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VACCINE AND SERUM THERAPY

INCLUDING ALSO A STUDY OF INFECTIONS, THEORIES
OF IMMUNITY, OPSONINS AND THE
OPSONIC INDEX

EDWIN HENRY SCHORER, B. S., M. D.,

"/

ASSISTANT PROFESSOR OF PARASITOLOGY AND HYGIENE, UNIVERSITY OF MISSOURI;

FORMERLY ASSISTANT ROCKEFELLER INSTITUTE FOR MEDICAL

RESEARCH, NEW YORK CITY.

ILLUSTRATED.

St. Louis

C. V. MOSBY CO.
1909.

COPYRIGHTED BY C. V. MossV Co., 1909.

CONTENTS.

I. INFECTION- Page

Course of infection 7

II. IMMUNITY.

Natural and acquired. Active and passive 12

Theories of immunity: Klebs and Pasteur, Chauveau, Metchnikoff, Salmon
and Smith, !''<>. lor, Brhrin^ and Kimrr, Ehrlich's side chain theory; opsnnin
theory, Wright and Douglas; Neufeld and Rimpau 14

III. OPSONIC INDEX.

Irishman's method of estimating phagocytic power 23

Wright and Douglas' opsonic index 23

Serum; leucocytes; bacterial emulsion; strength of bacterial emulsion; mix-
ture of bacteria, leucocytes and serum; smears, preparation, fixing and
staining; examination of smears; calculation of the opsonic index 24

IV. CRITICISMS AND MODIFICATIONS OF WRIGHT'S OPSONIC INDEX
DETERMINATIONS.

Mechanical technique; serum; bacterial emulsions; leucocytes; calculation
and determination of the opsonic index, Simon, Strong; leucocytes ex-
amined 37

V. OPSONIC INDEX IN HEALTH AND DISEASE.

Importance of opsonins in immunity and accuracy of the method of deter-
mining the opsonic index; changes in opsonic index in disease; changes in
opsonic index due to vaccines 49

VI. NATURE OF OPSONINS.

Specificity; effect of heat; structure of opsonins; relation to leucocytosis; rela-
tion to stimulins; opsonins and aggressins; influence of chemicals and
reaction; non-bacterial opsonins 58

VII. VACCINE THERAPY.

Bacterial Vaccines: dosage; organisms used in vaccination; preparation of

vaccines; control of injections, opsonic index and clinical 64

Staphylococcus, streptococcus, gonococcus, pneumococcus, tuberculosis, colon
Bacillus, Bacillus typhosus, Bacillus dysenteriae, cholera, and less com-
mon infections 73

Specific vaccines for diseases of unknown etiology: hydrophobia, small-pox. ... 81
Future of opsonins and vaccines 85

VIII. SERUM THERAPY.

Historical: Untoward effects of serum injections, serum disease and hyper-
susceptibility to serum; indications for immune serum injections; con-
centration and purification of serum; dried immune serum; oral adminis-
tration of serum 87

Anti-toxic serum: Diphtheria antitoxin; tetanus anti-toxin 99

Anti-bacterial serum: Anti-streptococcic syrum; anti-meningocoocic serum;
;inti-gonococcic serum; anti-pneumococcic serum; anti-typhoid serum;
anti-dysenteric serum; anti-staphylococcic serum 109

PREFACE

Treatment of individual diseases with medicines or by methods having
a selective curative action has until recently been limited. With the establish-
ment of the germ theory of certain diseases and the development of informa-
tion concerning immunity, new methods of specific treatment were made pos-
sible and are now practiced under the name of serum and vaccine therapy.
As might be expected, the medical profession has been much interested in these
methods of treatment, and applied them whenever possible. The develop-
ment of vaccine and serum therapy has been slow, the methods have had to
be revised and in some cases the results obtained have been found to be other-
wise than was at first expected. Because of this, much confusion has arisen.
The practitioner has not been able to keep pace with the developments and
literature on these subjects, and finally has been forced to depend on the state-
ments and recommendations coming from serum and vaccine laboratories,
enthusiasts and ever; exploiters.

In this work fin attempt has been made to state concisely and accurately
the present knowledge concerning vaccines and immune sera. An effort has
been made to establish theoretical and experimental evidence as well as clin-
ical application of the specific treatment of bacterial diseases. To accomplish
this, some space is given to infections in general, the theories of immunity,
with especial emphasis on the opsonic theory of immunity, as well as the par-
ticular methods of vaccine and serum therapy.

Considerable space has been given to opsonins, the opsonic index and the
importance of opsonins in health and disease. This has been done because
since 1904 no subject has appeared more prominently or frequently in medical
literature than that concerning opsonins, opsonic immunity, and bacterial
vaccines. In the first presentations of discoveries on this form of immunity
and specific treatment of bacterial diseases, great possibilities were promised.
Methods, which would, according to Wright, give uniform success in treat-
ment of the large class of bacterial infections and diseases, naturally received
immediate and general attention by the medical world and were at once quite
generally applied. This has been followed by much indiscriminate and un-
scientific use of these methods of specific treatment so that in the minds of

many, opsonins and vaccine therapy, have gone into disrepute as did tuber-
culin nearly twenty years ago. We now know tuberculin has many appli-
cations of importance in the diagnosis and treatment of tuberculosis, though
this knowledge has been gained some years later than it would have been had
it not been for improper exploitation. In the hope of avoiding a repetition
of such an effect as far as opsonin and bacterial vaccines are concerned, this
subject is given considerable attention. The opsonic index technique which
is given here is the one taught the writer by Dr. W. G. Ross, who was for two
years a pupil of Sir A. E. Wright.

An attempt has been made to bring the subjects taken up as nearly up
to date as is possible. It is hoped that this work may furnish to the medical
student and practitioner information which may lead him to a better under-
standing of the nature of infections and the subjects of immunity, and active
and passive immunization.

Columbia, Missouri, April, 1909.

CHAPTER I.

INFECTIONS

By the term infection we understand the entrance of micro-
parasitic living agents into the body tissue or substance, and
the occurence of definite symptoms of disease as the result of
the multiplication and action of the invading organisms. Infec-
tions do not always occur when pathogenic microorganisms are
present in the body tissues, certain conditions being necessary
for the establishment of a bacterial disease. These conditions
may be broadly divided into two classes; those dependent upon
the biological properties of the infecting organism, and those
dependent upon the conditions in the host and tissues invaded.

Of the biological characters of importance in the infecting
organism in the production of a bacterial disease, the most im-
portant are, that the microorganisms must be able to multiply
rapidly and greatly in the tissues of the body, and that they must
be able to produce poisons or substances harmful, either to some
or to all, of the tissues of the body. The number of organisms
necessary to cause disease varies with different species. Many
more staphylococci are necessary to cause the formation of a
furuncle than of anthrax bacilli to cause anthrax. The property
to produce substances poisonous to the body tissues generally
determines virulence. That virulence is of great importance is
evident from the fact that innumerable so-called saprophytic
bacteria which are present in the different parts of the body, grow
and multiply there but because in their growth sufficient poison
is not produced, cannot cause an infection. The virulence of an
organism, according to numerous investigators, depends upon
the presence of certain substances in the parasite which reduce
the resistance of the body and its tissues. These substances are
variously designated as lysins and aggressins.

