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Studies on staphylococcus enterotoxin Wilson, Robert J. 1937

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STUDIES OF STAPHYLOCOCCUS EiTTSROTOXIlT  J . W i l s o n , B. A,  A t h e s i s submitted t o the Department o f B a c t e r i o l o g y and P r e v e n t i v e M e d i c i n e , University o f B r i t i s h Columbia i n p a r t i a l f u l f i l l m e n t o f the requirements f o r the degree o f Master o f A r t s .  Hesearch d i r e c t e d by C  E . Dolman, M«B,,,, B.S. Lond,,. M.R.-CeP„ • Lond., DcP.H.  Thesis submitted" i n A p r i l , 1937.  S t u d i e s on Staphylococcus E n t e r o t o x i n *  Introduction.  W i t h i n r e c e n t y e a r s , voluminous r e p o r t s  have appeared  d e a l i n g w i t h the d i v e r s t o x i c p r o p e r t i e s of f i l t r a t e s , pared under s u i t a b l e c o n d i t i o n s , staphylococcus..  pre-  from c e r t a i n s t r a i n s of  The c o m p l e x i t y of these f i l t r a t e s was  i l l u s t r a t e d by the demonstration of a g a s t r o - i n t e s t i n a l  further irri-  t a n t i n f i l t e r e d b r o t h p r e p a r a t i o n s from s t r a i n s i m p l i c a t e d i n outbreaks o f f o o d - p o i s o n i n g . With the r e c o g n i t i o n of this m i c r o organism as a p o t e n t i a l e t i o l o g i c a l agent o f g a s t r o - e n t e r i t i s , a number of f o o d - p o i s o n i n g epidemics were r e p o r t e d i n which staphylococcus was  incriminated.  As e a r l y as 1907, Owen ( l ) d e s c r i b e d an outbreak i n which 19 persons s u f f e r e d from acute g a s t r o - e n t e r i t i s f o l l o w i n g a meal of d r i e d beef, contaminated by l a r g e numbers of s t a p h y l o cocci.  A milk-borne epidemic was r e p o r t e d by Barber (£) i n the  P h i l i p p i n e s i n 1914.  He observed acute g a s t r o - i n t e s t i n a l d i s -  t r e s s i n a nurfjber of persons who had i n g e s t e d m i l k contaminated by an a l b u s s t r a i n o f s t a p h y l o c o c c u s . I n t h i s case, more conc l u s i v e evidence was produced before the c a u s a t i v e micro-organism was i n c r i m i n a t e d .  Barber i n o c u l a t e d the organism i n t o  s t e r i l e m i l k , i n c u b a t e d i t at 36.5  0  G. f o r 8|- hours, and drank  a 50.0 c.c. p o r t i o n . Acute g a s t r o - e n t e r i t i s , s i m i l a r i n nature t o t h a t observed i n the other persons a f f l i c t e d , f o l l o w e d w i t h i n 2 hours.  A p o r t i o n o f the m i l k , r e f r i g e r a t e d immediately  (s) f o l l o w i n g I n o c u l a t i o n f o r the same l e n g t h o f time, did-not cause symptoms o f f o o d - p o i s o n i n g when i n g e s t e d .  The organism  was i s o l a t e d i n almost pure c u l t u r e from the udder of the cow producing the m i l k .  Barber's  c l a s s i c r e p o r t presented the  f i r s t c o n c l u s i v e evidence t h a t f o o d - p o i s o n i n g c o u l d r e s u l t from staphylococcal  contamination.  This work remained unconfirmed f o r some 16 y e a r s , u n t i l Back and h i s c o l l e a g u e s (3) i n v e s t i g a t e d a s i m i l a r due t o the e l a b o r a t i o n of a f i l t r a b l e  outbreak  p o i s o n by a.yellow  hemolytic- s t a p h y l o c o c c u s , i n a c r e a m - f i l l e d Christmas  cake.  P o r t i o n s o f the cake were i n g e s t e d by v o l u n t e e r s , and acute g a s t r o - e n t e r i t i s f o l l o w e d w i t h i n 3 hours.  A 40-hour b r o t h  f i l t r a t e of the y e l l o w staphylococcus i s o l a t e d from the cake produced i d e n t i c a l symptoms when i n g e s t e d by v o l u n t e e r s . poisonous substance  appeared t o be f i l t r a b l e  The  exotoxin,  causing a food i n t o x i c a t i o n . F o l l o w i n g the l a t t e r p u b l i c a t i o n , there appeared i n r a p i d s u c c e s s i o n numerous o b s e r v a t i o n s of f o o d - p o i s o n i n g  attributed  to the growth o f staphylococcus  (Table 1.)  i n food p r o d u c t s .  A study of the l i t e r a t u r e shows a u n i f o r m sequence of symptoms, c h a r a c t e r i z e d by a s h o r t hut d e f i n i t e i n c u b a t i o n p e r i o d o f 2 t o 4 hours f o l l o w i n g i n g e s t i o n o f the contaminated  food.  Towards  the end o f the i n c u b a t i o n o r l a t e n t p e r i o d , nausea and d i z z i n e s s appear, accompanied by sweating, c h i l l i n e s s and s a l i v a t i o n . The nausea i n c r e a s e s i n i n t e n s i t y and r a p i d l y g i v e s way t o  (3)  v i o l e n t paroxysmal v o m i t i n g , abdominal cramps, d i a r r h o e a and p r o s t r a t i o n ; t h e r e may be t e t a n i c muscular c o n t r a c t i o n s and i i n t e n s e muscular p a i n .  The more acute symptoms u s u a l l y pass  away w i t h i n . a few h o u r s , but g e n e r a l weakness may p e r s i s t f o r s e v e r a l days.  I t i s not u n u s u a l however, f o r the p a t i e n t t o  f e e l normal w i t h i n 24 hours a f t e r onset of the i l l n e s s .  These  symptoms have been f a i t h f u l l y reproduced on a number o f oceansi o n s when s t e r i l e b r o t h f i l t r a t e s o f suspected s t r a i n s o f staphylococcus have been i n g e s t e d by human v o l u n t e e r s .  ( 3 , 10,  14, 22.) A r e v i e w o f a v a i l a b l e data r e v e a l s t h a t there have been recorded a t l e a s t 47 epidemics a t t r i b u t a b l e t o the presence o f t h i s micro-organism i n food s t u f f s .  Of these 47 r e p o r t e d out-  b r e a k s , 40 have i n v o l v e d some 1400 p e r s o n s .  (Table 2.)  It is  a matter o f some s i g n i f i c a n c e t h a t t h i s type o f f o o d - p o i s o n i n g should show a p r e d i l e c t i o n f o r m i l k and m i l k p r o d u c t s .  The  r e p o r t o f Barber (2) i n 1914 has a l r e a d y been recounted. Ramsey and Tracy i n 1931 (4) d e s c r i b e d an epidemic a t t r i b u t e d to the presence o f an orange s t a p h y l o c o c c u s . They noted an " o f f f l a v o r ? d e s c r i b e d as a "malt" o r " c a r a m e l f l a v o r i n the n  m i l k i n which the organism was grown.  Tanner and Ramsey, (5)  working w i t h the same organism a t a l a t e r d a t e , s t a t e d t h a t s e v e r a l of a group o f 20 i n d i v i d u a l s v i s i t i n g the l a b o r a t o r y to t e s t the " o f f f l a v o r " were made i l l .  I t i s o f i n t e r e s t to  note i n t h i s c o n n e c t i o n , t h a t a stronglyjliemolytic golden s t a p h y l o c o c c u s , o b t a i n e d from the Department o f D a i r y i n g a t the  (4) •at. th-s U n i v e r s i t y o f B r i t i s h Columbia, and reputed to produce a "caramel f l a v o r " when grown i n m i l k , e l a b o r a t e s a p o w e r f u l gastro-intestinal  irritant.  A l i s t o f the foods i m p l i c a t e d i n the r e p o r t e d outbreaks 'shows t h a t o f 47 epidemics recorded, 27, or 57$ were t r a c e d t o m i l k and m i l k p r o d u c t s .  (Table 2.)  Reference t o c e r t a i n pub-  l i c a t i o n s might tend to e x p l a i n , in;.part, the f r e q u e n t i m p l i c a t i o n o f t h i s type o f f o o d .  S t a r k ( 6 ) , i n 1936, examined  b a c t e r i o l o g i c a l l y 25 c e r t i f i e d d a i r y cows. :  The m i l k from every  animal r e v e a l e d white s t a p h y l o c o c c i , nine s t r a i n s of which were  hemolytic.  I n a s e r i e s o f 260 c a t t l e examined, by ffwatkin and  h i s - c o l l e a g u e s , (7) 143 had m a s t i t i s , and 30 o f these were due to s t a p h y l o c o c c u s .  67.4% o f the s t r a i n s i s o l a t e d from these  cows were t o x i n . p r o d u c e r s t • a l s o produced  toxin.  20% o f s t r a i n s from normal udders  Crabtree and l i t t e r e r  (8) r e p o r t e d a  milk-borne epidemic due t o a h e m o l y t i c staphylococcus from m a s t i t i s i n two cows.  Over 242 cases o c c u r r e d amongst members  of a s c h o o l before the cows were s i n g l e d out from the s c h o o l herd.  Of 8 s t r a i n s i s o l a t e d a t random from raw m i l k b o t t l e d  f o r l o c a l d i s t r i b u t i o n , and t e s t e d i n t h i s l a b o r a t o r y , 6 p r o duced a g a s t r o - i n t e s t i n a l  irritant.  While cream p i e s , c h o c o l a t e e c l a i r s and c u s t a r d - f i l l e d p a s t r i e s appear t o p r o v i d e a s i n g u l a r l y f a v o r a b l e medium f o r growth and consequent t o x i n p r o d u c t i o n by t h i s organism, and f Toxin i n G w a t k i n ' s r e p o r t r e f e r s t o hemolysins.  (5)  have "been h e l d r e s p o n s i b l e f o r a l a r g e number of  outbreaks,  (Table 2.) i t i s i n c o n c e i v a b l e t h a t a l l o f these c o u l d be t r a c e d t o a contaminated m i l k supplyc  K e l l e r t (9) r e p o r t e d an  epidemic caused by bakery goods, contaminated from a b o i l on a baker's forearm.  I n v e s t i g a t i o n s on s t r a i n s i s o l a t e d from m i l k ,  food e p i d e m i o l o g i c a l l y i m p l i c a t e d , p a t h o l o g i c a l l e s i o n s and a number of other sources l e d Jordan (10) t o b e l i e v e t h a t a h i g h percentage o f these organisms are capable o f e l a b o r a t i n g a gastro-intestinal irritant.  A s t r a i n "B", used i n t h i s l a b -  o r a t o r y f o r a g r e a t e r p a r t o f the experimental  work caused an •  erythema i n the nose o f the person from which i t was " i s o l a t e d . I t has produced a p o w e r f u l over a p e r i o d o f a y e a r .  e n t e r o t o x i n of u n d i m i n i s h e d potency The f a c t t h a t s t r a i n s from sources  other than contaminated m i l k s u p p l i e s may cause  food-poisoning  i s f u r t h e r s u b s t a n t i a t e d by r e p o r t s of epidemics due t o growth of staphylococcus  i n c h i c k e n gravy ( 1 1 ) , ham ( 1 2 )  9  sweet  p o t a t o candy ( 1 2 ) , and tongue sandwiches ( 1 3 ) . With t h e r e c o g n i t i o n o f i n c r e a s i n g numbers o f t h i s type of p o i s o n i n g arose the n e c e s s i t y f o r a l a b o r a t o r y t e s t by means of which.those s t r a i n s p r o d u c i n g an e n t e r o t o x i n c o u l d be r e a d i l y recognized.  