplasm were associated with areas of demyelination. Extensive lesions in the cerebellum were bilateral and limited to the medulla, in cluding the dendate and fastigial nuclei. A section at the same level through the pons, disclosed some staining variations of the neurons In the area of the nuclei of the fifth nerve and superior vestibular nuclei. This area also showed numerous cavities, about the size of a neuron. It was difficult to believe that those cavities had contained edema fluid since the perivascular spaces of the area were not distended. A few gitter cells and some eosinophilic protein globules were also seen. The lesions were slightly more severe on the right side. Except for the external brachlum a transverse section through the right inferior colliculus was entirely affected. Some areas were hemorrhagic, others which were invaded by glia cells, and there were also areas of necrosis which contained numerous gitter cells. 60

Hemorrhages in the central area were diffuse and the vessels from which the hemorrhage originated were often hidden or necrotic. At the periphery the lesions were iden tical with those described in the cerebellum, i.e., solid perivascular hemorrhages, thrombosis or sludging in the vessels, and a very prominent necrosis of the vessel walla. The same purplish-red material was seen perivascu lar ly and magenta colored globules (£-25 microns in diameter) were found (Fig. 5) intra and extracellularly. As seen in Fig. 4 large gitter cells were very numerous and their cyto plasm sometimes contained large vacuoles, erythrocytes, or eosinophilic bodies. There was extensive demyelination. The few neurons seen were undergoing degeneration with shrinking of the cytoplasm and karyolysis of the nucleus. The larger arterioles showed proliferation of their endothe lial cells and microglia and gitter cells were seen in perivascular spaces. The superior eolliculi contained no lesions, but lateral to the descending limb of the brachium conjunctivum and extending in the medial lemniscus and rubrospinal tract was an area of rarefaction with numerous microglia, some of which had phagocytized debris. Several gitter cells con taining hemosiderin were seen in the perivascular space of a large vessel, and the surrounding nerve tissue was demyelinate d• 61

Transverse sections through other parts of the brain and spinal cord (especially the striatum) did not show hemorrhages or extensive necrosis but instead there was satellltosis, neuronophagia and degeneration of astrocytes. In this dog, as well as in the previous dogs, it was concluded that stllbamidine caused acute necrotizing hemorrhagic encephalopathy primarily in the medulla of the cerebellum, the adjacent pons, and the midbrain. Dog $387 (8-month-old female collie dog, weighing 8.2 kg) received stllbamidine the same as dog 55-318 for the first five days. Then, according to Table 1|, during the entire treatment period (total dosage of 311*8 mg) there were no nervous signs, change in behavior or other signs of toxicity, we decided to euthanize him. Microscopic examination of the tissues revealed no significant changes so it was concluded that stllbamidine at the dosage given was tolerated without toxic effect. Dog 5U-1252 (2-year-old male, weight 12 kg) received the same dose/kg of stllbamidine as dog 55-318, and a total of 66 mg. This dog was maintained on a low-protein diet to determine if the toxicity of stllbamidine could be influ enced by low protein intake.

Clinical findings: On the sixth day, at 8:00 a.m., the dog was found lying on his left side. There was no evidence of struggling. 62

In contrast to dogs 55-392 and 55-316, this animal's neck was not rigid, and there was no opisthotonos. The legs were extended laterally but did not present extensor rigidity. When held in a standing position with the front feet careful ly placed on the floor, the front legs stiffened in extensor rigidity; if the phalanges touched the floor first, the metacarpal articulation was flexed. Pulse and respiration were normal, but the tempera ture was down to 99*3° from an average normal of 101°. Placing, hopping, and righting reactions could not be elicited. Sensibility to touch and prickling was present in the four foot pads and in the distribution area of the trigeminal nerve. Corneal reflex was absent. The nictitat ing membranes were closed to a mere slit, and the conjunc tiva covered half of the cornea. Pupils were rather small and did not respond to light. A whistle or a sudden noise did not provoke any response in the dog. At 12:30 p.m. the dog was in the same general state, but more comatose with the eyes nearly closed; when the membrana nictitans were retracted, the eyes were rotated up ward and outward. His extremities were very cold, the temperature was still 99#3°» and. euthanasia was performed.

Necropsy findings: The lungs showed congestion, edema and emphysema. Some free blood was found In the small intestine and black 63 feces were seen In the cecum and colon. No evidence of Inflammation was found at the sites of Injection. The brain had a flattened appearance. The cerebel lum was farther from the cerebral hemispheres than normal, and two hemorrhagic foci, nearly 1 cm In diameter, were seen under the surface of both inferior eolliculi. On cross- section through the inferior eolliculi, those hemorrhagic foci measured 1 cm by 5 mm. Bilateral, softened, hemorrhagic foci were also seen in the superior eolliculi, the tegmentum, the cerebellar medulla, the pons (by extension from the cerebellum) and the superior upper medulla.

Microscopic findings: Both inferior eolliculi were a mass of softened necrotic tissue and blood. The lesions extended through the pons, Including the lateral lemniscus. At this level, we saw focal and diffuse hemorrhages of non-hemolyzed red cells, congestion, neutrophils infiltrating the parenchyma or accompanying glia cells around the larger blood vessels, severe vacuolation and neuronal degeneration. Some myelin sheath were swollen and demyelination was severe. We found a few basophilic bodies similar to those described in dog 55*392 and 55*316. The main structures affected were the

Inferior colliculus, the lateral lemniscus, the deep teg mental nucleus, and the tecto-olivary, tecto-spinal and rubrospinal tracts. 61*

A transverse section through the superior eolliculi and tegmentum showed the same type of lesions affecting the optic stratum and spinotectal tract of both eolliculi, the optic, red and tegmental nuclei, the tecto-olivary tract and the decussation of the rubrospinal tracts. Similar, severe lesions, were found in the lower pons including the area containing the lateral and medial vestibu lar nuclei. Extensive vacuolation was present in both superior oliVes and in the fibers between them, extending from the acoustic striae and medial lemniscus. The medulla, at the level of the upper part of the inferior olive, showed diffuse hemorrhages with necrosis of the ground substance. Several swollen myelin sheaths were seen, and demyelination was extensive. There was prolifera tion of endothelial cells, and perivascular cuffing with glia and neutrophils. Neurons in the immediate vicinity of the hemorrhages were destroyed, and satellitosis was present throughout. All lesions were bilateral but hemorrhages were more extensive on the left side. A large number of nuclei and tracts were affected: included were the descending vestibular nucleus, the solitary fasciculus, the uncinate fasciculus of the cerebel lum, the nucleus reticularis of the medulla, the medial reticularis spinal tract, the tecto-spinal, direct vestibulo spinal, and tecto-olivary tracts. 65

Similar lesions were very extensive in the medullary portion of the cerebellum; Purkinje cells were degenerated. The toxic disease was classified as acute necrotiz ing hemorrhagic encephalopathy. Clinical signs somewhat different from other dogs in this experiment was explained by more diffuse distribution of lesions and extensive in volvement of the medulla.

Discussion: Prom this experiment we must conclude that stilbamidine cannot be used in dogs at the lowest therapeutic level used in man. It is interesting to note that dog 55-367 did. not show central nervous system disturbances after a total of 311,6 mg of stilbamidine given in fifty-nine days, while dogs 55-316 and 5U"1252 had to be euthanized after receiving as little as 55 and. 66 mg of the drug. Apparently some dogs, for unknown reasons, are resistant to this toxic effect of the drug. Other salient features were the apparition of serious nervous disturbances in dogs 55-392 and 55-316 about forty-eight hours after the last treatment, and the onset of neurologic signs in dog 54“1252 twenty-four hours after a reduced dose of the drug. (Table l\.). These findings dis prove the theory that the toxic effects are caused by endogenous histamine released by stilbamidine. 66

9 The theory inferred by Bichowsky in which the diamidines combine with nucleic acids or the nucleoproteins and thus interfere with the utilization of an ’’essential metabolite" or enzymatic pathways seems more plausible to us. The manifestation of the clinical signs after the dose has been lowered or stopped and the ability of stilbamidine to form protein complexes, suggests the possibility of an antigen-antibody reaction.

D) Effects of high doses of hydroxystilbamidine

Objectives: It is recognized that hydroxystilbamidine in man does not produce the late fifth nerve neuropathy which is a common sequel to stilbamidine therapy. Since no report could be found on the use of hydroxystilbamidine in dogs it seemed important to test its toxicity for this species and to es tablish a suitable treatment schedule. In the first experi ment (D) hydroxystilbamidine was given intramuscularly to three dogs In doses slightly higher than the usual human therapeutic doses. Dog 5390 (1-year-old male dog weighing 8.2 kg) re ceived hydroxystilbamidine at the rate of 3*0, J+.0, 5*0, and 2 mg/kg on four con secutive days r e s p e c tiv e ly , (to t a l lllj..5> mg.) No unusual signs were noticed but on the fifth day at 8 a.m ., I t was found dead. The sta te of the bedding in i t s TABLE 5. EFFECTS OF HIGH AND LOW DOSES OF HYDROXYSTILBAMIDINE

Day 1 2 3 4 5 6 7 Body T otal Dog No. wt/kg Dose mg/kg dose/mg

55390 8.2 3 4 5 2 D 114.5 55388 7.3 3 4 5 2 D 102.2 55393* 11.0 3 4 5 NX 11+4.0 55313 11.0 1 2 3 1.5 1.5 1.5 On alternate day to day 808.5 34, daily to day 56, 2 mg/kg daily to day 62, E 5588 8.2 1 2 3 1.5 1 .5 1 .5 Same as 55313 to day 62, 819.6 2.5 mg/kg daily today 71 55391* 11.0 1 2 3 1.5 N D 82.5

D - died from drug toxicity. E - euthanasia, no signs of toxicity. N - nervous signs. X - euthanasia because of irreversible CNS disease. - received a low protein diet. Pig. 5 - Dog 55-390. Sagittal section of brain showing extensive hemorrhagic encephalomalacia of the colliculi which compresses the cerebel lum. Hemorrhage is also seen in the medullary portion of the cerebellum and the upper medulla oblongata. These lesions are the result of hydro xy stilbami dine toxicity.

Pig. 6- Dog 55-1861. Sagittal section of brain showing hemorrhagic encephalomalacia of the colliculi and hemorrhage in the medullary portion of the cerebellum, resulting from stilbamidine

toxicity.

68 69

P ig . 5 70 cage indicated that the dog had convulsions before death.

Necropsy findings: Lungs showed edema, congestion and hemorrhages, and the kidneys were congested. Some blood was seen in the duo denal portion of the stomach, and some black mucoid feces were present in the jejunum and ileum. Focal interstitial hemorrhages and edema was present at the site of injection in the thigh muscles. A sagittal section through the brain showed that all four colliculi were reduced to a pulpy, hemorrhagic mass (Fig. 5)* This lesion caused compression on the anterior lobes of the cerebellum. Large bilateral hemorrhagic areas, 5 by 3 n®1* were seen in the medulla of the cerebellum. A smaller bilateral area of hemorrhages and necrosis was seen in the floor of the fourth ventricle and petechial hemorrhages were found in the pons.

Microscopic findings: Microscopically, the lesions were quantitatively and qualitatively similar to those produced by a lower dose of hydroxystilbamidine and by all levels of stilbamidine. The dendate nuclei showed hemorrhages and vacuolation. These lesions extended to the areas of the medial vestibular nuclei and the medial longitudinal fasciculus. There were focal hemorrhages in the pons, and slight satellitosis throughout the brain. The colliculi presented extensive 71 recent coalescent focal hemorrhages, with necrosis of the vessel walls, vacuolation, cuffing of the larger blood vessels, neutrophilic infiltration, neuronal degeneration and satellitosis. Those lesions extended beyond the brachium conjunctivum into the area of the central acoustic, tecto- olivary and rubrospinal tract. Dog 55-388 (1-year-old female beagle weighing 7»3 kg) received the same relative dosage of hydroxystilbamidine as dog 55-390, totalling 102.2 mg.

Clinical findings: No toxicity was apparent until the fourth day when, one hour after treatment, she became weak, walked with un- certainity, and the hind legs were abducted. Because the phalanges of the left hind foot could not be extended she walked on their dorsal surface (knuckled). The only abnor mality detected upon neurologic examination was failure to react to a needle prick through the left hind leg foot pad; although pressure in the same area caused retraction. A few hours later, those signs disappeared. On the next day, at 8:00 a.m., we found the dog dead. Its head was In opistho tonos, and the tail curved upward; the bedding was not dis turbed, indicating death without prior convulsions. 72

Necropsy findings: We found congestion and hemorrhages in the lungs, and congestion in the kidneys, liver, tonsils and uterus. Edema and interstitial hemorrhages were present between the thigh muscles. Extensive hemorrhagic brain lesions were similar in character and distribution to those seen in the previous dog.

Microscopic findings: Severe lesions, identical in nature to those described before, were found bilaterally in the medulla of the cerebellum, in the colliculi, the area of the spino tectal tract, the oculomotor nucleus and the central teg mental fasciculus. The red nucleus showed slight satellitosis. Lesions were also found in the area of the tecto- olivary tract, the medial vestibular nucleus, and to a lesser degree, in the area of the hypoglossal and gracilis nuclei, and the inferior olive. Other structures of the brain showed slight congestion and satellitosis. Dog 55-393 (1.5-year-old male weighing 12 kg) was maintained on a relatively low protein diet. It received, on four consecutive days respectively, 3t 4 > 5 mg/kg of hydroxystilbamidine, totalling 144 mg.

Clinical findings: At noon, on the fourth day, the dog was in convul sions. The animal rolled continually sometimes to the left 73 side at other times to the right side for 30 min. He then became quieter, rolling only when excited. The animal pre sented a very severe dyspnea, the neck was In opisthotonos, the tail curved upward, and his legs were In extensor rigidity. His pupils were dilated but reacted readily to light stimulus. For humane reasons the dog was destroyed at 3:00 p.m. the same day.

Necropsy findings: The lungs showed emphysema and congestion. The brain was not as extensively involved as in most dogs seen previously. The only gross lesions were three small hemorrhagic foci in the medulla of the cerebellum; one located on the right side, two on the left side. No gross lesions were visible in the colliculi. Histopathology: The lesions in the cerebellum were extensive and similar to those previously described. Other structures of the brain were essentially free of lesions. Acute necro tizing hemorrhagic encephalopathy seen in this dog further emphasized that 2-hydroxystilbamidine produces lesions In dogs identical to those caused by stilbamidine.

Discussion: All three dogs in this trial died after treatments; the dog on a low-protein diet was first to show signs. 7b

E) Effects of low doses of hydroxystilbamidine

Objectives: Concurrently with the last experiment, having the same objectives in mind, hydroxystilbamidine was tested for toxicity at doses comparable to about half the human dose. Dog 55-313 (1-year-old male dog of mixed breeding, weighing 11 kg) received on five consecutive days respective ly, 1.0, 2.0, 3«0i 1.5* and 1*5 mg of 2-hydroxystilbamidine per kg body weight. Thereafter 1.5 mg/kg was given every other day for a total of li+ injections. Since no abnormal signs were elicited we decided to continue administration of the drug at the daily rate of 1.5 mg/kg for twenty-one days and 2 mg/kg daily, for six more days. During this entire course, the dog received a total of 808.5 mg of hydroxy stilbamidine. No abnormal clinical signs or change in habitus was noticed. During this experiment, the heart rate became slightly lower than normal after atropine, and slightly above normal after hydroxystilbamidine. The dog was destroyed sixty-two days after the beginning of the treatment.

Necropsy findings: No gross lesions were found. 75

Microscopic findings: No significant lesions were found in the brain and spinal cord, The adrenal glands showed some congestion and hemorrhages in the zona reticularis and medulla. The liver presented albuminous degeneration, and bile retention. At the site of the intramuscular injections there were macro phages loaded with hemosiderin, and plasma cells. The surface of the muscles showed hyaline bundles, edema, fibro plasia, histiocytes, plasma cells and lymphocytes. This in filtration did not extend deep into the muscles. Dog month-old female collie weighing 8.2 kg) was a llttermate of dog 55-387 already described, and dog 55-90 in experiment (P). Dog 55-88 received the same pro portional dosage of hydroxystilbamidine during the first seven days as dog 55-313* The dose and schedule of treat ment are listed in Table l±. Within a period of seventy-one days the dog received a total of 819.6 mg of 2-hydroxystilbamidine. She remained in good health with no signs or change of habitus. Dog 55-391 (2-year-old male, weight 11 kg) was main tained on the same relatively low protein diet as dogs 5U-1252 and 55-314.

Clinical findings: This dog received 1.0, 2.0, 3*0 and 1.5 mg/kg of body weight of hydroxystilbamidine on four consecutive days, 76 respectively, and on the fifth day, at 1;:00 p.m., it pre sented central nervous disturbances. Consequently, the in jections were stopped after a total dosage of 62.5 mg. When seen at J+tOO p.m., the dog was walking with the front legs extended, rigid, and slightly abducted. He tended to sway from side to side. At ij.:i|5 p.m., he stood with great difficulty, continually rocking from one side to the other as well as forward and backward. Tremors were observed in the thigh muscles. He struggled backward instead of forward, trailing his abducted legs on the ground. Some times he lost his balance and turned over backward; when ex cited, he not only turned over backward but also violently rolled to the left. In spite of his precarious equilibrium, he leaned against the wall in a persistent effort to remain standing. The dog also kept its tail curved upward and moaned continually. The placing and righting reactions were present, but the hopping reactions were absent in the hind legs. The dog responded to a needle-prick through its foot pads and the trigeminal area. The sense of hearing was also present. The eyes showed vertical nystagmus. Prom a normal mean of 101.2°F. its body temperature was down to 99.2°P. The sixth day, at 8:00 a.m., the dog was found dead, lying on its right side, the head in opisthotonos, tail erected, legs extended and jaws opened. The state of the 7? bedding indicated that convulsions had occurred before death.

Necropsy findings: The dog had lost its rigor mortis and had probably died during the night. Lymph nodes of the cervical region and the lungs presented congestion and hemorrhages. Con gestion was also found in the liver, kidneys, thyroids, parotids, prostate and mesenteric lymph nodes. Liver showed evidence of post-mortem degeneration. Bilateral hemorrhagic foci measuring 3 by 4 Dim were seen in the medulla of the cerebellum; the lesion on the right side was somewhat further forward. The tegmentum was diffusely petechiated and the thalamus and cerebrum were congested.

Microscopic findings: Congestion was found in most structures of the brain, but extensive hemorrhagic and necrotic lesions (like those produced by stilbamidine in experiments (B) and (C) ) were seen only in the medulla of the cerebellum.

