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Studies on staphylococcus enterotoxin Wilson, Robert J. 1937

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STUDIES OF STAPHYLOCOCCUS EiTTSROTOXIlT J . Wilson, B. A, A t h e s i s submitted to the Department of Bacteriology and Preventive Medicine, U n i v e r s i t y of 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 of the requirements f o r the degree of Master of 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. Studies on Staphylococcus Enterotoxin * I n t r o d u c t i o n . Within recent years, voluminous reports have appeared dealing 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 , pre-pared under s u i t a b l e c o n d i t i o n s , from c e r t a i n s t r a i n s of staphylococcus.. The complexity of these f i l t r a t e s was f u r t h e r 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 i r r i -tant i n f i l t e r e d broth preparations from s t r a i n s implicated i n outbreaks of food-poisoning. With the r e c o g n i t i o n of this micro-organism as a p o t e n t i a l e t i o l o g i c a l agent of g a s t r o - e n t e r i t i s , a number of food-poisoning epidemics were reported i n which staphylococcus was i n c r i m i n a t e d . As e a r l y as 1907, Owen ( l ) described an outbreak i n which 19 persons suffered 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 large numbers of staphylo-c o c c i . A milk-borne epidemic was reported 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 ingested m i l k contaminated by an albus s t r a i n of staphylococcus. In t h i s case, more con-c l u s i v e evidence was produced before the causative micro-organ-ism was incriminated. Barber inoculated the organism i n t o s t e r i l e m i l k , incubated 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 to that observed i n the other persons a f f l i c t e d , followed w i t h -i n 2 hours. A p o r t i o n of 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 length of time, did-not cause symptoms of food-poisoning when ingested. The organism was i s o l a t e d i n almost pure culture from the udder of the cow producing the m i l k . Barber's c l a s s i c report presented the f i r s t conclusive evidence that food-poisoning could r e s u l t from staphylococcal contamination. This work remained unconfirmed f o r some 16 years, u n t i l Back and h i s colleagues (3) i n v e s t i g a t e d a s i m i l a r outbreak due to the elab o r a t i o n of a f i l t r a b l e poison by a.yellow hemolytic- staphylococcus, i n a c r e a m - f i l l e d Christmas cake. Portions of the cake were ingested by volunteers, and acute g a s t r o - e n t e r i t i s followed w i t h i n 3 hours. A 40-hour broth f i l t r a t e of the yellow staphylococcus i s o l a t e d from the cake produced i d e n t i c a l symptoms when ingested by volunteers. The poisonous substance appeared to be f i l t r a b l e exotoxin, causing a food i n t o x i c a t i o n . Following 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 succession numerous observations of food-poisoning a t t r i b u t e d to the growth of staphylococcus i n food products. (Table 1.) A study of the l i t e r a t u r e shows a uniform sequence of symptoms, characterized by a short hut d e f i n i t e incubation p e r i o d of 2 to 4 hours f o l l o w i n g i n g e s t i o n of the contaminated food. Towards the end of the incubation or 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 increases i n i n t e n s i t y and r a p i d l y gives way to (3) v i o l e n t paroxysmal vomiting, abdominal cramps, diarrhoea and p r o s t r a t i o n ; there may be t e t a n i c muscular contractions and i intense muscular pain. The more acute symptoms u s u a l l y pass away w i t h i n . a few hours, but general weakness may p e r s i s t f o r several days. I t i s not unusual however, f o r the patient to 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 of oceans-ions when s t e r i l e broth f i l t r a t e s of suspected s t r a i n s of staphylococcus have been ingested by human volunteers. (3, 10, 14, 22.) A review of a v a i l a b l e data reveals that there have been recorded at l e a s t 47 epidemics a t t r i b u t a b l e to the presence of t h i s micro-organism i n food s t u f f s . Of these 47 reported out-breaks, 40 have involved some 1400 persons. (Table 2.) I t i s a matter of some s i g n i f i c a n c e that t h i s type of food-poisoning should show a p r e d i l e c t i o n f o r m i l k and milk products. The report of Barber (2) i n 1914 has already been recounted. Ramsey and Tracy i n 1931 (4) described an epidemic a t t r i b u t e d to the presence of an orange staphylococcus. They noted an " o f f f l a v o r ? described as a "malt" or"caramel n f l a v o r i n the m i l k i n which the organism was grown. Tanner and Ramsey, (5) working with the same organism at a l a t e r date, stated that several of a group of 20 i n d i v i d u a l s v i s i t i n g the laboratory to t e s t the " o f f f l a v o r " were made i l l . I t i s of i n t e r e s t to note i n t h i s connection, that a stronglyjliemolytic golden staphylococcus, obtained from the Department of Dairying at the (4) •at. th-s U n i v e r s i t y of B r i t i s h Columbia, and reputed to produce a "caramel f l a v o r " when grown i n mi l k , elaborates a powerful g a s t r o - i n t e s t i n a l i r r i t a n t . A l i s t of the foods implicated i n the reported outbreaks 'shows that of 47 epidemics recorded, 27, or 57$ were traced to milk and mi l k products. (Table 2.) Reference to 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 frequent i m p l i -c a t i o n of t h i s type of food. Stark (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 milk from every :animal revealed 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 ser i e s of 260 c a t t l e examined, by ffwatkin and his-c o l l e a g u e s , (7) 143 had m a s t i t i s , and 30 of these were due to staphylococcus. 67.4% of the s t r a i n s i s o l a t e d from these cows were toxin.produce r s t 20% of s t r a i n s from normal udders • also produced t o x i n . Crabtree and l i t t e r e r (8) reported a milk-borne epidemic due to a hemolytic staphylococcus from m a s t i t i s i n two cows. Over 242 cases occurred amongst members of a school before the cows were s i n g l e d out from the school herd. Of 8 s t r a i n s i s o l a t e d at random from raw mi 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 tested i n t h i s l a b o ratory, 6 pro-duced a g a s t r o - i n t e s t i n a l i r r i t a n t . While cream p i e s , chocolate 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 to provide a s i n g u l a r l y favorable medium f o r growth and consequent t o x i n production by t h i s organism, and f Toxin i n Gwatkin ' s report r e f e r s to hemolysins. (5) have "been held responsible f o r a large number of outbreaks, (Table 2.) i t i s inconceivable that a l l of these could be traced to a contaminated m i l k supplyc K e l l e r t (9) reported 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) to believe that a high percentage of these organisms are capable of elaborating a g a s t r o - i n t e s t i n a l i r r i t a n t . A s t r a i n "B", used i n t h i s l a b -oratory f o r a greater part of the experimental work caused an • erythema i n the nose of the person from which i t was " i s o l a t e d . I t has produced a powerful enterotoxin of undiminished potency over a period of a year. The f a c t that s t r a i n s from sources other than contaminated m i l k supplies may cause food-poisoning i s f u r t h e r substantiated by reports of epidemics due to growth of staphylococcus i n chicken gravy (11), ham (12) 9 sweet potato candy (12), and tongue sandwiches (13). With the re c o g n i t i o n of inc r e a s i n g numbers of t h i s type of poisoning arose the necessity f o r a laboratory t e s t by means of which.those s t r a i n s producing an enterotoxin could be read-i l y recognized. In many of the e a r l i e r epidemics, the s t r a i n s concerned were tes t e d on human volunteers, or t h e i r i n c r i m i -nation remained unconfirmed, and they were merely suspected. In h i s i n v e s t i g a t i o n s i n 1930, Jordan (10) used human volunteers and i t was c l e a r l y evident that f o r a number of reasons the human was neither a convenient nor s a t i s f a c t o r y laboratory (6) animal. A recent paper by Dack et a l . (14) reports feeding experiments on a number of humans. I t would i n d i c a t e that 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 considerably, and inasmuch as extreme care must be exercised i n administering the t o x i n , the dosages are n e c e s s a r i l y so small as to not show t y p i c a l r e -actions i n many instances. Furthermore, the number of v o l -unteers a v a i l a b l e i s frequently l i m i t e d to such an extent that experiments performed are scarcely of s i g n i f i c a n c e . The use of M. rhesus monkeys, f i r s t reported by Jordan and McBroom i n 1931 (15), has since been confirmed by Woolpert and Dack (16). 