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The antigenic properties of Clostridium botulinum type E toxoids. Strasdine, George Alfred 1958

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THE ANTIGENIC PROPERTIES OF CLOSTRIDIUM BOTULINUM TYPE E TOXOIDS by GEORGE A. STRASDINE B.A., University of B r i t i s h Columbia, 1956.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF M. Sc. i n the Department of Bacteriology and Immunology  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1958.  -i-  ABSTRACT The conditions responsible for the preparation of crude, activated and purified toxins of Clostridium botulinum Type E and the antigenic properties of toxoids prepared from these toxins, are described. Optimum toxin production and toxin activation are seen to be c r i t i c a l l y dependent on conditions such as hydrogen-ion concentration, and incubation time and temperature. Immunization of human volunteers with the formolized toxoids demonstrated that the highest level of immunity occurred i n those individuals who received the crude toxoids.  In presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. agree that permission f o r extensive  I further  copying o f t h i s t h e s i s  for s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e .  I t i s understood  that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  Department of  ^ C ^ ^ ^ L ^ A X ^ y  4  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. Date  ^T^y  ^  /?iTS-.  -iiTABLE OF CONTENTS Page I.  Problem and D e f i n i t i o n of Terms Used.  I I . Introduction.  1 3  I I I . H i s t o r i c a l Review of the Literature. IV. Preliminary Experiments.  5 27  A. The effect of surface moisture on the i s o l a t i o n of variants.  27  B. Production of t o x i c and non-toxic f i l t r a t e s . 1. Production of crude t o x i n . (a)  Effect of pH  2. Production of activated t o x i n .  29 30 32 35  (a)  Dialysate t o x i n .  36  (b)  Combination of f i l t r a t e s .  37  (i)  39  (c)  Effect of incubation temperature.  ( i i ) Effect of pH.  41  Activation with t r y p s i n .  45  (i)  45  Toxin activation  ( i i ) I n h i b i t i o n of the a c t i v a t i o n phenomenon. 3. Production of non-toxic f i l t r a t e s . C. P u r i f i c a t i o n of Type E t o x i n . 1. P u r i f i c a t i o n with phosphates.  49 51 52 53  (a)  Procedure.  53  (b)  Results.  56  -iiiPage 2. P u r i f i c a t i o n by alcohol p r e c i p i t a t i o n . (a)  Procedure.  (b) Results. D.  Immunization of rabbits with Type E toxoids.  57 58 59  1. Toxoids.  60  2.  61  Immunization schedule.  3. Results. V.  57  63  Human Immunization.  66  1. Preparation of toxoids.  66  2. S t e r i l i t y t e s t s .  67  3.  68  Immunization schedule.  4. Results. VI. Discussion. V I I . Summary.  69 72 77  V I I I . Bibliography.  79  IX. Appendices.  87  -ivDiagrams  Page  I.  Production of crude (OT) toxin.  31  II.  The effect of an elevated pH on toxin production.  33  I I I . The effect of a delayed drop i n pH.  34  IV.  The effect of an early decrease i n pH.  35 (a)  V.  Toxin activation by mixed culture.  38  VI.  Toxin activation by a s t e r i l e proteolytic f i l t r a t e .  40  V I I . Effect of temperature on toxin activation . V I I I . Effect of pH on t o x i n a c t i v a t i o n .  42 44  LX.  Toxin activation by t r y p s i n .  47  X.  Rabbit immunization.  64  XI.  D i a l y s i s apparatus f o r toxin production.  95  Plates I  88  II  94  ACKNOWLEDGEMENTS  I would like to take this opportunity to express my appreciation for assistance i n this investigation to Dr. C . E . Dolman, to the staff of the Department of Bacteriology and Immunology, and to the Defence Research Board of Canada, I should also like to thank the many individuals who voluntarily received the toxoid injections i n the human immunization programme.  I.  The Problem and Definition of Terms Used.  Clostridium botulinum species, responsible for botulinum food poisoning i n man and animals, give rise to five different types of toxin, designated A, B, C, D and £ , each of which has been shown to be serologically distinct. Dolman, i n 1956, reported the variation of type E strains into spore— l y t i c , proteolytic and toxigenic variants (designated " 0 " , " T " and "0T" respectively), capable of isolation and propagation i n pure cultures. The further observation by Dolman, that toxin potency could be increased by the addition of proteolytic variants, has led  to intensive research on  the antigenicity of these "activated" products. 1.  The Problem (a)  Statement of the problem.  It i s the purpose of this inves-  tigation to study the antigenic properties of Clostridium botulinum type E toxins and toxoids i n animals and man, with the end result being the preparation of a toxoid suitable for human immunization. (b)  Importance of the Investigation.  The discovery that a r e l a -  t i v e l y low potency toxin such as type E could be activated to a potency of 300 to 300,000-fold led to the preparation of toxoids from these activated products, the assumption being that antigenicity would increase with potency. The importance of this investigation lay i n determining quantitatively the immunity conferred i n animals and man by the injection of crude, trypsinactivated, and purified toxoids. During the course of animal immunization a non-toxic, proteolytic variant (T) was seen to produce a low, but definite, immunity against the  -2crude and activated toxins.  This observation was considered important  enough to warrant further investigation, and was included i n the programme of human immunization. 2.  Organization of the Remainder of the Thesis. The remainder of the thesis i s arranged to cover a review of the  literature, experimental studies, discussion and summary.  Since there were  many aspects of the study, i . e . minimal lethal dose determinations, p u r i f i c ation techniques, optimum temperature and hydrogen ion concentration for growth e t c . , certain of these aspects w i l l be discussed separately for the sake of c l a r i t y .  The f i n a l discussion w i l l be reserved for the more  general issue of antigenicity.  -3-  I I . Introduction. I t i s remarkable that, with the exception of the diphtheria b a c i l l u s , the organisms forming powerful exotoxins belong almost e n t i r e l y t o the group of anaerobic spore-forming b a c i l l i .  Two of these, Clostridium botulinum and  Clostridium t e t a n i , give r i s e to toxins more potent than any other substances with which we are acquainted.  Table I l i s t s the diseases, most of them  toxaemic, that are associated with some species of C l o s t r i d i a ( l ) . Table  I.  Organism  Disease  CI. botulinum, types A-E  Botulism i n man and animals  CI. chauvoei  Blackleg i n c a t t l e  CI. haemolyticum  B a c i l l a r y haemoglobinuria (red water) in cattle.  CI. histolyticura  Sometimes associated with other C l o s t r i d i a i n gas gangrene i n man.  CI. oedematiens type A II ti ti it B it II II it C  Gas gangrene i n man Black disease and bradsot of sheep. B a c i l l a r y osteomyelitis of buffaloes.  CI. septicum  Blackleg and braxy of sheep; gas gangrene i n man.  CI. tetani  Tetanus.  CI. w e l c h i i type A II  II  n  II  It  II  It  it  ti  D  It  ti  it  ji  B  Q  Gas gangrene i n man. Lamb dysentery. "Struck" of sheep. Infectious enterotoxaemia of sheep. E n t e r i t i s necroticans i n man  Study of the anaerobic spore formers, before the war of 1914—18, was undertaken f i t f u l l y and by imperfect methods: much attention had been paid to t h e i r pathogenicity, but l i t t l e to t h e i r general b i o l o g i c a l characteristics.  There  was confusion i n nomenclature, and many organisms with the same name belonged to  different species.  The only two about which no doubt existed were those that  formed a highly potent toxin, namely C I . tetani and CI. botulinum.  With the  development after the war of new techniques, such as the use of the MclntoshFildes anaerobic jar, the obscurity around this group began to disperse. The and of  earlier work of Fildes (2), Fildes and Knight (3), Pappenheimer (4),  Stickland (5), on the essential nutrients of the C l o s t r i d i a and their methods u t i l i z a t i o n has been developed to the point where i t is clear that the  majority of the pathogenic C l o s t r i d i a are heterotrophs, requiring a large number of  amino-acids, vitamins, and carbohydrates for growth i n a r t i f i c i a l media.  The  energy-producing mechanisms, especially of those C l o s t r i d i a that depend mainly on amino-acid breakdown for their energy, have been studied i n detail by Gale (6), Clifton (7) and Guggenheim (8).  Most C l o s t r i d i a grow best at 3 7 ° C , but there  are exceptions. Apart from their toxic a c t i v i t y many of the anaerobes, such as CI. tetani, CI.  welchii and CI. septicum, produce filterable haemolysins.  Kerrin (9), i n  1930, stated that atoxic strains of C I . tetani produce as powerful a haemolysin as do the toxic strains, and that normal rabbit, horse and human serum have a very strong antihaemolytic effect.  Fibrinolysins and leucocidins also may be  produced by some species, as reported by Reed, Orr and Brown (10).  -5III.  A Historical Review of the Literature  Through the pioneer work of the Belgian bacteriologist van Ermengem i n 1895, the serious and often fatal form of food poisoning termed "botulism" (formerly known as a l l a n t i a s i s , ichthyosism, or Wurstvergiftung) i s now known to result from the ingestion of a toxin produced by one of the five Types of Clostridium botulinum.  The intoxication was originally attributed to the  consumption of sausages, preserved fish and other prepared foods of animal origin which had become contaminated with some poison possessing distinct pharmacological properties.  Later outbreaks however, especially those i n  the Western Hemisphere, have followed the consumption of home-canned vegetables as well as the aforementioned vehicles.  Van Ermengem (22) conceived the  disease to be a form of intoxication when he made an investigation of the E l l e z e l l e s outbreak near Ghent.  He succeeded i n not only isolating the  organism for the f i r s t time, but also demonstrated i t s a b i l i t y to liberate one of  the most powerful exotoxins known. This family of anaerobic spore-forming bacteria are inhabitants of s o i l ,  and stagnant water i n which vegetation i s decaying.  In contrast with many  other pathogenic C l o s t r i d i a , Clostridium botulinum i s not a common inhabitant of  the intestine of domestic animals ( l l ) .  Meyer and Dubovsky (12, 13, 14,  15) have recorded the occurrence of these spores i n s o i l samples taken from various kinds of cultivated and uncultivated land i n North America, Western Europe and China. In  spite of this widespread distribution i n s o i l s , botulism spores, i n  contrast with those of tetanus and gas gangrene, f a i l to germinate readily i n the tissues.  A few instances of wound complications are mentioned i n the  literature and w i l l be noted l a t e r .  -6Although botulism was f i r s t described i n man, comparable neurological disorders were encountered i n several species of domestic and wild animals early i n the twentieth century.  The same syndrome, with rapid onset and  high mortality, suggested the occurrence of botulism i n animals, and was soon established when toxic f i l t r a t e s from cultures of Clostridia from animals were prepared and studied immunologically.  The most common of these naturally-  occurring intoxications i n animals are:-  "lamziekte" amongst cattle i n South  Africa (16), and horses, cattle and sheep i n Western Australia (17); of "meningo-encephalitis" of horses i n Prance (18); many parts of the world (19);  a form  "limber-neck" i n fowl i n  and a paralytic condition amongst wild ducks on  reservations i n the Western United States (20),  In cattle and other herbivora  i n South Africa and Australia, the intoxication arises from the habit of sarcophagia and osteophagia of carrion which animals grazing on the veldt country develop to satisfy a craving for the phosphorous deficiency i n their ordinary diet (16).  In France, botulism among horses i s caused by ingestion of hay and  silage which has been contaminated with the remains of small rodents and birds (21),  Farmyard birds may become infected through eating the larvae of the  "green bottle" and other blowflies which are often present on contaminated carcasses.  Wild ducks become intoxicated through the consumption of marsh  weeds growing i n places i n which oxygen-consuming micro-organisms, notably Pseudomonas aeruginosa, flourish and create the anaerobic conditions which makes possible the proliferation of the C l o s t r i d i a (20). Intoxication In typical epidemics, a high proportion of the persons infected by consuming the food develop c l i n i c a l botulism.  About 18-24 hours or even longer  after the meal, the affected persons develop a syndrome which begins with  -7muscular weakness and ocular disturbances of which diplopia, lack of power of accommodation, and loss of the light-reflex occur.  Trouble i n articulating  and swallowing soon follow, brought about by weakness and inco-ordination of the pharyngeal muscles and partly by cessation of salivation.  Gradually a l l  the voluntary muscles weaken, and death generally takes place as a result of paralysis of the respiratory muscles. breathing has ceased.  The heart often continues to beat after  These symptoms and signs of intoxication have been des-  cribed by McClasky (23). Pood Vehicles Human cases, of which the history i s known, result from foods which have been smoked, salted, spiced, or canned, allowed to stand for a period, and consumed without cooking.  No cases are known to have been caused by the consump-  tion of fresh foods, cooked or uncooked.  In Europe most cases have been due  to sausages, ham, spiced meats, game pastes, potted meats, and i n Russia to salt fish.  In every recorded instance these foods have been eaten without prior  reheating.  In the Western Hemisphere, on the other hand, most cases have been  due to canned vegetables and f r u i t s , such as olives, string beans, corn and peas. A few cases have also been due to cooked meat, cheese and fish (24). majority of cases the foods contaminated were noticeably spoiled. had swollen ends and on opening demonstrated gas bubbles. were often paler and softer than normal.  In the Cans often  Meats such as ham  In some instances, however, foods  have been reported as apparently well—preserved.  More recently there has been  a number of reports on the isolation of Type E botulism from f i s h , f i s h products and other marine l i f e (25, 26, 27, 28, 29, 30, 31).  This type does not seem  peculiar to either the Western or Eastern Hemispheres and isolations have been reported from many points around the world.  