Discussion: Hydroxystilbamidine was not toxic for two dogs (maintained on the basic control diet) which received the drug for an unusually long course, i,*©^ a total dose of 808.5 sixty-two days, and 819.6 mg in seventy-one days, 78 respectively. The same daily dose (mg/kg) given to the third dog, maintained on a low-protein diet, caused severe toxicity and death after I4. daily doses or a total of only 82.5 mg of hydroxystilbamidine. In the latter dog extensive lesions were seen in the medullary portion of the cerebellum. It is evident that the majority of clinical signs were attributable to vestibu lar and cerebellar disturbances, with a terminal decerebel- late rigidity. This experiment indicated that hydroxystil bamidine is toxic at relatively low doses when given to a dog on a diet low in animal protein. Although a low-protein diet reportedly increases the therapeutic effects of both drugs, experiments (B), (G), (D), and (E) indicate that this diet also increases the toxicity of the drugs or lowers the resistance of the dogs to their effects. Of the twelve dogs used (experiments (B), (G), (D), (E), three animals given low doses of the drugs were com pletely resistant even though they received a very high total dose, comparable to the total dose given man. Prom this limited data, it appears that a series of low doses protects against subsequent high doses. 79

F) Schedule modification and therapeutic trial using dosage of dlamidines

Objectives: Dogs which were resistant to the toxicity of the diamidines (55-68, 55-313, 55-38?) were treated at every other day after the critical first five days; therefore it appeared logical (in the search to discover a safe thera peutic dose) to try a new schedule with high doses of stilbamidine given every other day. The dose and schedule of treatment is given in Table 6. If any of these regimens could be followed without toxicity, it was planned to test it therapeutically in dogs with blastomycosis. Dog 55-90 (8-month-old male collie, weighing 10 kg) was a littermate of dogs 55-68 and 55-387* and received hydroxystilbamidine at the rate of 2 mg/kg for three consecu tive days, then every other day for seventeen days. There were no signs of toxicity so we gave the drug daily for twenty more days. No change in habitus or unusual clinical signs were noticed, so he was further challenged by a larger dosage, 2.5 mg/kg daily for seven days, and then 3 mg/ kg daily for another week. This challenge caused no signs of toxicity. Treatment was discontinued at this time when the dose totalled 1,020 mg during a course of fifty-four days. The dog remained healthy and was included in a later TABLE 6. SCHEDULE MODIFICATION OF A HIGH DOSAGE OF THE DIAMIDINES

Day 1 2 3 k 5 6 7 8 Body 1 Total Dog No . wt/kgl Dose mg/kg Dose/mg HYDROXYSTILBAMIDINE 5590 10.0 2 2 2 2 2 On alternate day to day 20, daily to day 4 0 , 2.5 daily to day 47* 3 daily to day Sk 1,020.0

STILBAMIDINE

551862 7.3 i 2 2 2 2 On alternate day to day 2 7 , daily to day 37, 3 daily to day I4.7 760.2 551863 8.3 2 2 2 2 Same as dog 551863 656.2 5580 11.8 * 2 2 N ND 1*7.2

551861 9.5 *2 2 2 N ND 57.0

D - died from drug toxicity. N - nervous signs. ■a - Blastomyces dermatitidis inoculation.

oo o 81 experiment, when after Intratracheal inoculation with Blasto myces dermatitidis, he received another course of hydroxy stilbamidine that brought its total dose to lj.,065 mg; no signs of toxicity were detected. Dog 55-1862. (2-year-old female, weight 7*3 kg) received stilbamidine at the rate of 2 mg/kg every other day for a period of twenty-seven days, and daily during ten sub sequent days. As no sign of toxicity was evident, we used 2.5 mg/kg daily for ten additional days when a total of 760.2 mg had been given in forty-seven days. She remained healthy after this course of stilbamidine. Dog 55-1863 (2-year-old male, weight 6.3 kg) received the same relative dosage of stilbamidine as dog 55-1862. The dog showed no evidence of toxicity although a total dose of 656.2 mg was given during the forty-seven day course. Dog 5 5 -8 0, (2-year-old female, weight 1 1 .8 kg) was inoculated intratracheally with 0.5 ml of packed yeast of Blastomyces dermatitidis. The same day, the dog received stilbamidine at the rate of 2 mg/kg. This dose was repeated on the third day but discontinued on the fifth day because the dog showed signs of toxicity, similar to those previously described. Next morning, (day 6 ), the dog was found dead in the cage after a total dose of lj.7«2 mg of stilbamidine. 82

Necropsy findings: Severe lesions in the train were identical to those caused by stilbamidine in previous experiments. A longitudi nal section showed all colliculi to be hemorrhagic and necrotic. A transverse section disclosed hemorrhages and necrosis in the medulla of the cerebellum, and a 0.5 hemorrhagic area in the pons. Pneumonia was attributed to Blastomyces.

Histopathology: Extensive perivascular hemorrhages, demyelination, vacuolation and cuffing of the larger blood vessels were seen in the area of the dendate and fastigial nuclei, in the colliculi and cerebral peduncle, including the oculomotor nucleus, the central tegmental fasciculus, the tegmental nucleus and the tecto-olivary tract. The red nuclei showed only vacuolation. Similar lesions were also found in a more posterior section of the pons. Dog 55-1861, (1-year-old male, weighing 9-5 kg) was also inoculated with Blastomyces dermatitidis, like dog 55“80» and received stilbamidine at the rate of 2 mg/kg on three alternate days. The animal presented clinical signs of toxicity on the seventh day of the experiment, after a total dosage of 57 mg. 83

Necropsy findings: The necropsy disclosed lesions (Pigs. 6, 7) nearly identical to those found in dog 55-80.

Histopathology: Lesions were identical to the ones described previous ly, and were found in the same general area (Pig. 8); they included the dendate and fastigial nuclei, the nucleus of the spinal tract of the fifth nerve, and the spinto-tectal tracts. Bilateral lesions were also observed in the collicu li, and the thalamo-olivary tracts. The reticular substance and the area of the vestibular spinal nucleus were also affected.

Discussion: The schedule of hydroxystilbamidine used for dog 55-90 was completely successful, since no toxicity signs were seen after a total dosage of 1,020 mg of the drug. However, we were sceptical in the interpretation of the results, since both his litter mates, dogs 55-08 and 55-387* were also found to be resistant to the drug toxicity. It is tempting to theorize that a certain innate factor had protected these three dogs against the toxic effects of the drugs. Both dogs 55-1862 and 55-1863 were not related genetically and survived a rather extensive course of stil bamidine, without showing any sign of toxicity. The schedule Pig. 7“ Transverse section of the cerebellum and pons

of dog 5 5 -1 8 6 1 , showing diffuse hemorrhage in the cerebellum and a more localized hemorrhagic focus in the vestibular area of the pons.

Fig. 8 - Low magnification of tissue in figure 7 . The light

gray areas, including the dendate, fastigial and vestibular nuclei, are characterized by vacuolation, demyelination, necrosis, peri vascular and parenchymal hemorrhage. H & E, 5 X

81*.

86 used had a certain peculiarity that should be noted: the doses were never lowered, but were always equal or higher than the last until they were stopped. This is different from the schedules used previously. Could the survivals point to an antigen-antibody reaction, since the antigen being kept constantly at a high level might hinder such a reaction? There were no signs of toxicity after the treat ment was stopped, when such an antigen-antibody reaction should have taken place. This explanation is refuted by the fact that diamidines are excreted very slowly from the body and are stored in diverse organs for long periods; this re serve of the drug might keep the antigen at a level high enough to hinder an antigen-antibody reaction. Again, we must mention that both dog 55“90 and another previous sur vivor, dog 55-68, remained completely healthy during the observation period of more than a year after the stilbamidine course. When the stilbamidine regimen proved non-toxic for two days in this experiment it appeared to be promising for therapeutic uses, and was tried on two dogs experimentally infected with Blastomyces dermatltidis. Unfortunately, these two animals succumbed to stilbamidine toxicity in six and eight days, respectively. We may conclude that either dogs 55-1862 and 55-1863 were naturally resistant to the drug toxicity, or the inoculation of Blastomyces dermatltidis 87 did impede in some way the advantages of the new schedule. The new dosage was not tested further in therapeutic trials. Although atropine was not used throughout this experiment, no disadvantage could be noted and it was not used in subsequent experiments.

G) Stilbamidine and hydroxystilbamidine for the treat ment of canine blastomycosis

Objectives: According to the results of the preceding experi ments we were forced to use, in the therapeutic trials on canine blastomycosis, doses of stilbamidine and hydroxystilbamidine comparable respectively to one-fourth and one- half the dose used in man. Six dogs received stilbamidine and ten dogs, hydroxystilbamidine. All animals were of mixed breeding and most were about 1-year old. We did not employ atropine before ad ministration of the drugs. Bass 55-1031, 55-1032, 55-101tl, 55-1035, and 55-1036, received stilbamidine according to Table 7» Five dogs died from acute blastomycosis in three- to nineteen days without showing sign of drug toxicity after receiving a total rang ing from 28.7 to 1*9.7 mg of stilbamidine. The sixth dog (55-1036) recovered from blastomycosis; there were no signs of drug toxicity although treatment was continued until day TABLE 7. STILBAMIDINE REGIMEN

8 11 13 15 1? Body Total Dog No, wt/kg Dose mg/kg Dose/mg 49 J3 *-* 2 o 551031 8.2 0.5 0.5 32.8 a B >1A 551032 8.2 2 0.5 » 28.7 urtc\j • «■> o 551041 8.2 2 0.5 0.5 32.8 Mfl ^

551034 6.4 2 0.5 0.5 0.5 1.5 0.5 0.5 0.5 0.5* 44.8 Q> £i~ 551035 7.7 2 0.5 0.5 0.5 ■>.5 0.5 0.5 0.5* 49.7 ° n u«» 6 551036 7.7 2 0.5 0.5 0.5 on alternate day to 308.1 ♦>trs rt • day 152 **o +> w d

* - died from acute blastomycosis.

CO CD 89

152 when he was destroyed for necropsy. A total of 308.1 mg of stilbamidine had been given.

£ogs $5-1037, 55-1038, 55-1039, 55-1071)., 55-1033, 55-101+2, 55-1072 and 55-1067. received hydroxystilbamidine according to Table 8. Eight dogs died from acute blasto mycosis in seven to twenty-one days, without showing any sign of drug toxicity. Dog 55-1067 survived and was treated for 150 days, receiving a total dose of 681+ mg. After 181 days of further observation without signs of any illness the dog was destroyed for laboratory study. Dog 55-90, which had received 1,020 mg of hydroxystilbamidine five months previously, was inoculated with Blastomyces dermatltidis. For seventy days, the dog was treated with a high dose of hydroxystilbamidine according to Table 8. No sign of toxicity was noticed and the dog was destroyed 275 days later. At necropsy all dogs in experiment(0) were free of gross and microscopic lesions ascribable to drug toxicity.

Discussion: The therapeutic results will be discussed later. It is evident that the drugs were well tolerated by all dogs at the dosage used. TABLE 8. HYDROXYSTILBAMIDINE REGIMEN

Dav 1 2 3 k 5 n ? is 9 I10 In |12 !■? Il7 T 8 19 L Body Total Dose/mg Doa No. Dose m a/k9 - ...... - wt/kg «

H 551037 5.0 1 2 3 1.5 1.5 * 37.5 * s > •UN k9.2 •HCVJ £51038 8.2 1 2 3 1.5 9 o 551039 6.8 1 2 3 1.5 * k0.8 * 177.0 fH 551070 12.0 1 2 3 1.5 t.5 1.5 1.5 1.5 1.5 1.5 • s > 55107k 12.3 1 2 3 1.5 1.5 1.5 # 110.7 l-l o • * 112.2 551033 6.8 I 2 3 1.5 t.5 1.5 1.5 1.5 1.5 1-5 1.5

a H H S 5510k2 8.6 1 2 3 1.5 1.5 1.5 1.5 90.3 i-tlTk t 551072 5.0 1 2 3 1.5 1.5 1.5 * k5.o O

551067 6.0 1 2 3 1.5 1.5 1.5 0 n alternate day t0 day 150 68k. 0 (1 (1 ml) Sub.Cut,

* - died from acute blastomycosis. v£> O 91

THE PATHOGENESIS OP STILBAMIDINE AND HYDROXYSTILBAMIDINE TOXICITY

The objectives of this study were as follows: 1. to further characterize the clinical and pathologi cal aspects of the syndrome caused by the stil bamidine and hydroxystilbamidine toxicity in dogs. 2. to test several theories that could possibly explain the pathogenesis and predilection of their toxic action for the central nervous system.

MATERIALS, METHODS AND RESULTS

Delay in toxicity after fatal dosage of diamldines

Objectives: Since the dogs used in the preceding experiments never showed nervous signs before the fourth day after the first treatment two dogs were each given one large dose to determine if there will be a latent period before the onset of nervous signs. One of the dogs will also be given trypan blue intravenously early after the apparition of nervous signs, to determine if the blood brain barrier is affected before the occurrence of massive hemorrhage. Dog 55-1266 (6-month-old female weighing I}, kg) re ceived a single 12 mg/kg dose of hydroxystilbamidine. A few seconds after the injection the mean blood pressure, taken TABLE 9. LIST OF ALL TEST ANIMALS FOR STUDY OF PATHOGENESIS

Animal No. Specie Age/Mo. Sex Weight/kg Experiment 55-1054 C 12 M 7.0 Course of Histamine Phosphate. 55-1266 C 6 F 4.0 Single Injection of Hydroxystilbamidine. A451 C 12 M 6.8 Cortisone with Stilbamidine. A483 F 24 F 4.0 Feline Thiamine Deficiency. A484 F 12 F 2.4 Feline Thiamine Deficiency. A4-85 F 12 F 3.4 Feline Thiamine Deficiency. A4-86 F 24 F 4.0 Feline Thiamine Deficiency. Aij.87 F 4 M 1.3 Feline Thiamine Deficiency. A488 F 5 M 1.75 Feline Thiamine Deficiency. A5i5 C 12 F 8.2 Canine Thiamine Deficiency. A516 c 12 F 7.3 Canine Thiamine Deficiency. A517 G 60 M 7.7 Canine Thiamine Deficiency. A518 C 12 F 9.5 Canine Thiamine Deficiency. A520 G 12 F 8.6 Cortisone with Hydroxystilbamidine. A696 C 12 F 6.4 Canine Thiamine Deficiency. A779 C 12 F 6.4 Antihistamine with Hydroxystilbamidine. A780 C 12 M 9.5 Cortisone with Stilbamidine. A781 C 12 F 9.0 Cortisone with Stilbamidine. A782 C 12 M 6.0 Cortisone with Hydroxystilbamidine. A783 c 12 M 10.0 Cortisone with Stilbamidine. A858 c 12 M 12.0 Antihistamine with Stilbamidine. A862 G 12 M 12.0 Trypan blue and 4 doses of Stilbamidine. A865 C 12 M 10.0 Trypan blue and 1 dose of Stilbamidine.

vO ro TABLE 10. DELAY IN TOXICITY AFTER FATAL DOSAGE OF DIAMIDINES

Total Day dose/mg Body Dog No. wt/kg 1 2 3 4 5 6

551266 4 Hydroxystilbamidine 48 N NX 46 A865 10 Stilbamidine 120 m x 120

A862 12 Stilbamidine 24 36 18 18 N*X 96

N - nervous signs. X - euthanasia because of irreversible CNS disease. * - trypan blue intravenously.

vO 94 at the femoral artery, declined from 132 to $0, but an hour later, rose to 120 mm. Hg. The animal did not show nervous signs until the fifth day, when it was found lying prostate on its right side. Her head was flexed and the tail was be tween her hind legs. After touching the dog or exciting it in some way, the head was thrown backward and the legs showed extensor rigidity. The dog was seen crawling on her sternum and hitting her head on the floor on either side; often the toes extended fan-like and she made bicycling movements. The animal was conscious but no placing or hopping reactions could be elicited; knuckling was seen in the front legs. The postural thrust and righting reflexes were demonstrated, and the animal responded to a needle prick through its paws and the trigeminal area. The next day, the dog was in a comatous state; the eyes were half closed and did not show reflexes. No rigidity was observed except in the lumbar spine. The dog retained sensitivity to a needle prick and the righting re actions were still present. Breathing became very dyspneic and euthanasia was performed that same day.

Necropsy findings:

The only significant findings were in the brain. Several foci of softening were present in the medulla of the cerebellum, superior colliculi, medulla oblongata and the 95 left caudate nucleus. The gross appearance differed from the lesions previously seen; they were yellowish in color without extensive hemorrhage.

Histopathology: The head of the left caudate nucleus presented ex tensive degenerative changes, a few perivascular hemorrhages and an infiltration of neutrophils. Similar degenerative lesions were found involving the dendate and fastigial nuclei of the cerebellum, the central reticular substance, the in ferior olive, and most of the tracts in the medulla oblongata. The superior colliculi and the red nuclei were also involved, more extensively on the left side than on the right. This dog presented lesions of acute necrotizing encephalopathy, without hemorrhages, and some of those lesions were in a new location, the head of the left caudate nucleus. Dog &865 (weighing 10 kg) was given a single in jection of 12 mg/kg of stilbamidine. On the fourth day he exhibited a severe ataxia and a few minutes later prostra tion and generalized extensor rigidity were present. He was immediately given an intravenous injection of lj.0 ml of a trypan blue solution. Twenty minutes later, the dog was

deeply anesthetized with nembutal and bled to death from the axillary vessels. 96

Necropsy findings: All tissues except the brain were intensely blue. The meninges, pituitary, and adjacent hypothalamus were also blue. Three control dogs injected with trypan blue presented similar changes. Coronal sections from the control dogs did not disclose any lesion. Dog A865 showed an extensive blue staining of the medullary portion of the cerebellum and hemorrhage. Non-hemorrhagic small foci of blue stain ing were also present in the vestibular area of the pons. Larger non-hemorrhagic foci of blue staining (0.5 cm in diameter) were seen in both inferior colliculi; smaller blue staining foci were seen in the pedunculi, the thalamus and the hypothalamus.

Histopathology: Extensive vacuolation, focal hemorrhage, neutrophilic infiltration and some perivascular cuffing were found in the medulla of the cerebellum. Small foci of vacuolation were seen in the region of the vestibular nuclei and in the in ferior colliculi. A larger focus of vacuolation as well as perivascular hemorrhages were seen in the left peduncle. The brain was fixed in Susa’s fluid in an attempt to demonstrate the trypan blue histologically. We were not successful even in portions of tissue which grossly were heavily stained. 97

D o r A862 received a daily dose of stilbamidine (2.0, 3.0, 2.0 and 1.5 mg/kg respectively) for four consecutive days. A few hours after the onset of nervous signs on the fifth day the animal was given trypan blue (1+ mg/kg) intra venously and thereafter was handled like dog A865« Figure 9 shows diffusion of trypan blue throughout the inferior colliculi, before the occurrence of hemorrhages.

Discussion: The appearance of nervous signs only on the fourth and fifth day after a single dose, may be due to a slow accumulation of the drug. The latent period could also be required for the drug to destroy the blood brain barrier. A blood brain barrier damage was well demonstrated by trypan blue in dog A865 and A862. The tissues were stained in an area of predilection for lesions (the inferior colliculi) before the actual appearance of hemorrhage and necrosis. Naturally, the areas with lesions (especially the medulla of the cerebellum) were also stained. This would suggest that the first change attributable to the drug is damage to the blood brain barrier, especially in some areas of predilec tion. Necrosis and hemorrhages then occur as the result of damage to the blood-brain barrier. Another peculiarity common to dog A865 and especially to dog 55-1266, is the slight amount of hemorrhages through out the lesions. The most extensive hemorrhages were usually 98 seen In dogs that died soon after their last injection of diamidines. The known ability of stilbamidine to liberate histamine from mast cells explains the sudden severe hemorrhage after the last of a series of diamidine doses. Histamine release is sizable soon after a stilbamidine in jection and produces a constriction of the efferent venules 50 and an increase in the capillary permeability, and thus causes massive hemorrhages through the already damaged brain capillaries. After a single Injection, the brain capillaries are probably not extensively damaged until the fourth or fifth day thereafter. Although histamine is un doubtedly released immediately after the absorption of the stilbamidine it causes no hemorrhage at that time because the blood-brain barrier has not yet been seriously altered.

I) Effect of concurrent adminlstratlon of cortisone (10 mg/kg) and diamidines

Objectives: Stilbamidine has a particular affinity for nucleo- 63,6k proteins and may form complexes with ribose nucleic acid.'*'^,^ ^ ,^‘*'^ Although the drug Is not antigenic in It self, one might suspect that some of its complexes could be antigenic. Since Snapper found stilbamidine complexes in the

myeloma cells of some of his patients treated with 99 stilbamidine, we may theorize that the symmetrical lesions found in our dogs could be due to the formation of similar antigenic complexes. To prove or disprove the allergic nature of the lesions we used concurrent injections of 167 cortisone. Farrell and Cole inhibited the allergic aspects of histoplasmosis in dogs with 100 mg of cortisone daily. Cortisone at the level used should cause involution of the lymphoid tissue and hinder the production of antibodies.