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 volunteer. Meyer (17), i n 1934, Stated; "Contrary to experiences made by Jordan and McBroom, and Woolpert and Dack, feeding t e s t s on monkeys have y i e l d e d inconclusive r e s u l t s . " Furthermore, monkeys are d i f f i c u l t to handle, and an experiment on even^moderate scale would be expensively i m p r a c t i c a l . Dack and h i s colleagues (3) inoculated f i l t r a t e s i n t r a -venously into r a b b i t s . Complications that arose due to the presence of the Hemolytic, l e t h a l and other t o x i c substances caused the death of several animals; vomiting was not observed, although loose stools i n some cases ?/ere recarr§.e&. There was obviously 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 (18), reported a method of preparation that rendered guinea pigs 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 enterotoxin. Ho vomiting or diarrhoea occurred, and the post-mortem find i n g s strongly suggested death from the -A other exotoxic products of staphylococcus. Attempts made i n t h i s laboratory to reproduce the r e s u l t s have so f a r been un-s u c c e s s f u l . 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 , biochem-i c a l reactions and chromogenicity of a large number of s t r a i n s implicated i n food-poisoning 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-in g enterotoxin could be recognized. A c u l t u r a l method f o r detecting food-poisoning s t r a i n s of staphylococci has been described 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. Li 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 enterotoxin. A recent m o d i f i c a -t i o n by the same author includes agar i n the medium, and ammo-nium sulphate s o l u t i o n as a developer. The presence of c l e a r zones around a colony i n d i c a t e an enterotoxin producer. No confirmation of these r e s u l t s has been published, and the few experiments performed i n t h i s laboratory have y i e l d e d r e s u l t s inconcordant with 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 conclusive statments have been made regarding the properties 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 . Jordan and Dack (20), and Jordan and Burrows (21), r e c e n t l y reported the (8) f o l l o w i n g " c h a r a c t e r i s t i c s : 1. The active 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 d i a l y s a b l e . 3. I t i s markedly unstable to ff/lOO ITaoH. 4. I t i s unstable- to 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 with the hemolytic substances present i n many staphylococcus f i l t r a t e s , nor does i t produce a s k i n r e a c t i o n . 6. 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 with e t h y l ether or chloroform, as judged by our method of assay. 7. I t may be extracted 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 ether or chloroform 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 to mask such removal. 8. The g a s t r o - i n t e s t i n a l poison i s not completely des-troyed when exposed to a temperature of 100° C. f o r 30 minutes e 9. 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 long as 67 days but i s perhaps somewhat weakened. Monkeys i n j e c t e d intravenously with 5.0 c c . of s a l i n e s o l u t i o n of a c i d ether e x t r a c t of potent f i l t r a t e s , vomited and manifested the usual signs of acute d i s t r e s s of the gastro-i n t e s t i n a l t r a c t . The same s o l u t i o n given intravenously to guinea p i g s , r a b b i t s , cats and dogs produced no i l l e f f e c t s whatsoever. (9) Several wear.leers agreed that no tolerance was induced "by-repeated i n g e s t i o n of the food-poisoning substance. Woolpert and Dack i n 1931 (16), stated that a degree of active immunity could be induced by repeated parenteral i n o c u l a t i o n , but a l l attempts at passive immunity f a i l e d . Woolpert and Back (16), using monkeys as t e s t animals, and Dolman (22), 1934, i n human feeding experiments, demon-st r a t e d that the hemolysins and diverse other t o x i c properties of staphylococcal f i l t r a t e s are d i s t i n c t from the food-poison-, ing substance. I n the l a t t e r ' s experiments, humans ingested r e l a t i v e l y large amounts of f i l t r a t e s containing potent hemo-l y s i n s and t h e i r a l l i e d t o x i c substances with impunity, while much smaller amounts of f i l t r a t e s prepared from one p a r t i c u l a r s t r a i n evoked symptoms of acute food-poisoning i n several volunteers. This s t r a i n produces potent enterotoxin. 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 enterotoxin has hampered i n v e s t i g a t i o n of i t s chemical nature and antigenic s t r u c t u r e . Consequently the conditions favoring i t s production, and i t s mode of a c t i o n on the animal system are as yet undefined. Meyer (17), i n a di s c u s s i o n on staphylococcus food-poisoning s t a t e s ; "However, i n view of the w e l l known f a c t that the enterotoxic 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 of a staphylococcus, i t i s not admissible to consider a given 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) i r r i t a n t . While heating g r e a t l y reduces the k i l l i n g t o x i n , and leaves the enterotoxic capacity unimpaired, the separation of •A the two f r a c t i o n s i s frequently fraught with d i f f i c u l t i e s . The t e s t s on human volunteers are conclusive hut not without r i s k s . Suitable monkeys are not always a v a i l a b l e , and the preparations of small laboratory animals by the methods described by..Borth-wick are not always e f f e c t i v e . A simple physiologic-pharma-co l o g i c t e s t to detect and to separate the enterotoxic substance from the k i l l i n g poison of a staphylococcus i s , therefore, urgently needed." The work undertaken i n t h i s laboratory 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 con-venient that 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 pro-ducing enterotoxin. I t was f u r t h e r deemed desi r a b l e that the t e s t be adequately s e n s i t i v e i n detecting the presence of 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 that i t might be employed i n study-in g the various p r o p e r t i e s , mode of a c t i o n and requirements for the production of the irritantsufestajxce. (11) Experimental. The s t r a i n used i n the greater part of our experimental work, designated as s t r a i n "B", was i s o l a t e d from a human nasal mucous membrane, and caused a chronic erythema of that organ, Toxic f i l t r a t e s of s t r a i n IT^" contain potent vC &n(L/3 hemolysins, and t h e i r a l l i e d exotoxic products, and by v i r t u e of t h i s pro-perty were at one time included w i t h the pooled preparations from a number of organisms during the process of producing staphylococcus toxoid at the Connaught Laboratories, Toronto„T Small subcutaneous doses of these toxoids caused nausea and vomiting i n a number of human subjects; but when s t r a i n "B" was removed from the preparations the violence of the reactions dimin'shed. Ingestion experiments on human volunteers confirmed the suspected presence of enterotoxin, g a s t r o - e n t