I n many o f the e a r l i e r e p i d e m i c s , t h e s t r a i n s  concerned were t e s t e d on human v o l u n t e e r s , or t h e i r i n c r i m i n a t i o n remained unconfirmed, and they were merely suspected. I n h i s i n v e s t i g a t i o n s i n 1930,  Jordan (10) used human v o l u n t e e r s  and i t was c l e a r l y evident t h a t f o r a number o f reasons the human was n e i t h e r a convenient nor s a t i s f a c t o r y l a b o r a t o r y  (6) animal.  A r e c e n t paper by Dack et a l . (14) r e p o r t s f e e d i n g  experiments on a number of humans.  I t would i n d i c a t e t h a t  i n d i v i d u a l s u s c e p t i b i l i t y v a r i e s c o n s i d e r a b l y , and inasmuch as extreme care must be e x e r c i s e d i n a d m i n i s t e r i n g the t o x i n , the dosages are n e c e s s a r i l y so s m a l l as t o not show t y p i c a l r e a c t i o n s i n many i n s t a n c e s .  F u r t h e r m o r e , the number of v o l -  u n t e e r s a v a i l a b l e i s f r e q u e n t l y l i m i t e d to such an e x t e n t t h a t experiments performed are s c a r c e l y of s i g n i f i c a n c e . The use of M. rhesus monkeys, f i r s t r e p o r t e d by Jordan and McBroom i n 1931 ( 1 5 ) , has s i n c e been confirmed by Woolpert Dack ( 1 6 ) .  and  S i m i l a r d i f f i c u l t i e s a r i s e here as i n the case of  the human v o l u n t e e r .  Meyer ( 1 7 ) , i n 1934, S t a t e d ; "Contrary  to e x p e r i e n c e s made by Jordan and McBroom, and Woolpert  and  Dack, f e e d i n g t e s t s on monkeys have y i e l d e d i n c o n c l u s i v e results."  Furthermore, monkeys are d i f f i c u l t to h a n d l e , and  an experiment on even^moderate s c a l e would be e x p e n s i v e l y impractical. Dack and h i s c o l l e a g u e s (3) i n o c u l a t e d f i l t r a t e s venously i n t o r a b b i t s .  intra-  C o m p l i c a t i o n s t h a t arose due t o the  presence o f the H e m o l y t i c , l e t h a l and o t h e r t o x i c substances caused the death of s e v e r a l animals; v o m i t i n g was not observed, a l t h o u g h l o o s e s t o o l s i n some cases ?/ere recarr§.e&.  There  was  o b v i o u s l y no s p e c i f i c i t y i n such a t e s t and i t was abandoned. Borthwick, i n 1933 ( 1 8 ) , r e p o r t e d a method of p r e p a r a t i o n t h a t rendered guinea p i g s and r a b b i t s s u i t a b l e f o r i n g e s t i o n exper-  (7)  iments on the e n t e r o t o x i n .  Ho v o m i t i n g  or d i a r r h o e a  occurred,  and the post-mortem f i n d i n g s s t r o n g l y suggested death from the -A  other e x o t o x i c products of s t a p h y l o c o c c u s .  Attempts made i n  t h i s l a b o r a t o r y to reproduce the r e s u l t s have so f a r been unsuccessful. A d e t a i l e d study of the a g g l u t i n a t i v e p r o p e r t i e s , biochemi c a l r e a c t i o n s and chromogenicity of a l a r g e number of s t r a i n s i m p l i c a t e d i n f o o d - p o i s o n i n g outbreaks (£7)  f a i l e d to r e v e a l  any  constant c h a r a c t e r i s t i c s by which those organisms produc-  ing  e n t e r o t o x i n c o u l d be  recognized.  A c u l t u r a l method f o r d e t e c t i n g f o o d - p o i s o n i n g s t r a i n s of s t a p h y l o c o c c i has been d e s c r i b e d by Stone (19).  The  suspected  organisms are grown i n a 3% b e e f - e x t r a c t - g e l a t i n medium. L i q u e f a c t i o n of the g e l a t i n i n 24 hours i s taken as an i n d i c a a t i o n that  s t r a i n produces e n t e r o t o x i n .  A recent  modifica-  t i o n by the same author i n c l u d e s agar i n the medium, and ammonium sulphate  s o l u t i o n as a d e v e l o p e r .  The presence of c l e a r  zones around a colony i n d i c a t e an e n t e r o t o x i n p r o d u c e r . confirmation  of these r e s u l t s has been p u b l i s h e d ,  No  and the  few  experiments performed i n t h i s l a b o r a t o r y have y i e l d e d r e s u l t s inconcordant w i t h animal and human t e s t s . Due  to l a c k of s u i t a b l e experimental animals and  adequate  tests, few c o n c l u s i v e statments have been made r e g a r d i n g p r o p e r t i e s of t h i s g a s t r o - i n t e s t i n a l i r r i t a n t .  the  Jordan and Dack  ( 2 0 ) , and Jordan and Burrows (21), r e c e n t l y r e p o r t e d  the  (8) following"characteristics: 1.  The a c t i v e p r i n c i p l e w i l l not d i s t i l .  2.  I t i s not r e a d i l y  3.  I t i s markedly u n s t a b l e t o ff/lOO ITaoH.  4.  I t i s unstable- t o heat i n If/lOO H G l s o l u t i o n .  5.  I t i s not i d e n t i c a l w i t h the h e m o l y t i c substances  dialysable.  p r e s e n t i n many s t a p h y l o c o c c u s f i l t r a t e s , n o r does i t produce a s k i n 6.  reaction.  I t i s completely removed from a c i d aqueous s o l u t i o n by e x t r a c t i o n w i t h e t h y l ether o r c h l o r o f o r m , as judged by our method o f assay.  7.  I t may be e x t r a c t e d  from a l k a l i n e s o l u t i o n w i t h e t h y l  e t h e r o r c h l o r o f o r m but the d e l e t e r i o u s  e f f e c t of  a l k a l i tends t o mask such removal. 8.  The g a s t r o - i n t e s t i n a l p o i s o n i s not completely dest r o y e d when exposed t o a temperature o f 100° C. f o r 30 m i n u t e s  9.  e  The t o x i c q u a l i t y doe s not disappear a f t e r storage at low temperatures f o r as l o n g as 67 days but i s perhaps somewhat weakened.  Monkeys i n j e c t e d i n t r a v e n o u s l y s o l u t i o n of a c i d ether e x t r a c t  w i t h 5.0 c c . o f s a l i n e  o f potent f i l t r a t e s , vomited  and manifested the u s u a l s i g n s of acute d i s t r e s s o f the gastrointestinal tract.  The same s o l u t i o n g i v e n i n t r a v e n o u s l y  guinea p i g s , r a b b i t s , c a t s and dogs produced no i l l whatsoever.  to  effects  (9)  S e v e r a l wear.leers agreed t h a t no t o l e r a n c e was induced "byrepeated  i n g e s t i o n of the f o o d - p o i s o n i n g  substance.  Woolpert  and Dack i n 1931 ( 1 6 ) , s t a t e d t h a t a degree o f a c t i v e immunity c o u l d be induced by repeated p a r e n t e r a l i n o c u l a t i o n , but a l l attempts a t p a s s i v e immunity f a i l e d . Woolpert and Back ( 1 6 ) , u s i n g monkeys as t e s t animals, and Dolman (22), 1934, i n human f e e d i n g experiments, demons t r a t e d t h a t the hemolysins and d i v e r s e other t o x i c p r o p e r t i e s of s t a p h y l o c o c c a l f i l t r a t e s are d i s t i n c t from the i n g substance.  food-poison-,  I n the l a t t e r ' s experiments, humans i n g e s t e d  r e l a t i v e l y l a r g e amounts of f i l t r a t e s c o n t a i n i n g potent hemol y s i n s and t h e i r a l l i e d t o x i c substances w i t h impunity,  while  much s m a l l e r amounts o f f i l t r a t e s p r e p a r e d from one p a r t i c u l a r s t r a i n evoked symptoms o f acute f o o d - p o i s o n i n g volunteers.  i n several  This s t r a i n produces potent e n t e r o t o x i n .  The l a c k of a convenient and s p e c i f i c t e s t f o r the presence of the e n t e r o t o x i n has hampered i n v e s t i g a t i o n of i t s chemical nature  and a n t i g e n i c s t r u c t u r e .  Consequently the c o n d i t i o n s  f a v o r i n g i t s p r o d u c t i o n , and i t s mode of a c t i o n on the animal system are as y e t u n d e f i n e d .  Meyer ( 1 7 ) , i n a d i s c u s s i o n on  staphylococcus  states;  food-poisoning  "However, i n v i e w o f the  w e l l known f a c t that the e n t e r o t o x i c substance i s d i s t i n c t from the k i l l i n g t o x i n o f a s t a p h y l o c o c c u s ,  i t i s not a d m i s s i b l e t o  c o n s i d e r a g i v e n s t r a i n as an offender i n a food-poisonfg  out-  break without a d i r e c t demonstration of the g a s t r o - i n t e s t i n a l  (10) irritant.  While h e a t i n g g r e a t l y reduces the k i l l i n g t o x i n , and  l e a v e s the e n t e r o t o x i c c a p a c i t y u n i m p a i r e d , the s e p a r a t i o n of •A  the two f r a c t i o n s i s f r e q u e n t l y f r a u g h t w i t h d i f f i c u l t i e s . t e s t s on human v o l u n t e e r s are c o n c l u s i v e hut not without S u i t a b l e monkeys are not always a v a i l a b l e , and the  A simple  c o l o g i c t e s t to detect and t o separate  risks.  preparations  of s m a l l l a b o r a t o r y animals by the methods d e s c r i b e d wick are not always e f f e c t i v e .  The  by..Borth-  physiologic-pharma-  the e n t e r o t o x i c substance  from the k i l l i n g p o i s o n of a staphylococcus  i s , therefore,  u r g e n t l y needed." The work undertaken i n t h i s l a b o r a t o r y was p r i m a r i l y an attempt to f i n d a r e l i a b l e b i o l o g i c a l t e s t s u f f i c i e n t l y conv e n i e n t t h a t i t might be c a r r i e d out i n any b a c t e r i o l o g i c a l laboratory- as a means of i d e n t i f i c a t i o n of those s t r a i n s p r o ducing e n t e r o t o x i n .  I t was  f u r t h e r deemed d e s i r a b l e t h a t the  t e s t be adequately s e n s i t i v e i n d e t e c t i n g the presence of the gastro-intestinal irritant  t h a t i t might be employed i n study-  ing  the v a r i o u s p r o p e r t i e s , mode of a c t i o n and requirements  for  the p r o d u c t i o n of the irritantsufestajxce.  (11) Experimental. The  s t r a i n used i n t h e g r e a t e r p a r t o f our experimental  work, designated  as s t r a i n "B", was i s o l a t e d from a human n a s a l  mucous membrane, and caused a c h r o n i c erythema o f t h a t organ, Toxic f i l t r a t e s o f s t r a i n ^" c o n t a i n potent IT  vC &n(L/3  hemolysins,  and t h e i r a l l i e d e x o t o x i c p r o d u c t s , and by v i r t u e o f t h i s p r o p e r t y were at one time i n c l u d e d w i t h the p o o l e d  preparations  from a number o f organisms d u r i n g the process o f producing staphylococcus  t o x o i d at t h e Connaught L a b o r a t o r i e s , Toronto„T  Small subcutaneous doses of these t o x o i d s caused nausea and v o m i t i n g i n a number of human s u b j e c t s ; but when s t r a i n "B" was removed from t h e p r e p a r a t i o n s the v i o l e n c e o f the r e a c t i o n s dimin'shed.  