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Studies on spore resistance and toxigenic characteristics of Clostridium botulinum, type E Munro, Elma Joan 1953

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STUDIES ON SPORE RESISTANCE AND TOXIGENIC CHARACTERISTICS OF CLOSTRIDIUM BOTULINUM, TYPE E by ELMA JOAN MUNRO A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of BACTERIOLOGY AND IMMUNOLOGY We accept t h i s thesis as conforming to the standard required from candidates for the degree of MASTER OF SCIENCE Members of the Department of Bacteriology and Immunology. THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1953 ABSTRACT A b r i e f h i s t o r i c a l review of Type E botulism i s presented. Emphasis i s placed on the role of f i s h i n the epidemiology. Experimental d e t a i l s are given of tests carried out on the spore resistance of these Type E strains i n comparison with the other types of CI. botulinum. The data obtained indicate that they are more thermolabile than any of the other types, e s p e c i a l l y A or B. In addition, evidence i s presented which shows that the Type E strains seem to be d i v i s i b l e into two groups on the basis of t h e i r spore s t a b i l i t y . The importance of t h i s thermolability i s stressed i n regard to i s o l a t i o n s of the seemingly rare Type E from suspected foodstuffs or i n routine surveys. Details are given also of experiments conducted on the Type E toxins. The effect of dextrose, certain peptones, and colony type on toxin production i s discussed. Some experiments on the storage properties of the toxins are pres-ented. Active immunization experiments on mice indicate that Type E toxoids are poor antigens. In only two groups did the mice exhibit demonstrable immunity. Even i n these groups the l e v e l of immunity was exceedingly v a r i a b l e . By contrast, a Type A toxoid proved a good antigen, protecting mice to a uniformly high degree against the homologous t o x i n . E f f o r t s to increase the an t i g e n i c i t y of Type E toxoids are discussed. In v i t r o cross-neutralization tests with four Type E toxins and t h e i r antitoxins are described. On the basis of these i n vivo and i n v i t r o t e s t s , i t i s concluded that Type E toxins are not homogeneous: a conclusion sup-ported by the evidence that some Type E toxins contain a chicken-lethal factor, while others do not. This evidence i s discussed i n r e l a t i o n to the problem of human immuniza-t i o n . ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. C. E. Dolman for his continuous encouragement and guidance throughout the course of t h i s research. I should also l i k e to thank the many other people connected with,this department, esp e c i a l l y Miss Helen Chang, for t h e i r i n t erest and f r i e n d l y help and advice. TABLE OF CONTENTS Page I. Introduction • • . . . . . . . . . . . . . . . 1 II* Historical review . . . 3 H i e Source of-cultures 10 TV. Experimental Studies . . . . . . 11 A. Thermal stability of spores . . . . . . . .11 1* Standardization of inoculum . 11 2. Procedures for determining the thermal resistance of the spores . . . . . . . . . . . 13 3. Results . . . . . . . . . . . . . . . . . . . 15 4. Discussion . 21 B. Toxigenic Studies . . . . . . . . . . . 26; 1. Toxin production 26 2„ Toxin stability . . . . . . . . . . 34 3. Active immunization of mice . . . . . . . . . . 35 4. Gross-neutralization tests • • 43 5. Discussion . . . . . . . . . 50 BIBLIOGRAPHY . . . . . . . . . 52 APPENDIX I - Media . . . . . . . . . . . . 54 APPENDIX II - Methods of anaerobiosis . . . . . . . . . . . 57 - i i -LIST OF TABIES Page I. Known isolations of Clostridium botulinum, Type E . . . . . 9 II. Thermal stability of Cl> botulinum spores ^ methods a & b) . 16 III. Thermal st a b i l i t y of CI. botulinum spores (method c) . . . 17 IV. Thermal stability of CI. botulinum spores (method d) . . . 18 V. Thermal stability of CI. botulinum spores (method e) . . . 19 VI. The effect of dextrose on the toxin production of Type E strains of CI. botulinum 28 VII. Titres of'five-day toxins of E74 and P34 on different occasions30 VIII. Time required for detoxification of various tpxoidsfwith formalin . . . . . 36 IX. Immunity produced in mice by Nanaimo chicken and Beluga toxins . . . . . . . . . . . . . . . . . . . . 41 X. Results of type E toxin-antitoxin cross-neutralization tests 44 XI. Number of ID/50's of toxin neutralized by various antitoxins 48 XII. Comparative neutralizing capacities of various antitoxins 48 - i i i -LIST OF ILLUSTRATIONS Page Figure 1* Suryey of thermal s t a b i l i t y of CI. botulinum spores, from experimental results . . . . . . . . . . 20 Figure 2. The effect of dextrose on the toxin production of Type E strains of CI» botulinum • 29 Figure 3. Decline i n toxigenic capacity of Type E strains . . . 30(a) - 1 -I . INTRODUCTION Although botulism i s a r e l a t i v e l y rare disease, i t s incidence i s apparently not de c l i n i n g . Apart from i t s c l a s s i c a l association with food i n t o x i c a t i o n , several i n c i -dents of botulism due to wounds have recently been reported. Clostridium botulinum as a po t e n t i a l weapon i n bacteriolog-i c a l warfare i s a subject that has recently received much notice. Since botulism i s almost always f a t a l , more ade-quate methods of prophylaxis and treatment must be found. Man has been immunized against types A and B (1) of CI.  botulinum but l i t t l e work has been done with type E. One of the major problems undertaken i n t h i s research was to determine whether the type E toxins are homogeneous or heter-ogeneous. This i s , of course, an important consideration i n developing a preparation capable of protecting humans against a l l type E toxins. The spore resistance of types A and B of CI. botulinum has been given a great deal of attention; type E has received p r a c t i c a l l y none. Numerous experiments were therefore per-formed to elucidate t h i s question of spore resistance. Provided that type E spores were less heat resistant than types A and B spores, the precautions normally taken i n can-ning processes to ensure destruction of the l a t t e r types w i l l also destroy the former. On the other hand, i f type E.spores were especially heat-labile, customary methods of isolating Gl. botulinum from suspected materials f i r s t treated by heat might lead to type E strains being overlooked. Thus, while type E strains appear to be rare they are probably widely distributed. I I . HISTORICAL REVIEW In the early nineteenth century, the term botulism (Latin botulus, a sausage) was applied i n Germany;to a complex of neuroparalytic symptoms supposed to be caused by the con-sumption of spoiled sausages. Van Ermengem ( 2 ) , i n 1896, isolated the causative organisms from a ham incriminated i n an outbreak of botulism i n E l l e z e l l e s , Belgium. He called the organism B a c i l l u s botulinum and showed that i t produced a toxin which gave r i s e to the same symptoms i n cats as had the o r i g i n a l ham toxin. Since the experimental work reported here i s mostly concerned with CI. botulinum, Type E, a review of the outbreaks and i s o l a t i o n of t h i s type i s i n order. The f i r s t report of Type E i n the l i t e r a t u r e came from Gunnison, Cummings, and Meyer ( 3 ) , i n 1936. They described the morphology, biochem-i c a l reactions, thermal resistance of the spores, and the toxin production, of two cultures sent by Dr. L. Bier of the Bacteriologic I n s t i t u t e at Dniepropetrowsk, i n the Russian Ukraine. These workers showed, by t o x i n - a n t i t o x i n tests made by i n j e c t i o n of guinea-pigs, that a n t i t o x i n of Types A, B, C, and D i n doses adequate to protect against 250 to 170,000 minimum l e t h a l doses (m.l.d.) of homologous toxin f a i l e d to neutralize 2 to 5 . m.l.d.'s of t h i s toxin. They also showed that a n t i t o x i n made with these cultures f a i l e d to protect x .u - 4 -against at least 2 to 3 m.l.d.'s of the toxins of Types A, B, C, and D. On t h i s basis they concluded that these organisms must represent a new type, Type E. E a r l i e r , i n 1927, Zlatogoroff and Soloviev (4) reported that previous cases of ichthyism or the disease caused by " f i s h poison" could, i n t h e i r opinion, d e f i n i t e l y be considered to be botulism. Although t h i s may not be true i n a l l cases, i t i s probable that the majority of ichthyism cases were fish-borne botulism, possibly of Type E. These workers state that, i n Soviet Russia', botulism i s due c h i e f l y to s a l t , smoked, or dried f i s h , which i s used as food without b o i l i n g or roasting. Such f i s h as s a l t