e r i t i s appear-ing w i t h i n 2-2-|- hours a f t e r consumption of as l i t t l e as 1.0 c c . A subcutaneous dose of 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 patient d i d not completely recover for several 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 pa t i e n t ' s nose, i t y/as shown to have retained 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 t h i s l a b o r -atory i t has c o n s i s t e n t l y produced an enterotoxin of undimin-ished potency f o r oyer one year. The stock-culture i s maintain-ed i n bee 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 preparation employed by Dolman (22), and Dolman and E l t c h i n g (24) i n producing staphylococcus toxoid f o r c l i n i c a l use i s followed. Plates of semi-solid b e e f - i n f u s i o n agar (0.3%.agar) are inoculated with a f i r e - h o u r broth culture of the organism. A f t e r 40 hours incubation at 37* C. i n an • atmosphere of 70% oxygen and 30% carbon di o x i d e , the contents of the pl 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 funnels containing coarse f i l t e r paper. The f i l t r a t e i s cent-r i f u g a t e d at high speed f o r some 2-g— 3 hours and s t e r i l i z e d by passing through a S e i t z E.K. d i s k . The preparation from s t r a i n "B" contains potent hemolysins against r a b b i t and sheep ery-throcytes, has high l e t h a l and demo-necrotic powers as w e l l as the various 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 of staphy-lococcus f i l t r a t e s . The <<• l y s i n ( r a b b i t ' c e l l ) may measure 2500 u n i t s and t h e / 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 of hemolysin t i t r a t i o n described by Dolman (25). The removal of these tbxicjsubstances i s ef f e c t e d by the addi -U.S.P. t i o n of 0„3% s o l u t i o n of formaldehyde' Aand incubation at 37° C. u n t i l the aC and/3 hemolysins are no longer detectable (24). One now has a f i l t r a t e devoid of the deleterious l e t h a l hemo-l 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 to 1.0 c c . i n t r a p e r i t o n e a l l y occasioning no observable harm to mice. The preparation was tested f o r enterotoxin on a human volunteer,, who ingested portions on two occasions. Protocol 1. I . 1.0 c c . was ingested i n 30 c c of milk. Four hours (13) l a t e r , nausea and headache, accompanied by di 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 very 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 hours. P r o t o c o l 2. 2. 1.5 c c . of the same f i l t r a t e was ingested 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 of the preparation nausea appeared, increased i n i n t e n s i t y , and was accompanied by con-siderable g a s t r i c discomfort and sweating. Within 30 minutes of the appearance of nausea, v i o l e n t paroxysmal vomiting occurred, follovred by extreme weakness and d i s a b i l i t y . Abdom-i n a l pains and a general f e e l i n g of discomfort p e r s i s t e d for some S hours f o l l o w i n g the vomiting, during 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 profuse. Abdominal discomfort was r e l i e v e d with passage of the stools but a .feeling of nausea p e r s i s t e d f o r several hours. The f o l l o w i n g day aversion to food, and general weakness were evident. Recovery was complete w i t h i n 48 hours.. These tv/o experiments, combined with, the reports of sim-i l a r instances c i t e d above i n r e l a t i o n to experiences with s t r a i n "B" i n the preparation of toxoids at the Gonnaught Lab-ora t o r i e s i n Toronto i n d i c a t e d that (a) the enterotoxin was more stable to 0.3% formalin at 37° C. than were the hemolytic, dermo-necrotic and l e t h a l p r o p e r t i e s , (b) The enterotoxin was present i n appreciable 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 properties had fceen e n t i r e l y removed. This method of separation of 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 , hemolytic and derrao-necrotic p r o p e r t i e s has been Aalmost e n t i r e l y i n the ease of strain-,' TB" and some others with s i n g u l a r success throughout the i n v e s t i g a t i o n . (14) The f i r s t point of i n v e s t i g a t i o n was to determine the ef f e c t of the f i l t r a t e containing only the enterotoxic p r i n -c i p l e on guinea pigs, using Borthwick's method of preparation (18 j . Tifis procedure i s outlined 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 rabbits and guinea pigs was -a t t r i b u t e d by Borthwick to the d e s t r u c t i o n of the gastro-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 of the stomaciu Accordingly the stomach contents were adjusted to pH' > 7.3 wit h HaHCO 3 5.0% and H/lO HG1, t i t r a t i o n s being c a r r i e d out by means of a stomach tube. When the desired reaction was obtained, the t o x i c f i l t r a t e was introduced and the stomach tube withdrawn. ±sorthwick reported death i n a number of animals w i t h i n one hour of admin i s t r a t i o n of the t o x i c substances. In t h i s laboratory, i t was deemed advisable to use a concentrated phosphate buffer to obtain the desired p H , rather than carry out the somewhat lengjiay procedure oi t i t -r a t i o n . An example of the method followed i n standardizing the hu f f e r i s o u t l i n e d . Protocol 5. Pig 1. 10 c c * of X2HP04, i n a concentration of 80gi per l i t r e , p.R\ 8.83 were introduced i n t o the stomach by mean of a stomach tube. Small samples were aspirated at i n t e r -v a l s of 5, 10,15, 20, minutes and the pff", of each tested on a quinhydrone electrode. 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 aspirated at 5 minutes 7^43 « « " 10 « 7.35 "« " « 15 « 7.30 «« it II 20 « 7 * 32 P r o t o c o l 4 t P i g 2.- 15 c c . of K2HP04 i n a concentration of 80g. per l i t r e . p^ 'H. 8,83, were introduced i n t o the stomach. Samples were aspirated at 2, 5, 10, 15, minutes and the p,H„ tested on a quinhydrone electrode. o r i g i n a l p..Hv of buf f e r s o l u t i o n 8.83 Sample aspirated at 2 minutes 7.55 « H II " « « 10 II 5 w 7i35 7.35 " 15 « 7.35 A suspension of MgC03, 10 g. P e r l i t r e , was also used •as a buf f e r s o l u t i o n . P r o t o c o l 5. (15) Pi g 1. 12.5 c c . MgC03, (10g. per l i t r e ) were i n t r o -duced by means of a stomach tube. . Samples were aspirated at 2, 5, 10, JL5, 20 minutes. Sample aspirated at 2 minutes 7.2 » " " 5 " 7i43 « ". " 1 0 " 7.30 n » " 15 " 7.30 « " » 20 '« 7.30 . The t o x i c f i l t r a t e was administered about 2 minutes af-ter the b u f f e r , i n q u a n t i t i e s of 2*0 to 7*0 c»CiWithout prod-ucing any symptoms other, than s l i g h t nervous twitching. A p o r t i o n of f i l t r a t e "B" was concentrated to about 1/10 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 at 37° G. Doses equivalent to 70 and 80 c.c. were administered enter-a l l y to guinea pigs without any untoward effects An abdominal i n c i s i o n was made i n a guinea pig, and 2.0 c.c; of 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 of a syringe. The i n c i s i o n was s t i t c h e d . The animal recovered from the anaesthetic w i t h i n 20 minutes, - became a c t i v e and remained so f o r a week, when i t was des-troyed; At no time did 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 believe that 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 provide no protection* One was i n t e r e s t e d as to whether, i f the t o x i n were absorbed from the gastro-i n t e s t i n a l t r a c t of the guinea-pigs, the c h a r a c t e r i s t i c mani-f e s t a t i o n s of vomiting and diarrhoea were p o s s i b l e . A, guinea-pig was given intramuscularly, o.05 grains of apomorphine. S l i g h t s a l i v a t i o n , but no vomiting occurred. There was 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 stimulus. S i m i l a r l y , 0.05 mgm. of histamine occasioned no upset i n a guinea-pig. Large qu a n t i t i e s of phosphates administered as buffer solutions^ i n s i g n i f i g a n t and a l s o c a s t o r - o i l produced/changes m the nature of the s t o o l s . One was prompted to believe that, i f vomiting were possible i n a guinea-pig i t occurred very r a r e l y , and not under the usual s t i m u l i a f f e c t i n g humans. 