Pood vehicles for Type E toxin  w i l l be discussed more f u l l y under the heading "Type E Toxin".  -8C e l l Pathogenicity Orr (32) reported i n 1922 that Clostridium botulinum could be recovered from the intestinal organs of animals which had been fed or injected with toxic cultures and toxin-free spores; and stated that c e l l s of Clostridium botulinum were capable of proliferating and producing toxin i n the body of a guinea p i g .  The presence of the toxin was demonstrated by precipitation as  well as direct toxicity tests. Clostridium botulinum as a complicating factor i n wound infections, by growing with other anaerobes and producing toxin, was also reported by Hampson (33) and Mattman (34).  These instances are exceedingly rare, but nevertheless  should be anticipated. Introduction to Types Early i n experimental studies of botulism i t was found that the typical syndrome of the intoxication could be produced by injection of culture f i l t r a t e s which, though identical i n behaviour, could be separated and distinguished immunologically by the use of specific antisera (35).  On the basis of cross-  neutralization tests with antitoxin, five toxins, now termed Types A, B, C, D and E , have been identified, and the variant strains from which they are obtainable have been named. Although the typing of the botulinum toxins is of importance both for immunological differentiation and relative pathogenicities for a wide range of animals, there are good reasons for believing that a l l five Types produce their toxicological effects through the same kind of tissue injury.  Prevot and  Brygoo (36) performed experiments i n which they mixed appropriate  fractional  doses of botulinum toxin, using two, three or more different Types, and producing a single lethal dose.  They found that the mice which had received these  -refractional mixtures died from botulism, much as did the control mice which had received one lethal dose of a single Type of toxin.  Prom these results they  concluded that a l l five Types exhibited mutual summation, but not reinforcement of action. Pathogenicity of Types for Animal Species The botulinum toxins are highly pathogenic on parenteral injection for a wide range of both warm- and cold-blooded animals, i n a l l of which the intoxication follows one common pattern.  When these toxin Types are examined  individually, however, their relative pathogenicities for species varies widely. Throughout the literature there are many observations concerning animal suscept i b i l i t y , but changes i n nomenclature during the past causes one to treat these findings with caution. Values obtained by different workers are approximate, but nevertheless one may infer from them that mice, guinea pigs, rabbits and monkeys are notably susceptible to a l l types of toxin (except perhaps Type D for monkeys), while cats, dogs, fowl and pigeons are more refractory (36, 37, 38, 39, 40). Since naturally-occurring botulism i s primarily a food poisoning, a number of investigators have sought to compare the lethal dose of toxins when administered parenterally and by mouth.  Figures obtained are quite variable:  Gunnison and Meyer (41) and Gunnison with Coleman (42) compared the two for Types B, C and D toxin on mice, guinea pigs and rabbits, and found that for Type B toxin the two did not differ greatly.  Using Types C and I) toxins, how-  ever, the oral dose was from several hundred to several thousand times larger than that needed for injection.  Differences of a similar kind were found for  dogs by Graham and Erikson (43) for Type A toxin: a preparation which was lethal when injected subcutaneously i n a dose of 0.1 ml. failed to k i l l when given orally i n a dose of 100 m l . , unless the recipient animal had been starved for  -10two days prior to injection.  Knowledge as to what extent toxin i s destroyed  by gastric juices and other agents within the alimentary canal i s rather scarce.  In general i t seems there is wide variation from one set of experi-  mental conditions to another. Bronfenbrenner and Schlesinger (44) attempted to simulate gastric conditions by adding various quantities of hydrochloric acid and sodium hydroxide to broth containing Type A toxin, and found that between pH 2.3 and 5.0 there was l i t t l e or no change i n t o x i c i t y after 24 hours at 3 7 ° C . values were increased did potencies f a l l .  Only when pH  Nor did the addition of pepsin and  trypsin diminish the t o x i c i t i e s of the solutions.  Coleman (45), however,  obtained different results when he repeated this experiment employing a highly purified preparation of Type A toxin.  At the end of eight hours, the toxin  at pH 1.5 had lost l i t t l e t o x i c i t y while that at pH 6.5 had f a l l e n to one-fifth, and after seventy-two hours, both preparations had declined to about one-tenth of their original potency;  and solutions of pepsin and trypsin at pH values of  1.4 and 6.8 respectively, completely detoxified the solutions by the end of seventy-two hours.  Littauer (46) points out the p o s s i b i l i t y that the presence  of proteins and organic materials i n the broth employed by Bronfenbrenner and Schlesinger may have protected the toxin from digestion by the enzymes. Dach and Wood (47) and Dach and Gibbard (48) introduced Type A toxin into the upper ends of loops of dogs1 and rabbits* intestines, which they perfused with oxygenated blood, to determine at what point i n the alimentary canal the toxin i s absorbed.  They found l i t t l e indication of absorption.  Haerem, Dach  and Drogstedt (49), and Coleman (45) also failed to demonstrate absorption i n the ileum of dogs.  Dach and Hoskins (50) demonstrated macacus monkeys to be  very susceptible to Type A toxin by mouth, but doses of toxin introduced into the loops of the colon failed to produce botulism.  -11-  Resistance of Toxin to Destructive Agents Many studies have been undertaken concerning the resistance of t o x i n and spores of Cl» botulinum species to heat and other agents.  Spores of the  organism are as a whole highly r e s i s t a n t , depending on type of culture media, pH, etc.  Dolman (51) more recently reported that spores of Type E organisms  exhibit very low thermal s t a b i l i t y .  In general, however, spores of  Clostridium botulinum w i l l withstand 100°C. for 3 i to 5 hours, although i n 1920, Bigelow and Esty (52) reported strains which would withstand 100°C. f o r up to 22 hours.  As mentioned previously, the pH of the medium has a marked  influence on heat s t e r i l i z a t i o n of spores. Orr (53) examined the thermal destruction of ten t o x i n preparations obtained from ten strains of the bacterium. 80°C. i n less than f i v e minutes:  Nine of them were inactivated at  the tenth was more r e s i s t a n t to heat, and,  considering t h i s i n the l i g h t of today's knowledge, may have been a Type C or D toxin.  Bengston (54) studied the thermal s t a b i l i t y of Type C and recorded  i t s greater resistance to heat.  In general, Types A, B and E are r e l a t i v e l y  more thermolabile i n that they are destroyed by exposure at 70°C. for a few minutes, while Types C and D are more r e s i s t a n t .  Schoenholz and Meyer (55)  demonstrated that the same Type of t o x i n proves more r e s i s t a n t to heat when present i n vegetable juices than when i n broth.  The greater a c i d i t y of the  former apparently provides some degree of protection. Mechanism of Toxin Production Dozier, Wagner and Meyer (56),  studying the effect of glucose on b i o -  chemical a c t i v i t i e s , growth and t o x i n production of CI. botulinum, reported that i t s presence stimulated early reproduction, maximum growth, and a greater content of amino acid nitrogen i n 96 hours than when i t was omitted.  -12-  However, the presence of glucose did not exert any effect on the production of toxin, indicating their results to be i n harmony with the suggestion that the toxin was an autolytic product.  Boroff, Raynaud and Prevot (57), by  obtaining a toxin from the particulate bacteria, similar i n a l l respects to that produced from cultures, demonstrated that the soluble toxin may derive from the bacterial cytoplasm.  This evidence tends to f i x the site for the  formation of the exotoxin i n the interior or on the surface of the organism. The view of these workers was that the toxic a c t i v i t y is due to relatively simple groupings or to a spatial configuration of these groupings on the molecule of bacterial protein;  otherwise ease of detoxification, with sub-  stances having a f f i n i t y for some specific grouping,or by oxidation, would not be possible.  The unfolding and s p l i t t i n g of the toxin molecule may occur  while under refrigeration, liberating i n the process more of i t s toxic groupings.  This may account for the observed augmentation i n toxic a c t i v i t y , a  phenomenon noted with crude extracts. colloidal and soluble forms.  These workers obtained toxin i n  The soluble toxin (exotoxin) may be s p l i t off  the c o l l o i d a l particle by ultrasonic waves i n the presence of catalase. assumed the colloidal toxin to be a precursor of the soluble form.  They  Boroff  (58), studying the relation of autolysis to toxin production i n Type D, noted that maximum amounts of toxin appear i n the culture f i l t r a t e s after ten days incubation.  At this time the culture i s past the logarithmic growth phase.  The culture f i l t r a t e then contains very l i t t l e , i f any, toxin;  yet i f the  cells are removed and cracked, large quantities of toxin are released.  This  lag period between maximum growth and toxin production, he suggested, was due to a required build-up of autolytic enzymes.  -13Site of Action of Toxin The neurological character of the symptoms of botulism directed workers to seek evidence of i n j u r i e s i n the central nervous system.  Many of these  studies were based on autopsy records of patients who had died i n some botulism outbreak.  This work was later supplemented with observations on  experimentally infected animals.  Many complications arose i n examination  of material due to the slow death of the animal from respiratory f a i l u r e and progressive anoxaemia.  These complications confused the study of i n i t i a l  morphological characteristics of the toxic action.  Cowdry and Nicholson  (59) recognized many of these d i f f i c u l t i e s and observed the disorder to be e s s e n t i a l l y a "biochemical l e s i o n " . Probably the most extensive studies concerning the action of botulinum toxin were performed by Dickson and Shevky (60, 61) who mapped the f i e l d of i n t o x i c a t i o n i n both the viscera and skeletal muscle, and pointed out the s i m i l a r i t i e s between the s i t e s of action of botulinum t o x i n and acetylcholine. They demonstrated that once botulism had become established i n dogs, cats and rabbits, progressive impairment developed i n the vagus nerve which induces cardiac i n h i b i t i o n and evokes salivary secretion;  i n the nervus erigens which  causes contraction of the bladder; and i n the oculomotor nerve which constricts the p u p i l .  These results led them to conclude that the t o x i n affects the  autonomic nervous system and that impulses along these f i b r e s become blocked at some point at the periphery.  They also reported i n t h e i r second paper the  cause of the paralysis that develops i n skeletal muscles as the i n t o x i c a t i o n advances.  I t was apparent that the weakness, so characteristic of i n t o x i c a t i o n  i n man and animals, was not due t o loss of contractile power of the muscle fibres themselves, since they reacted as promptly and e f f i c i e n t l y as normal  -14rauscles when e l e c t r i c a l s t i m u l i were applied.  A second observation demon-  strated that the musculature paralysis was not due to the i n a b i l i t y of the nerve f i b r e s to conduct an e l e c t r i c a l impulse.  The most c h a r a c t e r i s t i c  feature was revealed when they observed that when a threshold stimulus was applied the muscle of an intoxicated animal became fatigued very r a p i d l y as compared to the same muscle of a normal animal.  From a l l these observa-  tions they concluded that the weakness of the voluntary muscles i n botulism i s dependent upon the i n t o x i c a t i o n of some end organ and not upon any l e s i o n i n the central nervous system. The p o s s i b i l i t i e s offered by CI. botulinum as an agent i n b a c t e r i a l warfare during the Second World War led to much further research on the mode of action of the t o x i n , e s p e c i a l l y at the myoneural junction i n s k e l e t a l muscles.  E a r l i e r observers regarded t h i s paralysis as s i m i l a r to that which  follows the i n j e c t i o n of curare.  Guyton and MacDonald (62) were among the  f i r s t to draw attention to the points of difference i n the action.  By  injecting a small amount of acetylcholine i n t r a - a r t e r i a l l y they demonstrated that a muscle paralysed by botulinum t o x i n would contract.  This contraction  could not be brought about through the action of curare. The p o s s i b i l i t y that the release of acetylcholine at the junction might be impaired was examined by Burgen, Dickens and Zatman (40).  By a unique  apparatus they demonstrated that the amount of acetylcholine released i n a paralysed muscle after stimulation i s considerably smaller than when a normal muscle i s employed.  The manner i n which botulinum t o x i n interferes with  acetylcholine release at the neuromuscular junction after stimulation of the motor nerve has not as yet been reported.  -15The early studies on the paralytic action of botulinum toxin on the cholinergic components of the nervous system were made on animals suffering from generalized intoxications, and therefore disclosed chiefly the effect of the toxin on the post-ganglionic fibres.  In studies on local tissue intox-  ication i n the region of the c i l i a r y and superior cervical sympathetic ganglia, Ambache (63) has shown that the toxin i s also injurious to the preganglionic fibres. Considerable evidence is now available which shows that botulinum toxin acts very widely on a l l those portions of the peripheral nervous system that are cholinergic, regardless of whether they form pre- or post-ganglionic components of the autonomic nervous system.  There is no experimental  information to demonstrate that the toxin exerts any d i r e c t l y injurious effects on the brain and spinal cord.  Accounts of the terminal stages of  botulism i n man have recorded the f u l l retention of mental faculties when a l l the typical signs of the disease are f u l l y developed;  nor i s there any e v i -  dence that the toxin is more rapidly lethal or exhibits a higher potency when i t is injected intracerebrally than when inoculated parenterally by some other route  (39).  