or dried r e d - f i s h , salmon, sturgeon, smoked sturgeon, smoked herring or vimba are the usual ones involved. The organisms described by Gunnison, Cummings, and Meyer (3) r had been isolated from sturgeon i n t e s t i n e s . The next mention of Type E i n the l i t e r a t u r e comes from Hazen (5), i n 1937* Hazen described an organism i s o -lated from a t i n of German-canned sprats (Kielersprotten) which had caused f a t a l botulism in.New York State. This organism, s t r a i n E35396, "was described i n some d e t a i l . I t may be noted that the thermo-stability of the spores was r e l a t i v e l y low (e.g. 80° C. for 10 minutes) compared to other types of CI. botulinum. Hazen found that the toxin produced was not neutralized by CI. botulinus monavalent an t i t o x i c sera, types A, B, or C, and that the antiserum - 5 -produced against t h i s toxin did not neutralize the toxins of Types A, B, C, or D. The next year Hazen (6) isolated another s t r a i n , E36208, from smoked salmon caught and prepared i n Labrador which also caused f a t a l botulism i n New York state. In t h i s b r i e f report, Hazen mentions that type E strains may have been overlooked i n the past because methods suitable f o r the detection of Types A and B toxins were found to be inade-quate for the detection of Type E. Hazen reported the d i f f e r -e n t i a l c h a r a c t e r i s t i c s of her two s t r a i n s , i n 194-2 (7). She showed that toxic f i l t r a t e s of the salmon s t r a i n (E36208) were l e t h a l to white Leghorn chickens, but those of the sprat s t r a i n (E35396) had no demonstrable effect on chickens. These strains were also shown to belong to d i s t i n c t agglutinogenie groups. A further outbreak of botulism due to Type E was des-cribed by Geiger (8), i n 1941. The food incriminated was un-cooked, commercially canned mushroom sauce. The mushrooms came from Yugoslavia, but had been processed i n C a l i f o r n i a . The can was not a "swell" but normal i n taste, odour, and appearance. Only three of the s i x people who had eaten the sauce became i l l and but one of these died. No culture was i s o l a t e d , but the presence of type E toxin was demonstrated by Dr. Meyer of the Hooper I n s t i t u t e . Dolman and Kerr ( 9 ) , i n 1947 > reported the next out-break of Type E botulism. This i s the f i r s t Type E episode - 6 -and the t h i r d reported outbreak of botulism i n Canada, a l -though probably there have been unreported cases (10 , 15)• Three f a t a l cases of botulism at Nanaimo, B r i t i s h Columbia, occurred i n 1944, following the consumption of home-canned salmon. Improperly processed t i n s of salmon and chicken found i n the household, as wel l as a sample of the garden s o i l , yielded CI. botulinum, Type E. Dolman et a l (11), i n 1950, described the i s o l a t i o n of another s t r a i n of Type E from a t i n y piece of home-pickled herring which had been incriminated i n two cases of botulism, one f a t a l , at Vancouver, B r i t i s h Columbia i n October, 1949. In 1951? Meyer and Eddie (12) described a small out-break of botulism among Eskimos i n Alaska i n 1950, due to beluga (white whale) f l i p p e r s cured i n o i l . They demonstra-ted the presence of type E t o x i n , while shortly afterwards a type E s t r a i n was isolated from a sample of the beluga, by Dolman and Chang (13). Prevot and Huet (14), i n a survey of organisms isolated from the' i n t e s t i n a l content of f i s h (163 fresh-water f i s h and 13 salt-water f i s h ) found, apart from other toxigenic and non-toxigenic microorganisms, one s t r a i n of CL botulinum, type E. This s t r a i n (P34), which was isolated from a perch, i s some-what si m i l a r to the VH s t r a i n of Dolman et a l ( 11) . Dolman (15) has presented the l a t e s t report of a Type E botulism outbreak. A 42-year old man of Natal, B r i t i s h - 7 -Columbia , developed the c l a s s i c a l symptoms of botulism within a few hours of eating home-pickled trout cutlets and died rl a f t e r 18 days. A type E s t r a i n was isolated from the non-to x i c contents of a j a r of the same batch as the one i n c r i m i -nated . In the entire history of Type E botulism there i s , as yet, only one instance i n which some sort of f i s h was not the incriminated foodstuff. On t h i s basis i t would seem that type E strains have marked p r e d i l e c t i o n for f i s h . 1 (see Table I ) . Two possible explanations of t h i s phenomenon should be considered. The f i r s t i s that f i s h ingest CI. botulinum from the water but are not harmed by the organisms. On the death of the f i s h certain chemical changes take place which allow the organisms to p r o l i f e r a t e and to produce toxin. This i s p a r t i c u l a r l y l i a b l e to happen i f the f i s h are not eaten immediately or are improperly processed. The findings of Prevot and Huet (14) seem to support t h i s f i r s t hypothesis. The second explanation i s that the f i s h are contaminated during the handling or processing. In t h i s case the source of the organism would be the s o i l . I t may be that Type E i s more prevalent i n some s o i l s than i n others, and that f i s h are treated with less regard f o r cleanliness before consumption than other foodstuffs. This hypothesis could be f i t t e d quite e a s i l y to the facts of the Nanaimo cases reported by Dolman 1 A recent paper from Japan (now i n the process of transla-: tion) describes fourteen cases of type E botulism with four deaths. The food incriminated was vinegared herring and r i c e cakes. - 8 -and Kerr (9) . However, othe^actors may contribute to t h i s apparent p r e d i l e c t i o n of Type E for f i s h . TABLE I KNOWN ISOLATIONS OF CLOSTRIDIUM BOTULINUM, TYPE E Year Reported by- Cases Deaths Place of Occurrence Source of Culture 1935-36 Gunnison, Cummings 0 0 and Meyer (1936) 1934- Hazen (1937) 3 1 1934 Hazen (1938) 3 1 1941 *Geiger (1941) 3 1 1944 Dolman and Kerr (1947) 1949 Dolman, Chang, Kerr and Shearer (1950) 2 1 1950 »Meyer and Eddie 5 0 (1951) Dolman and Chang (1951) 1951 Pre'vot and Huet 0 0 ( 1 9 5 D 1952 Dolman (1953) 1 1 Soviet Ukraine Westchester County, New York Cooperstown, New York San Francisco, Cal* i f o r n i a . Nanaimo, B r i t i s h Columbia Vancouver, B r i t i s h Columbia Point Hope, Alaska France l a t a l , B r i t i s h Columbia. Russian sturgeon in t e s t i n e s . German commercially canned sprats. Labrador smoked salmon. Yugoslav mushrooms canned i n C a l i f o r n i a Home-canned l o c a l salmon (also home-canned chicken and s o i l of chicken run). Home-pickled l o c a l herring (also jejunal contents of f a t a l case). Uncooked, l o c a l beluga (white whale) f l i p p e r s . Fresh water perch i n t e s t i n a l contents Home-pickled l o c a l freshwater trout * Culture not i s o l a t e d . Type E tory Section, Canadian Public toxin i d e n t i f i e d . From:(l6) Dolman, C.E. Address to Labora-Health Association, Christmas 1952. vo - 10 -III. SOURCE' OF CULTURES Although the major part of the experimental work was done on type E cultures of Clostridium botulinum, some strains of types A, B, C, and D were studied comparatively. A l l strains were maintained as stock cultures i n Greenberg's semi-solid reduced medium (see Appendix 1). They were sub-cultured at least twice a month. The three type A s t r a i n s , A38, A73, and A1959? were i stock cultures maintained i n the department. The A1959 s t r a i n , and two type B s t r a i n s , (B39 and B1960) were received from Dr. K. F. Meyer. Strains B7949 and B lamanna were sent by Dr. Reed of Queen's University, Kingston. Other strains used were from a c o l l e c t i o n i n the Western D i v i s i o n , Connaught Medical Research Laboratories. - 1 1 -IV. EXPERIMENTAL STUDIES A. Thermal S t a b i l i t y of Spores. 