I n g e s t i o n experiments on human v o l u n t e e r s  confirmed  the suspected presence o f e n t e r o t o x i n , g a s t r o - e n t e r i t i s appearing w i t h i n 2-2-|- hours a f t e r consumption o f as l i t t l e as 1.0 c c . A subcutaneous dose o f 0.1 c c . caused acute symptoms i n a human w i t h i n 1-|- hours a f t e r i n o c u l a t i o n ; the p a t i e n t d i d not completely  recover f o r s e v e r a l days.  When s t r a i n "B" was r e i s o l a t e d one year l a t e r from the same p a t i e n t ' s nose, i t y/as shown t o have r e t a i n e d i t s . a b i l i t y to produce a powerful  g a s t r o - i n t e s t i n a l poison..  In this labor-  a t o r y i t has c o n s i s t e n t l y produced an e n t e r o t o x i n o f undimini s h e d potency f o r oyer one y e a r . The s t o c k - c u l t u r e i s m a i n t a i n ed i n b e e f - i n f u s i o n agar. t Tersotval commute catton. from. Tjr. C.E.t5oLma.n.  (IS) Preparation. The method of p r e p a r a t i o n employed by Dolman ( 2 2 ) , and Dolman and E l t c h i n g (24) i n p r o d u c i n g staphylococcus c l i n i c a l use i s f o l l o w e d .  toxoid f o r  Plates of semi-solid beef-infusion  agar (0.3%.agar) are i n o c u l a t e d w i t h a f i r e - h o u r b r o t h c u l t u r e of the organism.  A f t e r 40 hours i n c u b a t i o n a t 37* C. i n an •  atmosphere o f 70% oxygen and 30% carbon d i o x i d e , the contents of t h e p l a t e s are squeezed through cheese c l o t h i n t o f i l t e r i n g f u n n e l s c o n t a i n i n g coarse f i l t e r paper.  The f i l t r a t e i s c e n t -  r i f u g a t e d a t h i g h speed f o r some 2-g— 3 hours and s t e r i l i z e d by p a s s i n g through a S e i t z E.K. d i s k .  The p r e p a r a t i o n from s t r a i n  "B" c o n t a i n s potent hemolysins against r a b b i t and sheep e r y throcytes,  has h i g h l e t h a l and d e m o - n e c r o t i c powers as w e l l  as the v a r i o u s other t o x i c p r o p e r t i e s c h a r a c t e r i s t i c o f staphylococcus  filtrates.  2500 u n i t s and t h e /  The <<• l y s i n ( r a b b i t ' c e l l ) may measure 3  l y s i n (sheep c e l l ) 1600 u n i t s as c a l i b r a t e d  by the method o f h e m o l y s i n t i t r a t i o n d e s c r i b e d by Dolman ( 2 5 ) . The  removal o f these tbxicjsubstances i s e f f e c t e d by the a d d i U.S.P.  t i o n o f 0„3% s o l u t i o n o f formaldehyde' and i n c u b a t i o n a t 37° C. A  u n t i l the aC and/3 hemolysins are no l o n g e r d e t e c t a b l e  (24).  One now has a f i l t r a t e devoid of t h e d e l e t e r i o u s l e t h a l hemol y t i c and dermo-necrotic p r o p e r t i e s , q u a n t i t i e s up t o 1.0 c c . i n t r a p e r i t o n e a l l y o c c a s i o n i n g no observable harm t o mice.  The  p r e p a r a t i o n was t e s t e d f o r e n t e r o t o x i n on a human volunteer,, who i n g e s t e d p o r t i o n s on two o c c a s i o n s . P r o t o c o l 1. I.  1.0 c c . was i n g e s t e d i n 30 c c o f m i l k .  Four hours  (13) l a t e r , nausea and headache, accompanied by d i z z i n e s s appeared, Tk!ey passed away w i t h i n one hour. Two hours l a t e r a v e r y loose and f o u l - s m e l l i n g s t o o l was passed. Recovery was complete w i t h i n a few h o u r s . P r o t o c o l 2. 2. 1.5 c c . o f the same f i l t r a t e was i n g e s t e d i n 30.0 c c of m i l k . Two hours a f t e r i n g e s t i o n o f the p r e p a r a t i o n nausea appeared, i n c r e a s e d i n i n t e n s i t y , and was accompanied by cons i d e r a b l e g a s t r i c d i s c o m f o r t and sweating. W i t h i n 30 minutes of the appearance of nausea, v i o l e n t paroxysmal v o m i t i n g o c c u r r e d , follovred by extreme weakness and d i s a b i l i t y . Abdomi n a l p a i n s and a g e n e r a l f e e l i n g o f d i s c o m f o r t p e r s i s t e d f o r some S hours f o l l o w i n g the v o m i t i n g , d u r i n g which, time 3 s t o o l s were passed. They were almost e n t i r e l y f l u i d , f o u l - s m e l l i n g and p r o f u s e . Abdominal d i s c o m f o r t was r e l i e v e d w i t h passage of the s t o o l s but a .feeling o f nausea p e r s i s t e d f o r s e v e r a l h o u r s . The f o l l o w i n g day a v e r s i o n t o food, and g e n e r a l weakness were e v i d e n t . Recovery was complete w i t h i n 48 hours.. These tv/o experiments,  combined with, the r e p o r t s o f sim-  i l a r i n s t a n c e s c i t e d above i n r e l a t i o n t o experiences  with  s t r a i n "B" i n the p r e p a r a t i o n o f t o x o i d s a t the Gonnaught Labo r a t o r i e s i n Toronto i n d i c a t e d t h a t (a) the e n t e r o t o x i n was more s t a b l e t o 0.3% f o r m a l i n a t 37° C. than were the h e m o l y t i c , dermo-necrotic  and l e t h a l p r o p e r t i e s ,  (b) The e n t e r o t o x i n  was present i n a p p r e c i a b l e q u a n t i t i e s i n f i l t r a t e s from which the aforementioned p r o p e r t i e s had fceen e n t i r e l y removed.  This  method o f s e p a r a t i o n o f the g a s t r o - i n t e s t i n a l i r r i t a n t from the k i l l i n g , h e m o l y t i c and derrao-necrotic p r o p e r t i e s has been almost A  e n t i r e l y i n the ease o f strain-,' B" and some others w i t h s i n g u l a r T  success throughout the i n v e s t i g a t i o n .  (14) The f i r s t p o i n t o f i n v e s t i g a t i o n was t o determine the e f f e c t of the f i l t r a t e c o n t a i n i n g o n l y the e n t e r o t o x i c p r i n c i p l e on guinea p i g s , u s i n g B o r t h w i c k ' s method o f preparation (18 j . Tifis procedure i s o u t l i n e d i n b r i e f . .•The i n s u s c e p t i b i l i t y of r a b b i t s and guinea p i g s was - a t t r i b u t e d by B o r t h w i c k t o the d e s t r u c t i o n o f the g a s t r o i n t e s t i n a l i r r i t a n t by the a c i d r e a c t i o n o f the stomaciu A c c o r d i n g l y the stomach contents were adjusted t o pH' > 7.3 w i t h HaHCO 3 5.0% and H/lO HG1, t i t r a t i o n s b e i n g c a r r i e d out by means o f a stomach tube. When the d e s i r e d r e a c t i o n was obtained, the t o x i c f i l t r a t e was introduced and the stomach tube withdrawn. ±sorthwick r e p o r t e d death i n a number o f animals w i t h i n one hour of a d m i n i s t r a t i o n o f the t o x i c substances. In t h i s l a b o r a t o r y , i t was deemed a d v i s a b l e to use a concentrated phosphate b u f f e r to o b t a i n the d e s i r e d p H , r a t h e r than c a r r y out the somewhat lengjiay procedure o i t i t ration. An example o f the method f o l l o w e d i n s t a n d a r d i z i n g the huffer i s outlined. P r o t o c o l 5. P i g 1. 10 c c * o f X2HP04, i n a c o n c e n t r a t i o n o f 80gi per l i t r e , p.R\ 8.83 were i n t r o d u c e d i n t o the stomach by mean of a stomach tube. Small samples were a s p i r a t e d a t i n t e r v a l s of 5, 10,15, 20, minutes and the pff", of each t e s t e d on a quinhydrone e l e c t r o d e . o r i g i n a l p>H/of b u f f e r s o l u t i o n 8.83 Sample a s p i r a t e d a t 5 minutes 7^43 « « " 10 « 7.35 "« " « 15 « 7.30 «« it II 20 « 7 * 32 Protocol 4 t P i g 2.- 15 c c . o f K2HP04 i n a c o n c e n t r a t i o n o f 80g. per l i t r e . p^'H. 8,83, were i n t r o d u c e d i n t o the stomach. Samples were a s p i r a t e d a t 2, 5, 10, 15, minutes and the p,H„ t e s t e d on a quinhydrone e l e c t r o d e . o r i g i n a l p..Hv of b u f f e r s o l u t i o n 8.83 Sample aspirated a t 2 minutes 7.55 « H II 5 7i35 " « « 10 II 7.35 " 15 « 7.35 w  A suspension o f MgC03, 10 g. e r l i t r e , was a l s o used •as a b u f f e r s o l u t i o n . P  Protocol  5.  (15) P i g 1. 12.5 c c . MgC03, (10g. per l i t r e ) were i n t r o duced by means o f a stomach tube. . Samples were a s p i r a t e d a t 2, 5, 10, JL5, 20 minutes. Sample a s p i r a t e d at 2 minutes 7.2 » " " 5 " 7i43 « ". "10 " 7.30 n » " 15 " 7.30 « " » 20 '« 7.30 . The t o x i c f i l t r a t e was a d m i n i s t e r e d about 2 minutes a f t e r the b u f f e r , i n q u a n t i t i e s o f 2*0 to 7*0 c»CiWithout produ c i n g any symptoms other, than s l i g h t nervous t w i t c h i n g . A p o r t i o n of f i l t r a t e 1/10  "B" was  concentrated to about  i t s o r i g i n a l volume by d i s t i l l a t i o n  i n vacuo a t 37°  G.  Doses e q u i v a l e n t to 70 and 80 c.c. were a d m i n i s t e r e d e n t e r a l l y to guinea p i g s w i t h o u t any untoward e f f e c t s An abdominal i n c i s i o n was made i n a guinea p i g , and 2.0  c.c; o f f i l t r a t e  "B" introduced' d i r e c t l y i n t o the duo-  denum by means o f a s y r i n g e .  The i n c i s i o n was  stitched.  animal recovered from the a n a e s t h e t i c w i t h i n 20  The  minutes,  - became a c t i v e and remained so f o r a week, when i t was destroyed;  At no time d i d i t e x h i b i t symptoms a t t r i b u t a b l e to  the presence  of the t o x i c f i l t r a t e *  These experiments  l e d one to b e l i e v e t h a t the  p,H<  of the stomach might be of l i t t l e importance; and indeed^ i n humans, i t seems to p r o v i d e no p r o t e c t i o n * as to whether, i f the t o x i n were absorbed  One was  interested  from the g a s t r o -  i n t e s t i n a l t r a c t of the g u i n e a - p i g s , the c h a r a c t e r i s t i c manif e s t a t i o n s of v o m i t i n g and d i a r r h o e a were p o s s i b l e . A, g u i n e a - p i g was g i v e n i n t r a m u s c u l a r l y , o.05 of apomorphine. There was  grains  S l i g h t s a l i v a t i o n , but no v o m i t i n g occurred.  l i t t l e evidence  that the animal was  i n any way a c t -  (16) -ting|abnornially under t h i s s t i m u l u s .  S i m i l a r l y , 0.05  of histamine occasioned no upset i n a g u i n e a - p i g .  mgm. Large  q u a n t i t i e s o f phosphates a d m i n i s t e r e d as b u f f e r s o l u t i o n s ^ insignifigant and a l s o c a s t o r - o i l produced/changes m  the nature of the  stools. One was prompted to b e l i e v e t h a t , i f v o m i t i n g were p o s s i b l e i n a g u i n e a - p i g i t occurred v e r y r a r e l y , and under the u s u a l s t i m u l i a f f e c t i n g humans.  not  Thus, one of the  most c h a r a c t e r i s t i c symptoms of staphylococcus  food-poisoning  would p o s s i b l y be absent i f t h i s animal were used i n l a b o r a t o r y t e s t s ; and i n the experience of the author, on no o c c a s i o n has a g u i n e a - p i g g i v e n arty i n d i c a t i o n of showing the u s u a l l y prominent symptoms of g a s t r o - e n t e r i t i s . method t h e r e f o r e was  This  abandoned.  