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 -oratory t e s t s ; and i n the experience of the author, on no occasion has a guinea-pig given 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 . This method therefore was 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 excretary habits, and both are known to vomit on occasion, i t seemed l o g i c a l to investigate the s u s c e p t i b i l i t y of these animals to the gastro-i n t e s t i n a l i r r i t a n t . 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 cats, using the f i l t r a t e of s t r a i n "B" tested on a human. (Protocols 1 and 2.) P r o t o c o l 6. Oat No.l 2 months old, weight about one kilogram. 50.0 c.c. of f i l t r a t e "B" were administered i n the animal's milk when i t was fed-. Time 12.35 P.M. 1 Hr.35 Min* The animal became somnolent and drowsy; i t preferred to 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 very marked. 1 Hr.50 Min. The cat vomited. I t seemed much, r e l i e v e d , became more a c t i v e and played a l i t t l e . 2 Hr. 05 Min. Vomiting was repeated; merely g a s t r i c secretions. 2 E r . 15 Min. The cat became unsteady on i t s . feet and refused food. 3 Hr. 10 Min. Giddiness was apparent; the animal swayed from side to side when i t sat up. 5 Hr. 15 Min. V i o l e n t vomiting recurred, 3 times. The'.an-imal 's c o n d i t i o n seemed-much improved a f t e r vomiting. 4 Hrs. The cat accepted food. 4 Hr. 15 Min. The animal was p l a y i n g and seemed quite r e -covered. There was no s i g n i f i g a n t diarrhoea; recovery was complete w i t h i n 24 hours. P r o t o c o l 7. Gat, Wo.2. 2 months o l d , weight about one kilogram. 50.0 c c . of entero^toxic f i l t r a t e were ingested i n m i l k . Time: 12:35 P.Me 1 Hr.55 Min. The animal regurgitated; 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 of the l i p s . 2 Hr.05 Min. Giddiness was apparent. The animal swayed d i z z i l y when standing or s i t t i n g up. 2 Hr.40 Min. G a s t r i c d i s n t r e s s increased; 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 Min. Another wave of nausea occurred, the animal shuddered, spasmodically; 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 to come at i n t e r v a l s . 3 Hr«25 Min. The general c o n d i t i o n of the animal seemed im-proved. 4 Hr.50 Min. A large bulk of f l u i d feces, 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 consistency. The animal recovered completely w i t h i n 24 hours. P r o t o c o l 8o Oat No. 3 large 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 given i n m i l k . The aose was equivalent 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.Me 2 Hrs. The animal remained quiet a i l afternoon. At 5.00 P„M<, i t became a g i t a t e d , c r i e d frequently and l i c k e d itS: l i p s e 2 Hrs.40 Min. I t vomited v i o l e n t l y . The animal seemed r e -l i e v e d a f t e r vomiting. 3 Hrs. V i o l e n t vomiting was repeated, and diarrhoea followed s h o r t l y a f t e r . A large bulk of f o u l - s m e l l i n g feces, mucoid i n consistency, was expelled. The animal recovered completely. 2 ' on (18) The above pr o t o c o l s , merely representative of a number of others i n which the manifestations of 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 greater 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 food-poisoning i n humans. Vomiting occurred from 2 to 2-g 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 and followed by abdominal d i s t r e s s and diarrhoea. Heoovery was rapid and'complete i n a l l cases. As some workers have observed i n humans, diarrhoea m^ y, be present without vomiting or vomiting may occur alone, although as a general r u l e both symptoms are marked* The dose required to produce these t y p i c a l manifestations of food-pcdsoning i n cats when given e n t e r a l l y i s about 50 times that required to cause vomiting and diarrhoea 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 , (Protocols no... 1 and 2) The enteral a d m i n i s t r a t i o n of such large doses to laboratory animals i s neither convenient nor accurate. Inasmuch as the enterotoxic preparation used was innocuous, the hemolysins, l e t h a l and dermo-necrotic substances having been previously destroyed by formalin, parenteral i n s o c u l a t i o n was employed., Protocol 9. 3.0 c.c. of Time: 11 :20 15 Mins; 20 Mins. 30 Mins . ] 40 Mins* A . . * V wj.^y . Vomiting occurred several times, tepeated vomiting occurred, very loose f o u l - s m e l l i n g s t o o l , mucoid i n Consistency 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 preferred 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 recovery were evident. The animal be-came more, a c t i v e and in t e r e s t e d i n i t X s surroundings. Complete r e c o v e r y followed w i t h i n 24 hours. Pr o t o c ol 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 given 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 unnaturally 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 followed by repeated vomitings I lir.10 Mins. Vomiting was repeated* 1 Hr. 25 Mins. A loose f o u l - s m e l l i n g s t o o l was passed, i t was profuse, mucoid i n consistency and l i g h t colored* Retching and vomiting were repeated over a period of an hour, accompanied by several loose s t o o l s . The animal appeared weak and somnolent* 5 Hrs.3 0 Mins. The cat seemed to be recovering, f o r i t became more a c t i v e and accepted food. Recovery was complete i n 24 hours. Protocol 11. Cat Mo.5 - 6 wdcs old, weight" 400 grams* 0.5 OiC. f i l t r a t e "B" were inoculated 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. Vomiting was. repeated. 24 Mins. Vomiting was repeated* The animal remained somnolent and l e t h a r g i c up to 6.00 P.M* Diarrhoea occ-urred some time l a t e r . I t w i l l be observed that i n parenteral i n o c u l a t i o n of 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 that occurring when enteral administration 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 5-10  minutes, depending i n p a r t , upon the s i z e of the dose ad-ministered. 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 increased rate of r e s p i r a t i o n and even panting has been observed, p r i o r to the onset of vomiting. S a l i v a t i o n i s marked; the vomiting i s p r o j e c t i l e (HO) and recurs at frequent i n t e r v a l s . Stools are frequent, and a f t e r the f i r s t one or two, sometimes contain no f e c a l material. They c o n s i s t e n t i r e l y of a c l e a r mucoid discharge resembling ,egg-albumin. Recovery sets i n , i f not too large a dose 13 administered, w i t h i n a few hours, u s u a l l y 5 or 6, and i s most frequently complete w i t h i n 24 hours. As one c o n t r o l measure, animals were inoculated i n t r a p e r i -t o n e a l l y with s t e r i l e broth containing q u a n t i t i e s of form-a l i n (0.3$) and merthiolate (1:10,000) equivalent to those pre-sent i n the f i l t r a t e s prepared 6 10 d i f f e r e n t animals were i n -oculated with q u a n t i t i e s 0 f r o m 1.0 to 5.0 c.c. In no case was there a suggestion of the symptoms c h a r a c t e r i s t i c of the syn-drome produced by the enterotoxin present i n the f i l t r a t e of s t r a i n "B". As a further c o n t r o l measure, f i l t r a t e s of strain , rWood 46", prepared under conditions i d e n t i c a l with those of s t r a i n "B", were inoculated i n t o cats on a number of occasions;doses v a r i e d from 1.0 to 5.0 c.c. The animals remained normal i n every case. F i l t r a t e s of "Wood 46" have been ingested by human volunteers i n r e l a t i v e l y large amounts and have occ-asioned no upseto In a l l , some 50 i n d i v i d u a l experiments have been performed on cats with f i l t r a t e "B", using 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 varying ^rom 0.4 to 3.0 c.c. were employed, and i n every case, symptoms of 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 greater or l e s s e r i n t e n s i t y , depending (21) upon the s i z e of the lose and. the