1 . Standardization of inoculum. In the f i r s t tests done on the thermal s t a b i l i t y of the Type E spores, no attempt was made to standardize the inoculum. Esty ( 1 7 ) found that types A and B spores exhib-it e d d i f f e r e n t degrees of heat resistance, Aof the number of spores present. He also found that the heat resistance of a given spore s t r a i n i s markedly influenced by the number of spores heated. This seems l o g i c a l when one considers the fact that growth of a heated spore suspension depends on the sur v i v a l of only one or two spores and that with a larger number of spores there i s l i k e l y to be a comparatively large number of resistant spores. Differences were noted i n the thermal s t a b i l i t y of these Type E s t r a i n s , and sometimes i n various samples of the same s t r a i n . These differences were considered possibly due to inconsistencies i n the number of viable spores i n the inoculum. Therefore, an attempt was made to correlate the number of viable spores i n the inoculum with the a b i l i t y of the sample to withstand the heat treatment. I t was necessary to employ a r e l a t i v e l y simple but accurate method of determin-ing the viable spores present i n a mixture of both l i v i n g and dead vegetative c e l l s and spores. After three or four - 12 -attempts te determine the number of spores t u r b i d i m e t r i c a l l y t h i s method was abandoned as unreliable. A method s i m i l a r to that of Wynne and Foster (18) was adopted. The spores suspensions which had been heated at 55-60°G. for 30 minutes to destroy the vegetative c e l l s were s e r i a l l y d i l u t e d , i n t r i p l i c a t e , i n the counting medium. (See Appendix I) After the medium had s o l i d i f i e d , 2 to 3 ml. of an anaerobic seal were placed i n each tube. This seal was 2$ agar with 0.1$ sodium t h i o g l y c o l l a t e . Wynne and Foster used pork infusion agar with starch and t h i o g l y c o l l a t e supplements. In the experiments reported here a si m i l a r medium substituting beef in f u s i o n f o r pork was used. After t h i s work was begun, Sugiyama reported (19) using a beef infusion agar medium. These tubes were incubated i n a i r at 37° C. The c o l -onies were counted at 24 and 48 hours. I f more than 3 or 4 strains were being tested at one time, as was usually the case, i t was found necessary to standardize the spore suspen-sions one day and to perform the actual spore resistance tests the next day. This prolongation of the procedure did not seem to have any deleterious effect on the accuracy of the t e s t . Stumbo et a l (20) found that s i m i l a r spore suspen-sions of CI. botulinum did not have detectable variations i n counts as a resu l t of storage at 38-40°F fpr as long as 18 months. -13-2. Procedures for determining the thermal resistance of the spores. Several somewhat di f f e r e n t procedures, outlined below, have been used throughout the course of these experiments on spore resistance. I t was found necessary to make improvements from time to time, for the sake of both speed and accuracy. a. Three-day cultures i n glucose-peptone-beef infusion broth with added meat (G.P.B.I.) were f i l t e r e d through s t e r i l e absorbent cotton, eentrifuged, and the toxin d i s -carded. The c e l l s were resuspended i n s t e r i l e d i s t i l l e d water and 1 . 5 ml. amounts of each suspension were subject-ed to d i f f e r e n t temperatures f o r various times. Then 0.5 ml. amounts of the heated suspensions were inoculated o into G.P.B.I. These subcultures were incubated at 37 C and examined d a i l y for from one week to ten days and then discarded. b. Three-day cultures i n G.P.B.I. were placed i n a b o i l -ing water-bath. From each tube two samples of 0.5 ml. each were removed at specified i n t e r v a l s and inoculated into G.P.B.I. c. Three-day cultures i n G.P.B.I. were f i l t e r e d through absorbent cotton into s t e r i l e pyrex s l a n t - s i z e tubes. Two tubes for each s t r a i n were placed i n a b o i l i n g water bath. From each tube two samples of 0 .5 cc. each were removed at the desired time.and inoculated into G.P.B.I. -14-d. Three-day cultures i n G.P.B.I. were f i l t e r e d through absorbent cotton. These were centrifuged and washed three times with M/15 pH 7.0 phosphate buffer. Excess-i v e l y heavy suspensions were reduced to a #12 McFarlane t u r b i d i t y standard. Three tubes of each suspension were placed i n water-baths of the desired temperature. The thermal s t a b i l i t y of each spore suspension was then t e s t -ed by withdrawing two 0.25 nil. samples from each tube, aft e r i t had been heated f or the required time, and trans-planting into G.P.B.I. e. The l a s t method i s a modification of that used by Meyer (21) i n his early experiments on the thermal s t a b i l -i t y of spores of Types A, B, and C. Brain heart infusion broth was inoculated from Greenberg subcultures of the test s t r a i n s . After being incubated i n a Mclntosh-Fildes anaerobic j a r for ten days, the cultures were centrifuged at 2500 rpm. for one hour. The supernatant toxins were drawn o f f and the c e l l s resuspended i n a s t e r i l e M/15 pH 7.0 phosphate buffer. These suspensions were placed i n Erlenmeyer flasks containing glass beads. The flasks were shaken with a rotary motion f or 4 to 5 minutes. The suspensions were then f i l t e r e d through glass wool into s t e r i l e Erlenmeyer f l a s k s . These l a s t two steps were necessary to break up the spore clumps. The spore suspensions were dispensed i n 1.5 ml. amounts i n s t e r i l e Pyrex tubes. These tubes were heated - 15 -at 100° C. for various lengths of time. Several tubes were done for each time. One minute was allowed for the tube contents to reach 100° C. This time was determined by a previous experiment. Each tube, on removal from the b o i l i n g water bath, was placed i n an ice-bath f or rapid cooling. One ml. was taken from each tube and subcultured into brain heart infusion broth with th i o g l y -c o l l a t e supplement. Each subculture was layered over with p a r a f f i n and incubated aer o b i c a l l y . These subcultures were observed for a period of ten days and then discarded. Prolonged dormancy or slow germination of spores, a phenomenon which was reported by Burke (22) as early as 1923? was not taken into account i n these experiments. 3 . Results The results of these spore tests are given i n Tables I I to V. Figure 1 i s a survey of the thermal s t a b i l i t y of the Type E spores from these experimental r e s u l t s . "TABLE TT T w e H M A * . o r C u (3oTOM«ur-v 5 PoR<4 lrlfcT»t<>M ». A M * b . ) STRAIN M E T H o O a. METHOD b. A38 J t t f - f cO Vvl-lX* A73 i/4 - ixo A W t o > 3 o > 1 X . O b3 ci < 3 0 > V \ a *ti4 - b O > 3o > H O ' / x - I O ' A . - I O */-4 -'S <»/*! - i O E 8 «tfx-S o/x- l O < 3 ^ - 3 »/x-3 »iT. - .o < iS •»/«4 - i » " E i S " l - /x-r ' / x - 3 • / x - 5- < Eion • / x . - is- ' /x-'} SO -£35*39^ •lx.->< - * /x -^ 4/4- S" ii*-!? "/il- i a »<4 - lo £-5^10 a Vv- i f »/».- io J / j . . fO <f». - s-'/j. - io . 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BOTULINUM SPORES, FROM EX -PERIMENTAL RESULTS SOR.WI VtD - 2 1 -4. Discussion. ' " The method of counting the spore inoculum was found to be r e l a t i v e l y simple and reasonably accurate. A plate count would no doubt have yielded as good result s but such a method would have been impractical mainly because the num-ber of plates required, not to mention the number of anaerobic j a r s , would have been p r o h i b i t i v e . Andersen (24) has devel-oped a fast plate method for counting spores of Cl» botulinum. The method involves the use of a p e t r i dish which i s made anaerobic with a glass plate and anaerobic agar overlying the inoculated medium. The chief reason why t h i s method was not used i s that matched p e t r i dishes and s p e c i a l l y molded glass plates were required. The main d i f f i c u l t y encountered with the beef in f u s i o n agar was i n dispensing the medium both quickly and accurately. However, t h i s was soon overcome by using a 50-ml. burette attached to an open glass b o t t l e which served as a reservoir. In t h i s way several l i t r e s could be dispensed i n an afternoon. Counts i n the medium were done i n t r i p l i c a t e and i t was found that each of the three tubes of a certain d i l u t i o n gave result s of the same order. This was taken as an i n d i c a t i o n that the method was not grossly inaccurate. Since a l l strains were treated i n the same way the counts were r e l a t i v e to one another; - 22 -absolute counts could hardly be expected. In any event, a r e l a t i v e picture was a l l that was desired. An attempt to correlate the number of spores i n a test sample with the thermal resistance of that sample i n d i - _ cated that the resistance was independent of the number of spores heated. Since high concentrations were used, large numbers of resistant spores would be present i n a l l such concentrations. Perhaps the previously mentioned findings of Esty (19) would have been applicable i f some much lower concentrations of spores had been used as w e l l as the higher concentrations. I f an attempt had been made to determine the actual number of spores surviving the heat treatment, i t i s conceivable that the size of the inoculum could be corre-lated with the survivors. However, the p r i n c i p a l concern i n these experiments was to determine how long a suspension con-taining spores of CU botulinum, Type E could be heated and s t i l l survive on