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TREASURY DEPARTMENT. 
: Health and Marine-Hospital Service of the United States. 

Walter Wyman, Surgeon-General. 



iYGIENIC LABORATORY— BULLETIN No. 29. 

Tvl. J. I^JOSICNAII, Oirector. 

April, 1906. 



TDY OF THE CAUSE OF SUDDEN DEATH 
FOLLOWING THE INJECTION OF 

HORSE SERUM. 



BY 



]VI. J. ROSENAU 

AND 

JOHN K. ANIDERSON. 




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WASHINGTON: 

GOVERNMENT PRINTING OFFICE 

I 906. 



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4 



ORGANIZATION OF HYGIENIC LABORATORY. 

Walter Wyman, Surgeon-General , 
United States Public Health and Marine-Hospital Service. 

ADVISORY BOARD. 

Major Walter D. McCaw, Surgeon, U. S. Army; Surgeon John F. Urie, U. S. Navy; 
Dr. A. D. Melvin, Chief of U. S. Bureau of Animal Industry, and Milton J. Rosenau 
U. S. Public Health and Marine-Hospital-Service, ex officio. 

Prof . William H. Welch, Johns Hopkins University, Baltimore, Md.; Prof. Simon 
Flexner, Rockefeller Institute for Medical Research, New York; Prof. Victor C. Vaughan, 
University of Michigan, Ann Arbor, Mich.; Prof. William T. Sedgwick, Massachusetts 
Institute of Technology, Boston, Mass., and Prof. Frank F. Wesbrook, University of 
Minnesota, Minneapolis, Minn. 

LABORATORY CORPS. 

Director. — Passed Assistant Surgeon Milton J. Rosenau. 
Assistant director.^— Passed Assistant Surgeon John F. Anderson. 
Pharmacist. — Frank J. Herty, Ph. G. 
Acting librarian. — E. B. K. Foltz. 

DIVISION OP PATHOLOGY AND BACTERIOLOGY. 

Chief of division. — Passed Assistant Surgeon Milton J. Rosenau. 

Assistants. — Passed Assistant Surgeons John F. Anderson, W. W. King, and H. A. 
Stansfield. 

DIVISION OP ZOOLOGY. 

Chief of division. — Ch. Wardell Stiles, Ph. D. 

Assistants.— Passed Assistant Surgeon Joseph Goldberger, Philip E. Garrison, M. D., 
and Arthur L. Murray, M. D. 

DIVISION OP PHARMACOLOGY. 

Chief of division. — Reid Hunt, Ph. D., M. D. 

Assistants. — Ren^ de M. Taveau, A. B., and Madison B. Porch, B. S. 

DIVISION OF CHEMISTRY. 

Chief of division. — Joseph H. Kastle, Ph. D. 
A6si$Uxvi. — H. L. Amoss, B. S, 

(3J 



98743 



TABLE OF CONTENTS. 



Page. 

Introduction 7 

I. Control experiments 9 

The action of normal hoi-se seium upon normal guinea pigs 9 

The action of antitoxic horse serum upon normal guinea pigs 10 

II. Horse serum is poisonous to a " treated " or ''used " guinea pig 11 

The symptoms caused by the injection of horse serum into a susceptible 

guinea pig 14 

The poisonous action of horse serum acts upon the respiratory center. . 14 

III. The toxic action bears no relation to diphtheria 16 

The poison is not toxon 16 

Guinea pigs can not be rendered susceptible with previous infections with 

the B. diphtherise 18 

Diphtheria toxine can not render guinea pigs susceptible 19 

Diphtheria antitoxin plays no r6le in this action 21 

IV. The toxic principle '. 25 

Is the poison specific? 25 

Are other blood serums equally toxic? 28 

The relation of hemolysis 31 

The relation of precipitin to the toxic action 35 

The effect of heat upon the toxicity of horse serum 38 

The toxic principle in horse serum is filterable through porcelain 38 

Drying does not injure the toxic principle 39 

The toxic principle can not be separated by precipitation and dialysis. . . 39 
The effect of various chemical substances upon the toxic principle of 

horse serum 39 

The influence of antiseptics 42 

The effect of old horse serum upon susceptible guinea pigs 42 

The effect of X rays upon the toxic principle 43 

V. The influence of time 45 

The time necessary to render a guinea pig susceptible 45 

The guinea pig remains susceptible a very long time 45 

VI. Dosage as a factor 47 

The minimal amount of horse serum necessary to render a guinea pig 

susceptible 47 

The minimal amount of horse serum necessary to poison a susceptible 

guinea pig 48 

The influence of large quantities in rendering guinea pigs susceptible 50 

VII. The sensitizing substance 51 

Guinea pigs may be sensitized with precipitated and dialyzed serum 51 

Drying docs not injure the sensitizing substance 51 

Small quantities of horse serum may render guinea pigs more susceptible 

than large quantities 52 

The sensitizing substance is not free in the blood serum 54 

The effect of heat upon the sensitizing substance 54 

(5) 



6 

> 

PEMzre 

Vm. The action of horse serum upon man and other animals 55 

Man •. 55 

* Other animals 57 

IX. Inmiunity 59 

Active immunity 59 

Guinea pigs may be immunized against the toxic substance in horse 

serum : 59 

Passive immunity 62 

The effect of normal guinea pig blood and organs upon the toxicity 

of horse serum 62 

The neutralizing effect of the blood and organs of an immunized 

guinea pig upon the toxicity of horse serum 64 

X. Feeding experiments 67 

Guinea pigs may be sensitized by feeding horse serum 67 

Guinea pigs may be sensitized by feeding horse meat 69 

May guinea pigs be slightly sensitized by feeding beef ? 70 

XI. Hereditary transmission of the susceptibility in guinea pigs 73 

XII. The toxic action of other albuminous substances 75 

XIII. A review of the literature relating to our work 77 

XIV. Summary and conclusions 91 



A STUDY OF THE CAUSE OE SUDDEN DEATH EOLLOW- ' 
ING THE INJECTION OF HORSE SERUM. 



By Milton J. Rosenau, 

Passed AssislanI S'li'^fon, IHreclnr Hygiemc Laboratory, U. S. Pvblic Bealth aiid Marine 

Ho^ljritaJ Service. 

John F. AsDeRaos, 

Passed AsgiidaTd Surgeon, Assistant Director Hygienic LaboTatory, JJ. S. Public Health and 

MaiiTie-Hospiial Service. 



It has long been known that the blood of certain animals is 
poisonous when transfused or injected int-o certain other species. 

Many instances might be cited showing that the blood serum of 
one animal has poisonous properties when injected into an animal 
of another species. But the blood serum of the horse apparently 
lacks such poisonous action. Very large quantities of the blood 
serum of the horse may be injected into man, rabbits, guinea piga, 
and many other animals without serious inconvenience, except 
occasionaUy a slight reaction at the site of inoculation. 

In a certain proportion of cases the injection of horse serum into 
man is followed by urticarial eruptions, joint pains, fever, swelling 
of the lymph nodes, edema, and albuminuria. This reaction, which 
appears after an incubation period of eight to thirteen days, has 
been termed by Pirquet and Schick the "serum disease." 

In exceptional instances sudden death has followed an injection of 
horse serum in man. 

These studies were taken up in October, 1905, in order to throw 
light upon the cause of this unfortunate accident. We have shown 
that ordinarily horse serum is a comparatively bland and harndess 
substance when injected into certain animals; but these animals may 
be rendered so susceptible that an injection of horse serum may 
produce sudden death or severe symptoms. For example, large 
quantities of horse serum may be injected subcutaneousiy or into the 
peritoneal cavity of a guinea pig without apparently causing the 
animal tlie least inconvenience. However, if a guinea pig is injected 
with a small quantity, say jld c, c, of horse serum and after the 



I 



expiimtion of a certain interval is again injected witli horse serum 
the result will probably be fatal. The first injection of horse serum 
itized the animal in such a way as to render it very suscep- 
tible to a toxic principle in horse serum. It is probable that when 
the guinea pig is injected with the first, or sensitizing, quantity of 
.serum the strange protcid contained in tlie horse serum develops in 
j.the body of the guinea pig "antibodies" which, when brought into 
. contact with more horse serum given at a second injection, produce 
either a union or a reaction, which causes the toxic action. 

A certain time is necessary to elapse between the first and second 
injections of horse serum before this toxic action is able to manifest 
itself. This "period of incubation'' is from ten to twelve days, 
and corresponds suggestively with the period of incubation of the 
serum disease which Pirquet and Scliick place at eight to thirteen days. 

Guinea pigs may be sensitized with exceedingly small quantities 
of horse serum. In most of our work we used quantities less than 
^5 c. c. and we found in one instance that luoJ.irEff c. c. of horse 
serum was sufficient to render a guinea pig susceptible. 

It also requires very small quantities of horse serum, when given 
in a second injection, to produce poisonous symptoms. One-tenth 
c. c. injected into the peritoneal cavity is sufficient to cause the death 
of a half-grown guinea pig. One-tenth c. c. of horse serum injected 
subcutaneously is sufficient to produce serious symptoms. The fact 
that this toxic action may be developed by such small quantities 
of serum and the fact that exceedingly small quantities are sufficient 
to produce symptoms and death upon a second injection, a priori 
places both the sensitizing and the toxic principle in the horse serum 
in the "haptin group" of substances in the sense used by Ehrlich. 

A still further indication that the side-chain theory in its broadest 
sense may be applicable is the further fact that immunity may be 
produced against the toxic action by multiple injections of the 
serum. 

While at first we thought that diphtheria antitoxin had some 
relation to this action, we are now able to state positively that it 
has nothing whatever to do with the poisonous action of horse serum; 
further, tliat diphtheria antitoxin in itself is absolutely harmless. 
The toxic action which we have studied is caused by a principle in 
normal horse serum and is entirely independent of the antitoxic 
properties of the serum. 



¥ I 



Part L 
CONTROL EXPERIMENTS. 

ACTION OF NORMAL HORSE SERUM UPON NORMAL GUINEA PIGS. 

Normal horse serum when injected into normal guinea pigs causes 
no symptoms. Large amounts, such as 6 or 10 c. c, may be injected 
into the peritoneal cavity of a guinea pig without any apparent 
inconvenience to the animal. When normal horse serum is injected 
subcutaneously into the guinea pig it is sometimes either absorbed 
very slowly or there is a slight local reaction, as indicated by edema 
and induration of the subcutaneous tissue at the site of injection. 

G. P. No. 2.0 Six c. c. normal horse serum (roan horse) mjected into the peritoneal cavity. 

No symptoms. 
G. P. No. 3. Ten c. c. normal horae serum (roan horse) injected into the peritoneal cavity. 

No symptoms. 
G. P. No. 63. Six c. c. normal horse serum (Sp. horse) injected into the peritoneal cavity. 

No symptoms. 
G. P. No. 64. Six c. c. normal horse serum (Jane horse) injected into the peritoneal cavity. 

No symptoms. 
G. P. No. 66. Six c. c. normal horse serum (Sam horse) injected into the peritoneal cavity 

No symptoms. 
G. P. No. 65. Six c. c. normal donkey serum (donkey No. 58) injected into the peritoneal 

cavity. No symptoms. 
G. P. No. 296. Six c. c. normal donkey serum injected into the peritoneal cavity. No 

symptoms. 
G. P. No. 297. Do. 

G. P. No. 298. Six c. c. normal horse serum (Sp.) injected into the peritoneal cavity. No 

symptoms. 
G. P. No. 299. Do. 

Specimens of serum from each one of the normal horses used in this 
work appear in the above series of guinea pigs, which serve as controls. 

Donkey serum is also harmless for normal guinea pigs and is just as 
toxic as horse serum to a sensitized guinea pig. 

Uhlenhuth (Zur Kenntniss der giftigen Eigenschaften des Blut- 
serums, Ztschr. f. Hyg., v. 26, 1897, p. 384) found that normal horse 
serum, when injected intravenously into rabbits, is harmless. He 
injected as large quantities as 60 c. c. per 1 kilogram of rabbit and 
found that this caused no reaction at all. On the other hand he 
found that 11 c. c. of sheep serum, 12 c. c. of hog serum, or 6 c. c. of 
cattle serum per 1 kilogram of rabbit, when injected intravenously, 
caused the death of the animal. 

The nontoxic action of horse serum on animals was further shown 
by Hermann Pfeiffer (Ueber die nekrotisirende Wirkung normaler 

oG. P., abbreviation for guinea pig. The numbers refer to the laboratory serial numbers. 

(9) 



10 

ipifn, Ztsehr. f. Hyg. u. Infectionskrankh., t. fil, (2), pp. 181-196, 
f HHI.5), who fu-st confirmed the above work of nhlpnhuth and then 
f showed lliat ihv subcutaneous inoculation of largo quantities of horse 
f wnun into guinea pi^B produced at most a slight local reaction. He 
1 found on I lie other hnnd that the serum of certain other animals, e. g., 
[ wilt l('. Iiog, man, wlien injected subcutaneously into guinea pigs, pro- 
I duced a local reaction and in large quantities caused necrosis. 

Wo took the temperature of a number of guuiea pigs twice daily 
I for i-ifjlitcpn days followmg the injection of large quantities of horae 
[ aonim eubcutaneously, in order to determine whether a febrile reac- 
[ tion followed. No marked deviation from the normal temperature 

tan noted. 

ACTION OP ANTrrOXIC HORSE SERUM UPON NORMAL GUINEA PIGS. 

It w It well-known fact that large quantities of horse serum contain- 

■ ing high antitoxic potency may be injected subcutaneously or mto the 

I peritoneal cavity of a normal guinea pig without causing symptoms. 

r We use this method of testing the diphtheria antitoxin bought upon 

the open market to determine its freedom from bacterial and toxic 

impurities. Of the many guinea pigs thus inoculated none has shown 

untoward effects except in the case of bacterial or toxic contamina- 

\ tions. This method of testing the purity of antitoxin is also used by 

the manufacturers almost daily. We may therefore state that 

[ diphtheria antitoxic serum when injected into a normal giiinea pig 

[ CRUBea no symptoms. 

However, we present the following experimental data, which serve 
tftS controls for this work, because the normal guinea pigs in the follow- 
I ing sei'ies have been inoculated into the peritoneal cavitj' with large 
I quantities of the antitoxic horae serums .subsequently used in this 
I Work. 



fil. P, No. 300. Six e, c. sntitojiip horse st 

p«vitj. No symptoma. 
"J.P.No.301. Do. 

i. p. Nu. 302. Six c. c. antitodc! horse s 

^y. No symptoma. 
I. P. No. 303. Do. 

p. P. No. 304. Six c. c- aotitoiiu horae aur 

No symptoms. 
1. P. No. 305. Do- 

i. P. No. 306. Six c. c. nntitoxic. horao sei 
toneal pavity. No symptoms. 
te. P. No. 307. Six li. c. antitoiic horae sei 
toneal cavity. No aynptoms. 
[ G. P. No. 308. Six c. p. antitoxic hoisB at 
c&vity. No symptoms. 
G. P. No. 309. Six c. c. antitoxic horae be 
cavity. No symptoma. 
t e. F. No. 310. Do. 



im (Natl, rx. 191 i"jpr>ted into the perittmed 
UQi (Natl. V. 14) iiijefled into the p-.ritoneal 
n (Natl. VIII. 18) injectc(i into the peritoneal 

m (NYBH lOB, Gilisoii) injected into the peri- 
m (NYBII 13C, Gibson) injected into the peri- 
im (Alc's. A 211) injected into the peritoneal 
im (Natl. IX. 17} injected into the peritoneal 



fl 



Partn* 

HORSE SERUM IS POISONOUS TO A "TREATED" OR **USED" GUINEA PIG. 

It has long been known by some of the manufacturers of diph- 
theria antitoxin that when antidiphtheric serum is inoculated into 
a used guinea pig the result is almost invariably death. By a '^ used '^ 
or ^ treated ^^ guinea pig we mean one that has recovered from the 
effects of an inoculation of the toxine-antitoxin mixture used in test- 
ing the potency of diphtheria antitoxin. For example: 

G. P. No. 4191. Six c. c. antitoxic horse serum (Alex. 192) injected into the peritoneal 
cavity. Death in 15 minutes. 

[Previous treatment: 26 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+^J^ c. c. antitoxic horse serum (PD&Co 08033).] 

G. P. No. 4103. Nine c. c. antitoxic horse serum (Alex. 193) injected into the peritoneal 
cavity. Dead in 5 minutes. 

[Previous treatment: 31 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+^J^ c. c. antitoxic horse serum (Alex. 190).] 

G. P. No. 4330. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into peritoneal 
cavity. Dead in 2 minutes. 

[Previous treatment: 26 days prior, inoculated subcutaneously with 0.139" c. c. toxine 
No. 5+3fi^ c. c. antitoxic horse serum (Alex. 185).] 

G. P. No. 6B. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into peritoneal 
cavity. Dead in 27 minutes. 

[Previous treatment: 86 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B25).] 

G. P. No. 4381. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 10 minutes. 

[Previous treatment: 11 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+7}^ c. c. antitoxic horse serum (Natl. V. 10).] 

G. P. No. 4336. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 12 minutes. 

[Previous treatment: 21 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+ jj^ c. c. antitoxic horse serum (PD&Co 07635).] 

G. P. No. 4369. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 21 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+jl^ antitoxic horse serum (Cutter 1461).] 

G. P. No. 4377. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 21 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+j^ c. c. antitoxic horse serum (Natl. V. 7).] 



12 

G. P. No. 4526. Six c. c. antitoxic h6rse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 30 minutes. 

[Previous treatment: 17 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

G. P. No. 4491. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 41 minutes. 

[Previous treatment: 18 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

G. P. No. 4494. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 37 minutes. 

[Previous treatment: 19 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

G. P. No. 75. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 3 hours. 

[Previous treatment: 15 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+.2io <'• <'• antitoxic horse serum (Natl. IX. 17).] 

G. P. No. 88. Six c, c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 15 days prior, inoculated subcutaneously with 0.006 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Natl. IX. 17).] 

G. P. No. 4522. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 20 minutes. 

[Previous treatment: 20 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

G. P. No. 4500. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 21 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

G. V. No. 4471. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 8 minutes. 

[Previous treatment: 22 days prior, inoculated subcutaneously with 0.22 c, c. toxine 
No. 7+ 2 Jo ^' ^' antitoxic horse serum (Natl. IX. 17).] 

G. P. No. 4503. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. No symptoms.^ 

[Previous treatment: 22 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

G. P. No. 4499. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 20 minutes. 

[Previous treatment: 24 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

G. P. No. 4465. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 40 minutes. 

[Previous treatment: 26 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+7r^^ c. c. antitoxic horse serum (Natl. VIII. 17).] 

G. P. No. 4387. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 28 days prior, inoculated subcutaneously with 0.2 c. c. toxine No. 
58+1 unit antitoxic horse serum (Standard B27).] 



a Explanation of occasional irregularities, page 63. 



13 

G. P. No. 4509. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 25 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

G. P. No. 143. Six c. c. antitoxic horse serum (Natl. IX. 17) injected into the peritoneal 
cavity. Dead in 35 minutes. 

[Previous treatment: 42 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+yJ^ c. c. antitoxic horse serum (Steams 1351).] 

G. P. No. 73. Six c. c. antitoxic horse serum (Natl, VIII. 18) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 57 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+250 units antitoxic horse serum (Natl. IX. 17).] 

G. P. No. 82. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 57 days prior, inoculated subcutaneously with 0.006 c. c. toxine 
No. 7+^^^ c. c. antitoxic horse serum (Natl. IX. 17).] 

G. P. No. 139. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected into the peritoneal 
cavity. Dead in 15 minutes. 

[Previous treatment: 49 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+ jj^ c. c. antitoxic horse serum (Steams 1351).] 

All of the above pigs were first given the toxine-antitoxin mix- 
tures in order to test the strength of diphtheria antitoxic serum bought 
on the open market in accordance with the law of July 1, 1902. 

Horse serum is also toxic when injected subcutaneously and in 
small quantities, as will be seen from the following experiments : 

G. P. No. 4951. Six c. c. normal horse serum (roan) inoculated subcutaneously. Dead in 
35 minutes. 

[Previous treatment: 40 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+yJ^ c. c. antitoxic horse serum (NYBH lOB).] 

G. P. No. 5047. Same injection. Dead in 40 minutes. 

[Previous treatment: 51 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+5^J-^ c. c. antitoxic horse serum (Alex. A205).] 

G. P. No. 4950. Same injection. Symptoms; recovered. 

[Previous treatment: 40 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+yJ^ c. c. antitoxic horse serum (NYBH lOB).] 

G. P. No. 5046. Same injection. Dead in 40 minutes. 

[Previous treatment: 51 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+ jj^ c. c. antitoxic horse serum (Alex. A204).] 

G. P. No. 4524. Six c. c. antitoxic horse serum (Natl. VIII. 17) inoculated subcutane- 
ously. Symptoms; recovered. 

[Previous treatment: 28 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+^^^ c. c. antitoxic horse serum (Alex. 192).] 

G. P. No. 4623. One c. c. antitoxic horse serum (Natl. VIII. 17) inoculated subcutane- 
ously. Symptoms; recovered. 

[Previous treatment: 28 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+j-J-^c. c. antitoxic horse serum (Alex. 192).] 

G. P. No. 4512. One c. c. antitoxic horse serum (Natl. VIII. 17) inoculated subcutane- 
ously. Dead in 81 minutes. 

[Previous treatment: 42 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 imit antitoxic horse serum (Standard B27).] 



14 

G. P. No. 4502. Six c. c. antitoxic horse serum (Natl. VIII. 17) inoculated subcutane 
ously. Symptoms; recovered. 

[Previous treatment : 42 days prior, inoculated subcutaneously with 0.19 c. c. toxine No. 
7+1 unit antitoxic horse serum (Standard B27).] 

G. P. No. 4514. Two-tenths c. c. antitoxic horse serum (Natl. VIII. 17) inoculated subcu- 
taneously. Symptoms; recovered. 

[Previous treatment: 42 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

SYMPTOMS CAUSED BY THE INJECTION OF HORSE SERUM INTO A SUSCEPTIBLE 

GUINEA PIG. 

Very characteristic symptoms are produced by horse serum, either 
normal or antitoxic, when injected into a susceptible guinea pig. The 
symptoms are apparently the same whether the injection is made 
subcutaneously or into the peritoneum, or whether normal or anti- 
toxic horse serum is used. In five or ten minutes after injection the 
pig manifests indications of respiratory embarrassment by scratch- 
ing at the mouth, coughing, and sometimes by spasmodic, rapid, or 
irregular breathing; the pig becomes restless and agitated. This 
stage of exhilaration is soon followed by one of paresis or complete 
paralysis. The pig is unable to stand or, if it attempts to move, falls 
upon its side; when taken up it is limp. Spasmodic, jerky, and con- 
vulsive movements now supervene. 

Pigs in this stage with complete paralysis may fully recover, but 
usually convulsions appear, and are almost invariably a forerunner 
of death. Symptoms appear about ten minutes after the injection 
has been given; occasionally in pigs not very susceptible they are 
delayed thirty to forty-five minutes. Only in one or two instances 
of the several hundred pigs which we have observed have the symp- 
toms developed after one hour. Pigs developing symptoms as late 
as this are not very susceptible and do not die. The chain of symp- 
toms is exceedingly characteristic. The 'symptoms do not always 
follow in the order given. Death usually occurs within an hour and 
frequently in less than thirty minutes. 

THE POISONOUS ACTION OF HORSE SERUM ACTS UPON THE RESPIRATORY 

CENTERS. 

Judging from the symptoms produced by the injection of horse 
serum into a susceptible guinea pig we assumed that the poison acted 
upon the nervous system. Autopsies done immediately after the 
death of the guinea pigs showed invariably that the heart continued 
to beat long after respiration had ceased. In some instances the 
heart would continue to beat a full hour when exposed. This would 
seem to indicate that we were dealmg with a poison which caused 
death through the neivous control of the respiration, and the follow- 
ing experiment shows that this effect is certainly not local. 



15 

. -P. No. 4526. Six c. c. antitoxic horse serum (National VIII. 17) were injected into the 
peritoneal cavity. Death in 30 minutes; 

[Previous treatment: 17 days prior, inoculated subcutaneously with 0.19 c. c. of toxine 
No. 7+1. unit antitoxic horse senmi (Standard B27).] 

- Immediately after death the phrenic nerve was exposed and stimu- 
.ted high up, causing contractions of the diaphragm both upon mak- 
ig and breaking the galvanic current. The contractions were also 
aused with the induced current. The contractions of the diaphragm 
-aused by stimulating the phrenic nerve in this way were produced 
^th a weaker current than those required to cause similar contrac- 
tions in a normal control guinea pig. 



Part m. ~J1 

THE TOXIC ACTION BEARS NO RELATION TO DIPHTHERIA. 



2t occurred to ua that possibly diphtheria antitoxic serum may t 
1 free toxone which, when injected into a "used" or "treati 
I pig, might produce acute effects upon the nervous system of ■ 
"" such a pig, which, on account of the previous effects of diphtheria i 
toxine, had been rendered pecuharly susceptible. It is well knowa I 
that the toxone does not cause the acute death of a guinea pig, but 
only produces a local edema and late paralysis. This fact, first dem- 
onstrated by Eiu-Iich, may readily be demonstrated by inoculating 
guinea piga with toxine and antitoxin mixed in such proportions so 
that the toxine is but partly neutralized. We. were further led to 
believe that the poisonous effects of horse serum upon a treated guinea 
pig might he a manifestation of the toxonea because the symptoms so ■ 
plainly point to the nervous system. The pigs almost invariably I 
show paresis or paralysis. 