degree of ac t i v e immunity acquired by the animal due to previous i n j e c t i o n s . T h e ' f i l t r a t e "B" producing 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 parenteral a d m i n i s t r a t i o n i n cats was inoculated i n t o a dog, 3 months of age, 3.0 c.c. were Injected i n t r a -p e r i t o n e a l l y . Yaomiting began 5 minutes a f t e r i n o c u l a t i o n and continued at i n t e r v a l s f o r over 3 hours; several f l u i d s t o o l s were passed w i t h i n one hour of i n o c u l a t i o n . The animal appeared dazed and giddy, and was too weak to stand 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 refused food; recovery seemed complete however, w i t h i n 48-60. hours. The s e n s i t i v i t y of dogs to the enterotoxin has been demonstrated on several occasions. In every case the symptoms are i d e n t i c a l with those produced i n eats by i n o c u l a t i o n of the gastro-i n t e s t i n a l . i r r i t a n t . During the course of i n v e s t i g a t i o n , c e r t a i n observations were recorded that tendeAto confirm 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 (21), Woolpert and Dack (16) and l a t e r Dolman (22) produced evidence to show that the enterotoxic p r i n c i p l e was not i d e n t i c a l w ith the hemolysins, l e t h a l or. other recognized t o x i c products of sta 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 of preparing enterotoxic f i l t -rates devoid of these substances provides f u r t h e r data i n support of t h i s contention. The author wishes to present a d d i t i o n a l experimental evidence i n favor of the f a c t that the entero-t o x i n i s a separate t o x i c product of staphylococcus. (22) Despite 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 detectable i n f i l t -rates of "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 inoculated into mice, and 5 to 10c*c<, q u a n t i t i e s into guinea-pigs occasioned no harmful r e a c t i o n ; some cats died f o l l o w i n g parenteral i n o c u l a t i o n of the enter-otoxic f i l t r a t e s * Several protocols are presented to i l l u s t r a t e the phenomenon. Protocol 12. . Cat Ho.l 1-2 months old. Y/eight 600 g. 2.0 c.c. f i l t r a t e "B" were inoculated 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. Violent vomiting occurred. Vomiting and d i a -rrhoea recurred at i n t e r v a l s f o r several hours* The animal was weak, dazed, and suffered acute abdominal d i s t r e s s . Death occurred at 12:15 the f o l l o w i n g day, 25 hours a f t e r inoculation. Just before death the animal appeared markedly emaciated and i n a state of extreme p r o s t r a t i o n . Mucus was discharged i n v o l u n t a r i l y from the anus at frequent i n t e r v a l s o The animal died during a spasm of convulsive 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; Emaciation was very marked. The peritoneum and s i t e of 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 contracted, blanched and very f i r m i n consistency. Internal examination of the gut revealed an excessive secre-t i o n of a thi c k yellow mucus throughout the en t i r e leng^th* The stomach contained a large quantity of gas. The g a l l • bladder was enormously engorged, and d i f f u s i o n of b i l e p i g -ments stained the viscera, over a wide area. The u r i n a r y bladder was completely contracted and f i r m . Uo further macroscopic abnormalities were apparent. Proctocol 13. Gat ITo.2 1 month old. Weight 350 g. J u l y 2,1936. 0.1 c.c. of f i l t r a t e "B" was administered 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 administered 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 several 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 "Bt? were administered i n t r a p e r i t o n e a l l y . There was general 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. Stools occurred frequently, often at EO minute i n t e r v a l s , and expulsion was involuntary i n most instances. Death occurred on J u l y 13. Autoptsy: The t a i l and hind legs were s o i l e d with 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 period 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 large and small i n t e s t i n e s were contracted, 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 length. The stomach contained a large quantity of mucuous s e c r e t i o n . The g a l l bladder was engorged; d i f f u s i o n of b i l e pigments stained the abdominal v i s c e r a over a wide area. The u r i n a r y bladder was completely Contracted and f i r m . There were no further macroscopic abnormalities. These examples are t y p i c a l , of some zo reactions observed when f i l t r a t e "B" has been inoculated i n t r a p e r i t o n e a l l y into c a t s . In a l l cases, the only constant abnormalities noted on autopsy have been those presented i n the above p r o t o c o l s . In no instance were the signs of general vascular 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 exotoxins, present to any observable degree. This-phenomenon! has also 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 occurss about 24 hours a f t e r the f i r s t dose, and continues with i n c -reasing s e v e r i t y u n t i l death. Vomiting however, has not been observed on subcutaneous administration of enterotoxic f i l t r a t e s . A large dose of f i l t r a t e T ,B , f, or the frequent adminstration of smaller doses, produces an exaggerated form (24) . of the usual 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 of the c h a r a c t e r i s t i c post-mortem symptoms of acute death from potent exotoxic f i l t r a t e s as described by Dolman (25) were observed 4- s t r o n g l y i n d i c a t e s that 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 i s a substance e n t i r e l y separate from any_o_f the recognized t o x i c products of staphylococci. The heat s t a b i l i t y of staphylococcus enterot.oxin has been recognized f o r several years* Jordan, et a l . (20) using human subjects as t e s t animals stated; "The t o x i c substance present 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 completely destroyed by exposure f o r 30 minutes to the temperature of b o i l i n g water. Some diminution i n t o x i c power may, however, p o s s i b l y be caused by heating even at temperatures below 100 ° C.TT F i l t r a t e s of s t r a i n »B" heated at 100 0 C. f o r 30 minutes have, on 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 i n cats, 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 enterotoxin. Some diminution 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 are required to produce t y p i c a l symptoms a f t e r heating at 100° G. f o r 30 minutes, than are necessary a f t e r heating at 100" C. f o r 10, 15, or 20 minutes. The t o x i n i s , however, not completely destroyed by heating at 100" G. f o r 30 minutes, when assayed by the " k i t t e n - t e s t " . (25) E a r l i e r p u b l i c a t i o n s notedthat the enterotoxin i s stable to storage at low temperatures. In t h i s laboratory preparations from s t r a i n "B" containing 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 diminution i n t o x i c power a f t e r 5 months. Incubation at 57° 0. i n the presence of 0.5%  formalin f o r 78 days did not destroy the enterotoxin 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 that no p e r c e p t i b l e degree of immunity or tolerance could be acquired by repeated i n g e s t i o n of enterotoxic f i l t r a t e s . Woolpert and Dack (16) stated 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 against the enterotoxin could be induced by repeated parenteral i n o c u l a t i o n , but a l l attempts at passive immunity f a i l e d . Working with cats i n t h i s laboratory, i t came to one's a t t e n t i o n t hat, a f t e r a number of i n j e c t i o n s , an animal was able to receive with impunity several times the dose of f i l t -rate "B'f that p r e v i o u s l y caused symptoms of acute gastro-e n t e r i t i s . During the course of i n v e s t i g a t i o n t h i s phenomenon! was c o n s i s t e n t l y i n evidence. The active immunity described by e a r l i e r workers could, then, be induced by repeated parent-e r a l i n o c u l a t i o n of f i l t r a t e "B". Further 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 enterotoxin of f i l t r a t e "B". Serum was obtained from a c t i v e l y immunized cats and incubated with f i l t r a t e "B,T at 37° C. f o r !