8 VACCINE AND SERUM THERAPY.

Besides conditions in the causal organism, certain conditions
must exist in the host or body so that infection can occur. Cer-
tain animals are insusceptible to the action of certain species of
microorganism, thus for example, the horse and other domestic
animals are naturally immune to venereal diseases. Again cer-
tain organisms can only produce disease when they are present
in certain parts of the body and have entered the body by certain
portals of entry. The spirillum cholerae can only produce its
typical form of disease when it has gained entrance through the
small intestine. Invasion of the dermis or epidermis by this or-
ganism produces no disease. The bacilli producing tuberculosis as
well as certain other organisms, can cause infection when they
have entered through the skin or through the mucous membrane.
Certain organisms as Bact. diphtheriae, B. tuberculosis, and B.
typhosus, show predilections for certain tissues but will also pro-
duce diseased conditions in other parts of the body.

In addition to the non-susceptibility of the host, the body
possesses certain natural barriers to disease which must be over-
come before infections can be produced. The unbroken skin
usually offers a barrier to infection. Age, sex, race, occupation,
etc. , at times account for certain resistance to invasions of organ-
isms. The resistance furthermore varies for the different organ-
isms, as is evidenced by the fact that for B . tetanus a wound
is necessary to produce lock-jaw, while for the glanders bacillus
the slightest abrasion of the muccus membrane will furnish a
focus for an infection. The body fluids, lymph glands, phagocytes,
all offer resistance to infection. These barriers are overcome in
various ways and under different conditions, so that while indi-
viduals may be immune at one time they may, at another time,
be susceptible to the same infectious agent.

COURSE OF INFECTIONS.

From the foregoing it is evident that not only must the in-
fectious organism come into contact with the body tissues to
produce disease, but the organism must be able to grow, multi-
ply, produce its poison, and overcome the resistance of a body
susceptible to its action. The symptoms and signs of infections
do not appear until a certain time after the invasion by the micro-

INFECTIONS. 9

organism. This period is known as the "incubation period, " and
varies according to the biological characters of the infecting or-
ganism, but is also influenced by the number and virulence of
the organisms and the individual susceptibility of the host.

The course of a disease is determined by conditions produced
by the specific organism and partly by the distribution of these
organisms. But even here it is to be noted that the course of
the disease varies. Many of these variations we are by no means
able to explain.

The symptoms and signs produced by an organism will vary
as the organism acts locally or generally. In local infections
the most marked disturbance occurs at the portal of entry of the
microorganism, while in general infections the reaction manifests
itself in all, or a large part, of the body. The infecting microor-
ganism may produce disease either in a mechanical way because
of large numbers, or, as is the case of most all infections, because
of toxins which may act locally or be distributed over a large part
or the whole, of the body. Some organisms, as the bacilli of diph-
theria and of tetanus, produce extracellular toxins, while other
organisms, as those causing typhoid fever and cholera, have
intracellular toxins which are liberated supposedly when the
organisms disintegrate.

After organisms have entered the body the distribution varies
with the species. Thus we see that staphylococci form furuncles,
carbuncles and pustules in localized areas, typhoid bacilli though
causing lesions in the intestine are usually present in "the blood
stream, while the tetanus bacilli form a toxin which is distributed
through the entire body. At times microorganisms do not remain
localized at the portal of entry but pass on to the lymphatic
glands and other parts of the body, here producing pathological
conditions. In some infections the microorganisms gradually
involve more and more of the tissues of the body. Microorgan-
isms may first lodge in one part of the body and produce disease
in that part and from these primary lesions, other parts of the
body may become infected. When the blood stream is not only
the carrier of microorganisms but becomes the place for growth
and reproduction of the same, a septicaemia arises. While almost
any of these conditions may be produced by any of the pathogenic
bacteria, still certain organisms are more likely to produce one

10 VACCINE AND SERUM THERAPY.

or another of these conditions. Resultant of this tendency of
microorganisms to produce particular kinds of conditions, the
prognosis for infections varies with the species of microorganism
producing the lesions or condition.

The same species of microorganism will not always be distri-
buted in an identical manner; the location of the lesion and the
condition produced varying with the properties of the particular
strain in question and the particular part of the body infected.
The appearance of general symptoms will vary greatly with the
rapidity of the absorption of the toxin. Experimentally, intra-
venous and intraperitoneal injections of microorganisms are fol-
lowed by symptoms earlier and more consistently than are sub-
cutaneous injections.

The number of bacteria invading the tissues is of some im-
portance. If few bacteria are present they may die without
producing -infections, while if larger numbers are present some
of them will be likely to grow and produce disease.

At the point of infection, or portal of entry, the effects pro-
duced will vary with the infecting organism, some producing
specific forms of inflammation along with those common to many
bacteria. Even with the same species of microorganism the
reaction will vary with the prevalence and virulence of the invad-
ing organism and with the anatomical structure of the part of
the body involved. The local reaction, however, is not only pro-
duced when the bacteria enter the tissues of the body but also
when the "toxins produced by the microorganism are present in
the tissues. The most commonly seen reaction at the portal of
entry is that which results in pus formation, in which, due to
chemotaxis, leucocytes accumulate. While a local reaction is
usually produced at the portal of entry of bacteria, the reaction
at times may be slight or absent entirely. A local reaction when
produced is generally regarded as of importance in the protection
of the body against the invasion of bacteria.

Besides the local reaction, general reactions usually follow
in all severe infections. General reactions occur as a result of
the action of toxins produced by the microorganisms when they
are absorbed by body tissues. The general symptoms produced
vary according to the location of the primary lesion, the extent
of the process, the peculiarities of the organism, and the resistance

INFECTIONS. 1 1

of body tissues. The more common general reactions are fever,
digestive disturbances, effects on the nervous system, leucocytosis,
anaemia, and enlargement of the spleen and oilier glands.

Infections and infectious diseases may be divided into two
classes; one in which the disease runs a self-limited course, recovery
coming after a definite period of time has elapsed, while in the
other class the course may be extended over a very indefinite
period of time.

The elimination of the casual or etiological factor of infections
and infectious diseases occurs in various ways. When the seat
of the disease is on the surface of the body, the organisms are elim-
inated with the diseased tissues, but when the organisms are
found in parts of the body where this is impossible, the microor-
ganisms must enter the general circulation, be allowed to pass
through the glandular tissues and be eliminated with the secretions
and excretions. In the blood and the body tissues, bacteria
are in many cases destroyed by bacteriolytic and bactericidal sub-
stances. Sometimes after an apparent recovery from the disease,
viable organisms may remain and at a later time again give rise
to disease.

CHAPTER II.

IMMUNITY

Immunity may be defined as, non-susceptibility to a disease,
or as the ability to resist the action of the causes of the disease.
The body may be immune because of inherited properties or
because it has become so during life. Immunity because of in-
herited properties is called "natural" immunity, while the im-
munity acquired during life is called "acquired" immunity.

Natural immunity is demonstrated by the non-susceptibility
of the hen to the action of the tetanus bacillus. It is an im-
munity of race or species. This immunity at times may be re-
duced or removed by hunger, exhaustion, exposure to cold, etc.
Certain closely related races or species of animals sometimes show
a natural immunity and at times a natural susceptibility to the
same infecting agent. This non-susceptibility is frequently called
a natural resistance and at times is only an apparent immunity,
depending in these cases on the common natural barrier to the
entrance and development of disease producing organisms. Again,
what may be regarded as a natural immunity is, in part at least,
only a resistence to infection due to the inability of organisms to
reach viable tissues. This is the case when the acidity of the
stomach is sufficient to kill cholera organisms before they reach
the epithelium of the intestine.

Acquired immunity only results after a pathological condi-
tion exists or has existed. The individual becomes immune,
because he has survived a natural course of the disease, as is the
case following an attack of scarlet fever; because he has gone
through a modified form of the disease, as is the case in vaccination
against small-pox; or, because he receives substances prepared by
some other individual or animal that has gone through a natural

IMMUNITY. 13

or modified course of the disease. Acquired immunity may be
either active or passive.