inas^much as cats and dogs resemble humans more c l o s e l y than do rodents i n t h e i r d i e t a r y and e x c r e t a r y h a b i t s , and b o t h are known to vomit on o c c a s i o n , i t seemed l o g i c a l to i n v e s t i g a t e the s u s c e p t i b i l i t y of these animals to the g a s t r o intestinal  irritant.  P r e l i m i n a r y i n v e s t i g a t i o n s were c a r r i e d out on c a t s , u s i n g the f i l t r a t e o f s t r a i n "B" t e s t e d on a human. ( P r o t o c o l s 1 and 2.) P r o t o c o l 6. Oat No.l 50.0 c.c. o f m i l k when i t Time 12.35 1 Hr.35 Min* p r e f e r r e d to  2 months o l d , weight about one k i l o g r a m . f i l t r a t e "B" were a d m i n i s t e r e d i n the animal's was fed-. P.M. The animal became somnolent and drowsy; i t l i e down and moved very k i t t l e *  (17) 1 Hr.45Min. The oat became a g i t a t e d . I t l i c k e d i t 3 l i p s v i o l e n t l y ; - s a l i v a t i o n was v e r y marked. 1 Hr.50 M i n . The cat vomited. I t seemed much, r e l i e v e d , became more a c t i v e and p l a y e d a l i t t l e . 2 Hr. 05 M i n . V o m i t i n g was repeated; merely g a s t r i c s e c r e t i o n s . 2 E r . 15 M i n . The c a t became unsteady on i t s . f e e t and r e f u s e d food. 3 Hr. 10 M i n . G i d d i n e s s was apparent; the animal swayed from s i d e to s i d e when i t s a t up. 5 Hr. 15 M i n . V i o l e n t v o m i t i n g r e c u r r e d , 3 t i m e s . The'.ani m a l 's c o n d i t i o n seemed-much improved a f t e r v o m i t i n g . 4 Hrs. The cat accepted f o o d . 4 Hr. 15 M i n . The animal was p l a y i n g and seemed q u i t e r e covered. There was no s i g n i f i g a n t d i a r r h o e a ; recovery was complete w i t h i n 24 h o u r s . P r o t o c o l 7. Gat, Wo.2. 2 months o l d , weight about one k i l o g r a m . 50.0 c c . of e n t e r o ^ t o x i c f i l t r a t e were i n g e s t e d i n m i l k . Time: 12:35 P.M 1 Hr.55 M i n . The animal r e g u r g i t a t e d ; there was some g a s t r i c d i s t r e s s . S a l i v a t i o n was evidenced by l i c k i n g o f the l i p s . 2 Hr.05 M i n . G i d d i n e s s was apparent. The animal swayed d i z z i l y when s t a n d i n g or s i t t i n g up. 2 Hr.40 M i n . G a s t r i c d i s n t r e s s i n c r e a s e d ; v i o l e n t p e r i s t a l t i c movements were observed through the abdominal w a l l . 3 Hr.05 M i n . Another wave of nausea o c c u r r e d , the animal shuddered, s p a s m o d i c a l l y ; v i o l e n t p e r i s t a l t i c movements were observed through the abdominal w a l l . Nausea appeared t o come at i n t e r v a l s . 3 Hr«25 M i n . The g e n e r a l c o n d i t i o n of the animal seemed improved. 4 Hr.50 M i n . A l a r g e b u l k of f l u i d f e c e s , f o u l - s m e l l i n g and l i g h t i n c o l o r was passed. I t was mucoid i n c o n s i s t e n c y . The animal recovered completely w i t h i n 24 h o u r s . e  P r o t o c o l 8o Oat No. 3 l a r g e female, f u l l y grown. 6.0 to 8.0 c c of f i l t r a t e "B", concentrated by d i s t i l l a t i o n i n vacuo to about l / l O volume, were g i v e n i n m i l k . The aose was e q u i v a l e n t to 60-80 c c of the o r i g i n a l f i l t r a t e . Time 5.00 P.M 2 H r s . The animal remained q u i e t a i l a f t e r n o o n . At 5.00 P„M<, i t became a g i t a t e d , c r i e d f r e q u e n t l y and l i c k e d itS: l i p s 2 Hrs.40 M i n . I t vomited v i o l e n t l y . The animal seemed r e lieved after vomiting. 3 H r s . V i o l e n t v o m i t i n g was repeated, and d i a r r h o e a f o l l o w e d s h o r t l y a f t e r . A l a r g e b u l k o f f o u l - s m e l l i n g f e c e s , mucoid i n c o n s i s t e n c y , was e x p e l l e d . The animal recovered c o m p l e t e l y . e  e  (18) The above p r o t o c o l s , merely r e p r e s e n t a t i v e of a number o f others i n which the m a n i f e s t a t i o n s o f acute g a s t r o - i n t e s t i n a l d i s t r e s s were evident i n g r e a t e r or l e s s e r i n t e n s i t y , - are a complete p i c t u r e of the symptoms of  staphylococcus  f o o d - p o i s o n i n g i n humans. Vomiting occurred from 2 to 2-g 2  hours a f t e r i n g e s t i o n of the f i l t r a t e s , preceded by s a l i v a t i  ' on  and f o l l o w e d by abdominal d i s t r e s s and d i a r r h o e a . was r a p i d and'complete i n a l l cases.  Heoovery  As some workers have  observed i n humans, d i a r r h o e a m^y, be present without v o m i t i n g or v o m i t i n g may  occur alone, although as a g e n e r a l r u l e both  symptoms a r e marked* The dose r e q u i r e d to produce these t y p i c a l m a n i f e s t a t i o n s of food-pcdsoning i n c a t s when g i v e n e n t e r a l l y i s about 50 times t h a t r e q u i r e d to cause v o m i t i n g and d i a r r h o e a i n humans on i n g e s t i o n of the same f i l t r a t e s , ( P r o t o c o l s no... 1 and  2)  The e n t e r a l a d m i n i s t r a t i o n of such l a r g e doses to l a b o r a t o r y animals i s n e i t h e r convenient nor a c c u r a t e . e n t e r o t o x i c p r e p a r a t i o n used was innocuous, l e t h a l and dermo-necrotic  Inasmuch as the the  hemolysins,  substances having been p r e v i o u s l y  destroyed by f o r m a l i n , p a r e n t e r a l i n s o c u l a t i o n was employed., P r o t o c o l 9. 3.0 c.c. of Time: 11 :20 15 Mins; . . * V wj.^y . 20 Mins. Vomiting occurred s e v e r a l times, 30 Mins . ]tepeated v o m i t i n g occurred, 40 Mins* A very l o o s e f o u l - s m e l l i n g s t o o l , mucoid i n C o n s i s t e n c y was passed* 2 Hr.55 Min. The animal appeared to be weak and somnolent. 3 Hr.55 Mini.The cat seemed dazed and giddy when standing or s i t t i n g up. I t p r e f e r r e d to l i e down, and was com-  (19) - p l e t e l y d i s i n t e r e s t e d i n i t s ' surroundings, 4 Hr*2Q Min, Signs of r e c o v e r y were e v i d e n t . The animal became more, a c t i v e and i n t e r e s t e d i n i t X s surroundings. Complete r e c o v e r y f o l l o w e d w i t h i n 24 hours. :  P r o t o c o l 10. Cat iJo.g. 2-3 months o l d , weight about 1 kilogram* 1.5 Ci. c. f i l t r a t e "B" were g i v e n i n t r a p e r i t o n e a l l y . Time: 2.50 P.M. 25 Min Si, . The animal seemed nauseated, uncomfortable» 37 Mins. The animal l a y on the f l o o r of the cage; i t s head hung u n n a t u r a l l y w i t h the nose r e s t i n g on the f l o o r . 1 Hr. S a l i v a t i o n , accompanied by l i c k i n g of the l i p s was f o l l o w e d by repeated vomitings I l i r . 1 0 M i n s . Vomiting was repeated* 1 Hr. 25 Mins. A l o o s e f o u l - s m e l l i n g s t o o l was passed, i t was p r o f u s e , mucoid i n c o n s i s t e n c y and l i g h t c o l o r e d * R e t c h i n g and v o m i t i n g were repeated over a p e r i o d of an hour, accompanied by s e v e r a l l o o s e s t o o l s . The animal appeared weak and somnolent* 5 Hrs.3 0 Mins. The c a t seemed to be r e c o v e r i n g , f o r i t became more a c t i v e and accepted food. Recovery was complete i n 24 hours. P r o t o c o l 11. Cat Mo.5 - 6 wdcs o l d , weight" 400 grams* 0.5 OiC. f i l t r a t e "B" were i n o c u l a t e d i n t r a p e r i t o n e a l l y . Time; 2;07 15 Mins. Vomiting occurred'. 25 Mins. V o m i t i n g was. repeated. 24 Mins. V o m i t i n g was repeated* The animal remained somnolent and l e t h a r g i c up t o 6.00 P.M* D i a r r h o e a occu r r e d some time l a t e r . I t w i l l be observed t h a t i n p a r e n t e r a l i n o c u l a t i o n o f f i l t r a t e "B", the syndrome i s merely an i n t e n s i f i e d form of t h a t o c c u r r i n g when e n t e r a l a d m i n i s t r a t i o n i s employed. The l a t e n t - p e r i o d i s shortened from 2-3 hours to as l i t t l e as minutes, depending ministered.  i n p a r t , upon the s i z e of the dose ad-  The symptoms, i n the author's experience, are  more v i o l e n t and d e f i n i t e .  An i n c r e a s e d r a t e of r e s p i r a t i o n  and even p a n t i n g has been observed, p r i o r to the onset of vomiting.  5-10  S a l i v a t i o n i s marked; the v o m i t i n g i s p r o j e c t i l e  (HO) and r e c u r s a t frequent i n t e r v a l s .  S t o o l s are f r e q u e n t , and  a f t e r the f i r s t one or two, sometimes c o n t a i n no f e c a l m a t e r i a l . They c o n s i s t e n t i r e l y o f a c l e a r mucoid discharge  resembling  ,egg-albumin. Recovery s e t s i n , i f not too l a r g e a dose 13 a d m i n i s t e r e d , w i t h i n a few hours, u s u a l l y 5 or 6, and i s most f r e q u e n t l y complete w i t h i n 24 h o u r s . As one c o n t r o l measure, animals were i n o c u l a t e d i n t r a p e r i toneally alin  w i t h s t e r i l e b r o t h c o n t a i n i n g q u a n t i t i e s o f form-  (0.3$) and m e r t h i o l a t e (1:10,000) e q u i v a l e n t t o those p r e -  sent i n the f i l t r a t e s p r e p a r e d  6  10 d i f f e r e n t animals were i n -  o c u l a t e d w i t h q u a n t i t i e s f r o m 1.0 to 5.0 c . c . 0  I n no case was  there a s u g g e s t i o n o f the symptoms c h a r a c t e r i s t i c o f the s y n drome produced by the e n t e r o t o x i n present strain  i n the f i l t r a t e  of  "B".  As a f u r t h e r c o n t r o l measure, f i l t r a t e s o f strain Wood 46", ,r  p r e p a r e d under c o n d i t i o n s i d e n t i c a l w i t h those o f s t r a i n "B", were i n o c u l a t e d i n t o c a t s on a number o f v a r i e d from 1.0 t o 5.0 c.c. every case.  occasions;doses  The animals remained normal i n  F i l t r a t e s o f "Wood 46" have been i n g e s t e d by  human v o l u n t e e r s i n r e l a t i v e l y l a r g e amounts and have occa s i o n e d no u p s e t o I n a l l , some 50 i n d i v i d u a l experiments have been performed on c a t s w i t h f i l t r a t e "B", u s i n g i n t r a p e r i t o n e a l i n o c u l a t i o n . Doses v a r y i n g ^rom 0.4 t o 3.0 c . c . were employed, and i n every case, symptoms o f g a s t r o - i n t e s t i n a l d i s t r e s s have been present i n g r e a t e r or l e s s e r i n t e n s i t y , depending  (21) upon the s i z e o f the l o s e and. the degree of a c t i v e immunity a c q u i r e d by the animal due to p r e v i o u s i n j e c t i o n s . T h e ' f i l t r a t e "B" p r o d u c i n g g a s t r o - e n t e r i t i s by both e n t e r a l and p a r e n t e r a l a d m i n i s t r a t i o n i n c a t s was i n o c u l a t e d i n t o a dog, 3 months o f age, 3.0 c . c . were I n j e c t e d i n t r a peritoneally.  