-§- hours. The mixture (26) was Inoculated i n t r a p e r i t o n e a l l y i n t o cats* Protocol 14. -a Gat No.'l 1.5 c.c. f i l t r a t e "B"+ 2.0 c.c. immune serum incu-- • bated at 57° G. f o r hours were inoculated i n t r a p e r i t o n e a l l y . The animal remained normal, played, and ate h e a r t i l y whenever food was o f f e r e d , No symptoms of 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 present. 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 at '67 ° o. f o r !-§- hours were inoculated i n t r a p e r i t o n e a l l y . The animal showed symptoms of acute g a s t r o - i n t e s t i n a l upset. Diarrhoea continued f o r some 5-7 hours. Gat No.5 1.5 c.c. of s a l i n e + 2.0 c.c.immune serum incubated at '67 9 0. f o r 1-g- hours were inoculated i n t r a p e r i t o n e a l l y . The animal remained normal. . . Pr 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 at 37" C. f o r 1-g- hours were inoculated i n t r a p e r i t -oneally. The animal remained normal. Gat No. the fo l l o w i n g day - 0.5 c.c, f i l t r a t e "B" were ino-eu'lated~intraperitoneally, Vomiting and diarrhoea occurred. This animal, apparently s e n s i t i v e to 0.5 c.c. of f i l t r a t e , received with impunity three times that amount mixed with immune serum. Rabbits were given repeated parenteral inoculations of f i l t r a t e "B", and the serum was tested f o r immune substances against hemolysins. I n t h i s experiment, a f i l t r a t e of s t r a i n ir "B" containing a l l of the t o x i c substances was used. I t was not d e t o x i f i e d with 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 to n e u t r a l i z e the hemolysins. The mixture was i n -cubated overnight at 37° G», the p r e c i p i t a t e removed and the . supernatant tested on c a t s . P r o t o c o l 16. Gat No.l 1.5 c.c. t o x i n "B" heated at 100* G. f o r 10 minutes (to destroy the cC and p and l e t h a l toxins,) were inoculated 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 . . Vomiting and diarrhoea recurred at i n t e r v a l s f o r some time. Gat No.2 4.2 c.c. of t o x i n - a n t i t o x i n mixture (5.0 c.c. t o x i n  "B", 1.2 c.c. a n t i serum) were inoculated 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 at 100 ° C. f o r 10 minutes (to destroy the oC , fi hemolysins and l e t h a l toxins,) plus 1.5 c c normal rab b i t serum incubated overnight at 37 ° C. were inoculated i n t r a p e r i t o n e a l l y . V i o l e n t vomiting and diarrhoea appeared i n 10 minutes and recurred frequently over a period of 2-3-hours. The s t o o l was a c l e a r mucoid secretion containing no f e c a l m a t e r i a l . An antiserum was prepared by immunizing a rabbit with f i l t -rates of s t r a i n "Wood 46", containing a l l of the t o x i c products of staphylococcus except the enterotoxin. The antiserum was capable of n e u t r a l i z i n g i n v i t r o the oC and (S hemolysins. P r o t o c o l 16 (cont'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 to n e u t r a l i z e the oC and (* hemolysins) incubated at 37° C. overnight, were inouulated i n t r a p e r i t o n e a l l y . With-i n 10 minutes, v i o l e n t vomiting occurred, followed by d i a -rrhoea. The animal was i l l throughout the day. Death occurred the f o l l o w i n g day, and autopsy revealed only the symptoms t y p i c a l of those produced by the* e n t e r o t o x i n e I t would seem evident that a n t i s e r a prepared against f i l t r a t e "B" are capable of n e u t r a l i z i n g i n v i t r o , the gastro-i n t e s t i n a l i r r i t a n t contained i n that f i l t r a t e . Gat No.2 (protocol 16), received with impunity twice the dose that 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 that n e u t r a l i z e s the enterotoxin i s not present i n normal r a b b i t serum, (cat 3) noran a n t i s e r a prepared against f i l t r a t e s containing a l l of the known t o x i c products of staphy1ococcus except the enterotoxic 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 point 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 tested by human volunteers, and known *to produce a powerful enterotoxin. The s e n s i t i v i t y • of cats 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 various sources., 1. In an epidemic of food-poisoning reported by Dolman (26), large numbers of hemolytic staphylococci were i s o l a t e d from the suspected pastry, custard f i l l e d " v a n i l l a s l i c e s " . I n t r a p e r i -toneal i n o c u l a t i o n i n cats of s u i t a b l y prepared 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 gastro-e n t e r i t i s described e a r l i e r i n t h i s paper. 2. Hemolytic staphylococci i s o l a t e d from a cream p i e , suspected of causing food-poisoning, y i e l d e d a powerful e n t e r o t o x i n . ^ c .c. . of the f i l t r a t e inoculated i n t r a p e r i t o n e a l l y i n t o a 3 months old 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 reported to cause "caramel f l a v o r " i n m i l k , produced a potent enterotoxin as w e l l as high 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 c l a s s -i f i e d as "Tetracoccus l i q u e f a c i e n s " (Orla-Jensen). In 10 exper-iments with 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 to 3.0 c c , and death occurred i n 3 instances, from doses as low as 1.5 c c A siS-------ar instance has been described 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 high t i t r e s ofo£andj3 hemolysins as w e l l as a powerful g a s t r o - i n t e s t i n a l (29) i r r i t a n t . 8 experiments were c a r r i e d out wi t h appropriately prepared f i l t r a t e s . Doses as low as 0.5 c.c. produced p o s i t i v e r e s u l t s , and death occurred i n 3 instances from doses as low as 2.0 c.c. 5. From a t o t a l of 8 s t r a i n s picked at random from raw mi 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 , 6 produced a powerful entero-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 issues involved i n the study of staphy-l 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. Since 1928 there have "been reported some 47 outbreaks of staphylo-coccus food-poisoning; 40 of these involved more than 1400 persons of both sexes and a l l age groups. The f a i l u r e to recognize 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 to the influence exerted by the exhaustive studies of Savage (28) and Etkeles and Standfuss (29) on the Salmonella group; and while i n certain instances staphylococci were known to be present i n foods incriminated e p i d e m i o l o g i c a l l y , t h e i r presence was not reported by the laboratory. Furthermore, a t r a n s i e n t d i s a b i l i t y such as that caused, by the enterotoxin would only too frequently be diagnosed by the layman as " i n t e s t i n a l f l u " ornsummer d i a -rrhoea" one would expect r e l a t i v e l y few such cases to be r e -ported 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 consider-a t i o n s , combined with the fact that the texture, appearance and taste of food h e a v i l y contaminated by staphylococci remain un-a l t e r e d , would lead one to believe that staphylococcal gastro-e n t e r i t i s i s more common than reports i n d i c a t e . The high percentage of outbreaks a t t r i b u t e d to milk and milk products i s a, p u b l i c health issue 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 that 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 staphylococci i n m i l k obtained from apparently healthy cows may play a part i n (31) outbreaks of food-poisoning (6,$). Inasmuch as t h i s source of contmlnation'cannot he r e a d i l y c o n t r o l l e d by any method of pro-duction, the only e f f e c t i v e preventative measure would be univer-s a l 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 the a d d i t i o n a l hazard of droplet i n f e