The fact that neither toxone nor any other constituent in the toxine 
plays any r61e in the poisonous action of horse serum is demonstrated 
in the following experiment: 

.01) was injected into the peritoneal cavity., 
I, reaultitig Iroin previous treatmen 
listed aubcut*ueously with 0.22 c 
(Mulford's No. 942),J 



the ti 



a. p. No. 4161. 0.1 c. c. toxine No. 15 (MLD^ 

No Bymptoms, Completely paralyzed 

IPrevious treatment: 31 days previously 

.bNo. 7+j|sC. c. diphthe 



One hour 50 inin, later this same pig wm mnciilated 
serum (P. D. & Co. No. 08022) into the peritoneal cavity. 

It ia well known that overpowering doses of toxine injected either- j 
Bubcutanoously or into the peritoneal cavity'of a guinea pig require I 
about twenty hours to kill the animal. These large doses of toxina 
do not cause immediate symptoms. 

It occurred to us, however, that while toxino alone may not have 
this power, nevertheless when it is mixed with antitoxic horse s 
and when large doaea of" the mixture are injected into a guinea pig ( 
there maj' be a sufficient liberation of toxone or other poisonous ' 
substances capable of producing symptoms and sudden death. 

(17) 



28337— No. 29—08 2 



18 

The following mixture of toxine and antitoxin represents these two 
substances in the proportion of the L** dose: 

G. P. 4483. 1.6 c. c. toxine No. 7+11-5 units antitoxic serum (standard B27) injected sub- 
cutaneously. No symptoms. 

18 days later 6 c. c. antitoxic horse serum (National VIII, 17) injected into the peri- 
toneal cavity of the same pig. Death 30 minutes. 

In a similar way the effects of mixing large doses of the diphtheria 
toxine with normal horse serum were tested, as follows: 

G. P. No. 40. Six c. c. normal horse serum (roan horse) + 0.3 c. c. diphtheria toxine No. 5 
mixed in vitro and injected into the peritoneal cavity. No immediate symptoms. Died 
2 days later. 

GUINEA PIGS CAN NOT BE RENDERED SUSCEPTIBLE BY PRIOR INFECTIONS WnH 

THE BACILLUS DIPHTHERIAS. 

When this work was first undertaken it was thought to have a bear- 
ing upon sudden deaths in children suffering with diphtheria. The 
work was begun in order to discover, if possible, the cause of this acci- 
dent. We thought that possibly by injecting small quantities of a 
virulent culture of the EZlebs-LoflBer bacillus into guinea pigs, in that 
way imitating mild cases of diphtheria, some of these pigs could be 
rendered susceptible to subsequent inoculations of antitoxic horse 
serum. This, however, is not the case for guinea pigs, as is plainly 
seen in the following experiments, in which a number of guinea pigs 
were treated with sufficient quantities of a culture of the Klebs- 
Loffler bacillus to cause slight symptoms and at varying intervals of 
time were inoculated again into the peritoneal cavity with large 
quantities of antitoxic horse serum. In no instance did the guinea 
pigs show any ill effects from this latter treatment. 

G. P. No. 41. Six c. c. antitoxic horse serum (Natl. VIII, 17) injected into the i>eritoneal 
cavity. No symptoms. 

[Previous treatment: -4 days prior, inoculated subcutaneously with 0.1 c. c. suspension.] 

G. P. No 9. Six c. c. same serum and injection as g, p. 41. No sj^mptoms. 

[Previous treatment: 7 days prior, inoculated subcutaneously with 1 gtt. of 24-hour 
suspension.] 

G. P. No. 51. Six c. c. same serum and injection as g. p. 41. No symptoms. 

[Previous treatment: 7 days pt^ior, inoculated subcutaneously with 0.1 c. c. of suspen- 
sion. 

G. P. No. 42. Six c. c. same serum and injection as g. p. 41. No symptoms. 

[Previous treatment: 9 days prior, inoculated subcutaneously with 1 gtt. 24-hour 
suspension.] 

G. P. No. 56. Six c. c. same serum and injection as g. p. 41. No symptoms. 

[Previous treatment: 9 days prior, inoculated subcutaneously with 0.1 c. c. susj>ension.] 
G. P. No. 43. Six c. c. same serum and injection as g. p. 41. No symptoms. 

[Previous treatment: 12 days prior, inoculated subcytaneously with 1 gtt. 24-hour 
suspension.] 

G. P. No. 45. Six c. c. same serum and injection as g. p. 41. No symptoms. 

[Previous treatment: I4. days prior, inoculated subcutaneously with 1 gtt. 21-} oir 
suspension.] 



19 

G. P. No. 58. Six c. c. same serum and injection as g. p. 41. No symptoms. 

[Previous treatment: 21 days prior, inoculated subcutaneously with 0.1 c. c. susDen- 
sion.] 

G. P. No. 44. Six c. c. same serum and injection as g. p. 41. No symptoms. 

[Previous treatment: 24. days prior, inoculated subcutaneously with 1 gtt. 24-hour 
suspension.] 

G. P. No. 52. Six c. c. same serum and injection as g. p. 41. No symptoms. 

[Previous treatment: 27 days prior, inoculated subcutaneously with 0.1 c. c. 24-hour 
suspension.] 

G. P. No. 50. Six c. c. same serum and injection as g. p. 41. No symptoms. 

[Previous treatment: 32 days prior^ inoculated subcutaneously with 1 gtt. 24-hour 
suspension.] 

DIPHTHERIA TOXINE CAN NOT RENDER GUINEA PIGS SUSCEPTIBLE. 

For the same reason that guinea pigs were treated with cultures 
of the Bacillus diplitheri^^ we also attempted to render them suscep- 
tible by the injection of small quantities of diphtheria toxine. On 
account of the great importance of this subject a large number of 
guinea pigs were used to demonstrate the role which the diphtheria 
toxine may play in the subsequent action of horse seium. Our 
experiments demonstrate conclusively that diphtheria toxine alone 
plays absolutely no r61e in rendering guinea pigs susceptible to the 
toxic action of horse serum; for in no instance, as will be seen in the 
following series, does the guinea pig show the slightest effects when 
subsequently injected with large quantities of serum. 

G. P. No. 4584. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 2 days prior, inoculated subcutaneously with 0.001 c. c. toxine 
No. 7.] 

G. P. No. 4593. Six c. c. same serum and injection as g. p. 4584. No symptoms. 
[Previous treatment: 3 days prior, as g. p. 4584.] 

G. P. No. 4534. Six c. c. same serum and injection as g. p. 4584. No symptoms. 

[Previous treatment: 4. days prior, inoculated subcutaneously with 0.002 c. c. toxine 
No. 7.] 

G. P. No. 4550! Six c. c. same serum and injection as g. p. 4584. No symptoms. 
[Previous treatment: 6 days prior, as g. p. 4534.] 

G. P. No. 4548. Six c. c. same serum and injection as g. p. 4584. No symptoms. 
[Previous treatment: 6 days prior, as g. p. 4534.] 

G. P. No. 4585. Six c. c. same serum and injection as g. p. 4584. No symptoms. 
[Previous treatment: 6 days prior, as g. {>. 4584.] 

G. P. No. 4432. Six c. c. same serum and injection as g. p. 4584. No symptoms. 
[Previous treatment: 7 days prior, as g. p. 4534.] 

G. P. No. 4434. Six c. c. same serum and injection as g. p. 4584. No symptoms. 

[Previous treatment: 7 days prior, inoculated subcutaneously with 0.004 c. c. toxine 
No. 7.] 

G. P. No. 4426. Six c. c. same serum and injection as g. p. 4584. No symptoms. 
[Previous treatment: 8 days prior, as g. p. 4534.] 

G. P. No. 4583. Six c. c. same serum and injection as g. p. 4584. No symptoms. 
[Previous treatment: 9 days prior, as g. p. 4534.] 



w 


20 


^^H 


^ft G. F. No. 4579. Six <>. 
^H [Previoua treatment 


. same serum and injection as g. p. 4584. 
iOtfaygprior, Bsg. p.4534.] 


Nosym^H^^I 


^H G. F. No. 45S2. Six c. 
^^H [Frevioua treatment 


same serum and injection as g. p. 4584. 
11 days prior, us g. p. 4534.] 


No symptoms.^^H 


^H G. P. No. 4430. Six o. 
^^H [Previous treatment 


same serum and injection as g. p. ^584. 
IS days prior, es g. p. 4534,] 


No aymptoiiw.^^H 


^H O.P. No. 4572. Sine. 
^^1 [Previous treatment 


same serum and injection as g. p. 4584. 
12 days jmor, as g. p. 4,534.] 


No Bymptoms,^^^! 


^B 0. F. No. 456S. Six c. c 
^H [Pceyiou.-^ treatment 


same serum and injection as g. p. 4,584. 
13 doj/B prior, as g. p. 4534.] 




^H G. P. No. 4576. Six f- c 
^H [Previous treatment 


same serum and injection as g. p. AFSi. 
li daye prior, as g. p. 4534.] 


No symptomsJ^^^I 


^H Q. P. No. 4577. Six c. c 


same serum and injection aa g. p. 45S4. 
16 days prior, as g. p. 4534.] 


No symptonu^j^^^l 


^H G. P. No. 1535. Six r. c 
^H [Previous treatment 


same serum and injection as g. p. 45S4. 
17 daya frior, as g, p. 4534.] 


symptomB.^^1 


^H G. P. No. 4M7. Six c. r 
^H [Previous treatment 


same serum and injection as g. p. 4584. 
18 days prior, as g. p. 4534.] 


No symptonia.^^^1 


^H G. P. No. 4580. Six c. c 
^H [Previous treatment 


same serum and injection as g. p. 4584. 
19 days prior, aa g. p. 4534.] 


No symptfima. ^^H 


^H 0, P. No. 4556. Six c. 
^H [Previous treatment 


same serum and injection as g. p. 4584. 
30 day> prior, aa g. p. 4534.] 


No symptoma^^^^^l 


^H G. P. No. 455S. »x c. r 
^H [Previous treatment 


same serum and Injection as g. p. 45S4. 
31 da>is prior, as g. p. 4534.] 


No a jmptoDiK;^^! 


^H G. P. No. 4545. Six c c 
^H [Previous treatment 


same serum and injection as g. p. 4584. 
m days prior, aa g. p. 4,534.] 


No symptoma. ^^^H 


^H G. P. No. 4555. Six c. c 


same serum and injection as g. p. 4584. 
U day) prior, as g. p. 4531.1 




^H G. P. No. 4541. Six c. c 
^H [Previous treatment 


same sonim and injection as g. p. 4584. 
2(5 days prior, aa g. p. 4534.] 




^H G. P. No. 4404. Six c. c 

^M No. 


same serum and injection as g. p. 4584. No symptoms. 


^V G. P. No, 4454. Six c. c 
^B [Praviou» treatment 
^1 No. 19.] 


same serum aud injection as g. p. 4584. No symptoms. 

S7 days prior, inoctdated subcutaneouslj with 0.01 c c. toxEU 


^H G. P. No. 451)5, Six e. c 
^H [PreTiouB treatment 


same serum and injection as g. p. 4,584. 
27 days prior, as g, p. 4534.] 


No symptoms. ^JH 


^1 G. P. No. 4554. Six e. c 
H [Previous treatment 


same serum and injection as g. p. 4584. 
30 days prior, oa g. p. 4534.] 


No symptoms. .^^H 


^B 0. P. No, 4578. Six r. c 


same serum and injection as g. p. 4684. 
31 days prior, as g, p, 4534.] 


No symptatns..^^^| 


^^ G. P. No. 4542. Sixc. c 


same serum and injection as g. p, 4584. 
35 daye prior, aa g. p. 4.534.] 




^H G.F.No.4.^. Sixi^.r. 


antitoxic horse serum {Natl. VIII. IS) inj 


ctcd inlEi tbt- peritDuc 1 


^^^^ cavity. No symptoms 
^^B p>roviou9 treatmeut: 


Ji6 days prior, as g, p. 4534.] 


1 


^^R 0. P. No. 4428. Do. 




y 


^Ht 0. p. No. 4427. Do. 




1 


^^B 0. P. No, 4431. Do. 




J 



21 

G. P. No. 4567. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 65 days prior, as g. p. 4534.] 

G. P. No. 4554. Do. 

G. P. No. 4553. Do. 

DIPHTHERIA ANTITOXIN PLAYS NO r6LE IN THIS ACTION. 

Before we realized that the diphtheria toxine or antitoxin played 
no r61e in rendering guinea pigs susceptible to subsequent injections 
of horse serum we prepared the following series of guinea pigs, in 
order to eliminate the antitoxin as a factor. These pigs were injected 
subcutaneously with a mixture of 1 c. c. of normal horse serum and 
a small quantity of diphtheria toxine, slightly less than a minimal 
lethal dose. This was done for comparison with the susceptibility 
produced by the toxine-antitoxin mixtures. 

G. P. No. 91. Six c. c. normal horse serum (roan) injected in£o the peritoneal cavity. 
Symptoms. Died 12 hours later. 

[Previous treatment: 65 days prior, inoculated subcutaneously with 0.002 c. c. toxine 
No. 7+1 c. c. normal horse serum (Sam).] 

G. P. No. 296. Six c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Dead in 60 minutes. 

[Previous treatment: 71 days prior, inoculated subcutaneously with 0.002 c. c. toxine 
No. 7+1 c. c. normal horse serum (Sam).] 

G. P. No. 96. Do. Dead in 40 minutes. 

G. P. No. 93. Do. Symptoms; recovered. 

G. P. No. 90. Do. 

It will be seen from our experiments that guinea pigs are quite as 
sensitive when previously treated with toxine-nomial serum as with 
the toxine-antitoxin mixtures. 

In order to compare the toxic action of normal serum with anti- 
toxic serum upon pigs so treated, four animals of the above series 
were inoculated with antitoxic horse serum, as follows: 

G. P. No. 92. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms. Died 12 hours later. 

[Previous treatment: 15 days prior^ inoculated subcutaneously with 0.002 c. c. toxine 
No. 7+1 c. c. normal horse serum (Sam).] 

G. P. No. 98. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected into the peritoneal 
cavity. Dead in 53 minutes. 

[Previous treatment: 58 days prior, inoculated subcutaneously with 0.002 c. c. toxine 
No. 7+1 c. c. normal horse serum (Sam).] 

G. P. No. 95. Do. Dead in 43 minutes. 

G. P. No. 94. Same injection. Symptoms; recovered. 

[Previous treatment: 65 days prior, inoculated subcutaneously with 0.002 c. c. toxine 
No. 7+1 c. c. normal horse serum (Sam).] 

It is evident from the above that fresh normal serum is quite as 
poisonous to a sensitized guinea pig as is antitoxic horse serum. 



22 

. In order further to eliminate the antitoxin as a factor in this prob- 
lem, the following series of used or treated guinea pigs were injected 
with normal horee serum: 

G. P. No. 4100. Six c. c. normal horse serum (roan) injected into the peritoneal cavity. 
. Dead in 15 minutes. 

[Previous treatment: 36 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7-|-^J^ c. c. antitoxic horse serum (Alex. 190).] 

G. P. No. 4105. Same injection. Dead in 30 minutes. 

[Previous treatment: 36 days prior, inoculated subcutaneously with 0.22 c. c. toxme 
No. 7-|-7^ c. c. antitoxic horse serum (Alex. 189).] 

G. P. No. 4106. Same injection. Dead in 5 minutes. 

[Previous treatment: 36 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+^J^ c. c. antitoxic horse serum (Alex. 189).] 

G. P. No. 4331. Same injection. Dead in 90 minutes. 

[Previous treatment: 25 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+^^^ c. c. antitoxic horse serum (Alex. 185).] 

G. P. No. 4351. Same 'injection. Dead in 40 minutes. 

[Previous treatment: 18 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5-\-^^jj c. c. antitoxic horse serum (PD&Co 08021).] 

G. P. No. 3950. Six c. c. normal horse serum (Sam) injected into peritoneal cavity. Dead 
in 10 minutes. , 

[Previous treatment: 51 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+ ^1^7 c. c. antitoxic horse serum (Hubbert 1).] 

G. P. No. 3951. Six c. c. normal horse serum (Jane) injected into the peritoneal cavity. 
Dead in 30 minutes. 

[Previous treatment: 51 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+^ii7 c. c. antitoxic horse serum (Hubbert 1).] 

G. P. No. 3952. Six c. c. normal horse serum (Sp.) injected into the peritoneal cavity. 
Dead in 38 minutes. 

[Previous treatment: 51 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+TjJ^ c. c. antitoxic horse serum (Hubbert 1).] 

G. P. No. 3953. Six c. c. normal donkey serum (No. 58) injected into the peritoneal cavity. 
Dead in 60 minutes. 

[Previous treatment: 51 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. S-f^^Tir c. c. antitoxic horse serum (Hubbert 1).] 

G. P. No. 4109. One c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Symptoms: recovered. 

[Previous treatment: 37 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+ ^j^c. c. antitoxic horse serum (Alex. >88).] 

G. P. No. 4945. Six c. c. normal horse serum (roan) inoculated subcutaneously. Symp- 
toms; recovered. 

[Previous treatment: 34 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+5^ c. c. antitoxic horse semm (Wellcome 478).] 

G. P. No. 4934. Six c. c. normal horse serum (roan) inoculated subcutaneously. Symp- 
toms; recovered. 

[Previous treatment: 34 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+TiTr ^' ^- antitoxic horse serum (Wellcome 479).] 
G. P. No. 4930. Six c. c. normal horse serum (roan) inoculated subcutaneously. Symp- 
toms; recovered. 

[Previous treatment: 34 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5-\-jI^ c. c. antitoxic horse serum (Wellcome 475).] 



23 

• • 

G. P. No. 4931. Six c. c. normal horse serum (roan) inoculated subcutaneously. Dead in 
5 minutes. 

[Previous treatment: 34 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+^^^ c. c. antitoxic horse serum (Wellcome 475).] 

G. P. No. 4941. Six c. c. normal horse serum (roan) inoculated subcutaneously. Dead in 
20 minutes. 

[Previous treatment: 34 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. S+yJj^ c. c. antitoxic horse serum (Wellcome 474).] 

All of the above animals and most of those which appear upon the 
subsequent pages of this bulletin were first used to test the strength 
of the diphtheria antitoxin bought on the open market in accordance 
with the law of July 1, 1902. ^ 

The following series of guinea pigs were given small quantities of 
antitoxic horse serum, some subcutaneously and come intraperitone- 
ally, in order to compare the sensitizing action bj' these two methods 
of inoculation. At the second injection they were all given 6 c. c. 
of normal horse serum into the peritoneal cavity. 

G. P. No. 276. Six c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Dead in 25 minutes, 

[Previous treatment: 17 days prior, inoculated subcutaneously with ^l-^ c. c. antitoxic 
horse serum (Natl. V. 14).] 

G. P. No. 277. Do. Dead in 30 minutes. 

G. P. No. 278. Do. Dead in 55 minutes. 

•G. P. No. 279. Do. Symptoms; recovered. 

G. P. No. 280. Do. Symptoms; recovered. 

G. P. No. 281. Six c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Symptoms; recovered. 

[Previous treatment: 17 days prior, ^l-^ c.c. antitoxic horse serum (Natl. V. 14) 
injected into the peritoneal cavity.] 

G. P. No. 282. Do. Dead in 23 hours. 

G. P. No. 283. Do. Symptoms; recovered. 

G. P. No. 284. Do. Dead in 60 minutes. 

G. P. No. 285. Do. Symptoms; recovered. 

From the above series it will bo seen that guinea pigs may be 
rendered as susceptible by intraperitoneal as by subcutaneous 
inoculations. 

Finally, in order to demonstrate without doubt that neither anti- 
toxin nor diphtheria toxine plays any part in this action, and to show 
that this action is a property of normal horse serum, we treated a 
number of guinea pigs giving them at both the first and second 
injection fresh normal horse serum, with the following results: 

G. P. No. 101. Three c. c. normal (roan) liorse serum injected into the peritoneal cavity. 
Dead in 35 minutes. 

[Previous treatment: 55 days prior, inoculated subcutaneously with jl^ c. c. normal 
horse serum (Jane).] 



u 

G. P. Ko. 105. One c. c. normal horse serum (roan) inoculated subcutaneously. Sj 
toms; recovered. 

[Previous treatment: As above.] 

G. P. No. 104. One c. c. normal horse serum (roan) injected into the peritoneal ca 
Symptoms; recovered. 

[Previous treatment: As above.] 

G. P. No. 102. Three c. c. normal horse serum (roan) inoculated subcutaneously. S3 
toms; recovered. 

[Previous treatment: As above.] 

G. P. No. 286. Six c. c. normal horse serum (roan) injected into the peritoneal ca 
Dead in 25 minutes. 

[Previous treatment: 17 days prior, inoculated subcutaneously with ^J^- c. c. no 
horse serum (roan).] 

G. P. No. 287. Do. Dead in 50 minutes. 

G. P. No. 288. Do. Symptoms; recovered. 

G. P. No. 289. Do. do do 

G. P. No. 290. Do. do do 



Part IV* 
THE TOXIC PRINCIPLE. 

At one time we made efforts to isolate the active principle in horse 
serum which causes the symptoms, but as soon as we realized that 
the toxic principle present in horse serum exerts its action in quan- 
tities so minute as to place it almost in the category of the ferments, 
and, further, when we concluded from our work that this toxic prin- 
ciple is doubtless one of those highly organized and complex proteid 
substances belonging to the ^^haptin^' group, we recognized how 
hopeless it would be with present technique to isolate this substance. 
Nevertheless, we devoted much time and study to the relation of 
this toxic principle to various chemical, physical, and electrical influ- 
ences. The practical importance of eliminating or neutralizing this 
toxic principle from horse serum is at once evident. 

IS THE POISON SPECmC ? 

Whether the poisonous substance in horse serum is specific may 
be determined in several ways. We first endeavored to determine 
whether guinea pigs treated with horse serum are susceptible to the 
serums of other aimals; and, secondly, whether guinea pigs treated 
with the serums of other animals are susceptible to subsequent injec^ 
tions of horse serum. 

The following is a list of animals treated with horse serum and sub- 
sequently tested with the blood serums of other animals. 

It will be noticed that in this list some of the guinea pigs were 
treated with donkey serum instead of horse serum, but as the donkey 
serum in our hands has shown itself to act the same as horse serum 
we have used them interchangeably. 

(25) 



26 



Table showing the effect of alien serums upon guinea pigs sensitized with horse serum. 



No. of 


Kind and amount of 
normal serum. 




Previous treatment. 




guinea 
pig. 


Days 
prior. 


Inoculated subcutaneously with— 


Result. 


178 


Cattle, 6 c. c 


34 


-in c. c. donkey serum 


Symptoms, recovered. 
Do. 


149 


do 


41 


0.22 c. c. toxine No. 7+3J0 c. c. horse 
serum (Steams 1351). 


180 


Hog, 6 c. c 


34 


2 Jo c. c. donkey scrum 


No symptoms. 
Symptoms, recovered. 


138 


do 


41 


0.22 c. c. toxine No. 7+3 Jo c. c. horse 
serum (Stearns 1351). 


4743 


Hoe, 1 c. c 


41 


0.139 c. c. toxine No. 5+3^0 c c. horse 
serum (Alex. 192). 


Do. 








179 


Sheep, 6 c. c 


34 


-In c. c. donkey serum 


Do. 


152 


do 


41 


0.22 c. c. toxine No. 7-f sio c. c. horse 
serum (Steams 1351) . 


Do. 


177 


Cat, 6 c. c 


34 


«in c. c. donkey serum 


Do. 


124 


do 


41 


0.22 c. c. toxine No. 7+3J0 c- c horse 
serum (Steams 1351). 


No symptoms. 


4744 


Cat, 1 c. c 


41 


0.139 c. c. toxine No. 5+3J0 c. c. horse 
serum (Alex. 192). 


Symptoms, recovered. 






121 


Rabbit, 6 CO 


41 


0.22 c. c. toxine No. 7+3^0 c c. horse 
serum (Steams 1351). 


Do. 


4380 


do 


19 


0.22 c. c. toxine No. 7+aJo c c. horse 
serum (Natl. V. 10). 


Do. 


4374 


do 


19 


0.22 c. c. toxine No. 7+j4o c. c. horse 
8emm(NatI. V. 7). 


Do. 


153 


Doe, 6 c. c 


41 


0.22 c. c. toxine No. 7 +3^0 c. c. horse 
serum (Steams 1351). 


Do. 








4379 


do 


19 


0.22 c. c. toxine No. 7+2^0 c. c. horse 
serum (Natl. V. 10). 


Do. 


4742 


Doc, 1 c. c 


41 


0.139 c. c. toxine No. 7+3^0 c- c horse 
serum (Alex. 192) . 


Do. 








4375 


Guinea pig, 3 c. c . - . 


19 


0.22 c. c. toxine No. 7 +3^0 c. c. horse 
serum (Natl. V. 7). 


No symptoms. 