Immunization Employing as antigen single c e l l strains of CI. botulinum which had been dissolved by alternate freezing and thawing, Dack and Starin (86), i n 1924, demonstrated that i t was possible to detect the presence of complementbinding bodies i n the serum of animals immunized with these same strains. These reactions were specific to A and B Types of CI. botulinum, and by employing quantitative methods i t was possible to demonstrate  subgroups  -16within the Types.  This study was not undertaken f o r immunization purposes,  but was developed by the authors for the detection of C l . botulinum i n canned vegetables. Graham and Thorp i n 1929, following the work of Ramon i n preparing formolized diphtheria toxins, performed a series of experiments to determine the antigenic value of formalin-treated botulinum toxins A, B and C.  The  protective character of formolized heat-treated cultures and culture f i l t r a t e s was demonstrated i n horses, mules, r a b b i t s , guinea pigs and chickens (87). They employed formalin to a concentration of 0.5 per cent at 37°C u n t i l d e t o x i f i c a t i o n was complete. In the following ten years studies concerning immunological reactions with C l . botulinum became rather scarce, and the small number of reports that are available pertain mostly to studying past observations of immunological importance. Rice et a l (88) reported i n 1947 the r e s u l t s of a systematic study on the preparation of highly antigenic formolized and alum-precipitated toxoids of Types A and B toxins.  Their investigations showed that formalin added to a  f i n a l concentration of 0.3 to 0.6 per cent produced higher a n t i g e n i c i t y than when concentrations below or above t h i s range were employed.  Immunization of  mice with alum-precipitated toxoids conferred a much higher degree of immunity than when formolized toxoids were employed.  They also stated that mice appear  to be s a t i s f a c t o r y test animals i n t i t r a t i n g the t o x i c i t y of the f i l t r a t e s and the immunizing properties of the material, and also i n determining the antitoxic t i t r e of sera from immunized guinea pigs.  Results obtained i n a  later experiment (89), comparing the action of alum-precipitated and formolized toxoids of Types A and B on mice and guinea pigs, proved interesting i n that  -17Type B toxoids showed a much lower protective potency than those of Type A. Investigating the immune response when Types A and B toxoids are combined, Rice (90) indicated that mixing CI. botulinum Type A toxoid with Type B toxoid improved the immunizing properties of the l a t t e r f o r mice and to a lesser degree f o r guinea pigs, notwithstanding the fact that the two antigens when used singly induced no cross protection. In the same year the Camp Detrick workers (91) took the f i r s t step i n producing toxoids suitable f o r human use.  They developed a medium for the  production of t o x i n by removing those fractions of a l l constituents of the medium which d i d not seem to contribute to t o x i n y i e l d , especially those of large molecular s i z e .  During t h i s work these workers observed that the a n t i -  genicity of the toxoid was not d i r e c t l y proportional to the t o x i c i t y of the culture from which the toxoid was prepared.  As a r e s u l t , higher L f values  were obtained i n a medium containing 4 per cent Pepticase than when 2 per cent Pepticase was employed, although the t o x i c i t y of the l a t t e r was much higher than that of the former.  They assumed that although the t o x i n produced i n the  former medium was of much lower potency, there was no corresponding reduction i n combining power as r e f l e c t e d i n i t s f l o c c u l a t i o n t i t r e . In 1954 Boroff and Cabeen (92) reported the induction and formation of antibodies against toxic forms of Type C strains by i n j e c t i o n of an atoxic variant of Type C.  This phenomenon i s mentioned here to substantiate an  observation i n t h i s laboratory i n which i t was shown that an atoxic variant of Type E would produce a low but d e f i n i t e immunity to other Type E toxic s t r a i n s i n rabbits and humans.  -18Immvinization experiments with Type E toxoids began recently when Baron and Reed (93) presented a systematic study of methods f o r the preparation of crude alum-precipitated Type E toxoids employing cultures grown i n cellophane bags.  These workers reported a similar phenomenon to that described by  Rice (90) with Types A and B toxoids, v i z , the augmentation of a n t i g e n i c i t y of Type E when mixed with Type A and B toxoids. In 1957 the Camp Detrick workers (94) succeeded i n preparing formolized toxoids from crude and 'activated* Type E toxins, and obtained protection of mice approaching that offered by Types A and B toxoids.  Their results were  similar t o those reported by Batty and Glenny (95) f o r epsilon toxins and toxoids of Clostridium w e l c h i i .  Both groups of workers found that a c t i v a t i o n  of the respective toxins by t r y p s i n increased the antigenicity i n rabbits and guinea pigs, despite a considerable loss i n combining power. In reviewing the l i t e r a t u r e concerning experimental immunization of man with botulinum toxoids, one finds experience with i t s use i s indeed scarce. In 1936 Velicanov (96) prepared Types A and B toxoid by treatment of t o x i n with formalin.  These toxoids were used on a small scale experiment to  immunize human volunteers.  The author concluded that h i s preparation of  toxoid immunized man against the botulinum toxins, and that a single booster i n j e c t i o n a year after the i n i t i a l immunization caused a decided increase i n immunity. Bennetts and H a l l (17) used a Type C alum-precipitated botulinum toxoid i n performing  a f i e l d experiment to test the value of immunizing sheep  against Type C i n t o x i c a t i o n .  Their experiment proved very successful i n that  out of 1,249 treated animals 10 died of botulism, an incidence of 0.8 per cent. On the other hand there was an incidence of 6 per cent deaths from botulism among 3,432 non-immunized sheep.  Reames et a l (97) immunized human volunteers with Type A and B toxins on a large scale and obtained comparable results with formolized and alum-precipitated toxoids.  Within 5 months after starting immunization on a schedule of  four bi-weekly injections of fluid toxoid, over 90 per cent of the individuals had protective levels.  The importance of the spacing of the injections was  illustrated i n the experiments with a combined Type A and B alum-precipitated toxoid.  A schedule consisting of two doses 8 weeks apart was more effective  than one that called for two doses 3 to 4 weeks apart. Action of Serum and Other Materials on Toxin Increases i n the l e t h a l i t y of botulinum toxins by the addition of serum and other protein-containing materials has been recorded with horse serum (64, 65), rabbit and sheep serum (66), and guinea pig leukocytes (67). Sommer (68) reported this phenomenon and evaluated their findings.  Sommer and Using a  Type A toxin, they found that exposure to 2 per cent Witte's peptone at 3 7 ° C for several hours could double or t r i p l e the potency, while similar treatment with horse serum might raise i t even higher.  However, with a l l agents, the  period of increase was only for a few hours at the most and was then quickly followed by a rapid decline i n toxic a c t i v i t y . The manner i n which these agents cause increases i n potency remains obscure.  Bronfenbrenner (41) drew attention to the i n s t a b i l i t y of greatly  diluted toxins, especially when highly purified, and to the p o s s i b i l i t y that any added serum might be protective against oxidative or destructive  influences.  Other Toxins of Clostridium Botulinum While studying the properties of highly concentrated toxin of CI. botulinum Type A, Lamanna (69) found that his preparation could agglutinate  -20the red c e l l s of chickens, r a b b i t s , guinea pigs, sheep and man.  Since then  t h i s characteristic has received further study (70, 71), from which i t has become apparent that the neurotoxic and the haemagglutinative properties of highly p u r i f i e d materials from Types A and D cultures are i n many respects independent of one another. A haemolysin for sheep red c e l l s has been found by Guillaumic and Kreguer (72) i n culture f i l t r a t e s from C and D organisms.  Lecithinase  a c t i v i t y has been demonstrated i n c e r t a i n culture f i l t r a t e s of Types A and B organisms (73). Type A Toxin Lamanna and h i s colleagues c u l t i v a t e d a highly toxigenic s t r a i n of C l . botulinum Type A on a simple medium containing casein hydrolysate and corn-steep liquor (74, 75).  After s t e r i l i z i n g the cultures i n an autoclave,  they were able to recover a highly t o x i c component by repeated precipitations with HC1 at pH 3.5 and re-solution i n various saline buffers of specified composition.  Prom the l a s t of the concentrates they were able to c r y s t a l l -  ize a globulin which, on parenteral i n j e c t i o n into mice, had the high t o x i c i t y of 220 x 10^ LD|JQ per mg. of N .  On hydrolysis t h i s protein yielded 19  d i f f e r e n t amino acids, among which microbiological assays disclosed an unusually high proportion of aspartic acid, tyrosine and threonine  (76). When  examined by electrophoresis, d i f f u s i o n methods and sedimentation i n the u l t r a centrifuge, the t o x i n behaved as a homogenous protein whose apparent molecular weight was about 900,000. Abrams, Kegeles and Hottle (77) also used t h i s highly toxigenic s t r a i n of C l . botulinum i n the p u r i f i c a t i o n of Type A t o x i n .  Their method of  fractionation d i f f e r e d i n some respects from that of Lamanna et a l . Starting  -21with a sequence of precipitations with the acid at pH 3.5 and re-solution i n phosphate buffer, they completed t h e i r p u r i f i c a t i o n with p a r t i a l l y saturated ammonium sulphate.  Their c r y s t a l l i n e f i n a l product had a t o x i c i t y higher  than that obtained by Lamaima.  The i s o e l e c t r i c point was 5.6, and the  molecular weight was about 1,130,000. Both values for molecular weight were i n the range of 1,000,000.  Since  the t o x i n i s of such a high molecular weight, i t i s d i f f i c u l t to imagine how t h i s can be absorbed through the i n t e s t i n a l w a l l .  However, Wagman and Batman  (78) found that when a solution of Type A t o x i n i s brought to a pH of 7.5, a s i g n i f i c a n t proportion of the large molecules, which are t y p i c a l of the protein i n acid solution, undergo disruption into much smaller ones whose weight they estimated to be about 70,000.  This "dissociated" t o x i n s t i l l possessed the  same l e t h a l i t y for mice as the complex t o x i n , and may prove to be the form i n which the t o x i n i s absorbed i n naturally-occurring botulism. Type B Toxin Lamanna and Glassman (37), again separated the t o x i n as an electrophoretically homogenous protein.  Immunologically, chemically and physically  i t d i f f e r e d from Type A t o x i n , although i t had a potency of 160 x 10^ mouse LD^Q per mg. of nitrogen, not much below that of the Type A t o x i n .  The most  noteworthy difference between the two Types of t o x i n was that the Type B had a molecular weight of 60,000, a value very close to that of the dissociated form of Type A toxin referred to by Y/agman and Batman. Type C Toxin No attempt has been made as yet to fractionate toxic f i l t r a t e s from Type C cultures that can be compared with those undertaken f o r Types A and B.  -22Sterne and Wentzel (79) have made a potent toxin preparation by c u l t i v a t i n g a toxigenic s t r a i n inside a cellophane sac immersed i n a large volume of nutrient medium.  By t h i s technique they prevented the dispersal throughout  the entire culture of the t o x i n which was formed, thus producing a concentrated product.  The greatest potency of t h e i r preparations, which they termed  "dialysate toxins", was about 3 x 10^ mouse M.L.D. per mg. of N. Type D Toxin Using the same cellophane sac culture technique f o r C l . botulinum Type D as they used for Type C, Sterne and Wentzel (79) produced a highly toxic concentrate which, without further treatment, contained 130 x 10^ mouse M.L.D. per mg. of N.  Starting with t h i s preparation and employing successive  p r e c i p i t a t i o n and re-solution i n ammonium sulphate solutions, they obtained an electrophoretically homogenous material which from d i f f u s i o n measurements appeared to have a molecular weight of about one m i l l i o n .  When dissolved i n  a d i l u t e g e l a t i n phosphate buffer solution at pH 6.2, t h i s material had a 12 t o x i c i t y of 4 x 10  mouse M.L.D. per mg. N (80).  An attempt to concentrate the Type D t o x i n has also been made at the Pasteur Institute by Boroff, Raynaud and Prevot (57).  Their most potent  preparation, however, contained only about 14 x 10 mouse LD^^ per mg. N, a value f a r different from that obtained by Sterne and Wentzel, but not so very d i f f e r e n t from that obtained with the Type A and B toxins. Type E Toxin Clostridium botulinum Type E was f i r s t reported i n 1936 by Gunnison, Cummings and Meyer (25), after they isolated the organism from the intestines of sturgeon.  They described the morphology, biochemical reactions, thermal  resistance of spores, and the t o x i n production of two cultures sent by Bier  -23of the Institute at Dnepropetrovsk, i n the Russian Ukraine.  They showed  that the a n t i t o x i n of Types A, B, C and D f a i l e d to protect guinea pigs against t h i s t o x i n , and that a n t i t o x i n made with these Type E cultures f a i l e d to protect against at least 2 to 3 M.L.D. of the toxins of Types A, B, C and D. One year l a t e r , Hazen (26) isolated an organism from a t i n of Germancanned sprats which had caused f a t a l botulism i n New York State.  Hazen noted  that the thermc—stability of the spores of t h i s s t r a i n (E35396) was very low i n comparison with other Types of Clostridium botulinum.  In 1938, Hazen (27)  isolated a second s t r a i n of Type E botulinum from salmon,caught i n Labrador and smoked, which had caused a f a t a l case of botulism i n New York State.  In  t h i s same report the author mentions that Type E strains i n the past may have been overlooked due to the low thermo-stability of the spores. F i r s t mention of Type E botulism i n Canada came i n 1947 when Dolman and Kerr (29) reported the i s o l a t i o n and i d e n t i f i c a t i o n of Type E t o x i n from homecanned salmon which had caused 3 f a t a l cases of botulism i n Nanaimo, B r i t i s h Columbia.  