c t i o n by staphylococci from those handling the m i l k during production; and since milk seems to provide an e x c e l l e n t medium f o r the rapid 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 enforced. The frequent i n c r i m i n a t i o n of cream pies and c u s t a r d - f i l l e d 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 that droplet i n f e c t i o n , and the transmission 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 products. Slow cool i n g i n large open k e t t l e s at room temperature p r i o r to f i l l i n g pastry s h e l l s enhances the r i s k of contamination, and provides admirable temperature conditions f o r rapid m u l t i p l i c a t i o n . By v i r t u e of the large batches of f i l l e r prepared i n bakeries, the number of persons involved i n outbreaks from t h i s source i s frequently high. (Tablel.) Meyer(17) reports an increased incidence i n the Southern United States during 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 health a u t h o r i t i e s i n that region have, during the warmer months, r e s t r i c t e d the sale and d i s t r i b u t i o n of c r e a m - f i l l e d (32) bakery goods to bakeries with properly 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 cases. An u n o f f i c i a l report i n d i c a t e s that enforcement of the l a t t e r measure has a c t u a l l y reduced the .incidence of staphylococcal g a s t r o - e n t e r i t i s i n the area concerned. Methods of preparation could be discussed at 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 products, (b) frequent s t e r i l i z a t i o n of 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 production, (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 ordinary r u l e s of hygiane by those handling 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 conclusions regarding the mode of a c t i o n of the enterotoxin on the animal system, there are c e r t a i n i n t e r e s t i n g points which might tend to throw some l i g h t on the matter. Despite the f a c t that the more obvious reactions produced by the t o x i n are c l i n i c a l signs of a gastro-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 large i n t r a p e r i t o n e a l inoculations of f i l t r a t e s con-t a i n i n g only the enterotoxic p r i n c i p l e does not reveal signs of acute i n t e s t i n a l inflammation. There i s an excessive secretion 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 rapid on-set of the symptoms, frequently w i t h i n 5 minutes of i n t r a p e r i -toneal i n o c u l a t i o n , would scarcely 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 of the signs of l o c a l inflammation at the point 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 extensive damage through contact between the t o x i n and ex-posed t i s s u e s . The mode of a c t i o n would appear to be through the nervous system. This would necessitate absorption of the t o x i c sub-stance, and tr a n s p o r t a t i o n by the blood to the nerve centres attacked. The p o s s i b i l i t y that 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 length of the l a t e n t period, and by the s e v e r i t y Of the symptoms varying with the route of admini-s t r a t i o n . I f the substance be ingested with food, the l a t e n t period i s 3 hours, which may be taken to correspond with the period required f o r d i g e s t i o n and subsequent absorption of the ingested m a t e r i a l . 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 period i s shortened to as l i t t l e as 5-10 minutes, due to more rapid, absorption by that route. 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 that, the slower absorption i n the l a t t e r case, combined with the f a c t that much of the t o x i c m a t e r i a l may be discharged i n vomltus and feces, only a small part of the ingested dose i s a c t u a l l y absorbed. This factor/is eliminated by parenteral administration, 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. In subcutaneous i n o c u l a t i o n symptoms are l e s s severe, and appear a f t e r 18-S4 hours. The onset-of symptoms would seem to demand a c e r t a i n p r i o r concentration of (34) t o x i n i n the blood stream, thelatent period being the time r e -quired f o r that concentration to be reached, and the length of the l a t e n t period depending upon the rate ox absorption. The nerve centres attacked by the enterotoxin are not known. In " post-mortem examination, the urina r y bladder has been c o n s i s t e n t l y found i n a state of complete co n t r a c t i o n , which would discount the p o s s i b i l i t y that p e r i p h e r a l nerve endings i n the gastro-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, that f a c t that the vomiting and diarrhoea which fol l o w the administration of the enterotoxin to laboratory animals are u s u a l l y associated with unsteadiness and apparent v e r t i g o , strongly suggests that the substance concerned may act upon adjacent centres i n the mid-b 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 discovery of a c t i v e immunization methods with staphy-lococcus toxoid necessitated the establishment of a r b i t r a r y standards f o r innocuity and potency to which preparations f o r c l i n i c a l use could be r e f e r r e d before release from the labora-tory (24). Since the i n c l u s i o n i n the toxoids of f i l t r a t e s con-t a i n i n g enterotoxin might give r i s e to unpleasant reactions, pre-cautions must be observed to prevent t h e i r being used i n prepar-ations f o r human immunization. The suggestion has already been made (30) that an a d d i t i o n a l standard f o r innocuity should be appended to those recommended by Dolman and H i t c h i n g (24), v i z . ; ,T3.0 c.c. of the toxoid , inoculated i n t r a p e r i t o n e a l l y into a normal k i t t e n should occasion no deleterious symptom'r„ ( 3 5 ) But s t r a i n s of staphylococci i s o l a t e d from human le s i o n s and subsequently found able to produce enterotoxin, may con-ceivably -owe t h e i r pathogenicity i n partto t h i s l a t t e r property, The r e a c t i o n Of the host to continued absorption of small quan-t i t i e s of enterotoxin, such as may occur i n acute staphylococcal 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 of our knowledge of the deleterious e f f e c t upon cats under .certain circumstances of repeated parenteral i n o c u l a t i o n , i t i s possible that the enterotoxin might play 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 enterotoxin i s antigenic i t would seem desirable that e f f o r t s should be made to render the antigen innocuous to humans so that i t might be incorporated with other staphylococcal antigens i n materials d i s t r i b u t e d f o r the acti v e immunisation of man. Further work Is desirable an the problem of the antigenic properties and r e l a t i o n s h i p s of the enterotoxin, and methods of assay of an t i - e n t e r o t o x i c sera require i n v e s t i g a t i o n . In the meanwhile, f i l t r a t e s - of s t r a i n s producing enterotoxin might to advantage be included i n the pooled antigens used f o r the imm-u n i s a t i o n of horses i n the preparation of staphylococcus a n t i -t o x i c sera. (36) Summary. 1. C e r t a i n s t r a i n s of staphylococcus produce a g a s t r o - i n t e s t -i n a l poi&on i n conjunction with various 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 necrotoxin by f o r m a l i n i a a t i o n at 57 ° C. u n t i l the oC and^ hemolysins are no longer detectable. 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 of small amounts of these formalinized f i l t r a t e s i n cats and dogs provides a te 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 of the presence of the ent-erotoxin 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 staphylococcal food-poisoning. Guinea-pigs, rabb i t s and mice are not s e n s i t i v e to o r a l or par-enteral a d m i n i s t r a t i o n of 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 parenteral 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 that produced by o r a l administration; excessive dosage may prove f a t a l . Post-mortem changes d i f f e r from those present i n animals dying from the e f f e c t s of the potent exotoxins of staphylococcus. 