As the above series of guinea pigs previously treated with horse 
serum showed unmistakable susceptibility to subsequent injections 
of the blood serum of other animals, we are justified in saying that 
the action is not specific; but there is a very evident quantitative 
difference. It will be noticed that none of these guinea pigs died, 
and it was a matter of observation that, for the most part, they had 
characteristic but mild symptoms. 

We next took up the question as to whether a guinea pig may be 
sensitized to horse serum with the blood serum of animals other than 
the horse. The following is a list of guinea pigs which had been 
treated by the subcutaneous inoculation of small quantities (g^o c. c.) 
of blood serum from the rabbit, rat, donkey, cat, dog, hog, sheep, 
chicken, cattle, and man. Each pig was subsequently injected with 
6 c. c. of antitoxic horse serum (Natl. IX. 17) into the peritoneal cavity. 



27 



Table showing the results of injecting 6 ex. of antitoxic horse serum {Nail. IX. IT) irUo the 
peritoneal cavity of guinea pigs treated from 25 to 3^ days prior with 2^0 ^' ^' of the blood 
serum of other animals. 



No. of 
guinea 

pig- 



171 
172 
174 
167 
168 
169 
170 
176 
191 
192 
193 
214 
215 
212 
185 
18G 
182 
184 
196 
197 
206 
207 
210 
201 
202 



Previous treatment. 



Kind of serum. 



Rabbit aJoCC. 

— do do 

do do 

Rat do 

do do 

do do 

do do 

Donkey do 

Cat do 

— do do 

do do 

Dog do 

do do 

do do 

Hog « do 

do do 

Sheep do 

do do 

Chicken do 

do do 

Cattle do 

do do 

do do — 

Man do 

do do 



Days 
prior. 



25 
34 
34 
25 
34 
34 
34 
25 
25 
34 
34 
26 
'35 
.35 
30 
30 
30 
30 
26 
26 
31 
31 
31 
31 
31 



Result. 



No symptoms. 

Do. 
Symptoms, recovered. 
No symptoms. 

Do. 

Do. 

Do. 

Do. 

Do. 
Symptoms, recovered. 
Symptoms (?), recovered. 
No symptoms. 

Do. 
Symptoms, recovered. 
No symptoms. 
Symptoms, recovered. 

Do. 
No symptoms. 
Symptoms, recovered. 

Do. 
No symptoms. 

Do. 

Do. 
Symptoms, recovered. 
No symptoms. 



As the above series of guinea pigs was tested with antitoxic horse 
serum, we tested another series, similarly sensitized, with normal 
horse serum, as shown in the table on the following page, the results 
in all respects being similar. 



28 

Tdble showing the results of injecting ^ c. c. of normal Tiorse serum (roan) into the pertttmeal 
cavity of guinea pigs from 47 to 56 days prior with, gio c.c.ofthe blood serum qfothr 
animals. 



No. of 


Previous trfsatment. 




guinea 
pig- 


Kind of 


serum. 


Days 
prior. 


Result. 


173 


Rabbit 


aioC.e.. 


55 


Symptoms, recovered. 


175 


do 


do 


55 


Do. 


195 


Cat 


do 


55 


Do. 


213 


Dog 


do 


56 


Do. 


211 


do 


do 


56 


Do. 


187 


Hog I 


do 


51 


Do. 


189 


do 


do 


51 


Do. 


190 


Sheep 


do 


47 


No symptoms. 


183 


do 


do 


51 


Symptoms, recovered. 


181 


do 


do 


51 


Do. 


199 


Chicken 


do 


47 


No symptoms. 


198 


do 


do 


47 


Symptoms (?), recovered. 


200 


do 


do 


47 


Symptoms, recovered. 


203 


Man 


do 


52 


No symptoms. 


204 


do 


r do 


52 


Symptoms, recovered. 


205 


do 


do 


52 


Do. 



We think it is plain from the above that this action is not strictly 
specific for horse serum; that is, guinea pigs treated with horse 
serum are rendered somewhat susceptible to the subsequent injection 
of the serum of certain other species and that guinea pigs treated with 
the serum of other animals are slightly sensitive to the toxic action of 
horse serum. However, it is also perfectly plain from this series of 
experiments that the action is quantitatively specific, for it will be 
noticed that of the many guinea pigs sensitized with the blood serum 
of animals other than the horse, none died from subsequent injections 
of large quantities (6 c. c.) of horse serum. The same may be said 
of the action of these other serums upon guinea pigs previously 
treated with horse serum. While most of these serums produced 
symptoms, none of the guinea pigs died. 

In other words, there is plainly a distinct quantitative diflFerence, 
both in the results of the power of horse serum to render guinea pigs 
susceptible to other serums and in its toxic action upon guinea pigs 
sensitized by other serums. 

The action of horse serum upon other animals than the guinea pig 
is treated of in another chapter (p. 55). 

ARE OTHER BLOOD SERUMS EQUALLY TOXIC? 

We now know that guinea pigs previously treated with small 
injections of horse serum become very susceptible to subsequent 
injections of horse serum, but only slightly susceptible to the blood 
serum of other animals. It becomes important to determine whether 



Lea pigs treated with the serum of other animals (dog, hog, rat, 
<5attle, sheep, cat) become as highly sensitized to a second injection 
of the homologous serum. 

The following series of guinea pigs were all sensitized with 1/250 

<3. c. of the blood of various animals. Subsequently they were injected 

"with horse serum to which they either did not respond or, after 

^characteristic symptoms, recovered. These guinea pigs were finally 

injected with large quantities of the same kind of serum as that used 

at the first injection. 

Dog serum. 

G. P. No. 214. Six c. c. antitoxic Iwrse serum (Natl. IX. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 26 days prior, inoculated subcutaneously with ^\^ c. c. dog 
serum.] 

5 hrs. later 6 c. c. dog serum injected into the peritoneal cavity. Symptoms. 

G. P. No. 215. Six c. c. antitoxic Iwrse serum (Natl. IX. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 35 days prior, inoculated subcutaneously with 5^^ c. c. dog 
serum.] 

22 days later 6 c. c. dog serum injected into peritoneal cavity. Severe symptoms. 

G. P. No. 212. Six c. c. antitoxic Iwrse serum (Natl. IX. 17) injected into the peritoneal 
cavity. Mild symptoms. 

[Previous treatment: 35 days prior, inoculated subcutaneously with ^^^ c. c. dog 
serum.] 

22 days later 6 c. c. dog serum injected into peritoneal cavity. Severe symptoms. 

G. P. No. 211. Four c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Symptoms. 

[Previous treatment: 56 days prior, inoculated subcutaneously with 5^^ c. c. dog 
serum.] 

1 day later 6 c. c. dog serum injected into the peritoneal cavity. Dead in 35 minutes 

Hog serum. 

G. P. No. 186. Six c. c. antitoxic Iwrse serum (Natl. IX. 17) injected into the peritoneal 
cavity. Severe symptoms. 

[Previous treatment: 30 days prior, inoculated subcutaneously with ^^^ c. c. hog 
serum.] 

22 days later 6 c. c. hog serum injected into the peritoneal cavity. Severe symptoms. 

G. P. No. 185. Six c. c. antitoxic horse serum (Natl. IX. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 30 days prior, inoculated subcutaneously with ^^^ c. c. hog 
serum.] 

22 days later 5 c. c. hog serum injected into the peritoneal cavity. No symptoms. 

G. P. No. 187. Four c. c. antitoxic horse serum (Natl. IX. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 51 days prior, inoculated subcutaneously with ^\j^ c. c. hog 
scrum.] 

1 day later 6 c. c. hog serum injected into the peritoneal cavity. Symptoms. 

G. P. No. 189. Same injection and previous treatment. No symptoms. 

1 day later 6 c. c. hog serum injected into the peritoneal cavity. Symptonjs. 



30 

G. P. No. 206. Six c. •\ antitoxic /^--w j<.--ni Xi:!. IX. IT^ injected into the paitouil 
cavity. No symptoms. 

[Previous treatment: 31 days prior. ir.<«^i:k:ed aubcutABeoushr with v^ c.c oA 
serum.] 

22 days later 6 c. c. ca^i^- senzr, ir:j<cr;c«i i=.io the pentoneal cavity. Dead in 13 
minutes. 

G. P. No. 210. Six c. c. antitoxic ?•-.■-« ser-i^i Xitl. EX. 17 > injected into theperitooMl 

cavity. No symptonL<. 

[Previous treatment: 31 days prior. :r.>.i:la:ed suhcutaDeoushr with yj^ c. c coflk 
serum.] 

22 days later c. c. catih serum iii-t-i.^te^i ir.:o iht- periioneaJ cavity. Dead in 45 minuto. 

G. P. No. 207. Six c. c. antitoxic ao-*? s^.-n:::l Xs:I. LX. 17 injected into the peritoneil 
cavity. No symptoms. 

[Previous treatment: 31 days prior. irAXulate-.i suWuianeously with ^J^ c. c. eal^ 
serum.] 

22 days later c. c. cattle serum injected \v.io :be pc-.iioneal cavity. Dead in 90 mhrnt M. 

G. P. No. 184. Six c. c. antitoxic /n*r.vt s^^rum iX.-iil. IX. 17* injected into the peritoneil 
cavit}'. No symptoms. 

[Previous treatment: 30 days prior, im^-ulaiod suK*utaneously with ::r^|^ c. c. duef 
scrum.] 

22 days later 6 c. c. atheep st^rum injected into the peritoneal canity. Seveie aymptontt; 
dead in 23 hours. 

G. P. No. 182. Six c. c. antitoxic horsf semm -Natl. IX. 17^ injected into the peritoneil 
cavity. Severe symptoms. 

[Previous treatment: 'SO days prior, inoculated subcutaneously ¥rith ^r^ c. c duep 
scrum.] 

22 days later G c. c. *7<a/) senim injected into the peritoneal cavity. Severe 8jmptoio& 

G. P. No. HK). Four o. c. antito.xic horse semm tXail. IX. 17"^ injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 47 days prior, in^^culatt'd subcutaneously with srijj c. c. sfteep 
serum.] 

1 day later C c. c. sheep serum injected into the peritoneal cavity. Severe symptons. 

G. P. No. 183. Four c. c. antitoxic horse senim (^Natl. IX. 171 injected into the peritoneai 
cavity. Symptoms. 

[Previous treatment: 51 days prior, insulated sulx-utaneously with ^^ c^ c. 6Ae«p 
serum.] 

1 day later C c. c. sh^ep serum injected into the peritoneal cavity. Dead in 18 houra. 

G. P. No. 181. Same injection and previous treatment. Mild symptoms. 

1 day later 6 c. c. sTieep serum injected into the peritoneal ca^-ity. Severe symptoms. 
Dead in 21 hours. 

Cat serum. 

(i. P. No. 192. Six c. c. antitoxic horse serimi (Natl. IX. 17> injected into the peritoneal 
cavity. Severe symptoms. 

[Previous treatment: 34 daj's prior, inoculated sul>cutaneously with ^r^ c. c. cat 
senun.] 

22 days later c. c. cat serum injected into the jx?ritoneal cavity. Severe symptoms. 



( 



I^. No. 195. Four c. c. antitoxic Tiorse serum (Natl. IX. 17) injected into the peritoneal 
<5«ivity. Symptoms. 

[Previous treatment: 55 days prior, inoculated subcutaneously with ^J^ c. c. cat 
^»«rum.] 

1 day later 6 c. c. cat serum injected into the peritoneal cavity. Dead in 35 minutes. 

Rat serum. 

^- P. No. 168. Six c. c. antitoxic horse serum (Natl. IX. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 34 days prior inoculated subcutaneously with ^i^ c. c. rat 
serum.] 

2 hours later 2 c. c. rat serum injected into the peritoneal cavity. Severe symptoms. 
Dead in 3 hours. 

We infer from the above series that guinea pigs treated with the 

scrums of various animals and subsequently injected are much more 

Qnsceptible to homologous serums than to heterologous serums. We 

-liave here another indication that this toxic action of blood serum is 

^quantitatively specific. 

We expect to take up the work with the serums of other animals 
xnore in detail, and the results will be reported at another time. 

THE RELATION OF HEMOLYSIS. 

So much work has recently been done indicating that the toxic 
property of blood serum is closely allied with its hemolytic action 
that we made some experiments in order to determine what connec- 
tion exists between the hemolytic and toxic action of horse serum 
upon guinea pigs. As a result of these studies we believe that we 
have shown very clearly that blood serum may contain an acute 
poison entirely independent of any hemolytic action. 

Landois " in his work on transfusion showed long ago (1875) that 
the blood serum of many mammals dissolves the red blood cells 
of other mammals. Landois . specifically demonstrated that the red 
blood corpuscles of rabbits, when mixed with alien blood serum, are 
readily dissolved, while, on the other hand, the red blood corpuscles 
of cats and dogs are much more resistant. If large quantities of red 
blood corpuscles are dissolved, there follows an excretion of hemo- 
globin through the kidneys, intestines, and also into the serous cavi- 
ties. If the serum is injected into the blood stream of a rabbit, 
some of the erythrocytes of the rabbit are dissolved in vivo, as 
may readily be confirmed microscopically; but before the corpuscles 
are dissolved there is an agglutination of the blood corpuscles into 
clumps, by means of which large capillary areas may become plugged. 
The same process is seen by the further extraction of hemoglobin 
from the mass of the stroma of the corpuscles which fills the capil- 

a Landois: Lehrbuch der Physiologie des Menschen, 1896. Die Transfusion des Blutes. 
Leipzig, 1875. 



I 
I 



I 



32 

laries. The dissolved heniogKibin now cjiuscs further blocl 
the vessels by means of clots, which are formed through the 
of the hemoglobin upon the leucocytes, freeiiip the fibrin feraiem,' 
From this somewhat complicated process there may result aerioiB 
symptoms, as dyspnea, convulsions, etc., in proportion to the amo\ii4] 
of bjood injected. If the amount of blood is large, death may follow, 
as Landois has shown, with phenomena of asphyxiation. (I,'hl«i-' 
huth: Zur Kenntniss der giftigen Eigenschaften des Blutsenuna. 
Zeit. f. Hyg., vol. 26, 1897, p. 384.) 

Hermann Pfeiffer," confirming the work of Uhlenhuth upon the 
necrotic action of blood serums when injected subcutaneously into 
guinea pigs, believes that the toxic substance producing the necrosis 
is the same as the hemolytic agent found in the blood serum. 

In recent years hemolysis has been studied more particularly by 
Ehrlich, Morgenroth, Bordet, Metchnikoff, and many others. Much 
of the work done appears to corroborate the parly observations of 
Landois, who believed that the toxic action of blood serum when 
injected intravenously depends upon a " globulicidal " action upon the 
red blfiocl cells and the plugging of the capillaries. On the other 
hand, we have the views of other authors based in part upon experi- 
mental data that the toxic action of blood serum is dependent upon 
albuminous substances independently of the lytic substance present 
in the scrum. 

Creite '> in 1869 was aware of Landois's view concerning hemolysis, 
but separated the toxic power from the blood serum by means of 
coagulation (heat). 

Also, Weiss ' in 1896 showed by separating albuminous substances 
from the blood scrum by means of heating or absolute alcohol that 
the serum has no longer the toxic power and concluded, therefore, 
that the toxic action is brought about by the albumins. 

Albu ** separated the albuminous substances from blood serum by 
precipitation with ammonium sulphate, which he dialyzed out, as it 
is itself poisonous, and came to the same conclusion as Creite and 
Weiss. 

Rummo ' believed that the toxic substance is a tox-albumin which, 
according to his views, is formed in the living cells of the respective 
animals. 

" llermniin PfeilTer: Uljer die nekrotisirendeWirkang nonoaler Seren. Zeit. L Hyg.,. 
1905, Band 150. p- 183. 

* Creite: Versiicbe Qber die Wirkung des Serumweisses liach Injection in das Blut. Zeit. 
^ retionelie Med. Bd. 36. S. 00. 
«W«ia9: Uebar die Wirkungpn von BliitHomm-Inji'ctiuiicn in's Blut. FflQger's Arch., 
jL6^ 1896. 

|tf Jtlbu: Uuterauchungen flber die Toxicitat normaler u. pathol. Serumfliifsigkcit. Vit- 
jnr^AKh., Bd. 149, 3. 

U Jluiiunii: Veher die Giltigkeit dea Blutsemms bei Mcnschen iind Thiereu ini nurnuJen 
lo und bei Inloctionakrajikheiten. Wiener med. Woch., 1891, nr. 19-20. 



Mairet and Bosc " were able to distinguish in blood serum a sab- 
stance capable of prtiducing coagulation and another substance capa- 
ble of producing poisonous symptoms. They believed both of these 
substances to be albumins. They were able to eliminate the coagu- 
lation-producing substance by heating from 52° to 53° C. or by the 
addition of sodium chloride and sodium sulphate. 

Pearce * believes that it appears to be definitely established that 
red blood corpuscle thrombi occur in the experimental lesions caused 
by sera possessing hemagglutinative power. He demonstrated that 
the capillaries of certain areas in the liver are entirely occluded by 
fused red cells, and he also showed the inability of the products of 
hemolysis to produce such action and necrotic areas in the liver. 

L. Camus and E, Gley ^ first endeavored to separate the hemolytic 
action from the toxic action of bloofi serum by means of heating. 
They found that heat ilestroys the hemolytic action; but while it 
diminishes it does not entirely destroy its general toxic action. Thia 
work was done with eel serum. In their previous work the two 
actions seemed about parallel. For instance, eel serum is very hemo- 
lytic for rabbits and guinea pigs and equally poisonous for these ani- 
mals. On the other hanti, the red blood cells of the pigeon and those 
of the porcupine are very resistant to eel serum, and these animals 
also resist doses which are fatal for the rabbit and the guinea pig. 
However, these authors finally found an animal, the marmot, for 
which eel serum had slight hemolytic action but was extremely 
toxic, and they believed that these researches upon the marmot have 
permitted them to separate the toxic action of eel serum from its 
hemolytic action better than did their previous work by means of 
heating. 

We found horse serum to have practically no hemolytic action 
upon the corpuscles of the guinea pig. This applies with equal force 
to antitoxic horse serum as to normal horse serum, as may be seen 
from the following table: 

HimiAysis. 
G. P. No, 2. Normal pig. Serum obtftined by coagulation. 
G. P. Nn. I , Normal pig. Copusclos obtained hy (.■eotrifugiition, etc. 

a Mairet and Bosc: R<'cberc)jE» sur Igs causes de la toxicity du scrum du sang. Compt. 
rend., t. 110, 4, p. 292. Toxicity du serum du sang de I'hninine sain., Compt. rend., 1897. 
Dea effects de la cbaleur Bur la toxicity du Bfrum. Compt. rend. July 7, 1894. 

b Pearce, Ritliard M.; A further study of the experimental production of liver n 
by tbe injection of hemagglutinative sera. J. M. Research, v. 14, Apr., 1906, \ 
Pearce, H. M., and Winne, C. K., jr.; Concerning hemagglutins of bacterial origin and thair ' 
relation to hyaiin thrombi and liver necroses. Am. J. M. Sci., v. 128, 1904, p. 6 

c Camus, L., and Gley, E.: Comparison between the hemolytic and the toxic action of j 

eel's serum in the marmot {Arctomya marmota). Arch, intemat. de pharmacodyn. et da J 

therapie, tome IS, fasc. 1 and 2, 1905. 

^ 28337— No. 29—06 3 



I 
I 



^^P G. P. No. 4517. Serura obtainBd by coagulation. 

^H No. 7+1 -JoIt horse serum (Standard B27).l 

^^V G. P. 4S10. Corpu^Ies abtained bj c«nbrirugatioD, etc. 

^^H [Previoua treatment: 35 daya prior, inoculated Bubcutaneousty with 0.19 u. c. toiine 

^^M No. 7+1 unit horse serum (Standard B27),] 


^^M 


normal guinea pig No. 1 


(wABlied). 




Do. +1 
r?mnTr 


Do. +1 






^uiT 




Palnt^S 

6 

TraoB. 



Faintest 

Trace, 


Fabitast 
tnwe. 





Traca. 



Falnt«t 

Traoa. 






^^M Antltoilo barw Hnim (NaU, VIII, 17),. 
^^1 AntltoxlDhnrM wrum Natl. tX. 18}.... 

^^H AntltoxIcbormHrmu JNatl, V. 141 

^H Antltailo br.r« nriiui (A1a;<, AWS, Glb- 




^H First the corpusclps werp pla«!ed in the teat tubes, then the various 
^H horse serums, and finally the drop of guinea-pig serum. 
^^V The corpuscles were obtained by whipping the blood and washing in 
^H the usual way four times and suspended so as to make the so-called 
^H 5 per c«nt suspension. After the corpuscles and the serum were 
^H mixed they were well shaken and placed in the incubator, again 
^H shaken at the end of one hour, again at the end of two hours, and at 
^^B the end of three hours taken out of the incubator and placed in the ice 
^^1 chest. The readings were taken the next morning. 
^H This work was repeated several times until we were satisfied that 
^^B neither the normal horse serum nor the antitoxic horse serum had 
^^^ practically any power of dissolving the hemoglobin from the guinea- 
pig corpuscles. There can therefore be no doubt that there is a toxic 
substance in the blood serum of horses entirely independent of any 
hemolytic action. As a further corroboration we may state that 
autopsies upon guinea pigs which have died from the results of the 
injections of horse serum showed none of the usual effects, indicat- 
^_ ing that hemolysis took place within the body. 

^B Further, horse serum heated to 60° for six hours is as toxic as 
^H unheated serum. 

^^H As a further study of the relation of hemolysis to the toxic action 
^^^ and also of the relation of the toxic principle to the haptin group 
^^H we made the following experiments: 
^^H G. F. No. 249, Four subcutBneous injections of 1.5 c c. each on successive days, using 

^^^H developed a high degree of hemolytic power against horse corpusclea. The blood serum 
^^^f of this Ruinea pig was mixed with equal quantities of normal horse Berum and the ini»- 
^^F ture injected into the following three guinea pigs: ^^^1 



85 

6. P. No. 4903. Two c. c. of the above mixture injected into the peritone*! cmvitT. Dead 
ID 40 minutes. 
[FtoTious treatment: 28 days prior, inoculated subcutaneously with 0.22 c« c. Uxnaob 

^0- 7+5^ c. c. antitoxic horse serum (Alex. A206).] 

6. P. No. 4068. Same injection. Dead in 30 minutes. 

[Previous treatment: 28 days prior, inoculated subcutaneously with 0.22 c. c. toxme 
No. 7+rhr ^* ^* antitoxic horse.serum (Alex. A202).] 
6. P. No. 4002. Same injection. Dead in 35 minutes. 

[Previous treatment: 28 days prior, inoculated suboutaneuosly with 0.22 c. c. toxme 

No. 7+7^7^ c. c. antitoxic horse serum (Alex. A208).] 

The above experiment was repeated as follows: 

6. P. No. 248. Four subcutaneous injections of 1.5 c. c. each on successive days were made, 
using roan corpuscles washed three times. The blood serum of this guinea pig developed a 
hi^ degree of hemolytic power against horse corpuscles. The serum was mixed with 
equal quantities of normal horse serum and the mixture injected into the following guinea 
p^: 

6. P. No. 4013. Four c. c. of the above mixture injected into the peritoneal cavity. Dead 
in 10 minutes. 

[Previous treatment: 41 days prior, inoculated subcutaneously with 0.130 c. c. of toxine 
No. 5-|-Tiv ^' c. antitoxic horse serum (Steams 12D).] 
G. P. No. 4014. Two c. c. same serum injected into the peritoneal cavity. l)ead in 20 
minutes. 
[Previous treatment: Same asG. P. No. 4013.] 

THE RELATION OF PRECIPITIN TO THE TOXIC ACTION. 

The precipitins are so specific and so closely related to the cyto- 
toxins that it natually occurred to us to determine what relation there 
may exist between the toxic power of the blood serum of the horse and 
of the guinea pig, and the power bi these two serums to precipitate 
each other. 

If we iAject horse serum several times into a guinea pig we would 
expect to find that the blood serum of a guinea pig so treated would 
have a power of precipitating horse serum. This we found to be the 
case; but we could not find that the injection of a single small dose of 
horse serum into the guinea pig developed this power in the guinea 
pig's blood serum. We might, however, conjecture that the union of 
the two serums under the conditions named causes a precipitation 
which is not visible, or causes a chemical union between two highly 
organized albuminous substances having toxic properties. In order to 
test this subject more thoroughly we first made some experiments to 
detennine the precipitating action of normal and antitoxic horse 
serum upon normal guinea-pig serum, as follows : 

One c. c. of horse serum in dilution of 1:21 was placed in a small 
test tube. The 0. 1 c. c. of normal guinea-pig serum was added to 
each tube. These were thoroughly shaken, placed in the incubator, 
and the results read after twenty-four hours. 



86 



Normal G. P. No. 1. Killed the same day with chloroform, blood defibrinated, uid the 

serum separated in the centrifuge. 

Normal G. P. No. 2. EiUed by chloroform «3 days previously and the serum allowed to sqia- 

rate by coagulation. 

(Two teste made of each senim.) 



1 c. c. hone serum (1:21). 



Normal hone serum (Sam) 

Normal horse serum (Jane) 

Antitoxic horse serum (Natl. VIII, 17) 
Antitoxic horse serum (Hubbert op. 1) 

Antitoxic horse serum (Natl. IX, 17) . . 
Antitoxic horse serum (Stearns 1110) . . 