The i s o l a t i o n of another s t r a i n from home-pickled herring i n  Vancouver, B r i t i s h Columbia, by Dolman i n 1950 (30), was Canada's second reported incident  of Type E botulism.  Meyer and Eddie i n 1951 (15) described a small outbreak of botulism among Eskimos i n Alaska due t o white whale f l i p p e r s cured i n o i l .  A s t r a i n of Type  E was l a t e r isolated from the infected f l i p p e r s by Dolman and Chang (31). Outbreaks of Type E botulism have since been reported from Canada (81, 82), Japan (83), France (84) and Greenland (85). i n common:  These occurrences held one p e c u l i a r i t y  they were a l l isolated from marine foodstuffs, indicating a marked  predilection for t h i s type of food.  -24Data concerning the physical and chemical properties of purified Type E toxin are not available to date.  A method of purification has been described  by Duff et a l . ( 9 4 ) i n 1 9 5 7 , i n which they obtained a product containing 56  x  10  3  IAJQ  P  E  R  mg*  N  for non-activated toxin and a value of  per mg. N for a trypsin-activated product.  19  x  10  6  IAJQ  In each case, preliminary u l t r a -  centrifugation studies indicated that this product was not homogenous, although the value obtained, 1 9 x 1 0 ^ , is approaching that obtained for purified Types A and B toxins.  One interesting observation concerning the purification of  Type E toxin i s the i n a b i l i t y to precipitate the toxin with acid, a procedure which i s fundamental i n the purification of Types A and B toxins.  Instead,  precipitation with alcohol, followed by extraction of the toxin with calcium chloride solutions, has proved to be  successful.  Variation The phenomenon of variation i n Type strains of Clostridium botulinum was f i r s t mentioned i n 1 9 2 8 by Schoenholz ( 9 8 ) , while studying colonial growth of various strains on blood agar medium.  When 4 2 stock cultures were streaked  on these plates he obtained two to three variants i n each strain, and noted five different variants i n a l l .  However, i t should be noted that i n these  five variants he succeeded i n isolating and propagating i n pure culture, no mention was made of a non-toxic variant. One year later Gunnison and Meyer ( 9 9 ) demonstrated that toxic and nontoxic variants of C l . botulinum may exist side by side i n mass culture from contaminated foods;  but the authors stated that i t was more l i k e l y that these  non-toxic organisms were actually a Clostridium sporogenes contaminant rather than non-toxic variants of Clostridium botulinum.  -25In the same year Townsend (101) reported what would seem to be the f i r s t systematic study of non-toxic and t o x i c variants of botulinum species, although his conclusions that i t was not possible to d i f f e r e n t i a t e between t o x i c and non-toxic strains of CI. botulinum by c u l t u r a l , biochemical or serological methods were l a t e r demonstrated to be erroneous. I t was not u n t i l 1957 that the phenomenon of v a r i a t i o n i n Clostridium botulinum was f u l l y c l a r i f i e d .  At t h i s time, Dolman (51) demonstrated  v a r i a t i o n i n Type E strains and succeeded i n d i f f e r e n t i a t i n g the i s o l a t e d variants both morphologically and biochemically.  These variants consisted  of a t o x i c , non-proteolytic variant ("TQX") which produced a mosaic pattern colony by transmitted l i g h t ;  an atoxic, non-proteolytic, sporolytic variant  ("OS"), y i e l d i n g opaque colonies;  and a p r o t e o l y t i c , non-toxic variant ("TP")  which produced a f l a t , transparent colony.  The author states that the t o x i -  genic Type E strains tend to degenerate into the sporolytic and less often into the proteolytic variants, consequently losing t h e i r t o x i c i t y ; that though both the "TP" and "OS"  and secondly,  forms are non-toxic i n themselves, or,  combined together, they may revert to the toxic phase under the proper conditions.  The importance of t h i s observation to the production of toxins of  Type E, and i n the i s o l a t i n g of Type E strains from infected food material cannot be overestimated. Activation A marked difference between Type E toxin and the toxins produced by other Types of Clostridium botulinum i s the r e l a t i v e l y low potency of the former under ordinary laboratory conditions.  Potencies exceeding one m i l l i o n  M.L.D. per ml. are not uncommon f o r Types A and B toxins, whereas potencies 3,000 to 5,000 M.L.D. per ml. are maximum for Type E.  Despite t h i s .  of  -26c h a r a c t e r i s t i c , deaths from Type E toxin occur almost as quickly as with those of Types A and B (51).  An explanation of t h i s phenomenon has been put forward  by Dolman, after observing that high potency Type E t o x i n can be obtained by the combination of a toxic variant with a non-toxic, proteolytic variant (51). This increase (10 - 100-fold) can be brought about by culturing the two variants together, or by mixing s t e r i l e f i l t r a t e s of these cultures and incubating them for specified periods of time. Rapid a c t i v a t i o n of Type E toxin has been reported by Duff et a l (102) employing crude and p u r i f i e d t r y p s i n solutions as the activating substances. Activation by t h i s method has also been shown with the epsilon t o x i n of Clostridium w e l c h i i by Turner and Rodwell (100), who suggested the increase i n potency was due to the presence i n the culture of an inactive toxin or pro-toxin which became active on addition of t r y p s i n , thus allowing a more potent toxin to be produced.  -27iy.  Preliminary Experiments.  The production of botulinum toxins for the purpose of preparing toxoids suitable for human immunization involved much preliminary experimentation. Before these toxoids could be prepared, i t was necessary to study the conditions required for optimum toxin production, the minimal lethal dose of the toxin for laboratory animals, the complex phenomenon of toxin a c t i vation and the effect of temperature, hydrogen-ion concentration, etc. of the crude and activated toxins. A.  The effect of surface moisture on the isolation of variants. In the preparation of potent toxins of Clostridium botulinum Type E  strains, one must be certain that the toxigenic strain employed has not degenerated into a non-toxic, sporulating state.  This point is emphasized  when one considers that incubation periods may i n some instances be as high as 12 days, as i s the case with concentrated dialysate  toxins.  It i s conceivable that when one sets about to obtain this information by culturing aliquots of the toxic suspension, a false picture concerning the state of the strain may be realized due to inadequate techniques i n culturing the sample.  For example, the use of a wire loop for streaking a non-toxigenic,  proteolytic culture on agar plates i s sometimes conducive to the growth of nontoxigenic colonies which resemble the mosaic pattern of the toxic variant when examined by indirect l i g h t i n g .  However, i f the inoculum is streaked with the  smooth, round end of a glass rod, thus not disturbing the surface of the medium, this phenomenon does not occur. A second and more important determinant for producing characteristic colonies and for ease of isolation is the amount of surface moisture on the  -28medium to be inoculated.  To obtain some degree of standardization, an  experiment was performed to determine the optimum length of time culture plates should be dried before inoculation. Brain heart infusion plates were poured and allowed to dry at 3 7 ° C . for varying lengths of time.  After the desired drying time had elapsed,  plates were removed and streaked with a toxic, non-proteolytic, nonsporeforming (OT) strain and others with a non-toxic, proteolytic, nonsporefarming (T) strain.  The plates were then incubated at 3 7 ° C for 24  hours, i n Mclntosh-Fildes anaerobic j a r s .  The following table l i s t s the  results of this experiment, the c r i t e r i a for evaluation being the amount of strain variation, and ease of i s o l a t i o n .  The results shown are an average  of those from three separate experiments. TABLE II. Drying Time  Culture  Results  0  OT T  No isolated colonies. II it n  5 min.  OT T  Colonies f a i r l y well isolated. No isolated colonies.  10  "  OT T  Colonies well isolated. Few isolated colonies.  20  "  OT T  Colonies well isolated, some OT-0 variation. Well isolated colonies.  30  "  OT T  Isolated OT colonies, considerable 0 variants. Isolated T colonies, considerable variation to the 0 form. Appearance of some variation to colonies resembling OT i n morphology.  Determination of optimum drying-time for plates.  -29The optimum drying time of the plates is dependent on the variant being cultured*  For toxigenic variants this is approximately 5 to 10 minutes at  3 7 ° C . , whereas for the non-toxigenic. proteolytic variants 10 to 20 minutes would seem to be optimum.  If mixed cultures are to be plated the plates  should be dried 10 minutes at 3 7 ° C .  It should also be noted that when the  (T) variant is streaked on plates dried for 30 minutes or longer colonies appear which do not conform to those described for (OT) variants (see Appendix A ) .  However, i f these colonies are picked and inoculated into glucose-  peptone-beef -infusion media ( G . F . B . I . ) , the growth i s indicative of normal (T) variants. B.  Production of toxic and non-toxic  filtrates.  The production of botulinum toxoids involves the preparation of two types of toxin, designated "crude" and "activated". Crude toxin is that toxin produced by growing a pure toxigenic variant (OT) of a Type E strain and harvesting the f i l t r a t e after the required incubation period.  This toxin normally has a potency of 2,000 to 5,000  M.L.D. per ml. of culture f i l t r a t e , depending on the medium employed, pH and the condition of the strain i t s e l f . Activated toxin is crude toxin which has an a r t i f i c i a l l y increased potency.  This increase may be brought about by (a) culturing together a  proteolytic, non-toxic variant (T) with an (OT) variant, (b) by combining sterile filtrates  of (OT) and (T) cultures and allowing them to incubate,  and (c) by the addition of trypsin to a crude toxin followed by incubation (51).  The potency of this activated product may range from values s l i g h t l y  above that of the crude toxin to as high as 100 million of mouse M.L.D. per m l . , again depending on conditions such as incubation time, pH and the state of the crude toxin per se. 1.  Production of crude toxin. For the production of crude toxin, the (OT) variant of strain "Iwanai"  was employed.  Stock cultures were prepared i n G . P . B . I . media and stored at  4°C. Erlenraeyer flasks containing 4 l i t r e s of G . P . B . I . media at pH 7.8 were inoculated with 10 ml. of a 20 hour culture of the (OT) variant grown at 32°C.  The flasks were incubated at 3 2 ° C . for 7 days followed by r e f r i g -  eration at 4 ° C . for 24 hours.  At the end of this period, the f i l t r a t e was  removed and s t e r i l i z e d by f i l t r a t i o n through a Seitz f i l t e r .  Before  fil-  tration of the toxin, 100 ml. of s t e r i l e nutrient broth at pH 5.2 was passed through the f i l t e r pad.  The toxin was then titrated by intraperitoneal  injections i n mice, and normally had a potency of 2,000 to 3,000 mouse M.L.D. per ml. TABLE III Days incubation 1  Potency mouse M.L.D. per ml. 2  pH 7.2  2  10-30  6.7  3  50-100  6.1  4  100-300  5.7  5  500-700  5.3  6  2,500-3,000  5.2  7  2,500-3,000  5.2  -32-  Diagram I indicates the rise i n toxin potency and pH decline i n the preparation of a crude toxin.  This particular toxin had a potency of  2,500 to 3,000 mouse M.L.D. per m l . , or 2,272 L D 5 0 per ml. Crude Type E botulinum toxin i s very stable at refrigeration temperatures; potency.  samples stored at 4 ° C . for 2 years demonstrated no alteration i n As shown i n Diagram I, the pH of the culture i s of extreme  importance from the standpoint of toxin potency and, as w i l l be shown later, for toxin activation. (a)  Effect of pH on production of crude toxin.  "*  The pH of the incubating culture undergoes the same increase i n each batch of toxin, and a number of experiments were undertaken to study this action more closely.  Diagram II i l l u s t r a t e s the effect of an elevated pH  on toxin production. In this experiment the pH of the culture was not allowed to f a l l below a value of 6.0.  This was accomplished by adding N/l0 NaOH to the culture  beginning on day 3.  As seen i n this graph,  beyond 300-500 mouse M.L.D. per ml.  toxin potency did not increase  Furthermore, microscopic observation  of the culture throughout the incubation period did not demonstrate the characteristic decrease i n Gram-positive cells between days 4 and 5, as i s noticed with normal cultures. When the above experiment was repeated and the pH allowed to decline on day 7, toxin potency was seen to again increase to a f i n a l t i t r e of 750 1,000 mouse M.L.D. per ml. on day 10. would not increase this potency.  Further incubation or decreased pH  These results are shown i n diagram  III.  3ooo-l  pH  48  M.L.D.  0-  2.000 4  +7  o j 6. (Lf 0  looo J  5  +5  O AYS D i a q r a m  H . .  The  iNCUSATVON effecT  of  <3.n e l e v a t e d  R-T p!4  3 ^ on  C  +oxin  prodo  n  -35Diagram IV i l l u s t r a t e s the effect on toxin potency when the pH i s lowered to 5.2, on day 2, by the addition of N/lO HC1. Toxin production, i n this instance, increased normally to a potency of 10 to 30 mouse M.L.D. per ml. on day 2, but failed to increase to any extent during the remaining 5 days of incubation.  Adjusting the pH again to 7.0 on  day 7 and prolonging the incubation did not cause a potency increase. From these experiments i t was evident that the pH plays an important role in the production of toxin.  Not only must the pH reach the required acidity,  but i t must attain this value at a particular time during the growth of the culture. I believe these findings also substantiate the theory that the toxin i s an exotoxin released from c e l l s on autolysis as described by Boroff (58) for Type D toxin.  Boroff*s suggestion that the long lag period between maximum  growth and toxin production is due to a required build-up of autolytic enzymes might also be applicable here. The importance of these pH studies i s again emphasized under the heading of toxin activation. 2.  The production of activated toxin. The production of activated toxin, as stated e a r l i e r , may be accomplished  by one of three methods.  