Y a o m i t i n g began 5 minutes a f t e r i n o c u l a t i o n  and continued a t i n t e r v a l s f o r over 3 hours; s e v e r a l s t o o l s were passed w i t h i n one hour o f i n o c u l a t i o n .  fluid The animal  appeared dazed and g i d d y , and was too weak t o s t a n d or s i t up. The f o l l o w i n g day i t walked u n s t e a d i l y and r e f u s e d food; r e c o v e r y seemed complete however, w i t h i n 48-60. hours.  The  s e n s i t i v i t y o f dogs to the e n t e r o t o x i n has been demonstrated on s e v e r a l o c c a s i o n s .  I n every case the symptoms are i d e n t i c a l  w i t h those produced i n eats by i n o c u l a t i o n o f the g a s t r o intestinal .irritant. D u r i n g the course o f i n v e s t i g a t i o n , c e r t a i n o b s e r v a t i o n s were recorded t h a t tendeAto c o n f i r m statements made by other workers, as w e l l as by the author e a r l i e r i n t h i s paper. Jordan and Burrows ( 2 1 ) , Woolpert and Dack (16) and l a t e r Dolman (22) produced evidence t o show t h a t the e n t e r o t o x i c p r i n c i p l e was not i d e n t i c a l w i t h the h e m o l y s i n s , l e t h a l or. other recognized t o x i c products of s t a p h y l o c o c c i .  The method  employed i n t h i s i n v e s t i g a t i o n o f p r e p a r i n g e n t e r o t o x i c  filt-  r a t e s devoid o f these substances p r o v i d e s f u r t h e r d a t a i n support of t h i s c o n t e n t i o n . The author wishes t o present a d d i t i o n a l e x p e r i m e n t a l evidence i n f a v o r o f the f a c t t h a t the e n t e r o t o x i n i s a separate t o x i c product of s t a p h y l o c o c c u s .  (22) D e s p i t e the f a c t t h a t : (a) the h e m o l y s i n s a n d  (3 were no longer d e t e c t a b l e i n f i l t -  r a t e s o f "B" employed i n the  experiments;  (h) rfo s k i n r e a c t i o n could he demonstrated; ( c ) 1.0  c.c. q u a n t i t i e s i n o c u l a t e d into mice, and 5 to 10c*c<,  q u a n t i t i e s i n t o guinea-pigs occasioned no harmful r e a c t i o n ; some cats d i e d f o l l o w i n g p a r e n t e r a l i n o c u l a t i o n of the e n t e r otoxic f i l t r a t e s * S e v e r a l p r o t o c o l s are presented to i l l u s t r a t e the phenomenon. P r o t o c o l 12. . Cat Ho.l  1-2 months o l d .  Y/eight 600  g.  2.0 c.c. f i l t r a t e "B" were i n o c u l a t e d i n t r a p e r i t o n e a l l y . Time: 11:10 A.M. 30 Mins. A . t h i n watery s t o o l was passed. 1 Mr. 5 Mins. V i o l e n t v o m i t i n g occurred. Vomiting and d i a r r h o e a r e c u r r e d a t i n t e r v a l s f o r s e v e r a l hours* The animal was weak, dazed, and s u f f e r e d acute abdominal d i s t r e s s . Death occurred a t 12:15 the f o l l o w i n g day, 25 hours a f t e r i n o c u l a t i o n . J u s t b e f o r e death the animal appeared markedly emaciated and i n a s t a t e o f extreme p r o s t r a t i o n . Mucus was d i s c h a r g e d i n v o l u n t a r i l y from the anus a t f r e q u e n t i n t e r v a l s o The animal d i e d d u r i n g a spasm of c o n v u l s i v e p e r i s t a l s i s i n v o l v i n g boththe oesophagus and i n t e s t i n a l t r a c t . Autopsy; E m a c i a t i o n was very marked. The peritoneum and s i t e o f i n o c u l a t i o n showed nothing abnormal. The i n t e s t i n e s were completely c o n t r a c t e d , blanched and very f i r m i n c o n s i s t e n c y . I n t e r n a l examination o f the gut r e v e a l e d an e x c e s s i v e s e c r e t i o n of a t h i c k y e l l o w mucus throughout the e n t i r e l e n g ^ t h * The stomach contained a l a r g e q u a n t i t y of gas. The g a l l • b l a d d e r was enormously engorged, and d i f f u s i o n of b i l e p i g ments s t a i n e d the viscera, over a wide area. The u r i n a r y bladder was completely c o n t r a c t e d and f i r m . Uo f u r t h e r macroscopic a b n o r m a l i t i e s were apparent. P r o c t o c o l 13. Gat ITo.2 1 month o l d . Weight 350 g. J u l y 2,1936. 0.1 c.c. o f f i l t r a t e "B" was a d m i n i s t e r e d i n t r a p e r i t o n e a l l y . :'There we're no untoward symptoms except • somnolence*  (23) J u l y 3, 1936. 0.2 c c . of f i l t r a t e "B" were a d m i n i s t e r e d i n t r a p e r i t o n e a l l y . Some g a s t r i c discomfort and d i z z i n e s s accompanied by somnolence p e r s i s t e d f o r s e v e r a l hours f o l l o w i n g i n o c u l a t i o n . J u l y 4, 3,936. 0.3 c c of f i l t r a t e "B were administered i n t r a p e r i t o n e a l l y . There was g e n e r a l l a s s i t u d e and discomfort for several hours. < J u l y 5, 1936. Diarrhoea was evident on J u l y 5, and continued f o r 8 days. S t o o l s occurred f r e q u e n t l y , o f t e n at EO minute i n t e r v a l s , and e x p u l s i o n was i n v o l u n t a r y i n most i n s t a n c e s . Death occurred on J u l y 13. t?  Autoptsy: The t a i l and h i n d l e g s were s o i l e d w i t h mucus d i s charged from the anus. The animal was p i t i f u l l y emaciated; the l o s s i n weight over the 11 day p e r i o d exceeded 40%. The peritoneum was normal; there was no r e a c t i o n at the s i t e of i n o c u l a t i o n . Both l a r g e and s m a l l i n t e s t i n e s were c o n t r a c t e d , hard, blanched, and, on i n t e r n a l examination, showed excessive s e c r e t i o n of mucus throughout t h e i r e n t i r e l e n g t h . The stomach contained a l a r g e q u a n t i t y of mucuous s e c r e t i o n . The g a l l b l a d d e r was engorged; d i f f u s i o n of b i l e pigments s t a i n e d the abdominal v i s c e r a over a wide a r e a . The u r i n a r y bladder was completely C o n t r a c t e d and f i r m . There were no f u r t h e r macroscopic a b n o r m a l i t i e s . These examples are t y p i c a l , of some zo when f i l t r a t e cats.  r e a c t i o n s observed  "B" has been i n o c u l a t e d i n t r a p e r i t o n e a l l y i n t o  I n a l l cases, the only constant  a b n o r m a l i t i e s noted on  autopsy have been those p r e s e n t e d i n the above p r o t o c o l s .  In  no i n s t a n c e were the s i g n s of g e n e r a l v a s c u l a r engorgement and p e t e c h i a l hemmorhages, c h a r a c t e r i s t i c of r a p i d death from the e f f e c t s of staphylococcus  e x o t o x i n s , present t o any  observable  degree. This-phenomenon! has a l s o been produced by repeated subcutaneous i n o c u l a t i o n of the f i l t r a t e .  Diarrhoea  about 24 hours a f t e r the f i r s t dose, and continues reasing s e v e r i t y u n t i l death.  with inc-  V o m i t i n g however, has not been  observed on subcutaneous a d m i n i s t r a t i o n of e n t e r o t o x i c A l a r g e dose of f i l t r a t e adminstration  occurss  T,  B , or the ,f  filtrates.  frequent  of s m a l l e r doses, produces an exaggerated form  (24) . o f the u s u a l temporary g a s t r o - i n t e s t i n a l upset..  The absence -  of the hemolysins, dermo-necrotic and l e t h a l substances i n t & e o r i g i n a l f i l t r a t e employed, combined -with the f a c t that none o f the c h a r a c t e r i s t i c post-mortem symptoms o f acute death from potent e x o t o x i c f i l t r a t e s as d e s c r i b e d by Dolman (25) were observed - s t r o n g l y i n d i c a t e s that t h e g a s t r o - i n t e s t i n a l 4  i r r i t a n t i s a substance e n t i r e l y separate from any_o_f t h e r e c o g n i z e d t o x i c products o f staphylococci. The heat s t a b i l i t y o f staphylococcus enterot.oxin has been r e c o g n i z e d f o r s e v e r a l years*  Jordan, et a l . (20) u s i n g  human s u b j e c t s a s t e s t animals s t a t e d ; "The t o x i c substance p r e s e n t i n staphylococcus f i l t r a t e s , causing g a s t r o - l n t e s t i n a l derangement, i s not c o m p l e t e l y destroyed by exposure f o r 30 minutes t o the temperature o f b o i l i n g water.  Some d i m i n u t i o n  i n t o x i c power may, however, p o s s i b l y be caused by h e a t i n g even a t temperatures below 100 °  C.  TT  F i l t r a t e s of s t r a i n »B" heated a t 1 0 0 have, on i n t r a p e r i t o n e a l  inoculation  0  C. f o r 30 minutes  i n c a t s , produced the  g a s t r o - i n t e s t i n a l syndrome c h a r a c t e r i s t i c of the e n t e r o t o x i n . Some d i m i n u t i o n i n t o x i c power i s evidenced by the f a c t that l a r g e r doses of the same f i l t r a t e a r e r e q u i r e d to produce t y p i c a l symptoms a f t e r h e a t i n g a t 100° G. f o r 30 minutes, than a r e necessary a f t e r h e a t i n g a t 1 0 0 " C. f o r 10, 15, or 20 minutes.  The t o x i n i s , however, not completely destroyed  by h e a t i n g a t 1 0 0 " G. f o r 30 minutes, when assayed by the "kitten-test".  (25) E a r l i e r p u b l i c a t i o n s notedthat  the e n t e r o t o x i n i s s t a b l e  to storage at low temperatures.  In t h i s laboratory  preparations  from s t r a i n "B" c o n t a i n i n g 0,3^  f o r m a l i n , maintained  at r e f r i g -  .erator temperatures, show l i t t l e d i m i n u t i o n i n t o x i c power a f t e r 5 months.  I n c u b a t i o n at 57° 0. i n the presence of  f o r m a l i n f o r 78 days d i d not destroy the e n t e r o t o x i n  0.5%  although  some d e t o x i f i c a t i o n took p l a c e . Barber (2) i n 1914  and Dack et a l . (23) i n 1928  reported  t h a t no p e r c e p t i b l e degree of immunity or t o l e r a n c e could a c q u i r e d by repeated  i n g e s t i o n of e n t e r o t o x i c  be  filtrates.  Woolpert and Dack (16) s t a t e d i n a l a t e r p u b l i c a t i o n that a degree of a c t i v e immunity a g a i n s t the e n t e r o t o x i n c o u l d be induced by repeated p a r e n t e r a l i n o c u l a t i o n , but a l l attempts at p a s s i v e immunity f a i l e d . Working w i t h c a t s i n t h i s l a b o r a t o r y , i t came to one's a t t e n t i o n t h a t , a f t e r a number of i n j e c t i o n s , an animal  was  able to r e c e i v e w i t h impunity s e v e r a l times the dose of  filt-  r a t e "B'  f  t h a t p r e v i o u s l y caused symptoms of acute g a s t r o -  enteritis. was  D u r i n g the course of i n v e s t i g a t i o n t h i s phenomenon!  