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Studies on Brucella abortus Mathias, Douglas Gordon Browne 1940

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STUDIES ON BRUCELLA ABORTUS. by D.GUB!. HATHIAS, B..A. A Thesis Submitted as Part of the Requirements Towards the MASTER OF ARTS DEGREE l a y , 1940.. ASKMOWEEaBSEMBtETS. The v^r i t e r wishes t o express h i s appreciation*, to Doctor C. S. Dolman f o r t h e ; i n t e r e s t shown and the guidance received throughout t h i s work* The, numerous volunteers who made possible c e r t a i n portions of t h i s work are thanked f o r t h e i r co-ODeration. Il r . M. Darrach i s thanked f o r h i s assistance i n r e l a t i o n to the chemical aspects." G Q M E M T S . I * Some H i s t o r i c a l Considerations. I I . I n t r o d u c t i o n . I I I , Experimental* A. Raw M i l k Surveys.. B3* Adminis/bration of Vaccine to Humans* G. The Value of Vaccinia in. P r o t e c t i n g Mica Against Active I n f e c t i o n . D» The Production of B r u c e l l i n * I . The E f f e c t of pH on the V i a b i l i t y of B r u c e l l a abortus. IV:. Diacu&sion. . V» Summary. VI» Bib l i o g r a p h y . STUDIES,. ON, BRUGELLA ABORTUS. " I. SOME. HISTORICAL:CONSIDERATIONS• With the discovery by Bruae (1) i n 1886 of the "Micrococcus m e l l t e n s i a " and h i s subsequent :proof that i t was the causal microorganism of Mediterranean, Malta, or undulant f e v e r , there began the study of a genus of b a c t e r i a the inertness of which renders i t s i n v e s t i g a t i o n most d i f f i c u l t . The c h r o n i c i t y of the disease u s u a l l y r e s u l t i n g from i n f e c t i o n i s .another d i f f i c u l t o bstacle* The o r i g i n a l nomenclature applied to t h i s genus was misleading. Bang (2),.the Danish v e t e r i n a r i a n , i n 1897 was the f i r s t t o recognize the r o d - l i k s character- of a eocco-bacillus which he. proved t o be the causative agent; of "contagious a b o r t i o n " (Bang's disease) i n . c a t t l e and which!-he. c a l l e d " B a c i l l u s .abortus The two generic names "micrococcus" and " b a c i l l u s " misled bac-t e r i o l o g i c a l i n v e s t i g a t i o n s of the human disease and the disease i n c a t t l e i n t o separate channels f o r two decades. In 1918, Evans (3 ) , while en-gaged i n studying the b a c t e r i a l f l o r a of freshly-drawn milk, recognized the close r e l a t i o n s h i p between Bang's " b a c i l l u s " and Bruce's "micrococcus". P r i o r t o Evans* discovery, Traum (4) i n 1914 i s o l a t e d from an aborted pig foetus a b a c i l l u s he considered t o be "Bacterium abortus"; i n 1916 Good and Smith (5) reported t h i s b a c i l l u s as the e t i o l o g i c a l f a c t o r i n the production of i n f e c t i o u s abortion of swine. Meyer and Shaw (6) i n 1920, and Feusier and Meyer ( 7 ) , i n the same year, suggested the generic name " B r u c e l l a " i n honour of S i r David Bruce,- a suggestion since widely accepted. Huddleson's (8) studies on the b r u c e l l a group resulted i n his conclusion that there are three d i s -t i n c t species, .Brucella m e l i t e n s i s of. the goat,, B r u c e l l a , abortus of c a t t l e , and B r u c e l l a auls of swine, with other hosts aside from the prim-ary host i n each case. Huddles on *s nomenclature has been generally adopted and t h i s has freed the l i t e r a t u r e of misleading terminology. The term " b r u c e l l o s i s " i s used extensively now with reference t o the human manifestations of the disease previously c a l l e d Malta, Mediterranean, or undulant fever. I t i s a l s o f i n d i n g acceptance i n r e -l a t i o n t o the animal diseases. - IT. . IKtRODUCTIOII. B r u c e l l o s i s i s a public- health problem of considerable propor-f t i o n s and one that i s becoming more s e r i o u s . It ha3 spread a l l over the c i v i l i z - e d world and: a f f e c t s persons of a l l ages and both sexes regardless of occupation. As yet no s u c c e s s f u l s p e c i f i c or n o n - s p e c i f i c therapeutic agent has been found f o r the treatment of the disease. Brucellosis: i s a disease communicable to man from the lower animals by d i r e c t or i n d i r e c t contacts or by i n g e s t i o n of i n f e c t i v e an-imal products. Because of the high incidence of "contagious a b o r t i o n " (Bang's disease) i n the c a t t l e of the Fraser Valley and the dependence of Greater Vancouver on t h i s source of supply f o r i t s d a i r y products, l o c a l cases of human b r u c e l l o s i s might be a n t i c i p a t e d . During the past few years many such cases have been c l i n i c a l l y recognized, and v e r i f i e d by laboratory evidence. The inc r e a s i n g i n t e r e s t shown i n the epidemiological and immunological problems raised by these cases of* human, b r u c e l l o s i s led t o the present i n v e s t i g a t i o n which was be-gun i n 1937. . At the outset i t was deemed des i r a b l e t o obtain evidence r e l a t -ing to the extent of the r i s k of contracting human, b r u c e l l o s i s , i n terms of the. degree of contamination w i t h Br u c e l l a s of the l o c a l raw milk s u p p l i e s . . Such evidence was sought by conducting c u l t u r a l surveys at i n t e r v a l s on a l l pooled raw milk d i s t r i b u t e d i n the c i t y of Vancouver. A s e r i e s of experiments was