Each of these has been studied i n some d e t a i l i n an  attempt to determine the conditions responsible for toxin activation, and secondly, to determine which method should be employed for the production of an activated product suitable for the preparation of a toxoid.  pH  Zoool M.L. D.  \7  o_  \ooo  1  6  III  o  2  D A Y S Diagram  17  The  I N C U B A T I O N effect  of  an  early  a1 d e c r e a s e  3$ ° in  C.  pH.  0_  -36(a)  Dialysate toxin. The production of activated toxin by culturing a toxic (OT) variant  with a proteolytic, non-toxic (T) variant i n dialysis tubes has been employed i n this Department for some time (51).  A complete description of  the apparatus and i t s mode of operation is contained i n Appendix C. For the preparation of this toxin the apparatus was inoculated with 2 0 m l . of 16 hour cultures of "Iwanai" (OT) and "VH" (T), grown i n G . P . B . I . media at 3 2 ° C . Samples of the toxic f i l t r a t e were removed each day throughout the incubation period for the determination of pH and toxin potency. results of these determinations are l i s t e d i n Table IV. TABLE TV Days incubation at 3 2 ° C . 0 1  Mouse M.L.D./ml. 0 300 - 1,000  pH 7.8 7.3  2  10,000 - 30,000  7.0  3  50,000 - 66,000  6.6  4  66,000 - 100,000  6.2  5  100,000 - 150,000  6.0  6  150,000 - 200,000  5.8  7  200,000 - 250,000  5.6  8  1,000,000 - 2,000,000  5.3  9  2,000,000 - 3,000,000  5.3  10  2,000,000 - 3,000,000  5.3  11  2,000,000 - 3,000,000  5.3  The  -37Refrigeration of toxin on day 11 for 24 hours caused a further increase i n potency to 3 million - 4 million mouse M.L.D. per ml.  This latter  charac-  t e r i s t i c has been demonstrated many times with the (T) + (OT) toxin. The toxin suspension was s t e r i l i z e d by Seitz f i l t r a t i o n and stored at 4°C.  Activated toxin produced by this method is r e l a t i v e l y stable compared  with the other types. Diagram V shows the characteristic curves for pH and potency obtained throughout the incubation period. (b)  Combination of  filtrates.  Toxin activation by the combination of s t e r i l e f i l t r a t e s of (OT) and (T) cultures has been reported i n a recent publication by Dr. C . E . Dolman of this Department.  The potency of activated toxin produced i n this manner i s much  more variable than when the organisms are cultured together.  On some  occasions a rapid activation to a potency 50 times that of the original toxin w i l l result, whereas on other occasions l i t t l e or no a c t i v i t y follows combination and incubation.  Preliminary experiments indicate that such con-  ditions as time, pH, temperature and the conditions under which the two f i l trates themselves are produced are very c r i t i c a l . The following experiment entails the production of activated toxin by this method. Two hundred ml. of a s t e r i l e f i l t r a t e of "Iwanai" (OT) toxin (2,500 3,000 mouse M.L.D. per ml.)were mixed with 25 ml. of a s t e r i l e "VH" (T) trate.  fil-  The mixture was incubated at 3 2 ° C . and gently swirled every few hours  to assure a homogenous mixture. removed and titrated i n mice.  At designated intervals, small aliquots were Table V contains the results of this experiment.  2  4  D A Y S Diagram  2  6 I N C U B A T I O N  Toxin  a CI i vat i o n  10  8  by  rffixed  at  3 2°  culture-  12 C-  -39TABLE V Time  Mouse M.L.D./ml.  0  2,000 - 3,000  2 hrs.  5,000 - 10,000  4  "  20,000 - 33,000  6  "  33,000 - 50,000  8  "  50,000 - 75,000  10  "  75,000 - 100,000  12  "  100,000 - 125,000  16  "  125,000 - 150,000  20  "  150,000 - 175,000  24 ."  175,000 - 200,000  28  200,000 - 250,000  32  "  200,000 - 250,000  As stated e a r l i e r , i t was not always possible to repeat these r e s u l t s . Some experiments gave higher f i n a l potencies, while others demonstrated no activation.  Consequently, a number of experiments were undertaken to  examine more closely the effect of incubation temperature and pH.  Ti-  trations of the toxin after maximum potency had been attained indicated a gradual decline i n t i t r e both at incubation temperature as well as at 4 ° C , though the latter was much slower. (i)  Effect of incubation temperature. Three flasks containing 25 ml. each of "Iwanai" (OT) toxin and "VH" (T)  f i l t r a t e were incubated at temperatures of 2 8 ° C , 3 2 ° C . and 3 7 ° C . respectively.  Titrations of toxin potency were made every 8 hours for 136 hours.  Results are tabulated i n Table V I .  300,000  E  8  16  I N C U B A T I O N DlaqromTZr  Toxin  at  activation  24  3 2 ° by  C. sterile  32  in  hours. proteolytic  filtrate.  -41TABLE VI. Time i n hrs*.  Effect of temperature on toxin activation. Mouse M.L.D./ml. 32°C.  28°C.  0  1,000 - 2,000  1,000 - 2,000  1,000 - 2,000  8  10,000 - 20,000  33,000 - 50,000  33,000 - 50,000  16  10,000 - 20,000  100,000 - 150,000  100,000 - 150,000  24  20,000 - 30,000  100,000 - 150,000  150,000 - 200,000  32  30,000 - 50,000  150,000 - 200,000  150,000 - 200,000  40  50,000 - 100,000  150,000 - 200,000  50,000 - 100,000  48  50,000 - 100,000  50,000 - 100,000  50,000 - 100,000  56  50,000 - 100,000  50,000 - 100,000  30,000 - 50,000  64  30,000 - 50,000  30,000 - 50,000  10,000 - 30,000  72  10,000 - 20,000  10,000 - 20,000  3,000 - 5,000  80  3,000 - 5,000  3,000 - 5,000  1,000 - 2,000  88  1,000 - 2,000  500 - 1,000  96  500 - 1,000  104  100 - 200  112  50 - 100  120  10 - 30  128  2 - 10  -  136  (ii)  37°C  100 - 300  -  300 - 500 50 - 100  -  Effect of pH Five flasks, each containing 25 ml. of "Iwanai" (OT) toxin and 25 ml.  "VH" (T) f i l t r a t e , were prepared and adjusted to pH values of 4.8, 5.2, 5.6, 6.0 and 7.0 respectively and incubated i n a water-bath at 3 2 ° C .  At specified  intervals, aliquots were removed from each flask and titrated i n mice for potency determinations.  These results are shown i n Table V I I .  TIME DlaqramTZIE:  Effect  of  In  hour 8  temperature  on toxin  activation.  TABLE VII  Effect of. pH on toxin activation  Incubation time i n hrs.  pH 4.8  pH 5.2  0  1,000 - 2,000  1,000 - 2,000  2  1,000 - 2,000  4  Mouse M.L.D. per ml. pH 5.6  pH 6.0  pH 7.0  1,000 - 2,000  1,000 - 2,000  1,000 - 2,000  10,000 - 20,000  2,000 - 3,000  1,000 - 2,000  1,000 - 2,000  1,000 - 2,000  20,000 - 33,000  3,000 - 5,000  1,000 - 2,000  1,000 - 2,000  8  1,000 - 2,000  33,000 - 50,000  5,000 - 10,000  2,000 - 3,000  1,000 - 2,000  16  500 - 1,000  100,000 - 150,000  20,000 - 30,000  2,000 - 3,000  1,000 - 2,000  24  500 - 1,000  150,000 - 200,000  20,000 - 30,000  3,000 - 5,000  1,000 - 2,000  32  500 - 1,000  200,000 - 250,000  20,000 - 30,000  3,000 - 5,000  1,000 - 2,000  40  500 - 1,000  150,000 - 200,000  10,000 - 20,000  5,000 - 10,000  1,000 - 2,000  48  500 - 1,000  100,000 - 150,000  10,000 - 20,000  5,000 - 10,000  1,000 - 2,000  56  500 - 1,000  50,000 - 100,000  5,000 - 10,000  3,000 - 5,000  1,000 - 2,000  8  16  24  T I M E D i a g r a m W  Effect  of  in pH  32 hours on  toxin  at  4 0 32°C. activation.  4 8  56  -45The production of activated toxins by combining f i l t r a t e s  of toxic  and proteolytic variants i s dependent on the pH of the mixture and the incubation temperature.  The pH is seen to be very c r i t i c a l and variations  of 0.5 pH units on either side of the optimum (pH 5.2) i s detrimental i f not inhibitory to toxin activation.  The incubation temperature of the reaction  mixture is not as c r i t i c a l , activation occurring from temperatures of 3 2 ° C . to 3 7 ° C .  Results are indicative of activation by enzymatic substance or  substances contained i n the (T) f i l t r a t e . (c)  Activation with trypsin. Duff et a l . (102) have recently reported the marked activation of  Clostridium botulinum Type E toxin by treatment with trypsin.  The authors  state that when crude toxins were incubated with 1 per cent trypsin for 45 minutes, potencies could be increased to as high as 50 f o l d . Experiments i n this Department have confirmed and extended these findings.  These studies involved determinations of optimum conditions for  activation, inhibition of activation and the s t a b i l i t y of the activated products at temperatures required for toxoiding. (i)  Toxin activation. F i f t y ml. of crude "Iwanai" (OT) toxin were incubated at 3 7 ° C . with 1  per cent trypsin (Difco 1:250).  Aliquots were removed at specified inter-  vals and t i t r a t e d for potency i n mice.  The results are shown i n Table V I I I .  -46TABLE VIII  Time  Mouse M.L.D./ml.  0  PH  - 3,000 10,000 - 30,000 50,000 - 100,000 500,000 - 1,000,000 30,000,000 - 50,000,000 50,000,000 - 100,000,000 50,000,000 - 100,000,000 30,000,000 - 50,000,000 10,000,000 - 30,000,000 5,000,000 - 10,000,000 1,000,000 - 5,000,000 10,000 - 50,000 5,000 - 10,000 5,000 - 10,000 3,000 - 5,000 1,000 - 3,000 2,000  30 min. 1 hr. 2 hrs. 3  II  4  II  5  II  6  II  8  II  10  it  20  II  40  ti  50  it  60  ii  120  II  240  II  480  n  1  5.3 5.3 5.4 5.4 5.5 5.6 5.6 5.4 5.4 5.3 5.3 5.2 5.0 5.0 4.8 4.8 4.8  The effect of trypsin on the crude toxin at a pH well below the o p t i mum for this enzyme was very surprising.  As seen i n the above table, the  toxin i s very unstable and once the high potency i s attained i t decreases very rapidly.  This detrimental action is retarded somewhat at refrigeration  temperatures but s t i l l remains comparatively rapid. The activation i t s e l f w i l l not occur with trypsin at a pH of 7.5 (optimum for trypsin) nor with pepsin at pH values of 5.2 or at i t s optimum pH of 2.5, as demonstrated i n Table IX.  TABLE IX  Time i n hrs.  Activation of, .toxin-with crude trypsin and pepsin  1% Trypsin pH 5.2  pH 7.5  pH 5.2  Vfc  Pepsin'  pH 2.5  0  2,000 - 3,000  2,000 - 3,000  2,000 - 3,000  2,000 - 3,000  1  30,000 - 50,000  2,000 - 3,000  2,000 - 3,000  1,000 - 2,000  2  100,000 - 300,000  1,000 - 2,000  2,000 - 3,000  1,000 - 2,000  3  ljoooyooo - 3,000,000  1,000 - 2,000  2,000 - 3,000  500 - 1,000  4  10,000,000 - 30,000,000  500 - 1,000  2,000 - 3,000  300 - 500  8  10,000,000 - 30,000,000  300 - 500  2,000 - 3,000  100 - 200  16  1,000,000 - 3,000,000  50 - 100  2,000 - 3,000  10 - 30  24  300,000 - 500,000  10 - 30  2,000 - 3,000  1  -49(ii)  Inhibition of the activating phenomenon. The apparent i n s t a b i l i t y of this activated toxin, as shown by i t s rapid  decrease i n toxin potency, does not allow for i t s use as such i n the preparation of toxoids. Duff, i n a recent publication (102), employed purified trypsin as the activating substance and succeeded i n inhibiting toxin activation by adding purified soya i n h i b i t o r .  Consequently, experiments were undertaken i n this  laboratory to determine i f destruction of the activated toxin could be inhibited by treating the product with soya i n h i b i t o r .  Preparation of the  solutions employed may be found i n Appendix B. Twenty ml. of a 0.02 per cent purified trypsin solution were added to an equal volume of crude "Iwanai" (OT) toxin and the mixture incubated at 3 7 ° C . At times 1, 2, 4, 8 and 16 hours, 4 ml. of the mixture were removed and added to 4 ml. of a 0.02 per cent soya inhibitor solution.  The aliquots were re-  incubated and titrated i n mice for toxin potency after 16 hours and after 3 days further incubation. The results shown i n Table X demonstrated that the inhibitor protected activated toxin from decreasing i n potency after maximum activation for a minimum of 16 hours, and allowed only a slight decrease after 3 days at 3 7 ° C . Meanwhile, activated toxin without added inhibitor had diminished to a potency of 1 m i l l i o n - 3 million i n 16 hours of further incubation and to as low a potency as 3,000 - 5,000 after 3 days.  TABLE X Effect o':f'-„pjar.if.Aed soya inhibitor on activation of toxin with trypsin.  Time inhibitor added 1 hour  !  Activated toxin (no inhibitor)  Activated toxin (at 16 h r . incubation)  +  Inhibitor (at 3 days)  100,000 - 300,000  100,000 - 300,000  1^000,000 - 3,000,000  1,000,000 - 3,000,000  300,000 - 1,000,000  50,000,000 - 100,000,000  50,000,000 - 100,000,000 10,000,000 - 30,000,000  2  "  4  "  8  "  10,000,000 - 30,000,000  10,000,000 - 30,000,000  16  »  1,000,000 - 3,000,000  1,000,000 - 3,000,000  3 days  3,000 - 5,000  -  ; 100,000,000 - 300,000,000  100,000 - 300,000  300,000 - 1,000,000  -  -51The activated toxins, with and without added i n h i b i t o r , were refrigerated at 4 ° C . for a period of one month and re-titrated.  In this time toxin potency  had only s l i g h t l y diminished i n the samples with added trypsin i n h i b i t o r , while those without the inhibitor had become non-toxic. 3.  Production of non-toxic  filtrates.  For the production of non-toxic, proteolytic f i l t r a t e s the (T) variant of the "VH" strain was employed.  Early i n the investigational studies a  number of media were tested to determine which one supported the best growth and enzyme production for this "VH" (T) strain.  Evaluation of media was based  on the a b i l i t y of the proteolytic f i l t r a t e produced to activate crude toxin. Media tested were as follows:#1  Brewer's thioglycollate anaerobic medium.  #2  Beef-heart infusion medium with added thioglycollate.  #3  G-.P.B.I. medium.  #4  Yeast extract medium.  Preparation of the above media i s discussed i n Appendix B. One hundred ml. aliquots of each medium were prepared i n Erlenmeyer flasks and inoculated with 0.3 ml. of a 20-hour culture of "VH" (T). incubated at 3 2 ° C . for 48 hours.  The flasks were  Following incubation the proteolytic  fil-  trates were removed from the flasks containing media #1, #3 and #4. (Medium #2, beef-heart infusion with added thioglycollate, would not support growth of the "VH" strain).  The proteolytic f i l t r a t e s were s t e r i l i z e d by Seitz  fil-  tration and 10 ml. added to 50 ml. quantities of crude "Iwanai" (OT) toxin. Mouse titrations for toxin activation were performed at intervals of 8, 16, 32, 64 and 128 hours.  Table XI contains the results of the experiment.  -52TABLE XI  Incubation time with crude toxin (values i n thousand mouse M.L.D. per ml.) Media  8  16  32  64  128  # 1  3 - 10  10 - 30  30 - 50  30 - 50  30 - 50  # 3  30 - 50  50 - 75  100 - 125  100 - 125  100 - 125  #4  50 - 75  50 - 75  100 - 125  100 - 125  50 - 75  Yeast extract and G . P . B . I . media both supported good growth and proteolytic a c t i v i t y of the "VH" (T) strain, as indicated by toxin activation. Brewer , s thioglycollate medium, under the conditions stated, did not produce an active proteolytic f i l t r a t e comparable to the two aforementioned media. Both types of media were employed i n later experiments, depending on the desired function of the (T) f i l t r a t e . C.  Purification of Type E toxin. Two procedures were employed for the preparation of Type E toxin for  *  subsequent toxoiding.  