Material: We used six dogs, males and females of mixed breed ing, weighing approximately 10 kg. Dog A783 was given a Rosenthal*s liver function test before the experiment which proved to be within normal limits.

Methods: Three dogs received stilbamidine and two dogs, hydroxystilbamidine, according to the same dosage used in experiments (B) and (D) on toxicity, in which three dogs showed irreversible nervous signs in six, five and four days, when receiving stilbamidine, and three other dogs, in five, five and four days, when receiving hydroxystilbamidine. (Tables 3 and £). The sixth dog was given I4. mg/kg of stil bamidine daily for ten days. TABLE 11. CONCURRENT ADMINISTRATION OF CORTISONE '(10 mg/kg) AND DIAMIDINES

Day 1 2 3 b 5 6 7 8 Weight/ Total/ kg mg Dog No. Dose mg/kg and results

±IjD AHU.JO.L.HU!. Al+51 2* 3* 1.5# 1.5* 1* * # No sign of toxicity. 6.8 61.2 A780 2* 3* 1.5* 1.5* 1* •» N 9.5 85.5

A781 2* 3* 1.5* 1.5* 1* N 9.0 81.0

A783 k* U* 4* 4* 1** 2it N 10.- 220.0 HYDRCIXYSTILBAMIDINE

A782 3* [j.* 2* a N 6.0 82+. 0 A520 3* 4* 5* 2* ■» * ■3t Challenged by twice repeat 8.6 931.2 ing the course of hydroxy stilbamidine, omitting cor tisone. Died after 6 fur ther consecutive daily doses of 10 mg/kg, omitting cortisone. * - received cortisone 10 mg/kg. N - irreversible nervous signs. 100 101

Pig. 9- Cerebellum, pons and inferior colliculi of dog A862• Note the trypan blue staining of the parenchyma prior to hemorrhage.(Stilbam idine toxicity).

Pig.10- Necrosis of a blood vessel in the caudate nucleus of dog A783 (Stilbamidine toxicity). Pig. 11- Transverse sections of brain from dogs A78O and A78I at the level of the superior colliculi, inferior colliculi and cerebellar medulla. Both dogs were concurrently receiving corti sone and stilbamidine. The bilateral hemorrha gic lesions are especially x*ell illustrated in the dendate and fastigial nuclei of dog A780.

Pig. 12- Three transverse sections of the thalamus of

dog A858 showing the distribution of the encephalomalacia. This animal was concurrently treated with antihistamine and stilbamidine.

102 Fig. 11

Fig. 12 101+

Daily, concurrently with the injection of drug, and for three days after the end of treatment, we gave 10 mg/kg cortisone acetate intramuscularly.

Results: One dog survived the course of stilbamidine; three other dogs on this drug developed irreversible nervous dis turbances on the sixth day. One dog, receiving hydroxystilbamidine, also showed CNS disturbances on the sixth day, but the second dog was resistant to three similar courses of hydroxystilbamidine; cortisone was given concurrently with only the first series. Only peripheral disturbances were manifested after receiving a fourth course of stilbamidine, consisting of six daily injections of 10 mg/kg. This dog died two days after the latter challenge. Dog Ali.51 (mongrel beagle, weighing 6.8 kilograms) did not show central nervous system disturbances. He was destroyed twelve days after the last injection of cortisone. No gross or microscopic lesions were found in the central nervous system. An abscess in the right thing was attributed to the multiple intramuscular injections. Dog A780 was treated for five days with stilbamidine and cortisone according to the regimen listed in Table 11 and developed irreversible nervous signs the seventh day. He laid prostrate, in opisthotonos and with the legs in ex treme extensor rigidity. The front toes were extended in a 105 fan-like fashion. He was conscious but most of his reflexes were absent. The pupillary reflexes were present and a vertical nystagmus was seen. The paws and skin showed sensi bility to pin pricking. Euthanasia and necropsy were performed. Grossly there were bilateral foci of hemorrhagic softening in the inferior and superior colliculi, the medulla of the cerebellum, and similar but unilateral lesions in the right vestibular area of the pons (Fig. 11). Microscopically, the lesions consisted of large zones of recent hemorrhage, rarefaction, diffuse infiltration of neutrophils, destruction of vascular endothelium, with sludging and some perivascular cuffing of red blood cells and neutrophils. Dog A78l was treated in a manner identical to dog A780 and developed CHS signs on the day after the five-day treatment. He paddled continually with rigid front legs; this sign was more pronounced when he was suspended slightly above a table top. The toes were extended fan-like. The righting reaction was present and the knee jerk overactive. The hind, legs were very weak, the toes being turned downward. Muscular tonus was present and the extensor postural thrust could be elicited in the hind legs. At 3:00 p.m. the dog was in a decerebrate rigidity position, and bicycled continually. At JzOO p.m. the animal was comatous and was 106 destroyed for necropsy. Necropsy findings of acute hemorrhagic encephalopathy were similar to the previous dog (A780) Fig. 11, but in addition there were extensive bilateral lesions in the thal amus (level of mammillary bodies) adjacent to the superior colliculi, in the reticular substance, and extending for a distance of 10 mm. in the pons. Dog A783 (weight 10 kg) received a total of 220 mg of stilbamidine in six days. On the seventh day it was in a state of rigidity (Table 11). The dog's clinical signs were very similar to those of dog A780; however, nystagmus was absent. The eyes were turned upward and showed only a slight tactile reflex when at 10:00 p.m. euthanasia and necropsy were performed. Grossly, the entire right anterior caudate nucleus was an hemorrhagic mass, the left side showed less exten sive hemorrhage. The medulla of the cerebellum and both superior colliculi hemorrhagic. The latter lesion extended slightly in the inferior colliculi. Microscopically, the extensive hemorrhagic lesions consisted of diffuse perivascular' hemorrhages, neutrophilic infiltration, vascular necrosis of endothelium (Fig. 10) and edema. Dog A782 (weight 6 kg) received cortisone and a

total dose of 8I4. mg of hydroxystilbamidine in four days. 107

(Table 11). The dog was in a state of rigidity on the morn ing of the sixth day. The gross and microscopic lesions, similar to the preceding cases, were located in the medulla of the cerebel lum, the inferior colliculi, the left superior colliculus, the pons and lower medulla oblongata. Dog A5>20 (weight 8.6 kg) received the same treatment as dog A782. Eight days after the end of the four-day treat ment, the animal had not shown any signs of illness. We then challenged her with two consecutive courses of hydroxystilbamidine identical to the first treatment, except that cortisone was omitted. No abnormal clinical signs were seen excepting for swelling at the site of the intramuscular in jections. A liver function test disclosed no abnormal re tention. This animal was considered refractory to the toxic effects of hydroxystilbamidine. After an observation period of twenty days, another course of hydroxystilbamidine was given at very high level to determine if her resistance to toxicity could be broken. After 10 mg/kg were given daily for six consecutive days, she developed continual knuckling of the rear legs. She walked with extreme difficulty, be came very weak, and finally prostrate with no apparent sensitivity. The animal died two days after the last dose of hydroxystilbamidine, or a total dose of 927 mg in forty- seven days. 108

Grossly, the liver, kidneys and lungs were exten sively congested. Both thighs presented several abscesses, and the sciatic nerves, especially the left one, lay in an area of suppurative myositis. A cavity, 3 mm long and 1*5 mm wide was found in the anterior medulla, on the left side in front of the obex. A red area, 3 mm in diameter, was found in the left inferior colliculus. Microscopically, the smooth edged cavity was in the area of the left vagal nuclei with no lesion in the vicinity. An area of swollen myelin sheaths, neutrophilic infiltration and endothelial swelling was seen in the right reticular area. Posterior to the cavity in the medulla there was a delimitated lesion consisting of a large center of necrosis and neutrophils surrounded by an area of perivascular hemorrhages, necrosis of vessels, cuffing, and myelin sheaths. A similar extensive lesion was seen in the left inferior colliculus. In the area of the cerebellar nuclei, we saw several small foci of vacuolation and gliosis. One of these foci contained numerous neutrophils, proteinaceous globules and necrotic capillaries, surrounded by palisading glia cells. Similar hemorrhagic lesions were found in the putamen and in very unusual situations, the internal capsule and the corpus callosum. 109

Discussion: Cortisone did not prevent drug toxicity and associ ated irreversible signs and lesions in four of six dogs. The two survivors may have been naturally resistant since one of them, dog A520 remained healthy (Table 11), when challenged with two series of hydroxystilbamidine treatments without cortisone twelve days after the combined treatment. The animal died two days after an extreme challenge with six consecutive daily injections of 10 mg/kg. The signs and lesions indicate that even a naturally resistant dog may be subject to the effects of toxicity if given large enough doses. It is probable that dog Aij.5l was similarly resistant and we must conclude that cortisone failed to prevent the toxic effects of either diamidine. Note in Table 11 that the first signs of toxicity appeared on the sixth or seventh day after the first injection. Nervous signs cannot be induced earlier by giving a higher dosage. This fact is illustrated by dog A783 who received a dose 2j§- times greater than dogs A?80 and A78l, but reacted to the toxic effects no earlier than the latter dogs.

J) Effect of histamine phosphate

Stilbamidine releases histamine and causes a fall in blood pressure. This action has been incriminated in some of the toxic effects of stilbamidine. We therefore attempted to produce central nervous system disturbances in a dog 110 using histamine phosphate. Dog 55-105U (weight 7 kg) received intravenously 2.75 mg of histamine acid phosphate daily for five days and 5.5 mg daily for three subsequent days. The dog usually collapsed during the injections, but recovered very rapidly. A generalized severe hyperemia of the skin was evident. The dog was destroyed two days after the last dose; no gross lesions were found at necropsy. Since histamine causes dilatation of the peripheral vessels the extreme cutaneous hyperemia was expected. No lesions were detected in the nervous system which could be attributed to histamine.

K) Effect of concurrent administration of antihistamine and diamidines

Objectives: Although we were unable to produce severe brain 50 lesions using histamine phosphate, we like Halpern at aT believed that histamine liberated at the cellular level may behave differently from outwardly applied histamine. Con siderable increase in capillary permeability was produced 50 in the dog by an histamine releaser. Therefore, it seemed

logical to study the effects of an antihistamine compound on the toxicity of the diamidines, when administered concurrently. Ill

Materials and Methods: Two dogs were prepared as for the preceding experi ment. The antihistamine compound administered subcutaneously was dimethylamine-ethoxy-methyl-benzyl-pyridine succinate (Jen-Sal Co. ). Dog A779 received the same dosage of hydroxystil- bsmidine used in experiment (I). In addition, it received each day 10 mg/kg of the antihistamine compound. In the protocol dog A8£8 was scheduled to receive 1) the same dosage of stilbamidine used in three dogs of experiment (I) plus 2) 10 mg/kg of antihistamine concurrently and three days thereafter.

Results: After three injections (on the fourth day of the ex periment) dog A858 showed nervous signs and died at noon the same day. A total of only 78 mg of the diamidine had been given. Dog A779 was given the first course of the drug, without showing any clinical sign. Twenty-one days after this first course, the dog was again submitted to a similar treatment, but without the antihistamine compound. After this second course no clinical signs were noticed, but the dog died the day after a subsequent injection of 2 mg/kg of hydroxystilbamidine. The dog had received a total of 219 mg of the diamidine. The dog died in the characteristic position seen in this syndrome, i..e_. opisthotonos with the TABLE 12. CONCURRENT ADMINISTRATION OP ANTIHISTAMINE (10 mg/kg) AND DIAMIDINES

Weight/ Total/ Day 1 2 3 4 5 6 7 kg mg Dog No, Dos© mg/kg and results

STILBAMIDINE

A858 2* 3* 1.5* N L2.0 78.0

E[YDR OXYSTILBAMIDINE k m 3* 4* 5* 2* *- * Beginning day 19 challenged 6.4 192.0 by repeating a course of hydroxystilbamidine, omitting antihistamine. Results: fatal toxic encephalopathy.

*■ - received antihistamine. N - irreversible nervous signs. 112 113 four legs extended. Grossly, dog A779 showed a few lesions in the medulla, inferior and superior colliculi. In dog A858 gross lesions were seen mainly in the medulla, the cerebellum (including the flocculi), and were especially severe in the thalamus. In both dogs, the lesions in the cerebellar flocculi were hemorrhagic; other lesions were well delimitated dark gray areas. Microscopically, the lesions were typical of encephalomalacia, but with less hemorrhage and few neutrophils than observed in our other cases of toxicity. Dog A858 had very extensive and diffuse focal hemorrhages throughout the gray and white matter of the Inferior cerebellar flocculus and in the nuclei area of the thalamus.

Discussion: One dog (A779) survived a course of hydroxystll- baraidine treatment when antihistamine was given concurrently. Subsequent treatment without protective antihistamine caused fatal toxicity thus demonstrating that the dog was not inately resistant. Antihistamine did not protect dog A85>8 against stil bamidine toxicity. Ilk

L) Comparison of thiamine deficiency in cats and dog3 with diamidine toxicity in dogs

Objectives: In 19l|.2, Evans e_t al 37 described the microscopic findings and distribution of lesions in Chastek paralysis, a disease of foxes. This disease was believed to be related to the polioencephalitis hemorrhagic superior of Wernicke, a disease seen In alcoholics and believed to be caused by a 168,169 thiamine deficiency. Later it was discovered that fresh fish containing thiaminase was the etiologic agent of Chastek paralysis. The histopathology and distribution of lesions In these diseases resemble our findings in diamidine toxicity in dogs. Experimental thiamine deficiency has been produced in cats and dogs using three different types of diets. Swank , 121 et al in 19i|.l used In dogs a diet consisting mainly of casein, corn starch and autoclaved yeast. In cats, a diet 108 of raw fish has been used by Smith and Proutt. In 1941+, 38 Everett published an excellent clinical study of thiamine deficient, cats. His diet, which we used in our experiment, contained autoclaved canned dog food and brewer *s yeasts supplemented by oral and parenteral vitamins. We found no report of thiamine deficiency in cats or dogs produced by antivitamins B1 like neopyrIthiamine or 115

oxythiamine. Most of the researchers added small quantities of thiamine to their diet to produce chronic thiamine deficiency. They were trying to correlate the animal deficiency with beriberi in man. We were not interested in producing cardiac failures or peripheral nerves degeneration and consequently, did not add thiamine to our diet. We produced thiamine deficiency in both cats and dogs to compare the brain lesions of this avitaminosis and the lesions of diamidine toxicity in dogs. The manifestation of diamidine toxicity resembled those of thiamine deficiency in animals. It seemed logical to suspect that the pathogenesis of diamidine deficiency might be dependent upon some anti thiamine property of the diamidines.

Materials and methods: Six cats and five dogs (males and females) varying from four months to five years of age, were caged individual ly for baseline studies while on the control diet for at least two weeks. The oats were weighed daily and the dogs on alternate days. The latter were vaccinated against dis temper and canine hepatitis. During the two-week observa tion period all animals received the same diet that was used throughout the experiment, except that the canned dog food and brewer's yeast was not autoclaved. 116

The cats received the following diet daily: auto claved canned dog food (Hill's regular) 225 gm, autoclaved brewer's yeast 0.5 gm, pyridoxine hydrochloride O.lj. mg, calcium pantothenate 1 mg, riboflavin 0 . 5 mg; weekly, they received 2,000 units vitamin A, vitamin D, alpha tocopherol

5 mg; weekly, they also received parenterally, pyridoxine hydrochloride 0 .1]. mg, calcium pantothenate 1 mg, riboflavin

0 . 5 mg. The dogs received daily; autoclaved canned dog food (Hill's regular) 1 lb, autoclaved brewer's yeast 1 gm»

calcium pantothenate 6 mg, riboflavin 1 mg, pyridoxine 1 mg; weekly, they received orally ij.,000 units of vitamin A and i|.,000 units of vitamin D; parenterally, they received

calcium pantothenate 5 mg, riboflavin 1 mg, pyridoxin 1 . 2 5 mg, alpha tocopherol 2 0 mg, menadione 3 * 6 mg. The canned food and brewer's yeast were autoclaved

six hours under 2 0 pounds of pressure. At first, this diet was consumed voluntarily. When inappetence developed, the feed was mixed with water and given to the dogs through a stomach tube or to the cats with a syringe. The animals were examined daily for CNS disturbances or any change in habitus. Electrocardiograms of the dogs were made monthly. Routine neurologic examinations were conducted on all animals with nervous signs. After death we followed the same procedure used in previous experiments. 117

R e su lts: Gats Aii.83* kkQkt Alt86, Alj.88 died in forty-eight-to seventy days after they were placed on the deficient diet. Gat Aii85> was destroyed after eighty-five days. A resume of the clinical findings is listed in Table 13. The cats gradually lost their appetite and inappe- tance became complete after being on the thiamine deficient diet for four weeks. By that time, they began to wobble on their hind legs in an unsteady fashion and assumed a large posterior base. At this stage or some days later, cats Alj.83>, Aij.87, Alj.88 and especially Aij.86, assumed a crouching position in walking, with the spine flexed somewhat ventrally, and the stiffle joint at a more acute angle. Cat AJ4.85 and ALj.86 had respectively three and four kittens after forty-seven and twenty-five days on the deficient diet. The kittens were found dead, the latter being severely mangled by the mother. Cat AI4-8I4. favored a very convex spine; he walked on the tips of his feet, lifting the hind feet rather high. Ataxia, in this cat, became progressively worse. Cat Al}.83 did not show nervous disturbances before the forty-sixth day when she showed convulsions. Two cats, Aij.86 and Al+87, were found dead after being on the deficient diet for sixty-three and fifty-eight days without having shown any convulsion or irreversible central nervous system disturbances. 118

Gat Al+85 3 howed typical convulsions after fifty-one days on the deficient diet. Suddenly, her head flexed ventrally and her nose was pushed hard against the floor with clonic contractions of the neck and shoulders. 1’he cat laid on her sternum, the front legs being maximally abducted and participating in the clonic contractures. These convulsions lasted about three seconds, then, the cat would waver a few seconds and regain its habitual ataxic gait. The next day, the cat did not show convulsions but presented a much more ataxic gait, especially after exertion. If the cat was lowered towards the floor, headfirst, she would invariably hit the floor with the back of her head. On the fifty-

third day, she was given 1 mg of thiamine, intramusclarly• Recovery was gradual, the ataxia improved slightly, and soon, her alertness and appetite returned. The animal was destroy ed after being on a deficient diet for eighty-five days.

Gats Ai4-81+ and Al+ 8 8 showed convulsions (like those described for cat Aij.85) of two- to four seconds duration, after being on the diet forty-eight and sixty-seven days. The onset of convulsions preceded death by three- to five days. After their onset, the convulsions were less frequent

until death. Gat AI4.8 I4. showed serious CNS disturbances in addition to the convulsions. The animal often fell on one side and started rolling wildly by propelling itself with the front feet. The hind legs were very weak. The animal often 119 emitted cries of distress. He was neither blind nor deaf and no nystagmus was seen; the pupils did not respond to light. The vestibular disturbances became progressively worse, and before death, the animal was very weak and not conscious of his surroundings. Abnormal posture was caused by ventral flexion of the neck, a convex spine and rigid front legs• Cat Aij.83 did not show the typical clonic convulsions, but presented severe weakness of the hind legs, ventral curvature of the spine, an acute head tilt to the left, a wide forward base and claws extended fan-like. Her pupils did not respond to light. Occasionally she rolled wildly or her legs became rigid. Her hind quarters became paralyzed. Before death, the cat laid prostrate on the right side, the front legs In extensor rigidity with spread-out claws, the head flexed ventrally and the back convex. Apparently, the animal was blind and deaf.