0.1 c. c. serum 


0.1 


c. c. semm 


normal guinea 


normal guinea 


Pigl- 




P«2. 


NU. 




Nfi. 


NU. 




NU. 


NU. 




NU. 


NU. 




NU. 


NU. 




NU. 


NU. 




NU. 


NU. 




NU. 


Slight precipi- 




NU. 


tation(7). 






NU. 




NU. 


NU. 




NU. 


NU. 




NU. 


NU. 




NU. 



From this and other similar experiments we are justified in conclud- 
ing that neither normal horse serum nor antitoxic horse serum has any 
power to precipitate normal guinea-pig serum. 

We next made some tests to determine whether a guinea pig pre- 
viously treated with one small injection of horse serum developed 
precipitms. 

One c. c. of horse serum both normal and antitoxic in dilution of 
1 : 21 was placed in small tubes, as before, and 0.1 c. c. of a sensitized 
guinea-pig serum added to each tube, as shown in the table. Thejr 
were shaken and placed in the incubator and results read after 
twenty-four hours. 

G. P. No. 4617. [Previous treatment: 35 days prior, inoculated subcutaneously with 0.19 

c. c. toxine No. 7+1 unit horse serum (standard B27).] 
G. P. No. 4510. ' [Previous treatment: same.] 



1 CO. horse serum (1:21). 



Normal horse serum (Sam) 

Normal horse serum (Jane) 

Normal horse serum (S. P.) 

Normal donkey serum 

Antitoxic horse serum (Natl. VIII, 17) 
Antitoxic horse serum (Natl. V, 14) . . . 
Antitoxic horse serum (Natl. IX, 17) . . 
Antitoxic horse serum (Stearns 1110) . 
Antitoxic horse serum (Stearns 1429) . 



0.1 c.c. normal 
guinea-pig serum. 


0.1 c.c. 
guinea-pi 


Centrif- 
ugation. 


Coagula- 
tion. 


Guinea 
pig 4617. 
























































+ (?) 



























Guinea 
pig 4510. 













It would therefore appear that guinea pigs which have previously 
been inoculated with one injection of a very small amount of horse 
serum sufficient to render them highly susceptible to a second injec- 
tion of horse serum do not contain sufficient precipitin in their 






S7 

blood aerum to cause a visible precipitation when added to horse 
senim. 

The following studies show still more clearly that the specific 
precipitins have nothing to do with this toxic reaction. 

We prepared guinea pigs by injecting them many times at short 
intervals with horse serum, so as to develop a high degree of pre- 
cipitin in their blood serum against horse serum. The blood serum 
of guinea pigs so prepared was then added to horse serum in the 
proportions of about one of the former to two of the latter and 
allowed to stand until the precipitating action was complete. This 
guinea-pig serum was so active that very large quantities of the pre- 
cipifcum settled to the bottom of the test tubes. The mixture was 
now filtered, so as to separate the precipitum entirely, and the 
filtrate injected into sensitized guinea pigs and found to be quite 
as toxic as untreated horse serum. 



iDJeetbtis at I c- c. each normal horae ierum (roan) 
honte senim (Natl. IX, 19) were gtvea 



ith 3 c. c. of the at^nim uf thia guinea 
temperature. The large amouot of pre- 
ijected into the following two 



0. P. No, 107. SfiTenteen subcutani 

and one peritoneal injection of 6 

betweeo February 8 and March 11. 
Six e. t. normal horse serum (roan) w 

pig and allowed to staud about 3 houis 

cipitum which developed was filtered oil and the lilt 

guinea pigs: 
O. P. No. 4996. Two c. c. of the above liltrate iiije.-ted into peritoneal cavity. Dead in 20 

[Previous treatment: 27 days prior, inoculated aubcutaneouslj with 0.22 e, c. loxine 
No. 7+Tij c. fi. antitoiio honie serum (Wellcome 474A),] 
G. P. No. 5000. Five c. c. ot the above filtrate iniected into the peritoneal cavity. Dead 



[Previous 
No. 7+Ti, 



28 days prior, inorula(«d subcutaneously with 0.22 c. 
[: horae serum (Wellc.ome 477B).] 



These experiments were repeated under varying conditions, espe- 
cially allowing the two serums to remain in contact a longer time 
before filtering, and similar results obtained. 

The results of this part of our work are strengthened by the con- 
clusions of Pirquet and Schick, who found that the antibodies, what- 
ever they may be, producing symptoms of the serum disease, are not 
identical with the precipitins. Thej found, for instance, that the 
symptoms of the serum disease appear within eight to thirteen days 
following the first injection of horse seritni into man, whereas it 
requires about three weeks for precipitins to appear in the blood in 
children after the injection of horse serum. 

The formation of precipitins does not take place as readily in 
man following the injection of horse serum as it does in rabbits. 
fact, Pirquet and Schick found that sometimes even after the injec- 
tion of 200 c. c. there was no production of precipitins, and, further, 
b the length of time that the precipitin reaction remains in the 



lh^ ihi 



J 



38 

blood of man following the injection is variable. It sometimes 
disappears in the fourth week, and sometimes is still markedly 
present in the ninth week. 

Finally, we know that the precipitins are strictly specific, whereas 
the toxic action is only quantitatively specific. 

EFFECT OF HEAT UPON THE TOXICmr OF HORSE SERUM. 

One of the first questions which required solution was whether 
the toxic substance in blood serum is thermolabile. That it is able 
to withstand a comparatively high degree of heat for a long time, 
viz, 60*^ C. for six hours, is evident from the following experiments: 

G. P. No. 4349. Six c. c. antitoxic horse serum (Natl. VIII. 17) heated to 60** C. for 30 
minutes, then injected into the peritoneal cavity. Dead in 30 minutes. 

G. P. No. 60. One c. c. same serum and injection as g. p. 4349. Dead in 10 minutes. 
[Previous treatment: With toxine-antitoxin mixture.] 

G. P. No. 61. Do. Dead in 10 minutes. 

G. P. No. 62. Do. Symptoms; recovered. 

G. P. No. 4897. Injected with 5 c. c. of an extract of normal horse serum (roan) heated to 
60** C. for 6 hours. This degree of heat coagulated the serum. About 1 c. c. of the coagu- 
lum was added to 9 c. c. of salt solution, thoroughly shaken up, filtered, and the filtrate 
injected into the peritoneal cavity. Symptoms; death in about 3 hours. 

[Previous treatment: 41 days prior, injected subcutaneously with 0.139 c. c. toxine 
No. S+tJtt c. c. horse serum (Alex. No. 192).] 

G. P. No. 4508. Six c. c.of an extract of antitoxic horse serum (Natl. IX. 19) heated to 
100^ C. for 15 minutes, injected into the peritoneal cavity. The coagulum was ground up 
with a few c. c. of salt solution and strained through gauze before being injected. No 
symptoms. 

One and a half hours later about 8 c. c. of the coagulum was ground up and the entire 
mass, not strained, injected into the peritoneal cavity of the same guinea pig. No 
symptoms. 

G. P. No. 5048. Six c. c.of the coagulum of normal horse serum (roan) heated to 100** C. 

for 15 minutes and ground up with salt solution (0.85) injected into the peritoneal cavity. 

No symptoms. 

[Previous treatment: 20 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+5J77 c. c. antitoxic horse serum (Alex. A204).] 

From the above it is evident that while a temperature of 60*^ C. 
continued for six hours has practically no influence upon the toxic 
substance in horse serum, the toxicity is entirely destroyed by a 
temperature of 100° C. for fifteen minutes. 

THE TOXIC PRINCIPLE IN HORSE SERUM IS FILTERABLE THROUGH PORCELAIN. 

The following experiment is suflB.cient to demonstrate that the 
close-grained pores of a Pasteur-Chamberland filter B do not retain 
the toxic principle of horse serum: 

G. P. No. 4521. Six c. c. of antitoxic horse serum (Natl. VIII. 17) filtered through a Pas- 
teur-Chamherland filter B and injected into the peritoneal cavity. Dead in 35 minutes. 
[Previous treatment: 32 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit horse serum (Standard B27).] 



89 

DBYING DOES NOT INJURE THE TOXIC PRINaPLE. 

We have on hand a large quantity of dried antitoxic horse serum 
which has been prepared and preserv^ed under sp>ecial conditions for 
the preparation of the standard antitoxic unit. This serum was 
carefully dried under bacteriological precautions at a temp>erature 
below 37^ C, as described in Bulletin No. 21." 

That such dried and redissolved serum still retains its toxic prop- 
erty is evident from the following exj)eriment: 

Q. P. No. 4376. Six c. c. antitoxic horse serum (B 1+12) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 19 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+1 J^f c. c. horse serum (Natl. V. 7).] 

THE TOXIC PRINCIPLE CAN NOT BE SEPARATED BY PRECIPFFATION AND 

DIALYSIS. 

We made several experiments to determine whether precipitation 
by ammonium sulphate and subsequent dialysis to remove the sul- 
phate and other inorganic salts would also remove the toxic principle. 
For this purpose we used antitoxic horse serum prepared and dialysed 
in accordance with Gibson's method.^ It is plain from the following 
experiments that precipitation and dialysis do not remove the toxic 
principle: 

G. P. No. 4104. Six c. c. antitoxic horse serum (NYBH A) injected into the peritoneal 
cavity. Dead in 5 minutes. 

[Previous treatment: 37 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. T+iJtt c. c. horse serum (Alex. 190).] 

G. P. No. 4506. Five c. c. antitoxic horse serum (Alex. A 203/N YBH) injected into the 
peritoneal cavity. No symptoms, c 

[Previous treatment: 25 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7-|-l unit horse serum (Standard B27).] 

G. P. No. 4513. Five c. c. antitoxic horse serum (Alex. A 203/N YBH) injected into the 
peritoneal cavity. Dead in 39 minutes. 

[Previous treatment: 26 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7-|-l unit horse serum (Standard B27).] 

EFFECT OF VARIOUS CHEMICAL SUBSTANCES UPON THE TOXIC PRINCIPLE OF 

HORSE SERUM. 

Various substances of widely different chemical nature were added 
to horse serum, in order to see whether any of them had the power 
of precipitating, oxidizing, or neutralizing the toxic property of the 
serum. The following substances were used: Succinic acid per- 

aRosenau, M. J.: The immunity unit for standardizing diphtheria antitoxin (based on 
Ehrlich's normal serum); official standard prepared under the act approved July 1, 1902. 
Bulletin 21, Hygienic Laboratory, U. S. Public Health and Marine-Hospital Service. 
Washington, Government Printing Office, April, 1905. 

b Gibson, R. B.: On the practical concentration of diphtheria antitoxin. Am. med., 
vol. 10, 1905, p. 915. 

c £2xplanation of occasional irregularities, p. 63. 



40 

oxide (alphozone), butyric acid^ hydrogen dioxide^ permanganate of 
potash, alcohol, ammonium sulphate, and citrate of soda. 

Butyric acid. 

N 
G. P. No. 4368. Ten c. c. of a |q butyric acid solution were added to 25 c. c. antitoxic 

horse serum (Natl. IX. 17); the mixture was kept at 15^ C. for 40 hours, at the end of 
which time the fine precipitate which developed was filtered off and 3 c. c. injected 
into the peritoneal cavity. Dead in 2 hours 10 minutes. 

[Previous treatment: 30 days prior, inoculated subcutaneously with 0.22 c. c. toxino 
No. 7+yJ^ c. c. horse serum (Cutter 1461).] 

Hydrogen dioxide. 

G. P. No. 4371. Five c. c. of hydrogen dioxide (Oakland) were added to 25 c. c. antitoxic 
horse serum (Natl. IX. 19) ; the mixture was kept at 15^ C. for 40 hours, at the end of which 
time a very little fine precipitate had formed, but the mixture efifervesced in filtering; 
3 c. c. of the filtrate was injected into the peritoneal cavity. Symptoms (?); recovery. 
[Previous treatment: 30 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+ff+ir c. c. horse serum (Cutter 1504).] 

G. P. No. 4917. Five c. c. hydrogen dioxide (McKesson & Robbins) were added to 25 c. c. 
antitoxic horse serum (Natl. IX. 17); the mixture was kept at 15^ C. for 40 hours, at the 
end of which time the fine precipitate which developed was filtered ofif, and 6 c. c. injected 
into the peritoneal cavity. Symptoms; recovered. 

[Previous treatment: 41 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7-{- xiv c* c* antitoxic horse serum (Stearns 16H).] 

PermanyanaU of potash. 

G. P. No. 4370. Three c. c. of a 1 per cent solution of permanganate of potash were added to 
25 c. c. antitoxic horse serum (Natl. IX. 19); the mixture was kept at 15® C. for 40 hours, 
and as no precipitate had formed in that time it was not filtered; 3 c. c. of the mixture 
inoculated into the peritoneal cavity. Dead in 45 minutes. 

[Previous treatment: 30 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+ jy^ c. c. horse serum (Cutter 1461).] 

Succinic peroxide add {alphozone). 

G. P. No. 4372. Five c. c. of a 1 per cent solution of alphozone were added to 25 c. c> antitoxic 
horse serum (Natl. IX. 19); the mixture was kept at 15® C. for 40 hours; the precipitate 
which formed was filtered off and 3 c. c. of the filtrate injected into the peritoneal cavity. 
No symptoms. « 

[Previous treatment: 30 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+yi7r c. c. horse serum (Cutter 1504).] 

G. P. No. 5A. Ten c. c. of the same filtrate injected into the peritoneal cavity. Dead in 
35 minutes. 

[Previous treatment: 30 days prior, inoculated with 0.22 c. c. toxine No. 7-t-l unit of 
horse serum (Standard B27).] 

As we found that 2 c. c. of a 1 per cent solution of succinic peroxide 
acid (alphozone) injected into the peritoneal cavity of a normal 
guinea pig produced transient irritating eflFects these experiments 
were repeated, using smaller quantities of the acid, as follows: 



a Explanation of occasional irregularities, p. 63. 




41 

G. P. No. 451S. Ooe c. c. oFa I per cent solution of alpliozone wa9addedto25a.i 

horaa aerum (Natl. IX. 19); the mixture was kept at 15 C. for -iO* houre; the 

which formed waa fiitered off and 6 c. c. of the filtrate injected into the perilon 

Dead b 24 minutes. 

[Previous treatment: 28 daja prior, inoculated Hubeutaiieoualy with 0.19 c. 

No. 7 + 1 unit horao aerum (Standard B27).] 
G. P. No, 4518. Fire c. c. added as above ojid 6 c. <■. uf the filtrate injected into the 

cavity. Dead iu 25 minutes. 

|Previous treatment: 28 daya prior, inoculated siihcutantMiusly with 0.19 r 

No. 7 + 1 unit horee serum (Standard B27).] 

Ahohoi, 

O. P. No. 4373. Five >■- c- o( 9.'> per rent alcohol were added to 25c. c. antitoxic li 

(Natl. IX. lU); the mixture waa kept at 15°C. for 40 hours; the precipitate which formed j 
was filtered olT and 3 c. c. of the filtrate injerted into the peritoneal cavity. SymptoniB; 
recovered. 

{Previous treatment: 30 days prior, inoculated suhcutaneoiisly with 0.22 c. c. toxi 
No. 7+7I5 c. c. horse aerum (Cutler 1504).] 
O. P. No. 5E. Seven and a half c. c. added as ahove and 3 e. c. o( the filtrate injected ir 
the peritoneal cavity. No aymptoraH.a 

jPrevioua treatment: 30 daya prior, inoculated aubcutsneously with 0.22 c. c. toxi 
No. 7+1 unit horse serum (Standard B27).] 
G. P. No. 156. Seven and a half c. c. added aa alxive and 6 c. <:. uf the hltrate injected ir 
the peritoneal cavity. Dead in 90 minutes. 

[Provioua treatment: 60 days prior, inoculated subcutaneously with 0.22 c. c. toxi 
No. 7+ j^ac.c,. antitoxic horse eerum (Steoma 1351).] 
O. P. No. 4303. Ten c. c. added aa above and 3 c. c. of the filtrate injected into the perito- I 
neal cavity. Symptoms, recovered. 

[Previous treatment: 30 days prior, inoculated subcutaneously with 0.22 c. c. toxin 
No. 7+j-iB c. c. horse scrum (P D & Co .07173-G).] 
G. P. No. 4C. Twenty-five c. c. added as above. This caused practically solid coagulatioi 
on the senim. The coagulant was vigorously shaken up with salt solution (0.S5) and filtered 
by the aid of a vacuum, and 6 e. r. of the opalescent filtrate injected into the peritoneal 
cavity. Dead in 4 hours 10 tninutea. 

[Previous treatment: 30 daya prior, inoculated subcutaneously with 0.22 c. 
No. 7+1 unit horse aerum (Standard B27).l 

Ammoniam suliAah. 

We know from the work done with serum precipitated with a 
nium suljjhate and subsequently dialysed that the ammonium ( 
sulphate lias no action upon the toxic principle found in horse serum, i 
(See guinea piga Nos. 4104, 4106, and 4513, p. 39.) 

Cilnxti of soda. 

We know that citrate of soda has no injurious action upon the 1 
toxic principle found in horse serum because some of the s 
used was prepared by adding 1 per cent solution of citrate of soda I 
to the whole blood as it was drawn from the horse in order to prevent j 



42 

clotting and the clear serum subsequently pipetted off. The citrate 
of soda was afterwards removed by dialysis (Gibson's method) and 
the plasma proved just as toxic to treated guinea pigs as other horse 
serum. We also have the following experimental data upon this 
point: 

G. P. No. 4925. Six c. c. normal horse serum (roan) containing 1 per cent citrate soda 
injected into the peritoneal cavity. Dead in 15 minutes. 

[Previous treatment: 39 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 54-7^7^ c* ^' antitoxic horse serum (Cutter 1465).] 
G. P. No. 4918. Injected as above. Dead in 5 minutes. 

[Previous treatment: 40 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 54- 7^7 c.c. antitoxic horse serum (Steams 16 H).] 

G. P. (control). The above guinea pigs were controlled by injecting 6 c. c. of the same 
serum containing 1 per cent citrate of soda into the peritoneal cavity of a normal pig. 
The only effects of this inoculation were symptoms of irritation which soon passed away. 

INFLUENCE OF ANTISEPTICS, ETC. 

As much of this work was done with horse serum preserved by 
means of chloroform and trikresol (0.4 per cent) we made a control 
experiment, using horse serum as fresh as possible without the addi- 
tion of any substance. The following experiment eliminates these 
factors as having any influence upon the toxicity of the serum: 

O. P. No. 125. Three c. c. normal horse serum (roan) freshly drawn, centrifugized and 
injected into the peritoneal cavity as soon as the serum separated. Dead in 45 minutes. 
[Previous treatment: 49 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+tJ^ c. c. antitoxic horse serum (Steams 1351).] 

EFFECT OF OLI> HORSE SERUM UPON SUSCEPTIBLE GUINEA PIGS. 

At one time in our work it apperaed to us that the toxic properties 
of horse serum gradually diminished in toxicity with the age of the 
serum. It was, however, soon proved by the following experiments 
that very old serums may be quite as poisonous as those freshly 
drawn. 

G. P. No. 4496. Four c.c. antitoxic horse serum (Natl. V. 2) injected into the peritoneal 
cavity. The serum was at least one and a half years old when injected. Dead in 20 
minutes. 

[Previous treatment: 25 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit of horse serum (Standard B27).] 

G. P. No. 4921. Six c. c. antitoxic horse serum over S years old (P. D. & Co .0495 prepared 
Dec. 15, 1902) inoculated into the peritoneal cavity. Symptoms; recovered. 

[Previous treatment: 39 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+yJij c. c. antitoxic horse serum (Cutter 1465).] 

G. P. No. 4922. Same injection. No symptoms.^ 

[Previous treatment: 39 days prior, inoculated subcutaneously with 0.139 c. c. toxino 
No. 5+f J^ c. c. antitoxic horse serum (Cutter 1465).] 

G. P. No. 4899. Same injection. Dead in 6 minutes. 

[Previous treatment: 48 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+yii7 c.c. antitoxic horse serum (Alex. 192).] 

^ Explanation of occasional irregularities, p. 63. 



The following guinea pigs were inoculated with horse serum eight 
s&d one-half years old : 

0* P. No. 258. Six c. c. antiamariUic serum injected into the peritoneal cavity. Dead in 
40min. 

(Pk«yiou8 treatment: 24 days prior, inoculated subcutaneously with j^jf c c. normal 
lK>rBe serum (roan) heated to 60^ C. for two hours.] 

^' ?. No. 256. Same injection. Symptoms., recovered. 

[Previous treatment: 20 days prior, inoculated subcutaneously with -^ c. c. normal 
hoise serum (roan) heated to 60® C. for two hours.] 

From the above experiments we see that blood serum over eight 
^d one-half years old is as poisonous to sensitized pigs as fresh 
Serum. 

EFFECT OF X-RAYS UPON THE TOXIC PRINCIPLE. 

Normal horse serum (roan) was exposed to the action of X rays, 
40 amperes, 4 inches from the tube for forty minutes, then injected 
into a susceptible guinea pig. It was foimd as poisonous as horse 
serum not so treated. 

Q. P. No. 6A. Six c.c. above serum injected into peritoneal cavity. Dead in 10 minutes. 
[Previous treatment: 33 days prior, inoculated subcutaneously with 0.293 c. c. toxine 
No. 7-j-l unit antitoxic horse serum (Standard B28).] 



Part V* 
THE INFLUENCE OF TIME, 

TIME NECESSARY TO RENDER A GUINEA PIG SUSCEPTIBLE. 

• 

We know from our preliminary work that some time is necessary 
after the first injection of horse serum before a guinea pig is rendered 
susceptible to the toxic action of a second injection. The following 
series demonstrates that this time is about ten days: 

G. P. No. 4464. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 5 days prior, inoculated subcutaneously with 0.19 c. c. toxine No. 
7+1 unit horse serum (Standard B27).] 

G. P. No. 4485. Six c. c. same serum and injection as g. p. 4484. No svmptoms. 
[Previous treatment: 6 days prior, as g. p. 4484.] 

G. P. No. 4486. Six c. c. same serum and injection as g. p. 4484. No symptoms. 
[Previous treatment: 7 days prior, as g. p. 4484.] 

G. P. No. 4533. Six c. c. same serum and injection as g. p. 4484. No symptoms. 
[Previous treatment: 8 days prior, as g. p. 4484.] 

G. P. No. 4530. Six c. c. same serum and injection as g. p. 4484. Symptoms; recovered. 
[Previous treatment: 10 days prior, as g. p. 4484.] 

G. P. No. 4532. Six c. c. same serum and injection as g. p. 4484. Symptoms; recovered. 
[Previous treatment: 11 days prior, as g. p. 4484.] 

G. P. No. 4488. Six c. c. same serum and injection as g. p. 4484. Dead in 10 minutes. 
[Previous treatment: 12 days prior, as g. p. 4484.] 

G. P. No. 4525. Six c. c. same serum and injection as g. p. 4484. Dead in 45 minutes. 
[Previous treatment: 12 days prior, as g. p. 4484.] 

G. P. No. 4487. Six c. c. same senmi and injection as g. p. 4484. Dead in 60 minutes. 
[Previous treatment: 13 days prior, as g. p. 4484.[ 

G. P. No. 4531. Six c. c. same serum and injection as g. p. 4484. Dead in 45 minutes. 
[Previous treatment: 13 days prior, as g. p. 4484.] 

G. P. No. 4476. Six c. c. same serum and injection as g. p. 4484. Dead in 10 minutes. 
[Previous treatment: 14 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No 7+^^ c. c. antitoxic serum (Natl. EX. 18).] 

G. P. No. 4490. Six c. c. same serum and injection as g. p. 4484. Dead in 46 minutes. 
[Previous treatment: 14 days prior, as g. p. 4484.] 

G. P. No. 4529. Six c. c. same serum and injection as g. p. 4484. Dead in 47 minutes. 
[Previous treatment: 16 days prior, as g. p. 4484.] 

THE GUINEA PIG REMAINS SUSCEPTIBLE A VERY LONG TIME. 

That the guinea pig remains susceptible to the toxic action of horse 
serum a very long time is indicated in the following experiments in 
which 160 days elapsed between the first treatment and the second 

(45) 



46 

injection. How much longer this action may continue will be ascer- 
tained and reported upon at a subsequent time. 

G. P. No. 4507. Six c. c. antitoxic horse serum (Natl. IX. 17) injected into the perito- 
neal cavity. Dead in 20 minutes. 

[Previous treatment: 60 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+1 unit horse serum (Standard B27).] 

G. P. No. 4A. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected into the peritoneal 
cavity. DeiCd in 22 minutes. 

[Previous treatment: 84 days prior, inoculated subcutaneously with 0.293 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

G. P. No. 3827. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected into the perito- 
neal cavity. Dead in 33 minutes. 

[Previous treatment: 160 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7-\-sijf c. c. horse serum (Jordan No. 201).] 



Part VL 
DOSAGE AS A FACTOR* 

MINIMAL AMOUNT OF HORSE SERUM NECESSARY TO RENDER A GUINEA PIG 

SUSCEPTIBLE. 

A large part of our work was done upon guinea pigs which had 
been used to measure the strength of antitoxic serums. As the 
strength of diphtheria antitoxin runs between 250 and 1,000 units 
to the c. c. most of our guinea pigs were therefore rendered sus- 
ceptible by doses of horse serum varying from ^\jf to j-^^^f c. c. It 
now becomes interesting to know how small a quantity of horse 
serum injected into a healthy guinea pig would render it susceptible 
to the toxic action of horse serum. 