planned to throw l i g h t on various possible means of producing a c t i v e immunity to B r u c e l l a s . A f t e r determining the average l e v e l of b r u c e l l a a g g l u t i n i n s i n a group of normal healthy s t u d -ents, and of mice, the e f f i c a c y of h e a t - k i l l e d suspensions, prepared from l o c a l l y - i s o l a t e d s t r a i n s , i n evoking production of s p e c i f i c agglut-i n i n s , was i n v e s t i g a t e d . The degree of p r o t e c t i o n thus afforded against l i v i n g B r u c e l l a s was tested i n mice.' F i n a l l y a few experiments were performed w i t h a view to determining the possible use of " B r u c e l l i n " as an immunizing agent, and the f e a s i b i l i t y of producing b r u c e l l a v a r i a n t s of unusual a n t i g e n i c e f f i c i e n c y . The i n v e s t i g a t i o n has involved study o f a number of s t r a i n s of B r u c e l l a e i s o l a t e d l o c a l l y , . Methods c u r r e n t l y a v a i l a b l e f o r d i f f e r e n -t i a t i o n show a l l these s t r a i n s to have been of the " B r u c e l l a abortus" v a r i e t y * I I I . , BXPERIHENTAL. A* Raw H i l k Surveys. Dolman and Hudson (9) presented evidence bearing upon the b r u c e l l o s i s hazard e n t a i l e d by the Consumption of raw milk i n and around Vancouver. Their report was based on a two-year period during which f i f -teen l o c a l cases of c l i n i c a l l y - a c u t e b r u c e l l o s i s were shown by s e r o l o g i c a l , c u l t u r a l , and epidemiological methods, to have been milk-borne and due to B r u c e l l a abortus. Following the above f i n d i n g s , i t was thought d e s i r a b l e t o under-take a more comprehensive survey which would involve examination of routine samples from a l l raw milk d i s t r i b u t o r s i n the c i t y of Vancouver. Two complete surveys; and.one d a i l y survey were made. Routine. The medium used i n c u l t u r i n g milk f o r B r u c e l l a abortus was •Huddleson's beef l i v e r i n f u s i o n agar containing 1$200 ?000 c r y s t a l v i o l e t . Plates of this, medium were poured 24 hours before they were spread w i t h three or four l o o p f u l a of 24-hour standing cream, centrifuged cream, and centrifuged'sediment r e s p e c t i v e l y , obtained from 15 0 . 0 . of milk sample, three plates being used f o r each f r a c -tion... Plates were incubated f o r s i x days at 37°G. i n ah atmosphere containing approximately twelve per cent carbon d i o x i d e . Suspicious colonies were then picked, t h e i r i d e n t i t y being confirmed by micro-sc o p i c examination, t h e i r carbon dioxide requirements, t h e i r s p e c i f i c a g g l u t i n a b i l i t y i n the presence of an anti-abortus serum of high t i t r a , and by t h e i r s u s c e p t i b i l i t y t o f u c h s i n , t h i o n i n , and pyronin. Results of the Summer Survey. The f i r s t survey was done over the period June 1 to August 10, 1938, during which time two milk samples were examined from every dairy purveying raw milk i n Vancouver. In a l l ^ 1,197 plates were spread, of which 161 were overgrown. Of the 1,036 readable p l a t e s , nine had t y p i c a l b r u c e l l a colonies on them, the t o t a l number of b r u c e l l a colonies on the nine plates being f i f t e e n . Results of the F a l l Survey. The l o c a l health a u t h o r i t i e s decided that the human b r u c e l l o s i s hazard could be e f f e c t i v e l y c o n t r o l l e d by the e l i m i n a t i o n of i n d i v -i d u a l "reactrars" from every herd supplying raw milk t o the c i t y . Three months f o l l o w i n g the adoption of t h i s p o l i c y , a second survey was begun i n an endeavour to assess the e f f i c a c y of t h i s p o l i c y . The second survey covered the period November and December, 1938* A t o t a l of 828 plates were spread of which 173 were over-grown. The 655 readable plates did not y i e l d a s i n g l e b r u c e l l a colony. Concurrently, Miss Hudson of the P r o v i n c i a l Board of Health lab o r a t o r i e s , Vancouver, d i d a p a r a l l e l survey. Her r e s u l t s are set f o r t h i n a report by Dolman, Hudson, and Mathias (10). The apparent discrepancy between the two surveys made upon sim-i l a r samples i s n©t r e a d i l y explained. A l l c o n s t i t u t e n t s of the medium were c a r e f u l l y checked and rechecked. The only v a r i a b l e s were personal technique and f l u c t u a t i o n s In the temperature of i n -cubation. The former f a c t o r was.dismissed f o l l o w i n g the obtainment of p r a c t i c a l l y i d e n t i c a l r e s u l t s by both t e c h n i c i a n s concerned, i n an experiment c a r r i e d out under i d e n t i c a l c o n d i t i o n s . Fluctuations i n the e l e c t r i c incubator used i n the greater part of t h i s work ex-tended over a range of s i x degrees from 35°C. t o 41°C., the range depending on the atmospheric temperature* Results of the Daily Survey on Two D a i r i e s * To gain information as to the number of days i n a month a raw milk supply would contain v i a b l e Brucellas, d a i l y samples were c u l -tured from two d a i r i e s over a period of one month, one having p r e v i o u s l y y i e l d e d very few p o s i t i v e s and one being c o n s i s t e n t l y p o s i t i v e . Each day of February, 1939, samples from these two d a i r i e s were