Necropsy and histopathology: Grossly, the brains were somewhat congested, and a few petechiae could be seen. No gross hemorrhage or soften ing comparable to the lesions of stilbamidine toxicity In

dogs could be demonstrated. Only cats Alj.83 and AI4.QI4. showed severe microscopic changes. Cat Ai}.83 presented some large areas of vacuolation involving the dendate and fastigial nuclei, the lateral TABLE 13. THIAMINE DEFICIENCY IN CATS AND DOGS Clinical findings in post-inoculation days; total weight loss in per cent.

Complete W t/loss CNS No. Sex inappetence Per Cent Disturbances Death Cat: A483 F .. 31 46 48 Ai|8i|. M 28 36 48 53 Aij.85* F 28 52 51 85 (euthanasia) Al|.86«* F 21 57 not seen 63 A487 M 28 30 not seen 58 Ali.88 M 28 60 67 70

Dog: A515 F 32 5 not seen 97 (euthanasia) A516 F 27 25 79 79 A517 M 28 12 56 57 A518 F 18 5 18 24 A696 F - (gain, 13 per cent) not seen 97 (euthanasia)

*had k itten s on day 47* ■^^had kittens on day 25* 120 Pig. 13- Gat AJ4.8 3 . Section of pons showing vacuolation, pyknosis of a nerve cell and swollen vas cular endothelium. H & E, 375 X

121 122

Fig. 13 123 vestibular nuclei and especially the medial vestibular nuclei. Edema and swelling of the endothelial cells were also prominent (Pig. 13)• The whole area of the superior olivary nuclei showed extensive gliosis, swollen endothelial cells, and some perivascular cuffing with lymphocytes and glia. Similar changes were found in the area of the cochlear nuclei. We saw a single small focus of hemorrhage in the vestibular area. In the medulla, at the level of the obex, we noticed edema in the area of the vagal nuclei, and rarefac of tissue with hemorrhages in the central nucleus of the dorsal horn. The inferior colliculi showed lesions similar to those described in the area of the superior olivary nuclei. Congestion and minimal perivascular hemorrhages were found in the cerebral cortex, the thalamus, the striatum and the spinal cord. Cat showed less extensive but similar lesions, consisting of edema, hyperemia slight perivascular hemorrhages, gliosis and swollen myelin sheaths. These lesions were found in the gray matter of the lower medulla oblongata, the medulla of the cerebellum, in the area of the trigeminal and vestibular nuclei, in the area of the red nuclei, the striatum and the marginal gyrus of the cortex. We did not demonstrate any microscopic lesions in

cats Alj.85 a n d AI4.8 6 . Cat Ai|87 presented some vacuolation in 1214. and gliosis in the putamen while cat AI4.88 showed vacuolation in the right inferior colliculus and putamen. No sciatic nerve demyelination was found in any of the animals. The absence of tonicity in the myocardium of the right ventricle (present in all cats) suggested cardiac failure. No microscopic lesions were demonstrable in the heart.

Dog A515 (weight 1 8 lb) showed complete inappetence and lost 2 lbs in weight from day 3 2 to day 5 2 on the deficient diet. Thereafter she began to recover her appetite, no CNS disturbances developed and after being on the defi cient diet 9 7 days she was destroyed. At necropsy the animal weighed 17 lb; no gross lesions could be demonstrated. The histopathologic study disclosed disintegration of tissue, necrosis and swollen myelin sheaths in both gracilis and cuneatus tracts at the level of the cervical cord. The origin of this necrobiosis is not known but could be due to circulatory disturbances. Other organs did not show significant lesions. Dog A5l6 (weight 16 lb) showed complete inappetence after being on the deficient diet for 27 days. She vomited most of the food given by stomach tube. She first gained

lb in weight and kept it until day 5 8 when she began to lose weight. On day 72 she weighed only 1)4 lb, walked with a wide posterior base and was generally inooDrdinated. She 125

developed a head tilt to the left and became prostrate,

lying in a decerebrated position, after 7 9 days of dieting. She was euthanized. At necropsy the animal weighed only 12 lb. No gross lesions were demonstrated except reduced tonicity of the myo cardium of the right ventricle. Lesions in the brain consisted of vacuolation and

endothelial swelling similar to those described in dog A5 l7 » The lesions were not hemorrhagic as. in the latter dog and included both inferior colliculi, the oculomotor nuclei, the right red nucleus and the vestibular area of the pons. Dog A517 (weight 17 lb) showed complete inappetence

after 28 days on the deficient diet. On day 58 the animal presented serious CNS disturbances. He walked with a wide posterior base, swayed, sidestepped, and showed hypermetria. His eyes were bulging. The animal came to command but took a roundabout way, bumping into objects. The neck was slightly more rigid than normal. Tremors were often seen in the posterior legs. Flexion was usually not provoked by pinching the foot pads, the legs being kept rigid. When lowered down headfirst, the head hit the floor before his feet, and the dog remained in that posture without moving, indicating very wesk righting reflexes. The dog became progressively worse and in a few hours, incoordination and rigidity were extensive. Knuckling was 126 frequent. Tremors of the head and neck were seen while he continually balanced his head. On day 57» the dog laid prostrate in opistothonos. Breathing was strenuous. Periodically the opistothonos position became extreme with clonic movements of the legs. At the same time generalized tremors appeared with excessive salivation and chewing. Most reflexes were absent. For humanitarian reasons the dog was euthanized.

Necropsy findings: Wood shavings from the litter were found in the trachea and bronchi. Heart was in diastole and the right ventricular wall very flabby. The inferior colliculi dis

closed hemorrhagic foci 1 . 5 Dm in diameter on left side, 0 * 2 mm on right side. A section at the level of the superior colliculi also presented small well circumscribed hemorr hagic foci in the areas of the oculomotor and red nuclei. Hemorrhage was seen in the anterior flocculus of the cere bellum. The medulla showed bilateral hemorrhagic areas, 1 by

9 mm on left side, 1 by 5 mm on right side, running longi tudinally through the brain stem in the area of the direct vestibular nuclei, as far back as the obex (Figs. 1 i| and 15)•

Histopathology: The brain lesions were characterized primarily by extensive vacuolation of the parenchyma and swollen Pig. ll]_- Dog A517. Transverse section at the level of the cerebellar and inferior and superior collicu li. Hemorrhagic lesions are seen in the vermis, the left inferior colliculus, the oculomotor and red nuclei.

Pig. 15- Dog A517. Hemorrhagic lesions are seen following the course of the descending vestibular and vagal nuclei in the medulla, as far back as the obex. One section show a large hemorrhage in the area of the right arcuate fibers.

127 METR|IC 1 2 3 4 5 1 „ _£J iniTinrirmTTTTWTnr rmtnn milmi A 5ir F ig . 1U-

METRIC 1

Fig. 129 endothelial cells. Perivascular hemorrhage, often extensive, was a common but inconstant finding. They were always bi lateral but varied in intensity. The main structures involved were the inferior colliculi, the oculomator, red, direct vestibular, hypoglossal and fastigial nuclei, and the anterior cerebellar flocculus.

Dog Agl8 presented periodic excessive salivation ac companied by chewing (2-3 minutes duration) on days V~> and 16 of the deficient diet. This dog had a good appetite

until day 1 8 when she displayed a complete change in habitus. The tame and gentle dog suddenly became vicious. She growled continually and lunged at us snapping her jaws. It was impossible to handle her without a snare. An hour after this display of viciousness, the animal appeared frightened and laid back in her cage. On days 19, 20 and 21 the animal looked anxious and snapped at you if you tried to touch her. She had trouble to keep her balance, swaying in the cage. On day 22, the dog did not show any aggressiveness. Her hopping reaction was late and hypermetric in the front legs and some intermittent knuckling was seen In both legs. The righting reflexes were overactive. The dog put her nuzzle In water when trying to drink. Later that day, she fell suddenly on her side; her jaws were widely opened and her feet were bicycling wildly. The convulsions lasted 3 minutes.

It was repeated a few times after which the dog was weaker. 130

On day 23, photophobia of the right eye was noticed, and the next day, the right pupil was smaller, blurred, and did not react to light. The dog often circled to the left. She be came prostrate and died later that day. The animal had only lost one pound in weight.

Necropsy findings: The stomach presented a few erosions around the cardia. A reddish fluid material, containing some blood and bile, was seen throughout the intestines. The mesenteric lymph nodes were hypertrophic. The lungs showed several calcified nodules 1 to 2 mm in diameter. The heart was in systole. Coagulated blood was found in both frontal sinuses. The brain stem, at the level of the inferior olive showed an extensive hemorrhagic lesion on the left side. This focus

of hemorrhage (1 by ij. mm) extended 5 nun toward the obex.

Histopathology: Most of the organs were congested. Granulomas in the lungs consisted of central caseation necrosis surrounded by reticulo-endothelial cells. Coagulation necrosis in the bronchial node and lymphoid hyperplasia of the mesenteric lymph nodes were ascribed to subclinical chronic histoplas mosis. The frontal lobe showed satellitosis, conglutination of the intravascular erythrocytes, edema, areas of 131 rarefaction, and diffusion of an eosinophilic material into the tissue. Edema was noticed in the superior colliculi and the thalamus, the latter showing some cuffing adjacent to the third ventricle. Vacuolation was seen in the area of the vestibular nuclei and around the fourth ventricle. Gliosis and slight hemorrhage were seen in the cerebellar gyri. The brachium conjunctivum showed an area of demyelination. The brain stem disclosed extensive recent hemorrhage disrupting the parenchyma and affecting the ventral and inferior olive, the first cervical nerve, the arcuated fibers, the direct vestibulo-spinal tract, the ambiguus and lateral nuclei. The gracilis nuclei presented extensive vacuolation. Dog A696 (weight 15 lb) received the same diet as the preceding dogs but was given a weekly intramuscular injection of 5 mg of thiamine. This control animal maintained a good

appetite and gained 2 lb while on the diet. No clinical signs were noticed and no gross or micro scopic lesions were found when the dog was euthanized after

being 9 7 days on the special diet supplemented with thiamine.

Discussion: The terminal clinical signs of some thiamine deficient cats and dogs resembled those of diamidine toxicity. Not only were the lesions produced by thiamine deficiency and diamidine toxicity identical in location but histopathological studies 132

also revealed the following qualitative similarities in the brain lesions: swelling or hypertrophy of the vascular endo thelium, hemorrhage, vacuolation of the brain parenchyma considered to be edema, and minimal necrosis and perivascular cuffing. The following differences were also noted: the neutrophilic infiltration, excessive perivascular cuffing, severe parenchymal necrosis, necrosis of blood vessels and the presence of numerous gitter cells which were prominent features in diamidine toxicity were absent in thiamine deficiency. It would appear that the early lesions caused by diamidines can also be produced by a deficiency of thiamine. Perhaps diamidines interfere in some manner with the path ways of thiamine metabolism and the damage to the blood- brain barrier is (at least in part) result of the "blocking*1 effect of diamidines on thiamine. The above hypothesis is further supported by the fact that the signs and lesions of toxicity did not appear until a latent period of days after dogs had received one large dose of stilbamidine. This latent period may represent l) the time required for absorp tion and distribution of the drug, plus, 2) the time for the body cells to suffer from lack of thiamine. Possibly, the early thiamine deficiency (resulting from vascular damage) produced by the diamidines allows the passage of the drugs through the blood-brain barrier with subsequent increase in the intensity of the lesions. 133 Location of the Most Frequent Lesions Produced With High Dosage of Stilbamidine

Dog 55—69

Dog 55“ '386

Dog 55—314

■ 1 Extensive Hemorrhages 8 Softening Legend ■ Slight Hemorrhages, Vacuolation, Neuronophagia A-Level of Superior Colliculi B~Level of Inferior Colliculi C“Level of Dendate Nuclei 8 Pons D-Medulla Fig. 16

Location of the Most Frequent Lesions Produced With Low Dosage of Stilbamidine

Dog 55—3 16 Dog 55—387CD CO Dog 54— 1252 t# [Extensive Hemorrhages 8 Softening Legend ■ Slight Hemorrhages, Vacuolation, Neuronophagia ArLevel of Superior Colliculi B-Level of Inferior Colliculi OLevel of Dendate Nuclei 8 Pons D-Medulla Fig. 17 13L Location of the Most Frequent Lesions Produced With High Dosage of ^“Hydroxy stilbamidine A A. B Dog 5 5 —39 0 « C3 O Dog 55—388 <55>

Dog 5 5 - 3 9 3

■■Extensive Hemorrhages tt Softening Legend Hi Slight Hemorrhages, Vacuolation, Neuronophagia A-Level of Superior Colliculi B-Level of Inferior Colliculi C-Level of Dendate Nuclei 8 Pons D-Medulla Pig. 1 8

Location of the Most Frequent Lesions Produced With Low Dosage of 2“ Hydroxy stilbam idine

Dog 5 5 —313

< £ > C O “ ‘ Dog 5 5 —6 8 © n Dog 5 5 —391

■■Extensive Hemorrhages 8 Softening Legend ■■Slight Hemorrhages, Vacuolation, Neuronophagia ArLevel of Superior Colliculi B-Level of Inferior Colliculi C-Level of Dendate Nuclei 8 Pons D-M edulla Fig. IS 135

EFFECTS OF STILBAMIDINE AND 2-HYDROXYSTILBAMIDINE ON EXPERIMENTAL CANINE BLASTOMYCOSIS

OBJECTIVES 1. To evaluate the effectiveness of stilbamidine and 2-hydroxystilbamidine therapy in experimental blasto mycosis induced in dogs by the intravenous, subcutan eous and intratracheal inoculation of Blastomyces dermatitidis. 2. To investigate the protective effect of the above drugs when given prior to the inoculation of Blasto myces dermatitidis. 3* To determine the clinical signs, lesions, patho genesis and distribution of the fungus in canine blastomycosis and thus establish criteria for clini cal and laboratory diagnosis of the disease*

MATERIAL AND METHODS The inoculum.— The yeast phase of Blastomyces derma titidis isolated from a naturally infected dog, was grown on brain-heart infusion agar with blood at 37 C. The organisms were transferred twice a week. The yeast phase used in the experiments was obtained from 3-day-old cultures which were washed with saline and packed by centrifugation at 1,^00 rpm for 10 minutes. Yeast cells were counted by the dilution and plating technique. 136

The culture was pure and contained 122 x 10? viable organisms per ml of packed cells. Immediately preceding Inoculation the packed cells were suspended in saline at a ratio of 1 :9 .

Drugs.— Stilbamidine and 2-hydroxystilbamidine isethionate were obtained from the Wm. S. Merrell Company, Cincinnati, Ohio. The salts were diluted in 2 per cent pro caine, as explained on page 3>b»

Animals.— Thirty-one healthy dogs selected for this experiment were all about 1-year-old, and weighed approxi mately 20 pounds each. Base line studies were conducted during an observa tion period of at least three weeks. During this time they were Immunized with a killed virus vaccine against canine distemper and infectious hepatitis. They were also sub jected to histoplasmin and blastomycin skin tests, and treated for Internal and external parasites. Other base line studies included urinalysis, hematology, ophthalmo scopic examinations, physical examinations, chest radio grams and daily recording of temperature, pulse and respira tory rate.

Infection.— A complete inoculation schedule will be found In Table II4.• The dogs inoculated intratracheally were anesthetized with pentothal sodium. The packed yeasts were 137 diluted in saline to a total volume of 5> ml and introduced into the trachea with a male dog catheter. The dogs were rotated, while injecting the inoculum to insure distribution of the yeast cells to all bronchia.

Treatment.— The complete schedule of treatment has been outlined Tables 7 and. 8 . The drugs were prepared within one hour before use (as mentioned In the drug toxicity experiments) and were in jected intramuscularly after dilution to 2 ml with 2 per cent procaine hydrochloride. The diluted drugs were kept In amber vials, and protected from light at all time. The dogs were housed in a room with low light intensity. The treat ment was initiated on the day of Infection.

Clinical examination.— During the experimental period the animals were subjected to daily physical examina tions with special emphasis on the respiratory, digestive, integumentary and nervous systems. The dogs were weighed weekly. The mean arterial blood pressure was recorded on selected dogs once before infection and weekly thereafter.

Radiology.— Chest radiograms were obtained one day before infection, at the time when pulmonary signs became apparent (i|- to 12 days post-inoculation), at weekly inter vals as long as pulmonary signs persisted and within 138

21| hours preceding death. We used the routine technique for lateral chest radiograms. Radiograms of the moderately inflated lungs were ob tained when the organs were removed at necropsy. We used non-screen film for these post-mortem radiograms which were taken at O.lj. second and $Q ma. The kilovolts were used according to the thickness of the lungs.

Blood counts, blood culture, serology;— Hematologic studies were made on post infection day 2 , 7 * 10* 1^4- and weekly thereafter. The blood cells remaining in the tube after centrifugation to remove plasma were cultured for fungi at each of the above intervals. Dermal sensitivity tests with human blastomycin, canine histoplasmin and human torulin were made once a month. Direct microscopic and cultural (Sabouraud's glucose and brain-heart infusion blood agar) examinations of exudates, feces and scrapings of cutaneous lesions were performed. Plasma was collected and frozen weekly for subsequent serological studies.

Uecropsy and mi crop at ho logy.— Necropsy was performed on control animals shortly after death. Animals that sur vived were euthanized several months after the disappearance of all clinical signs. Micropathology was done on grossly visible lesions as well as on representative sections of the following organs: eyes, skin at site of the subcutaneous TABLE 1^. SUMMARY OF ROUTE AND DOSE OF B. DERMATITIDIS AND TREATMENT

Dose ml Dog No. Age/mo. Sex packed yeast Route Treatment

551031 25 M 0.25 intravenous stilbamidine 551032 12 M 0.25 intravenous stilbamidine 55105.1 12 F 0.25 Intravenous stilbamidine

551037 12 M 0.25 Intravenous hydroxystilbamidine 551038 12 M 0.25 intravenous hydroxystilbamidine 551039 12 F 0.25 Intravenous hydroxystilbamidine

551071 25 F 0.25 intravenous untreated control 551070 12 M 0.10 intravenous hydroxystilbamidine 551075- 25 M 0.10 intravenous hydroxystilbamidine 551069 25 M 0.10 intravenous untreated control 551073 12 F 0.10 Intravenous “untreated control 551067 36 F *1.00 subcutaneous hydroxystilbamidine 551065 12 M *1.00 subcutaneous untreated control 5510311 6 F 0.50 intratracheal stilbamidine 551035 12 M 0.50 intratracheal stilbamidine 551036 25 M 0.50 intratracheal stilbamidine

^Inoculum was divided: 0.5 ml subcutaneously in the thoracic region, 0.5 ml sub cutaneously over the sacral region. TABLE 14— Continued

Dose ml Dog No. Age/mo. Sex packed yeast Route Treatment 551033 12 P 0.50 intratracheal hydroxystilbamidine 551042 12 M 0.50 intratracheal hydroxystilbamidine 551072 6 P 0.50 intratracheal hydroxystilbamidine

551044 12 M 0.50 intratracheal untreated control 551045 12 M 0.50 intratracheal untreated control 551066 6 M 0.50 intratracheal untreated control

5568 24 P 0.50 intratracheal #untreated control 5590 24 M 0.50 intratracheal 4K*hydroxys t ilbamidine

551540 12 M 0.25 intratracheal untreated control 551541 12 P 0.25 intratracheal untreated control 551862 24 P 0.50 intratracheal 4BBipr etr eated 551863 24 M 0.50 intratracheal ■iHHtpretreated 551865 24 M 0.50 intratracheal untreated control

5580 24 P 0.50 intratracheal stilbamidine 551861 12 M 0.50 intratracheal stilbamidine

551053 2 P 0.50 intratracheal eulicin

#hydroxystilbamidine 62 days, then 14-day interval before infection, ’^^hydroxystilbamidine 54- days, then 14-day interval before infection. stilbamidine 4-7 days, then 7-day interval before infection.