c o n s i s t e n t l y i n evidence.  The  a c t i v e immunity d e s c r i b e d  by e a r l i e r workers c o u l d , then, be induced by repeated e r a l i n o c u l a t i o n of f i l t r a t e  parent-  "B".  F u r t h e r i n v e s t i g a t i o n s were c a r r i e d out to determine whether the serum of those animals a c q u i r i n g a c t i v e immunity i n t h i s manner would n e u t r a l i z e the e n t e r o t o x i n of "B".  Serum was  filtrate  obtained from a c t i v e l y immunized c a t s  incubated w i t h f i l t r a t e  "B  ,T  at 37° C. f o r !-§- hours.  and The  mixture  (26) was I n o c u l a t e d i n t r a p e r i t o n e a l l y i n t o  cats*  P r o t o c o l 14. -a  Gat No.'l 1.5 c . c . f i l t r a t e "B"+ 2.0 c . c . immune serum i n c u - - • bated a t 57° G. f o r hours were i n o c u l a t e d i n t r a p e r i t o n e a l l y . The animal remained normal, p l a y e d , and a t e h e a r t i l y whenever food was o f f e r e d , No symptoms o f g a s t r o - i n t e s t i n a l d i s t r e s s •:e were p r e s e n t . Gat No.2 1.5 c.c f i l t r a t e "3"+ 2.0 c.c. s a l i n e incubated a t '67 ° o. f o r !-§- hours were i n o c u l a t e d i n t r a p e r i t o n e a l l y . The animal showed symptoms o f acute g a s t r o - i n t e s t i n a l u p s e t . D i a r r h o e a continued f o r some 5-7 h o u r s . Gat No.5 1.5 c . c . o f s a l i n e + 2.0 c.c.immune serum incubated at '67 0. f o r 1-g- hours were i n o c u l a t e d i n t r a p e r i t o n e a l l y . The animal remained normal. 9  . .  P r o t o c o l 15.  Gat No. 1.0 c . c . f i l t r a t e "B"+- 1.5 c . c . "immune serum" i n cubated a t 37" C. f o r 1-g- hours were i n o c u l a t e d i n t r a p e r i t oneally. The animal remained normal. Gat No. the f o l l o w i n g day - 0.5 c . c , f i l t r a t e "B" were i n o eu'lated~intraperitoneally, V o m i t i n g and d i a r r h o e a o c c u r r e d . This a n i m a l , a p p a r e n t l y s e n s i t i v e to 0.5 c . c . o f f i l t r a t e , r e c e i v e d w i t h impunity three times t h a t amount mixed w i t h immune serum. R a b b i t s were g i v e n repeated p a r e n t e r a l i n o c u l a t i o n s o f f i l t r a t e "B", and the serum was t e s t e d f o r immune substances a g a i n s t hemolysins. I n t h i s experiment, a f i l t r a t e o f s t r a i n "B" c o n t a i n i n g a l l o f the t o x i c substances was used. I t was not d e t o x i f i e d w i t h f o r m a l i n . S u f f i c i e n t serum was added to the t o x i n t o n e u t r a l i z e the hemolysins. The mixture was i n cubated o v e r n i g h t a t 37° G», the p r e c i p i t a t e removed and the . supernatant t e s t e d on c a t s .  ir  P r o t o c o l 16. Gat N o . l 1.5 c . c . t o x i n "B" heated a t 100* G. f o r 10 minutes (to d e s t r o y the cC and p and l e t h a l toxins,) were i n o c u l a t e d i n t r a p e r i t o n e a l l y . Severe g a s t r o - i n t e s t i n a l symptoms appeared 10 minutes a f t e r i n o c u l a t i o n . . V o m i t i n g and d i a r r h o e a r e c u r r e d at i n t e r v a l s f o r some t i m e . Gat No.2 4.2 c . c . o f t o x i n - a n t i t o x i n m i x t u r e (5.0 c.c. t o x i n "B", 1.2 c . c . a n t i serum) were i n o c u l a t e d i n t r a p e r i t o n e a l l y . The animal remained normal.  • (27) Gat No* 5 1.5 c c . t o x i n "B" heated a t 100 ° C. f o r 10 minutes (to d e s t r o y the oC , fi hemolysins and l e t h a l toxins,) p l u s 1.5 c c normal r a b b i t serum incubated overnight a t 37 ° C. were i n o c u l a t e d i n t r a p e r i t o n e a l l y . V i o l e n t v o m i t i n g and d i a r r h o e a appeared i n 10 minutes and r e c u r r e d f r e q u e n t l y over a p e r i o d o f 2-3-hours. The s t o o l was a c l e a r mucoid s e c r e t i o n c o n t a i n i n g no f e c a l m a t e r i a l . An antiserum  was prepared by immunizing a r a b b i t w i t h  filt-  r a t e s o f s t r a i n "Wood 46", c o n t a i n i n g a l l o f the t o x i c products of staphylococcus  except the e n t e r o t o x i n .  The antiserum  was  capable o f n e u t r a l i z i n g i n v i t r o the oC and (S h e m o l y s i n s . P r o t o c o l 16 ( c o n t ' d ) . Gat ITo.4 1.5 c c t o x i n "B" 3.0 c c antiserum "W" ( s u f f ? i c l e n t t o n e u t r a l i z e the oC and (* hemolysins) incubated a t 3 7 ° C. o v e r n i g h t , were i n o u u l a t e d i n t r a p e r i t o n e a l l y . W i t h i n 10 minutes, v i o l e n t v o m i t i n g o c c u r r e d , f o l l o w e d by d i a r r h o e a . The animal was i l l throughout the day. Death occurred the f o l l o w i n g day, and autopsy r e v e a l e d only the symptoms t y p i c a l o f those produced by the* e n t e r o t o x i n e  I t would seem evident t h a t a n t i s e r a prepared a g a i n s t f i l t r a t e "B" are capable o f n e u t r a l i z i n g i n v i t r o , the g a s t r o i n t e s t i n a l i r r i t a n t contained  i n that  filtrate.  Gat No.2 ( p r o t o c o l 1 6 ) , r e c e i v e d w i t h impunity  twice the dose  t h a t produced v i o l e n t g a s t r o - e n t e r i t i s i n animals 1 , 3 and 4. The substance t h a t n e u t r a l i z e s the e n t e r o t o x i n i s not present i n normal r a b b i t serum, ( c a t 3) noran a n t i s e r a prepared a g a i n s t f i l t r a t e s c o n t a i n i n g a l l o f the known t o x i c products of staphy1ococcus except the e n t e r o t o x i c p r i n c i p l e (cat 4 ) .  (28)  The i n v e s t i g a t i o n s recorded, up to t h i s p o i n t have been c a r r i e d out on f i l t r a t e s of a s t r a i n t e s t e d by human v o l u n t e e r s , and known *to produce a powerful e n t e r o t o x i n .  The  sensitivity  • of c a t s and dogs to the g a s t r o - i n t e s t i n a l i r r i t a n t has been uti.r' l i z e d i n examining s t r a i n s from v a r i o u s sources., 1.  I n an epidemic of food-poisoning r e p o r t e d by Dolman ( 2 6 ) ,  l a r g e numbers of h e m o l y t i c s t a p h y l o c o c c i were i s o l a t e d from the suspected p a s t r y , c u s t a r d f i l l e d " v a n i l l a s l i c e s " . t o n e a l i n o c u l a t i o n i n c a t s of s u i t a b l y prepared  Intraperi-  f i l t r a t e s from  t h i s s t r a i n produced the unmistakeable symptoms of g a s t r o e n t e r i t i s d e s c r i b e d e a r l i e r i n t h i s paper. 2.  Hemolytic  s t a p h y l o c o c c i i s o l a t e d from a cream p i e ,  suspected  of c a u s i n g food-poisoning, y i e l d e d a p o w e r f u l e n t e r o t o x i n . ^ c .c. . of the f i l t r a t e i n o c u l a t e d i n t r a p e r i t o n e a l l y i n t o a 3 months o l d pup evoked v i o l e n t g a s t r o - e n t e r i t i s .  The dog vomited more  than 18 times w i t h i n 30 minutes of i n o c u l a t i o n . 3.  A golden staphylococcus  r e p o r t e d to cause "caramel f l a v o r "  i n m i l k , produced a potent e n t e r o t o x i n as w e l l as h i g h t i t r e s of oC and/? hemolysins.  The  organism, obtained from the Department  of D a i r y i n g at the U n i v e r s i t y of B r i t i s h Columbia, was i f i e d as "Tetracoccus  l i q u e f a c i e n s " (Orla-Jensen).  class-  I n 10 exper-  iments w i t h t h i s s t r a i n p o s i t i v e r e s u l t s were obtained i n every , case w i t h doses of 1.0  t o 3.0  c c ,  i n s t a n c e s , from doses as low as 1.5  and death occurred i n 3 c c  A siS------- instance ar  has been d e s c r i b e d by Tanner and Ramsey ( 5 ) . 4.  An albus s t r a i n , i s o l a t e d from raw milk,produced  ofo£andj3 hemolysins as w e l l as a powerful  high t i t r e s  gastro-intestinal  (29) irritant.  8 experiments were c a r r i e d out w i t h a p p r o p r i a t e l y  prepared f i l t r a t e s .  Doses as low as 0.5 c . c . produced  positive  r e s u l t s , and death occurred i n 3 i n s t a n c e s from doses as low as 2.0 c . c . 5.  From a t o t a l o f 8 s t r a i n s p i c k e d a t random from raw m i l k  bottled  f o r l o c a l d i s t r i b u t i o n , 6 produced a p o w e r f u l e n t e r o -  t o x i n when assayed by the k i t t e n t e s t .  (30) " Discussion., The p u b l i c h e a l t h i s s u e s i n v o l v e d i n the study of staphyl o c o c c a l g a s t r o - e n t e r i t i s cannot "be r e a d i l y overlooked. 1928  Since  t h e r e have "been r e p o r t e d some 47 outbreaks of s t a p h y l o -  coccus f o o d - p o i s o n i n g ;  40 of these i n v o l v e d more than  persons of both sexes and a l l age groups. recognize  1400  The f a i l u r e to  the s l g n i f i g a n c e of the micro-organism i n such out-  breaks p r i o r to 19S8  has been a t t r i b u t e d by some authors p a r t l y  t o the i n f l u e n c e exerted by the exhaustive  s t u d i e s of Savage  (28)  and E t k e l e s and Standfuss (29) on the Salmonella group; and w h i l e i n certain i n s t a n c e s s t a p h y l o c o c c i were known to be  present  i n foods i n c r i m i n a t e d e p i d e m i o l o g i c a l l y , t h e i r presence was r e p o r t e d by the l a b o r a t o r y .  not  Furthermore, a t r a n s i e n t d i s a b i l i t y  such as t h a t caused, by the e n t e r o t o x i n would only too f r e q u e n t l y be diagnosed by the layman as " i n t e s t i n a l f l u " or summer d i a n  r r h o e a " one would expect r e l a t i v e l y few such cases to be r e p o r t e d to a u t h o r i t i e s f o r f u l l i n v e s t i g a t i o n . These c o n s i d e r a t i o n s , combined w i t h the f a c t t h a t the t e x t u r e , appearance and t a s t e of food h e a v i l y contaminated by s t a p h y l o c o c c i remain una l t e r e d , would l e a d one to b e l i e v e that s t a p h y l o c o c c a l g a s t r o e n t e r i t i s i s more  common than r e p o r t s i n d i c a t e .  The h i g h percentage of outbreaks a t t r i b u t e d to m i l k  and  m i l k products i s a, p u b l i c h e a l t h i s s u e of s p e c i a l s l g n i f i g a n c e . I t has been demonstrated t h a t contamination  a r i s i n g from staphy-  l o c o c c a l i n f e c t i o n s of the udder (2, 8) and perhaps s t a p h y l o c o c c i i n m i l k obtained from a p p a r e n t l y h e a l t h y cows may  play a part i n  (31) outbreaks of food-poisoning contmlnation'cannot  (6,$).  