plated. The r e s u l t s obtained i n our laboratory were much lower than those obtained concurrently at the P r o v i n c i a l l a b o r a t o r i e s . Twelve of the twenty-eight samples from the " p o s i t i v e " d a i r y y i e l d e d B r u c e l l a ^ the other dairy being c o n s i s t e n t l y negative. The con-current survey at the P r o v i n c i a l Laboratories y i e l d e d , correspon-d i n g l y , twenty-one p o s i t i v e out of twenty-eight samples and four p o s i t i v e out of twenty-eight. Conclusions. Results of the summer survey showed the presence of v i a b l e Brucellar i n c e r t a i n raw milk samples. The low incidence of p o s i t i v e cultu r e s obtained must i n part be a t t r i b u t e d to small inocula being spread on the plates t o compensate f o r the higher t o t a l colony counts obtained during the.hotter months. I t would be advisable to i n v e s t i g a t e f u r t h e r the e f f e c t of temperature f l u c t u a t i o n s on the growth of Brucellaefrom raw milk. The apparent discrepancy between r e s u l t s obtained i n our lab o r -a t o r i e s and the P r o v i n c i a l Laboratories during the d a i l y survey was i n a l l p r o b a b i l i t y due to the same f a c t o r as i n the f a l l survey --the incubator. B'-» The Administration of .Vaccine to Humans. " Preliminary Tests on Normal I n d i v i d u a l s . (a) The average a g g l u t i n i n t i t r e of the blood serum of the normal i n d i v i d u a l . Before commencing the i n v e s t i g a t i o n of the value of vac-cine i n inducing a high a g g l u t i n i n t i t r e i n humans, i t was nec-essary t o a s c e r t a i n the average t i t r e of a representative group of apparently normal i n d i v i d u a l s i n t h i s area. • Blood was taken by venepuncture from the median c u b i t a l veins of seventy unselected healthy volunteers and a g g l u t i n a t i o n t e s t s done on the serum. The antigen used was prepared according to Huddleson's method (8) as f o l l o w s t The surface of the l i v e r i n f u s i o n agar i n Roux f l a s k s was inoculated f a i r l y h e a v i l y from a 48-hour cul t u r e of B r u c e l l a abortus. (The c u l t u r e s used must be t y p i c a l s t r a i n s of B r u c e l l a abortus which have shown no tendency t o spontaneous a g g l u t i n a t i o n and which have manifested normal a g g l u t i n a b i l i t y . E i t h e r a monovalent or polyvalent antigen may be employed.) The antigen b o t t l e s were incubated f o r 48 - 72 hours at 3V°C. The growth was washed-off with a phenolized s a l i n e s o l -u t i o n containing ,0,85$ sodium c h l o r i d e ( C P . ) and 0.5$ phenol c r y s t a l s ( C P . ) , It was put i n the icebox and allowed to stand u n t i l the microorganisms were k i l l e d . The suspension was^  kept w e l l mixed by shaking and was cu l t u r e d every 3 - 5 days f o r s t e r i l i t y . When s t e r i l e , i t was f i l t e r e d a s e p t i c a l l y through cotton and gauz e. For use, the antigen was d i l u t e d with normal s a l i n e to MacFarland f 2 standard of t u r b i d i t y . The standard p r a c t i c e f o r a g g l u t i n a t i o n t e s t s was t o make doubling d i l u t i o n s , the i n i t i a l tube having a f i n a l serum con-ce n t r a t i o n of 1:5, the f i n a l tube l t 6 4 0 . Adequate co n t r o l s were set up with each t e s t . The t e s t s were incubated f o r 48 hours i n the 37°C water-bath, removed and l e f t at room temperature f o r 24 hours. The t e s t s were read at 24, 48, and 72 hours ( f i n a l reading). The r e s u l t s obtained from the seventy apparently normal volunteers are set f o r t h i n Table I* TABLE I . AGGLUTININ TITRES OF SEVENTY APPARENTLY NORMAL INDIVIDUALS. Highest d i l u t i o n A g g l u t i n i n No. of Per cent of showing a g g l u t i n a t i o n t i t ra i n d i v i d u a l s i n d i v i d u a l s 1 . 5 0 2 2.8 l s 5 P 8.6 1«5 c 5 7.1 l t l O P 1 1.4 i t i o c 4 5.7 ls20 p 13 18.6 •'# ls20 c; 1 1.4 ls40 P 16 22.9 # T t 4 Q c 1 1.4 , lj'ao'. - P 13 18.6 1*160 P 5 7.1 ls320 P 3 4 . 3 c as complete a g g l u t i n a t i o n P SB p a r t i a l i) 0 SB no « Complete a g g l u t i n a t i o n i n a ls20 d i l u t i o n or higher was recorded as a p o s i t i v e r e s u l t . On t h i s b a s i s , only two i n d i v i d u a l s or 2,8% showed a p o s i t i v e t e s t . In no instance d i d t e s t s d e s i g -nated as " p a r t i a l * * show complete a g g l u t i n a t i o n i n lower d i l u t -i o n s . (b) The "Brucellergen" s k i n t e s t a p p l i e d to apparently normal i n d i v i d u a l s . As an a d d i t i o n a l check on the apparently normal volunteers, who we ra subsequently t o be given vaccine, the intradermal a l l e r g i c t e s t as developed by Huddleson ( l l ) was employed. The b r u c e l l a nucleoprotein s o l u t i o n , designated "Brucellergen", was obtained from his laboratory. The technique f o r the pre-par a t i o n of t h i s ether-washed antigen i s described i n " B r u c e l l a 10. Infections i n Animals and Man"(8)» The t e s t was performed on f i f t e e n healthy volunteers by i n -j e c t i n g intradermally 0.1 c.c. of the nucleoprotein s o l u t i o n i n the l a t e r a l surface of the forearm. Readings were made i n 24 and 48 hours i n order to exclude early non-specific reactions. The p o s i t i v e r e a c t i o n i s characterised by a c e n t r a l