G. P. No. 217. Six c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Symptoms; recovered. 

[Previous treatment: 45 days prior, inoculated subcutaneously with j^ c. c. antitoxic 
horse serum (Natl. IX. 19).] 

G. P. No. 219. Do. 

G. P. No. 216. Six c. c. antitoxic horse serum (Natl. IX. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 24 days prior, inoculated subcutaneously with -j^ c. c. antitoxic 
horse serum (Natl. IX. 19).] 

G. P. No. 222. Six c. c. antitoxic horse serum (Natl. IX. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 24 days prior, inoculated subcutaneously with jjhjf c. c. anti- 
toxic horse serum (Natl. IX. 19).] 

G. P. No. 223. Six c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Dead in 130 minutes. 

[Previous treatment: 45 days prior, inoculated subcutaneously with j-^jj c. c. anti- 
toxic horse serum (Natl. IX. 19).] 

G. P. No. 220. Do. Dead in 68 minutes. 

G. P. No. 225. Six c. c. antitoxic horse serum (Natl. IX. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 24 days prior, inoculated subcutaneously with jjfhrs c. c. antitoxic 
horse serum (Natl. IX. 19).] 

G. P. No. 224. Six c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Symptoms; recovered. 

[Previous treatment: 45 days prior, inoculated subcutaneously with 31^^177^ c. c. anti- 
toxic horse serum (Natl. IX. 19).] 

G. P. No. 227. Do. 

(47) 




48 

I. p. So. 228. Six (. c. uititmdc horeo Beniia (Natl. IX- 17) injerted otto (lie peritonei 
ravitj. SympUHns; recovrred. 

Qt: 24 dttvB prior, inocul&led subcutaneously with tsVWe c. c. ac:^. 
le senim (N'stl. IX. 19)-] 



1 (roan) injected into the peritoneal cav 
ilaled subcutaneoualj with rjnfBee t. c- t 



iti (Sail. IX. ir> injected into the periloneij 
ilated suboutftoeously with i3,4„j c. c. toti- 



n (Nail. Vin. 18) injected ii 



G. P. No. 230. Six c. c. nomial horse 9> 
SvmpComs; recorered. 
[PreTiouc rrealment: 45 days prior, li 
, lone horse wmm (Sail. IX- 19).\ 

G. P. No. 228. Do. 

[ O. P. No. 232. Six c. ('. anlitoiic horse b 
I ravtty. No symptoms. 
I (Previous treatment: 24 days prior, in 

toxic horse semm (Natl. IX. 19).| 
G.P. No. 235. Dq. 

G. P. So, 234- Sis c'. c. anli;oKic horse 
cavity. No Hymptom-'i. 

[PrevioUH treatment; 30 days prior, inoculated BubcutaneouBly with y^^^^ c. c anti- 
toxic boree serum (Natl. IX. 19).I 
G. P. No. 233. Do. Syniptoma; recovered. 

It is plain from the above series that guinea pigs may be scna- 
tized with very small amounts of horse serum. Guinea pig No. 233 
received only ■) unisinr of ^ <'■ c at the first injection and this quantity 
was sufficient to render it susceptible to a subsequent injection of 
horse aerum. 

MINIMAL AMOUNT OF I 

From the following series of guinea pigs it will be seen that the 
toxic principle must be present in exceedingly small quantities of 
horse serum. 

0. P. No. 4348. Four c. c. antiloxir horsB serum (Natl. VIII. 17) injected into the perito- 
neal cavity. Dead in 30 minutes. 

[Previoua treatment: 21 days prior, inoculated subcutaneously with 0.139 c. c. toxins 
No. 5-i-ji, antitoxii^ horae serum (PD 08021).] 
G. P. No. 4099. Three c. c antitoxic horse serum (Natl. Vm. 17) injected into the perito- 
neal cavity. Dead in 15 minutes. 

[Previous trctijneiot: 37 days prior, Inoculated subcutaneously with 0.22 c. c. toidiiB 
No. 7-l-Tio c. -. antitoxic horse serum (Alex. 190).] 
G. P. No. 3954. Three c. c. anlitoiic horse serum (Na'.i. VIII. 17) injected into the perilo- 
oeal cavity. Dead in 57 minutes. 

(Previous treatment: 51 days prior, inoculated subriitaneou.^lj with 0.139 c. c. toxme 
No. 5+,\„ V. r. antitoxic horse serum (Hubliert 1).] 
G. P. So. 4364. Throe c. o. antitoxic horae serum (Natl. IX. 19) injected into the perito- 
i Deal cavity. Dead in 45 minutes. 

\ [Previous treatment; 30 days prior, inoculated suiwutaneoualy with 0.22 c. c. toxins 
^ No. 7-l-xi, c. c, antilojde horse serum (PD (m73).[ 

■I. P. No.4SW, Two c. c. antitoxic horse serum (Natl. VIII. 17) injected into thoperito- 
K DhI cavity. Dead in 15 minutes. 

^L [h«viout Ireatmenl: 21 days prior, inoi'ulaled subcutaneously with 0.139 c. u. toson 
MfPa, g+^ c e. Mtitoxic horse serum (PD 08021).] 



49 

G. P. No. 4352. One c. c. antitoxic horse serum (Natl. VHI. 17) injected into the perito- 
neal cavity. Dead in 35 minutes. 

[Previous treatment: 21 days prior, inoculated subcutaneously with 0.139 c. c. toxins 
No. 5+5^J^ c. c. antitoxic horse serum (PD 08021).] 
G. P. No. 4101. One c. c. antitoxic horse serum (Natl. VIII. 17) injected into the perito- 
neal cavity. Symptoms; recovered. 

[Previous treatment: 37 days prior, inoculated subcutaneously with 0.22 c. c. toxins 
No. 7+^^^ c. c. antitoxic horse serum (Alex. 190).] 
G. P. No. 4511. One c. c. antitoxic horse serum (Natl. VIII. 17) injected into the perito- 
nea) cavity. Dead in 35 minutes. 

[Previous treatment: 42 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 
G. P. No. 4512. One c. c. antitoxic horse serum (Natl. VIII. 17) inoculated subcutaneously. 
Dead in 81 minutes. 

[Previous treatment: 42 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 
G. P. No. 4623. One c. c. antitoxic horse serum (Natl. VIII. 17) inoculated subcutaneously. 
Symptoms; recovered. 

[Previous treatment: 28 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+yJ^ c. c. antitoxic horse serum (Alex. 192).] 
G. P. No. 4514. Two-tenths c. c. antitoxic horse serum (Natl. VIII. 17) inoculated subcu- 
taneously. Symptoms; recovered. 

[Previous treatment: 42 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 
G. P. No. 4520. Two-tenths c. c. antitoxic horse serum (Natl. VIII. 17) injected into the 
peritoneal cavity. Dead in 65 minutes. 

[Previous treatment: 42 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 
G. P. No. 130. One-tenth c. c. antitoxic horse serum (Natl. IX. 17) injected into the4)eri- 
toneal cavity. Dead in 55 minutes. 

[Previous treatment: 45 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+yJ]y c. c. antitoxic horse serum (Stearns 1351).] 
G. P. No. 151. One-tenth c. c. antitoxic horse serum (Natl. IX. 19) inoculated subcuta- 
neously. Symptoms; recovered. 

[Previous treatment: 45 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+yi]j^ c. c. antitoxic horse serum (Steams).] 
G. P. No. 136. Do. Symptoms; recovered. 

G. P. No. 144. Do. No symptoms. 

G. P. No. 165. One hundredth c. c. antitoxic horse serum (Natl. IX. 17) inoculated subcu- 
taneously. No symptoms. 

3 hrs. 45 min. later, 0.1 c. e. Do. Symptoms; recovered. 

[Previous treatment: 45 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+yj5 c. c. antitoxic horse serum (Stearns 1351).] 
G. P. No. 148. One-hundredth c. c. antitoxic horse serum (Natl. IX. 17) injected into the 
peritoneal cavity. No symptoms. 

3 hrs. 45 min. later, 0.1 c. c. subcutaneously. Symptoms; recovered. 

It will be seen from the above series that one-tenth of a cubic 
centimeter of horse serum into the peritoneal cavity of a suscepti- 
ble guinea pig is sufficient to cause the death of the animal, whereas 
the same quantity subcutaneously causes symptons. 

28337— No. 29—06 4 




[4 SENDBimiG GUINEA PIGS 



The guinea pigs in the following series were first used as conl 
to show that the injection of large quantities of horse serum int:^^ 
the peritoneal cavity produces no effect. They were subsequent"^, 
injected with horse serum again in large quantities into the pe-*-,'. 
toneal cavity to determine their susceptibility: 
G. P. No. 206. Six c c^. normal liorae serum (roan) injected into the peritoneal cpvjijr. 

Symptoms; I'ecovered. 

[Previous treatmeDt: 14 days prior, <> •:. c. dcnkey sprum InjecMd into the periUmstJ 

I G. P. No. 297. Do. 

G. P. No. 298. Same injection. No Bymptoras. 

[Previous treatment; 14 days prior, 6 e. c. normal horse 
peritoneal eavity.] 
G. P. No. 299. Do. SyraptoraH; recovered. 

G. P. No. 300. S^o injection. Symptoms; recovered. 

[Previous treatment: 22 days prior, 6 c.c.antito:iir horse 
o tlie peritoneal cavity.] 



p.) injected into tb) 



Ti(NBtl, rX. I9)inj«tod 



G- P. No. 301. 

G. P. No. 302. Same injection. Syniptoini 

[Previous treatment: 14 days prior 6 c. c. antitoxic horse st 
into the peritoneal cavity.] 
G. P. No. 303. Do. Symptoma {t) 

O. P. No. 304. Same injection. Dead in 65 minutes. 

[Previous treatment: 14 days prior 6 e 
injected into the peritoneal cavity.] 
0. P. No. 305. Do, Dead in 95 minute 

G. P. No. 306. Same injection. Symptoms; 
[Previous treatment: 14 days prior, 3.6 c. 
injected into the peritoneal cavity.] 
G. P. No. 307. Same injection. Dead in 48 
[Previous treatment: 14 days prior, 2 c. c 
injected into the peritoneal cavity.] 
0. P. No. 308, Same injection. Dead in 43 
[Previous treatment: 14 days prior, 3 c. i 
into the peritoneal cavity.] 
G. P. No. 309., Same injection. Symptoms; 
[Previous treatment: 14 dayaprior,6 c. c 
into the peritoneal cavity.] 
0. P. No. 310. Do. Symptoms; recovered. 

It is plain, therefore, that guinea pigs may be rendered susceptible 
by the injection of lai^e amounta of horse serum into the peritoneal 
cavity. We have seen, in another part of this bulletin, that these 
a may also be rendered susceptible by the injection of large 
^mounts into the subcutaneous tissue. 
' We believe, however, that guinea pigs are rendered more suscep- 
[ble by the injection of the toxine-antitoxin mixture than by the 
lOrae aerum alone. 



n (Natl. V. 14) injeded 



n(NYBH10BGib6on) 



I (N1-BH 13C Gibson) 



ni(Alex.A211)injected 



n (Natl. IX. 17) injected 



Part Vn* 
SENSITIZING SUBSTANCE. 

We already know that guinea pigs may be sensitized with either 

^nnal or antitoxic horse serum. It is of course both interesting 

^i important to know whether the substance contained in horse 

^tvm which renders the guinea pigs sensitive is the same as the sub- 

*^ce which exerts its poisonous action upon the second injection. 

QUINEA PIGS MAY BE SENSmZED WITH PRECIPITATED AND DIALYZED SERUM. 

The following examples selected from our work demonstrate this 
Point: 

Q. P. No. 4972. One c. c. normal horse serum (roan) injected into the peritoneal cavity. 
SymptcMns; recovered. 

[Previous treatment: 22 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+7^7 ^* ^' antitoxic horse serum (NYBH 13C, precipitated and dialyzed in accord- 
ance with Gibson's method).] 

G. P. No. 4973. Do. 

G. P. No. 4955. Six c. c. same serum. Dead in 27 minutes. 

[Previous treatment: 40 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 54-Thr ®' ^' antitoxic horse serum (NYBH lOB).] 
G. P. No. 4957. Same injection. Symptoms; recovered. 

[Previous treatment: 40 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5-\-s^jf c. c. antitoxic horse serum (NYBH lOB).] 

DRYING DOES NOT INJURE THE SENSITIZING SUBSTANCE. 

A number of guinea pigs which were used by us in this work were 
sensitized with the antitoxic serum preserved under special condi- 
tions in this laboratory as the official standard in accordance with the 
law of July 1, ld02. This serum was reduced to dryness under special 
conditions and must be redissolved before using. Compare page 39. 

.The following are selected as experimental data proving that dry- 
ing does not injure the sensitizing substance: 

G. P. No. 6B. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Dead in 27 minutes. 

[Previous treatment: 86 days prior inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B25).] 

Q. P. No. 4488. Same injection. Dead in 10 minutes. 

[Previous treatment: 12 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

(61) 



Before wo learned that the antitoxin Rontained in the blood senim 

of the horse plays no r61o in rendering guinea pigs susceptible to 

subsequent inooulatious of horse aerum we planned and carried out 

I extensive series of experiments to determine just what relation 

I the susceptibility of guinea pigs bears to the antitoxic potency of 

I the horse serum injected. One series of guinea pigs was injected 

I subcutaneously with one unit of an antitoxic horse scrum containing 

[ 2S0 units to the c. c. This series, therefore, received j^u c. c. of horse 

I eerum. The second seritis was treated subcutaneously with 2fiO 

imits— that is, the guinea pigs received 1 c. c. of the horse serum. 

The third series of guinea pigs was treated with 1 ,000 units— that is, 

each guinea pig received 4 c. c. of the antitoxic horse serum, At 

varying intervals following these injections the pigs were given a 

second injection of horse serum in order to determine their sua-' 

ceptibility. 

The following guinea pigs received j^ts c. c. antitoxic horse serum 
I containing 1 unit: 

I 0. F. No. 18. Six c. c. nDtiloiiic- horee serum (Natl. VIII. IT) injticled into the peritoneal 

[Previous trentmeot: 1 day prior, inoculated sub(^ulBueously with 1 unit, viz, j^ c. 
horae BBmm (Natl. EX. 18).] No ^^ymptorns. 
G. P. No. 17. Same bjection aa g. p. 18. No Hymptoma. 
[Previous treatniBnt; 4 days prior, us g. p. 18.) 
I G. P. No. 11. Same iniBctioo as g. p. 18. Dead in 35 minulea. 

[Previous treatmsnt: 17 daya prior, as g. p. 18.) 
^ G. P. No. 16. Same iojeutioo as g. p. 18. Symptoms; recovered. 

[Previous treatment: 18 days prior, as g. p. 18.] 
I G. P. No. 15. Same injection as g. p. 18. Dead in 18 minul«a. 

[Previous treatment: 24 days prior, as g. p. 18.] 
I G. P. No. 19. Same injection as g, p. 18. Symptoms; recovered. 

[Previous treatment: 32 days prior, as g. p, 18.] 
' G. P. No. 12. Six c. r. antitoxic hoise senim (Natl. IX. 17) injected into peritoneal ct 
Dead in 44 minutes. 
[Previous troatment: 47 days prior, as g. p. 18.] 
' G. P. No. 10. Six c. c. antitoxic horao senmi (Natl. EX. 19) injected into tlie pertioneal 
cavity. No symptoms. 
[Previous treatment: 47 days prior, as g. p. 18.] 
■ G. P. No. 13. Six c. c. antitoxic horse seram (Nail. VIII. 18) injected into the peritoneal 
^^^ cavity. Symptoma; recovered. 

^^L [Previous treatment: 68 days prior, as g. p. 18-1 

^^H The following guinea pigs received 1 c. c. antitoxic horse serum 

^^B containing 250 units: 

^^B O. P. No. 29. Six c. c. antitoxic hoise serum (Natl. VIU. 17) injected into the peritonea] 

^^^K cavity. No symptoms. 

^^^^B [Previous treatment : 1 day prior, inocidated subcutaneously with 2t 

^^H horee serum (Natl. IX. 18).] 



leritoneal ctti(^^^| 






r 



13. p. So. 27. Sane BiMtioo ■> g, p^ 2L No nv 
I [Pnrioos tmtEMoi 4 <U_r« prior, ■« ;. p. 29ll 

Q-P. Nai.2K. Suw iii}KtK>D ks g. p. 29. SrmptaiM^ n>~oT«(«d. 
I {Rw iu » tmtnwQt 1 Jirx prwr *s f ■ P- 3£*-l 

t Q- P. No. as. Same mjeriKMi K iMi. 29. SjTupuwi*: iwtotmwI, 
1 [Fmtmim tRMmni!: ISilaT^ prior ^« g. p. 29.) 

I 0. F. No.^ Suneiiijfniooksg p. ^. Srmploms; rMOTMwl, 
[PnriiMB treat inrai IMdarsprtor.^g. p.29.) 
G. P. No. 23. Same injwtjoo aa g. p. 29. Symptoms: rMOvnvd. 

[Prariaos tieatniHil; 32 davs prior, as g. p. 29.| 
O. P. No. 24. Six c. c. aniiioxir: botse anum iKatl. IX. 191 iDJwinl bto iba prriloiMd 
caritj. Dead in 45 mimitea. 
[Previous trealmcDl : 47 davs prior, as g, p. 29.] 
O- P. N«- 22. Six c- r. antitoxic horse senim (Xatl. IX. 17) mjectMl bto (he prritoiwal 
Ckvitj. SynqNoms; recorered. 
[PrerioDS trealmpnt i7 days prior, as g. p. 29.] 
C- P. No. 21. Six < . r- entlioiii- horae serum (Natl. \1II. 18) injertrd into tho peritoneal 
CaTTty. Dead in 44 niiuiite^. 

[Ptevious treatment: 67 days prior, as g. p. 29.) 
^- P. No. 20. Foot c. c. DMioal borae senuu (man) iojerled into tb« perilo&Ml ravity. 
Symptoms; recovered. 

[Previous treatment: 83 days prior, aa g. p. 29.] 

The following guinea pigs received 4 c. c. of antitoxic horse serum 
^^ntaining 1,000 units: 

Q. P. No. 38. Six c. c. antitoxic borse serum (Natl. VIII. 17) injected into (he pt-ritowal 
cavity. No qtnptoms. 
[Previous treatment: I day prior, inoculated aubcutaneously with 1,000 uoits, via, 
4 c. c, antitoiic Lorse senijii (Natl. IX. 18).| 
Q. P. No. 35. Same injection as g. p. 33. No symptoms. 

[Previous treatment 4 days prior, as g. p. 38.) 
G. P. No. 36. Same injection as g. p. 38. Symptonu: recovered. 

[Previous trettlraeni 17 days prior jls g. p. SS.J 
O. P. No. 32. Same injection as g. p. 38. Symptoms; remvered. 

[Previous treatmfnl. 18 dara prior a.>ig. p. 38.) 
G. P. No. 34. Same injection aa g. p. f 

[Previous treutinent 24 days prior lis g, p. 38.) 
G. P. No. 37. Same injection as g. p. 38. No symplovw 

[Previous treatment: 32 days prior, as g. p. 38.] 
G. P. No. 39. Six c. c. antitoxic horse st 
cavity. No symptoms. 
[Previous treatment: 47 days prior, as g. p. 38.) 
0. P. No. 31. Six c. c. antitoxic horae serum (Natl. IX. 19) injerleil i[ 
cavity. Symptoms; recovered. 

[Previous treatment: 47 days prior, as g. p. 38.) 
Q. P. No. 33. Six .■ ,■. aatkoxi.' hoRe serum (Natl. VIII. 18) ini«-l.«l ii 
cavity. Dead in !<i niiiiiili.'!^. 
[Previous treatment: 68 days prior, as g. p. 38.] 
0. P. No. 30. Four c. c. normal horse serum (roan) injected into tlie ]H>riliiiu-Bt nivity. 
^n^toma; recovered. 

[Previous treatment: 83 days prior, as g. p. 38.] 



; recovered. 



a (Natl. IX. 17) injerlix) ii 



L) Llin jwritonMl 
[> tho peritoneal 
i> (lin [wriliilKial 



We see from the above series that it required 17 days (<»■ lew) 
for the guinea pigs to become susceptible. 

We leani from these three series that guinea pigs receiving 4 c. c. 
of antitoxic horse serum are apparently not quite so susceptible as 
those receiving the smaller amounts (jIj c. c. and 1 c. c); also, 
that those which received 1 c, c. are apparently not quite bo sns- 
eeptible as those which received only gij c, c. of the horse serum. 
From our work we know that it is the quantity of horse serum, and 
not the number of units contained in the serum, which plays the 
important rdle. If this relation of the amount of serum is true, we 
presume that animals receiving large quantities of horse serum for 
the first injection are less susceptible because they are rendered 
slightly immune, just as animals may be immunized against this 
action by daily repeated inoculations. When large quantities are 
given at the first injection, the horse serum is probably absorbed 
very slowly and acts somewhat in the same manner. 

It would also seem from the above that guinea pigs are not ren- 
dered quite as susceptible by first injections of horse serum as by 
injections of the toxine-anti toxin mixture. 

THE SENSITIZING SUBSTANCE IS NOT FREE IN THE BLOOD SEBUM. 

The following work seems to indicate that the substance whicli 
renders the guinea pigs susceptible to a second injection of horse 
aerum is not free in the blood serum of the guinea pig in the same 
sense that the free receptors are in diphtheria antitoxic aerum: 
G. P. No. 298 (Dormal). Eight e. c. of & mixture cootainiiig 4 c. e. serum uf susceptible 
guinea pigs aod 4 c. c nonnal home EFnini, injec^ted int« the peritoneul cavity; the 
aeniras were mixed and iiijepted at once. No symptoma. 
G. P. No. 299. Ten c. c. above mixture injected into the peritoneal cavity. No Bymptoma. 
O. P. No. 297. Plve c. c. above mixture injected into the peritoneal cavity. No symptoms. 
From the fact that the sensitizing substance does not appear to be 
- free in tho blood of the guinea pig and from the further fact thatwe were 
unable to transfer the immunity we might infer that this roat^tion may 
not reside so much in the blood serum as in the cells of the body.^^BJ 



THE EFFECT OF HEAT U 



SUBSTANCE. 



The following experiments plainly prove that the sensitizir^ sub- 
stance is very resistant to heat, for guinea piga previously treated 
with small quantities of horse scrum heated to 60° C. for six hours 
subsequently proved highly susceptible: 

: c. c. normal hotse serum (roan) injected 



G. P. No. 265 

Dead in 38 1 
[Previous 

^O. P, No. 266. 
?. No. 267. 



17 days prior, inoculated subuutaneously with 
i) heated to 60° C. for 6 houra.j 
Do, Dead in 20 minutes. 
Do. Dead in 25 loinutea. 



the peritoneal cavity. 
normal 




It may be that man can not be sensitized in the same way that 
We have shown is the case with guinea pigs. We made no human 
experiments, but have experimental data done by others which has 
a direct bearing on this question. 

Pirquet and Schick injected children with antitoxic horse serum I 
at intervals. 

It has been customary to immunize numbers of children when I 
exposed to diphtheria with antitoxic horse serum at intervals of from | 
three to four weeks. 

We have no doubt that there are many such instances on record 1 
and, so far as we know, this practice has never caused death. 

Repeated injections of horse serum into man is not an infrequent 
occurrence. Patients suffering with diphtheria are often given | 
injections of antitoxic serum at short and frequent intervals. It ii 
also not rare for persons to have several attacks of diphtheria at long | 
intervals and to be treated each time with antidiphtheric serum. 

Certain serums, for example, the antitubercle serum of Maragliani 
or the antirheumatic scrum of Menzer are habitually used by giving \ 
injections at intervals of days or weeks. 

In all of these cases of frequent and repeated injections the amount j 
which has been injected and the interval between the injections must 
be taken into account in relation to this work. Pirquet and Schick 
in their work on Serumkrankeit give the following instances in which 
children received two injections of horse serum at intervals of from 
sixteen to forty-two days between the first and second injections. 

L*op)W H.—Oetobec 3, 1902. 100 c. c. Bcarlet-fever serum (Moser). Eight days fol- i 
lowing this injection ajmptoma a! tbc serum diaeaae apjwurod nnd kstcd severei wi^eks. 

December 2, 1902. Fifty days after the first injection patient rerelTed 2 c. c. anti- 
diphtheric serum under skin o( arm. 

In fifteen minutes following this second injection stormy symptoms aet in. The boy 
began to cry and complained of nausea. Ederaa of the lip set in and soon ^read over 
the wbole Face. In several hours general urticaria. 
(55) 



I 
I 




66 

BeinriA K.—Thi^ jeara old. June 13, 1902. li 
eighth day following the injection, symptoms oF the 
unta tbe twenty-sixth day, 

July 7. Thirty-four days following the firat injection, 
injected into the left a-rai. The same afternoon urticaria ai 
next morning tbe arm v/aa highly edematous. 

Alexandrine K. Nine years old. May 28, 1902. 180 c. c. normal horse serum. Eleven 
days after the injection severe symptoms appeared, which lasted until June 17. 