Experimental active immunization is usually called vacci-
nation and generally produces in the individual a modified form
of the disease. All, or nearly all, of the symptoms are less severe
than in the natural course of the disease. The individual in this
case produces his own immunity. In artificial or intentional in-
oculation, the etiological factors, or more particularly, the causal
organisms injected must be so modified that the natural course
of the disease will not follow the inoculation or injection. Ex-
perimentally acquired active immunity is produced by the in-
jection of living or killed microorganisms, or of toxins produced
by these organisms. When living organisms are injected their
virulence is usually reduced by passage through animals, growth
at high temperatures, on artifical media, in the presence of chem-
icals, or growth in the presence or absence of oxygen, etc. Active
immunity may also be acquired as a result of injection of living
virulent organisms into such parts of the body where the disease
will not be produced. The first amounts injected are usually
smaller than those in subsequent injections. When bacteria are
injected the immunity produced is a bacterial immunity and
when toxins are injected a toxin immunity results.

In passively acquired immunity, the individual that becomes
immune does little or nothing toward obtaining this immunity.
It results from the injection of immunizing substances which
have been prepared by an actively immunized individual or
animal.

There are two classes of immunizing substances; those
that act on bacteria, said to be anti-bacterial; and those that
act on toxins, called anti-toxic. Of these two classes of immune
substances, the anti -toxic has been more efficient in passive im-
munization.

After the formation of immunizing substances they do not
remain indefinitely within the body, but are lost through the ex-
cretions either as immune bodies or as immune bodies broken
down by the body cells. On this the difference in persistance of
natural and acquired immunity is partly based, for in acquired
immunity the supply of immune bodies is exhausted, while in
natural immunity new substancss are constantly formed.

14 VACCINE AND SERUM THERAPY.

THEORIES OF IMMUNITY.

Acquired immunity, as has been stated, results because some
individual or animal has gone through a natural or modified
course of the disease. While acquiring immunity, the body and
its tissues have in some way been modified. Various theories
have been advanced to explain this phenomenon.

Klebs and Pasteur tried to explain the changes that occur
in the acquisition of immunity by assuming that in going through
a natural or modified course of disease, certain substances, nec^
essary as food for the parasites, are used up. As the result of
immunization, the food necessary7 for the microorganism is con-
sumed and the individual is immune to a certain organism be-
cause this organism cannot obtain the food it needs for its ex-
istence and production of the disease.

Chauveau assumes that in immunization certain products
of bacterial metabolism are retained in the body of the immun-
ized animal or individual, which products protect the body tissues
from further invasions by that particular parasite.

MetchnikofT, in 1883, formulated a theory according to which,
during immunization, certain white blood cells and cells of certain
organs acquire the ability to engulf and destroy the attenuated
bacteria.. This results in acquiring the ability to engulf and de-
stroy more virulent forms of this same species of microorganisms.

EHRLICH'S SIDE CHAIN THEORY OF IMMUNITY.

In 1887 Salmon and Smith, Foa and Bonome, Roux and
Chamberland, and others found that immunity could be produced,
not only bythe injection of bacteria, but also, as a result of the injec-
tion of the products of bacterial metabolism. As a result, a chemi-
cal theory, of immunity was advanced. According t<? this theory
the tissues of the body are chemically changed by immunization.

Fodor, in 1887, was the first to observe that the normal body
fluids, especially the blood, contain certain substances able to
destroy bacteria. Buchner, Behring, and Nuttall, soon after
Fodor's observation also recognized the bactericidal powers of
certain sera. In 1889 Buchner reported that the cell -free blood
serum contains certain substances which he called alexines.
Alexines, he found, have the property, of destroying bacteria.

BHRLICH 8 rHBORY OF IMMUNITY. 15

In isss Ifericourt and Richet, and in iss'.i Hal>es and Lepp re-
ported investigations which showed that by injections of blood
serum from animals having acquired immunity to certain diseases,
immunity to these same diseases can be conferred to other ani-
mals. Soon after this Behring and Kitasato reported successful
immunization of rats to tetanus by means of injections of blood
serum from rabbits immunized to tetanus. From the work of these
investigators there developed the humeral theory of immunity.

Behring and Knorr found that the toxic product of the meta-
bolism of the tetanus bacillus, could, without the presence of the
bacillus, be used in immunization. The serum thus produced, they
found would protect against the disease and the injection of the
by-products of the tetanus bacillus. They assumed from this
that toxin is neutralized by the immunizing substance in the m-
mune serum, as a base is neutralized by an acid.

Numerous theories have been advanced to explain the facts
observed and reported, but of all these theories one stands out
prominently — Ehrlich's side chain or receptor theory, advanced
by Ehrlich in 1897.

According to this theory, every living cell consists of a domi-
nating nucleous (Leistungskern) and of side chains or receptors.
The paradigm of this picture is to be found in the benzol ring
with its side chains. The side chains or receptors of a cell are of
many different kinds and serve usually to anchor and assimilate
the food stuffs. At times, however, by means of the receptors
the cells are combined with substances, not foods but cell poisons.
The combination of the receptors of the cell with the receptors of
foods differs from the combination of receptors of the cell and re-
ceptors of toxins. The combination of the receptors of the cell
with the receptors of foods is so loose that after undergoing certain
changes which are of value in assimilation of the food and the
nourishment of the cell the food stuff is again eliminated without
having injured the receptor. Toxins on the other hand, combine
so firmly with the receptor of the cell that they cannot ajjain
be separated. As a result of this, receptors anchoring toxins
are lost to the cell.

When a certain limit of anchoring of receptors by toxins has
taken place the cell peri hes, and if a sufficient number of cells
which have vital functions to perform are destroyed, death of

16 VACCINE AND SERUM THERAPY.

the individual follows When, however, the toxins do not anchor
enough receptors or are not potent enough to destroy the cell
and the organism, immunity results or follows.

In 1896, Weigert advanced the hypothesis that when tissue
is lost the regeneration that follows is not only sufficient to restore
the amount of tissue lost, but actually results in overproduction
of this tissue. This process is observed in the over-production
of segments in reptiles and of cell proliferation in granulating
wounds in man and the animals. Ehrlich assumes that when
the toxins combine with the receptors of the cell, the receptors
are lost to the cell and as a result the cell is stimulated to repro-
duce the destroyed receptors. The process does not stop, how-
ever, when all lost receptors have been reproduced, for more re-
ceptors will be produced than were anchored by the toxins. The
excessive production of receptors as a .result of the stimulus fol-
lowing the anchorage of the receptors by toxins, Ehrlich assumes,
leads to a disturbance of the equilibrium of the cell. As a result
of this, the surplus receptors are thrown off and constitute what
is known as the anti-body. This process can be actually ob-
served in the lower animals in which extra parts, produced
as a result of destruction of some parts, are cast off or lost.

The principal function of all receptors and side chains is to
provide for the nutrition and metabolism of the cells. Receptors,
and hence immune bodies, however, are not all of the same com-
position or even of the same general structure. In order to ex-
plain the different functions and actions of different immune
bodies, Ehrlich has assumed that the receptors may be of simple
constitution and structure or they may be complex. Any cell
of the body may have large numbers of the same and different
kinds of receptors. Ehrlich divides this large number of receptors
into three orders.

RECEPTORS OF THE FIRST ORDER.

These are receptors that are of relatively simple constitution
and structure and combine with substances that can be easily
and readily anchored. Bacterial toxins anchor receptors of
this order. The receptor here consists of only one haptophore,
or combining group, which combines directly with the hapto-
phore group of the bacterial toxin. This order of receptors and

RECEPTORS — FIRST AND SECOND ORDER.