red area at l e a s t 4 X 4 mm. with an outer p i n k i s h area at l e a s t 20 X 20 mm.s oedema u s u a l l y being present. Of the f i f t e e n i n d i v i d u a l s , f i v e , or one-third, showed a p o s i t i v e r e a c t i o n . Administration of Vaccine. (a) Source of c u l t u r e s . / Five s t r a i n s of B r u c e l l a abortus, a l l i s o l a t e d at the P r o v i n c i a l Laboratories, Vancouver, by blood cultures from f i v e human p a t i e n t s , were used i n preparing the polyvalent vaccine. (b) Preparation of vaccine. Each of the f i v e s t r a i n s was seeded on eight beef l i v e r i n -f u s i o n s l a n t s . A f t e r 72 hours incubation i n an atmosphere of approximat ely carbon d i o x i d e , the growth from, a l l these tubes was washed—off with s t e r i l e s a l i n e , the suspension being d i v i d e d i n t o two p a r t s , one f o r p a r e n t e r a l i n j e c t i o n s , the other f o r o r a l a d m i n i s t r a t i o n . Both f l a s k s were heated at 60°C. f o r 11. 1 hour. S t e r i l i t y t e s t a were done on both. The suspensions corresponded to a MacFarland // 6 t u r b i d i t y standard. (c) P arenteral i n j e c t i o n . Bight volunteers were given s i x subcutaneous i n j e c t i o n s at weekly i n t e r v a l s , the d i l u t i o n s of the doses, of the sus-pension as prepared i n (b) above, being 0.1 c.c. of 1:20, 0.1 c.c. of 1:10, 0.1 c.c. of 1:10, 0.15 c.c. of 1:10, 0.1 c.c. of l s 5 , and 0.15 c.c. of 1:5. A g g l u t i n i n t i t r e s were done on blood serum obtained from the above volunteers p r i o r t o the i n i t i a l i n j e c t i o n and again one week a f t e r the f i n a l i n j e c t i o n . The r e s u l t s are compared i n Table I I * •AgGUJtlNIM .TITRES BEFORE AND AFTER PARENTERAL INJECTION.-OF TABLE; I I . VACCINE. A g g l u t i n i n t i ' t r e before i n j e c t i o n s A g g l u t i n i n t i t re a f t e r i n j e c t i o n s c i n 1:40 p i n ls80 p i n 1:80 p i n 1:160 p i n 1:80 p i n 1:160 p. i n 1:40 p i n 1:80 c i n 1:320 c i n 1:160 c i n 1:160 c i n 1:160 p+ i n 1:160 p+ i n 1:160 p i n 1:320 p i n 1:30 c sr complete a g g l u t i n a t i o n . P + - about 50% " p — p a r t i a l " On a n a l y s i s i t i s seen that four of the volunteers showed a 12. marked, increase i n a g g l u t i n i n t i t r e s , two showed a s i g n i f i c a n t r i s e , one evidenced a s l i g h t r i s e , whereas one volunteer showed no change. (d) Oral a d m i n i s t r a t i o n . Eight healthy volunteers drank s i x doses of the suspension prepared as i n (b) above, at weekly i n t e r v a l s , the increasing doses being 1 c.c. of a 1:10 d i l u t i o n , 1 c.c. of 1:1, 2 c.c., 4 c . c , 6.5 c.c. and 10 c.c. Over the six-weeks'* period, a t o t a l of 23.6 c.c. of a suspension corresponding to a MacFarland //' 6 t u r b i d i t y standard, or the equivalent of over 210 b i l l i o n Brucellae abortus, was taken by mouth. None of the volunteers showed any detectable increase i n serum a g g l u t i n i n t i t r e . ' r Conclusions. In only one of the f i f t e e n healthy volunteers the b r u c e l l e r g s n s k i n t e s t and the a g g l u t i n a t i o n t e s t i n d i c a t e d a p o s i t i v e r e a c t i o n . Four i n d i v i d u a l s gave p o s i t i v e brucellergen s k i n reactions and negative a g g l u t i n i n t e s t s , whereas ten i n d i v i d u a l s were negative t o both. This lack of conformation i n d i c a t e d the p o s s i b i l i t y of the presence of a l o c a l h y p e r s e n s i t i v i t y to b r u c e l l a p r o t e i n without evidence of detectably increased a g g l u t i n i n s . Oral a d m i n i s t r a t i o n of a h e a t - k i l l e d polyvalent vaccine was proved to be i n e f f e c t i v e i n s t i m u l a t i n g a g g l u t i n i n production. Thus the consumption of pasteurized milk containing dead Brucellae would not induce s i g n i f i c a n t a g g l u t i n i n t i t r e s . 1 3 . The effectiveness of the h e a t - k i l l e d polyvalent vaccine, when administered p a r e n t e r a l l y , i n inducing an increased a g g l u t i n i n t i t r e has been shown. Six out of eight persons evidenced s i g n i f i c a n t i n -creases i n blood serum a g g l u t i n i n s . The a d v i s a b i l i t y of a much larger experimental group i s r e a l i z e d . Only then could a more comprehensive conclusion be drawn on the use of the vaccine. G-. The Value of Vaccine i n P r o t e c t i n g Mice Against .Active I n f e c t i o n . The value of increased a g g l u t i n i n t i t r e i n combatting a c t i v e disease was worthy of i n v e s t i g a t i o n . The a g g l u t i n i n t i t r e of the normal mouse. From 0.8 t o 1.0 c.c. of blood was obtained by cardiac puncture from each of eight normal mice under ether anaesthesia. The pooled sera gave no i n d i c a t i o n of any b r u c e l l a a g g l u t i n i n s . The above r e s u l t was confirmed by two a d d i t i o n a l experiments. The approximate Minimal Lethal Dose f o r mice. As the r e s u l t of preliminary experiments, i t was. found necessary to g i ve a heavy dose, due t o the v a r i a b i l i t y of the response to ex-perimental b r u c e l l a i n f e c t i o n i n mice, thus ensuring f a i r l y uniform r e s u l t s . At four weekly i n t e r v a l s , the # M.L.D. v/as checked on groups of f i v e mice each. The most consistent r e s u l t s were obtained by the