subculture. Although considerable time was spent i n modifying the methods used i n the thermal resistance t e s t , the procedure was e s s e n t i a l l y the same throughout. From the results ob-tained i t seems that a l l of the methods gave, for the same s t r a i n , the same general picture of thermal s t a b i l i t y . Method a. gave p a r t i c u l a r l y inconsistent resu l t s but t h i s might be explained by the p r o b a b i l i t y of considerable error - 23 -since these were the f i r s t tests attempted. In addition, the suspending f l u i d was d i s t i l l e d water which would not be l i k e l y to exert any buffering action on the spores. Stumbo ( 2 5 , 20) has devised a complicated apparatus for determining the resistance of b a c t e r i a l spores to a higher range of temperatures (220°F. - 2?0° F.). This new piece of equipment i s called a "resistometer". Spore sam-ples are placed i n the apparatus and heated at a constant temperature with steam. The time of exposure can be measured very accurately. Although t h i s machine i s adapted only for high temperatures (104.4°C. i s the lowest), Dr. Stumbo agreed to t r y some Type E strains as a check on the method used here. Accordingly, some cultures were sent but no word of the results has been heard as yet. Nevertheless, a few general observations may be made from the results obtained. Since the Type E'strains were the chief concern i n these experiments, the other types served mainly as controls. I t i s apparent that the Type E strains are much more heat l i a b l e than the Type A and Type B (except B39) s t r a i n s . I t i s int e r e s t i n g to note that s t r a i n B39 has a thermal s t a b i l i t y of the same order as that of the Type E. Indeed, studies carried out by Miss Helen Chang, of t h i s de-partment, indicate that B39 resembles the Type E s t r a i n agglu-ti n o g e n i c a l l y . Unfortunately t h i s s t r a i n has l o s t i t s - 24 toxigenic capacity so that i t was impossible to check i t s ' c l a s s i f i c a t i o n as a Type E. In any event, the statement that Type E spores d e f i n i t e l y would be destroyed by the precautions taken for Types A and B may be repeated here. I t seems that the Type C tested and D468 are simi-l a r to Type E i n thermal s t a b i l i t y and that % i s somewhat more heat stable. I t i s believed that data soon to be presented by Miss Janet B. Gunnison of San Francisco w i l l show that D468 i s a c t u a l l y a Type C s t r a i n . The Type E strains themselves appear to be divided into two groups, one very l a b i l e and the other not quite as l a b i l e . Figure 1 compares graphically the thermal s t a b i l i -t i e s of the Type E s t r a i n s . A study of t h i s i l l u s t r a t i o n shows that the very l a b i l e group includes E2, E8, E151, Nanaimo s o i l , HV, and Beluga, and that the other group i n -cludes E74, E3017, E35396, E36208, and Nanaimo chicken. Also i t i s seen that the results are more variable i n the very thermolabile group. No doubt t h i s can be p a r t l y explained by the fact that since there are fewer resistant spores t h e i r d i s t r i b u t i o n would be more random than i n the more thermo-stable s t r a i n s . In addition, a constant temperature appara-tus set at a lower temperature than 100° C. would probably have given more consistent resul t s f o r the very l a b i l e group. A few tests were done at 80° C. - 25 -On the basis of these experiments i t i s seen that, f o r the i s o l a t i o n of a suspected Type E s t r a i n of CUbotulinum, b o i l i n g for three to f i v e minutes a tube of G.P.B.I., or si m i l a r medium, which had been inoculated with a small piece of the incriminated foodstuff, i s the safest method to get r i d of non-sporulating contaminants without k i l l i n g the sus-pected Type E organism. Additional tubes could be boiled for ten and f i f t e e n minutes as w e l l . The r e s u l t i n g growth i n a l l these tubes could then be plated out on blood agar plates and these incubated anaerobically. From these plates the actual i s o l a t i o n s could be made. I - 26 -IV. EXPERIMENTAL STUDIES J3. Toxigenic Studies. 1. Toxin Production, a. Effect of dextrose. Two parallel sets of G.P.B.I. cultures of the Type E organisms were set up, one set with added dextrose (2%) and the other with no dextrose. These cultures were incubated at 37° C. in a Mclntosh-Pildes anaerobic jar. Toxin production was tested at 48, 96, and 156 hours by mouse i n -oculation. The pH of most of the cultures was reoorded at 156 hours. The i n i t i a l pH of the medium was 7»8. The results are shown in Table VI and Figure 2 on the following pages© b» Effect of the type of peptone and of L-cysteine. An attempt was made.to find a suitable (inexpensive and easy to prepare) f l u i d anaerobic medium which would allow good toxin production. A basal medium with the following ingredients was made: Bacto-beef extract . . . . . . . . . . . . . 5.0 grams Sodium chloride . . . . . . 2.5 " Disodium phosphate . . . . . . . . . . . . 2.0 " Sodium thioglycollate . . . . . 1.0 " D i s t i l l e d water . . . . . 1000 ml. • This was divided into three parts and the following peptones were added, to the- amount of b% by volume: Part I Neppeptone \ Part II Pfanstiehl peptone Part III Proteose^peptone $5 - 27 -Each of these was divided into two partsj one (a) was l e f t untreated and the other (b) had 0,015% L-cysteine added to i t . The pH of each medium was ad-justed to 7.8. The media were autoclaved for 20 minutes at 15 pounds. Be-fore inoculating the media, 2% dextrose was added aseptically. A l l six media were inoculated with strains E74, E35396, and HV. Toxicity tests were done by mouse inoculation at three and five days, ^he results indicated that medium II (b), Pfanstiehl peptone plus L-cysteine, produced toxins of the highest t i t r e in a l l cases. In the Neopeptone media the toxins produced consistently exhibited the lowest t i t r e . The addition of L-cysteine to the medium consistently increased the toxin production. c. Titration of toxins. The toxicity of culture f i l t r a t e s was titrated using serial ten-fold dilutions of the toxins in physiological saline. Mice of 15 to 20 grams weight were inoculated intraperitoneally. An approximate estimate of the M.LoD. was made using single mice; but when a more accurate determination of the LD/50 was required, several mice were used and results were calculated by the method of Reed and Muenoh (25). The biological variation among mice for the same dose of toxin was almost negligible, therefore one mouse tests were often employed when mice were not p l e n t i f u l . With the type E toxins, deaths usually occurred between 12 and 20 hours after the inoculation. No deaths from botulism occurred after the second day 0 d» Stability of toxin production. Type E cultures of CI. botulism were found to be very inconsistent pro-ducers of toxin. For example, the t i t r e s (in mouse M.L.D. per ml.) of E74 and P34 toxins produced on different occasions are shown in Table VII. - 28 -TASIE VI-THE EFFECT OF (Z%) DEXTROSE ON THE TOXIN PRODUCTION OF TYPE E STRAINS OF CLOSTRIDIUM BOTULINUM STRAIN DEXTROSE 48 Hrs. TOXICITY - mll.d's/co. 96 Hrs. 156 Hrs. pH at 156 hours E2 100 -300 less than 30 less than 30 30 -100 30 -100 30 -100 5.0 E8 less than 30 less than 30 E74 100 -30-100 -. 300 100 -300 less than 30 less than 30 5.0 6.8 more than 1000 more than 3000 more than .10,000 5.3 E151 less than 10 less than 3 less than 3 30 -100 30 -100 30 -100 4.9 E3017 300 -1000 less than 100 less than 30 300 -1000 .100 -300 100 -300 5.1 E35396 300 -1000 less than 100 less than 30 7.0 1000 -3000 3000 -10,000 3000 -10,000 5.2 E36208 300 -1000 less than 100 less than 30 7.1 300 -1000 1000 -3000 1000 -3000 5.1 (continued) - 28 (a) -STRAIN DEXTROSE 48 Hrs. Table VI (Cont'd) TOXICITY - mll.d's/cc. 96 Hrs. 156 Hrs. pH at 156 hours Nan. Soil less than 3 less than 3 less than 3 Less than 3 Nan. Chick. 30 -100 less than 30 less than 30 7.05 100 -300 less than 1000 300 - 5.3 VH 300 -1000 more than 3000 300 - 1000 30 -100 less than 30 less than 30 5.0 Beluga 300 -1000 less than 100 less than 30 6.8 more than 1000 1000 -3000 1000 -3000 5.1 Incubation temperature 37° I n i t i a l pH 7.8. F IGURE L O & of M o o s e ^ L t ) c t 1 I s H o O « S O F X N C O S J A I I C W CA e « t in c n tun «iti»a N«.,.CU;CI<.. »w tM~3o. T H E t F P E C T o f O E K T R o S C OH 1*1 TOniH P«.ofcO«-vi«»i o f T X P C £ S l U A l N l S tv.- fcolOv-iMOn-- 30 -TABLE VII TITEES OF FIVE-DAY TOXINS OF STRAINS E74 AND P34 ON DIFFERENT OCCASIONS DATE E74 P34 26/V/51 more than 10,000 not tested 16/VII/52 3000 - 10,000 less than 3000 23An/52 3000 - 10,000 more than 1000 30/VIl/52 300 - 1,000 300 - 1000 18/IX/52 3000 - 10,000 1000 - 3000 20/lx/52 approx. 