17

immune bodies is demonstrated in Figure 1, and represents the
type of receptor on which is based the action of bacterial toxin
and the formation of anti-toxin.

Fio. 1.

RECEPTORS OF THE SECOND ORDER.

The receptors of the second order are distinguished from
those of the first order in that the receptors here have in addition
to the haptophore group, a zymophore group. This zymophore
group acts on the larger food particles, making them more readily
assimilable. In a similar manner it acts on the bacterial cell.
Receptors of this kind, possessing haptophore and zymophore
groups, are broken off from the cells and circulate in the blood as
agglutinins and precipitins after immunization by the injection
of certain bacteria. The haptophore group of the immune body
in an agglutinating or precipitating serum combines with the
bacterial cells. The zymophore group, however, does not com-
bine with anything but exerts its influences entirely through the
haptophore group. The zymophore group is destroyed by age,
acids, heating to 65° C., etc. Receptors and immune bodies of
the second order are represented in Figure 2.

FIG. 2.

18

VACCINE AND SERUM THERAPY.

RECEPTORS OF THE THIRD ORDER

The receptors of the third order are likewise adapted to the
anchorage of bacteria. These receptors, however, have two com-
bining groups: one for the anchorage of cells or food substances
and the other for the anchorage of substances which, acting through
the receptors, can produce a ferment-like action. This latter
substance is called an activating substance and is present in nor-
mal sera. It is known either as complement or alexin.

Complement is easily destroyed by ageing, acids, heat, 55° C.,
being sufficient to destroy it. When the receptors of this order
are anchored by bacteria in numbers not sufficient to kill the cell
but to stimulate it to over-production of receptors, the extra
receptors are thrown off. The receptors thrown off constitute
the immune body, amboceptor or substance sensibilitrice of Bor-
det. They have two combining or haptophore groups, one for
the combination with bacterial or other cells and substances,
called the "cytophylic" group; the other for combination with
alexin or complement, called the "complementophore" group.
It is by means of complement that the amboceptor is able to
dissolve bacteria, red blood cells and other substances. Immune
sera containing receptors of the third order are bacteri-
olytic, haemolytic, or cytolytic, depending upon whether they,
together with complement, can dissolve bacteria, red blood cells,
or other cells. The receptors of the third order are graphically
represented in Figure 3.

FIG. 3.

It will be observed that receptors of the first, second or third
order can be produced in excess to form immune body or anti-

ANTI-BODIES. 19

body. Specific immune bodies are produced for many substances,
toxins, bacterial cells, red blood cells, ferments, etc. As a result
of this multiplicity of substances that can cause the cells to
produce anti- bodies, ant i- bodies may be anti- toxic, aggluti-
nating, precipitating, lytic, etc. To produce agglutination,
precipitation or lysis it is necessary that a ferment-like sub-
stance be a part of, or able to combine with, the immune body.
This ferment-like substance, as stated before, is labile and lost
on ageing, heating, etc. When this is lost the serum containing
the immune body is said to be inactive. In normal serum there
is present a substance called complement which can again reacti-
vate a lytic serum. The agglutinating power, however, cannot
be restored by the addition of fresh complement. Complement
can be preserved for long periods of time if the serum is dried
without heat soon after the blood is drawn. Whether there is
only one complement or a multiplicity of the same has not been
definitely decided, Ehrlich assuming that there is a multiplicity
of the same, while Bordet and Buchner claim there is only one.

It has been demonstrated that anti-toxic substances are im-
portant in preventing the action of toxins on the body cells, while
lytic substances have been found to protect the body by the
solution and destruction of bacteria. Whether the agglutinat-
ing or precipitating substances are of importance in destroying
bacteria, is by no means certain. Numerous investigators as-
sume that they injure and change the bacterial cells or substances
used in immunization, while others claim that they exert no
such action. It has been quite definitely observed that it is pos-
sible to cultivate and grow bacteria that have undergone ag-
glutination.

As has been stated earlier, immune bodies are formed only
for such substances as are able to combine firmly with the recep-
tors of the cells, and it is on this assumption, that Ehrlich explains
the impossibility of producing immunity to certain poisons, as
the alkaloids, strychnine and morphine.

The cells whose receptors anchor the substances and cells for
which immune substances are formed, are probably widely dis-
tributed throughout the body. The particular tissues in which
anti-bodies are formed has not been determined and probably
varies for the different substances to which immunization can be

20 VACCINE AND SERUM THERAPY.

obtained. It seems quite definite that in certain cases, internal
organs may contain more immune substances than the blood se-
rum. After immune bodies have been formed they do not remain
permanently in the body, but are gradually lost through destruc-
tion in the body or lost with the excretions.

Ehrlich's views on the formation of immune bodies are quite
generally accepted. His views, however, concerning the com-
bination of toxin and anti-toxin, agglutinable substances and
agglutinins, and substances capable of solution and lysins are
not as universally accepted. Ehrlich assumes that the union of
immune bodies and the specific substances is a chemical combina-
tion. Bordet assumes that in lytic sera the immune body acts
as a mordant which sensitizes the substance capable of solution
to the action of the complement. Field has explained the inter-
action of immune bodies and specific substance according to the
principles of colloid chemistry, so that for example in a toxin
and anti-toxin combination one is adsorbed (physical rather than
chemical) by the other.

OPSONIN THEORY OF IMMUNITY.

It has been stated earlier that in 1883, Metchnikoff advanced
his phagocytic theory of immunity. According to this theory
the presence or absence of immunity depends upon the, ability of
phagocytes to engulf and destroy bacteria. Metchnikoff had
given serum little consideration in immunity although he believed
that some sera contain substances which stimulate the leucocytes
to engulf bacteria. These substances he called "stmiulins." The
phagocytic theory, however, was practically replaced by the
humeral theory of immunity because Fudor, Buchner, Behring,
Nuttall and others had found that serum containing no cells
whatever can destroy bacteria by solution. It was also found
that antitoxin, which combines with toxin, is carried in serum.

From 1887 on the humeral theory of immunity gained a
strong foothold and it was not until 1895 that attention was again
called to the function of the leucocytes in immunity. In this
year Denys and LeClef reported experiments from which they
concluded that in immunization certian changes are produced
in the serum, which make it possible for the leucocytes to engulf
bacteria. In their conclusions they state that leucocytes from a

OPSONIN THEORY OF IMMUNITY. 21

normal rabbit on the addition of normal rabbit serum, have only
slight phagocytic action for the streptococcus. If, however,
leucocytes from a normal rabbit, or from a streptococcus-immune
rabbit are added to the serum from an animal vaccinated
with streptococci, the leucocytes actively engulf and destroy
the streptococci. When streptococci, able to produce erysipelas
in a normal rabbit, are injected under the skin of a rabbit vacci-
nated against streptococci, disease is not produced. This pro-
tection is due, according to these investigators, especially to the
ability of the leucocytes to destroy streptococci. They attribute
the increased phagocytosis to an action of the immune serum on
the leucocytes.

The results obtained and conclusions arrived at by Denys
and LeClef were repeated by Bordet, who was not able to verify
the results obtained by these investigators. Mennes found that
immunity to Mic. pneumoniae depends on the action of serum
which produces active phagocytosis but was not certain that
this action is due to stimulation of the leucocytes.