i n j e c t i o n of 0.2 c . c . of a (X 10) MacFarland § 2 t u r b i d i t y standard, j£ M.L.D s Minimal Lethal Dose. 14. of the Huyck s t r a i n of B r u c e l l a abortus, t h i r t e e n of twenty mice dying c o n s i s t e n t l y between 32 and 40 hours. The M.L.D. f o r p r a c t i c a l purposes was, t h e r e f o r e , 0.2 c c . of a (X 1$) MacFarland § 2 t u r -b i d i t y standard i n j e c t e d i n t r a p e r i t o n e a l l y * P a r e n t e r a l Immunization. Two groups of mice, comprising t h i r t e e n males and t h i r t e e n females r e s p e c t i v e l y , were given seven doses of the h e a t - k i l l e d polyvalent vaccine at weekly i n t e r v a l s , i n t r a p e r i t o n e a l l y . The suspension was analogous to that used i n the human experiments. The doses were 0.05 c . c , 0*1 c c , 0.1 c . c , 0.15 c . c , 0.2 c . c , 0.2 c c , and 0.2 c.c. Three mice per group were s a c r i f i c e d i n order to obtain, by cardias puncture, a s u f f i c i e n t quantity of pooled blood f o r a g g l u t i n a t i o n t e s t s . The t i t r e of the male blood was + f f i n 1.640, the female -fff- i n 1:320. The same day the remaining members of the two groups were each given 0.2 c c of a (X 10) MacFarland $ 2 t u r b i d i t y standard, i n t r a -p e r i t o n e a l l y , of the Huyck s t r a i n of B r u c e l l a abortus* This was the quantity found by previous experiment to represent 1 M.L.D. Of the males, one died from the effects of f i g h t i n g , the nine remaining appeared quite normal. Of the females, one died a f t e r 48 hours but the post mortem signs were not of the c h a r a c t e r i s t i c type described below. Of twelve normal c o n t r o l mica in j e c t e d w i t h the same suspension, eight died between 33 and 40 hours. Three of the four remaining died a f t e r about 56 hours. 15. Oral Administration. Two groups of mice were sel e c t e d as i n the experiment above. They were a l l fed seven doses, by graduated eye-dropper, of the same b a c t e r i a l suspension at weekly i n t e r v a l s . These o r a l doses were approximately 0.125 c . c , 0.25 c c , 0.125 c c , 0.125 c . c , 0.125 c c , 0.2 c c , and 0.2 c . c Three- mice per group were s a c r i f i c e d , as i n the former exper-iment,, f o r blood a g g l u t i n a t i o n t e s t s . The t i t r e of the male, blood was -f- i n 1*40, the female + i n l s 2 0 . The remaining members of the groups were each given 1 M.L.D. i n t r a p e r i t o n e a l l y . Nine of the t e n males died between 46 and 54 hoursj a l l the females died, nine between 33 and 37 hours T one at 97 hours. The t y p i c a l : post mortem signs, .inffche deaths- apparently r e s u l t i n g from the b r u c e l l a i n j e c t i o n s were. Hemorrhagic lungsj bloated stomach; pale l i v e r , kidneys and spleenj extensive subcutaneous hemorrhage and engorgement of the p e r i p h e r a l vessels$ enlarged adrenals and g a l l bladder. A l l the'mice were autopsied, pure c u l -tures of Brucellgejffere r e a d i l y obtained at: autopsy from the kidney, heart, spleen and l i v e r i n twenty-six out of t h i r t y - t w o attempts. Gonclusq ona. Normal mice have no demonstrable a g g l u t i n i n s f o r B r u c e l l a abortus. The M.L.D. can be ascertained f a i r l y accurately f o r mice, r e l a t i v e l y large numbers of Brucellas being necessary to produce a 16. f a t a l termination i n approximately 40 hours. . ; P a r e n t e r a l i n j e c t i o n , of a h e a t - k i l l e d polyvalent vaccine pro-duced s i g n i f i c a n t r i s e s i n t i t r e s of both male and female mice, the males showing the greater response. The enhanced a g g l u t i n i n t i t r e apparently protected both the male and female mice against a c t i v e i n f e c t i o n . Oral a d m i n i s t r a t i o n of the vaccine d i d not cause a demonstrable r i s e i n a g g l u t i n i n t i t r e i n e i t h e r the males or females; t h i s t r e a t -ment d i d not e l i c i t the response of a d d i t i o n a l p r o t e c t i v e forces against a c t i v e i n f e c t i o n . The males l i v e d on the average 12 hours longer than the females. P. The Production of B r u c e l l i n . The foregoing evidence, re l a t i n g s - t o the production of a g g l u t i n i n s by i n j e c t i o n of k i l l e d c u l t u r e s of Brucellas r a i s e d the question, as t o the e f f i c a c y of metabolic by-products of Brucellar i n lnduaing:.-the> production of s i m i l a r or a d d i t i o n a l a n t i b o d i e s . Huddleson (11) was the f i r s t t o develop and use a material,, analogous, to t u b e r c u l i n i n preparation, which he c a l l e d " B r u c e l l i n " . . The c l i n i c a l use of t h i s m a t e r i a l i s s t i l l i n the ear l y experimental stage. Apparently i n j e c t i o n of b r u c e l l i n evokes.a leucocytoais of the neutrophil© type, besides i n c r e a s i n g the s p e c i f i c phagocytic power of the blood. Media Employed. As i s generally recognized, the i n j e c t i o n of the complex p r o t e i n s o l u t i o n s i n common use f o r c u l t u r i n g b a c t e r i a often has undesirable a f t e r - e f f e c t s . A synthetic medium composed of amino-acids. plus. 17. ess e n t i a l Igrowth fact orsy which wculd s u s t a i n adequate growth of B r u c e l l a ^ obviously would be advantageous. Huddleson had employed peptic digest l i v e r broth medium f o