Sixteen days following the first injection, given 1 c. c. diphtheric antitoxic scrum in iho 
right forearm. Next morning, the hand swollen, very painful, and smartly to such an 
extent that the infection was considered of an erysipelatous nature. All redDeas and 
swelling disappeared in a few days. 

Elkabelh K. Six and one-half years old. May 3, 1902. Given 180 c. c. scarlet^tever 
serum. May 15, t«n days following, sudden rise of temperature, 39.4° C. Swelling of the 
lymph nodes. No exanthem. 

Nineteen days after the tirst injection 50 c, c. scarlet-fever serum, following which occurred 
severe and painful edema of the skin of the abdomen, which spread \a the labia and thighs, 
and disappeared in about a week. 

Franz Z. June ft, 1903. 1 c. c. aDtidiphtberic serum. June IS had urticaria, about 
nine days after the injection. August 2, eight weeks after first injection again given 5 c. c. 
antidipbtberic serum. Shortly following, urticaria and swelling of the face, 

Frieda Z. June 10, 1903. Ice. antidiphtheric serum. June 26, sixteen days following, 
the same bjeetion repeated and acted in all respecta similar to tbe above case, Franz Z., 
her brother. On August 2 she was sgain given 5 c. c. antidiphtheric serum. One hour 
later her face became red and swollen. The lids became so edematous that she could nol 
open her right eye. In two hours there was general urticaria. Temperature, 38.6" C, 
Moderate swelling, locally. 

EUiM. Four monlha old. First mjection June 20, 1904. 12 c, c. entidipbtberic 
serum. Ten days later sudden rise of temperature 39° C. and urticaria. The fever lasted 
two days. Eruption later became measle-like and remained several days. 

Nineteen days after the iirst injection 5 c. c. antidiphtheric serum. One hour after this 
severe general urticaria. From the site of injection, swelling of the ^in of the abdomen. 
Id the afternoon the temperature rose to 39.5" C. ; frequent vomiting. 

Leopoldi-ae E. December !, 1903. 5 c. c. horse serum. Twenty-seven daja following 
the drat injection, patient received 1 c. c. antidiphtheric serum. One hour following this 
la^ injection, definite sweQing of the right hand, which later spread to the entire arm.. 

All these eight cases show this in common, that after the first 
injection of horse serum, the symptoms of the serum disease appear 
after the normal period of iaeubation, namely, between the eighth 
to thirteenth day. But when the same individuals are again injected 
with horse serum after intervals of sixteen to forty-two days, there 
reappears at once, or at least within twenty-four hours, symptoms 
of the serum disease. 

Von Pirquet and Schick further give a list of 60 children who 
were injected with antitoxic horse serum at intervals from six days 
to seven and a half years between the first and second injections. 
They found that when the second injection was given from fourteen 
days to four months after the first injection thej' obtained, with 
great regularity, what they termed "the immediate reaction," but 




^^en the interval between the first and second injection is over 

'^Xir months they obtained little or no immediate reaction, but what 
*^ey termed "an accelerated reaction," for the fever, urticaria, and 
'^t^er symptoms of the serum disease appeared on the fifth, sixth, 
Seventh, or eighth day. It will be remembered that the normal 
period of incubation for the symptoms of the serum disease to appear 
after the first injection is between the eighth and thirteenth da_v. 
Von Pirquet and Schick lay special stress upon the phenomena of 
the "immediate" and "accelerated" reactions following the second 
injection. 

We might also conclude despite the suggestion contained in Part 
X of our work upon sensitizing guinea piga by feeding them with 
horse serum or horse meat that children may not be sensitized to 
the toxic action in horse serum by eating horse meat from the fact 
that horse meat is a favorite article of diet in certain European 
countries, and there is nothing on record to show that the injection 
of horse serum in those countries is fraught with more danger than | 
where this practice does not obtain. We must, however, remember 
that our work has shown that guinea pigs are sensitized with exceed- 
ingly minute quantities of the strange proleid, and that repeated 
injections cause an inmiunity; and it is possible that the same 
action may be true of feeding. 

Man reacts to the first injection of horse serum after a period of I 
incubation of eight to thirteen days. Guinea piga show practically 
no reaction following the first injection. Both react to a second 
, injection. The reactions in man and the guinea pig, however, differ 
both in severity and kind. The relation, therefore, that our o 
vfttions upon the guinea pig may have in its application to man 
must await further study. Of course, the fact that other animals 
besides man and guinea pigs react to a second injection of horse 
serum would seem to indicate that we are dealing with one and the 
same action. 

We have tested monkeys, rabbits, mice, dogs, cats, rata chickens, 
and pigeons to determine whether any of these animals may be sen- 
sitized to the action of horse serum. Thus far we have obtained a 
response in the dogs, rabbits, and cats. This work is still in progress 
and will be reported at a future date. 

Von Puquet and Schick also found that the first injection into J 
rabbits caused no cUnical effect, but that subsequent subcutaneous I 
injections caused immediate reaction in the production of local edema , 
which extended even to gangrene. Second injections, when intro- 
duced intravenously, produced symptoms of collapse and uvea 
death. 



58 

Arthus also found that the injection of horse serum into rabbits 
caused no symptoms, whether the horse serum was injected subcu- 
taneously or intravenously, but when he injected the serum ev.ery 
six days subcutaneously he obtained, after the fourth injection, 
edema and local reaction which continued to gangrene. 

A rabbit which von Pirquet and Schick had previously treated 
with 8 injections was then given 2 c. c. of horse serum into the ear 
vein. The rabbit reacted to this in a minute and, after characteristic 
symptoms similar to those we see in guinea pigs, died in about four 
minutes. 

Arthus obtained similar results with repeated injections of steril- 
ized milk. 



Part DC 
IHBilUNITY. 

ACTIVE IMMUNITY. 

GUINEA PIGS MAY BE IMMUNIZED AGAINST THE TOXIC SUBSTANCE IN 

HORSE SERUM. 

Guinea pigs may be immunized against this toxic substance, fol- 
lowing the lines of active immunity produced by repeated inocula- 
tions of bacterial toxins. For instance, a guinea pig that has received 
an injection daily for ten days of small quantities (1 c. c.) of horse 
Serum develops a high degree of immunity to subsequent injections 
of horse serum. Guinea pigs that have received more than ten inoc- 
ulations on ten successive days develop a still higher degree of 
immunity. 

G. P. No. 109. Six c. c. antitoxic horse serum (Natl. IX. 19) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 15 to 27 days prior, 10 subcutaneous inoculations, 1 c. c. each, 
of antitoxic horse serum (Natl. IX. 19).] 

G. P. No. 111. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 36 to 48 days prior; 10 subcutaneous inoculations, 1 c. c. each, 
normal horse serum (Sam).] 

G. P. No. 107. Six c. c. antitoxic horse serum (Natl. IX. 19) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 15 to 27 days prior, 10 subcutaneous inoculations, 1 c. c. each, 
normal horse serum (Sam).] 

G. P. No. 106. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 36 to 48 days prior, 10 subcutaneous inoculations, 1 c. c. each, 
normal horse serum (Sam).] 

G. P. No. 108. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Prior treatment: 37 to 50 days prior, 10 subcutaneous inoculations, 1 c. c. each, 
normal horse serum (Sam).] 

6 days later, 1 c. c. normal horse serum (roan) inoculated subcutaneously on each of 12 
successive days. No symptoms. 

6 days later, 6 c. c. normal horse serum (roan) into peritoneal cavity. No symptoms. 

G. P. No. 110. Six c. c. antitoxic horse serum (Natl. VIII. 18) injected intothe peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 37 to 50 days prior, 10 subcutaneous inoculations, 1 c. c. each, 
antitoxic horse serum (Natl. IX. 19).] 

6 days later, 1 c. c. normal horse serum (roan) inoculated subcutaneously on each of 12 
successive days. No symptoms. 

6 days later, 6 c. c. normal horse serum (roan) into peritoneal cavity. No symptoms. 

(59) 



60 

We noticed that the subcutaneous injection of horae serum in"^:.c 
guinea pigs sometimes occasions a local reaction, as indicated fczsy 
edema at the site of inoculation, but the above guinea pigs Noa. 1 ^J8 
and 110, which received alarge number of injections, seemed to re«.<tt 
less each time. 

We give below a aeries of 1 1 guinea pigs injected with large quan- 
tities of horse serum at irregular intervals. It is plain from these 
experiments that a rather high degree of immunity may be induced 
by repeated injections. 
O. P. No. 4101. One c. c. anlitoxic horse serum (Natl. VIII. 17) injected bto the pi 

ne»l c»vity. Sjmptoms; recovered. 

[Previous treatment; S7 daja prior, inoculated sulicutanfioo.'ily with 0.22 o. c 

No. 7+ik c. c. antitoxic horse Benim <Alejt. 190).] 

8 days later, 1 c. e. antitoxic horae serum (Natl. VIII. 17) into peritoneal cavity. Symp- 

tonw, les9 severe. 

16 days later, 6 c. t. into peritoneal tavity. No symptoms. 
6 days later, 6 c. r. into peritoneal cavity. Symptoms. 

9 days later, 6 c. c into peritoneal cavity. No symptoms. 
34 days later, 6 c. c. intn peritoneal cavity. Symptoms (]). 
20 days later, 6 c. c. antitoxic horse serum (Nat!. VIII. IS) into peritoneal cavity. 



* 



O. P. No. 4530. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the pentoneal 
cavity. Symptoms; recovered. 

[Previous treatment; 10 days prior, inoculated subrutaneoualy with 0.19 c. e. toxine 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

15 days later, B c. c. antitoxic horse serum (Natl. VIII. 17) into peritoneal CKTity. 
Symptoms. 

2 days later, B c. c. into peritoneal cavity. RestlesB. 

6 days later, 6 c. c, into peritoneal cavity. Symptoms (1). 

9 days later, 6 c. c. into peritoneal cavity. Symptoms (t). 

33 days later, A c. e. antitoxic horse serum (Natl. VIII. IS) into peritoneal cavity. No 
symptoms. 

21 days later, 6 c. e. into peritoneal cavity. No symptoms, 

G. P. No. 4532. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

(Previous treatment: 11 days prior, inoculated subcutaneously with O.IS c. c. loxine. 
No. 7+1 unit antitoxic horse serum (Standard B27).] 

14 days later, 6 c. c. antitoxic horse aermn (Natl. VIII. 17) into peritoneal cavity. No 
symptoms. 

S days later, 6 c c. into peritoneal cavity. Symptoms (1). 
9 days later, 6 c. c. into peritoneal cavity. No symptoms. 

34 days Uter, 6 c. c. antitoxic horse serum (Natl. VIII. 18) into peritoneal cavity. 
Symptoms; recovered. 

20 days later, 6 c. c. into peritoneal cavity. Symptoms; recovered. 
G. P. No. 4432. Six c. c. antitoxic horse serum (Natl, VIII. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 7 days prior, inoculated subcutaneously with 0.002 c. c. toxine 
No. 7.] 

22 days later, fl c. u, antitoxic horse serum (Natl. VIII. 17) into peritoneal cavity. No 
aymptoma. 

t£ dkya later, 6 o, c. into peritonaal cavity. Symptoma. 



33 days later, 6 c. c. antitoxic horse serum (Natl. VIII. 18) into peritoneal cavity. No 
symptoms. 

21 days later, 6 c. c. into peritoneal cavity. No symptoms. 

^- P. No. 4426. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 8 days prior, inoculated subcutaneously with 0.002 c. c. tcntine 
No. 7.] 

27 days later, 6 c. c. antitoxic horse serum (Natl. VIII. 17) into peritoneal cavity. 
Symptoms (?). 

9 days later, 6 c. c. into peritoneal cavity. No symptoms. 

34 days later, 6 c. c. into peritoneal cavity. Symptoms. 

20 days later, 6 c. c. antitoxic horse serum (Natl. VIII. 18) in peritoneal cavity. No 
symptoms. 

G. P. No. 4593 Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 3 days prior, inoculated subcutaneously with 0.001 c. c. toxine 
No. 7.] 

18 days later, 6 c. c. antitoxic horse serum (Natl. VIII. 17) into peritoneal cavity. 
Symptoms. 

8 days later, 6 c. c. into peritoneal cavity. Symptoms. 

9 days later, 6 c. c. into peritoneal cavity. No symptoms. 
34 daysiater, 6 c. c. into peritoneal cavity. Symptoms. 

20 days later, 6 c. c. antitoxic horse serum (Natl. VIII. 18) into peritoneal cavity. No 
symptoms. 

G. P. No. 4377. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms; recovered. 

[Previous treatment: 21 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7+y^ c. c. antitoxic horse serum (Natl. V. 7).] 

Same day, 6 c. c. antitoxic horse serum (Natl. VIII. 17) into peritoneal cavity. Symp- 
toms, less severe. 

10 days later, 6 c. c. into peritoneal cavity. No symptoms. 

2 days later, 6 c. c. into peritoneal eavity. No symptoms. 
15 days later, 6 c. c. into peritoneal cavity. No symptoms. 
34 days later, 6 c. c. into peritoneal cavity. No symptoms. 

20 days later, 6 c. c. antitoxic horse serum (Natl. VIII. 18) into peritoneal cavity. No 
symptoms. 

G. P. No. 4. Six c. e. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. Symptoms, recovered. 

[Previous treatment: 27 days prior, inoculated subcutaneously with 6 c. c. antitoxic 
horse serum (Alex. 189).] 

3 days later, 6 cc. antitoxic horse serum (Natl. VIII. 17) into peritoneal cavity. No 
symptoms. 

19 days later, 6 c. c. into peritoneal cavity. Symptoms. 

6 days later, 6 c. c. into peritoneal cavity. No symptoms. 

9 days later, 6 c. c. into peritoneal cavity. No symptoms. 

G. P. No. 4534. Six c. c. antitoxic horse serum (Natl. VIII. 17) injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 4 days prior, inoculated subcutaneously with 0.002 c. c. toxine 
No. 7.] 

10 days later, 6 c. c. antitoxic horse serum (Natl. VIII. 17) into peritoneal cavity. No 
symptoms. 

18 days later, 6 c. c. into peritoneal cavity. Symptoms (?). 
9 days later, 6 c. c. into peritoneal cavity. No symptoms. 




horse Benrni (Nsti. VIII. 18) into peritoneal 

nto peritoneal (»vity. No aympton 
in) injected in 
ByinptomH. 

[Previoua treatment: Hon?.] 

3 days later, 6 o. c. antitoxic- horse serum (Natl, VIII. 17) ir 
■ymptoms. 

19 days later, 6 c. c. into perit<ine»l ravity. Symptoma. 
6 days later, 6 r. c, into peritoneal cavity. No symptoms. 
43 days later, G c. e. antitoxiu horse semm (Natl. VIII. 18) into peritoneal Cl 

20 days later, (i c. t. into peritoneal cavity. No symptoms. 
G. P. No. 3. Ten u. c. normal horse senira (roan) injected into the peritone 

Bymptoms. 

[Previous treatment: None.] 

2 days later, 6 c. c. same serum into ptiritoneal c^avity. No aymptomn. 

3 days later, 6 c. e. antitoxic horse serum (Hatl. VIII. 17) into peritoneal cavity, 
symptoms. 

9 days later, fi t. c. into peritoneal cavity. No symptoms. 

10 days later, 9 c. c, into peritoneal cavity. No ayraptomi 




I 
I 



As a normal untreated guinea pig is entirely insusceptible to the 
toxic substances found in horse serum, it naturally occurred to us to 
try to ascertain whether the blood aerum or the juices from the 
various organs of such a normal guinea pig had a restraining, neutral- 
izing, or immuniziog action when mixed with horse serum and the 
mixture inoculated into susceptible guinea pigs. 

Normal O. P. A large, full-grown female guinea pig was chloroformed, then exsanguinated. 
Various orgaoa, mentioned liclon, were mjnced and ground separately b a mort«r with 
10 c. c. of antitoxic horee wrum (National VIII. 17). The nvixturea were allowed to 
'stand at room temperature 3 hours, then strained through cheese cloth, and injected into 
the peritoneal cavity of auaceptible guinea pigs, as followa : 
Q. P. No. 4365. Six c. c. of a mixture containing equal parts of antitoxic horse serum and 
normal guinea-pig serum injected into the peritoneal cavity. Death in 40 miniltea. 

[Previous treatment: 31 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 74-iii 0. c. antitoxic serum {PD&Co, No. 07173).] 
G. P. No. 4463. Six c. c. of a mixture containing equal parts of antitoxic horse serum 
(Natl. VIII. 17) and normal guinea-pig liver injected into the peritoneal cavity. Symp- 
toms, recovered. 

[Previous treatment: 26 days prior, inoculated aubcutaneously with 0.22 c. c. toxbis 
No. 7+ihi "■ c. antitoxic aerum (Natl. Vin. 17).] 
G. P. No. 4393. Seven c. c. of a mixture containing equal parts of antitoxic horse sanun 
(Natl. VIII, 17) and normal guinea-pig liver injected into the peritoneal cavity. Symp- 
toms, recovered. 

[Previous treatment: 28 days prior, inoculated aubtutaneoualj with 0.2 c, c. toxins 
No. se-l-1 unit antitoxic serum (Standard 827).] 





>F*Na 4470. Six c. c. ol a mixture containing equal parts of antitoxic hone serum 

(RitL VnL 17) and normal guinea-pig spleen injected into the peritoneal cAvity. No 

^Diptoms. For occasional irregularities see below, 
^^f fPimom treatment: 26 days prior, inoculated subcutaneously with 0.22 c. c. toxine 

^ ^'f i^ c. c. antitoxic serum (Natl. IX. 17).] 

6 cc antitoxic horse serum (Natl. VIII. 17) injected into peritoneal cavity of same pig 
' ^ fl one day later. Symptoms, recovered. 

[H P. No. 4475. Six c. c. of a mixture containing equal parts of antitoxic horse serum 
(Natl. Vm. 17) and normal guinea-pig ovary injected into the peritoneal cavity. Death 
in 45 minutes. 
^^' [Prerious treatment: 26 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
^o-TfyJiy c. c. antitoxic serum (Natl. IX. 18).] 

^. p. ^0^ 4466. Six c. c. of mixture containing equal parts of antitoxic horse serum 
*'| (^fttl. vm. 17) and normal guinea-pig kidney injected into the peritoneal cavity, 
^th in 45 minutes.' 

[Previous treatment: 26 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
^0. 7+yJ^ c. c. antitoxic serum (Natl. VIII. 17).] ' 

I "• r. No. 4378. Six c. c. of a mixture containing equal parts of antitoxic horse serum 
(^atl. vm. 17) and normal guinea-pig suprarenal injected into the peritoneal cavity. 
*^th in 10 minutes. 

[Previous treatment: 31 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
^o. T-friry c. c. antitoxic serum (Natl. V. 10).] 

* P. No. 4464. Six c. c. of mixture containing equal parts of antitoxic horse serum 
(Natl. vm. 17) and normal guinea-pig thyroid injected into the peritoneal cavity. 
Symptoms, recovered. 

[Previous treatment: 26 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No- 7+iiTy c. c. antitoxic serum (Natl. VIII. 17).] 

Q. P. No. 4469. Six c. c. of a mixture containing equal parts of antitoxic horse serum 
(Natl. vm. 17) and normal guinea-pig brain injected into the peritoneal cavity. Symp- 
toms, recovered. 

[Previous treatment: 26 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
No. 7-1-7^7 c. c. antitoxic serum (Natl. IX. 17).] 

We may conclude from the above that neither the blood serum nor 
the organs of a normal guinea pig have any neutralizing action upon 
the toxicity of horse serum when injected into a sensitized guinea pig. 

Guinea pig 4470, which received an injection of horse serum plus 
the spleen pulp of a normal guinea pig, showed no symptoms. We 
therefore repeated this experiment as follows : 

G. P. No. 4493. Six c. c. of a mixture containing equal parts of antitoxic horse serum (Natl. 
IX. 19) and normal guinea-pig spleen injected into the peritoneal cavity. Symptoms, 
recovered. 

[Previous treatment: 27 days prior, inoculated subcutaneously with 0.19 c. c. toxine 
No. 7 H- 1 unit horse serum (Standard B27).] 

We believe that occasional irregularities like this which have excep- 
tionally occurred in this work may be accounted for by the fact that 
sometimes when the stomach and intestines are full and distended the 
inoculating needle enters the intestinal tract instead of the peri- 
toneal cavity. While we suspected this possibility, it was proved to 
our satisfaction one day; after injecting a guinea pig, the intestinal 



64 

contents flowed back into the barrel of the syringe, owing to accidentaC^ 
withdrawal of the piston. This particular guinea pig showed ncz» 
symptoms as a result of the injection; whereas we knew it to be sus — 
ceptible, because a subsequent operation, done a few hours later — 
caused characteristic symptoms. 

For intraperitoneal inoculations the needle should be short anc^i 
blunt to avoid this accident and the injection should be given ove^B 
the region where the coils of small intestine he close to the abdomina-^ 1 
walls. 

NEUTRALIZING EFFECT OF THE BLOOD AND ORGANS OF AN IMMUNIZED GUINEtf<^ 

PIG UPON THE TOXICmr OF HORSE SERUM. 



We found that repeated injections of horse serum into a ^^uinea pi 
produces a certain degree of immunity to the subsequent action o^ 
horse serum, and it naturally occurred to us to try whether such 
guinea pigs contained in their body juices an immunizing substance 
in sufficient quantity to neutralize the poisonous substance found i^ 
horse serum. 

G. P. No. 109 (immunized). This guinea pig had received 10 subcutaneous inoculations of 
1 c. c. each of antitoxic horse serum (Natl. EX. 19) on 10 successive days from Feb. 8 to 20; 
15 days later, viz, March 7, it was inoculated with 6 c. c. antitoxic horse serum (Natl. IS- 
19) into the peritoneal cavity. No symptoms. 

Twenty-one days later, viz, March 28, it was again tested by injecting 6 c. c. of antitoxic 
horse serum (Natl. VIII. 18) into the peritoneal cavity. No symptoms. 

As this guinea pig showed a high degree of resistance to the toxic action of horse serum 
it was killed two days later, March 30, with chloroform. About 15 c. c. of the blood was 
taken from the heart and defibrinated, the various organs were removed and placed in 
separate mortars and ground up with 10 c. c. of antitoxic horse serum (Natl. VIII. 18). 
This mixture of horse blood with organ juices of the guinea pig was allowed to stand about 
3 hours at room temperature and strained through cheese cloth. It was then inoculated 
into the guinea pig. 

G. P. No. 166. Six c. c. antitoxic horse serum (Natl. VIII. 18) mixed with suprarenal of 
g. p. 109 injected into the peritoneal cavity. Dead in 9 minutes. 

[Previous treatment: 49 days prior, inoculated with 0.22 c. c. toxine No. 7-|-yJ^ c. c 
horse serum (Steams No. 1351).] 

G. P. No. 158. Six c. c. same serum as g. p . 166 mixed with whole (defibrinated) liood^ of 
g. p. 109 injected into peritoneal cavity. Dead in 15 minutes. 
[Previous treatment: As g. p. 166.] 

G. P. No. 126. Six c. c. same serum as g. p. 166 mixed with thyroid of g. p. 109 injected into 
peritoneal cavity. Dead in 16 minutes. 
[Previous treatment: As g. p. 166.] 

G. P. No. 132. Six c. c. same serum as g. p. 166 mixed with kidney of g. p. 109 injected into 
peritoneal cavity. Symptoms, recovered. 
[Previous treatment: As g. p. 166.] 

G. P. No. 164. Six c. c. same serum as g. p. 166 mixed with spleen of g. p. 109 injected into 
peritoneal cavity. Symptoms, recovered. 
[Previous treatment: As g. p. 166.] 



^* No. 129. Six c. c. same serum as g. p. 106 mixed with liver of g. p. 100 injected into 
^loHoiieal cavity. No symptoms. 

tPnrious treatment: As g. p. 166.] 

^ Iff. 45 min. later. To test susceptihUity. 6 c. c. same serum as g. p. 166 injected into 
l^^'Hoiieal cavity. Dead in 30 minutes. 

^* No. 147. Six c. c. same serum as g. p. 166 mixed with brain of g. p. 100 injected into 
^•HtoDeal cavity. No symptoms. 

(IWious treatment: As g. p. 166.] 

1 hr. 20 min. later. To test susceptibility. 6 c. r. same senim as g. p. 166 injeote<l into 
PoiUnieal cavity. Dead in 35 minutes. 

^* I*. No. 139 (control). Six c. c. same serum as g. p. 166 injected alone into peritoneal 
^vity. Dead in 15 minutes. 
[Previous treatment: As g. p. 166.] 

[ Owing to the fact that guinea pigs 129 and 147 Hhowod no «yinp- 
^ »oins, the experiment was repeated, using organs from a similarly 
^l^ated pig as follows : 

^' P. No. 108 (immunized). This guinea pig had received 10 Hul)cutanooUN imnmlat ions of 
1 c. c. each of normal horse serum (Sam) on 10 successive days from Feb. 8 to 20; 37 days 
later, viz, March 29, it was inoculated with 6 c. c. antitoxic horse serum (Natl. VI 11. 18) 
into the peritoneal cavity. Symptoms, recovered. 

Beginning six days later, viz, April 4, and continuing 12 days, it was given a daily sul>- 
cutaneous inoculation of 1 c. c. normal horse serum (roan). No symptoms. 