In 1903 Wright and Douglas pointed out that there are cer-
tain substances in serum that so affect bacteria that they are
more easily taken up and disposed of by the leucocytes. This
substance they called "opsonin." They found opsonin present in
normal and immune serum and from their investigations decided
that the amount present in serum is variable, and can be increased
or decreased by the injection of killed cultures of bacteria. To
determine the amount of opsonin present in blood they modified
the method of estimating phagocytic power introduced by Leish-
man in 1902. According to their method the average number
of bacteria taken up by leucocytes, when bacteria, leucocytes and
serum are mixed together and allowed to remain together for a
certain time, is determined. The ratio of the average number
of bacteria per leucocyte when patient's serum and serum from
a healthy individual determine the phagocytosis, they called the
opsonic index. The methods employed in the determination of
this opsonic index will be taken up later. %

In 1904 Neufeld and Rimpau, entirely independently of
Wright and Douglas referred to the two well-known elements,
antitoxin and bactericidal substances, in immune serum and stated
that in anti-streptococcic and anti-pneumococcic serum they had

22 VACCINE AND SERUM THERAPY.

found a third factor of importance in immunity. This element,
according to these investigators, sensitizes the corresponding
bacteria and so modifies them that they are more readily engulfed
by the leucocytes. This third factor in immunity they called
"bacteriotropin." It is to be noticed that Neufeld and Rimpau
worked with virulent streptococci and pneumococci. These or-
ganisms are not ingested by the leucocytes in a mixture of leuco-
cytes, virulent streptococci or pneumococci and normal serum.
However, when, instead of normal serum, a specific anti-serum
takes part in the mixture there is ingest ion even of cultures of
virulent strains of these organisms. They furthermore showed
that the serum does not act on the leucocytes but exerts its in-
fluence on the bacteria, sensitizing them to the action of leuco-
cytes.

There seems now to be but little doubt that opsonin and
bacteriotropin are the same substances, the greatest difference
that can be assigned, being that bacteriotropins are probably
what Wright and Douglas have called "immune opsonins."

Most investigators now take an intermediate position on the
phagocytic and humeral theories of antibacterial immunity. It is
quite generally accepted that both serum and leucocytes contain
substances, which, acting after the manner of ferments, are able
to dissolve bacteria. Just as all important manifestations of life
are found in the normal and pathological cellular elements, so also
the means of defense against harmful agents is probably closely
related to the condition and functions of cells which prepare and
secrete the protective substances by means of which bacteria and
other harmful agents are destroyed or neutralized.

CHAPTER III.

THE OPSONIC INDEX

From the foregoing chapter it is seen that since the work
of Denys and LeClef in 1895, it has been known that serum is of
importance in phagocytosis. Little attention, however, was
given to the various observations on this subject until the work
of Wright and Douglas was presented. Although Neufeld and
Rimpau discovered probably the same substances as Wright and
Douglas, these investigators are seldom referred to in the discusion
of the substances that change bacteria so as to prepare them tor
ingestion by leucocytes. There are two definite scientific reasons
for the great prominence given to the work of Wright and Doug-
las: one is that they advanced and improved a technique for the
determination of the amount of phagocytosis, by means of which
the opsonic index could be determined; the other is to be found
in the widespread interest in the methods they advanced for
active immunization of patients by the injection of killed cultures.

In 1902 Leishman presented a method for the quantitative
determination of phagocytosis. He mixed equal quantities of
patient's blood and bacterial emulsion, which he then incubated
for a time in a moist chamber. After incubation he made cover
glass spreads which he dried, fixed and stained. On these slides
he counted the number of bacteria ingested by the leucocytes,
from which the average number per leucocyte was determined.
This average he compared with the average per leucocyte obtained
when normal blood instead of patient's was added to the bacterial
emulsion. Leishman did not take into consideration the action
of serum on bacteria or leucocytes but devised merely a "method
of estimating phagocytic power."

Wright and Douglas, in their work on the determination of
the opsonic index modified Leishman's method to meet their
theory on phagocytosis. According to their observation opsonin

24 VACCINE AND SERUM THERAPY.

is a substance present in all serum. The amount varies in the
different sera and as the amount varies so also will the amount of
phagocytosis vary. To determine the phagocytic power it is
necessary to have the three factors of phagocytosis in the mix-
ture. These three factors are found in the blood serum, leuco-
cytes and bacteria. A mixture of these three must be allowed
to remain together for a definite period of time, after which the
average number of bacteria taken up by the polymorphonuclear
neutrophiles must be determined. This average number of bac-
teria per leucocyte is called the "phagocytic index." The
phagocytic index obtained when serum from the patient is mi^ed
and incubated with leucocytes and bacteria, is compared with
the phagocytic index obtained when serum from a healthy in-
dividual is added to and incubated with a similar amount of the
same emulsions of bacteria and leucocytes. The result obtained
is called the "opsonic index."

The technique here given is essentially the one developed
by Wright and Douglas and demonstrated in New York City in
1906 by Wright and taught by his associate, Dr. Ross. It is
given somewhat in detail inasmuch as it is the one most generally
used, even though many modifications have been suggested and
followed by different investigators.

SERUM.

Blood from the individual whose serum is to be used in the
determinations of the opsonic index, is collected as follows: A
small glass capsule with one curved capillary limb is made as is
indicated in Fig. 4. This tube is brought to a needle's point at
"b" by heating over the pilot flame of a Bunsen burner. This

FIG. 4.

point is later used to puncture the finger or lobe of the ear. The
end "a" of the capsule is left open. One of the fingers of the

COLLECTION OF SERUM. 25

hand is cleaned with alcohol and water, after which a bandage,
handkerchief or rubber band is quite firmly tied around this fin-
ger. The windings of the bandage, handkerchief or rubber band
are started at the base of the finger and run gradually toward
the tip of the finger as more turns are put on. In this way there
is produced an accummulation of blood in the veins and capil-
laries. Now, with the pointed end of the capillary bulb, the fin-
ger is pricked at a point about a quarter of an inch back of the
nail, the needle end is broken off enough to open the capillary
end. The open end "a" is now held close to the drop of blood
which gradually fills the bulb; this is indicated in Fig. 5.

Fio. 5.

When enough blood has been drawn and collected in the bulb, the
bulb is held between the fingers at "a" and "c" and the end"b" is
heated and sealed off. As the end "b" cools the blood will draw
away from the end "a" and finally all the blood will collect in
the bulb proper. After this the bulb is again taken hold of at
the curve "c" and by a rapid swing of the arm, as is practiced
in shaking down a clinical thermometer, the blood is thrown down
into the sealed end "b" as is indicated in Fig. 6. The blood is
now allowed to clot in order to expel the serum. The serum
may also be separated by hanging the bulb at "c" over the arm
of the centrifuge, and centrifuging until the clot and serum have

26 VACCINE AND SERUM THERAPY.

well separated. The tube may now be opened by filing and break-
ing at "c."

FIG. 6.

According to the original method of Wright and Douglas,
blood was obtained from the patient and from a number of
healthy individuals. Equal parts of the normal serum were taken
and mixed together or "pooled." This was done so as to get a
better normal serum. After it has once been established that
serum from a certain healthy individual has an opsonic index
of 1 . 0 for a particular organism when compared with pooled
serum, this individual's serum replaces the pooled serum. After
this has been established, the opsonic index of any patient for
this organism is determined by dividing his phagocytic index
by the phagocytic index of the "normal individual," as he may
now be called.

LEUCOCYTES.

The leucocytes used in the determination of the opsonic
index are usually obtained from the blood of supposedly healthy
individuals, most frequently from the blood of the investigator
himself. No particular stress, however, is laid upon the individ-
ual from whom the blood is obtained.