r h i s preparation of b r u c e l l i n (12). In the present i n v e s t i g a t i o n s , the accepted medium f o r the c u l t i v a t i o n of Brucellag.beef l i v e r i n f u s i o n brothi at a pS of 6.8, was one type of medium used. In h i s recent p u b l i c a t i o n , Huddleson (13) has adopted.this medium in:the production of b r u c e l l i n . I t was found i n preliminary experiments that a s l i g h t l y modified form of Gladstone's s y n t h e t i c medium (14)., which he had developed f o r the c u l t i v a t i o n , of Staphylococci, would support the growth of three s t r a i n s of B r u c e l l a abortus without the necessity of t r a i n i n g the cultures to grow on the medium. Therefore,it was used i n the pro-duction of b r u c e l l i n . The composition i s given below. Composition.per L i t r e ; of the Synthetic Medium. . KHigP% , ' '4:.5 gm0., Water 500.G c.c. N/1 NaOH 26.0 c.c. 5% ami no-acid base 200.0 c c . Ferrous ammonium sulphate M/500 i n M/50 HG1 25.0 c c MgS%.7H 20 . " M/60 10.0 C C . NaM$3 •'• .. H/5 '" •<•.>• io,o c c Vitamin B i (Thiamin) M/20Q0 i n l/lOOO HOI d i l u t e d l/lOO 20.0 c c N i c o t i n i c acid, (amide) H/20Q d i l u t e d l / l O 20.0 c c Glucose M/2, 25.0 c c Adjust pH to*7.4 with N/5 NaOH. Water t o make 1000.0 c c . S t e r i l i z e by Se i t z f i l t r a t i o n . #The amino-acid base was obtained from proteose-peptone by hydrolyzing $f<> protsbse-peptone f o r 40 hours i n b o i l i n g 2.5 N HgSO^. The H 2 S O 4 must be exactly n e u t r a l i z e d with Ba(0H)2 so that no barium or sulphate ions remain. The r e s u l t i n g p r e c i p i t a t e was separated by Buchner f i l -t r a t i o n . 18. In a d d i t i o n to the above c o n s t i t u e n t s , Gladstone incorporated other'amino-acids i n h i s medium, and used nicotinamide i n place of n i c o t i n i c a c i d . The a d d i t i o n a l amino-acids were omitted as being un-necessary. Routine. Two rec e n t l y i s o l a t e d smooth s t r a i n s of B r u c e l l a abortus were used i n the production of b r u c e l l i n , the Huyck s t r a i n i s o l a t e d from the blood of a human s u f f e r i n g from b r u c e l l o s i s , and the S 12 s t r a i n Isolated from raw milk. Each s t r a i n ; was inoculated into separate 250 c.c. amounts of both the beef l i v e r i n f u s i o n broth and the synthetic medium. A generous l o o p f u l from a 48-hour beef l i v e r i n f u s i o n agar slan t -was the inoculum i n each instance. The f l a s k s were incubated at 37.5°G. .in an atmos-phere with ah increased carbon dioxide tension of about twelve per cent by volume above that of atmospheric a i r . I f , at the end of f i v e days, growth f a i l e d to appear i n any f l a s k , i t was re-inoculated. Every f i v e days each f l a s k was examined f o r growth, shaken c a r e f u l l y , and re-incubated under the above c o n d i t i o n s . In twenty-four days a l l f l a s k s evidenced f a i r l y heavy growth. Each was checked as- to the p u r i t y of the growth by p l a t i n g s u i t a b l e q u a n t i t i e s on beef l i v e r i n f u s i o n agar. No contaminating b a c t e r i a appeared on the plates i n f i v e days. Each f l a s k of broth culture was now Se i t z f i l t e r e d . S t e r i l i t y t e s t s were devoid of growth over a seven-day period. Merthiolate,, l/5000, was added to each f l a s k and a f t e r standing 2 hours the b r u c e l l i n was dispensed into s t e r i l e v i a l s . To prove the innocuous nature of the b r u c e l l i n , 1 c.c. of each of the four preparations was inoculated i n t r a p e r i t o n e a l l y i n t o each 19. of three* mice. The b r u c e l l i n s prepared with the s y n t h e t i c medium gave no evidence of causing discomfort} the beef l i v e r i n f u s i o n broth b r u c e l l i n s apparently caused a t r a n s i t o r y discomfort. A l l mice re-mained i n normal health. Use of B r u c e l l i n . The opportunity arose f o r the t r i a l of b r u c e l l i n i n a human ap-parently s u f f e r i n g from b r u c e l l o s i s . Because of the o r i g i n of the c u l -t u r e , the b r u c e l l i n produced by the Huyck s t r a i n grown on the synthetic medium was s e l e c t e d f o r the t r i a l . To t e s t the r e a c t i o n of the normal human to the i n j e c t i o n of t h i s b r u c e l l i n preparation, two volunteers were each given 0.1 c.c. i n t r a -muscularly. No discomfort whatsoever was experienced by e i t h e r volun-t e e r . Three days l a t e r the same two i n d i v i d u a l s received 0.3 c.c. of the same preparation intramuscularly. No apparent re a c t i o n occurred. The p a t i e n t (Miss McL.) was diagnosed as suffering-from b r u c e l l o s i s on the c l i n i c a l symptoms of general l a s s i t u t e , weakness, lack of energy, easy t i r i n g , c h i l l s , headaches, leucopenia, and loss of weight, and was supported by the laboratory f i n d i n g of a blood serum a g g l u t i n i n t i t r e f o r B r u c e l l a r of j- in-.'1.320. She was con-f i n e d t o bed. • Her physician administered the b r u c e l l i n intramuscularly i n con-secutive weekly doses of 0.1 c . c , 0.2 c c , 0.3 c . c , 0«5 c.c. and 0.9 c . c , a t o t a l of 2 c c The only symptom evidenced from the i n -j e c t i o n s was a a l i g h t t r a n s i t o r y r i s e i n temperature 24 hours a f t e r each i n j e c t i o n . Following the fourth i n