4=“ o 11+1 inoculation, skin and muscles at site of the drug injections, peripheral lymph nodes, parotid gland, tongue, tonsils, thyroids and parathyroids, mediastinal lymph nodes, bronchial node, heart, all seven lobes of the lungs, spleen, stomach, liver with gallbladder, pancreas, small intestine, cecum, colon, mesenteric lymph node, adrenals, kidneys, bladder, prostate, ovaries, uterus, testicles, brain (cerebellum, mid brain, cerebrum, pons and brain stem), spinal cord (at several levels), bone and bone marrow. Hematoxylin and eosin stain and Bauer’s, Bridley’s and Grocbtt’s histochemical methods were employed. At the time of necropsy, Wright *s-stained smears of lesions and of lungs and bone marrow, were examined for the presence of yeasts. Gross lesions as well as the lungs, bronchial node, liver, spleen, mesenteric lymph node, heart blood, feces and urine were routinely cultured for fungi.

RESULTS

M) Dogs inoculated intratracheally with 0.25 ml of packed cells 1. Untreated: The clinical course of the two dogs in this group was similar until the eighteenth day, when dog 55-l51|l died; dog 5£“l£l+0 progressed to a chronic and localized disease. 11+2

Dog 55-151+1 received 0.25 ®1 of packed cells lntra- tracheally•

Clinical findings: From a basal of 102 F. the temperature curve started to go up sharply on the tenth post-inoculation day (p.i.d.), reaching its peak (105.2 F.) on the twelfth p.i.d., and gradually going down to 100 F. on the seventeenth p.i.d. Dyspnea was first noticed on the sixth p.i.d., and cough appeared on the eighth. Blastomyces yeasts were found microscopically in the sputum and feces on the eleventh and sixteenth p.i.d. The white blood cell count (w.b.c.) became slightly elevated shortly after inoculation, but attained its peak of 22,050 on the fourteenth p.i.d. A radiogram, taken on the eleventh day, showed diffuse coalescing focal opacities radiating from the hilura.

Necropsy findings: The right diaphragmatic lobe of the lung was light cream, solidified and twice the size of the left diaphragmat ic lobe. On section, both diaphragmatic lobes showed caseation necrosis. The other lobes were of a vivid red and diffusely sprinkled with pale foci measuring up to 3 mm in diameter. The bronchial lymph node was yellow and measured 3.5 cm in length. The thyroids, pancreas, heart, and especially the kidneys, showed several tan foci, 1 to 3 mm in diameter. All the cortex of the kidneys contained 1 h3 these small nodules. Typical Blastomyces dermatitidis yeasts were seen on smears of blood, spleen, lungs, bronchial node, feces, pancreas, mesenteric node and kidneys (see Table 18). Blastomyces were cultured from the above tissues and feces, liver an urine.

Histopathology: The yeasts were identified in sections of most tissues (Table 18). The tan areas of the lungs showed diffuse caseation necrosis sprinkled with foci of neutrophils. A granulomatous reaction predominated in some areas. Blastomyces derma titidis were seen throughout the parenchyma and in bronchiolar purulent exudate. Areas (red grossly) consisted of conges tion, edema, reticulo-endothelial cells, neutrophils, epithelioid giant-cells and blastomyces yeast. A few large bronchioles were distended and filled with a purulent exudate mixed with red blood cells. The bronchial lymph nodes were undergoing extensive caseation necrosis. Small granulomas (about 1 mm) were seen in most organs. They consisted of well demarcated foci with a caseous center containing a few blastomyces and some neutro phils, surrounded by a zone of epithelioids and reticulo endothelial cells, epithelioid giant-cells, blastomyces yeasts and lymphocytes. The granulomatous lesions were noted in the following organs: kidney, spleen, liver, heart, 144 pancreas, adrenals, thyroids, tonsils, parotid, ovaries, uterus, brain, tongue, epiglottis, and both eyes. A granuloma (2 by 0.5 mm) was seen in the ciliary body of the left eye, and another (2 by 0.2 mm) was present in the choroid, close to the ora serrata. Smaller granu lomas were seen throughout the choroid. In the yeast cells, several of them budding, had been engulfed by reticulo endothelial and epithelioid cells. Fibroblasts were also numerous, especially at the periphery. Giant-cells formed by the macrophages, engulfed yeasts and melanin pigment. Granulomatous and fibrous choroido-cyclitis best described the lesions in the left eye; a similar granulomatous choroiditis was seen in the right eye.

Dog 55-1540 inoculated like dog 1541* had an ©arly clinical course similar to the latter but overcame the in fection and was destroyed 151 days after inoculation.

Clinical findings: Soon after inoculation, the temperature started to go up gradually, reaching a peak of 105.6 F. on the eleventh p.i.d. The same day, the dog showed depression, inappetence, rales, dyspnea and cough. The feces and sputum contained blastomyces. The animal lost weight gradually until the fortieth p.i.d., when its temperature settled to a normal level. The total w.b.c. count became slightly elevated soon after inoculation, reaching a peak of 32,1^50 on the twenty- second p.i.d., and returning to the normal level in a few days. On the twenty-fifth p.i.d., both eyes showed cloudi ness of the cornea. Exophthalmos and conjunctivitis soon became severe but a culture of the ocular exudate was nega tive for blastomyces. A few days later, hemorrhage could be seen in both cornea, and a small elevation was present in the upper sclera of the right eye. The animal continually searched with his head and scratched his eyes with legs or paws. He gradually gained weight, had another period of coughing on the ninth and tenth week after inoculation, and soon was free of clinical signs, except for irreversible blindness. Direct examinations of the feces for blastomyces were positive from the sixth to the seventy-fifth day.

Necropsy findings: The pupils of both eyes were brown, opaque with a white center. The left eye had a reddish brown spot in the middle of the white center. The right diaphragmatic lobe was adherent ventrally to the diaphragm. This lobe also showed an empty crescent shaped cavity measuring 1.5 cm x 0.8 mm x 1 cm. The right kidney contained an old infarct.

Histopathology: The lung was the only organ in which blastomyces were found. They consisted mainly of a cell wall without the central body. The cavitations had thick fibrous walls, containing a few small granulomas consisting of palisading reticulo-endothelial cells and a caseous center with degener ated yeast. Both eyes showed cataract, detachment of the retina and glaucoma. The hyperplastic retina was dissociated from the choroid; the lens itself was very big and pushed forward into the anterior chamber, close to the cornea. The anterior chamber contained eosinophilic material, a few round cells and pigment. The posterior cavity (between the detached retina and choroid) contained a pale eosinophilic acellular fluid. The lens presented disintegrated lens matter, small spheres of Morgagni, and several vesicular cells and vacuoles, especially at the posterior portion of the lens; all those changes were compatible with a diagnostic of cataract.

N) Dogs inoculated intratracheally with0.50 ml of packed cells 1. Untreated; Three of the four dogs in this group became acutely ill and died from eight to fourteen days after inoculation. One dog recovered and was destroyed 85 days after inoculation. Dogs 55-10UU. 55-10U5. 55-1066 showed similar clini cal responses yhich are presented in Table 15* On the thirteenth p.i.d., dog 55-1066 showed a few pustules (0.75 cm in diameter) on the left inside flankfold TABLE 15. CLINICAL SUMMARY OF DOGS INFECTED I.T. WITH 0.5 ML YEAST

Post Infection day first noted Treatment Stilbamidine Hydroxystilbamidine Untreated Dor No . 103k 1035. 10£6 IO33 10U2 1072 10kk 10US 1066 1865 Death 17 13 218* 20 10 10 13 9 Ik 85# Temperature initial rise 3 3 9 5 3 5 k k 6 5 maximum F. IOI4. • 2 101). 8 102.3 105.2 lOk.O 103.1* 106.2 10k. 6 105.2 10k .C maximum k 5&9 10 9 6 5 13 8 11 10 Pulmonic signs dyspnea 2 2 9 2 2 5 h it {* 1* cough 3 3 10 5 3 3 9 5 0 10 Chest radiopacities hilar 6 6 5 5 5 5 k 5 k Ik diffuse 6 6 - 12 11 k k focal - - 5 - - - - - subsiding - - 67 -- - — - mm 72 Leucocytes initial rise 7 7 2 10 10 10 2 - 7 2 initial decline - 2 - k 2 2 - 2 - maximum (thous./mm3) 33. 25. 31. 35 .8 26.3 3^.6 21*. 1 ik 63 25.9 maximum 1 Ik 10 7 14 10 10 10 7 Ik 2

* destroyed. - none. 4=rH - 3 llj.8

and the hairless area of its abdomen. Aspiration of pus re vealed blastomyces on culture but not in smears. Blasto myces was demonstrated in smears of sputum and feces of all three dogs.

Necropsy findings: The lesions found in these three dogs were very similar. At least 75 P©n cent of the lung parenchyma was consolitated, tan in color, and contained caseous exudate. The apical lobes were the most extensively affected. The other lobes presented focal granulomas, sometimes coalescing to form large areas of consolidation. Lungs of dog 55-1066 showed numerous adhesions with the costal pleura. The bronchial lymph nodes were very large; one of them measured

4.5 x 1.5 cm. The kidneys of dog 55-1066 showed numerous white nodules (1 to 3 ™ ) in the cortex. In dog 55-10lj.li, blastomyces was cultured from the lungs only but we found yeasts in smears of blood, feces, spleen, lungs and bronchial nodes. The cultural findings are listed in Table 20.

Histopathology: The microscopic findings of the lungs and other organs were very similar to dog 55-l5i|-0. A resume of the lesions is found in Table 1.7. Log 55-1066 presented an acute suppurative conjunctivitis of the palpebral and bulbar Iks conjunctiva. No granulomatous reaction or blastomyces could be demonstrated. Dog only showed only granuolmas in the eyes and brain. Small foci of granulomatous inflamma tion containing organisms were seen in the cerebral cortex and hippocampus. A focal granulomatous choroiditis was pres ent in the left eye and a small area of necrosis (containing epithelioid cells and a few blastomyces) was seen in the episcleral tissues of the right eye, close to the bulbar conjunctiva. Dog 5>5>-l865 showed a one degree temperature rise the day after inoculation. A peak 101}. P. was recorded on the tenth p.i.d. and a high temperature was maintained until the seventeenth p.i.d. The dog showed no depression. From a basal of 15,850 w.b.c. the blood picture showed 25,900 w.b.c. the second day after inoculation and a gradual diminution in the subsequent weeks. The dog showed early dyspnea and rales but the coughing was minimal. Smears of the feces and sputum showed blastomyces on the eleventh and twenty-eighth p.i.d.; they were negative thereafter. A radiogram taken on the fourteenth day showed coalescing focal opacities in the hilar area and on the seventy-second p.i.d., only slight evidence of opacities were noticeable. The dog was destroyed on the eighty-fifth p.i.d. i$o

Necropsy findings: There was a fibrous adhesion between the left apical lobe and the pericardium. The left apical, cardiac, and part of the left diaphragmatic lobes were nodular, bluish gray and contained several cavitations. Yellow foci (1 mm) were seen in the parenchyma around these cavitations. The right lobes and other organs appeared normal.

HIstopathology: Hemorrhages were seen throughout the lungs and most of the alveolar walls were thickened and very collagenous; a thin fibrous capsule surrounded the cavitations. G-ranuolmas, containing poorly stained blastomyces, were seen close to the cavities. Some showed a caseous center with small deposits of calcium salts and cholesterol crystals. 2. Dogs treated with stilbamidine: The treatment regimen is listed in Table 7* Dogs 55-103U and f?5-1035> showed similar clinical signs (Table 1$). The animals died In eighteen and fourteen days, after losing respectively 33 euid. 18 per cent of their body weight.

Necropsy findings: About 60 per cent of the lungs were consolidated, light gray, and adherent to the costal pleura. Dog 5f?-103lj. presented a few pustules on the hairless area of the skin, inside the thighs, but smears and cultures were negative for 151 blastomyces. A diffuse granulomatous pericarditis was pres ent in both dogs. Blastomyces dermatitidls were demonstrated on smears and cultures as noted in Table 20.

Hi s topathology: The lungs of both dogs showed diffuse granulomatous and suppurative pneumonia, accompanied by a diffuse granulo matous pleuritis. Caseation necrosis was also extensive. Dog 55-1035 showed a diffuse necrotic and granulomatous in flammation in the bone marrow of the left ilium with numerous blastomyces. Granulomas and dermatitidis were present in several organs as noted in Tables 17 and 20. Both dogs showed granulomas in the choroid, especially numerous near the ora serrata. In dog 55-1035* fibroblasts formed a significant portion of the granulomatous choroiditis. Dog 55-1036 was infected and treated like dog 55-103l| and 55-1035* The temperature was never very high with peaks of 102*8 F. and 103*2 F. on the tenth and twenty-fourth p.i.d. The white blood cell count went up to 31*000 on the seventh p.i.d. and stayed at this level for about ten days. From the tenth day to the twenty-fifth day, the animal showed depres sion, poor appetite, dyspnea, cough and rales. During this period the feces and sputum contained blastomyces. After the twenty-fifth day the clinical signs disappeared and the hilar opacities were subsiding. The first three weeks after inocu lation the animal lost 3 lb. At the end of the fifth week, 152 the dog had regained his original weight of 17 lbs and when destroyed 232 days after inoculation, he weighed 20 lb.

Necropsy findings: The only lesions were adhesions between the left apical and cardic lobes.

Histopathology: We found lymphoid hyperplasia of the bronchial, mesenteric and medial retropharyngeal lymph nodes and tonsils. Some bile duct hyperplasia of the liver and a few fibrous foci in the kidneys were also seen. Irregular areas of con solidation were found in the lungs around some bronchi or large vessels which contained mostly fibroblasts, reticulo- endothelial cells and monocytes. No fungi were demonstrated. Some congestion, a few lung cavitations and fibrous pleuritis were also present. Dogs 55-80 and 55-1861 received higher doses of stilbamidine than the two previous dogs and died from the drug toxicity in five and seven days. Dog 55-80 did not show fever or leucocytosis. Dog 55-1861 developed a fever and leucocytosis on the third p.i.d. (103 F.) Besides the toxicity lesions already described, dog 55-80 showed a few granulomas in both kidneys (1 mm) and pneu monia in the left lobes. The extensive granulomatous reaction In the lungs consisted of reticulo-endothelial, epithelioid, 153 monocytic and polymorphic cells and epithelioid giant cells. Blastomyces were numerous in the cardiac lobe only. The lungs of dog 55-1861 were much more extensively involved, all lobes being filled with granulomas. (1 mm to 1 cm). These pale gray nodules with sometimes a dark red aureole coalesced to form large lesions in the diaphragmatic lobes. Foci of extensive early granulomatous Inflammation were seen around the bronchioles. The unbroken alveoli were distended with epithelioid cells, giant-cells and numerous yeasts. The fungi were well stained, single or budding, sometimes en gulfed by giant-cells. Neutrophils were numerous throughout.

3. Dogs treated with hydroxystilbamidine: Dogs 55-1033, 55-10^2, 55-1072 were treated accord ing to the regimen in Table 8 and died in twenty, ten and ten days after inoculation. Their clinical signs are listed in Table 13. On the ninth p . i . d . , dog 55-1072 presented some pustules on the abdomen essentially similar to those seen on dog 55-1066 and 55-103U* Cultures and smears were negative for blastomyces. The feces and sputum of all animals (except the sputum of dog 55-1072) contained yeasts on ante-mortem examination.

Necropsy findings: The lungs of dog 55-1033 were very extensively in volved, 2-g- times larger than normal, tan in color, completely consolidated and necrotic. Adhesions between the lobes and 154

Fig.20- Lungs of dog 55-1035 (I.T. infected and stilbamidine-treated), Note the massive consoli dation.

Fig.21- Brain of dog 55-1033 (IT infected and hydroxystilbamidine-treated). Granuloma containing a few giant cells and some yeast. 155 with the costal pleura were diffuse. The right lobes con tained several yellow nodules (1 mm to 1 cm). This animal also showed numerous 1 mm yellow foci in the myocardium and endocardium; similar granulomas, some as large as 5 mm in diameter, were found in the cortex of the kidneys. Fifty ml of a pink, purulent fluid, containing a multitude of B, dermatitidis, were present in the thoracic cavity. Dogs 55-10^2 and 55-1072 died easily and their lungs were not extensively involved. The right lobes, especially of 55-1072, were twice their normal size, consolidated, mottled with red and gray. No granulomas were seen in other organs. Organisms were recovered in numerous organs as noted in Table 20.

Histopathology: Dogs 55-10^2 and 55-1072 presented focal or diffuse suppurative and necrotic granulomatous pneumonia; dog 55-1033 showed extensive necrotic granulomatous pneumonia without suppuration. The necrosis was either of the coagulation or caseation type, often containing faintly stained yeasts. Giant cells containing organisms were numerous throughout the lungs. The histopathology and mycology are summarized in Tables 17 and 20.

1|. Dogs treated prior to infection: Dogs 55-1862 and 55-1863 received respectively 750.2 and 656.2 mg of stilbamidine in forty-seven days previous to 156

TABLE lfc. CLINICAL SUMMARY OF PRETREATED DOGS INFECTED I .T . WITH 0.5 ML YEAST

Post infection day first noted Dog No. 5590 556B 551662 551663 Diamidine Hydr. Hydr. Stilb. Stilb. pretreatment/mg 1,020 819.6 760.2 656.2 concurrent treatment 3,045 -- - euthanasia 345 345 14* 52 Temperature initial rise 9 9 5 10 maximum F. 104.2 104.4 104.4 104.2. maximum 10 9 & 16 10 17 Pulmonic signs dyspnea 9 9 6 12 cough - 9 9 14 Chest radiopacities hilar 14 15 14 14 diffuse - - 14 - focal 25 25 14 subsiding 32 60 - 46 Total leucocytes Initial rise 2 7 - 2 initial decline — — 2 - max imum (thous•/mm3) 29.2 32.1 10.5 31.4 maximum 42 35 4 29 Total neutrophils ? maximum (thous . /mmr ) 21.3 30.6 6.3 31.0

Incoordination - - - 50

Reaction to blastomycin 73 73 - -

■anatural death.. - none. 157 infection. No abnormal clinical signs were shown. After a post-treatment interval of one week 0.5 nil of packed blasto myces yeast was introduced into the trachea of each dog. No further treatment was given. Dogs 55-68 and 55-90 received total doses of 819.6 and 1,020 mg of hydroxystilbamidine in seventy-one and fifty- four days respectively. (Table 6). After a 2-week rest period, both dogs were inoculated with 0.5 ml of packed blastomyces. Dog 55-68 did not receive further treatment but dog 55-90 was given 2.5 mg/kg of hydroxystilbamidine, daily, during seventy days (a total of 3*014-5 mg). The cumu lative dose was i|.065 grams of 2-hydroxystilbamidine isethionate for both courses of treatment. Dog 55-1862 died on the fourteenth post-inoculation day. Details of the clinical course will be found in Table 1.6.

Necropsy findings: A bright yellow consolidation, made up of coalescing granulomas, involved 75 per cent of the lungs. The left diaphragmatic lobe though not consolidated was peppered with small granulomas. Adhesions with the costal pleura were ex tensive. Purulent granulomatous pericarditis, myocarditis, endocarditis, and nephritis were characterized by a sprink ling of granulomas which measured about 2 mm in diameter. 158

Blastomyces were cultured from several tissues, including the blood. Organisms were also seen directly on smears of most organs as noted in Table 20.