To obtain blood, preferably, the middle finger is cleaned, con-
gested and punctured in the same manner as was followed in the
collection of serum. Ten drops of blood are collected in about
lOc.c. of a normal salt solution containing one per cent of sodium
citrate. The sodium citrate is added to keep the blood from
clotting. The tube containing the mixture is then centrifuged
in a centrifuge of not too great speed. When the speed exceeds
1,200 to 1,500 revolutions per minute, the cells are too closely

LKUCOCYTES EMULSION. 27

packed together and form clumps. The mixture is centrifuged
until the corpuscles are thrown to the bottom of the tube and
the fluid above is clear, although it may be slightly straw
colored. The supernatent fluid is then drawn off with a
pipette. The corpuscles are washed free from serum and sodium
citrate by again filling the tube with normal salt solution, mix-
ing thoroughly and centrifuging until there again is a clear super-
natent fluid. If the material in the centrifuge tube is now ex-
amined, one sees a clear fluid above and a red fluid in the lower
part of the tube. The red fluid below, however, is not of the
same shade throughout. The uppermost part consists of a gray-
ish red layer, the leucocytes. These are layered above the red
blood cells because of the difference in specific gravity of the
leucocytes and red blood cells. It is from the gray layer that
the leucocytes, to be used in the determination of the opsonic
index, are obtained.

To obtain the leucocytes the clear fluid above is drawn off
with a pipette and then with a clean pipette of about 1 . 6m. m.
inside diameter, the leucocytes are removed. To do this the
pipette firmly held in the hand, all of the air in the nipple
is expelled, and then as the pressure on the nipple is gradually
released, the open end of the pipette is held on the surface of the
uppermost layer of grayish red color. This fluid will contain
many red blood cells and also a relatively large number of leuco-
cytes. After this fluid has been drawn into the larger part of
the pipette the capillary end is sealed off. This fluid is called
"leucocytic cream." Wright and Douglas state that in their
experience there is no variation of the ability to engulf bacteria
within the space of a few hours but that after three days the
phagocytic power decreases to one-half or one-third of what it
was when freshly drawn.

f
BACTERIAL EMULSION.

Inasmuch as Wright had early decided that opsonins are
specific, such species or varities of bacteria are used in the deter-
mination of the opsonic index as may be of importance in the
bacterial infection. In the selection of the particular culture to
be used, there are two sources — either the different cultures iso-
lated from the lesion are used or else the same species and varieties

28

VACCINE AND SERUM THERAPY.

of microorganisms are taken from a stock culture. Usually liv-
ing freshly grown bacteria are used to make emulsions. The
principle, or practically only exception to this has been in the
determination of the opsonic index for the bacillus of tubercu-
losis, for which often killed and old cultures of the organism have
been used to make the bacterial emulsion.

In all cases it is intended that the bacteria shall be well sep-
arated and suspended uniformly in salt solution. The tech-
nique for making the bacterial suspension varies with the differ-
ent organisms, some of which it will be necessary to consider
separately. The organisms for which the opsonic index is de-
termined may be divided into three classes.

A. Many of the organisms belonging to this
class grow on the ordinary media, while for
others, media containing blood or other body
fluids are necessary. The organisms occur
singly or in pairs and when present in larger
groups the cell aggregates can be easily
broken up.

(a). Mic. pyogenes aureus, albus and citreus,
B. coli, B. typhosus, B. pyocyaneus, etc., are
grown on slant agar for twenty-four hours
at 37° C. Various investigators, including
Wright, have used four to five hour cultures
of these organisms.

(b). Mic. gonorrhoeas has usually been
grown on hydrocele or human blood agar.
Cole and Meakins obtained their cultures from
growth on agar in which 0.5 c.c. of fresh
blood had been added to 10 c.c. of ordinary
agar. The age of the cultures used has varied
from four to twenty-four hours incubation
at 37° C.

(c). Mic. meninigitidis, Mic. pneumonias,
etc., produce good growth on sheep serum
agar. The cultures have usually been incubated at 37° C. from
twelve to twenty-four hours.

To each culture of the proper age about ten drops of sterile
normal salt solution are added. With a platinum loop the cul-

FIG. 7.

BACTERIAL EMULSION. 29

ture is washed off the media and with a thick walled capillary
pipette, with the end broken off squarely, the suspension is drawn
into the pipette. The open end of the pipette is firmly pressed
against a watch glass, as indicated in Fig. 7. When this is done
only a small crevice will remain between the end of the pipette
and the watch glass. The bulb is now compressed and the bac-
teria suspended in the salt solution are forced out through the
small crevices. This is done to break up the small clumps of bac-
teria.

B. The organisms belonging to this class grow on the same
kind of media as do those belonging to the first group. The or-
ganisms of this group form cell aggregates that are not easily
broken up. The streptococci are the most important organisms
in this class. Streptococci frequently grow in long chains,
varying from two to thirty cocci. It is evident that a leuco-
cyte may be able to engulf one or more cocci without being able
to engulf a chain consisting of twenty or thirty cocci. The method
for breaking up ordinary clumps will not suffice for breaking up
chains of streptococci.

While observing the opsonic index for streptococci in ery-
sipelas, the writer found it necessary to adopt some method to
break up the long chains of streptococci frequently isolated from
the lesions in erysipelas. This method consisted in the addition
of 2 to 3 c.c. of sterile salt solution to each twenty-four hour cul-
ture on glycerine, glucose agar. After washing off the growth,
the emulsion was put into a small test tube containing sterile
sea sand; the tube was sealed in a flame and shaken for one and
one-half hours in the shaking machine. The sand and emulsion
in the tube were centrifuged for one minute and the supernatent
fluid drawn off. In this way short chains, from two to four
cocci, were obtained.

Another method was devised by Wright of Harvard University.
This method differs only from the one of Wright and Douglas in the
substitution of a paraffine block for the watch glass. When the
open end of the pipette is held against the paraffine the crevices
very small so that the chains are more completely broken up.
This method gives very satisfactory results.

C. This class includes practically only one species of mi-
croorganisms, the bacillus of tuberculosis. The preparation of

30 VACCINE AND SERUM THERAPY.

suspensions of tubercle bacilli is attended by numerous difficul-
ties, because this organism when grown on artificial media forms
conglomerated masses. According to Wright and Douglas the
living tubercle bacilli are heated to 100° C. before breaking up
the clumps. Later Wright modified this techinque by heating
the bacilli to 100° C. on three successive days. The clumps after
this are ground up in an agate mortar or in a watch glass, two or
three drops of 0 . 1 per cent salt solution being added at a time until
two or three c. c. have been added. One-tenth per cent salt
solution is used in making this suspension because with greater
concentration the bacilli are again clumped. Later Wright added
1 . 5 per cent salt solution in making up the emulsion of tubercle
bacilli because he found that this concentration is necessary to
prevent spontaneous phagocytosis. When the heated bacilli are
* thoroughly rubbed and suspended in 1 . 5 per cent salt solution
a homogeneous mixture, containing but few clumps and many
isloated bacteria, results. This mixture is then centrifuged at
high speed for about ten minutes. After this the supernatent
fluid is drawn ofT, and enough salt solution added to get the right
concentration of the emulsion.

While this method gives a fairly homogenous suspension of
tubercle bacilli, still in an emulsion made in this way many of
the tubercle bacilli are broken up. In determining the opsonic
index it is necessary to count the number of bacilli taken up by
the leucocytes, and when there is fragmentation of bacilli it is
necessary either to count each fragment as one bacillus, or else to
determine the fractional part of a bacillus. Either of these
methods is most unsatisfactory.

Sellard and Jeans have emulsified the living tubercle bacilli
in the same manner that has been used for the emulsification of
other bacteria. After this they have killed the bacilli by exposing
the emulsion to sunlight for a number of hours — ten hours being
sufficient to kill all tubercle bacilli present. In such emulsions
they have gotten no spontaneous clumping, no fragmentation, nor
spontaneous phagocytosis.