j e c t i o n the patient regained apparently-normal h e a l t h . At the present time, approximately eight 20. months, a f t e r treatment, the patient has had no recurrence of symptoms. .Conclusions. The s y n t h e t i c medium supported the growth of two s t r a i n s of B r u c e l l a abortus. B r u c e l l i n preparations proved innocuous to mice and the prepar-a t i o n obtained by-growth of the Huyck s t r a i n on the synthetic medium gave no de l e t e r i o u s e f f e c t s when i n j e c t e d into two normal human v o l -unteers . -In one c l i n i c a l l y . t y p i c a l case of subacute b r u c e l l o s i s , adminis-t r a t i o n of b r u c e l l i n derived from a synthetic medium seemed to bring about d e f i n i t e improvement. " 5. The E f f e c t .of; pH on the ".'Viability' of B r u c e l l a abortus.' The r e s u l t s of the raw milk surveys, provided ample evidence that many persons i n Vancouver d a i l y were consuming thousands of v i a b l e B r u c e l l a e and yet the number of cases of b r u c e l l o s i s apparently occurring remained s m a l l . Consideration was given to the p o s s i b i l i t y that the a c i d of normal human g a s t r i c j u i c e proved l e t h a l t o the greater number of Brue e l l a e and such s u r v i v o r s as there were proved too few to produce d i s -ease. '""-'-Experiments ?/era devised to a s c e r t a i n the pH range of tolerance of one s t r a i n of B r u c e l l a abortus, i n an adequate medium. Routine. , . In these experiments the '-"Barons 4" s t r a i n of B r u c e l l a abortus ,21. was used throughout. The procedure consisted In seeding uniform i n -ocula into 10 c.c. al i q u o t s of beef l i v e r i n f u s i o n broth, the pH of which had been adjusted to cover a range from approximately 1 to 9. The standard inoculum f o r each tube was thre6 l o o p f u l s of a five-day beef l i v e r i n f u s i o n broth c u l t u r e , which corresponded to a HaeFarland # 4 t u r b i d i t y . Determinations, of pH were made with a standard Leads and Northrup quinhydrone potentiometer. Flasks of the medium v/ere I n i t i a l l y ad-justed w i t h concentrated MaOH or concentrated HGl to- cover the range 1 to 9. These f l a s k s were incubated f o r 48 hours to allow s t a b i l i z -a t i o n of the pH, a f t e r which the pH determinations on respective samples were 1.1, 2.3, 2.9, 3.8, 5.2, 6.1, 7.1, 7.9, and 8.7. Thd standard inoculum was'' evenly dispersed i n each t e s t broth. Sub-cultures were taken i n t r i p l i c a t e from each tube at 15 minutes, 30 minutes, 1, 2, 4, and 16 hours, and plated om beef l i v e r infusion-agar at pH 6.8. Control plates were prepared i n t r i p l i c a t e w i t h s u i t a b l e d i l u t i o n s , of the inoculum to prove the v i a b i l i t y of the or-ganisms. A l l plates were incubated f o r 96 hours at 37 ° C , under the standard conditions of increased carbon dioxide t e n s i o n of the atmos-phere, to ensure that a l l v i a b l e organisms would form c o l o n i e s . Results . No growth occurred from any subcultures from broths of pH 1.1, 2.3, and 2.9 r e s p e c t i v e l y . Subcultures at 15 minutes, .30 minutes-, and 1 hour, from the broth at pH 3.8, sho\?ed decreasing amounts of growth; those taken at l a t e r i n t e r v a l s were negative. Subcultures from the broths at a l l other pH l e v e l s t e s t e d y i e l d e d abundant growth. 22. Three, d i s t i n c t colony types were obtained. Two ;of these were a t y p i c a l and were produced from the 15 minute, 30 minute, 1 and 2 hour subcultures. Theae were much l a r g e r (5-10 mm.), one being opaque and. . a buff colour, the other s l i g h t l y s m aller, t r a n s l u c e n t , and e x h i b i t i n g two zones, an. inner b u f f colour and a periphery showing pale green. In every plate showing growth, t y p i c a l colonies we're found. These were s m a l l , almost c o l o u r l e s s , and t r a n s l u c e n t . No apparent change occurred in. microscopic morphology. Conclusions. . "The l i m i t i n g pH f o r the v i a b i l i t y of the *Barona..'4* s t r a i n of B r u c e l l a abortus apparently l i a s between 2.9 and 3.8.. The pH obtaining i n the stomach, when milk i s ingested alone or with other foods i s c o n j e c t u r a l . Some means of mechanical p r o t e c t i o n w i l l a l s o be afforded B r u c e l l a e by•foodstuffs i n t h e i r passage through, the stomach. I t i s c l e a r l y d i f f i c u l t t o evaluate the pro-t e c t i o n afforded by the g a s t r i c j u i c e , but, a l l other circumstances being equal, ingested B r u c e l l a e w i l l more than l i k e l y survive contact with a g a s t r i c j u i c e of abnormally low a c i d i t y . ; The a t y p i c a l colonies obtained seemed t o be intermediate i n form between -the t y p i c a l l y smooth and those described by s e v e r a l workers as rough. They p o s s i b l y f a l l under the **I v a r i a n t " of Brucellae des-crib e d by Henry' (16). 