4. The substance producing gastro-intestt" rial derangement i n cats posess"* properties s i m i l a r to those ascribed by o the ^ work-ers to the enterotoxin 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 hemolytic, demro-necrotic or l e t h a l substances; heat-i n g at 100°C. f o r 30 minutes does not completely destroy the enterotoxic p r i n c i p l e ; i t i s stable when stored at low temper-atures J i t w i l l not d i s t i l } and a degree of active immunity can be induced by repeated parenteral i n o c u l a t i o n . (37) 5. The serum of an a c t i v e l y immunized animal apparently neut-r a l i z e s the enterotoxin i n v i t r o . 6. Strains of staphylococci from raw m i l k and pa t h o l o g i c a l l e s i o n s i n humans may, under appropriate conditions, produce a powerful enterotoxie substance. Acknowledgements . I t i s indeed a pleasure to acknowledge my indebtedness to Br. C E . Dolman, Department of Bacteriology and Preventive Medicine, U n i v e r s i t y of 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 valuable suggestions i n the d i r e c t i o n of t h i s work, and to Dr. H.J. Gibbons, Connaught Laboratories, Western D i v i s i o n , f o r h i s kind i n t e r e s t and assistance. Bibliography, 1. Owen; Phy 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 Jour. Sc., Sect.B., 1914, 9:515. 3. Dack, G.M., Gary, W.E., Woolpert, O.G ., and diggers, H.; Jour. P r e r . Med., 1930, 4:167. 4. Ramsey, R.J., and Tracy, P.H.; Proc, Soc. Exper. B i o l , and Med., 1931, 28:390. 5. Tanner, If.W., and Ramsey, R.J.; Am. Jour. Med, Sc., 1932, 184:80. 6. Stark, H.; Jour. I n f . D i s . , 1926, 39:114. 7. Grwatkin, R., Hadwen, S., and l e G-ard, H.M., Can. Pub. Health Jour. 1956, 27:391. 8. Grabtree, J.A., and l i t t e r e r , Wm.; Am. J our. Pub. Health, 1934, 24:1116. 9. K e l l e r t , E.; Am. Jour. Pub. Health, 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.; Jour. Prev. Med., 1931, 5:387. 12. Costa Mandray, A.; Puerto Rico Jour. Pub. Health 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.; Jour, Bact., 1937, 33:49. 15. Jordan, E.O., and McBroom, J . ; Proc. Soc. Exper. B i o l , and Med., 1931, 29:161. 16. Woolpert, 0 .C ., and Dack, G- ,H.; Jour. I nf. Dis ., 1933, 52:6. Bibliography (cont'd). 17. Meyer, K.F.; Sonderabdruck aus Sangger-Festschrift Seite 278-279, 1934, Hascher and C i e . A.-G., Yerlag, Z u r i c h . 18. Borthwick, G.R.; Br. Jour. Exper. Path., 1933, 14:236. 19. Stone, R.Y*; Proc. Soc. Exper. B i o l , and Med., 1935, 33:137. 20. Jordan, E.O., and Hack, 0 «M.; Jour. Prey. Ivied., 1931, 5; 383. 21. Jordan, E.O., and Burrows, W,; Proc. Soc. Exper. B i o l . and Med., 1933, 30:448. 22. Dolman, C.E.; Jour, I n f . Dis., 1934, 55:172. 23. Dack, G.M., Jordan, E.O., and Woolpert, O.C.; Jour. Prev. Med., 1931, 5:151. 24. Dolman, C,E., and E i t c h i n g , J „S.; Jour. Path, and Bac t . , 1935, 41:137. 25. Dolman, C.E.; Can. Pub. Health J our., 1932, 23:125. 26. Dolman, O.E.; Can. Pub. Health J our., 1936, 27:494. 27. Jordan, E.O., and Burrows, W»; Jour. I n f . D i s . , 1935, 57:121, 28. Savage; Med. Res. Coun c i l , Spec. Report Series Ho. 91, 1925, & Jotir. Prev. Med., 1932, 6:425. 29. E l k e l e s and Standfuss; Handb. d. pathog. Mikroorg, 3, ed., 1931. I l l , part 2, p. 1681. 30. Dolman, C.E., asd Wilson, R.J., and Oockcroft, W.H.j Can. Pub. Health Jour., 1936, 27:489. TABLE 1. Date 1.1-907 2,1914 3.1927-4.1928 5.1929 6. 1929 7.1929 8.1930 9.1930 10.1930 11.1930 12.1931 13*1931 14.1931 15.1931 16.1931 17.1931 16.1931 19.1932 20.1932 Ii 21.1932 22.1932 23-1932 j24.1932 ! 25-1932 :26.1932 I 27.1932 : 28.1933 Eeported by Owen Barber 28.Artb.ur French Kelson Jordan Jar dan Dack, Gary, Woolpert and Wiggene Jordan N.Y. state Dept. of Health a it or i*. » Jor dan Jordan & H a l l Jor dan: Meyer Meyer ILeyer Meyer Ramsey & Tracy Ramseyt Tanner Meyer, Krueger Mep'er, .Simpson Grieger , Gray, Meyer Jor dan,Bur r owe to It u ir n- n Buchanan, EcKer Jor dan , Bur r ows 29.1933 ! McBurney Epidemics of Staphylococcus Food-poisoning. . ( D i r e c t l y or i n d i r e c t l y reported.) • Fates This l i s t makes no cla i m f o r completeness* Jo u r n a l ^Location Physician and Surg., I907J •'29*28.9-P h i l i p p i n e Jour. Science,] Sect.B., 1914,9:51^; P h i l i p p i n e s Few Eng. MTed» Jour M*-* . J.A.M.A.,rf«-30, <?4: 16*8 J.A.ir.A . ,1130,9* - / 6 * 8 Jour. Prev. Med., 1930, 41167 J.A.M.Ae 1930, 4 t l 6 7 N-Y- Haalffv News, Ccth 1930-„ , i . Dec., )^50« J.A.M.A.. 1930 B 9711704 Jour tt Prev . Med.,. 1931, 5*387 Proc. af the Soc « f o r Exp. & B i o l . med o s 1931, 28s390 Am. Jour. S c . 1 9 3 2 , 80-85s184 Pasadena G a l . Boston Mass. Chicago, I i i . Chicago, 111. Chicago,. I l l • Porto Rico Long I s l a n d Few Jersey Milwaukee Panama Jersey C i t y C a l i f * C a l i f . Los AngelesCalJ C a l i f s . "Fo." Involved 19 sev ex a l Caused by dr i e d beef milk v a n i l l a i c e cream cream pie chocolate cream pie a p p r o D F . 1 5 0 Wedding cake 20 l a y e r cake & cream puff (custard f i l l e d ) Laboratory report staph* Am. Jour. Hygiene, 1934» 20,, F o e 3 8604-610 » a n n n n ir 9 II » » 15 « It It-It 11 it ii- » n it Am. Jour. Hygiene, 1934» 2 0 , Fo. 3s604-610 J.A.M.A., 1933* 100:1999 C a l i f . C a l i f • Washington San Francisco Milwaukee Chicago Milwaukee Chicago Cleveland Winona j Minn. IT. of Alabama 4 4 125 4 2 16 •••3 42 . 5 2 ?• 20 2c,-: 200 35 54 31 7 ii & ;several hundred [several j 150 white yellow yellow I yellow i yellow sponge cake, cream, f i l l e r yellow cheese cake i cream-puffs l D e v i l ' s food cake j chicken gravy j l a y e r cake ^ j chocolate e c l a i r s j head cheese sandwiches I coc'anut cream pie I chbalate e c l a i r s ! m i l k milk kippers j ground meat sandwiches I chocolate e c l a i r s custard f i l l e d p a s t r i e s cream f i l l e d p a s t r i e s custard f i l l e d coffee cake I c u s t a r d - f i l l e d doughnuts) j cream pie j- y jchocolate e c l a i r s yellow yellow Orange Or ange yellovj II 11 « n 11 n i» II tv 11 II » ii-it » I! n «i it T R B L E l l Copta. 30 1933 Meyer 31 1933 Meyer & ffynns 32 1933 11 . tt 11 33 1933 Meyer 3k 1933 Meyer & Stdne 35 1933 Costa!; Mandray 36 193k Meyer 37 193k n 38 . 193k Meyer & Krueger 39 193U C r a b t r e e & . L i t t e r e r 1*0 1935 Dack J : Bowman & Harger i a 1936 . Shaughnessy & ".' • ' Grubb 1)2 1936 Dolman. . A3 1736 Oujen 44 1936 45 1936 M e y e r 19 3T Thotnpsom.<^  Isaacs 47 1935 FoKhali P o r t o R i c o ^ P u b . H. & Trop. Med., 1933, 9-.kk Am. j . P. H., l§3k, 2ii.rl.l6' U. A. M. A., 1935, 105:1598 J.Itif. D i * . , 1936, 58:318 Can. Pub. H. J-., 1936,27, Wk Jour. Batt., 193.7, 33:56. Rm. Jour. "Bit. Health-, 1935, 2,5:93>3 Oakland, C a l i f . s e v e r a l Cocoanut Cream P i e Y e l l o w Staph. .Oakland, C a l i f . 6 C h o c o l a t e : E c l a i r s it it San F r a n c i s c o " k C u s t a r d f i l l e d Cake it 11 F r e s n o , C a l i f . 6 P a s t r y & P i e s : .. ; it it G l e n d a l e , " 66 B u l k I c e Cream n it P o r t o R i c o ? Ham Staph. C a l i f , . 8 ¥anana Cream;, P i e Y e l l o w Staph. San F r a n c i s c o 1 P o t a t o S a l a d tt « it » 5 Cream P u f f s Staph. Tennessee 2i|2 M i l k w 1 Tongue Sandwiches it 25 Cream F i l l i n g it Vancouver, B r i t i s h tt Columbia 3 C u s t a r d F i l l e d P a s t r y to 1 t> ca.ke. .ti Portland, Oregon ? Cream ?ie 11 Calif, ? Pastry i\ 31 Ice-cream Custard 11 1Z Gastard. filled, cake T A B L E Z.. Food Involved Humber of epidemics reported. Cream or Cust-a ^ f i l l e d 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 Cake and pastry^ type unspecified [Milk, cheese, ice cream tteat a, gravy Fumber of persons involved, 600; i n ad d i t i o n to several reports, number unspec-i f i e d . i n a d d i t i o n to several reports, number unspec-i f i e d . also reports, number un s p e c i f i e d , i n a d d i t i o n to several large epidemics,numbers u n s p e c i f i e d , plus several large e p i -demics ,numbers unspec-x .fx © cL © 

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