Histopathology: Lesions of diffuse and/or focal suppurative granulo matous inflammation accompanied by necrosis, were similar to lesions seen in previous disseminated cases. Dissemination of organisms was more extensive as seen in Table 20. We found several small granulomas in the choroid of both eyes, sometimes extending into the zoiua of rods and cones of the retina. These ocular granulomas, as well as those seen in previous dogs, were mostly found in the choroid close to the ora serrata. One larger granuloma showed extensive caseation necrosis (Pig. 22). The numerous granulomas seen throughout the cortex of the kidneys had caseous or purulent centers with surrounding blastomyces, epithelioid cells and reticulo-endothelial cells. At the periphery some lymphocytes extended between the tubules. These granulomas were not encapsulated and were located between or replaced the renal tubules. Many glomeru li were destroyed; others were partially destroyed and filled with blastomyces. This picture indicated that the renal lesions began in the glomeruli as the result of blastomyces emboli. 159

Dog; 551863. The onset of fever and pulmonic signs was later than in dog 551862; there was very little cough ing. Fever was recorded from day 17 to day 22; dyspnea persisted. Leucocytosis occurred on day 15 and reached a peak of 31,k00 °n day 30* The dog was apparently recovering, but on the forty-second p.i.d. the animal showed depression. On the forty-fourth day we noted a marked drop in the temperature to 100.2 F. In spite of subsiding lung radiopacities the animal became prostrated. The heart beat was fast and weak, the respiration slow and deep. The head was extended and the legs showed extensor rigidity. The dog was conscious of his environment, showed sensitivity to pin prick3 but seemed unable to move. By the forty-ninth day the temperature was down to 96 F. and the dog was paralyzed except for the eyes. The dog was destroyed.

Necropsy findings: We found nests of cavitations In the left apical and cardiac lobes, the largest cavity being 1 cm in diameter. Other lobes showed only a few granulomatous nodules. Both kidneys showed granulomas, the largest being 1.5 cm in diameter and extending from the cortex to the pelvis. The brain presented extensive hydrocephalus with atrophy of the cerebral cortex. An abscess, discharging a greenish pus, was seen in the area of the optic chiasma, hypothalamus and pituitary gland. Blastomyces were seen on direct smears from Pig. 22- Granuloma in the choroid of the Mght eye of dog 55-1862, showing blastomyces, caseation necrosis and peripheral fibrosis.

Bauer, 210 X

Fig. 23- Dog 55-1863. Granulomatous inflammation in the lateral ventricle adjacent to the caudate nucle us. Note the perivascular cuffing in the cau date nucleus. H & E 160 X

1 6 0 Fig* 22

Fig, 23 162 the urine, kidneys and brain, and cultured from the kidneys and brain.

Histopathology: Blastomyces were seen only on sections of lungs, kidneys and brain. Neutrophils lined the lung cavities and extended into the thick granulomatous walls to form micro- abscesses. Central areas of coagulation necrosis contained faintly stained organisms. The cavities had no epithelium and were distinct from the bronchi. The kidneys showed a few large areas of coalescing suppurative granulomas divided by strands of collagenous fibers. The brain was extensively involved; a lining of granulomatous tissues covered the lateral, third and fourth ventricles, and extended into the parenchyma to form a series of granulomas (Pig. 23). The cells were mostly glial cells, with a few neutrophils and gitter cells, and some scarce yeasts. Deeper in the brain parenchyma we saw cuff ing of the blood vessels. Similar lesions were found in the hypothalamus. A microabscess (no yeast) was found in the ciliary body of the left eye near the left fornix. Dogs 3568 and 3590 followed very similar clinical courses. Fever occurred on the ninth p.i.d. (lOij.• I4. P.) and persisted until the twenty-second day; a secondary peak was 163 recorded on the thirty-first day. Clinical details are pre sented in Table 16. Sputum and feces of dog 5568 showed blastomyces during the third week. A skin test with a commercial antigen diluted to 1:100 (Blastomycin, Parke-Davis) performed on the seventy- third post-inoculation day was negative after twenty-four hours. At forty-eight hours, we saw a 5 111111 induration in dog 5568 and a 3 111111 induration in dog 5590, both reactions had a 1 cm erythematous periphery. At seventy-two hours, the erythema was regressing. All prior and further skin tests were negative. The dogs were destroyed 3^5 days after ino ciilation.

Necropsy findings: The right apical lobe was pale pink and was traversed by blue areas.

Histopathology: Both dogs showed similar pulmonary lesions consist ing of fibrosis around some bronchi, bronchioles and vessels. When this consolidation was adjacent to the pleura, the latter was thick and fibrotic. These areas contained several large macrophages filled with vacuoles and sometimes hemo siderin. No B^ dermatitidis was seen. The kidneys also showed areas of fibrosis. In dog 5590 the renal cortex con tained several empty spaces lined by a single thin layer of an endothelial type of cells. 16]|

TABLE 17. COMPARISON OP UNTREATED, STILBAMIDINE AND HYDROXYSTILBAMIDINE TREATED DOGS INFECTED I.T. WITH 0.5 ML YEAST

Spinal Mesenteric Brain Cord Nodes Pancreas

Foe granFoe gran Foe granFoe Foe gran

1 0 6 6

1 0 3 4 © Foe gran Foe gran Lymph hyp •H 1035 Foe gran Foe gran Foe gran Lymph hyp Cong n Foe necr 1—1 -P 1 0 3 6 CO Lymph hyp

10 33 t Foe gran Foe gran Foe gran rH •H O P fj ra -h 1 0 4 2 Foe gran Cong Lymph hyp Cong K *H 0 ^ ^ n 1072 &

Dlff • Diffuse Foe • Focal Gran : Granulomatous inflammation Lymph hyp: Lymphoid hyperplasia Supp : Suppuration Necr : Necrosis FIbr : Fibrosis Cong : Congestion 165

TABLE 1.7 — Continued

Bronchial Lungs Nodes Spleen Kidneys Heart iokk Diff gran Diff gran Foe gran Foe gran Supp Necr 1066 1 Diff gran Diff gran Hyp Foe gran Diff gran Supp Necr Supp Supp

1031*. Diff gran Diff gran Foe Necr Foe gran Diff gran Supp Necr Necr

1035 Cong Lymph hyp Foe gran Foe gran Diff gran Diff gran Supp Necr Supp Necr Necr Stilbamidine 1036 Foe gran Lymph hyp Cong Fibr Fibr

1033 © Diff gran Diff gran Lymph hyp Foe gran Foe gran •H Necr Necr Foe gran t J Necr

101*2 H& Foe Diff Lymph hyp •H gran -P ra Supp Necr K 1072 o Foe Diff Foe gran Lymph hyp 3 Gran Supp 1 6 6

TABLE 17— Continued

Retropharyn Mandibulai Liver Thyroids Tonsils geal nodes nodes

10U4 Foe Necr Lymph hyp Lymph hyp Foe gran

1034 Diff gran Diff gran Necr

1035 Cong Foe gran Cong Necr

1036 Stilbamidine Bile duct Lymph hyp Lymph hyp hyp

1033 Fatty Cong Foe gran Lymph hyp degen Foe gran Cong Necr

101+2 Fatty Lymph hyp Lymph hyp

dine degen

1072 Lymph hyp Hydr oxys t ilbami- t oxys Hydr 167

TABLE 17--Continued

Bone Thoracic Adrenals Parotids Marrow £tomach nodes 1 Okb Foe grsm

•3<0 -P Cong 1045 aJ © Foe gran fH

1066 § Diff gran Supp Necr

1034 © Diff gran •Ha Supp Necr Foe gran Diff gran 1035 ,a Necr H •H 4-* 1036 CO Fibr

1033 Foe gran

IOI4.2 bamidine 1072 Hydroxystil- 168

0) D o r s Inoculated intravenous1y with 0.2$ ml of p a c k e d

cells 1. Untreated: Dog $$1071 lived only four days after infection (Table 10). Direct examination of sputum and culture of the blood were positive for blastomyees.

Necropsy findings: The lungs were completely and uniformly consolidated. The color varied from a dark red to a purplish hue and the whole organ was speckled with innumerable, small (0.$-l mm) focal embolic granulomas. Table 23 shows the cultural findings.

Histopathology: The lungs showed hyperemia, edema, and the alveoli were filled with organisms. The septa of these alveoli were intact. Some yeasts were ingested by the alveolar lining cells. Necrosis and a neutrophilic infiltration were also present. A resume of the dissemination of lesions and organ isms is presented in Table 19 and 23. 2. Dogs treated with stilbamidine: Dogs $$1031, $$1032, $$10Ul received stilbamidine according to the regimen listed in Table 7« The disease was similar in all dogs (Table l8). There was no leucocytosis during the short course; instead there was some decline in TABLE 18. CLINICAL SUMMARY OF DOGS INFECTED I.V. WITH 0.25 ML YEAST

Post in fe c tio n dayfirst noted Treatment Stilbamidine Hydroxystilbamidine Untreated Dog No. 1031 1032 10U2 _ 103? 1038 1039 1071 6 6 6 Death _. 5. .5. ... $ h __ Temperature initial rise 2 k k 2 3 3 2 maximum °F 101;. 8 101;. 1 0 3 4 101;. 2 101; • 2 101;. 0 105.2 maximum 2 k k k 3 3 3 Pulmonic signs dyspnea 1 1 3 2 3 1 1 cough - 3 3 - 2 3 Chest radiopacities d iffu se 3 3 3 3 3 3 3 fo ca l ------Leucocytes - initial rise - - - 2 - - initial decline 2 2 2 - 2 2 2 maximum (th o u s./ 16.2 17.5 7.9 20.6 12.3 11.6 11.2

None.

o vO 170 leucocytes. Blastomyces were not cultured from the blood. All three dogs died on the fifth post-infection day.

Necropsy findings: The lungs were entirely consolidated except for the emphysematous dependent extremities of a few lobes. They were hyperemic and showed a characteristic mottling of wine red and gray. The bronchial, mandibular and medial retro pharyngeal lymph nodes were slightly enlarged and congested. The tonsils were hyperemic and swollen. A few white granulomas (1 mm or less in diameter) were seen in the endocardium and myocardium, especially in dog 551031 and 55l0lj.l. Blastomyces dermatltldls were cultured according to Table 23.

Histopathology: The lungs of all three dogs showed congestion, hemorrhage^ and edema with blastomyces, neutrophils, reticulo endothelial cells and epithelioid giant-cells forming a uni form sheet, broken only by some distended bronchioles, con taining a purulent exudate and yeasts. An outline of the lesions are summarized in Table 19; Table 23 shows the dissemination of Blastomyces dermatitldis. Both eyes of dog 551031 an

Bauer, 70 X

Pig. 25- Dog 55-1031. High magnification of figure 21+. Note the vacuoles in the organisms and the

hyphae formation. Bauer, 700 X

171 Pig. 2k

4* *•*«! Pig. 25 contained blastomyces and a few epithelioid cells and lympho cytes but no giant-cells. They sometimes extended to the zona' of rods and cones and dissociated the retina from the choroid at that level. The vessels of the choroid were dilated. A piece of skin taken from the thigh of dog ££1031 showed a granuloma in the deep subcutis, 0.5 cm from the skin surface. The granuloma was between a vein and a lymphatic, extending to these two structures (Pig. 20). Blastomyces were numerous in this intense granulomatous re action. The reticulo-endothelial cells and the yeasts had dissociated and eroded the wall of the large vein but had not extended to the lumen at this level. Inside the vein there was another smaller granuloma, separated from the lumen by a thin endothelial membrane. The adjacent large lymphatic was filled with hemolyzed blood, blastomyces, lymphocytes and a few reticulo-endothelial cells. Most of the yeasts contained round vacuoles some even distending the organisms. The budding forms were numerous with attempts to form hyphae

(Pig. 25). 3. Dogs treated with hydroxystilbamidine:

Dogs 551037, 551038, 551039 received hydroxystil bamidine according to Table 8. They showed very similar signs and lesions. The dogs lived one day longer than the stilbamidine-treated dogs and yeast was slightly more dis seminated. The clinical findings are seen in Table 18. 17k

Necropsy findings: The gross appearance of the lungs was identical to the lungs of the previous dogs. A few white pin-point nodules were seen in the cortex of the kidneys. A resume of the cultural findings will be found in Table 23.

Histopathology: Alveoli of the lungs were distended or broken and filled with yeast cells, several of them budding, forming large sheets of tightly packed organisms interspersed by strands of congested alveolar walls and a few aerated alveoli or bronchioles. The lungs were congested; most of the lumina of the bronchi were lined with exudate and yeasts, but were not occluded. These lungs, contrary to all intra venous cases seen previously, contained very few neutrophils. As in dog 55l0li.l the precedent group, numerous glomeruli were filled with blastomyces without provoking any cellular reaction. A few foci of granulomatous inflammation containing some blastomyces and numerous plasma cells were seen between the renal tubules. While only dog 551031 in the precedent group showed a small brain granuloma without visible organisms, all three dogs of the present experiment showed a few granulomas in the brain. Both eyes of dogs 551037 and 551039, and the left eye of dog 551038 contained granulomas in the choroid, and rarely in the retina. Retinal lesions appeared to be exten sions of a granuloma in the choroid. 175

TABLE 19. COMPARISON OP UNTREATED, STILBAMIDINE AND HYDROXYSTILBAMIDINE TREATED DOGS INFECTED I.V. WITH 0*25 ML YEAST

Mesenteric

-p Eyes Liver NodesBrain Pancreas 1071 Lymph hyp -p

Foe gran Foe gran Foe gran

1032 •H Cong

ioui •H Foe gran Cong

1037 1 Foe gran Foe gran Foe Necr Foe gran n rH •H *d K O 1039 Foe gran Foe gran Foe gran w

DIff : Diffuse Foe : Focal Gran : Granulomatous inflammation Lymph hyp: Lymphoid hyperplasia Supp : Suppuration Necr : Necrosis Fibr : Fibrosis Cong : Congestion 176

TABLE 19— Continued

Bronchial 'd • Lungs • Nodes Spleen Kidneys Heart pal ® 1071 Diff -gran Biff gran Lymph hyp Fo’c Necr • • -p Supp § •

c 1031 ' Diff gran Lymph hyp Fibr Foe gran Supp $ •H 9 1032 Diff gran Foe gran Foe gran Supp Cong & i•H H p 10i|l CQ Diff gran Fpc gran Cong Foe gran Foe gran Supp

1037 e Diff & , Diff gran Foe gran a Foe •H Gran

1038 & Diff gran Lymph hyp Foe gran Foe gran Foe gran H •H" Necr Necr P00 ® ® 1039 8- Diff gran Foe gran Foe gran Foe gran Necr Necr & 177

TABLE 1ST— Continued

Retropharyn Mandibulax Thyroids Tbnsils geal nodes nodes Ovaries •o -p 1071 aJ Biff gran Lymph hyp Lymph hyp 0 Supp u Necr • & .

1031 Lymph hyp

1032 Foe gran Lymph hyp Lymph hyp Lymph hyp Necr Cong Cong Stilbamidine 101*1 Poc Necr Lymph hyp Lymph hyp Cong Cong

1037 Poc gran Supp

1038 Diff gran Supp Supp Necr Necr

1039 Supp Cong Poc gran Poc Necr Hydroxystilbamidine Hydroxystilbamidine j 178

TABLE 19— Continued ■»

AdrenalsParotids Prostate Skin

1071

1037 1 Foe necr

Xi 1—1 © 1038 •h rt Foe .gran -p -h Poc gran Foe gran m Supp 0fr 1039 Foe gran W TABLE 20. DISSEMINATION OP B. DERMATITIDIS IN DOGS INFECTED INTRATRACHEALLY

Stilbamidine Hydroxyst ilb am - Pretreated treated. I dine Not treated treated Hydrdxy. Stilbamidine

Dog number 551 551 551 551 551 551 551 551 551 551 551 551 55 55 551 551 034 035 036 033 042 072 044 045 066 540 541 865 90 68 862 863 Ante mortem blood sputum DDD D D 0 - DD D DD 0 DDD feces DD D D DD D DD DC DC D - D DD urine - 0 - mm ------skin lesions X XX X X X X X C XXXX X XX

Post mortem blood c c mm H D c c mm DC mm DC bone marrow - H ------spleen C C - H - C D D c mm DCH m - - DCH mm lung DCH DCH - D H DCH DCH DCH DCH DCH H DCH D H -- DCH H bronchial node DCH DCH - D H DC DCH D H DC CH - DCH - -- DCH - tonsils - - H - mrn -- - - H - m H - parotids - H --- -- 0 -- H -- m H — feces D H H DCH D H D H D H D H- H mm DCH - - m DC - liver - C mm mm C - «• C mm CH -- - DCH - pancreas - mm - H - mm H --- DCH ■* ■* - 0 m mesenteric node CC ** *» mm ■* *“ ■* DC C mm-

D direct microscopic demonstration of the fungus C culture of the fungus H histopathologic demonstration of the fungus 0 not examined 179 - negative x none TABLE 20— 'Continued

Stilbamidine Hydroxy, Pretreated treated treated Not treated Hydroxy. Stilbamidine Dog number 551 551 551 551" 551 551 551 551 551 551 551 551 55 55 551 551 034 035 036 033 042 -072 044 045 066 540 541 865 90 68 862 863

heart HH - H - - H - H - H -- - H - kidneys HH - D H H • f t H - H - DCH - am - DCH DCH • bladder - mm - - *• mm - -- - « ■ - - --• •ftt uterus am X X • f t X - XX X X H X X - • X ovaries ** X X - X ■ XXX X H XX - H X testis X - - X ft* -- -- •M X -- X -- prostate X - X ------X -- X - - urine aft 0 ------C - C - -- - D

•» - -•• - am - brain HH HH . H - H H DCH thyroids - H • f t H -- - *• - - H - - - H ma parathyroids H - adrenals -- ' - mm - - H - -- H -• - - H - peripheral l.n. HHHH - H •ft -- H «• eyes H H - H ri - H • f t -- H -- - H *a diaphragm 0 H - 0 0 0 0 0 H 0 0 0 0 0 H -* peritoneum - - . - -- - * <■» ------• H mm epiglottis - - H •ft 0 - - H - - - 0 am tongue » 0 - H »- •ft -- mm H - am - 0 mm stomach • H - 180 181

P) Dogs inoculated intravenously with. 0.1 ml of packed cells 1. Untreated: Dogs 551069 and 551073 died in eight and nine days. The clinical response is listed in Table .21. Dog 551069 showed no leucocytic response while dog 551073 had a white blood cell count of 25»100 on the seventh p.i.d.

Necropsy findings: The lungs of both dogs were similar to the lungs of hog 551071* The bronchial lymph nodes and tonsils were en larged and hyperemic. A resume of the cultural findings will be found in Table. 23.

Histopathology: The tissues presented the same minimal reaction re corded in previous intravenously inoculated dogs. Dog 551069 did not show neutrophilic infiltration and there was minimal inflammatory reaction to the massive growth of the organisms. Blastomyces dermatitldls was found in the bone marrow of the left tibia of dog 551073* Organisms were not found in brain or eyes of either dog. A resume of the microscopic and cultural findings will be found in Tables 22 and 23* .2. Treated with hydroxystilbamidine: (Table 8) Dogs 551070 and 551074 died respectively in seven teen and nine days. Dog 551070 at first appeared to resist 182

TABLE 21. CLINICAL SULSMARY OF DOGS INJECTED I.V* WITH 0*10 ML YEAST

Post Infect zion dayfir s t n oted Hydroxystilb «unidLne Untreated 1070 107h. 1069 1073 Death 17 9 8 9 Temperature Initial rise 9 It k 2 maximum (F.) 10k*6 lOil-.Aj. 1034 10l|.2 maximum 16 7 h 7 Pulmonic signs dyspnea ^ 2 2 2 cough 9 7 7 7 Chest radiopacities diffuse 10 lj. 3 3 focal •m m

Leucocytes initial rise 2 k initial decline 2 2 maximum (thous./mm 3) 21.1 Uj.,9 10.7 25.1 maximum 7 7 2 7 183 the Infection since the lung opacities were not seen until days after infection. On the twelfth day the dog had a fever of 10li,.6 P., showed a very severe dyspnea, and died. The clinical findings will be found in Table 21.

Necropsy findings: The lungs of both dogs were uniformly mottled red and gray with a granular cut surface. A frothy pink exudate was present In the trachea. The bronchial lymph nodes were pale and measured 3.5 x 1.5' cbu Other lymph nodes were con gested and edematous. The spleen presented some small gray foci. A few pinpoint white foci were seen throughout the renal cortex. The heart showed a few white granulomas, es pecially in dog 551070, which exhibited nodules (0.75 cm in diameter) throughout the myocardium. Cultural findings will be found in Table 2 3.