Inasmuch as t h i s source  of  he r e a d i l y c o n t r o l l e d by any method of p r o -  d u c t i o n , the only e f f e c t i v e p r e v e n t a t i v e measure would be u n i v e r sal  p a s t e u r i z a t i o n * Where raw m i l k Is d i s t r i b u t e d there i s  a d d i t i o n a l hazard  the  of d r o p l e t i n f e c t i o n by s t a p h y l o c o c c i from  those h a n d l i n g the m i l k d u r i n g p r o d u c t i o n ; and s i n c e m i l k seems to  provide an e x c e l l e n t medium f o r the r a p i d m u l t i p l i c a t i o n of  the micro-organisms, adequate c o o l i n g and r e f r i g e r a t i o n methods should be The  enforced. frequent i n c r i m i n a t i o n of cream p i e s and  custard-filled  p a s t r i e s presents a problem of i n t e r e s t and importance .  Meyer (IV)  contends t h a t d r o p l e t i n f e c t i o n , and the t r a n s m i s s i o n of  staphy-  l o c o c c i from s u p e r f i c i a l l e s i o n s on the hands of employees are the most common methods of contaminating  these p r o d u c t s .  Slow  c o o l i n g i n l a r g e open k e t t l e s at room temperature p r i o r t o f i l l i n g p a s t r y s h e l l s enhances the r i s k of contamination, p r o v i d e s admirable  and  temperature c o n d i t i o n s f o r r a p i d m u l t i p l i c a t i o n .  By v i r t u e of the l a r g e batches of f i l l e r prepared  i n bakeries,  the number of persons i n v o l v e d i n outbreaks from t h i s  source  is frequently high. (Tablel.) Meyer(17) r e p o r t s an i n c r e a s e d i n c i d e n c e i n the Southern U n i t e d States d u r i n g the warm summer months.  Thorough cooking  and prompt r e f r i g e r a t i o n of the f i l l e r s and f i n i s h e d  products,  as w e l l as d a i l y s t e r i l i s a t i o n of u t e n s i l s have been advocated , Some h e a l t h a u t h o r i t i e s i n t h a t r e g i o n have, d u r i n g the warmer months, r e s t r i c t e d the s a l e and d i s t r i b u t i o n o f c r e a m - f i l l e d  (32) bakery goods to b a k e r i e s w i t h p r o p e r l y r e f r i g e r a t e d storage f a c i l i t i e s and d i s p l a y c a s e s .  An u n o f f i c i a l r e p o r t  indicates  t h a t enforcement of the l a t t e r measure has a c t u a l l y reduced the .incidence of s t a p h y l o c o c c a l g a s t r o - e n t e r i t i s i n the area concerned. Methods of p r e p a r a t i o n c o u l d be d i s c u s s e d a t great l e n t h , but i t should s u f f i c e to s t r e s s : (a) p a s t e u r i z a t i o n of m i l k and m i l k p r o d u c t s , (b) frequent s t e r i l i z a t i o n o f bakery u t e n s i l s , and c l e a n l i n e s s i n methods of p r o d u c t i o n , (c) adequate r e f r i g e r a t i o n f a c i l i t i e s ,  and,  (d) the s t r i c t e s t observance of the o r d i n a r y r u l e s of hygiane by those h a n d l i n g food f o r human consumption. While there i s as yet i n s u f f i c i e n t experimental  evidence  , a v a i l a b l e from which to draw d e f i n i t e c o n c l u s i o n s r e g a r d i n g the mode of a c t i o n of the e n t e r o t o x i n on the animal system, there are c e r t a i n i n t e r e s t i n g p o i n t s which might tend to throw some l i g h t on the m a t t e r .  D e s p i t e the f a c t t h a t the more obvious  r e a c t i o n s produced by the t o x i n are c l i n i c a l s i g n s of a g a s t r o e n t e r i t i s , post-mortem examination  of animals dying from the  e f f e c t s of l a r g e i n t r a p e r i t o n e a l i n o c u l a t i o n s of f i l t r a t e s cont a i n i n g only the e n t e r o t o x i c p r i n c i p l e does not r e v e a l signs of acute i n t e s t i n a l i n f l a m m a t i o n .  There i s an e x c e s s i v e s e c r e t i o n  of mucus; but t h i s c o n d i t i o n does not appear to r e s u l t from an e n t e r i t i s i n the p a t h o l o g i c a l sense.  Furthermore, the r a p i d on-  s e t of the symptoms, f r e q u e n t l y w i t h i n 5 minutes of i n t r a p e r i t o n e a l i n o c u l a t i o n , would s c a r c e l y be due to an  inflammatory  (35) proeess a r i s i n g from d i r e c t i r r i t a t i o n of the i n t e s t i n a l mucosa. •The absence o f the s i g n s o f l o c a l inflammation a t the p o i n t of i n o c u l a t i o n or i n the p e r i t o n e a l c a v i t y excludes the p o s s i b i l i t y of e x t e n s i v e damage through c o n t a c t between the t o x i n and exposed t i s s u e s . The mode o f a c t i o n would appear to be through the nervous system.  This would n e c e s s i t a t e a b s o r p t i o n of the t o x i c sub-  s t a n c e , and t r a n s p o r t a t i o n by the blood t o the nerve centres attacked.  The p o s s i b i l i t y t h a t t h i s may a c t u a l l y occur i s  supported by the v a r i a b l e l e n g t h o f the l a t e n t p e r i o d , and by the s e v e r i t y Of the symptoms v a r y i n g w i t h the route o f administration. period i s  I f the substance be i n g e s t e d w i t h food, the l a t e n t 3 hours, which may be taken t o correspond w i t h the  p e r i o d r e q u i r e d f o r d i g e s t i o n and subsequent a b s o r p t i o n of the ingested m a t e r i a l . period  i s shortened  rapid, absorption  I n i n t r a p e r i t o n e a l i n o c u l a t i o n the l a t e n t to as l i t t l e as 5-10 minutes, due to more  by t h a t r o u t e .  The r e a c t i o n i s more severe i n  i n t r a p e r i t o n e a l than i n o r a l a d m i n i s t r a t i o n , and i t i s profeable t h a t , the slower a b s o r p t i o n i n the l a t t e r case, combined w i t h the f a c t t h a t much o f the t o x i c m a t e r i a l may be d i s c h a r g e d i n vomltus and f e c e s , only a s m a l l p a r t o f the i n g e s t e d dose i s a c t u a l l y absorbed.  T h i s f a c t o r / i s e l i m i n a t e d by p a r e n t e r a l a d m i n i s t r a t i o n ,  and i n t r a p e r i t o n e a l i n o c u l a t i o n evokes the most r a p i d , v i o l e n t and p e r s i s t e n t symptoms. are l e s s severe, and  I n subcutaneous i n o c u l a t i o n symptoms  appear a f t e r 18-S4 h o u r s .  The onset-of  symptoms would seem t o demand a c e r t a i n p r i o r c o n c e n t r a t i o n o f  (34) t o x i n i n the blood stream, t h e l a t e n t p e r i o d being quired  the time r e -  f o r t h a t c o n c e n t r a t i o n t o be reached, and the l e n g t h o f  the l a t e n t p e r i o d depending upon the r a t e ox a b s o r p t i o n .  The  nerve c e n t r e s a t t a c k e d by the e n t e r o t o x i n are not known.  In "  post-mortem examination, the u r i n a r y bladder has been c o n s i s t e n t l y found i n a s t a t e o f complete c o n t r a c t i o n , which would  discount  the p o s s i b i l i t y t h a t p e r i p h e r a l nerve endings i n the g a s t r o i n t e s t i n a l t r a c t alone have been a f f e c t e d .  Moreover, t h a t f a c t  t h a t the v o m i t i n g and d i a r r h o e a which f o l l o w the a d m i n i s t r a t i o n of the e n t e r o t o x i n t o l a b o r a t o r y animals are u s u a l l y a s s o c i a t e d w i t h u n s t e a d i n e s s and apparent v e r t i g o , s t r o n g l y suggests t h a t the substance concerned may a c t upon adjacent centres i n the midb r a i n whose e x c i t a t i o n r e s u l t s i n the syndrome recorded. The d i s c o v e r y o f a c t i v e immunization methods l o c o c c u s t o x o i d n e c e s s i t a t e d the establishment  w i t h staphy-  of a r b i t r a r y  standards f o r i n n o c u i t y and potency t o which p r e p a r a t i o n s f o r c l i n i c a l use c o u l d be r e f e r r e d before r e l e a s e from the l a b o r a tory (24).  Since the i n c l u s i o n i n the t o x o i d s o f f i l t r a t e s con-  t a i n i n g e n t e r o t o x i n might give r i s e t o unpleasant r e a c t i o n s , p r e c a u t i o n s must be observed t o prevent t h e i r b e i n g used i n p r e p a r a t i o n s f o r human immunization.  The s u g g e s t i o n has a l r e a d y been  made (30) t h a t an a d d i t i o n a l standard  f o r i n n o c u i t y should be  appended t o those recommended by Dolman and H i t c h i n g (24), v i z . ; ,T  3.0 c.c. o f the t o x o i d , i n o c u l a t e d i n t r a p e r i t o n e a l l y i n t o a  normal k i t t e n should occasion no d e l e t e r i o u s symptom' „ r  (35)  But s t r a i n s o f s t a p h y l o c o c c i i s o l a t e d from human l e s i o n s and subsequently found able t o produce e n t e r o t o x i n , may conc e i v a b l y -owe t h e i r p a t h o g e n i c i t y i n p a r t t o t h i s l a t t e r The  property,  r e a c t i o n Of the host t o continued a b s o r p t i o n o f s m a l l quan-  t i t i e s o f e n t e r o t o x i n , such as may occur i n acute s t a p h y l o c o c c a l i n f e c t i o n s , i s not d e f i n i t e l y knovm; b u t i i n the l i g h t o f our knowledge o f the d e l e t e r i o u s e f f e c t upon c a t s under . c e r t a i n circumstances  o f repeated p a r e n t e r a l i n o c u l a t i o n , i t i s p o s s i b l e  t h a t the e n t e r o t o x i n might p l a y an important  r o l e i n human staphy-  l o c o c c a l i n f e c t i o n s . Since the e n t e r o t o x i n i s a n t i g e n i c i t would seem d e s i r a b l e t h a t e f f o r t s should be made t o render the a n t i g e n innocuous t o humans so t h a t i t might be i n c o r p o r a t e d w i t h other s t a p h y l o c o c c a l antigens i n m a t e r i a l s d i s t r i b u t e d f o r the a c t i v e immunisation o f man. F u r t h e r work I s d e s i r a b l e a n the problem of the a n t i g e n i c p r o p e r t i e s and r e l a t i o n s h i p s of the e n t e r o t o x i n , and methods o f assay o f a n t i - e n t e r o t o x i c s e r a r e q u i r e i n v e s t i g a t i o n . I n the meanwhile, f i l t r a t e s - o f s t r a i n s producing  e n t e r o t o x i n might to  advantage be i n c l u d e d i n the pooled antigens used f o r the immu n i s a t i o n of horses i n the p r e p a r a t i o n o f staphylococcus toxic sera.  anti-  (36) Summary. 1.  