3000 more than 3000 25/IX/52 less than 300 3000v- 10,000 26/IX/52 approx. 2000 3000 - 5000 4/X/52 3000 - 10,000 1000 - 3000 . 24/X/52 approx. 300 4/XI/52 1000 100 - 300 29/XII/52 10,000 - 30,000 approx. 300 30/lIl/53 3000 - 10,000 100 - 300 t i t r e in mouse MID. per ml The table indicates that some strains, e.g. E35396, E36208, Nanaimo chicken, and Beluga, lost their toxigenio capacities altogether, during the period in question. This decline i n the toxigenic property i s illustrated in Figure 3» At the same time as the toxigenic decline became evident, i t was noted by other workers in this laboratory that there were at least two types of colonies in most cultures: a granular opaque type of colony with irregular - 31 -edges, and a smooth translucent type with regular edges* It was thought that this phenomenon might account for the f a l l i n g off of toxin production. Accordingly, an effort was made to isolate each of the two colony types, and to conrelate colonial form with toxigenic capacity. A comparison'of the colony types of two cultures of the same strain, one of which had lost i t s toxigenic capacity and the other had not, yielded the following results: CULTURE TOXIGENIC NON-TOXIGENIC Beluga ' slightly less than almost a l l are granular half are smooth E35396 mostly granular almost a l l granular Nan. chick.. slightly less than mostly granular half smooth On the basis of this incomplete survey, i t would seem -that the smooth colonies are the more.toxigenic. Supporting this possibility, was the fact that the Nanaimo salmon strain, which had been kept as a lyophilized stock culture, showed entirely smooth colonies and was highly toxigenic. (After a period of three months and many subculturings, this strain also showed-both types.) However, against the hypothesis that smooth colonies are toxigenic i s the fact that the Nanaimo s o i l and E36208 cultures, which had been sub-cultured many times in G.P.B.I. and had become non-toxigenic, exhibited an excess of smooth colonies* Morphologically the smooth colonies are solid, non-sporing rods, whereas the granular colonies were composed of sporulating rods. This rule held true for a l l strains tested. - 32 -Even in the toxigenic strains the granular type of oolony predominates. Nevertheless, the smooth colony seems to be responsible for toxi c i t y . The Beluga strain was twice separated through several successive transfers into a non-toxigenic granular colony and a toxigenic smooth colony. On the f i r s t occasion, a four-day culture in G.P.B.I. of the smooth colony yielded a toxin with a t i t r e of between 1000 and 3000 mouse MID. per ml. A similar culture from a granular colony had less than 10 mouse MID. per ml. On the second occasion, a four-day culture of a smooth colony showed 300 to 1000 mouse MID. per ml., while the granular culture showed no toxic i t y . The E36208 culture was similarly separated, but neither the smooth nor the granular colony could be shown to produce toxin. This would indicate that another factor besides colonial form is responsible for the toxigenic degeneration of Type E cultures. Incidentally, a similar decline in toxigenic a b i l i t y was noted by Stevenson, Helson, and Reed (26) in cultures of 01.parabotulinum, types A and B. They also noted a change in colonial form accompanying this gradual decrease i n toxin production., which involved a slow conversion from the characteristic pebbled form with serrate to irregular margins to more nearly smooth colonies with entire margins. However, in the case of types A and •B, the rough colonies were toxigenic e< Discussion. It i s seen that dextrose enhances the toxin production of most Type E strains of CI. botulinum. An outstanding exception to this generalization 33 -is the HV strain, which shows greater toxigenicity in the absence of dextrose. Although only an incomplete check was made on the f i n a l pH of these cultures, i t i s evident from Table VI that those cultures with added dextrose had a much lower pH than those without dextrose. This is not unusual and is accounted for by the presence of a fermentable sugar. Since these cultures also had a higher toxicity (except for HV) than those without dextrose, i t would seem that Type E toxin i s not activated by acidity* The attempt to find a suitable anaerobic medium indicated that the basal medium plus the Pfanstiehl peptone plus L-cysteine was the best tested. The purpose was to have available a f l u i d medium which could be prepared i n large quantities for use in the cellophane sac method mentioned in section 3 (a). As previously mentioned, the Type E strains do not produce toxins of a constant t i t r e . An attempt made to correlate colony form with toxicity seemed to be successful i n some cases, e.g.: Beluga and Nanaimo salmon, and unsuccessful in others, e.g.: E36208 and Nanaimo s o i l . There are, of course, other factors which must be considered in this irregularity of toxin pro-duction. One of these i s the fact that the medium used (G.P.B.I.) was by no means exactly repoduoible, although great.care was taken to make i t as uniformly as possible. However, perhaps not too much emphasis should be placed on variations in the medium since type E cultures (e.g., Nanaimo chicken, BV, and Beluga) resembled a type % strain (A1959) in producing comparably high t i t r e toxins in mashes of cooked and raw beef and of herring in saline© Another factor was the variable inoculum. A considerable amount (0.5 - 2.0 ml.) of the Greenberg's stock culture (whose age ranged from one day to two weeks) was transferred to the G.P.B.I. by means of a pasteur pipette. - 34 -2!. Toxin s t a b i l i t y . The stability of toxins on storage i s of importance i f experiments with the same toxin are to be carried out during a considerable length of time. a* Storage of, Seitzed and lyophilized toxins. ; ( The toxins used in these experiments were Seitzed f i l t r a t e s of five-day cultures i n G.P.B.I. These were titrated after sterilization and re-titrated each time they were used. Although most toxins exhibited a f a i r l y constant t i t r e , others showed some f a l l i n g off. Because of this , re-titrations were deemed necessary. It'was thought that i f some method were available which would allow the toxins to be kept at a constant t i t r e for a few months at least, i t should be put into immediate use. Such a method would.save both time and mice. Lyophilization was attempted, Seitzed-filtered toxins being dispensed in 0.5 cc. amounts in 2 ml. v i a l s . These were frozen i n an acetone bath in the freezing compartment of the lyophilizing apparatus and attached to the manifold after a sufficient vacuum was registered. Twenty such vials could be lyophilized in one eight-hour period. B e c a u s e -the machine was not always available and because only 20 vials could be done at once, the pro-cedure was very slow. In addition, there was not a 100 per cent yield since some d i f f i c u l t y was experienced in sealing the v i a l s . Considerable frothing of the toxin took place during the f i r s t five or ten minutes of evacuating the v i a l s . This meant that at least an hour was spent i n putting the vials on the machine. After this time the apparatus could be l e f t unattended. A comparison of the t i t r e s of Seitzed-filtered and of lyophilized toxins - 35 -on storage at 4 ° to 6 ° C. was made. The results indicate that with the lyophilized toxins there was a sharp i n i t i a l drop in t i t r e after which the preparations declined slowly i n their toxioity. The Seitzed-filtered toxins exhibited some i n i t i a l f a l l i n g off in toxicity and then remained stable for as long as eight months. In addition, some Seitzed-filtered toxins exhibited a constant t i t r e , with no i n i t i a l drop, for as long as five months. A comparison of the results obtained shows that the lyophilized toxins appear to be much less stable on storage than the ordinary Seitzed-filtered toxins. Considering the extra time that must be spent in lyophilizing these toxins, the procedure may be regarded as worthless. It may be that over a period of a year or two the lyophilized toxins might prove to be more stable but the immediate problem was to have available toxins which would not need re-titrating. Perhaps i f purified toxins rather than crude ones had been used, this method might have proved to be more successful. 