Histopathology: The lungs of these dogs differed somewhat. Dog 551070 showed a diffuse focal suppurative and necrotic granulomatous inflammation characterized by numerous nodules separated by distended alveoli, often filled with fibrin. These nodules were made up of yeasts, epithelioid cells, giant cells, some reticulo-endothelial cells, monocytes, neutrophils and necrotic debris. The lungs of dog 55l07i|.

presented a similar but less intense response with no TABLE 22. COMPARISON OF TREATED AND UNTREATED DOGS INFECTED I.V. WITH 0.10 ML YEAST

Bronchial Spinal Lungs Nodes Spleen Kidneys Heart Brain Cord Eyes

1069

1073 -P Diff gran Diff gran Lymph hyp Supp § Necr •Necr

Diff & Foe Diff gran Foe gran Diff 2c Foe Foe gran Foe gran gran gran Necr Supp Supp Necr Necr

Diff gran Lymph hyp Foe gran Foe gran Foe gran Foe gran Foe Foe gran Supp Diff gran Necr Necr gran Necr & Diff : Diffuse Foe : Focal Gran : Granulomatous inflammation Supp : Suppuration Necr : Necrosis Fibr : Fibrosis Lymph hyp : Lymphoid hyperplasia

H CD -P" TABLE 22— Continued

Retropha Mesenteric ryngeal Mandibular Liver Nodes Pancreas Thyroids Tonsils Nodes Nodes Ovaries

1069 o Foe gran Foe gran Lymph hyp -p ClJ Necr Necr

1 1070 aJ Foe gran Lymph hyp Foe gran Diff gran Foe gran Diff gran Diff gran .a rH Foe gran Necr Supp Supp Supp •H © ■P a Fibr Necr Necr Necr 03 *rf

1074 8 * Foe gran Foe gran Lymph hyp Lymph hjp Lymph hyp Necr Foe gran Foe gran 4 Neer

CD vn TABLE 22— Continued

Adrenals Parotids Stomach Prostate Bone Marrow Intestines Skin Foe gran Necr

1070 1■S Foe gran Fibr X s 0 3 rQ 1071* T3

Q) Dogs inoculated subcutaneously with 1.0 ml of packed yeast 1. Untreated: Dog 551065 was inoculated subcutaneously in the left thoracic region and in the left sacral region with 0.5 ml. of packed yeast at each site. This dog died on the nineteenth day. There was an edematous, subcutaneous swelling at the thoracic site on the first p.i.d. The same day, the temperature was 103.8 P. and remained high for three days, when this huge shoulder abscess (12.5 cm in diameter) ruptured. The fungus was easily cultured from the exudate. An identical disease process was noted in the sacral region. Dyspnea, bronchial rales and 188

cough appeared very early in the course of the disease. Diffuse and focal radiopacities were seen on the radiograms. D irect smears of the sputum, feces and skin ulcers disclosed organisms. Leucocytosis reached26 J4.OO on the seventeenth p .i.d .

Necropsy findings: Several ulcers up to 1 cm in diameter, either granu lating or discharging were found at the inoculation sites. The prescapular lymph nodes on both sid es were abscessed. The lungs showed some scattered , depressed, w ell circumscribed dark-red areas (1-5 mm in diameter) of con solidation . A resume of the cu ltu ral findings is found in Table 23.

Hi s topathology: Subcutaneous, focal and coalescing suppurative granulomas undergoing fibrosis were noticed at the inocula tion sites. The underlying muscles and usually the dermis were involved. Skin papules resembling similar lesions seen in some intratrachealy inoculated dogs, were seen over the sternum. They contained colonies of bacteria and a few h istio c y te s and blastom yces. The lungs showed d iffu se, in t e r s t it ia l and massive suppurative granulomatous inflamma tion, with great number of organisms. The dissemination of yeast will be found in Table 23. 189

2. Dog treated with hydroxystilbamidine: Dog 551067 received the drug according to the regimen in Table 8 . It received a total dose of 2I+5 mg of hydroxy stilbamidine in 150 days. The dog appeared clinically cured and was destroyed after 330 post-infection days.

Clinical findings: The inoculation areas were swollen and edematous on the first day. The swelling in the thoracic area remained small, the swelling in the lumbar area progressed rapidly and measured 1| by 3 inches before it drained on the sixth post inoculation day. The next day the temperature was IOI1..2 F. The swelling increased in size and the temperature had another peak of IOI4..I4. just before it opened again in two places. The dog became depressed and lost 3 pounds dur ing the first two weeks. The total white blood cells count gradually increased to a maximum of i\.Q,$Q0 on the twenty-first day and decreased thereafter. The animal exhibited some coughing from the thirteenth day to the fiftieth day. Dyspnea was noticed on the thirtieth day and continued for two weeks after the coughing stopped. Organisms could be seen readily in the smears from the ulcers; the feces and sputum were also positive on the twentieth day. The thoracic abscess opened on the nineteenth day and on the twenty-second day both sites of inoculation 190 were healing. An ulcer appeared in the right pre-scapular lymph node area and several pustules developed in the skin below the ulcer. Forty days after inoculation the skin lesions were almost healed; the dog had a good appetite and was gaining weight in spite of persistent dyspnea and cough. Sixty-five days after inoculation, the animal appear ed to have completely recovered. After several negative blastomycin skin tests, the animal was destroyed on the 330th post-infection day.

Necropsy findings:

There was no evidence of lesions except fibroBis at the sites of inoculation but every lobe of the lungs con tained five or six disseminated calcified nodules. These nodules were white with a dark red periphery and sometimes a rede area in the center. They measured 2 mm or less in dia- § meter except one that measured ij. mm in diameter. A few white granulomas (1 mm or less in diameter) were found in the cortex of the kidneys.

Histopathology: We found a few areas of fibrosis in the lungs, ad jacent to some bronchi, and some fibrosing tuberculoid granulomas, with or without blastomyces, and calcification. A typical tuberculoid granuloma (2 by 1*£ mm) was found sub jacent to the pleura, in the left diaphragmatic lobe. It 191 consisted of a calcified center and a ring of caseation necrosis sprinkled with calcified deposits and containing very few neutrophils and blastomyces. The central bodies of these yeasts were absent or hyalinizedj some were budding at the time their growth was arrested. At the periphery there was another ring of reticulo-endothelial and epitheli oid cells and finally, fibroblasts. The left tonsil showed lymphoid hyperplasia and a focus with a calcified center and a granular and radiating basophilic necrotic periphery devoid of fibroblastic re action or blastomyces. A small fibrous granulomatous focus without blasto myces was found in the parotid. A tuberculoid type of granuloma with caseation necrosis and calcification was found in the cortex of the kidney. TABLE 23. DISSEMINATION OP B. DERMATITIDIS IN DOGS INFECTED INTRAVENOUSLY OR SUBCUTANEOUSLY

INTRAVENOUS SUBCUTANEOUS Stilbamidine Hydroxystilbamidine Hydr. Not treated treated Not treated treated

Dog number 551 551 551 551 551 551 551 551 551 551 551 s s r 551 031 032 041 037 038 039 070 074 069 073 071 067 065 Ante mortem bTood - -- - — - - C - C -- sputum 0 0 0 0 0 0 DD DD D DD feces - 0 0 0 0 0 D - D D - D D urine - 0 0 0 0 0 D 0 D --- - skin lesions XXXXXXXX X Xk D D Post mortem blood - - - C CH CH CH CH DCH c H - c bone marrow - -- « - - - H - H - - - spleen C c c H CH C C CH DCH C C -- lungs CDK DCH DCH DCH DCH DCH DCH DCH DCH DCH D H H DCH bronchial node CH H CH CH C CH DCH DCH DCH DCH D H - C tonsils D - - - DH - CH H - H D H M - parotids — —- H H H -- 0 - - feces - - — - - - D D H DD -- DC liver C c C H C CH C DC c

D direct microscopic demonstration of the fungus C culture of the fungus H histopathologic demonstration of the fungus - negative £ 0 not examined 10 x none TABLE 23-~Contimied

INTRAVENOUSSUBCUTANEOUS Stilbamidine Hydroxy st ilb ami dine Hydr. Not treat ed treated Not •treated treated Dog number 551 551 551 551 551 551 551 551 551 551 551 551 551 031 032 041 037 038 039 070 074 069 073 071 067 065 pancreas H H M H H H H H m mesenteric nodes C - C c C CH C - - C - - heart H H H - H H DCS H -- mm - - kidneys «•» - HHHH DCH H H - H - - bladder mm ------urine - -- C 0 0 - - C - -- - ovaries X X - XX H XXX - 0 - X testis - - X - - X -- - XX X - prostate - X - H X - H - X X X » brain - - - H H H H . H •» M --- eyes H H H HH HH - - --- thyroids H - H - mm H H HH mm -- parathyroids - ~ ------adrenals - • v H - H • - H -- - - — peripheral nodes - -- mm - - D H H 0 -- DCH peritoneum - -- mm - - mm - mm mm - -- stomach --- H --- H - - - - - skin lesions H X X X XX XX X X X X DCH pharyngeal muscl . 0 0 0 H 0 0 H 0 0 0 0 0 0 intestines « ■ H 193 19 If.

TABLE 2k. DAYS BETWEEN INFECTION AND DEATH

Treated dogs Control dogs

Dog No Stilbam- Hydroxy- Dog No idine stilb. Intravenous (0.25ml) 551031 5 551032 5 5510J+1 5 551071 551037 6 551038 6 551039 6 Intravenous (0.10ml) 551070 17 8 551069 5510714- 9 9 551073, i'ntratracheal (0.25ml) 151 x 55151+0 _3J— 55151+1 Intratracheal (0.5ml) 5510314- 17 551035 13 0 551036 218* 13 5510104- 551033 20 9 55io!+5 551014.2 10 H+ 55io66 551072 10 85*- 551865 5590** 314.5* 5568*-:;- 31+5* 551862*** 11+ 551863*** 52 5580 # 5 551861 # 7 Subcutaneous (1ml) 331* 17 551065 551061 . .

-sc clinically cured treated prior to inoculation with hydroxystilbamidine treated prior to inoculation with stilbamidine X clinically cured but blind # died from drug toxicity 19 S

TABLE 25. INFLUENCE OF ROUTE OF INOCULATION AND TREATMENT ON DISSEMINATION OF BLASTOMYCOSIS

Localized Disseminated Inoc. Route Dor No . Treatment disease disease INTRAVENOUS 1031 Stilbamidine X 1032 X 10U1 X 3.03 7 Hydr. Stil. X 1038 X 1039 X 1070 X 107U X 1071 Untreated X 1069 X 1073 X INTRATRACHEAL 103U St i lbami dine X 1035 X 1036 X 80 X 1861 X 1033 Hydr. Stil. X 101j.2 X 1072 X •iBBfr 90 X 10I+& Untreated X 10ll5 X 1066 X 15U0 X X 1865 X •JBH* 68 X #1862 X *1863 X SUBCUTANEOUS 1067 Hydr. Stil. X 10.® Untreated X

•apretreated with, stilbamidine. *3MSpre'treated with hydroxystilbamidine 196 TABLE 26. DISTRIBUTION OP Jh DERMATITI PIS IN TREATED AND UNTREATED DOGS

Fluids 19 treated 10 untreated Tissues dogs dogs Excreta No positive No positive Ante mortem blood 0 2 sputum 11 9 feces 12 9 urine 1 1 skin lesions 1 1 Post mortem blood 9 8 bone marrow 2 1 spleen 13 7 lung 17 10 bronchial l.n. 8 tonsil 3 parotid 5 1 liver 10 k pancreas 6 k mesenteric l.n. 8 3 heart 11 3 kidney 11 5 central nervous system 12 2 eye 12 2 ovarie 2/8 i/3 uterus 0 i/3 prostate 2/11 0/7 thyroid 7 3 adrenal k 2 peripheral l.n. 6 5 diaphragm 2 l tongue 1 1 pharyngeal muscles 2 0 epiglottis 1 1 stomach k 0 intestines 1 0 feces 8 7 urine 2 3 thoracic fluid 1 0 TABLE 27. DISTRIBUTION OF B^ DERMATITIDIS IN 19 TREATED AND 10 UNTREATED DOGS (expressed In percentages)

Treated Pretreated Untreated Stilb. Hvdr. Total Stilb. Hvdr. Total None Total No. of Doks: 6 10 16 2 1 ... 3 10 10 Ante mortem: blood _-- — - 20. 20. sputum 50. 50. 50. 100. 100. 100. 90. 90. feces 50. 60. 56.3 100. 100. 100. 90. 90. urine - 10. 6.3 -- - 10. 10. skin lesions 16.6 - 6.3 -- - 20. 20. Post mortem: blood 33.3 60. 50. 50. - 33.3 80. 80. bone marrow 16.6 10. 12.5 - - 0.0 10. 10. spleen 83-3 70. 75. 50. M 33.3 70. 70. lungs 83.3 90. 87.5 100. - 66.6 100. 100. bronchial 1. n. 83.3 80. 81.3 50. - 33.3 I 80. 80. tonsils 16.6 40. 31.3 50. - 33.3 30. 30. parotids 16.6 30. 25.0 50. - 33.3 10. 10. liver 50. 60. 56.3 50. - 33.3 l+o. l+o. pancreas 16.6 50. 37.5 - - 0.0 1+0. 1+0 . mesenteric 1. n. 50. kO. 43.8 50. - 33.3 30. 30. heart 83.3 50. 62.5 50. - 33.3 30. 30. kidneys 50. 60. 56.3 100. - 66.6 50. 50. central nervous sys. 50. 70. 62.5 100. - 66.6 20. 20. eyes 66.6 70. 68.8 50. - 33.3 20. 20. ovaries - 100. - 50. 33.3 33.3

25. ik.3 197 prostate • 33.3 20.0 0.0 0.0 TABLE 2.7— Continued

Treated Pretreated Untreated Stilb. Hydr. Total Stilb. Hydr. Total None Total No. of Does: 6 10 16 2 1 3. 10 10 thyroids 33.3 2*0 . 37.5 20. .. 33.3 30. 30. adrenals 16.6 2 0. 18.8 50. - 33.3 20. 20 . peripheral 1 . n. 16.6 2*0 . 31.3 50. - 33.3 50. 50. diaphragm 16.6 - 6.3 50. 9m 33-3 10. 10. tongue - 10. 6.3 - - 0.0 10. 10. pharyngeal muse. - 20 . 12.5 - mm 0.0 - 0.0 epiglottis - 10. 6.3 - - 0.0 10. 10. stomach » 30. 18.8 5o. - 33-3 - 0.0 intestines - 10. 6.3 -- 0.0 - 0.0 feces 33.3 50. 2*3.8 50. - 33-3 70. 70. urine - 10. 6.3 50. - 33.3 30. 30. uterus 0.0 0.0 33.3 33.3

H vO oo TABLE 2 8 . DISTRIBUTION OP DERMATITIPIS IN 23 DOGS WITH DISSEMINATED FATAL INFECTION

Intravenous Intratracheal Sub- ______cut. Route of Inoculation

3 5 3 2 3 k I 1 23 100 Ante mortem : blood 0 0 2 0 0 0 0 0 2 8 . 7 sputum 0 2 3 2 2 3 2 1 1 5 65.2 feces 0 1 2 2 3 1+ 2 1 1 5 65.2 urine 0 1 1 0 0 0 0 0 2 8.7 skin lesion 0 0 0 0 0 0 0 1 1 1+.3 Post mortem : blood 0 5 3 2 1 4 1 1 1 7 73.9 bone marrow 0 1 1 1 0 0 0 0 3 13.0 spleen 3 5 3 2 2 k 1 0 20 87.0 lung 3 5 3 2 3 k 2 1 23 100.0 bronchial node 3 5 3 2 3 1+ 1 1 22 95.6 tonsil 1 3 2 0 1 1 1 0 9 39.1 parotid 0 3 0 1 0 1 1 0 6 26.0 liver 2 5 2 1 1 2 1 0 A 6 0 . 9 pancreas 1 k 2 0 1 2 0 0 1 0 1+3 A mesenteric l.n. 1 1+ 1 2 0 2 1 0 1 1 1+7.8 heart 3 1+ 1 2 1 3 1 0 lb 65.2 kidney 1 5 2 2 1 3 2 0 16 6 9 . 6 bladder 0 0 0 0 0 0 0 0 0 0.0 testis 0 0 0 0 0 0 0 0 0 0.0 ovaries 0 l/l 0 0 0 i A 1/2 0 3/9 33.3 prostate 0 2/1+ 0 0 0 0 0 0 2 / A A . 3 CNS 1 5 0 2 2 2 2 0 A 6 0 . 9 eye 2 5 0 2 2 2 1 0 A 60.9 thyroid 1 3 2 1 1 1 1 0 1 0 1+3.1+ adrenal 1 2 0 0 0 2 1 0 6 26.0 peripheral l.n. 0 2 0 1 0 1+ 1 1 9 39.1 diaphragm 0 0 0 1 0 1 1 0 3 13.0 peritoneum 0 0 0 0 0 0 1 0 1 1+.3 skin 1 0 0 0 0 1 0 1 3 13.0 tongue 0 0 0 0 1 1 0 0 2 8.7 pharynge al mus c. 0 2 0 0 0 0 0 0 2 8.7 epiglottis 0 0 0 0 1 1 0 0 2 8.7 urine 0 1 1 0 0 2 1 0 5 21,7 stomach 0 2 0 0 1 0 1 0 1+ 17 A feces 0 2 2 2 3 1+ 1 1 A 65.2 200

TABLE 29, DISTRIBUTION OP B^ DERMATITIPIS SHOWING FUNGIS

TATIC ACTIVITY AND INCREASED OCULAR AND CEN TRAL NERVOUS DISEASE IN DIAMIDINE TREATED DOGS.