The f i r s t method was that employed by Prevot and  Raynaud of the Pasteur Institute, and involved essentially the precipitation of the toxin by purified sodium metaphosphate at a pH of 3.5 and a temperature of - 1 5 ° C .  The precipitate was re-dissolved i n an acetate buffer and  f i n a l precipitation was effected by addition of concentrated phosphate  buffer.  Reasonable results were obtained by this method, but the f i n a l concentrations of the various added components were found to vary with the toxin preparation being purified.  The f i n a l concentration of precipitating  agents required to purify one batch of toxin could not always be applied to a  -53secpnd l o t of toxin and, as a result, a number of t r i a l experiments were undertaken throughout the procedure. The second method employed for the purification of toxin was that reported by Duff et a l . (103), which involved the precipitation of toxin by the addition of 95 per cent ethanol.  The extraction of toxin from the precipitate was  effected with a calcium chloride solution and phosphate 1.  buffer.  Purification with phosphates, (a) Procedure Six l i t r e s of crude "Twanai" (OT) toxin (potency of 2,000 to 3,000 mouse  M.L.D. per ml.) were employed for the experiment. (1)  Sodium chloride was edded to the toxin to a f i n a l concentration of 30 per cent and the temperature lowered to - 1 5 ° C .  (2)  A 10 per cent solution of sodium metaphosphate (Difco purified Na3P04), i n d i s t i l l e d water, was added to make a f i n a l concentration of 1 per cent.  (3)  Normal sulphuric acid was added u n t i l the pH of the mixture was lowered to 3.5, and the mixture was refrigerated for 24 hours at - 1 5 ° C .  (4)  The precipitate was collected by centrifugation for 30 minutes at 2,000 r.p.m. at 0 ° C , and dissolved i n 0.015 M sodium acetate solution at pH 6.0 to one-tenth the original culture volume.  (Des-  ignated NaAc extract). To this point the process followed that reported by Prevot and Raynaud. However,  when 3.5 M K^HPO^-KH^O^ buffer was added to a concentration of 30  per cent, precipitation would not occur.  Aliquots of the NaAc extract were  -54then removed and precipitation was attempted using varying concentrations of phosphate buffer.  The results :of this experiment are contained i n Table XII(a)  TABLE XII (a) Tube Number  Percent 3.5 M Phosphate Buffer (ml.)  Distilled Water (ml.)  Toxic Complex (ml.)  1  :.5  8.5  1  2  10  8.0  1  3  15  7.5  1  4  20  7.0  1  5  25  6.5  1  6  30  6.0  1  7  35  5.5  1  8  40  5.0  1  9  45  4.5  1  10  50  4.0  1  11  55  3.5  1  12  60  3.0  1  13  65  2.5  1  14  70  2.0  1  15  75  1.5  1  16  80  1.0  1  A control series was prepared substituting d i s t i l l e d water for the toxic complex. Flocculation began i n tube #9 of the test series, i . e . 45, per cent concentration of phosphate buffer.  No flocculation occurred i n the control  series. Each tube i n the series was then f i l t e r e d through a #1 f i l t e r pad to leave the precipitate, i f any, on the pad.  The f i l t e r paper was then washed  -55three times with the same sample of sodium acetate at - 1 0 ° C .  The supernatant  and resuspended precipitate were then t i t r a t e d i n mice for M.L.D. determinations.  Results of these titrations are shown i n Table XII(b) TABLE XI 1(b)  Tube Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16  Toxicity i n M.L.D. per ml. (in thousands) Supernatant Precipitate  - 20 15 - 20 15 - 20 15 - 20 15 - 20 15 - 20 15 - 20 15 - 20 3 -5 1 - 3 1 -3  -  15  1  3  -  10  - 15  10  - 15  15  - 20  15  - 20  15  - 20  15  - 20  15  - 20  15  - 20  Titrations indicated that the phosphate buffer i f added to the toxincomplex to a concentration of 55 per cent yielded a precipitate containing a high percentage of toxin  -56(5)  3.5 M phosphate buffer was added to the toxic complex to a concentration of 55 per cent, and the mixture was refrigerated at -10°C.  (6)  The precipitate was collected by centrifugation and re-dissolved i n 1 N NaCl to one-tenth the original culture volume. (Designated NaCl extract).  (7)  Extraneous protein was then removed by p a r t i a l precipitation at - 1 0 ° C . with phosphate buffer to a concentration of 26 per cent.  (8)  The mixture was then centrifuged and the precipitate discarded. (NaCl-2 extract).  (9)  Phosphate buffer was added to the supernatant to a concentration of 28 per cent to precipitate the toxin, and the mixture allowed to remain at - 1 0 ° C . for 24 hours.  (10)  The precipitate was collected by centrifugation at 0 ° C . and redissolved i n 50 ml. of NaCl.  It should be noted that the concentrations of phosphate buffer employed i n steps 7 and 9 were determined by experiments similar to that shown i n step 5. (b)  Results. Nitrogen determinations on the various extracts were obtained by direct  nesslerization technique (104). average of two separate t r i a l s .  The results indicated i n Table XIII are an  -57-  TABLE XIII  Sample  Fold Cone. From Culture  Mouse M.L.D./ml. (in 1,000)  Crude  undiluted  3  NaAc  10  NaCl-1 NaCl-2 Final  2.  Mouse M.L.D./mg. N (in 1,000)  Per cent Recovery  Fold Purif.  5  -  -  22  40  70  8  10  15  125  50  25  10  12  350  38  70  120  40  375  11  75  Purification by alcohol precipitation, (a.) Procedure. Five l i t r e s of crude "Iwanai" (0T) toxin were employed for this experiment.  The potency of this toxin was 2,000 - 3,000 mouse M.L.D. per ml. (1)  Ninety-five per cent ethanol was added to the toxin to a concentration of 25 per cent.  Micron bentonite (Volcay Bentonite,  BC g r i t free, American Colloid Company), a highly refined diatomaceous earth, was added to a concentration of 1 gram per l i t r e of culture to aid settling of the toxin precipitate.  The mixture was  allowed to stand 48 hours at - 7 ° C . (2)  The supernatant was siphoned off and the precipitate collected by centrifugation for 30 minutes at 4,000 r.p.m. at - 7 ° C .  (3)  The precipitate was diluted with d i s t i l l e d water to one-sixth the culture volume and stirred for 1 hour at room temperature.  (Des-  ignated f i r s t alcohol fraction). (4)  The alcohol-precipitated toxin was then diluted to one-quarter the culture volume with d i s t i l l e d water, and 1.0 M CaCl2 solution to a f i n a l concentration of 0.075 M CaCl2.  The pH was adjusted to 6.0,  and the suspension stirred for 2 hours at room temperature, and then c l a r i f i e d by centrifugation at 2 0 ° C . for 30 minutes at 4,000 r.p.m. (5)  (The supernatant i s referred to as CaCl2 fraction).  After adding 95$ ethanol to a f i n a l concentration of 25 per cent, at - 7 ° C . , the CaCl2 fraction was allowed to settle overnight.  (6)  The precipitate was again collected by centrifugation at - 7 ° C . and 4,000 r.p.m. for 30 minutes, and dissolved i n 0.08 M phosphate buffer, pH 6.0.  The mixture was then c l a r i f i e d by centrifugation  at 2 0 ° C . for 30 minutes, at a speed of 4,000 r.p.m,  (Designated  second alcohol fraction). (7)  Ninety-five per cent ethanol was again added to a f i n a l concentration of 25 per cent at - 7 ° C . , the suspension was allowed to stand overnight and the precipitate collected by centrifugation at - 7 ° C . for  (8)  30 minutes and a speed of 4,000 r.p.m.  The precipitate was dissolved i n 0.2 M succinate buffer at pH 5.5 to a volume one-half that of the second alcohol fraction.  (b)  Results.  The average results of three separate purification attempts by this method are shown i n Table XIV.  Nitrogen determinations were made by direct nessler-  ization techniques, and potency determinations were performed i n mice.  -59TABLE XIV  Fraction Whole culture  Fold Cone. Mouse M.L.D./ml. Mouse M.L.D./mg. N Per cent Fold (in 1,000^-s) (in 1,000's) From Culture Recovery P u r i f . undiluted  3  4  mm  First ale. fraction  6  15  40  83  10  CaCl2  4  8  90  66  22  Second ale. fraction  16  28  325  58  81  Third ale. fraction  32  32  450  33  113  Purification of toxin by the alcohol-precipitation technique was found to give a purer product and a higher recovery of toxin i n comparison with the phosphate precipitation method.  This method was easily executed and the  results obtained were easily reproducible. Further experiments to determine the degree of purity obtained were not undertaken.  Preliminary ultracentrifugation  studies by Duff et a l . on the  third alcohol fraction indicated that i t was not homogeneous. D.  Immunization of rabbits with Type E toxoids. During the preliminary experiments on toxin activation and purification,  an experiment was undertaken to study the immunization of rabbits with a number of Type E toxoids which had been prepared up to that time.  Information  obtained from this experiment regarding toxoiding-time, animal response and duration of immunization aided considerably i n the preparation of toxoids for human immunization.  -601.  Toxoids, Four toxoids were prepared by the formolization of different toxins.  The  toxoids, designated A, B, C and D, were prepared as shown below. Type A - derived from an activated toxin produced by culturing together, i n dialysis sacs, strains "VH" (T) and "Iwanai" (OT), preparation was 1-3 million mouse M.L.D, per ml.  The potency of this  Detoxification of the toxin  was effected by the addition of formalin to 0.4 per cent f i n a l concentration, and incubation at 3 0 ° C .  The time required for complete detoxification,  indicated by mice injections, was 25 days.  as  Complete loss i n potency was  indicated by the failure of five mice to develop symptoms of intoxication and loss i n weight over a period of 2 weeks after receiving 0.5 ml. of the toxoid intraperitoneally.  Toxoids were stored at 4 ° C .  Type B - derived from an activated toxin produced by the combination of s t e r i l e f i l t r a t e s of "VH" (T) and "Iwanai" (OT) cultures.  The f i n a l potency  of the preparation was 200,000 to 250,000 mouse M.L.D. per ml.  Detoxification  was effected by addition of formalin to 0,3 per cent and incubation at 3 0 ° C . Detoxification time was 20 days. Type C -  derived from a crude toxin produced by the cultivation of strain  "Iwanai" (OT) i n G . P . B . I . medium as outlined under the heading of "Toxin production".  Potency of the crude toxin was 2,500 - 3,000 mouse M.L.D. per ml.  The toxin was detoxified by the addition of formalin to a f i n a l concentration of 0.4 per cent and incubation at 3 0 ° C . for 19 days.  C r i t e r i a for complete  detoxification were the same as for Types A and B. Type D - derived from a non-toxic "VH" (T) culture as outlined under production of non-toxic f i l t r a t e s . course, not required.  Formolization of this f i l t r a t e was, of  -61S t e r i l i t y tests were conducted on a l l four toxoids before animal injections.  Five ml. of each toxoid were inoculated into tubes of G . P . B . l .  medium and tryptose-phosphate broth, and were incubated aerobically and anaerobically for two weeks at 3 7 ° C .  Macroscopic and microscopic obser-  vations of each tube showed the toxoids to be s t e r i l e . 2.  Immunization schedule. Each toxoid was injected intravenously into 2 rabbits, designated #1  and #2.  Animal bleeds and injections were performed i n accordance with  the schedule shown i n Table XV.  TABLE XV Rabbit immunization schedule.  A  B  Toxoids  #2  D  #2  #1  #2  1  bleed  bleed  bleed  bleed  bleed  bleed  bleed  bleed  1  0.25 ml.  0.25 ml.  0.25 ml.  0.25 ml.  0.25 ml.  0.25 ml.  0.25 ml.  0.25 ml.  4  0.75 "  0.75 "  0.75 «  0.75 "  0.75 "  0.75 "  0.75 "  0.75 "  10  1.00 "  1.00 "  1.00 "  1.00 '»  1.00 "  1.00 "  1.00 »  1.00 "  20  bleed  bleed  bleed  bleed  bleed  bleed  bleed  bleed  50  2.00 ml.  2.00 ml.  2.00 ml.  2.00 ml.  2.00 ml.  2.00 ml.  2.00 ml.  2.00 ml.  60  bleed  bleed  bleed  bleed*  bleed  bleed  bleed  bleed  90  II  120  ii  150 200 300  #1  #2  C  Day  »  * Death of animal  -  #1  »  #1  "  -633.  Results, Rabbit serum was titrated for antitoxin by toxin-neutralization tests  i n mice;  the t i t r e s designated are the average values of anti-mouse M.L.D.  per ml. of rabbit sera.  Table XVI and Diagram X i l l u s t r a t e the results  of these t i t r a t i o n s .  TABLE XVI Anti-mouse M.L.D. per ml. A B C  Day  (in 1,000's) D  1  1  1  1  20  0.02  0.2  0.15  0.005  60  1.15  15.0  10.0  0.15  90  1.0  15.0  10.0  0.10  120  0.75  7.5  5.0  0.02  150  0.4  4.0  2.0  0.02  200  0.4  4.0  2.0  0.02  300  0.15  2.0  2.0  0.015  0  Over the period of the f i r s t three injections, a l l 4 toxoids e l i c i t e d a very slow immune response.  The anemnestic response was well indicated by a  marked increase i n serum anti-M.L.D. following the administration of the booster injection.  The level of immunity then decreased slowly and could s t i l l be  demonstrated eight months after the f i n a l injection. Activated toxoid (B) produced the highest level of circulating antibody. Animals immunized with the crude toxoid (C) demonstrated the second highest titre.  Activated toxoid (A) did not stimulate antibody production comparable  to toxoids B or C.  SO  IOO TIME  Diagram X  Rabbit  150 in  200 da y s .  immunization.  250  300  -65A rather unexpected result was obtained with toxoid D, prepared from a non-toxic culture.  Both animals receiving this toxin developed a low but  definite immunity to crude Type E toxin. When the four types of antisera were titrated against their homologous toxins, i . e . Type A antisera titrated against the activated toxin employed for the production of toxoid A, they demonstrated ranges not unlike those already shown for the titrations against the crude toxin.  Although a l l  these values are not l i s t e d here, i t should be mentioned that the crude toxoid (C) and the activated toxoid (B) produced almost identical antitoxin t i t r e s when titrated against a l l three toxins.  These values are shown i n Table  XVII. TABLE XVII  Anti-mouse M.L.D. per ml. (in 1,000's) Toxin A Toxin B Toxin C Antiserum B  10  15  15*  Antiserum C  10  15  10*  ^Reported i n Table XVI  -66-  V.  Human Immunization.  Five toxoids were employed for the immunization of human volunteers against Clostridium botulinum Type E toxin. The high t i t r e s obtained i n rabbits with toxoids B and C i n the previous experiment, as. well as the development of a low immunity i n these animals when injected with a non-toxic f i l t r a t e (D), led to the use of these 3 toxoids i n the human immunization programme. A toxoid prepared from a purified toxin, and a second, prepared from a trypsin-activated toxin, were also used. 1.  Preparation of toxoids. Toxoid #1 was" derived from an activated toxin produced by combining  sterile f i l t r a t e s of "Iwanai" (OT) and "VH" (T) and allowing the mixture to incubate 30 hours at 3 0 ° C .  The potency of the activated product was 200,000  to 250,000 mouse M.L.D. per ml.  One hundred per cent formalin*was added to  the toxin to a final concentration of 0.3 per cent.  