3. Active immunization of mice. a . Preparation of toxoids. Toxoids were prepared in large quantities i n the following manner. Three 200-ml. quantities of G.P.B.I. were inoculated with each strain. After five days incubation at 37° C. the broth was Seitzed-filtered and the f i l t r a t e titrated for toxicity by mouse i n o G u l a t i o n . Filtrates of suitable toxicity (at least 100 mouse mld's/cc.) were toxoided by the addition of O.Z%, 0,5%, and 1.0% formalin and suitable incubation a t 37° C. After each strain 36 -had been toxoided (i.e., 0.5 cc. no longer k i l l e d a mouse), i t was stored at 4° to 6° C. Table VIII shows that the length of time these toxins needed to be toxoided i s dependent on the original t i t r e of the toxin as well as on the amount of formalin added. An attempt to produce toxoids of greater antigenicity than those l i s t e d in Table VIII was made. Accordingly, efforts were directed towards preparing toxins of higher potency and toxoids of greater purity. Considerable success has been obtained by South African workers with Types C and D (27, 28) and by the Kingston workers with Types A and B (29) by the use of cellpphane sacs in the large scale production of high t i t r e toxins. Sterne and Wentzel (29) used an intussascepted cellophane tube, f i l l e d with saline, immersed i n a carboy of a corn steep liquor medium. This invaginated tube permitted dialysis on two surfaces. The inoculum is placed in this tube and the concentrated toxin harvested from i t . TABLE VIII TIME REQUIRED FOR DETOXIFICATION OF VARIOUS TOXOIDS WITH FORMALIN TOXOID DATE ORIGINAL TITRE FORMALIN DAYS TO MLD'S/CC. ADDED {%) TOXOID A1959 3Q/Vn/52 approx. 20,000 0.5 30 E74 (1) 30/vil/52 300 - 1000 0.5 3 (2) 27/VIII/52 approx. 300 0.3 4 (3) 4/XI/52 1000 - 3000 1.0 (19 hrs) E35396 7/VIII/52 300 - 1000 0.5 3 E36208 21/VIII/52 100 - 300 0.5 3 Nan. chick 7/VIII/52 30-100 0.5 3 (2) 3/XI/52 300 - 1000 0.3 11 (3) 3/XI/52 300 - 1000 .1.0 2 HV (1) 27/X/52 30 - 100 0.3 2 (2) 3/XI/52 approx. 1000 1.0 (19 hrs) - 37 -TABLE VIII (Cont'd) . . ORIGINAL TITRE FORMALIN DAYS TO TOXOID DATE MLD'S/CC. ADDED (%) TOXOID Beluga 3/XI/52 approx. 3000 0.3 11 (2) 3/XI/52 approx. 3000 1.0 2 ,P34 (1) 30/VII/52 300 - 1000 0.5 3 (2) 3/XI/52 100 - 300 1.0 (19 hours) (3) 22/l/53 approx. 300 0.5 3 In this research a method similar-to that of Sterne and ^entzel (29) was used. An intussuseepted sausage hag (Visking casing) with an outside diameter of 4 inches was placed in a glass vessel containing 2250 ml. of G.P.B.I. with approximately 1.5 inches of meat in the bottom. 450 ml. of saline was placed inside the double sac. Glass tubes for inoculating and sampling were inserted. The whole apparatus was autoclaved and allowed to cool. An inoculum of 5 cc. of a three-day ©.P.B.I. culture was placed inside the cellophane sac. The cultures used in this experiment were E74, E35396, Beluga, and P34. . The experiment was repeated on a somewhat smaller scale (250 ml. medium) with E74, E35396, and Beluga strains, using the Pfan-stiehl f l u i d anaerobic medium. The.results of both experiments were very similar. Very heavy growth resulted but the t i t r e s of the toxins were no higher than those usually obtained. Since this method was so much more time -consuming than the ordinary method of toxin production and yielded no better results, no further experiments were done using the double-surface dialysing membrane. A common method of increasing the immunizing power of a toxoid i s to precipitate that toxoid with alum. Increased antigenicity may result from either a slower rate of absorption or from the absence of non-specific inter-fering substances. Hottle, Nigg, and Lichty (30), working with %pe A and - 38 -B toxoids, found that there was an almost complete failure of f l u i d toxoids to protect mice against small doses of challenge toxins, but that alum-precipitated toxoids protected to a high degree. A. Type E alum-precipitated toxoid was therefore prepared by dividing a batch of P34 toxoid, with an original t i t r e of 300 mouse MLD per ml. into two lots; one was l e f t untreated, and the other was alum-precipitated by the method of Nigg and co-workers (31). One ml. of a sterile 10% potassium alum solution was added to each 10 ml. of toxoid and the whole was shaken and l e f t overnight at room temperature. The supernatant was siphoned off and the precipitate washed with fresh, sterile, physiological saline. This shaking, settling overnight, and washing pro-cedure was repeated. The precipitate was then resuspended in saline and dissolved by adding sterile IN. NaOH to pH 5.0. This material was tested in the experiments detailed below. b. Immunization and Challenge Programme. The f i r s t group of mice was inoculated with E74 ( l ) , E35396, E36208, and A1959 toxoids. The dosage schedule used was 0.2 cc. 0.3 c c , and 0.5 cc., with intervals of 3 and 5 days respectively between doses. Ten days after the last dose the preliminary challenge was carried out. For the preliminary challenge, in each immunized group, single mice were challenged with 30, 10, 3, and 1 mouse MLD respectively, using three toxins. Since the mice seemed to be only slightly immune (except for the A1959 group), another dose (0.5 cc.) of toxoid was given 12 days after the last dose. A. second preliminary challenge was done five days later. This indicated that the Type E groups were not yet immune. The &1959 group resisted more than 400 mouse mld's of the homologous toxin but less than three mld's of Type E toxins. The fi n a l challenge of - 3 9 -this group indicated that the mice were able to withstand a challenge of more than 3 0 0 0 mouse mld's of 4 1 9 5 9 toxin and that they were completely un-protected against P 3 4 and E 3 6 2 0 8 , Type E toxins. The Type E groups were given two more spaced 0 . 5 ml. doses. The f i n a l challenge was given five weeks after the immunization procedure had commenced. The results were very disheartening. The mice were protected against no more than three to five mouse mld's of Type E toxin. There was, of course, no protection afforded against Type A t oxin• The next immunization schedule consisted of four doses of 0 . 5 ml. each. There were three days between the f i r s t two doses and five days between the other doses. The preliminary challenge was given five days after the last dose. The toxoids used were B 7 4 ( 3 ) , Nanaimo chicken ( 3 ) , Beluga ( 2 ) , and P 3 4 ( 2 ) . (See Table VIIl) The results of the preliminary challenge i n -dicated that mice immunized with Nanaimo chicken could withstand more than 3 0 0 mouse mld's of challenge toxin, that mice immunized with Beluga could withstand more than 1 0 0 mfcuee mld's, and that mice immunized with E 7 4 and P 3 4 could not withstand three mouse mld's. Consequently, the Nanaimo chicken and Beluga groups were challenged the next day, while the other two groups were given several further inoculations in an unsuccessful attempt to raise their resistance. The results of the f i n a l challenge are given in Table IX.\^ A small scale experiment was done to determine i f alum-precipitation would increase the^ immunizing power of a Type E ( P 3 4 ) toxin. One group of mice was inoculated with untreated P 3 4 toxoid and another with alum-precipitated P 3 4 toxoid. The immunization schedule was 0 . 2 c c , followed by a three-day - 40 -interval, 0.3 oo. with a four-day interval, 0.5 cc. with a five-day interval, 0.5 cc. with a seven-day interval; and then the preliminary challenge. This challenge indicated that neither group of mice had been immunized. c. Discussion. • . The unsuccessful results obtained in attempts at raising the t i t r e of toxins by use of an intussuscepted cellophane sac might he explained by the fact that the experiment was tried on a very much smaller scale than those previously mentioned (27, 28), and that a different medium was used. Perhaps the method is suitable only for large-scale toxin production, or perhaps i t does not lend i t s e l f to enhancing Type E toxin production. It is l i k e l y that large quantities are needed to ensure anaerobiosis. With regard to the efficacy of alum-precipitation, l i t t l e can be said on the basis of the small scale experiment attempted. It would seem that alum-precipitation does not increase the immunizing power of Type E toxoids. On the other hand, improper techniques in the precipitation procedure, not to mention the low t i t r e of the original toxin, probably had some disadvan-tageous effect on the antigenicity. Furthermore, in vitro cross-neutrali-zation tests have subsequently shown (see Part B, section 4) that P34 is weak in protective power. On the basis of a l l these experiments i t would seem that mice were very poor animals to have chosen, since they exhibited demonstrable resistance in only a few cases. - 41 -TABLE IX IMMUNITY PRODUCED IN MICE BY NANAIMO CHICKEN AND BELUGA TOXOID CHALLENGE MOUSE . ACTUAL CUMULATIVE DEATHS 50^ OF MICE STRAIN MLD'S DEAD ALIVE DEAD ALIVE % PRO. AGAINST Nan. Nan. 1000 2 2 4 2 66.7 chick. chick. 