C e r t a i n s t r a i n s o f staphylococcus produce a g a s t r o - i n t e s t -  i n a l poi&on i n c o n j u n c t i o n w i t h v a r i o u s other t o x i c substances. This e n t e r t o x i n may be separated from these l e t h a l t o x i n s , hemolysins and n e c r o t o x i n by f o r m a l i n i a a t i o n a t 57 ° C. u n t i l the oC a n d ^ hemolysins are no l o n g e r d e t e c t a b l e . So  The i n t r a p e r i t o n e a l i n o c u l a t i o n o f s m a l l amounts o f these  f o r m a l i n i z e d f i l t r a t e s i n c a t s and dogs p r o v i d e s a t e s t s u f f i c i e n t l y s e n s i t i v e f o r demonstration o f the presence of the ente r o t o x i n by evoking unmistakeable symptoms s i m i l a r to those observed i n humans s u f f e r i n g from s t a p h y l o c o c c a l f o o d - p o i s o n i n g . Guinea-pigs, r a b b i t s and mice are not s e n s i t i v e t o o r a l or p a r e n t e r a l a d m i n i s t r a t i o n o f the f i l t r a t e s . 3.  The g a s t r o - i n t e s t i n a l syndrome i n p a r e n t e r a l i n o c u l a t i o n i s  an i n t e n s i f i e d form of t h a t produced by o r a l a d m i n i s t r a t i o n ; e x c e s s i v e dosage may prove f a t a l .  Post-mortem changes d i f f e r  from those present i n animals d y i n g from the e f f e c t s o f the potent e x o t o x i n s o f s t a p h y l o c o c c u s . 4.  The substance p r o d u c i n g g a s t r o - i n t e s t t " rial derangement i n  c a t s posess"* p r o p e r t i e s s i m i l a r to those a s c r i b e d by o the ^workers to the e n t e r o t o x i n a f f e c t i n g humans.  Thus, i t i s d i s t i n c t  from the h e m o l y t i c , demro-necrotic or l e t h a l substances; heati n g a t 100°C. f o r 30 minutes does not completely destroy the e n t e r o t o x i c p r i n c i p l e ; i t i s s t a b l e when s t o r e d a t low tempera t u r e s J i t w i l l not d i s t i l } and a degree of a c t i v e immunity can be induced by repeated p a r e n t e r a l i n o c u l a t i o n .  (37)  5.  The serum o f an a c t i v e l y immunized animal apparently  neut-  r a l i z e s the e n t e r o t o x i n i n v i t r o . 6.  S t r a i n s o f s t a p h y l o c o c c i from raw m i l k and p a t h o l o g i c a l  l e s i o n s i n humans may, under a p p r o p r i a t e powerful enterotoxie  c o n d i t i o n s , produce a  substance.  Acknowledgements . I t i s indeed a pleasure  t o acknowledge my indebtedness t o  B r . C E . Dolman, Department of B a c t e r i o l o g y and P r e v e n t i v e M e d i c i n e , U n i v e r s i t y o f B r i t i s h Columbia, f o r h i s k i n d i n t e r e s t and many v a l u a b l e suggestions i n the d i r e c t i o n o f t h i s work, and t o D r . H.J. Gibbons, Connaught L a b o r a t o r i e s , Western D i v i s i o n , f o r h i s k i n d i n t e r e s t and a s s i s t a n c e .  Bibliography, 1.  Owen; P h y s i c i a n and Surgeon, 1907, 29:289=  2.  Barber, M. A.; P h i l i p p i n e J o u r . Sc., Sect.B., 1914, 9:515.  3.  Dack, G.M., Gary, W.E., Woolpert, O.G ., and d i g g e r s , H.; J o u r . P r e r . Med., 1930, 4:167.  4.  Ramsey, R.J., and Tracy, P.H.; P r o c , Soc. Exper. B i o l , and Med., 1931, 28:390.  5.  Tanner, If.W., and Ramsey, R.J.; Am. J o u r . Med, Sc., 1932, 184:80.  6.  S t a r k , H.; J o u r . I n f . D i s . , 1926, 39:114.  7.  Grwatkin, R., Hadwen, S., and l e G-ard, H.M., Can. Pub. H e a l t h J o u r . 1956, 27:391.  8.  G r a b t r e e , J.A., and l i t t e r e r , Wm.; Am. J our. Pub. H e a l t h , 1934, 24:1116.  9.  K e l l e r t , E.; Am. J o u r . Pub. H e a l t h , 1931, 21:1352.  10.  Jordan, E.O.; J.A.M..A., 1930, 94:1648.  11.  Jordan, E.O ., and H a l l , J.R.; J o u r . P r e v . Med., 1931, 5:387.  12.  Costa Mandray, A.; Puerto Rico J o u r . Pub. H e a l t h and Trop. Med., 1953, 9:44.  13.  Back, G-.M., Bowman, G ,W., and Harger, R.H.; J .A.M. A., 1935  9  105:1598. 14.  Dack, G-.M., and Gary, W,E.; J o u r , B a c t . , 1937, 33:49.  15.  Jordan, E.O., and McBroom, J . ; P r o c . Soc. Exper. B i o l , and Med., 1931, 29:161.  16.  Woolpert, 0 .C ., and Dack, G- ,H.; J o u r . I n f . D i s ., 1933, 52:6.  B i b l i o g r a p h y (cont'd). 17.  Meyer, K.F.; Sonderabdruck aus S a n g g e r - F e s t s c h r i f t S e i t e 278-279, 1934, Hascher and C i e . A.-G., Y e r l a g , Z u r i c h .  18.  Borthwick, G.R.;  B r . J o u r . Exper. P a t h . , 1933, 14:236.  19.  Stone, R.Y*; P r o c . Soc. Exper. B i o l , and Med., 1935, 33:137.  20.  Jordan, E.O., and Hack, 0 «M.; J o u r . Prey. Ivied., 1931, 5; 383.  21.  Jordan, E.O., and Burrows, W,;  P r o c . Soc. Exper. B i o l . and  Med., 1933, 30:448. 22.  Dolman, C.E.; J o u r , I n f . D i s . , 1934, 55:172.  23.  Dack, G.M.,  Jordan, E.O., and Woolpert, O.C.;  J o u r . Prev.  Med., 1931, 5:151. 24.  Dolman, C,E., and E i t c h i n g , J „S.; J o u r . P a t h , and Bac t . , 1935, 41:137.  25.  Dolman, C.E.; Can. Pub. H e a l t h J our., 1932, 23:125.  26.  Dolman, O.E.; Can. Pub. H e a l t h J our., 1936, 27:494.  27.  Jordan, E.O., and Burrows, W»; J o u r . I n f . D i s . , 1935, 57:121,  28.  Savage; Med. Res. C o u n c i l , Spec. Report S e r i e s Ho. 91, 1925, & J o t i r . P r e v . Med., 1932, 6:425.  29.  E l k e l e s and Standfuss; Handb. d. pathog. M i k r o o r g , 3, ed., 1931. I l l , p a r t 2, p. 1681.  30.  Dolman, C.E., asd W i l s o n , R.J., and O o c k c r o f t , W.H.j Pub. H e a l t h J o u r . , 1936, 27:489.  Can.  TABLE 1.  Date  Epidemics of Staphylococcus F o o d - p o i s o n i n g . . ( D i r e c t l y or i n d i r e c t l y r e p o r t e d . ) • F a t e s This l i s t makes no c l a i m f o r completeness* "Fo." ^Location Involved Caused by Laboratory report  Eeported by  Journal  1.1-907  Owen  2,1914  Barber  P h y s i c i a n and S u r g . , I907J •'29*28.9P h i l i p p i n e J o u r . Science,] S e c t . B . , 1914,9:51^; P h i l i p p i n e s  3.1927- 28.Artb.ur F r e n c h Kelson 4.1928 Jordan 5.1929 6.  1929  7.1929 8.1930 9.1930  J.A.M.A.,rf«-30, <?4: 16*8  Jar dan  J.A.ir.A.,1130,9* -  Dack, Gary, Woolpert and Wiggene Jordan N.Y. s t a t e Dept. of H e a l t h  J o u r . P r e v . Med., 1930, 41167  Chicago,. I l l •  J.A.M.Ae 1930,  Porto Rico Long I s l a n d  10.1930 11.1930 12.1931  Jor dan Jordan & H a l l  13*1931 14.1931 15.1931 16.1931 17.1931 16.1931  Jor dan: Meyer Meyer ILeyer Meyer Ramsey & T r a c y  a  it  i*. »  or  *  Chicago,  8  4tl67  N-Y- Haalffv News, Ccth 1930„  Dec.,  , i .  )^50«  B  tt  111.  20.1932 Ii 21.1932 22.1932 23-1932 j24.1932 ! 25-1932 :26.1932 I 27.1932 : 28.1933  Ramsey  t  Tanner  4 4  Meyer, Krueger Mep'er, .Simpson G r i e g e r , Gray, Meyer Jor dan,Bur r owe  125  to u n-  It  ir n  Buchanan, EcKer Jor dan , Bur r ows  29.1933 ! McBurney  a n  II  »  »  It  11  it  9  n  n  n  ir  15  «  It  It-  ii-  »  n  it  Am. J o u r . Hygiene, 1934» 2 0 , Fo. 3s604-610 J.A.M.A., 1933* 100:1999  yellow  2  ?•  ! milk  Orange  milk  Or ange  20  Calif • Washington San F r a n c i s c o  2c,-: 200 35  Milwaukee  54 31 7 ii  yellow yellow  n i»  Calif.  Chicago Milwaukee Chicago Cleveland  11  j l a y e r cake ^ j chocolate e c l a i r s j head cheese sandwiches I coc'anut cream p i e I chbalate e c l a i r s  2  Califs.  »  II  « n 11  16 Calif* •••3 Calif. 42 Los A n g e l e s C a l J . 5  Am. J o u r . Hygiene, 1934» 20,, F o 3 8604-610  yellow yellow  i cream-puffs l D e v i l ' s f o o d cake j c h i c k e n gravy  4  P r o c . af the Soc « f o r Exp. & B i o l . med 1931, 28s390  e  cheese cake  Jersey C i t y  Am. J o u r . S c . 1 9 3 2 , 80-85s184  white  i yellow sponge cake, cream, f i l l e r y e l l o w  os  19.1932  staph*  milk v a n i l l a i c e cream cream p i e c h o c o l a t e cream p i e a p p r o D F . 1 5 0 Wedding cake 20 l a y e r cake & cream p u f f (custard f i l l e d ) I  Few J e r s e y Milwaukee Panama  J.A.M.A.. 1930 9711704 Jour Prev . Med.,. 1931, 5*387  d r i e d beef  sev ex a l  Pasadena G a l . Boston Mass. Chicago, I i i .  Few Eng. MTed» Jour M * - * . / 6  19  &  ;several hundred Winona j M i n n . [several IT. of Alabama j 150  II  tv 11  kippers j ground meat sandwiches I chocolate e c l a i r s custard f i l l e d  II  »  ii-  pastries  cream f i l l e d p a s t r i e s c u s t a r d f i l l e d c o f f e e cake I c u s t a r d - f i l l e d doughnuts) j cream p i e jy jchocolate e c l a i r s yellovj  it » I!  n «i  it  T R B L E l l Copta.  Meyer Meyer & f f y n n s  33 3k 35  1933 1933 1933 1933 1933 1933  36 37 38  193k 193k 193k  Meyer n Meyer & K r u e g e r Crabtree & . Litterer D a c k Bowman & Harger . Shaughnessy & ".' • ' Grubb Dolman. .  30 31 32  39  .  193U  1*0  1935  ia  1936  1)2  1936  A3  1736  44 45  1936 1936  11  . tt  C o c o a n u t Cream P i e Chocolate Eclairs C u s t a r d f i l l e d Cake Pastry & Pies .. ; B u l k I c e Cream  Yellow Staph.  Staph. Yellow Staph.  5  Ham ¥anana Cream;, P i e Potato Salad Cream P u f f s  2i|2  Milk  w  U. A. M. A., 1 9 3 5 , 105:1598  1  Tongue S a n d w i c h e s  it  J.Itif. D i * . , 1 9 3 6 , 58:318 Can. P u b . H. J-., 1 9 3 6 , 2 7 ,  25  Cream  it  3  Custard F i l l e d  Oakland, C a l i f . .Oakland, C a l i f . San F r a n c i s c o " Fresno, C a l i f . Glendale, "  11  Meyer Meyer & S t d n e Costa!; M a n d r a y  P o r t o R i c o ^ P u b . H. & T r o p . Med., 1 9 3 3 , 9-.kk  Porto Rico Calif,. San F r a n c i s c o  it  Am. j . P. H., l§3k,  »  Tennessee  2ii.rl.l6'  J:  Wk  Vancouver, Columbia  British  k  6 66 ? 8 1  to  Oujen Portland, Oregon Calif,  Meyer  19 3T  several 6  ? ?  :  :  11  Filling  t>  Pastry  ca.ke.  Cream ?ie Pastry  Jour. Batt., 193.7, 33:56.  31  Ice-cream Custard  Rm. Jour. "Bit. Health-, 1935,  1Z  Gastard. filled, cake  Thotnpsom.<^ Isaacs 47  1935 FoKhali  2,5:93>3  it it it n  tt Staph.  tt .ti 11  i\ 11  it 11  it it  «  T A B L E  Food I n v o l v e d  Humber o f epidemics reported.  Cream or C u s t ^ filled p a s t r i e s and cakes i n c l u d i n g choc olate e c l a i r s a  Cake and pastry^ type u n s p e c i f i e d [Milk, cheese, i c e cream tteat a, gravy  Z..  Fumber o f persons i n v o l v e d , 600;  i n addition to several r e p o r t s , number unspecified. i n a d d i t i o n to s e v e r a l r e p o r t s , number unspecified. a l s o r e p o r t s , number unspecified, i n addition to several l a r g e epidemics,numbers unspecified, plus several large e p i demics ,numbers unspecx .fx © cL ©  

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