Complete detoxification  required 23 days at 3 0 ° C . and was demonstrated by the failure of five mice to develop botulinum intoxication symptoms, or loss i n weight over a period of two weeks. Toxoid #2 was derived from a trypsin-activated "Iwanai" (OT) toxin to which had been added soya-inhibitor to prevent a rapid loss i n t o x i c i t y . The conditions required for optimum toxin activation were determined i n the preliminary experiments and applied to the production of the toxin for the preparation of this toxoid.  The potency of the activated product was 50  million to 100 million mouse M.L.D. per ml. *37.4 per cent HC00H  Detoxification was brought about  -67by the addition of formalin to a f i n a l concentration of 0.3 per cent.  Time  required for complete detoxification, as indicated i n mice, was only six days. Toxoid #3 was derived from a purified "Iwanai" (OT) toxin.  For the  preparation of this toxoid, the third alcohol-fraction, made up i n 0.2 M succinate buffer at pH 5.5, of the alcohol precipitation purification method was employed.  This toxin suspension had a potency of 30,000 to 40,000 mouse  M.L.D. per m l . , or 350,000 to 400,000 mouse M.L.D. per mg. of nitrogen.  De-  toxification was effected by the addition of formalin to a f i n a l concentration of 0.3 per cent.  Detoxification time was four days, again surprisingly short.  Toxoid #4 was derived from a crude "Iwanai" (OT) toxin f i l t r a t e . of this toxin was 2,000 to 3,000 mouse M.L.D. per ml.  Potency  Detoxification was  effected by the addition of formalin to a f i n a l concentration of 0.3 per cent.  Detoxification time for this crude f i l t r a t e was 23 days at 3 0 ° C . Toxoid #5 was derived from a non-toxic "VH" (T) f i l t r a t e .  Formolization  of this f i l t r a t e was not required. The five toxoids were dispensed i n s t e r i l e 100 ml. serum bottles and stored at 4 ° C . 2.  S t e r i l i t y tests. Three ml. of each toxoid were inoculated into each of 6 tubes of G . P . B . I ,  medium and 6 tubes of tryptose-phosphate medium.  Three tubes of each medium  were then incubated aerobically and three anaerobically, and observed for contamination every second day for a period of two weeks. S t e r i l i t y checks on the toxoids were also conducted i n guinea pigs.  -68Five ml. of each toxoid were injected intraperitoneally into each of two guinea pigs, and the animals observed for a period of three weeks.  None  of the animals showed symptoms of botulinum intoxication or loss i n weight during the three-week period. 3.  Immunization schedule. Twenty volunteers were available for the testing of the five toxoids.  These were divided into 5 groups (A, B, C, D and E) each group containing four subjects. Group A received toxoid #1 Group B received toxoid #2 Group C received toxoid §3 Group D received toxoid #4 Group E received toxoid #5 Toxoids were injected into the deltoid muscle and blood removed from a ; vein of the arm.  The immunization programme is contained i n Table XVIII.  TABLE XVIII Day 0  10 ml. of blood removed from each volunteer.  "  0  0.1 ml. toxoid injected.  "  7  0.2 ml. toxoid injected.  » 21  0.5 ml. toxoid injected.  " 35 » 115 " 125  10 ml. blood removed from each volunteer. 1.0 ml. toxoid injected. 10 ml. blood removed from each volunteer.  -694,  Results. The level of circulating antibody was measured by toxin-neutralization  tests i n mice, the t i t r e being expressed i n anti-mouse M.L.D. per ml. of serum. Blood titrations of the volunteers prior to injection of toxoid indicated the absence of circulating antibody against Type E toxin i n a l l subjects. The results of the human immunization programme are contained i n Table  XIX.  -70-  TABLE XIX Anti-mouse M.L.D. per ml. of serum Day 35 Day 125  Group  Subject  A  1  6-10  300 - 350  2  6  150 - 200  3  6-10  350 - 450  4  10 - 15  350 - 400  1  6  50-75  2  6  40-50  3  6  40 - 50  4  6  _*  1  6-10  50 - 75  2  6-10  30 - 40  3  10-15  30 - 40  4  6-10  40 - 50  1  20 - 30  _#  2  30-40  1,000 - 2,000  3  30-40  2,000 - 3,000  4  15 - 20  1  6  6-10  2  6  10 - 15  3  6  6-10  4  6  15 - 20  B  C  D  E  800 -  1,000  * Volunteers no longer available. Group D, receiving toxoid #4, prepared from crude "Iwanai" (OT) toxin, demonstrated the highest level of antibody production. Group A, receiving the toxoid prepared from combined f i l t r a t e s of toxic and proteolytic variants, displayed the next best antibody l e v e l .  -71Groups B and C, receiving the toxoids prepared from trypsin-activated and purified toxins respectively, f e l l far below Groups A and D i n antibody levels. Group E , receiving the non-toxic f i l t r a t e , i t y to Type E toxin.  developed very l i t t l e immun-  However, the values i n themselves are significant and  w i l l be discussed l a t e r . Time did not permit further observations concerning the duration and rate of decline of immunity i n the five groups.  -72V I . Discussion. Clostridium botulinum Type E toxin, produced by the cultivation of a crude (OT) variant, is a stable toxin of relatively low potency.  Preliminary  experiments would suggest that the toxin is either released from the c e l l s on autolysis,  or the toxin i s excreted outside the cells during l i f e i n a protoxin  form, the protoxin then becoming active by the action of the intracellular enzymes released on autolysis.  This latter explanation has been forwarded  by Boroff (57) after he noticed that the addition of a 10 day culture to a young culture resulted i n an immediate increase i n t o x i c i t y . ular autolysis,  Maximum c e l l -  and consequently toxin production, is dependent both on the  incubation period and the hydrogen-ion concentration; not normally attained u n t i l 5 - 7  this pH (5,2 - 5.5)  is  days of incubation at 3 2 ° C , long after the  culture i s past the logarithmic growth phase for this organism  (58).  The characteristic rise i n toxin potency which occurs when the culture is refrigerated for 24 hours following incubation may be the result of (a) a "shock" to the whole cells i n the culture which have not yet lysed, causing their dissolution, or (b) a folding or s p l i t t i n g of toxin molecules alreadypresent, thus liberating more of the toxic groupings  (57).  Activated toxins are r e l a t i v e l y unstable and of extremely high potency. As with the crude toxin, incubation time, temperature and pH are seen to be very c r i t i c a l . Several similarities are apparent when toxin i s activated by the following methods:(i)  the combination of s t e r i l e f i l t r a t e s of toxic and proteolytic variants,  (ii)  the growth of toxic and proteolytic variants i n mixed culture,  ( i i i ) the addition of trypsin. In method ( i i i ) activation of toxin is extremely rapid and the a c t i vated product very unstable, rapidly declining i n potency after attaining maximum a c t i v i t y .  In contrast to t h i s , activation by methods (i) and ( i i )  is r e l a t i v e l y slow and the activated product is r e l a t i v e l y stable. It should be mentioned here, however, that activation w i l l continue after the toxin produced by the growth of mixed cultures has apparently reached i t s maximum a c t i v i t y , and has been stored at refrigeration temperatures.  This slow activation continues u n t i l a maximum is reached and then,  as with the trypsin-activated toxin, a slow destruction of toxin follows. entire process may require a number of years.  The  This has been demonstrated by  the periodical titrations of stored toxin activated by this method. It has also been shown that stored toxin which had been activated i n this manner and had not attained maximum potency, could be rapidly activated by the addition of trypsin with the consequent rapid decline i n potency after maximum a c t i v i t y was attained. The different processes of activation, then, would seem to be basically the same, differing only i n rate of reaction and possibly the height of maximum potency.  In this process, purified trypsin i s seen to act faster than  crude trypsin, which i n turn acts faster than a proteolytic  filtrate.  The experiments on activation of toxin by trypsin indicated a definite rise i n pH during the period of toxin activation.  Since trypsin acts  specifically to break peptide bonds adjacent to either of the two basic amino  -74acids, arginine or lysine, i t is suggested that this activating process may be:(a)  a result of the removal of contaminating protein from the surface of the toxin molecule, thus revealing an increased number of toxic endings,  (b)  or  a cleavage of the toxin molecule by the breakage of the specific peptide bonds adjacent to arginine and lysine residues, thus producing a large number of smaller toxic residues, and at the same time an increased number of basic amino acid endings, resulting in an increase i n pH.  The ionization of acidic amino acid  endings, which would also have been produced would be suppressed by the acid pH of the f i l t r a t e . Should this latter concept of the cleavage of the toxin molecule be the cause of activation, one would expect a decrease i n weight of the toxin molecule on activation. Type E toxin as yet.  There is no information of this type available for However, Wagman and Bateman (78), studying the  absorption of Type A botulinum toxin, reported a decrease i n the molecular weight of Type A toxin from 900,000 to 60,000 after this toxin had been adjusted to a pH of 7.5 with trypsin, simulating the conditions of the upper small intestine. The experiments i n animal and human immunization indicated that a c t i vated toxin had l i t t l e , i f any, advantage over crude toxin i n the preparation of toxoids.  If a comparison is made of the crude and activated toxins, on  the basis of which one w i l l produce the best toxoid, the following points are noted.  (1) Crude botulinum toxin can be prepared with l i t t l e d i f f i c u l t y , and providing the same strains and medium are employed each time, cultures of uniform potency are obtained.  Activated toxin, on the  other hand, requires r i g i d control of such conditions as pH, temperature and duration of incubation, for i t s production. (2) The crude toxin i s very stable, undergoing no detectable change i n potency for over two years when stored at refrigeration temperatures. Activated toxin is not stable and even at refrigeration temperatures w i l l fluctuate i n potency, regardless of the means of activation.  The  amount of variation i n potency i s seen to depend on the method employed for the production of the activated toxin.  Each time this  activated  toxin is employed for the t i t r a t i o n of an immune serum, i t must be f i r s t tested for potency.  In the case of highly activated toxins this test  for potency should be conducted at the same time as the t i t r a t i o n of the serum. The addition of trypsin-inhibitor to these activated toxins moderates the potency decrease to a great extent;  nevertheless the  detrimental action w i l l s t i l l occur. (3) F i n a l l y , toxoids prepared from crude toxins were seen to produce a higher immunity i n man than those prepared from activated toxins. This may have been brought about by the presence of extraneous protein surrounding the toxin molecule similar i n antigenic structure to the toxin molecule per se.  Supporting evidence, concerning the property  of a toxoid prepared from a low-potency toxin producing a higher a n t i genic response i n animals than a toxoid prepared from a high potency toxin, is given by the work of Nigg et a l . (91).  These workers noted  -76that the antigenicity of Types A and B toxoids were not proportional to the t o x i c i t y of the culture from which the toxoids were prepared. That i s , antigenicity does not necessarily increase with potency. Higher antibody levels than were obtained were expected for the toxoids prepared from highly activated and purified toxins.  In view of the short  periods of detoxification with 0.3 per cent formalin shown for these toxins, i t might be suggested that the concentration of formalin employed was too high.  Since these toxins would be i n a more purified, and consequently more  vulnerable state than the crude toxin, the formalin may have acted to cause considerable antigenic destruction of the toxin molecule.  It i s therefore  suggested that lower concentrations of formalin should be employed for the detoxification of such activated and purified products. The production of antibodies against Type E botulinum toxin by the injection into animals and humans of a non-toxic f i l t r a t e , produced from the atoxic "VH" (T) variant, would suggest a close similarity i n molecular structure between the toxin of the (OT) f i l t r a t e , and the active substance i n the proteolytic f i l t r a t e (T).  It may be argued that the (T) f i l t r a t e  actually contains a very minute and undetectable concentration of toxin which would account for the production of antibody.  However, should this be the  case, one would expect that this low concentration of toxin would be detectable i n mice upon trypsin-activation of the (T) f i l t r a t e .  This, i n fact, was  attempted but s t i l l failed to demonstrate the presence of one mouse M.L.D. Immunization of animals with a non-toxic variant was also demonstrated by Boroff and Cabeen (92) with Clostridium botulinum Type C .  -77VII 1.  SUMMARY.  Crude Type E toxin prepared from a pure toxic variant (OT) is very stable and of relatively low potency.  2.  The activation of Type E toxin is enzymatic i n nature, and is  therefore  dependent on temperature, pH, enzyme concentration, and the condition of the substrate (toxin) per se. 3.  Activation of Type E toxin may be accomplished by one of three methods:(a)  Culturing together proteolytic and toxic variants of "VH" (T) and "Iwanai" (OT) respectively.  (b)  By combining s t e r i l e f i l t r a t e s of proteolytic (T) and toxic (OT) variants followed by incubation at 3 2 ° C .  (c)  The addition of crude and purified trypsin to a toxic  filtrate,  followed by the required incubation period. 4.  Trypsin activation of toxin and destruction of activated toxin was shown to be arrested by the addition of purified soya-inhibitor.  5.  Type E toxin was p a r t i a l l y purified by two methods, one involving the precipitation of the toxin by concentrated salt solutions, and the other involving toxin precipitation by ethyl alcohol.  The latter  method was more easily executed and yielded a purer product, as shown by nitrogen determinations on both products. 6.  Type E toxoids, suitable for human and animal immunization, were produced from crude, activated and purified toxins.  7.  Formolized  toxoid, produced from a crude toxic f i l t r a t e , was seen to  produce the highest level of circulating antibody i n man.  -788.  