Walker has recommended a method, according to which Dor-
sett's egg medium is heavily inoculated with an actively growing
culture of the bacillus of tuberculosis. After fourteen to eighteen
hours of incubation at 37° C., salt solution is squirted over the

MIXTURE OF BACTERIA, LEUCOCYTES AND SERUM. 31

culture, which is now rubbed off into the salt solution, the clumps
being broken up with a platinum needle. The suspension is then
filtered first through a loosely packed cotton filter and later through
a filter made with scraped filter paper. Filtration is repeated
until all clumps are removed, after which the clump-free suspen-
sion is heated to 75° C. for twenty to thirty minutes.

STRENGTH OF BACTERIAL EMULSION.

The strength of bacterial emulsion most commonly used has
a slightly opalescent appearance. It has been found that cloudi-
ness may be absent or be only very slight and still the emulsion
may contain too many bacteria. According to Wright's instruc-
tions the strength of the emulsion to be used is one which gives
an average count of from five to eight bacteria for leucocytes
when normal serum, leucocytes and the suspension of bacteria
are incubated for the proper length of time. Walker has obtained
better results with heavier suspensions of bacteria and diluted
serum. It is not very difficult to make an emulsion of staphy-
lococci, for after a bit of experience it can be determined by the
naked eye whether the suspension of this species is heavy enough
or not. The tubercle bacillus, however, presents greater diffi-
culties, it frequently being necessary to actually make a trial
test for the bacterial emulsion. Simon and others have proposed
the numerical determination of the number of bacteria per c. c.
in the bacterial emulsion.

MIXTURE OF BACTERIA, LEUCOCYTES AND SERUM.

From the definition of the opsonic index, it is evident that
similar quantities of the same factors must be taken to make com-
parative mixtures. Furthermore, according to the methods de-
vised by Wright, equal amounts of each factor are mixed to-
gether. When it is desired to use diluted serum equal volumes
may still be used if the serum be diluted properly before taking
the volume.

In order to get equal volumes Wright has made a capillary
pipette, the walls of the capillary part of which are thick. The
end of the pipette is broken off squarely. About a quarter or
one-half inch from the open capillary end a mark is made with a
soft wax pencil. This pipette is shown in Fig. 8. By means of

32 VACCINE AND SERUM THERAPY.

a rubber teat which is attached to the large end of the pipette,
leucocytic cream is drawn into the capillary end up to the mark.
Then the capillary end is withdrawn from the leucocytic emulsion
and the emulsion in the tube is drawn about one quarter inch
further up into the tube. After this the capillary end of the
pipette is immersed in the bacterial emulsion, which is taken into
the tube up to the mark. The tube is withdrawn and again a
small amount of air drawn into the capillary tube. After this the
serum is drawn in, again taking the amount necessary to fill the
capillary pipette to the mark. Walker has suggested that small
quantities of serum and leucocytic cream be put into small tubes
and that serum and leucocytes be taken only once from each of
these small tubes. This is done in order to avoid carrying ma-
terials from one tube into another.

The capillary pipette now contains equal volumes of leuco-
cytic cream, bacterial emulsion, and serum, as is shown in Fig. 9.

FIG. 9.

All three volumes are now forced out onto a glass slide, being drawn
in and out with the pipette in order to mix the three parts thor-
oughly. When this has been done the mixture is drawn into the
tube and allowed to come about half way up the capillary/part
when the open end is sealed off infa* pilot flame of the^Bunsen
burner. Walker mixes the three factors in -a small test j tube
taking precautions against air bubbles. Slides, however, have
seemed better adapted especially because air bubbles are more
easily avoided.

After sealing the capillary pipette, containing the mixture of
leucocytes, bacteria and serum, it is incubated at 37° C. Realiz-
ing that the pipette containing the mixture will not assume the
temperature of the thermostat if it is merely exposed to the air
in the thermostat, Wright originated a so-called opsonizer.

The length of time for which the mixture is incubated varies
for the different organisms. It is to be noted that up to a certain

SMEARS. 33

limit the longer the period of incubation the more marked will
be the phagocytosis. The incubation time must in some cases
be limited because of solution and agglutination of bacteria.
In all instances the mixture containing patient's serum and
that containing normal serum must be incubated at the same
temperature and for the same length of time. The time of in-
cubation varies from five to thirty minutes, depending upon the
species of microorganism and the properties of the blood serum
tested.

After incubation the nipple is removed and the capillary
end of the pipette is broken off, the nipple is then replaced and
the leucocytes, bacteria and serum are again thoroughly mixed
on a glass slide, after which smears are made.

SMEARS.

If reference again is made to the principle involved in the
determination of the opsonic index, it will be noted that the index
shows the ratio of the number of bacteria taken up by the poly-
morphonuclear neutrophiles when patient's serum is a part of
the mixture, to the number of bacteria taken up by the same
class of leucocytes when normal serum is a part of the mixture.
It is therefore necessary to count the number of bacteria in the
polynuclear leucocytes. In order to have an abundance of leuco-
cytes the grayish red layer is taken off the centrifuged blood.
This contains a relatively larger number of white blood cells than
does blood freshly drawn. If a drop of this leucocytic cream
is spread out by dropping a cover glass onto it, fixed, and stained,
many red blood cells and some white blood cells will be found in
the field of the microscope. It would, however, be a tedious
task to find fifty or one hundred polynuclear neutrophiles. Wright
and Douglas have devised an ingenious method to gather the leuco-
cytes together in certain parts of the slide. The method of doing
this is based on the difference in size of red blood cells, mononu-
clear and polynuclear leucocytes; the polynuclear and large mon-
onuclear cells being largest in size.

The method of Wright and Douglas is as follows: A slide is
cleaned and slightly scratched by rubbing with jeweler's emory
paper to slightly roughen the surface of the glass slide. On the
left end of this slide a drop of the incubated mixture, which has

34

VACCINE AND SERUM THERAPY.

been thoroughly mixed after incubation, is placed. Then a
slide with a smooth edge is made into a spreader by breaking
off a corner. This is done so that the two margins of the spread
shall be on the slide instead of running over the edges of the same.

FIG. 10.

The narrowed end of the spreader is now touched to the slide
and the drop of blood allowed to run to the under edges of the
spreader. The spreader, held at an angle of about 35° to 45°
is drawn over the slide as is indicated in Fig. 10. The spread
on the slide will have an outline as is indicated in Fig. 11.

The different steps taken in making the spread are essential
to secure good smears. When the spreader is pressed firmly
enough against the slide while it is being drawn over the same,

FIG. 11.

the polynuclear leucocytes, being larger than the red blood cells,
will slide out at the edge of the spreader or be drawn to the end
of the spread. Care must be taken not to make the smear too
thin.

FIXING AND STAINING OF THE SMEARS.

After the smear has been made it must be fixed and stained
for examination. Fixing and staining is usually accomplished
by the ordinary blood stains, Irishman's, Wright's, Jenner's all
giving good results, except when the index for the tubercle bacillus
is to be determined. The writer, however, has gotten more sat-

CALCULATION OF OPSONIC INDEX. 35

isfactory results for organisms, other than the bacillus of tuber-
culosis, by fixing the spread with methyl alcohol for one minute,
washing off with water and then staining with Loeffler's methy-
lene blue for three to five minutes. In staining slides on which
the index for the tubercle bacillus is to be obtained, a modification
of Wright's method has given most satisfactory results. The smears
are first fixed in a saturated solution of bichloride of mercury which
is then washed off with water. After this the slide is immersed
in a jar containing Ziehl's carbol-fuchsin . The stain is heated
by placing the jar in a heated water bath. After five minutes
of staining in hot carbol-fuchsin, the stain is washed off and the
smear is decolorized in a mixture of 97 parts of alcohol and :>
parts of concentrated hydrochloric acid. The smear is counter
stained in a solution of one-half grams each of sodium carbon-
ate and methylene blue in 100 c. c. of water. This counter-stain
acts rapidly and has the advantage that if the slide be over
stained in the process it can be easily decolorized with warm water.