Six days after the last inoculation the pig was given 6 c. c. normal horse serum (roan) 
into the peritoneal cavity. No symptoms. 

This guinea pig, having showed a strong resistance to the toxic 
action of horse serum, was killed with chloroform and the brain, part 
of the spinal cord, and liver were minced and ground separately in a 
mortar with 20 c. c. antitoxic horse serum (Natl. VIII. 18) . The mix- 
ture was allowed to stand about 3 hours at room temperature and 
strained through cheese cloth and then inoculated. 

G. P. No. 4998. Six c. c. antitoxic horse serum (Natl. VIII. 18) mixed with liifer of g. p. 
108 injected into the peritoneal cavity. Dead in 95 minutes. 

[Previous treatment: 39 days prior, inoculated subcutaneously with 0.22 c. c. toxino 
No. 7 + jhs c. c. antitoxic horse serum (Wellcome 474A).] 

P. No. 5044. Six c. c. antitoxic horse serum (Natl. VIII. 18) mixed with brain and 
portions of the spinal cord of g. p. 108 injected into the peritoneal cavity. Dead in 86 
minutes. 

[Previous treatment: 33 days prior, inoculated subcutaneously with 0.139 c. c. toxino 
No. 5 -j- -gi-ff c. c. antitoxic horse serum (NYBH 13C).] 

G. P. No. 252. Six c. c. antitoxic horse serum (Natl. VIII. 18) mixed with brain and por- 
tion of spinal cord of g. p. 108 injected into the peritoneal cavity. Symptoms, recovered. 
[Previous treatment: 53 days prior, inoculated subcutaneously with ji^ c. c. normal 
horse serum (roan) heated to 60° C. for 30 min.] 

G. P. No. 251. Six c. c. antitoxic horse serum (Natl. VIII. 18) mixed with liver of g. p. 108 
injected into the peritoneal cavity. Symptoms; recovered. 

[Previous treatment: 53 days prior, inoculated subcutaneously with ^iv c. c. normal 
horse serum (roan) heated to 60° C. for 30 min.] 

28337— No. 29—06 6 



66 

As a further proof that the blood senim of an immunized guin.^ 
pig can not transfer this immunity, we added such serum from 
eral guinea pigs inmiunized by repeated injections with normal hoTrse 
serum; but when this mixture is injected into susceptible pigs tlxey 
react in a characteristic manner. The experimental data upon this 
point will be seen in Part IV, page 37, upon the relation of precipitin 
to the toxic action. 

It therefore appears that the '* immune bodies,'' if such exist 
against the toxic action of horse serum, are not free in the blood axid 
body juices, contrary to the case in diphtheria. 



Part X* 
FEEDING EXPERIMENTS. 

GUINEA PIGS MAY BE SENSITIZED BY FEEDING HORSE SERUM. 

Our work up to this point seemed to carry us very far away from 

an explanation of the cause of sudden death in man following the 

injection of horse serum. A thorough study of the literature upon 

this subject discloses the fact that almost all the unfortunate cases 

of this kind have been due to the first injection of horse serum. If, 

therefore, man may be sensitized to a toxic principle in horse serum, 

^t must be by some other means than a previous injection of horse 

^rum. We have seen that von Pirquet and Schick, in their studies 

^n serum disease, have actually injected children at intervals of 

Several weeks without any such effect as we have found upon guinea 

pigs. Further, that on numerous occasions children have been 

given successive injections of diphtheria antitoxin at intervals of 

^hree or four weeks for purposes of immunization. 

In casting about for another explanation it occurred to us that 
Probably this action may take place through eating horse meat. We 
therefore carried on a number of feeding experiments upon guinea 
pigs. We first fed guinea pigs with normal horse serum by means 
of a stomach tube, with the following results: 

G. P. No. 116. One feeding of 5 c. c. normal horse serum (Jane) into stomach. 

27 days later inoculated with 6 c. c. antitoxic horse serum (Natl. IX. 19) into the peri- 
toneal cavity. No symptoms. 
G. P. No. 114. Four daily feedings, each of 5 c. c, same serum. 

27 days after first feeding and 23 after last, same inoculation as g. p. 116. No symp- 
toms. 
G. P. No. 112. Five daily feedings, 5 c. c. each, same serum. 

27 days after first feeding and 22 after last, same inoculation as g. p. 116. No symp- 
toms. 
Cx. P. No. 113. Six daily feedings, 5 c. c. each, same serum. 

27 days after first feeding and 21 after last, same inoculation as g. p. 116. No symp- 
toms. 
G. P. No. 115. Ten daily feedings, 5 c. c. each, same serum. 

27 days after first feeding and 15 after last, same inoculation as g. p. 116. Symptoms, 
recovered. 

Only one of these five pigs showed definite and characteristic symp- 
toms following the intrastomachal administration of the horse serum. 
This pig, No. 115, which was sensitive to the subsequent injections 

(67) 



68 

of horse serum, had received ten feedings of 5 c. c. each of noncM.^ 
horse serum daily. In view of the fact, however, that exceeding:!/ 
small quantities (TTTTriTrTrTj c. c.) of horse serum is sometimes suffi- 
cient to sensitize a guinea pig, it seemed to us that feeding animals 
with a stomach tube might cause slight abrasions, resulting in absorp- 
tion, so that pigs fed in this manner might really be sensitized 
through minute wounds of the mucous membrane. 

We therefore used another series of pigs and fed the animals with- 
out using artificial means. As guinea pigs will not drink horse serum, 
we found it necessary to dry the serum, reduce it to powder, and 
then mix it with ground carrots. In this way they took the serum 
unknowingly with their food. By this means definite amounts of 
the horse serum may be given and the quantity given to each animal 
accurately controlled. Half a gram of the dried normal horse serum I 
was thus fed to each pig, which was afterwards tested for susceptibility, | 
as follows : 

G. P. No. 4551 . Fed 0.5 gm. dried normal horse serum daily from March 15 to 28. Injected 
March 28 into peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. IX. 19), 13 days 
from first and days from last feeding. Symptoms; recovered. 

G. P. No. 245. Fed 0.5 gm. dried normal horse serum daily from March 15 to 28. Injected 
March 28 into peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. EX. 18), 13 days 
from first and days from last feeding. No symptoms. 

G. P. No. 4581 . Fed 0.5 gm. dried normal horse serum daily from March 15 to 29. Injected 
April 5 into peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. VIII. 18), 21 days 
from first and 7 days from last feeding. Symptoms; recovered. 

G. P. No. 4540. Fed 0.5 gm. dried normal horse serum daily from March 15 to 31 . Injected 
April 6 into peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. VIII. 18), 21 days 
from first and 6 days from last feeding. Symptoms; recovered. 

G. P. No. 4546. Do. 

G. P. No. 4575. Fed 0.5 gm. dried normal horse serum daily from March 15 to April 6. 
Injected April 6 into peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. VIII. 
18), 21 days from first and days from last feeding. Symptoms; recovered. 

G. P. No. 172. Fed 0.5 gm. dried normal horse serum daily from March 15 to April 5. 
Injected April 6 into peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. VIII. 
18), 21 days from first and 1 day from last feeding. Symptoms; recovered. 

G. P. No. 173. Do. 

G. P. No. 276. Fed 0.5 gm. dried normal horse serum daily from March 15 to April 9. 
Injected April 9 into peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. VIII. 
18), 25 days from first and days from last feeding. Dead in 55 min. 

There is no doubt from the above experiments that guinea pigs 
may be sensitized by feeding to them horse serun:. Of the nine pigs 
so fed in the above series eight reacted to subsequent injections of 
horse serum, one of these died. 

These remarkable results are not surprising when we remember 
that Uhlenhuth " showed the possibility of the intrastomachal inimu- 



a Uhlenhuth, — : Neuer Beitrag zum spezifischen Nachweis von Eiereiweiss auf biolo- 
gischem Wege. Deut. med. Woch., Bd. 26, 1900, p. 734-735. 



^■^^tion in working on antiproteid immunization. He fed rabbits 
daily for a number of weeks with egg albumin by means of a stom- 
ach tube. The rabbits' serum was examined every eight days. After 
^Wenty-four days it developed the power to precipitate egg albumin. 
Metalnikoff " reports that he attained immunization by feeding one 
animal with the blood of another. He fed horse's blood to white 
rats. After three or four feedings no agglutination or hemolytic 
action of the rats' blood upon the horse's corpuscles was noted. In 
one week the action appeared feeble and increased to decided strength 
after one or two months. 

GUINEA PIGS MAY BE SENSITIZED BY FEEDING HORSE MEAT. 

After demonstrating the possibiUty of rendering guinea pigs sus- 
ceptible by feeding them with horse serum, we next fed a series with 
horse meat. The following series was fed with indefinite amounts of 
horse meat, both fresh and dried ground up with carrots, as guinea 
pigs do not Uke fresh horse meat and must be very hungry in order to 
eat it. 

G. P. No. 242. Fed 9 days on horse meat from Feb. 15. Injected March 9 into peritoneal 
cavity with 6 c. c. antitoxic horse serum (Natl. IX, 17), 24 days after first and 15 days 
after last feeding. Symptoms; recovered. 

G. P. No. 237. Fed 9 days on horse meat from Feb. 15. Injected March 10 into peritoneal 
cavity with 6 c. c. antitoxic horse serum (Natl. IX, 17), 25 days after first and 16 days 
after last feeding. Symptoms; recovered. 

G. P. No. 238. Do. 

G. P. No. 239. Do. 

G. P. No. 240. Do. 

G. P. No. 244. Do. 

G. P. No. 241. Fed 9 days on horse meat from Feb. 15. Injected April 5 into peritoneal 
cavity with 6 c. c. antitoxic horse serum (Natl. VIII, 18), 49 days after first and 40 days 
after last feeding. Symptoms?; recovered. 

G. P. No. 243. Fed 9 days on horse meat from Feb. 15. Injected March 13 into peritoneal 
cavity with 10 c. c. aqueous extract dry horse meat, 26 days after first and 17 days after 
last feeding. No symptoms. 

As the above series was fed with indefinite amounts, another series 
of guinea pigs was placed upon definite weighed amounts of horse 
meat. Four grams of the dried ground horse meat were mixed with 
fresh carrots and fed to the pigs daily. Almost all the pigs placed 
upon this diet did poorly, their coats became shaggy and the animals 
lost weight. 

G. P. No. 246. Fed 4 gm. dried horse meat daily from March 15 to 28. Injected March 28 
with 6 c. c. antitoxic horse serum (Natl. VIII, 18), 13 days after first and day after last 
feeding. Symptoms?; recovered. 

G. P. No. 4562. Do. 

• Metalnikoff, — : Ueber hamolytisches Serum durch Blutfutterung. Centblt. f. Bakt., v. 
29, 1901, p. 531-533. 



70 

G. P. No. 4544. Fed 4 gm. dried horse meat daily from March 15 to 29. Injected April 5 
with 6 c. c. antitoxic horse serum (Natl. VIII, 18), 21 days after first and 7 days after 
last feeding. Symptoms; recovered. 

G. P. No. 4543. Fed 4 gm. dried horse meat daily from March 15 to April 6. Injec 
April 6 with 6 c. c. antitoxic horse serum (Natl. VIII, 18), 21 days after first and Oda; 
after last feeding. No symptoms. 

G. P. No. 4563. Do. Symptoms?; recovered. 

G. P. No. 4371. Do. No symptoms. 

G. P. No. 270. Do. Symptoms; recovered. 

G. P. No. 4549. Do. No symptoms. 

G. P. No. 4538. Fed 4 gra. dried horse meat daily from March 15 to 31. Injected April 6 
with 6 c. 0. antitoxic horse serum (Natl. VIII, 18), 21 days after first and 6 days after last 
feeding. Symptoms; recovered. 

G. P. No. 174. Do. Dead in 20 minutes. 

G. P. No. 275. Fed 4 gm. dried horse meat daily from March 15 to April 9. Injected 
April 9 into peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. VIII, 18), 25 days 
after first and days after last feeding. No symptoms. 

G. P. No. 4557. Do. 

G. P. No. 4574. Do. Symptoms?; recovered. 

G. P. No. 4569. Do. No symptoms. 

G. P. No. 4566. Do. Symptoms; recovered. 

It is perfectly plain from the above that guinea pigs may be sensi- 
tized by feeding them horse meat as well as horse serum. 

MAY GUINEA PIGS BE SENSITIZED BY FEEDING BEEF? 

Another series of experiments was then conducted by feeding 
guinea pigs with beef. The meat was dried, ground, and mixed in 
definite weighed amounts with ground carrots. 

G. P. No. 247. Fed 4 gm. dried beef daily from March 15 to 28. Injected March 28 into 
peritoneal cavity with 5 c. c. antitoxic horse serum (Natl. VTII, 18), 13 days after first and 
day after last feeding. Symptoms?; recovered. 

G. P. No. 269. Fed 4 gm. dried beef daily from March 15 to 29. Injected April 5 into 
peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. VIII, 18), 21 days after first and 
7 days after last feeding. Symptoms?; recovered. 

G. P. No. 171. Fed 4 gm. dried beef daily from March 15 to April 6. Injected April 6 
into peritoneal cavity with 6 c. c. antitoxic horse serum (Natl. VIII, 18), 21 days after 
first and day after last feeding. No symptoms. 

Five guinea pigs were placed in this series, but two of them died, 
apparently as a result of the diet. 

We conclude, as a result of these feeding experiments, that guinea 
pigs may be highly sensitized to the toxic action of horse serum by 
previous feeding with horse serum or horseflesh. So far as conclu- 
sions may be drawn from the few experiments done upon feeding 
guinea pigs with beef, we feel justified in stating that guinea pigs 
may also be rendered slightly susceptible in this way. Whether 
pigs fed with beef would be more susceptible subsequently to the 



71 

^^^Jection of cattle serum than horse serum is reserved for further 
atoidy. 

The fact that guinea pigs may be rendered susceptible by the feed- 
^xig of strange proteid matter opens an interesting question as to 
^v^hether sensitized guinea pigs may also be poisoned by feeding with 
"tie same serum. K man can be sensitized in a similar way by the 
bating of certain proteid substances it may throw Kght upon those 
interesting and obscure cases in which the eating of fish and other 
sea food, etc., by certain individuals habitually causes sudden and 
sometimes serious symptoms. 



Part XL 

HEREDITARY TRANSMISSION OF THE SUSCEPTIBIUTY IN GUINEA 

PIGS* 

So far as may be judged from the following data on this subject the 
susceptibility to the toxic effects of horse serum may be transmitted 
^I'om the mother guinea pig to her young: 

FAMILY NO. 1. 

-Mother g. p. No. 105. One c. c. normal horse] serum (roan) inoculated subcutaneously. 
Symptoms; recovered. 

[Previous treatment: 55 days prior, inoculated subcutaneously with ^^ c. c. normal 
horse serum (Jane).] 

The progeny of the above guinea pig, when about five days old, was 
t-ested as follows: 

" - P. No. 105A. One c. c. normal horse serum (roan) inoculated subcutaneously. Symp- 
toms; recovered. 
^^. P. No. 105B. One c. c. normal horse serum (roan) injected into the peritoneal cavity. 

Symptoms; recovered. 

FAMILY NO. 2. 

^'lother g. p. No. 66. Six c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Dead in 120 minutes. 

[Previous treatment: 85 days prior, 6 c. c. normal horse serum (Sam) injected into the 
peritoneal cavity.] 

The progeny of the above guinea pig, when about a week old, was 
"tested as follows: 

^. P. No. 66A. One c. c. normal horse serum (roan) injected into the peritoneal cavity. 

Symptoms. 
K}, P. No. 66B. Do. 

FAMILY NO. 3. 

^^ilother g. p. No. 103. Six c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Symptoms. 

[Previous treatment: 71 days prior, if^ c. c. normal horse serum (Jane) inoculated 
subcutaneously: 33 days later 6 c. c. antitoxic horse serum (Natl. IX, 17) injected into the 
peritoneal cavity. Symptoms.] 

The progeny of the above guinea pig, when about ten days old, was 
iiested as follows: 

^. P. No. 103A. One c. c. normal horse serum (roan) injected into the peritoneal cavity. 
Symptoms. 

FAMILY NO. 4. 

Hother g. p. No. 4463. Six c. c. normal horie serum (roan) mjected into the peritoneal 
cavity. Symptons (l). 

[Previous treatment: 99 days prior, inoculated subcutaneously with 022 c. c. toxine 
No. 7-f-rtiF ^- c- antitoxic hor.# senim (Natl. VIII, 17>. 26 days later, 6 c. c. antitoxic 
horae serum (Natl. VIII, 17)+liver pulp of normal guinea {Hg injected into the peritoneal 
cavity. Symptoms.] 

^73) 



The progeny of the ahi 
tested as follows: 
G. P. No. 4463A. One c. t. normal liur«- 
Symptom 



?4 
guinea pit;, when about ton days old, 



Do. 



FAMILY NO. 5. 

c. Dormal horse serum (rm 



i) injected 




Mother g. p. No. 14, Six c 

Symptoms; recovered. 

[Previoua treatment: &4 days prior, inoculated snbputaneously » 

horse «?runi (Natl. IX, 18-)] 

The progeny of the above guinea [jig, when five days old, was tested 
as follows: , 

G.P. No. UA. One <-, c. normal horse serum (rtiari) iujetted into Ihe peritoneal laivily 

Dead in » 
G. P. 14B. Dd. 

G. P. No. 14C, Do. Dead in 

It is plain from the above that young guinea piga bom of a suscep- 
tible mother are themselves susceptible, often more so than the mother 
herself. 

To control the effect of horse serum upon such young guinea pigs 
the following data are given: 

(All controls born of normal parantB.) 
Control g. p. No. 01. One c.c. normal horse serum (roan) inoeulaledsubeutaneoualy when I 

5 days old. No syniptoma. I 

Control g. p. No. 02. Two c. c. same serum when 1 week old. No sjmptom.i, i 

Control g. p. No. 03. Same injection when G days old. No symptoms. 
Control g. p. No. 04. Same injection when 4 days old. No aymptoma. 

These four controls were selected from four different litters. 

These results upon the hereditary transmission of the susceptibility 
to the poisonous action of horse serum in guinea pigs may throw light 
upon the well-known inherited tendency to tuberculosis in children 
born of a tuberculous parent. 

There are certain analogies between the action of tuberculosis and 
horse serum. Both produce a hypersensitiveness and also a certain 
degree of immunity. Now that we have proved that this hypersen- 
sitiveness or anaphylactic action in the case of horse serum may be 
transmitted liercditarily in guinea pigs, may it not throw Hght upon 
the fact that tuberculosis "runs in families?" While there are 
several recorded instances demonstrating that immunity to certain 
infectious diseases may be transmitted from a mother to her young 
this is, as far as we know, the first recorded instance in which hyper^ 
sensitiveness, or ana]>hylaxi3, has been experimentally shown to be 
hereditarily transmitted from a mother to her young. 



J 



Part Xn* 
TOXIC ACTION OF OTHER ALBUMINOUS SUBSTANCES. 

As we found that guinea pigs may be so highly sensitized to the 
^xic action of horse serum and to a certain extent the serum from 
oAer animals we made a few experiments to determine whether 
other albuminous substances possessed the same toxic properties 
^nder these conditions. For this purpose we used egg albumin, 
peptone, skimmed milk, hemoglobin and vegetable proteid. 

^' P. No. 4366. Six c. c. of a saturated solution of egg albumin were injected into the 
peritoneal cavity. No symptoms. 

[Previous treatment : 30 days prior, inoculated subcutaneously with 0.22 c. c. toxine 
Xo.7-f :f}ff c.c. horse serum (PD & Co. 08022-A)]. 

Two days later this guinea pig was tested for susceptibility by the inoculation of 6 c. c. 
antitoxic horse serum (Natl. VIII. 17) into the peritoneal cavity. Dead in 50 minutes. 
G. P. No. 4924. Six c. c. of a saturated solution of peptone injected into the peritoneal 
cavity. No symptoms. 

[Previous treatment: 40 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5+jJiy c. c. antitoxic horse serum (Cutter 1540)]. 

Three days later this guinea pig was tested for susceptibility by the inoculation of 6 c. c. 
antitoxic horse serum (Natl. IX. 17) into the peritoneal cavity. Symptoms; recovered. 

G. P. No. 4926. Six c. c. skimmed milk injected into the peritoneal cavity. No symptoms. 
[Previous treatment: 40 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. S-f-ji^F c. c. antitoxic horse serum (Cutter 1540)]. 

Three days later tested for susceptibility by the injection of 6 c. c. antitoxic horse serum 
(Natl. IX. 17) into the peritoneal cavity. Dead in 11 minutes. 

G. P. No. 4919. Ten c. c. vegetable proteid from oats injected into the peritoneal cavity. 
No symptoms. 

[Previous treatment: 40 days prior, inoculated subcutaneously with 0.139 c. c. toxine 
No. 5-\-2\jf c. c. antitoxic horse serum (Steams 10 H)]. 

Three days later tested for susceptibility by the injection of 6 c. c. antitoxic horse s^rum 
(Natl. IX. 17) into the peritoneal cavity. Dead in 25 minutes. 

G. P. No. 4925. Six c. c. hemoglobin solution in water from washed normal (roan horse) 
corpuscles injected into the peritoneal cavity. No symptoms (?) 

[Previous treatment: 40 days prior, inoculated subcutaneously with 0.139 o. c. toxine 
No. 5+7^^ c. c. antitoxic horse serum (Cutter 1540)]. 

Three days later tested for susceptibility by the injection of 6 c. c. antitoxic horse serum 
(Natl. IX. 17) into the peritoneal cavity. Symptoms; recovered. - 

G. P. (normal). Four and a half c. c. solution of hemoglobin (watery solution of washed 
red blood corpuscles of roan horse) injected into peritoneal cavity. No symptoms. 

(75) 



76 

G. P. No. 4989. Ten c. c. same injection. Symptoms (?) 

[Previous treatment: 25 days prior, inoculated subcutaneously with 0.22 c. c. to:3^^® 
No. 7+iiiy c. c. antitoxic horse serum (Alex. A202)]. 

To control the two preceding pigs a solution of hemoglol^i^j 
obtained by dissolving red blood corpuscles from a normal ho:*^® 
(roan), was injected into the peritoneal cavity of a normal guirm^^^ 
pig, with negative results. 

Whether guinea pigs treated with these albuminous substances 8»— re 
rendered susceptible to subsequent injections of the same albumin ^ 
now being studied. 



Part Xm. 
A REVIEW OF THE LITERATURE RELATING TO OUR WORIC 

We feel it incumbent upon us to compare our results with the 
work of others and, therefore, have introduced this discussion upon 
the toxic action of serums in general, especially in relation to the 
production of immunity and hypersensitiveness. 

Note. — In this historical development we have drawn largely upon the splendid review 
of the literature as given by Uhlenhuth in *' Zur Kenntniss der giftigen Eigenschaften des 
Blutserums'' (Zeit. f. Hyg., v. 26, 1897), and C. Frh. von Pirquet and B.Schick in "Die 
Serumkrankheit'* (K. K. Universitats-Kinder-Klinik, Wien, 1905). 

Transfusion was formerly used as a therapeutic measure. The 
first reference to this practice is June 15, 1667, when von Denis 
transfused lamb's blood (cited in Landois's article on transfusion, 
Eulenburg's Realenzyklopadie, 3. Auflage). This practice gradually 
fell into disuse but was again revived about the beginning of the last 
century, when transfusion was used for acute anemia and poisonings. 

The intravenous injection of lamb's blood was soon shown to be 
associated with great danger. High fever in half an hour, emboli, 
hemorrhages, hemoglobinuria, etc., were often noticed. The causes 
of these serious symptoms, as well as the scientific basis of our knowl- 
edge upon the effects of transfusion, were first made clear by the work 
of Landois and Ponfick in the years 1873 and 1875. These authors 
showed that the blood of alien species caused solution of the cor- 
puscles when brought into the circulation. Naunyn and Francken 
showed that the injection of dissolved hemoglobin into the circula- 
tion caused coagulation. Great numbers of the white corpuscles 
were so acted upon by the dissolved hemoglobin that the fibrin- 
forming elements were thus set free, resulting in the formation of 
clots within the vessels. When this coagulation is extensive, death 
may result from asphyxia. 

Dallera ^ in 1874 reported that urticarial eruptions may follow 
the transfusion of blood. 



a Dallera: Oonsiderazioni e casi clinici di transfusione del sangue. 11 morgagni, 1874, 7. 

(77) 



78 

Neudorfer** refers to the fact that urticaria frequently follows 
transfusion. 

Landois ^ also refers to this complication. 

In consequence of the severe injury which often resulted from the 
transfusion of lamb's blood the practice was entirely discontinued. 
Recently, however, Dominici ^ has again taken up the subject. 

In England at one time the transfusion of milk was practiced, but 
proved dangerous and without benefit, as stated by Montard, Mar- 
tin, and Richet. (Injections intra veineuses de lait et de sucre, Soc. 
de Biol., 1879.) 

Latterly the transfusion of lamb's blood has given place to phys- 
iological salt solution and also to the transfusion of defibrinated 
human blood. 