300 1 3 2 5 28.6 535 100 1 2 1 7 12.5 E35396 500 1 3 3 3 50 300 1 2 2 5 35 500 100 1 2 1 7 12.5 30 0 2 0 9 0 Beluga 2000 0 2. 4 2 66.7 1000 2 1 4 3 57.1 647 300 1 1 2 4 33.3 100 1 1 1 5 16.7 E74 3000 1 1-, 8 1 89 1000 3 0 7 1 87.5 100 300 2 1 4 1 80 100 2 0 2 2 50 P34 100 2 0 - - — Trout 0.3 cc. 1 0 - - — Beluga Beluga 2000 0 2 3 2 60 1000 2 2 3 4 23.2 1650 300 1 3 1 7 12.5 100 0 3 0 10 0 E74 3000 1 1 6 1 85.7 1000 1 2 5 3 62.5 300 • 2 2 4 5 ' 44.5 420 100 2 1 2 6 25 P34 100 0 1 30 0 1 Trout 0.3 cc. 0 1 - 42 -The A1959 group was the only one in which mice were protected to a high degree. This suggests that Type K toxoids are much better antigens than Type E toxoids. In the two groups in which Type E toxoids did afford demonstrable immunity the level of immunity was exceedingly variable, as the results in Table IX show. Calculation by the method of Reed and Muench (25) indicated that in the group immunized with Nanaimo chicken toxoid, f i f t y per cent of the mice were protected against approximately 500 mouse MLD of either the homologous Nanaimo chicken toxin or of E35396 toxin; against approximately 650 mouse Mill's of Beluga toxin; and against 100 mouse MLD of E74 toxin. In other words, mice immunized with Nanaimo chicken toxoid seemed to be protected to approximately the same degree against E35396 and Beluga as against the homologous toxin, but not as well against E74. In the group immunized with Beluga toxoid the mice were protected against 400 mld's of E74 and against four times this amount of homologous toxin. The Beluga toxoid also protected single mice against the toxins of P34 and Trout strains. Single mice which had received Nanaimo chicken toxoid were not immune to these toxins at the level tested. From the data obtained in these experiments no definite statements may be made regarding the homogeneity of Type E toxins. It would seem that $74 contains some antigenic component which is not present in Nanaimo chicken or Beluga. 4. Cross-neutralization Tests, a. Procedure. To supplement the active immunization experiments, in vitro cross-neutralization, tests were done. Four type E toxins and their antitoxins were chosen. These rabbit antitoxins, were kindly supplied by the Western Division of the Connaught Medical Research Laboratories. Undoubtedly more strains should have been tested but there were no mice available for further experiments. The four strains chosen were E74, Nanaimo chicken, Beluga, and P34. Toxin from each strain was in turn titrated against the homologous and the three heterologous antitoxins. A suitable amount of toxin was found to be 10 mouse MLD. Preliminary tests were done, mixing this constant amount of toxin with varied amounts of antitoxin. Usually four different amounts of antitoxin were used. These toxin-antitoxin mixitiires were shaken and allowed to stand at room temperature for 45 to 60 minutes. Mice were then inoculated with these mixtures and their survival or death used as a criterion of the presence or absence of neutralization. Toxin controls were included. In these preliminary tests, only one mouse was used for each mixture. In the f i r s t set of experiments, several preliminary t r i a l s were necessary i n order to determine the end-point with each antitoxin. However, a definite pattern was soon established which was applicable in most cases to the other toxins. After the preliminary titrations were done, they were repeated on a larger scale, at least three mice being inoculated with each mixture, in order to obtain a more definite end-point. b. Results. The results of these cross-neutralization tests are given in Table X. Tables XI and XII compare and contrast the overall results of these ex-periments. TOXIN T A B L E x RESULTS OF TYPE TOXIN-ANTITOXIN CROSS-NEUTRALIZATION T E S T S ANTITOXIN ACTUAL C U M U L A T I V E NO OF LD50 DEATHS 5Q>% END PROTECTED STRAIN cc. ALIVE DEAD ALIVE * DEAD % POINT AGAINST E74 E74 • .01 4 • 0 7 0 0 .003 cc .005 3 1 3 1 25 .00369 6765 =25 .002 0 4 . 0 5 100 cc. LD/50 Nan. .02 4 0 9 0 0 chick. .01 4 0 6 0 0 .005 1 3 1 3 75 .0059 . 4237-.002 0 4 0 7 100 cc. Beluga .05 8 0 15 0 0 .02 7 1 7 1 12.5 .01486 1680 .01 0 8 0 9 100 cc. .005 0 8 0 17 100 P34 .05 4 0 6 0 0 .035 2 1 2 1 33 .0304 822 .02 0 4 0 5 100 cc. .01 0 4 0 9 100 TOXIN CONTROLS .0003 cc 0 3 0 5 100 .0002 cc 1 2 1 2 67 .00012 .0001 cc 3 0 4 0 0 cc. (continued) - 45 -NO OF LD50 TOXIN ANTITOXIN ACTUAL CUMULATIVE DEATHS 50?? END PROTECTED STRAIN cc. ALIVE DEAD ALIVE DEAD % POINT AGAINST Beluga E74 .04 3 0 7 0 0 • .01 cc. .02 3 0 4 0 0 .01257 992 - 12.5 .01 1 3 1 3 75 cc. LD/50 .005 0 3 0 6 100 Nan. .025 3 0 8 0 0 chick. .01 4 0 5 0 0 .00508 2460 .005 1 21 1 2 67 cc. .0025 0 3 0 5 100 Beluga .05 4 0 7 0 0 .025 3 1 3 1 2:5 .01848 675 .01 0 4 0 5 100 cc. .005 0 4 0 9 100 P34 .1 3 0 6 0 0 .03535 353 .05 3 0 3 0 0 cc. .025 0 3 0 3 100 TOXIN CONTROLS .003 cc. 0 4 0 7 100 .001 cc. 1 3 1 3 75 .0007955 .0005 co • 4 0 5 0 0 cc. 46 NO OF LD50 TOXIN ANTITOXIN ACTUAL CUMULATIVE DEATHS 50% END PROTECTED STRAIN co. ALIVE DEAD ALIVE DEAD (%) POINT AGAINST Nan. E74 .02 3 0 6 0 0 chick. .01 3 0 3 0 0 .00707 1070 •01 cc. .005 0 3 0 3 100 cc. - 7.7 .0025 0 2J 0 5 100 LD/50 Nan.• • .02 3 0 ; '' 8 0 0 Chick. .01 3 0 5 0 0 .005 2 0 2 0 0 .00353 2140 .0025 0 3 0 3 100 cc. Beluga .05 3 _ 0 6 0 0 .025 3 0 3 0 0 .0158 479 .01 0 3 0 3 100 cc. .005 0 3 0 6 100 P34 .05 3 0 6 0 0 .025 3 0 3 0 0 .0158 479 .01 0 3 0 3 100 cc. .005 0 Z 0 5 100 TOXIN CONTROLS .003 cc. 0 3 0 4 100 .001 cc. 2 (t 2 1 33 .00132 .0003 cc. 3 0 5 0 0 cc. 47 TOXIN ANTITOXIN ACTUAL NO OF LD50 CTJMUIATIVE DEATHS 50% END PROTECTED STRAIN CO. ALIVE DEAD ALIVE DEAD POINT . AGAINST P34 E74 .05 3 0 7 0 0 .03 cc. .025 2 1 4 1 20 .01 740 = 7.4 .01 2 1 2 2 50 CO. LD/50 .005 0 3 0 5 100 Nan. .03 3 0 3 0 0 chick .01 0 3 0 3 100 .0173 428 .003 0 3 0 6 100 cc. Beluga .05 3 0 3 0 0 .025 0 3 0 3 100 .0354 209 .01 0 3 0 6 100 cc. P34 .1 3 0 5 0 0 .05 Z 1 21 1 33 .0419 181 .025 0 3 0 4 100 cc. TOXIN CONTROLS .01 cc. 0 3 0 4 100 .003 cc 2 1 2 1 33 .00406 .001 cc 9 3 0 5 0 0 cc. - 48 -TABUS XI NUMBER OF LD/50's OF TOXIN NEUTRALIZED BY VARIOUS ANTITOXINS TOXIN ANTITOXIN E74 NAN. CHICK. BELUGA P34 E74 6765 1070 922 470 Nan. chick. 4237 2140 2460 428 Beluga 1680 479 675 209 P34 822 479 353 181 TABLE XII COMPARATIVE NEUTRALIZING CAPACITIES OF VARIOUS ANTITOXINS TOXIN ANTITOXIN B74 NAN. CHICK. BELUGA P34 E74 8 2 3 4 Nan. chick. 5 4.5 6 2 Beluga 2 1 2 1 P34 1 1 1 1 Neutralizing capacity of antitoxin P34 expressed as 1; capacities of other antitoxins expressed as multiples of t h i s . - 49 c. Discussion* It is seen from Tables XI and XII that there is some cross-neutralization between a l l four toxins and antitoxins. The E74 toxin apparently contained more neutralizable 10/50's than did any other toxin; whereas the P34 toxin contained fewer neutralizable LD/50's than any other toxin* In addition, E74 antitoxin protected mice to an equivalent degree against a l l three heterologous toxins, but proved several times more protective against i t homologous toxin. To continue with the comparison, the P34 antitoxin ex-hibited consistently the least protective power* Nanaimo chicken antitoxin neutralized i t s own and the Beluga toxin better than did any other antitoxin, and was also quite effective against the other two toxins. The Beluga anti-toxin was not much more potent i n neutralizing power than P34 antitoxin: i t protected to the same degree as the latter against Nanaimo chicken and P34 toxins, but was twice as effective against E74 and Beluga toxins* These toxins and antitoxins were not made at the same time. Consequently one cannot assume that the toxins used i n these neutralization experiments are antigenically similar to those against which these antitoxins were made* This fact may account for some of the discrepancies observed here. However, the question of heterogeneity cannot be t o t a l l y ruled out* Table XI, as previously mentioned, indicates that E74 antitoxin protected against i t s own toxin several times more effectively than i t protected against any of the heterologous toxins. This evidence suggests that E74 toxin contains an antigenic factor i n addition to that shared in common with other toxins* The other findings are not as easy to interpret i n terms of antigenic constitution. It should be borne in mind that considerable experimental errors may be involved in these t i t r a t i o n s ! An attempt to perform neutrali-o zation experiments with a l l the other available type E toxins and antitoxins should be made in the near future. V. DISCUSSION. . Although the individual experiments on the toxigenic studies have been discussed separately, some attempt should be made to correlate the results. It i s also necessary to interpret these results in terms of possible pre-parations for the