Treated Untreated *-

Disseminated cases 15 26

Ante Mortem : % % Blood 0.00 23.1 Sputum 53.33 81+.6 Peces 53.33 80.8 Urine 6.66 23.1 Post Moi?tern : Central Nervous System 80.00 19.2 Eyes 80.00 19.2 Blood 60.00 88.5

ho * Including 18 dogs from unpublished data 201

DISCUSSION OF TREATMENT OF BLASTOMYCOSIS

Before effectiveness of drugs can be evaluated we have to set up criteria by which they will be judged. We had to choose objective criteria like the period of survival after Inoculation, the severity of the clinical signs, including the disappearance of dyspnea and cough accompanied by an improvement in the radiogram, the results of the mycologic cultures, and finally the state of destruc tion or repair of the organs as seen at necropsy and under the microscope. There are several factors influencing the therapeutic effects over which we have no control. Such a factor is the individuality of each dog. The medium (soil) is very important in all natural mycosis and we have no way of preventing this influence. A large number of animals will surmount the latter problem but we have only very limited facilities for housing dogs. We considered that a subjec tive appraisal of each dog accompanied by the objective laboratory findings would be a practical basis for evalua tion. Knowing, however, that accurate reproducible data (scientific data) must be obtained by objective controlled methods we designed the protocol for each experiment to in clude strictly measurable observations. For example an amount of dermatitidis and a route of infection was chosen which was uniformly fatal for all non-treated dogs. 202

Since a few dogs are resistant, we used an amount of blastomyces yeast which was overpowering for most dogs: they died in a few days with destruction of $0 to 80 per cent of their lungs. Such an acute overwhelming infection is an extreme challenge to any drug. For this reason we felt justified in beginning treatment on the first day of inoculation. Once the fungus gained a foothold in the animal, they reproduced very rapidly; therefore the size of the inoculum did not actually influence the severity of the infection. Another hindrance in the evaluation of the drugs was their unexpected toxicity for the central nervous system. We used a treatment regimen found to be safe in our early experiments. The dosage caused no signs or lesions of toxicity but it was comparable to only one-fourth of the stilbamidine and one-half of the hydroxystilbamidine dose used in human treatment. In man, favorable results have been obtained with a total dose of at least 3 to 7 gm of stil bamidine and usually over 8 gm of hydroxystilbamidine; the treatment was given for a period of several weeks and the favorable results were consequently assessed after a few months. On the latter basis an animal weighing 7 kg should have received 300 mg of stilbamidine or 800 mg of hydroxystilbamidine. In 15 dogs early death prevented giving more than 28 mg to 177 mg of the drugs. Two dogs were cured; one infected intratracheally, received 308.1 mg of 203 stilbamidine, and another inoculated subcutaneously was given

6 8 I4. mg of hydroxys tilbamidine. Two of four dogs treated prior to the intratracheal infection (total dose 7 6 0 . 2 mg and 6 5 6 . 2 mg of stilbamidine respectively were not protected and died of blastomycosis in fourteen and fifty-two days. Two dogs given 1,020 mg and

8 1 9 . 6 mg of hydroxystilbamidine respectively, (the former re ceived a further post-infection course of 3 >Oij.5 mg of the drug) were protected and recovered rapidly after mild clini cal manifestations of blastomycosis. The dog that died after fifty-two days was apparently cured clinically when it developed a diffuse blastomycotic meningitis accompanied by hydrocephalus and died in a few days. Out of the 22 dogs treated with diamidines, three inoculated intratracheally and one inoculated subcutaneously (1 8 . 2 per cent) recovered from the early signs of the disease and were considered cured.. One -untreated control dog infected intratracheally also recovered (12.5 P©r cent). It appears that these drugs possess limited therapeutic properties at the level used and on the types of fatal experimental canine blasto mycosis we have induced. In the dogs inoculated intravenously with 0.25 ml of packed yeasts, the control died in four days while the dogs treated with stilbamidine and hydroxystilbamidine died re spectively in five and six days. Since these seven dogs were 201}. treated at the same time, the weather conditions being the same, we might ascribe the differences noted to a slight beneficial action of hydroxystilbamidine. When compared with each other or with untreated controls there were no significant differences in life span between two treated dogs infected intravenously (0 . 1 0 ml yeast) and six treated dogs infected intratracheally (0.5 ml yeast). Only one dog, treated with stilbamidine, was considered cured when destroyed 218 days after inoculation. The dogs which lived longer in each group (except for the above mentioned dog) were those with the most spectacular and disseminated lesions. The extension of their life span cannot be ascribed to fungistatic effects of the drugs, but rather to the vitality of the individual dogs, which could live with more extensive lesions than their congeners. One might theorize that the dogs may have been more resistant to in fection allowing delayed genesis of the disease, but this would not account for the more extensive lesions. The clinical findings also disprove such a theory. The course of blastomycosis was similar to the find- k9 ings of Gross and Cole^" in previous work done in this department. The intravenous inoculation of 0.25 ml of packed yeast cells produced a very uniform clinical picture. This inoculum uniformly overpowered the resistance of all dogs. Their temperature rose from the second to the fourth day, 205 with maximum of 103*U to 105.2 F. Dyspnea often occurred on the day of inoculation but cough did not always have time to develop before death. On the third post-inoculation day, a radiogram evinced diffuse lung radiopacities in all dogs. In this group, six of seven dogs showed a decline in the leucocyte count. The microscopic picture usually showed a diffuse infiltration of proliferating blastomyces, tissue necrosis, suppuration and a minimal reticulo-endothelial response• The group of four dogs inoculated intravenously with

0 . 1 0 ml of packed yeast presented a less uniform clinical re sponse, with an initial rise in temperature from the second to the ninth day and maximum of IO3 .I4. to IOI4..6 F. from the fourth to the sixteenth day. Dyspnea usually appeared on the second day and coughing on the seventh. Two dogs evinced a decline in the leucocytes count, In contrast to the

two others with leucocytosis reaching 2 1 . 1 and 2 5 * 1 thousand/

-3 nunJ on the seventh post-inoculation day. The microscopic findings were similar to those of the preceding group, but

dogs 5 5 1 0 7 0 and 5 5 1 0 7 3 which showed an elevation of the leucocytes count, demonstrated a better reticulo-endothelial response•

The group of 1 6 dogs inoculated intratracheally with

0 . 5 ml of packed yeasts showed a somewhat variable but 1 characteristic clinical response. An elevation in 206 temperature was noticed from the third to the sixth post inoculation day with maximum of 1 0 3 .1| to 1 0 6 , 2 from the fourth to the thirteenth day. Three dogs recovered after treatment and one recovered without any treatment. The dogs which recovered showed a similar early clinical picture, excepting for dog 5 5 1 0 3 & treated with stilbamidine, which showed only a slight fever of 102.8 F. The dyspnea usually appeared from the second to the fourth day and coughing the following day. Hilar and diffuse chest radiopacities appear ed from the fourth to the sixth day, but in a few treated dogs, the diffuse opacities appeared only late in the second week or the opacities were only focal. The focal pulmonary opacities regressed from the thirtieth to the sixty-seventh day after inoculation. All dogs, except 55>10lj5 and 551862 evinced leuco-

cytosis reaching 2 i|.l to 6 3 thousand/mm , usually in the latter part of the second post-inoculation week. Three of the four dogs treated with the diamidines previous to the blastomyces infection presented maximum leucocytosis from the twenty-ninth to the forty-second day. Human blastomycosis is usually classified as systemic or cutaneous. One should be prudent before diagnosing blastomycosis of the cutaneous type in the dog. Blastomyces readily enters the blood stream and frequently lodges and multiplies in the lungs. For example, both dogs inoculated 207

subcutaneously, discharged blastomyces organisms in their sputum and feces during the acute phase of the disease, and showed granulomas of the lungs at necropsy; one of the dogs was clinically cured but after euthanasia (218 days after inoculation) revealed pulmonary lesions. Similarly, in dog

5 5 1 0 3 1 inoculated intravenously, we found an active granuloma apparently originating from a subcutaneous blood vessel and infiltrating a few lymph vessels close to the skin surface. There is no doubt that this granuloma could have been the origin of a suppurating ulcer, if the dog had lived longer. A cutaneous lesion should be considered as a primary or secondary lesion and the disease classified as a localized or disseminated blastomycosis. We have some interesting findings in the distribu tion of Blastomyces dermatitidis (Tables 26, 21, 28 and 29). It is significant to note that 12 out of 19 treated dogs pre sented granulomas in the central nervous system and eyes compared to only 2 out of the 10 untreated animals. The eye lesions were nearly always in the choroid with occasional ex tension to the retina. There was marked predilection of the infection for the anterior third of the choroid, close to the ora serrata. The brain lesions were similarly of hemato genous origin with granulomas disseminated in all parts of the brain, and rarely in the spinal cord. Dog 551863 pre sented hydrocephelus and an extensive granulomatous 208

•v encephalitis and meningitis with distribution by extension to all ventricles. In treated animals we found a lower percentage of cases with blastomyces in their blood, sputum, feces and urine, when examined ante-mortem. At post-mortem, only per cent of the treated animals showed Blastomyces dermatitidls in their blood, compared to 8 0 per cent of the untreated dogs.

Only J4-O and 10 per cent of the treated dogs showed blasto myces in their feces and urine, compared to 70 and 3 0 per cent respectively in the untreated animals. There were no significant differences between treated and non-treated dogs, in the distribution of blastomyces in other tissues or organs. One may conclude that the two diamidines possess mod erate fungistatic activity which was not sufficient to alter the course of the disease. Since the central nervous system and the eyes were more frequently affected in the treated ani mals, we may assume that the drugs facilitated the infection in these organs. In the treatment of experimental blastomycosis in mice with stilbamidine, Heilman found that he could con trol the pulmonary infection, but a few days later the mice would sometimes die from a brain infection. V/e cannot ascribe these findings to the absence of the drugs in the brain since Snapper showed the deposition of significant ajnount of stilbamidine and hydroxystiIbamidine in this organ. 209

We are rather inclined to believe that the drugs act adverse ly on the blood-brain barrier and thus facilitate the passage of the yeasts into the brain parenchyma. Experience in this investigation indicates that a diagnosis of canine blastomycosis can be quickly and accurately confirmed by demonstration of the fungus in smears of sputum, feces or exudate, and biopsy sections using the new fungi histochemical methods. Skin testing, which are very successful in detect ing histoplasmosis and ooccidioidomycosis in dogs, proved to be of no value in canine blastomycosis. Human blastomycin did not prove to be a satisfactory diagnostic method. Dogs with blastomycosis either do not develop skin hyper sensitivity or the human antigen is not satisfactory for this specie. Pulmonary lesions were demonstrated radiographically but the radiograms revealed no diagnostic criteria for differentiating blastomycosis from other systemic fungus in fections. Leucocytosis occurred with consistency and this laboratory finding should aid in differentiating blasto mycosis from histoplasmosis; the latter disease does not consistently produce leucocytosis. If the dog presents some suppurative skin lesion the diagnosis is readily confirmed histopathologically. Disseminated blastomycosis with bone lesions have been described in naturally occurring canine blastomycosis 210 but we have not encountered this lesion in our experimental cases. In some extensively disseminated acute cases, we found the organisms in the bone marrow. If the animals had lived longer perhaps the fungus might have invaded the cortex of the bone. 211

SUMMARY AND CONCLUSIONS

1. Stilbamidine at more than one-fourth, and hydroxystilbamidine at more than one-half the comparable human dose was toxic for dogs and produced charac teristic central nervous system signs and lesions. 2. Both stilbamidine (lit. dogs) and 2-hydroxystilbami

dine (9 dogs) caused an identical toxic disease. Typically, after a large single dose or several moderate daily doses of the diamidine, clinical signs appeared uniformly in three- to six days after the first injection. Suddenly the dogs evinced cerebelio-vestibular disturbances, followed one hour later by extensor rigidity of the neck and leg muscles and periodic convulsions. During a convul sion the dogs fell to one side in opisthotonos (head and tail extended); rigidity of the legs alternated with bicycling movements. The dogs rolled or rotated to one side, while chewing and salivating profusely. Between seizures the dogs had an anxious expression and leaned unsteadily against one wall of the cage. Soon the dogs became prostrate, assumed the opistho tonos position, bicycled periodically, and died with in a few hours to four days. There were no recover ies once the toxic reaction appeared. 212

3. The brain showed striking bilateral areas of hemorr hagic encephalomalacia involving the medullary por tion of the cerebellum, the vestibular area of the pons, the colliculi and less often, the medulla, the striatum and the thalamus. Consistent histopathologic changes included ex tensive perivascular and parenchymal hemorrhage, necrosis, infiltration of neutrophils, swelling or hypertrophy of vascular endothelium, necrosis of blood vessels, and gitter cells.

I4.. Among different dogs there is variable sensitivity to diamidines. Fourteen dogs died after a total dose of

stilbamidine varying from Lf. to 2 2 mg/kgj four other dogs were unusually resistant and remained healthy

after receiving J4O to IOI4, mg/kg. Similarly, seven dogs died after receiving a total dose of hydroxy-

stilbamidine varying from 7 . 5 to 3 0 mg/kg, while four other dogs remained healthy after a total dose

ranging from 7 h to J+06 mg/kg. 5. The drug resistance is only relative since a dog re sistant to a dose fatal to the majority of dogs had a fatal toxic reaction when challenged with large doses of stilbamidine. 6. Since ribose and desoxyribose nucleic acids inhibit the in vitro bacteriostasis of stilbamidine, a diet 213

low in protein is generally recommended in man con currently with the administration of diamidines. Pour dogs on a low protein diet developed signs of toxicity one or two days earlier than control dogs who received the same diamidine dosage while on a stock diet. 7. A single large dose of stilhamidine or hydroxystilbamidine did not immediately precipitate a toxic re action; instead, like all our dogs showing toxic effects, the fatal central nervous reaction occurred

3 to 6 days after the intramuscular injection.

8 . Two dogs tolerated lOlj. mg/kg of stilbamidine when ad ministered on alternate days; the same dosage given to two dogs infected with Blastomyces dermatitldls produced an irreversible toxic reaction after they

had received only i| and 6 mg/kg, respectively. 9. The toxic effects of stilbamidine have been attributed to the ability of stilbamidine to release histamine and thus reduced the blood pressure. The intravenous injection of histamine phosphate in a dog did not produce signs or lesions, therefore histamine alone does not seem to be a significant factor in the pathogenesis of stilbamidine toxicity. 10. The concurrent administration of antihistamine pre vented the onset of hydroxystilbamidine toxicity in 2114.

one dog, but was without effects on a dog treated with stilbamidine. 11. Stilbamidine is known to form complexes with ribose nucleic acid. Hypersensitivity was considered as a possible factor in the pathogenesis since such complexes might be antigenic. The concurrent intra

muscular administration of cortisone (10 mg/kg) with stilbamidine or hydroxystilbamidine did not prevent the fatal reaction; therefore, the toxicity syndrome is apparently not related to an antigen-antibody reaction. 12. Trypan blue, injected intravenously shortly after the apparition of toxic signs, localized in areas of the central nervous system identical to those sites where diamidine damage was expected. Since this localiza tion of trypan blue preceded hemorrhage and necrosis this technic demonstrated that hemorrhage and necrosis of the brain, caused by the two diamidines, are pre ceded by damage to the blood-brain barrier.

1 3 . Since thiamine deficiency of foxes (Chastek paralysis) is characterized by topographic and histopathologic changes similar to those of diamidine toxicity, we postulated that thiamine deficiency may be associated with the toxicity syndrome. Thiamine deficiency in duced in dogs and cats caused vacuolation in the brain parenchyma, swelling or hypertrophy of the 215

vascular endothelium and perivascular hemorrhage. The signs as well as the location of the lesions closely paralleled those caused by diamidines. Therefore, it is postulated that the pathogenesis of diamidine toxicity is based, at least in part, upon their inhibitory effect on the metabolic activity of thiamine. li+» Blastomyces dermatltidis (yeast) originally isolated from a naturally infected dog, was pathogenic for

1 0 0 per cent of 3 1 dogs infected by subcutaneous or intravenous or intratracheal routes. 15. Intravenous and intratracheal routes induced blasto mycosis with a uniform incubation period ranging from

two to six days which terminated fatally in 8 6 per cent of the untreated dogs. 16. The clinical course was characterized by fever, dyspnea, bronchial rales, cough and leucocytosis. Lung radiograms of intratracheally infected dogs showed early diffuse hilar shadows with subsequent spreading toward all lobes. Focal opacities were soon evident and gradually disappeared in chronic cases. In intravenously infected dogs, the lungs radiograms were characterized by diffusely and uni formly distributed focal opacities. 216

17• Human blastomycin proved to be an unsatisfactory intradermal antigen for dogs. 18. At necropsy, the lungs showed lesions in 100 per cent of the dogs. During the acute phase, this organ pre sented massive and diffuse consolidation and caseation necrosis while in chronic cases, focal and nodular lesions, fibrosis and sometimes cavitations were prominent.

Granulomas (1 to 5 in diameter) were commonly seen throughout the kidneys and myocardium and occasionally in the thyroids, tonsils, pancreas, liver, spleen, parotids and diaphragm. The lymph nodes, especially the bronchial nodes, were often en larged and gray in color. 19. Histopathologic studies revealed focal or diffuse suppurative granulomatous inflammation often accom panied by caseation necrosis. Single or budding yeast were extracellular or engulfed by epithelioid giant cells. In chronic cases, the granulomas be came fibrotic and sometimes calcified. Organisms were often observed without gross lesions in the following organs: brain, spinal cord, eyes, ovaries, uterus, prostate, adrenals, bone marrow, stomach, intestines, skin, tongue, pharyngeal muscles, epi glottis and parathyroids. 2Q. Since intratracheal inoculation of 0.25 ml of packed yeast caused fatal blastomycosis in 66 per centof the dogs, and 0»$ ml of yeast caused fatal infection in 80 per cent of the dogs the la tter inoculum was employed for experiments designed to evaluate the therapeutic efficacy of the diamidines. 21. Varying levels of the diamidines were employed in several different schedules until a safe regimen was devised for each drug. Stilbamidine was given on

consecutive days at the rate of 1 .0 , 2 .0 , and 0 . 5 mg/kg, then 0 .$ mg/kg on alternate days. Hydroxy- stilbamidine was given on consecutive days at the

rate of 1 .0 , 2 .0 , and 3 * 0 mg/kg and then, 1 . 5 mg/kg on alternate days. These regimens were used safely in six dogs treated with stilbamidine and nine dogs treated with hydroxystilbamidine. No signs of toxicity were noted even when we gave a total dose

as high as 3 0 8 . 1 mg of stilbamidine and 68[|..0 mg of hydroxys tilbamidine. 22. No significant difference in the number of days be tween infection and death was noticed in untreated- stilbamidine- and hydroxystilbamidine-treated dogs,

which were infected intratracheally with 0 . 5 ml of packed yeast. Only one dog, treated with stilbami dine, recovered after showing early signs of infection. 218

2 3 - Two dogs pretreated with 1,020 and 819.6 mg of hydroxystilbamidine and infected intratracheally with

0 , 5 ml of packed yeast, recovered after the early signs of the disease and were healthy when euthanized 31+3 days later. One of these dogs also received

3 , 0 4 5 mg of hydroxystilbamidine during the course of the infection but showed no significant differences from the pretreated dog.

2 4- The course of blastomycosis was 5 days in three dogs

treated with stilbamidine, 6 days in three dogs

treated with hydroxystilbamidine and 4 days in an un

treated control infected intravenously with 0 . 2 5 ml of yeast. 25. Two animals were infected subcutaneously with 1.0 ml of yeast; the untreated dog died on the nineteenth post-infection day and the dog treated with hydroxy stilbamidine recovered after showing signs of blasto mycosis. 26. Blastomycosis of the central nervous system and eyes occurred far more frequently in treated than in tin-

treated animals. Twelve of the 19 treated dogs (63 per cent) in contrast to only two of the ten un

treated control dogs (20 per cent), had blastomycosic lesions in the eyes and central nervous system. 219

2 7 . The eye lesions, frequent in both intravenously and intratracheally infected dogs, were nearly always in the choroid with occasional extension to the retina. A marked predilection was shown for the anterior third of the choroid, close to the ora serrata. 28. Blastomyces were recovered ante mortem from the blood, sputum, feces and urine of treated dogs less fre quently than from untreated controls. Post mortem blood of only 9 of 19 treated dogs yielded blastomyces in contrast to positive results in 8 of 10 control dogs. BIBLIOGRAPHY

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131. Yorke, W.: The Therapeutic Action of Aromatic Diamidines in the Treatment of Protozoal Infections of Man and Stock. Brit. Med. Bull., 2:60-61]., I9I4J4-. 132. Farrell, R., and Cole, C. R.: Unpublished Data.

133. Courvillei C. B.: Pathology of the Central Nervous System. Pacific Press Pub. Ass., California, Third edition, 1950. 13k • Phillips, G. B., Victor, M., Adams, R. D., and Davidson, C. S.: A Study of the Nutritional Defect in Wernicke’s Syndrome. J. Clin. Invest., 31:859-871, 1952. AUTOBIOGRAPHT I, AndW Laigaioa, waa born in Montreal, Quebec, the thirty-first day of Ma^, 1930. I received my secondary education in Montreal and St,Cesalre, Quebec, and one year1 of pre-university studies at the ”Institut Agricole d* Oka .” After a year in the Royal Canadian Air Force X registered in 19^9 at '’l’Ecole de Me^decine Ve'terlnadLre de la, Province de Quebec” and in 19!?i|- received the degree Doctor of Veterinary Medecine from lITTniversltef de Montreal..

In September, 1954> 1 registered in the Graduate Scliool of the Ohio State University and for three years have taken graduate work in the department of Veterinary Pathology,

232.