In the year 1894 the use of diphtheria antitoxin introduced the wide- 
spread practice of injecting horse serum. This practice diflFered from 
the previous work with transfusion in that the injections are generally 
not given intravenously, but subcutaneously, and the quantity of 
thte alien albuminous substance is relatively small. According to 
von Pirquet and Schick this accounts partly for the fact that in the 
majority of these injections no injurious consequences followed. 
Luebinski (Uber eine Nachwirkung des Antitoxins bei Behandlung 
des Diphtheric, Deut. med. Woch., 1894) was the first to call attention 
to a case of exanthematous eruption following the injection of antitoxin 
serum. Soon afterwards Schiitz,^ Cnyrim,^ Asch,/ Rembold,^ and 
Treymann ^ reported urticarial eruptions following the prophylactic 
injection of antitoxic horse serum. Following these came a great 
mass of evidence which made it clear that following the injection of 
antidiphtheric serum these sequelae were comparatively harmless. 

In relation to the cause of these symptoms it was generally held 
that the serum contained some toxic substance, probably a globulin. 
This explanation led manufacturers to the commendable practice of 
starving their horses about twenty-four hours prior to bleeding, in 
order to eliminate as far as practicable the freshly assimilated prod- 
ucts of digestion. 

o Neudorfer: Beitrage zur Bluttransfusion. Zeitschr. f. Chirurgie, Bd. 6. 

2> Landois: Die Transfusion des Blutes, Leipzig, 1875. : Transfusion. Eulenburg's 

Realencyclopadie, S.Aufl., 1900. 

c Dominici: Transfusion. Wiener med. Wochenschr., 1895. 

d Schutz: Discussionsbemerkung zu Piorkowski's Vortrage. Berl. med. Gesellsch., 1904. 

« Cnyrim: Zwei Falle von Erkrankungen nach Anwendung des Diphtherieserums. Deut. 
med. Woch., 1894, p. 898. 

/ Asch: Zur Casuistik der Heilserumexantheme. Berl. klin. Woch., 1894. . 

^Rembold: Zwei Falle von Erkrankung nach Anwendung des Heilserams. Deut. med. 
Woch., 1894, p. 963. 

ft- Treymann, Otto: Ein Fall von acuter hamorrhagischer Nephritis nach Anwendung deg 
Behring'schen Diphtherieheilserums. Deut. med. Woch., 1894, p. 952. 



79 

Heubner " and von Bokay * expressed the opinion that these mani- 
f stations were due to other properties than the antitoxin in the 
serum. 

Johannessen ^ beUeved that the agent in horse serum which pro- 
duced these symptoms was due to the introduction into the body of 
an alien serum, for he produced the same eflFects by injecting normal 
horse serum. In 22 cases fever resulted, and skin eruptions in 12. 

S6vestre^ referred the exanthems which appear in eight to ten 
days back to the serum which had been injected. However, he 
thought that the measle-like eruption and fever was the expression 
of a streptococcus infection. Netter, however, in the discussion, 
beUeved these symptoms to be caused by the serum. 

Almost all authors express the opinion that the symptoms caused 
by the injection of serum are of minor consequence. The fact that 
from time to time serious symptoms and even death followed the 
injection of diphtheria antitoxic serum does not alter the general 
impression that as a rule such injections are harmless. The henejits 
far outweigh the danger. 

Gottstein,^ in addition to the Langerhans case, collected 8 deaths 
following the injection of serum in those having diphtheria and 4 in 
those not sick with diphtheria. 

We have collected from the literature 19 cases of such unfortunate 
results, and know personally of several more which have not been 
reported. Von Pirquet and Schick express the opinion that a critical 
examination of these cases may be otherwise explained, and authors 
generally have not permitted this rare accident to counterbalance 
the great benefit possible from the use of diphtheria antitoxic serum. 

It seems to have been definitely shown that the skin eruptions and 
other symptoms following the injection of horse serum follow in 
direct proportion to the amount of serum injected. This has resulted 
in endeavors to concentrate the serum as much as possible. 

o Heubner: Praktische Winke zur Behandlung der Diphtherie mit Heilserum. Deut. 

med. Woch., 1894, p. 701. Klinische Studien uber die Behandlung der Diphtherie 

mit dera Behring'schen Heilserum. Leipzig, 1895. Uber die Anwendung des Heil- 

serums bei der Diphtherie. Jahrb. f. Kdheilk., Bd. 38, p. 231. 

b Von Bdkay : Die Heilserumbehandlung gegen Diphtherie im Budapeste Stefanie- 

ICinderspital. H. f. Kdh.,Bd. 44, 1897. Meine Erfolge mit Behring's Diptherieheil- 

serum. Deut. med. Woch., 1895, p. 233. Meine Erfahrungen mit dem Moser'schen 

polyvalenton Schariach-Streptokokkenserum. Deut. med. Woch., 1904. 

c Johannessen : Uber Injektionen mit antidiphtherischen Serum und reinem Pferdeaerura. 

l>eut. med. Woch., 1895. IJber Immunisierung bei Diphtherie. Deut. med. Woch., 

1895. 

d S^vestre : Des accidents causfe par le streptococque k la suite des injections de s^rum 
^e Roux. Bull, de la Soc. m^d. des hdpit^aux, July 19, 1895, Jan. 31, •1896. La m6d. 
"Tiodeme, 1896. 

« Gottstein : Uber Todesfalle, welche bei der Anwendung des Diphtherieheilserums beo- 
t^^chtet worden sind. Therap. Monatsh., 1896. 



I 



80 

Bujwid ^ believed that these inaiiifestatioiis were due largely to 
fresh horse serum, and favors the use of old seasoned antitoxm for 
therapeutic purposes. 

Hamburger and Moro ^ have shown that the injection of alien 
serums into man result in the formation of precipitins. 

Hamburger,*^ in a further work, drew a distinction between alien 
albuminous substances introduced through the digestive tract and by 
other channels. 

Upon the general toxic properties of blood we quote the following 
instances, taken largely from Uhlenhuth: ^ 

Stem,* 1893, found that the blood from a case of erysipelas in 
amounts of 0.5 to 1 c. c. killed white mice, while normal human blood 
required 3 c. c. or more to kill the animals. 

Rummo/ 1891, showed that blood serum from cases of typhoid 
fever, malaria, pneumonia, and eclampsia had a high grade of toxicity 
for rabbits when injected intravenously. 

Albu,^ 1897, confirm Rummo^s experiments, and found an increase 
of toxicity in persons suffering from pneumonia, severe chronic bron- 
chitis, epilepsy, uraemia, and puerperal sepsis. 

Upon the toxicity of the blood serum of man in health and disease 
further work has been done by Charrin,* Leclainche and R6mond/ 
Chambrelent and Tamier, > Ludwig and Savor,*Guinard and Dumarest/ 

o Bujwid, O.: Kann des Antidiphtherieserum sch&dlich seini Polnisch, ref . Virchow's 
Jarhb., 1897, II, p. 659. 

b Hamburger, F., and Moro: Uber die biologischen nachweisbaren Ver&ndertungen des 
menschlichen Blutes nach Seruminjektion. Wiener klin. Wocb., 1903. 

c Hamburger, F. : Zur Frage der Immunisienmg gegen Eiweiss. Wiener klin. Woch., • 
1902. : Arteigenheit und Assimilation. Wien, 1903. 

d Uhlenhuth : Zur Kenntniss der giftigen Eigenschaften des Blutserums. Zeit. f . Hyg., 
vol. 26, 1897, p. 384. 

6 Stem: Ueber einigo Beziehungen zwischen menschlichen Blutserum und pathogenen 
!Bakterien. (XII Congress fur innere Medicin, Wiesbaden, 1893): Deut. med. Woch., 
1893. 

/Kummo: Ueber die Giftigkeit des Blutserums bei Menschen und Thieren im normalen 
Zustande und bei Infectionskrankheiten. Wiener med. Woch., 1891. 

^Albu: Untersuchungen Uber die Toxicitat normaler u. pathol. SerumflOssigkeit. 
Virchow's Archiv., bd. 140, 1897. 

^ Charrin: Compt. rend. soc. biol., 1890. 

« Leclainche and R^mond: Compt. rend. soc. biol., 1890, p. 1037. 

;■ Ludwig and Savor: Monatschrift fur Geburtshulfe, bd. 1, 1895. 

k Tamier and Chambrelent: Note relative k la recherche de la toxicity du s^rum sanguis 
dans deux cas d'eclampsie puerp^rale. Soc. de biolog., 1892. 

^ Guinard and Dumarest: Note sur la determination de la toxicity du serum sangou^' 
Techniques et r^sultats. Compt. rend., 1897, No. 15. Variations de la toxkit-^ du s&mo 
sanguin dans certaines infections exp^rimentales. Loc. cit., No. 18. A propos de * 
determination physiol. et clinique de la toxicity du s^rum humain. Loc. cit.. No. 18. 



81 

Bar and R6iioii,« Picolini and Conti,*^ Mairet and Bosc.'' All of these 
authors determined the toxicity of the blood serum in accordance 
with Rnmmo's method of intravenous injections into rabbits. 

The poisonous properties of the blood serum from healthy men, 
according to the work of these authors, varies considerably, as may 
be judged from the following summary: 

Rummo found 10 c. c. as the fatal dose for 1 kilogram of rabbit. 

Lugwig and Savor, 8 to 9 c. c. 

Chambrelent and Tamier, 10 c. c. 

Albu, 9.5 to 11 c. c. 

Mairet and Bosc, 12.5 to 18.0 c. c. 

Guinard and Dumarest, 17 c. c. 

Leclainche and R6mond, 23 c. c. 

Charrin, 27 c. c. 

It will therefore be seen that the fatal dose of normal human 
serum, according to these different investigations, varies between 
8 c. c. and 27 c. c. per kilogram of rabbit. 

Uhlenhuth foxmd that the intravenous injection of from 7 to 10 
c. c. of normal blood serum per kilogram of rabbit regularly caused 
death within one-half hour. 

The blood serum of other animals was injected intravenously into 
rabbits and showed considerable differences in their toxicity. The 
quantity necessary to kill in proportion of 1 kilogram of rabbit was : 



c.c. 



Sheep serum 11 

Hog serum 12 

Cattle serum 6 

Horse serum on the other hand, when injected in such large quan- 
tities as 60 c. c. and more per 1 kilogram of rabbit, not only did not 
kill but caused no reaction at all. 

In 1888 Mosso ^ discovered that eel serum in doses as small as 
0.02 c. c. per 1 kilogram of rabbit caused death. 

Uhlenhuth, finding intravenous injections into rabbits unsatis- 
factory, used subcutaneous inoculations into guinea pigs. He 
found that the injection of small quantities (0.5 c. c.) of human, 

a Bar and K^non: De la toxicity du sang et de I'urine chez une femme atheinte de troubles 
gravido-cardiagues. Compt. rend. soc. biol., 1894. 

6 Picolini and Conti: Toxicity du s^rum sanguin et de Purine dans la pneumonie. 

c Mairet and Bosc: Recherches sur les causes de la toxicity du s^rum du sang. Compt. 
rend., v. 119, p. 292. Toxicity du s^rum du sang de Thomme sain. Comp. rend., 
1897. *Des effects de la chaleur sur la toxicity du s^rum. Compt. rend., 1894. 

d Mosso: Die giftige wirkung des serums der muraeniden. Arch. f. exper. pathol. u. 
pharmakol., bd. 25, p. 111. 

28337— No. 29—06 6 



The hastening power of the reaction must be regarded as an advan- 
tage that the organism has won for itself through the occurrence of 
the first disease. 

In applying tliis view to disease von Pirquet and Schick find that an 
organism in the stage of tlie free antibodies is an expi'ession of the 
immediate power of reaction or hypersensitiveness. This stage has 
only a limited duration. The free antibodies disappear; however, 
the individual remains immune. The fact of this immunity consists, 
however, no longer in the power of immediate reaction against the 
cause of the infection, but in a hastened power of reproduction of 
antibodies. An oiganism has the power to combat a newly intro- 
duced infection and localize it, as is well shown in the case of vac- 
cination. Von Pirquet and Schick believe that this property does 
not depend so miich upon free antibodies in the fluids as upon a 
property acquired by the cells through the first disease, in which we 
see an expression of cellular immunity. 

In Wassermann's" observation that the adaptation of the tissues 
to the existence of microorganisms which were naturally pathogenic 
"without any apparent reaction and production of antibodies we see 
an instance of immiojtity through insusceptibility. 

A series of diseases — for example, smallpox, measles, varicella, 
rothlen — cause after one attack the individual to remain more or 
less protected throughout the rest of his life. They have the com- 
mon clinical characteristic that, after a long and definite period of 
incubation, the disease runs a course in a definite and comparatively 
short time. If the organism does not die as a result of the disease, 
then it has won a complete victory over the cause of infection; the 
same is no longer able to produce harm in such an organism. 

According to von Pirquet and Schick the fact that the clinical 
immunity against this group ot diseases, for which we may take vac- 
cinia as a type, does not consist of an acquired nonsusceptibility 
against the cause of the infection, but the power of au accelerated 
reaction. They beUeve they have shown from their work that they 
have reawakened interest in the serum disease and that this syn- 
drome is not only of interest clinically but, from the standpoint of 
general pathology, is of the greatest importance. 

Our explanation of the cause of sudden death following the second 
injection of horse serum does not differ essentially from the theory 
(which has just come to our notice as our manuscript is being edited 
for press) published by von Pirquet and Schick, relative to the reac- 
tion of the serum disease in man, although these similar deductions 
were reached independently and from different premises. 

o Wassermann, A.. Uber naturlithe und kQnstlichc Immunirait. Zeit. f. Hy);., bH. 37, 

p. 173. Wpsen der Infeption, in Kolle und Wessermann. Handb. der pa,t)iag«i. 

UikroorgBnismeii, 1903. 




89 

We believe that the substance which sensitizes the animal is iden- 
tical with that which later poisons it, absurd as that may seem at 
first glance. However, the first substance must cause a reaction in 
the organism resulting in the production of ''antibodies'' and it is 
these antibodies combining with a substance in the horse serum that 
produce the toxic action. We have found that small quantities of 
serum produce, after a definite period of incubation, a condition of 
anaphylaxis. Large quantities probably produce a lesser grade of 
hypersusceptibiKty. Multiple or repeated injections produce immu- 
nity. We therefore possess in horse serum a substance capable of 
causing both anaphylaxis and prophylaxis. 



SUMMARY AND CX)NCLUSIONa 

Normal horse serum, when injected into the peritoneal cavity of a 
normal guinea pig, produces no symptoms. When injected subcuta- 
neously there may result at most a slight local reaction consisting of 
swelling and edema, which gi^adually disappears. 

Antitoxic horse serum is equally harmless for normal guinea pigs. 

Horse serum is, however, poisonous to a guinea pig which has pre- 
viously been injected with horse serum*. Tne '^ period of incubation'' 
or time necessary to elapse between the first and second injection is 
about ten days. Under these circumstances, horse serum is poison- 
ous whether injected subcutaneously or into the peritoneal cavity. 

The first injection of horse serum renders the guinea pig susceptible. 

The symptoms caused by the injection of horse serum into a sus- 
ceptible guinea pig are respiratory embarrassment, paralysis, and con- 
vulsions, followed by death. The symptoms come on usually within 
ten minutes after the injection, and when death results it usually 
occurs within one hour, frequently in less than thirty minutes, and 
sometimes within a few minutes. 

The poisonous principle in horse serum appears to act upon the 
respiratory centers. The heart continues to beat long after respira- 
tion ceases. 

The toxic action of horse serum bears no relation to diphtheria. 
The poison is not toxone. Guinea pigs can not be rendered suscep- 
tible by previous infections with the B. dipJitherise or by previous 
injections with diphtheria toxine. 

It seems from our work, however, that guinea pigs first injected 
with a mixture of diphtheria toxine plus horse serum are more sen- 
sitive to subsequent injections of horse serum than are guinea pigs 
sensitized with a first injection of horse serum alone. 

Diphtheria antitoxin plays no part in this poisonous action 
and in itself is harmless. 

As soon as we realized that the toxic principle in horse serum 
exerts its action in quantities so minute as to place it almost in the 
category of the ferments and, further, when we concluded from 
our work that this toxic principle is doubtless one of those highly 
organized and complex proteid substances belonging to the ''haptin 
group'' in the sense used by Ehrlich, we recognized how futile it 
would be with present methods to attempt to isolate this substance. 

(91) 




I 



evertheless we devoted much time and study to the relation of 
this toxic principle to various clieiiiical, phyaicai, and electrical 
influences. The practical importance of eliminating or neutraliz- 
ing this toxic principle in horse serum ia at once evident. 

It is probable that when the strange proteid is introduced into 
the guinea pig it causes a reaction resulting in a production of 
"antibodies," so that when a second injection of horse serum is 
given there is probably either a union or a reaction between the 
antibodies and a substance in the horse serum which produces the 
poisonous effect. 

This poisonous principle is quantitatively specific; that is, guinea 
pigs treated with horse serum are rendered -'lomewhat susceptible 
to the subsequent injection of the serum of another animal. Guinea 
pigs treated with the serum of another animal are slightly sensitive 
to the toxic action of horse serum. 

Guinea pigs treated with the serums of various animals and sub- 
sequently injected, are much more susceptible to homologous serums 
than to heterologous serums. 

This poisonous action has no relation to hemolysis. Our work 
proves that blood serum may contain an acute poison entirely inde- 
pendent of any hemolytic action. Normal horse serum has no lytic 
power upon the red corpuscles of the normal guinea pig. 

This poisonous action has no relation to the specific albuminous 
precipitins. 

The poisonous principle in horse serum is not affected by a tem- 
perature of 60° C. for 6 hours, but it is destroyed at 100° C. for 15 
minutes. 

The poisonous principle is filterable tluough porcelain, ia not 
injured by drying, and can not be separated by precipitation with 
anunonium sulphate and subsequent dialysis. 

The following chemical substances do not oxidize, neutralize, or 
precipitate the poisonous principle in horse serum: Butyric acid, 
permanganate of potash, citrate of soda, alcohol, succinic peroxide 
acid (atphozone), hydrogen dioxide, and ammonium sulphate. The 
presence of chloroform or trikresol (0.4 per cent) does not interfere 
with this poisonous action. 

Serums eight years old are as toxic as those freshly separated. 

Exposure to X-rays does not affect the poisonous action of horse 

It requires about 10 days after the first injection of horse serum 
for a guinea pig to show susceptibility to a second injection. A 
guinea pig remains susceptible a very long time, at least 160 days. 

As small a quantity as i^mmi c, c. of horse serum was sufficient 
I in one instance to render a guinea pig susceptible. Quantities 



98 

varying from yi^r to tVvt c. c. almost invariably render guinea pigs 
highly susceptible when given in the toxine-antitoxine mixture. 

One-tenth c. c. of horse serum injected into the peritoneal cavity 
of a susceptible guinea pig is sufficient to cause death. The same 
quantity inoculated subcutaneously may cause serious symptoms. 

There is some evidence to show that the sensitizing substance in 
horse serum is the same as the poisonous substance. The sensitiz- 
ing substance is not affected by precipitation with ammonium 
sulphate and dialysis. 

Guinea pigs may be sensitized with horse serum tliat has been 
dried and redissolved. 

The sensitizing substance is not affected by a temperature of 60** 
C. for 6 hours. 

It is probable that small quantities of horse serum render a guinea 
pig more susceptible than do large quantities. If this be true, it is 
due, perhaps, to the fact that large quantities, owing to slow absorp- 
tion or prolonged reaction, partly immunize the guinea pig at the 
same time that it is being sensitized. 

The sensitizing substance apparently is not free in the blood 
serum of guinea pigs. 

An active immunity against this toxic principle may readily be 
established by repeated injections of horse serum, at short intervals, 
into a guinea pig. Although guinea pigs may be immunized actively 
in this manner we have not yet succeeded in transferring this immu- 
nity in the blood serum or body juices to another guinea pig. It 
therefore appears that the immune bodies, if such exist against the 
toxic action of horse serum, are not free in the blood and body juices 
contrary to the case in diphtheria. 

Guinea pigs may be sensitized to the toxic action of horse serum 
by feeding them with horse serum or horse meat. 

The fact that guinea pigs may be rendered susceptible by the feed- 
ing of strange proteid matter opens an interesting question as to 
whether sensitive guinea pigs may also be poisoned by feeding with 
the same serum given after a proper interval of time. If man can 
be sensitized in a similar way by the eating of certain proteid sub- 
stances may not this throw light upon those interesting and obscure 
cases in which the eating of fish, sea food, and other articles of diet 
habitually cause sudden and sometimes serious symptoms ? 

The susceptibility to the toxic action of horse serum is transmitted 
hereditarily from the mother guinea pig to her young. 

These results upon the hereditary transmission of the susceptr- 
bility to the poisonous action of horse serum in guinea pigs m 
throw light upon the well-known hereditary tendency to tubercal 
in children born of a tuberculous parent. There are certain 
ogies between the action of tuberculosis and horse serum. 



94 

may produce a hypersusceptibility and also a certain degree of 
immunity. Now that we have proved that this hypersusceptibility 
or anaphylactic action in the case of horse serum may be transmitted 
hereditarily in guinea pigs, may it not throw Ught upon the fact that 
tuberculosis ''runs in famiUes?^' 

Demonstrations of the hereditary transmission of acquired charac- 
ters are comparatively rare in biology. While there are several 
recorded instances demonstrating that immunity to certain infectious 
diseases may be transmitted from a mother to her young, yet, as far 
as we know, this is the first recorded instance in which hypersensi- 
tiveness, or anaphylaxis, has been experimentally shown to be trans- 
mitted from a mother to her young. 

Other albuminous substances, such as skimmed milk, peptone, 
hemoglobin, egg albumin, and vegetable proteids possess no poison- 
ous action upon guinea pigs sensitized with horse serum. Whether 
guinea pigs are rendered susceptible to a subsequent injection with 
the same albuminous matter with which they have been sensitized 
will be reported in a later paper. 

We believe that the substance which sensitizes the animal is iden- 
tical with that which later poisons it. However, the substance must 
first cause a reaction in the organism resulting in a production of 
antibodies. We have found that small quantities of horse serum pro- 
duce, after a definite period of incubation, a condition of anaphylaxis; 
multiple or repeated injections produce immunity. We therefore 
possess in horse serum a substance capable of causing both anaphy- 
laxis and prophylaxis. 

It may be that man can not be sensitized in the same way that we 
have shown to be the case with guinea pigs. Children have, in a num- 
ber of instances, been injected with antidiphtheric horse serum at 
short and long intervals without, so far as we are aware, causing 
death. Certain serums, for example, the antitubercle serum of 
Maragliano and the antirheumatic serum of Menzer, are habitually 
used by giving injections at intervals of days or weeks. In all such 
cases of frequent and repeated injections the amount which has been 
injected and the interval between the injections must be taken into 
account in relation to our work. Von Pirquet and Schick have shown 
that a second injection of horse serum into children causes an '^ imme- 
diate '^ or an '' accelerated '' reaction. Both the immediate and the 
accelerated reaction in children are characterized by symptoms of 
''the serum disease. '' 

We might conclude that children may not be sensitized to the toxic 
action of horse serum by eating horse meat, for horse meat is a favorite 
article of diet in certain European countries and there is nothing upon 
record to show that the injection of horse serum in those countries is 
fraught with more danger than where this diet is not used. It should, 



95 

however, be borne in mind that our work has shown that guinea pigs 
may be sensitized with exceedingly minute quantities of a strange 
proteid, and that repeated injections cause an immunity, and it does 
not seem impossible that the same action may be true of food. 

Man reacts to the first injection of horse serum after a period of 
eight to thirteen days; guinea pigs show no reaction as a result of the 
first injection; both man and guinea pigs react to a second injection. 
The reactions in man and the guinea pig differ, however, both in sever- 
ity and in kind. The relation, therefore, that our observations upon 
the guinea pig may have in their apphcation to man must await fur- 
ther study. 

The fact that other animals beside man and the guinea pig react to 
a second injection of horse serum would seem to indicate that we are 
dealing with one and the same action. 

We beUeve that our results make it probable that man may be ren- 
dered sensitive to the injection of a strange proteid, as is the case with 
the guinea pig and other animals, and that this explanation must be 
considered as well as the status lympJiaticuSj which has heretofore been 
assigned as the cause of sudden death following the injection of horse 
serum. 

[Post Scriptum. — After the galley proof of this article had left our 
hands an article by R. Otto entitled ^^ Das Theobald Smithsche Pha- 
nomen der Serum-UeberempfindHchkeit," reprinted from Leuthold- 
Gedenkschrift, band 1, first came to our notice. His paper deals 
with some of the problems .we have studied and his results are in 
harmony with many of our conclusions.] 

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Nij. ill.— A Btiwly of (be cniisR of iuddBU death foUuwing tlie UiiecUoa of fa 
»ennii- By M. J. Rusenaii ami Jolin F. Andcreon. 

In dtii« these bu!lelina, bt^inning with Ha. 8, bibUogrftr^"" >A^ anUiCrn n 

nH|Uu«l«J [o adopt the following ulibi^vmlioaa: Ball No, , Uyg. Ij»1i.i XT. j 

.Pub. Unith A Mar.-Hosp. 8erv-, Wash., pp. . 

M4IX.IHQ LWT, 

The Sm-ri«o wUl enter iotn exchan»^ o( pnbliuitionB vlth roedkal twl tdenti^ 



A 



LANE MEDICAL LIBRARY 



This 1«)ok should be returned on or before 
the date last stamped below. 



V541 Hosenau, M.J. 98742 
R81 A study of the cause 
1906 of sudden death followJ