protection of humans against type E botulism. It would seem that the best method of preparing toxoids i s f i r s t of a l l to obtain a high-titre toxin which has been made in a suitable medium containing dextrose. For making this toxin, cultures which are mostly i n the smooth phase should be selected. The next step is to toxoid these toxins by 0.5$ formalin and suitable incubation at 37° C. When toxoided, they should be stored in the refrigerator. Although three spaced doses proved sufficient to immunize mice, experiments on other animals would have to be done before a dosage schedule for humans could be determined. It i s unlikely that human immunizations w i l l ever be deemed necessary, except for laboratory workers or in the event of some special emergency. On the other hand, production of horse antitoxin for typing or therapeutic purposes may certainly be considered practical. Since these experiments involving mice have indicated that there may be some degree of heterogeneity among the type E toxins, this possibility must be taken into account in the selection of the strain or strains for immunization - 51 -purposes. There i s an indication given in these experiments that E74 contains some antigenic factor which i s not present in the other toxins. However, the E74 toxoid used did not prove to be a good immunizing agent. On the other hand, the strain with the highest immunizing capacity (apparently Nanaimo chicken), does not contain the chicken lethal factor ( 9 ) . Hence i t i s sug-gested that both these strains, v i z . E74 and Nanaimo chicken, should be employed in preparing toxoids for human immunization purposes, or for the preparation of a horse antitoxic serum for typing or therapeutic use. - 52 -BIBLIOGRAPHY 1. Reames, H.R., P.J. Eadull, R.D. Housewright, and J.B. Wilson. J. Imm. 55, 309, 1947. 2. van Ermengem, E. Rev. Hyg. 18, 761, 1896. cited from Topley and Wilson's Principles of Bacteriology and Immunity, G.S." Wilson and A.A. Miles. Edward Arnold and Co. London. Third Edition. 1946. 3. Gunnison, J.B., J.R. Cummings, and K.F. Meyer. Proc. Soc. Exp. ' Biol. & Med. 3_5, 278, 1936. 4. Zlatogoroff, S.J. and M.N. Soloviev. J. Am. Med. Assn. 88, 2024, 1927. 5. Hazen, E.L., J. Inf. Dis. 60, 260, 1937. 6. Hazen, E.L., Science 87, 413, 1938. 7. Hazen, E.L., Proc. Soc. Exp. B i o l . & Med. 50, 112, 1942. 8. Geiger, J.C., J. Am. Med. Assn. 117, 22, 1941.' 9. Dolman, C.E. and D.E.. Kerr. Can. J. Pub. H. 38, 48, 1947. 10. Dolman, C.E. Can J. Pub. H. 34, 97, 1943. 11. Dolman, C.E., H. Chang, D.E. Kerr, and A.R. Shearer. Can. J. Pub. H. 41, 215, 1950. 12. Meyer, K.F. and B. Eddie. Zeitsche.f.Hyg. 133, 255, 1951. 13. Dolman, C.E. end H. Chang. Proc. 6th Annual Meeting International North-west Conference on Diseases of nature communicable to man, p. 127. t^'S'l'. 14. Prevot, A.-R. et H. Huet. Bull. Acad. Nat. Med. 432, 1951. 15. Dojman, C.E. Can. Med. Assn. J., in press. 16. Dolman, C.E. Address to Lab. Sect.,.Can. Pub. H. Assn., Christmas 1952. U, Esty, J.R. Am. J. Pub. E. 13, 108, 1923. 18. Wynne, E.S, and J.W. Foster. J. B a o t . 55, 61, 1948. - 53 -19. Sugiyama, H. J. Eact. 62, 81, 1951. 20. Stumbo, C.R., J.R. Murphy, and J. Cochran. Food Tech. 4, 321, 1950. 21. Meyer, K.F. Handbuck der pathog. Mikroorg. (Kolle, Kraus U. Uhlenhuth, 4th Edition) 4, 1268, 1928. 22. Burke, G.S, J. Inf« Dis. 33_, 274, 1923. 23. Andersen, A.A. J. Bact. 62_, 425, 1951. 24. Stumbo, C.R. Food Tech. 2_, 228, 1948. 25. Reed, L.J. and H. Muench. M. J. Hyg. 27_, 493, 1938. 26. Stevenson, J.W., V.A. Helson, and G.B. Reed, Can. J. Res. E, 25, 14,1947. 27. Wentzel, L.M. and M. Sterne/ Science 110, 259, 1949. 28. Sterne, M. and LaM. Wentzel, J. Imm. 65, 175, 1950. 29. Reed, G.B. Personal communication, 1953. 30. Hottle, G.A., C. Nigg, and J.A.-Lichty. J. Imm. 55, 255, 1947. 31. Nigg, C , G. Hottle, L.L. C o r i e l l , A.S. Rosenwald, and G.W. Beveridge. J. Imm. 55, 245, 1947. - 54 -APPENDIX I. - MEDIA A l l of the following media were routinely sterilized by autoclaving for 20 minutes at 15 pounds pressure. If they were not used on the same day as prepared, these media were boiled and cooled before use. It must be noted that they should not be reheated more than once. 1. Glucose peptone beef infusion broth, with added meat. (G.P.B.I.) (Reference -.James Morton, B.A. Thesis, 1944) Infuse one pound-of fat-free beef (mineed) in one l i t r e of tap water overnight in the refrigerator. Infuse at 60 to 65° C. for 45 minutes. Strain f i r s t through one thickness and then two thicknesses of cheese cloth. F i l t e r through $1 Whatman paper. Take the volume and add the following: Boil for three minutes and f i l t e r through #1 Whatman f i l t e r paper. Adjust to pH 7.8. Dispense in large tubes with about one inch of meat in the bottom and approxi-mately 25 ml. of broth. S t e r i l i z e . A sterile 50% glucose solution i s pre-pared. This i s added to the tubes, aseptically, before inoculating to give a f i n a l concentration of 2%. 2. Greenberg's Semi-Solid Reduced Medium. (Reference - L. Greenberg. Can. J. ?ub. H. 22, 314, 1941) Proteose peptone • . . . . . . . 10 grams Tryptone • 10 " Agar 3 " NaCl 0,5% Difco peptone 1.0% Na 2HP0 4.12H 2© 0,2% - 55 -Sodium thioglycollate . . . . . . . . . . . . . . . . . 1 gram Di s t i l l e d water . • • • 1000 ml* Boil, to dissolve ingredients. Adjust pH to 7.8, dispense and s t e r i l i z e . 3. Beef Infusion Broth (Difco). Infuse 50' grams of Bacto beef i n one l i t r e of d i s t i l l e d water at 50° C« for one hour. Heat to 80°C. for a few minutes. F i l t e r through #1 Whatman f i l t e r paper. Take the •wDlume and add: NaCl..-. .;• 0,5% Difco peptone . v . . . 1»0% Na2HP04.12H20 0,2% .Boil three minutes and f i l t e r . Adjust the pH to 7.8, dispense and s t e r i l i z e . 4. Beef Infusion Agar, with thioglycollate and starch supplements. (Reference - Wynne, E.S. and J.W. Foster, J. Eact. 55, 61, 1948). Beef infusion broth i s made in the usual way. The pH is adjusted to 8.0* Take the volume and add: Agar . • 1*5% Sodium thioglycollate • 0,5% Soluble starch . . 0,1% Dispense in 9 cc. amounts and s t e r i l i z e . 5. Brain Heart Infusion Broth ^Difco). Dissolve 37 grams of Bacto Brain-Heart Infusion in 1000 ml. of d i s t i l l e d water. Dispense and s t e r i l i z e . The pH i s 7.4. - 56 6. Fluid Anaerobic Medium* Bacto-beef extract . ' •• • 5.0 grams Pfanstiehl peptone . . . . . . . . . . . . . . . 20.0 " Sodium chloride • • • • • • • • • . » * . . . . . . . . . . 2 05 " Disodium phosphate • • • • • • • . . . . . . . . . . . . . * 2 o0 " Sodium thioglycollate 1.0 " L-cysteine . . . . . . . . . . . . . . . 0.75 " D i s t i l l e d Water 1000 ml. Boil three minutes and f i l t e r through #1 Whatman f i l t e r paper. Dispense and s t e r i l i z e . Add sterile glucose, aseptically, to 2%* APPENDIX II - METHODS OF ANAEROBIOSIS USED The following methods for obtaining anaerobiosis were routinely employed in the cultivation of the Clostridium botulinum cultures used in this research. 1. "Suitable" medium constituents for cultures incubated i n a i r . In G.P.B.I. the sterilized meat particles contain reducing substances which are effective i n maintaining anaerobiosis at the bottom of the tube. In addition, the ground meat acts as a mechanical block to air currents. In Greenberg's medium, a reducing agent, sodium thioglycollate, maintains the medium at a low Eh compatible with anaerobic growth. In addition. 0.3/2 agar is incorporated to reduce the diff\ision of oxygen in the medium. 2. MoIntosh-FiIdes' Anaerobic Jar. The original Mcintosh and Fildes method (Lancet, 1:768, 1916) for anaerobiosis consisted, br i e f l y , in enclosing the culture medium, in an a i r -tight vessel which had suspended in i t a piece of asbestos wool impregnated with palladium black. Hydrogen was then passed i n . This was occluded by the palladium and combined with any oxygen. The general principle has, of course, remained the same but the details of the procedure and apparatus have since been modified. (Fildes and Mcintosh, Br. J. Exp. Path. 2., 153, 1921j Cumming, Am. J. Pub. H. 22, 410, 1932) The jars used here were models 1085 and 1085-B ©£ the Arthur H. Thomas Co. Philadelphia, Pa. 3. Pyrogallic acid and sodium, hydcoxide Ten grams of dry pyrogallic acid were placed in a petri dish at the bottom of a copper jar and 10 ml. of IN. NaOH was added. The plates or other cultures - 58 -were quickly placed in the jar and the l i d screwed on tightly. Then illuminating gas was passed through. This method was not completely satisfactory and was used only when there were not enough Mclntosh-Fildes' jars. References} Rosenthal, L., J. Bact. 34, 317, 1937 Brewer, J.H., Science 95, 387, 1942. J 

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