A low but definite immunity was also produced i n human volunteers and rabbits by the injection of f i l t r a t e from a non-toxic proteolytic Type £ variant, indicating a similar antigenic relationship and structure between the toxic and proteolytic molecules.  -79VIII.BIBLIOGRAPHY 1.  Van Heyningen, W . E . , "Bacterial Toxins", Blackwell Scientific Publications, Oxford, 1950, p,14.  2.  F i l d e s , P . , "The tryptophane and 'Sporogenes vitamin 1 requirements of B. botulinus", B r i t . J . Exper. Path., 1935, 16, 309.  3.  F i l d e s , P . , and Knight, B . C . , "Tryptophane and the growth of bacteria", B r i t . J . Exper. Path., 1933, 14, 343.  4.  Pappenheimer, A.W., "The nature of the •Sporogenes vitamin'", J.Biochem., 1935, 29, 2057.  5.  Stichland, L . H . , "Studies i n the metabolism of s t r i c t anaerobes", J . Biochem., 1934-35, 28, 1746.  6.  Gale, E . F . , "Enzymes concerned i n the primary u t i l i z a t i o n of amino acids by bacteria", Bacteriol. Rev., 1940, 4, 135.  7.  C l i f t o n , C . E . , "The u t i l i z a t i o n of amino acids by C I . t e t a n i " , J . B a c t e r i o l . , 1942, 44, 179.  8.  Guggenheim, K . , "Investigations on the dehydrogenating properties of certain pathogenic obligate anaerobes", J . B a c t e r i o l . , 1944, 46, 313.  9.  Kerrin, J . C , "Studies on the haemolysin produced by atoxic strains of B. tetani". B r i t . J . Exper. Path., 1930, 11, 153.  10.  Reed, L . J . , Orr, P . F . , and Brown, H.J.,, "Fibrinolysins from gas gangrene anaerobes", J . B a c t e r i o l . , 1943, 46, 475.  11.  Easton, E . J . , and Meyer, K . F . , "Occurrence of Bacillus botulinus i n human and animal excreta", J . Inf. D i s . , 1924, 35, 207.  12.  Meyer, K . F . , and Dubovsky, B . J . , "The distribution of spores of Bacillus botulinus i n the Territory of Alaska and the Dominion of Canada", J . Inf. D i s . , 1922, 31, 595.  13.  Meyer, K . F . , and Dubovsky, B . J . , "The occurrence of spores of Bacillus botulinus i n Belgium, Denmark, England, the Netherlands and Switzerland", J . Inf. D i s . , 1922, 31, 600.  14.  Meyer, K . F . , and Dubovsky, B . J . , "The occurrence of spores of B. botulinus i n the Hawiian Islands and i n China", J . Inf. D i s . , 1922, 31, 610.  15.  Meyer, K . 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P . , and Lamanna, C , "Factors affecting the botulinal haemagglutination reaction and the relationship between heamagglutinating a c t i v i t y and t o x i c i t y of toxin preparations", Amer. J . Hyg., 1951, 54, 342.  72.  Guillaumie, M . , and Kreuguer, A . , "Nouvelles recherches sur les hemolysines oxydables", Ann. Inst. Pasteur, 1950 , 78 , 467.  73.  Owen, C.R., Langohr, J . L . , and Blakely, E . , "Adaptions of the Macfarlane l e c i t h o - v i t e l l i n test", J . Path. & Bact., 1947, 59, 261.  -84-  74.  Lamanna, C , Eklund, H.W,, and McElroy, O . E . , "Botulinum toxin (Type A ) ; including a study of shaking with chloroform as a step i n the isolation procedure", J . B a c t e r i o l . , 1946, 52, 1,  75.  Lamanna, C , McElroy, O . E . , and Eklund, H.W., "The purification and crystallization of C l . botulinum Type A toxin", Science, 1946, 103, 613.  76.  Lamanna, C , Buehler, H . J . , and Schantz, E . J . , "The elemental and amino acid composition of crystalline C l . botulinum Type A toxin", J . B i o l . Chem., 1947, 169. 295.  77.  Abrams, A . , Kegeles, G . , and Hottle, G . A . , "The purification of toxin from C l . botulinum Type A " , J . B i o l . Chem., 1946, 164, 63.  78.  Wagman, J . , and Batman, R., "Isolation and sedimentation study of the low molecular weight forms of Type A botulinus toxin", Arch. Biochem., 1954, 50, 104.  79.  Sterne, M . , and Wentzel, L . M . , "A new method for the large scale production of high t i t r e botulinum formol toxoid Types C and D", J . Immunol., 1950, 65, 175.  80.  Wentzel, L . M . , Sterne, M . , and Poison, A . , "High t o x i c i t y of pure botulinum Type D toxin", Nature, 1950, 166, 739.  81.  Dolman, C . E . , and Chang, H . , "The epidemiology and pathogenesis of Type E and fish-borne botulism", Canad. J . Pub. H l t h . , 1953, 44, 231.  82.  Dolman, C . E . , Darby, G . E . , and Lane, R . P . , "Type E botulism due to salmon eggs", Canad. J . Pub. H l t h . , 1955, 46, 135.  83*  Nakamura, Y . , Iida, H . , Soeki, K . , Kanzawa, K . , and Karashimada, T . , Jap. J . Med. S c i . & B i o l . , 1956, 9, 45.  84.  Prevot, A . R . , Huet, M . , B u l l . Acad. Nat. de Med., 1951, Nos. 25 and 26, 432.  85.  Pedersen, H . O . , "On Type E botulism", J , App. Bact., J.955, 18, 619.  86.  Starin, W.A., and Dach, G.M., "Complement fixation studies on C l . botulinum", J . Inf. D i s . , 1924, 34, 137.  87.  Graham, R., and Thorp, F . J r . , "The effect of formalin on botulinum toxins A, B and C " , J . Immunol., 1929, 16, 391.  88.  Rice, C . E . , P a l l i s t e r , E . F . , Smith, L . C , and Reed, G . B . , "Clostridium botulinum Type A toxoids", Canad. J . Res., 1946-47, E 24-25, 167.  -85-  89.  Rice, C . E . , "Clostridium botulinum Type B toxoids", Canad. J . 1946-47, E 24-25, 175.  Res.,  90.  Rice, C . E . , "A preliminary study of the antigenic a c t i v i t y of mixtures of CI. botulinum toxoid Types A and B", Canad. J . Res., E 24-25, 181.  91.  Nigg, C , Hottle, G . A . , C o r i e l l , L . L . , Rosenvrald, A . S . , and Beveridge, G.W., "Studies on botulinum toxoid, Types A and B", J . Immunol., 1947, 55, 245.  92.  Boroff, D . A . , and Cabeen, H . E . , "Studies of toxins of CI. botulinum. II. Immunization of rabbits with an atoxic variant of CI. botulinum Type C", J . B a c t e r i o l . , 1954, 67, 195.  93.  Baron, L . , and Reed, G . B . , "Clostridium botulinum Type E toxin and toxoid", Canad. J . M i c r o b i o l . , 1954-55, 1,, 108.  94.  Gordon, M . , Froch, M . A . , larinsky, A . , and Duff, J . T . , "Studies on immunity to toxins of C I . botulinum. III. Preparation, purification and detoxification of Type E toxin", J . B a c t e r i o l . , 1957, 74, 533.  95.  Batty, I . , and Glenny, A . T . , "The antigenic efficiency of CI. welchii epsilon toxin and toxoid after treatment with trypsin", B r i t . J . Exper. Path., 1948, 29, 141.  96.  Velicanov, I., "Immunization experimentale de Phomme centre le botulisme", Gironale de Batteriologia, 1936, e Imm., 17, 451.  97.  Reames, H.R., Kadull, F . J . , Housewright, R . D . , and Wilson, J . B . , "Studies on botulinum toxoids, Types A and B", J . Immunol., 1947, 55, 309.  98.  Schoenholz, P . , "Surface colony types of CI. botulinum on blood agar", J . Inf. D i s . , 1928, 42, 301.  99.  Gunnison, J . B . , and Meyer, K . P . , "The occurrence of non-toxic strains of CI. parabotulinum", J . Inf. D i s . , 1929, 45, 79.  100.  Turner, A.W., and Rodwell, A.W., "The epsilon toxin of CI. welchii Type D", Austral. J . Exper. B i o l . Med. S c i . , 1943, 21, 17.  101.  Townsend, C . T . , "Comparative study of non-toxic and toxic strains of CI. parabotulinum", J . Inf. D i s . , 1929, 45, 87.  102.  Duff, J . T . , Wright, G . G . , and Tarinsky, A . , "Activation of CI. botulinum Type E toxin by trypsin", J . B a c t e r i o l . , 1956, 72, 455.  103.  Milton, G . , Fiock, M . A . , Tarinsky, A . , and Duff, J . T . , "Studies on immunity to toxins of Clostridium botulinum. III. Preparation, purification and detoxification of Type E t o x i n , " J . B a c t e r i d . , 1957, 74, 533.  -86-  104.  Hawk, P . B . , Oser, B . L . , and Summerson, W.H., "Practical Physiological Chemistry", Blakiston Company Inc., New Tork, 13th E d . , 1954, p.545  -87IX.  Appendices  Appendix A. Two strains of Clostridium botulinum Type E were employed. Por the production of proteolytic f i l t r a t e s the (T) or non-toxic proteol y t i c variant of the '•Vancouver Herring" ("VH") strain was used.  Type E toxin  was produced by the cultivation of the toxic, non-proteolytic variant (OT) of the "Iwanai" s t r a i n . The terms " T " and "OT", referring to the non-gas producing, proteolytic, non-toxic variant and the toxic, non-proteolytic, gas-producing variant respect i v e l y , have frequently been employed throughout this manuscript.  As mentioned  i n the "Historical Review" this designation of variants was f i r s t employed by Dr. C . E . Dolman, and i n a recent publication (51) the colonial morphology and biochemical characteristics of these variants are described.  In his publication,  however, the author altered the terms " T " , "OT" and "0" to the more descriptive designation "TP", "TOX" and "OS" respectively.  To demonstrate these character-  i s t i c s more clearly, a picture of these two variants growing medium is given (Plate I ) .  in G.P.B.I.  Examination of tubes 1 and 3 i n this picture  clearly differentiate the gas-producing, non-proteolytic, toxic variant (OT) i n tube 1, from the non-gas producing, proteolytic, non-toxic variant (T) i n tube 3.  88  I  2  3  PLATE  II  Tube I  Non-proteolytic, gas-producing,  Tube 2  Control  Tube 3  P r o t e o l y t i e , n o n - g a s - p r o d u c i n g , non-toxic  toxic  v a r i a n t (OTl  variant(T).  -89Appendix B, 1.  Media employed. A l l the following media were s t e r i l i z e d by autoclaving at 15 pounds  pressure, the time being dependent on the volume.  Glass tubes containing  75 ml. of medium and Erlenmeyer flasks containing 4 l i t r e s of medium were sterilized for 20 minutes and 45 minutes respectively.  Medium which was  not used on the day i t was prepared was boiled 5 minutes and cooled before inoculation. (a)  Glucose peptone beef infusion medium, with added meat (G.P.B,!.) (Ref,  James Morton, B.A. Thesis, 1944). Infuse one pound of fat-free beef (minced) i n one l i t r e of tap-  water overnight i n the refrigerator.  Infuse at 6 5 ° C . for 45 minutes.  Strain f i r s t through one thickness and then two thicknesses of cheesecloth.  F i l t e r through #1 Whatman paper.  Take the volume and add  the following:NaCl  0.5 per cent.  Difco peptone  1.0 per cent.  Na2HP04.12H20  0.2 per cent.  Boil for 3 minutes and f i l t e r through #1 Whatman f i l t e r paper.  Adjust  to pH 7.8. Dispense i n large tubes with about one inch of meat i n the bottom and approximately 25 ml. of broth. glucose solution is prepared.  Sterilize.  A sterile 50 per cent  This is added to the tubes aseptically,  before inoculating to give a f i n a l concentration of 2.0 per cent.  -90(b)  Brain heart infusion broth (Difco). Dissolve 37 grams of Bacto-Heart Infusion and 10 grams of sodium thioglycollate i n 1,000 ml. of d i s t i l l e d water. sterilize.  The pH is 7.4.  Dispense and  F i f t y per cent s t e r i l e glucose is added  before inoculation to a f i n a l concentration of 2.0 per cent. (c)  Brain heart infusion agar. Preparation is the same as (b) with the addition of 1.5 per cent Bacto-Agar.  (d)  Yeast extract broth (Difco). (Ref. Duff et a l . J . B a c t e r i o l . , 1957, 72,  455).  Dissolve 20 grams of Difco Yeast Extract and 20 grams of BactoPeptone i n 1,000 ml. of d i s t i l l e d water. The pH is 7.2.  Dispense and s t e r i l i z e .  F i f t y per cent s t e r i l e glucose solution is added  before inoculation to a f i n a l concentration of 2.0 per cent. (e)  Brewer's thioglycollate anaerobic broth. Dissolve 40.5 grams of Brewer's medium i n 1,000 ml. of d i s t i l l e d water.  2.  S t e r i l i z e and dispense.  The pH i s  7.2.  Activation of toxin with crude trypsin. Add trypsin (Difco 1:200) to the toxin f i l t r a t e to a f i n a l concen-  tration of one per cent weight to volume, and swirl the flasks gently u n t i l a l l the trypsin is 3.  dissolved.  Activation of toxin with purified trypsin. (Ref. Duff et al.. J . B a c t e r i o l . , 1957, 72,  453.)  Dissolve 0.005 gms. of Crystalline Trypsin (Difco) i n 25 ml. of  -91d i s t i l l e d water, giving a f i n a l concentration of 0.02 per cent. to pH 6.0 with N/200 NaOH.  Adjust  For activation of toxin mix equal volumes  of toxic f i l t r a t e and trypsin solution. 4.  Purified soya inhibitor solution. (Ref. Duff et a l .  J . B a c t e r i o l . , 1957, 72,  453.)  Dissolve 0.005 grams of purified Difco Soya—Inhibitor i n 25 ml. of a 0.0025 M HC1 solution giving a f i n a l concentration of 0.02 per cent. to pH 6.0 with N/200 NaOH.  Adjust  For inhibition of the purified trypsin, add an  equal volume of the purified soya inhibitor solution. 5.  Activation of toxin with crude pepsin. Add Pepsin (Difco 1:250) to the toxin f i l t r a t e to a f i n a l concentration  of 1 per cent weight to volume and swirl gently u n t i l a l l the pepsin is dissolved.  -92Appendix C. The dialysis apparatus for the preparation of toxin i s assembled as shown i n Plate II and Diagram XI.  The apparatus is s t e r i l i z e d by auto-  claving for 2 hours at 15 pounds pressure.  It i s then cooled and assembled  i n the presence of an ultra-violet lamp, to reduce the chance of possible contamination, i n an incubator at 3 2 ° C . Flask (A) (see Diagram XI) contains 6 l i t r e s of G . P . B . I . medium and flask (B) 1,500 ml. of 0.85 per cent NaCl solution.  After assembly,  as  shown i n the diagram, clamps #3 and #4 are closed and clamp #1 i s opened. Media i n flask (A) i s placed under negative pressure, with a suction pump attached to the air tube of flask (C) u n t i l the media begins to flow into the discard flask.  At this time clamp #1 is closed allowing the media to  flow into the dialysis tube $3) surrounding the dialysis sac (D). When the dialysis tube (E) i s approximately four-fifths f i l l e d , clamp #2 i s closed.  Clamp #4 is now opened, allowing the saline to flow into  the dialysis sac (D) u n t i l the volume of saline is at an equal level to the media i n the surrounding dialysis tube.  Clamp #4 i s then closed.  The apparatus is then allowed to remain 24 hours as a s t e r i l i t y check. If, at the end of this period, there are no v i s i b l e signs of contamination the apparatus is inoculated through the i n l e t tube. The medium is removed every 24 hours by opening clamp #1 and allowing the expended medium to run into the discard flask (C).  Clamp #1 i s then  closed and clamp #2 opened, r e f i l l i n g the dialysis tube. This procedure is repeated every 24 hours for the entire incubation period.  Samples of toxin may be removed by opening clamp #3 at the base  of the apparatus and collecting the toxin i n a s t e r i l e tube. At the end of the incubation period the toxin culture is removed byopening clamp #3.  The suspension is refrigerated for 24 hours and then  c l a r i f i e d and s t e r i l i z e d by f i l t r a t i o n .  —94—  P L A T E IX  Diolysis  apparatus.  — 95  Diagram S I  Dialysis  apparatus  —  for  toxin  production.  *  (* Vinst, G . , and Frodetto, V . , "Apparatus for the culture of bacteria- i n c e l l o p h a n e t u b e s " , Science, 1 9 9 1 , 1 1 4 . 5 4 9 . )  

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