23 IT DISCUSSION". The raw milk surveys have supplied s u f f i c i e n t evidence t o show that* d a i l y , i n Vancouver, many people are ingesting large numbers of v i a b l e B r u c e l l a abortus i n certain, raw m i l k s . Despite t h i s , the apparent case i n -cidence i s low* The fac t o r s i n the host or of the microorganism, or both, which determine the onset of the disease i n some persons and the resistance of others to the i n f e c t i o n , under apparently i d e n t i c a l c o n d i t i o n s , remain '•unknown* The experiments with mice gave evidence that i n j e c t i o n s of k i l l e d vaccine protected the mice against subsequent l e t h a l doses of B r u c e l l a s * In human beings, although the i n j e c t i o n s r e s u l t e d i n an enhanced a g g l u t i n i n t i t r e , t h i s alone i s a poor index of immunity. The introduction, of a s y n t h e t i c medium f o r the c u l t i v a t i o n of B r u c e l l a a has many i n t e r e s t i n g p o s s i b i l i t i e s . In such a medium, devoid of p r o t e i n s , when, the b a c t e r i a l c e l l s are removed by S e i t z : f i l t r a t i o n , any proteins, i n the f i l t r a t e w i l l have re s u l t e d from the metabolic a c t i v i t i e s of the microorganisms or the a u t o l y s i s of the c e l l s themselves. Huddles oa, i n h i s most recent, p u b l i c a t i o n " B r u c e l l o s i s i n Han and Animals" (13), r e p o r t i n g on the treatment of 500 cases of b r u c e l l o s i s w i t h b r u c e l l i n , b e lieves t h a t , of a l l the s p e c i f i c and n o n - s p e c i f i c pre- ' paratlons used to date, b r u c e l l i n i s the one in, his experience which shows most promise of success. As has been mentioned, his b r u c e l l i n has been prepared u t i l i z i n g beef l i v e r i n f u s i o n broth. He r e a l i z e s that the bene-f i c i a l treatment r e s u l t i n g from t h i s complex p r o t e i n m a t e r i a l may r e s u l t from non-specific p r o t e i n shock* In the above publication: the f i n a l sen-tence concerning the use of b r u c e l l i n i n treatment readsj "Eventually a preparation, of B r u c e l l i n w i l l be a v a i l a b l e that i s free from the extraneous constituents that are t o be found in. the present product". B r u c e l l i n pre-pared from a synthetic- medium might prove to be the product sought. It I s unfortunate that circumstances have prevented the use of la r g e r groups of humans and animals. The experiments with b r u c e l l i n are not s u f f i c i e n t l y extensive to permit d e f i n i t e conclusions. Moreover, i t should be emphasized that elab-o r a t i o n of a synthetic medium f o r production; of b r u c e l l i n i s as yet i n an ea r l y stage. Further experiments are. planned i n v o l v i n g use of a syn t h e t i c medium i n which hydrolyzed edestin replaces the amino-acid base obtained from proteose-peptone, and t o which s e l e c t e d amino-acids are added. Some recent r e s u l t s obtained with t h i s medium gave promise of a superior product. V. SUMMARY. (1) Of 2,110 readable plates obtained during the raw milk surveys i n the period from June ± ? 1938, t o February 28, 1939, 41 had t y p i c a l b r u c e l l a c o l o n i e s . (2) Oral a d m i n i s t r a t i o n of a k i l l e d vaccine proved i n e f f e c t i v e i n s t i m - , u l a t i n g a g g l u t i n i n production i n humans and i n mice. (3) P a r e n t e r a l a d m i n i s t r a t i o n of a k i l l e d vaccine caused s i g n i f i c a n t increases i n blood serum agglutinins, i n s i x out of eight persons. (4) P a r e n t e r a l i n j e c t i o n of a k i l l e d vaccine produced s i g n i f i c a n t i n -creases i n blood serum a g g l u t i n i n t i t res of. both male and female mice. 25. This enhanced a g g l u t i n i n t i t r e was apparently associated with the . - ' * p r o t e c t i o n afforded both sexes against a c t i v e i n f e c t i o n . A s y n t h e t i c medium has been, developed which supports the growth of B r u c e l l a abortus. A b r u c e l l i n was prepared u t i l i z i n g the foregoing synthetic medium. This b r u c e l l i n preparation seemed to brin g about d e f i n i t e improvement i n one c l i n i c a l l y , diagnosed case of b r u c e l l o s i s . The l i m i t i n g pH f o r the v i a b i l i t y of the "Barons 41* s t r a i n of B r u c e l l a abortus apparently l i e s between pH 2.9 and 3.8. 26. vT.. BIBLIOGRAPHY. (1) Bruce, D. (1887) P r a c t i t i o n e r . Lon., 391 161. (As quoted by Huddles on, 1934, i n * " B r u c e l l a Infections i n Animals and Han'*.) (2) Bang, 3. (1897) Jour. Comp. Path, and Therap.,Xs 125. (As quoted by G i l t n e r , 1934, i n : " B r u c e l l o s i s , a P u b l i c Health Problem".) (3) Evans, A l i c e 0. (1918) Jour. I n f . Dis., 22g 580-593. (4) Traum, J.E. (1914) Rep. Chief Bur. Anim. Industry, U.S.D.A., 30. (5) Good, E.S., and Smith, W.V. (1916) Jour. Bact., It 415-422. (6) Meyer, K.F., and Shaw, E.B;. (1920) Jour. I n f . Dis., 27_t 173. (7) Feusior, M.L», and Meyer, K.F. (1920) Jour. I n f . Dis., _27s 185. (8) Huddleson, I.F. (1934) " B r u c e l l a I n f e c t i o n s i n Animals and Man." Commonwealth Fund, Mew York. (9) Dolman, C.E., and Hudson, V". (1938) Canad. Pub. Health Jour., 291 236. (10) Dolman, C.E., Hudson, V., and Mathiaa, D.G.B. (1939) Canad. Pub. Health Jour.. 301 100. (11) Michigan Technical B u l l e t i n No. 149 (1936)s 42. (12) Michigan Technical B u l l e t i n No. 149 (1935)s 43. (13 ) Huddles on, I.F. (1939) " B r u c e l l o s i s i n Man and Animals," Common-wealth Fund, New York. (14) Gladstone, G.P. (1937) B r i t . Jour. Exp. Path., 18s 322. (15) Carlson, A.J. (1923) P h y s i o l . Rev., 3.* !• (16) Henry, B.S. (1933) Jour. I n f . Dis.,52; 374. 

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