EXAMINATION OF SMEARS.

The spreads are examined by first going over the slide with
the low power of the microscope to determine whether the slide
has been properly stained and also to find the part of the slide
where the polynuclear leucocytes are most numerous. Usually
it is found that these are most abundant near the margins and
at the end of the smear. Wright has recommended that those
at the end of the smear be examined.

Under the high power of the microscope the number of bac-
teria engulfed by a definite number of polynuclear leucocytes is
determined. The number of leucocytes examined varies. Wright
based many of his determinations on examinations of twenty
leucocytes. Most investigators, however, have counted the num-
ber of bacteria in fifty or more polynuclear leucocytes.

The average number of bacteria per leucocytes is spoken
of as the phagocytic index.

CALCULATION OF THE OPSONIC INDEX.

The opsonic index of Wright is determined by dividing the
phagocytic index obtained when patient's serum is used with

36 VACCINE AND SERUM THERAPY.

a certain leucocytic cream and bacterial suspension, by the pha-
gocytic index obtained when serum from the healthy individual
is used with the same leucocytic cream and bacterial emulsion.
This may be illustrated in a concrete case as follows : If the aver-
age number of staphylococci phagpcytosed as determined by
counting the cocci in fifty leucocytes is eight when the patient
serum is used, and ten when normal serum is used, then eight-
tenths or 0.8 is the opsonic index.

CHAPTER IV.

CRITICISMS AND MODIFICATIONS

OF WRIGHT'S OPSONIC INDEX

DETERMINATIONS

Wright's publications on opsonins, the part they play in im-
munity and the method of determination of the opsonic index
lead to much investigation on these subjects. Many investiga-
tors followed Wright's methods closely while some have followed
what they supposed were Wright's methods. The first reports
of work done by investigators other than Wright and his pupils,
agree strikingly with the results obtained by Wright. Later
results, however, were not as favorable either before or after
Wright himself and some of his pupils had demonstrated and
taught his technique. For a time want of personal skill and
ability, improper methods of work, and lack of ability to manipu-
late were supposed to account for results which did not agree
with those obtained by Wright. Numerous investigators, how-
ever, after having received instructions from Wright and his
pupils on the methods of determining the opsonic index, seriously
questioned the methods of Wright. This list of investigators
includes Park, Simon, Baldwin, Cole, Moss, Potter, Bolduan,
Walker, and many others. On the other hand there are num-
erous investigators who have not questioned the technique and the
results obtained by following the methods of Wright. These
investigators followed principally the opsonic index in the various
bacterial diseases and made efforts to determine the nature of
opsonins, their importance and behavior in the various diseases.

The opsonic index, according to Wright, is obtained when
the average number of bacteria ingested per leucocyte in a mix-
ture of patient's serum, bacterial emuslion and leucocytes in-
cubated for a certain period of time, is divided by the average

38 VACCINE AND SERUM THERAPY.

number of bacteria ingested by the leucocytes in a mixture of
the same quantity of serum from a normal individual, the same
bacterial emulsion and leucocytic cream, incubated for the same
length of time at the same temperature. In the process of the
determination of the opsonic index mechanical technique, serum,
bacterial emulsion, leucocytic cream, and calculation of tile aver-
age number of bacteria ingested are of importance. Inasmuch
as the results obtained by the different investigators are so
discordant and inconsistent, all of these factors have been
considered, emphasized and, in the minds of some at least,
improved.

MECHANICAL TECHNIQUE.

The mechanical part of the technique of Wright has prob-
ably been less criticised than any other part of his method. Var-
ious investigators have reported their technique in full and some
at least have shown that they have not interpreted Wright's ex-
planations of the same correctly, while others have modified the
technique in order to give greater accuracy to the method of the
determination of the index.

Only some of the modifications suggested can be mentioned.
Barber, thinking that the mark on the pipette as made by the
wax pencil, is too wide and therefore leads to inaccuracies in the
amounts used in the mixtures, has suggested that the mark on
the capillary pipette be made with a glass hair dipped in Bismarck
black. This suggestion, however, is not generally followed as it
is possible for one experienced in handling the capillary pipette
to draw fluids to the upper or lower edge of the wax pencil mark
with as much accuracy as it is to take it to the mark made by the
glass hair.

Walker has suggested the use of a water bath for the incu-
bation of the mixture of serum, leucocytes, and bacterial emul-
sion. Undoubtedly this has advantages over incubation for
short periods of time in the air in the thermostat. Wright's op-
sonizer, however, is more convenient and almost immediately
brings the pipette to the temperature of the metal of the opsonizer.

For the collection of serum, Walker has suggested that the
capsule be held in position by inserting it into a slit in a slide
box. Unless one is taking his own blood, however, it is

NORMAL SERUM. 39

about as convenient to hold the capsule in the hand as to hold
the punctured finger near the table on which the slide box rests.

Walker has also advocated that small quantities of full
strength or diluted sera and leucocytic cream be put into separ-
ate small test tubes in order to avoid carrying bacterial emulsion
to the lencocytic cream from which other preparations are to be
made later. This method is certainly to be recommended.

Walker has also modified Wright's methods concerning the
capillary pipette containing the mixture. In making opsonic
index determinations, Walker breaks off the large end of the cap-
illary pipette and seals both ends of the capillary tube. When
this is done it becomes necessary to again fit the capillary part
of the pipette to a larger tube to which a rubber teat can be fas-
tened. This Walker accomplishes by fastening a thin rubber
sheet over the open end of the large pipette. Thisworks as a dia-
phragm and can be easily punctured by the capillary tube.

SERUM.

Immunity to and recovery from numerous infections accord-
ing to Wright's theory is due to the presence of opsonin. Opsonin
prepares bacteria for ingestion by the leucocytes and is a sub-
stance contained in the serum. In the determination of the
opsonic index the ratio of bacteria ingested by the leucocytes is
established by mixing bacteria and leucocytes in one case with
patient's serum and in another case with serum from healthy
individuals. It is thus evident that the serum is the important
and valuable factor.

Normal Serum.

In the determination of the opsonic index numerous in-
vestigators have been struck by the apparently unexplainable
indices found. This has lead to the investigation of the
opsonic index obtained with serum from the normal individual,
to determine whether the index does not vary in the normal, as
well as in the infected, individual.

If reference is made to the technique, as proposed to obtain
the normal phagocytic index, it will be observed that Wright
recognized that there is a difference in the amount of opsonin
present in serum from the normal individual. He states that

40 VACCINE AND SERUM THERAPY.

the opsonic index of a normal individual will vary from 0.8 to
1.2. To obtain the true amount of opsonin in normal serum, he
pools equal quantities of sera from several normal individuals.
However, after it has been established that a healthy individual
has an opsonic index of 1.0, as determined by estimation with
pooled normal sera, he uses serum from this individual as a normal
serum. Wright's own determinations, from which he concedes
that there are variations in the amount of opsonin present in
the blood of healthy .individuals, discredit the accuracy either
of the method of determining the opsonic index or the value of
determining the same. If the opsonic indices of two normal in-
dividuals is 0 . 8 and 1 . 2 respectively on the same day as deter-
mined by co parison with pooled normal serum, then as com-
pared to each other their opsonic indices vary from 0.66 to 1.5
respectively. It is thus evident that from Wright's concession
the index of a normal individual may vary from 0 . 66 to 1.5
when one serum is used as a control for the other.

Indices for normal serum as actually determined will not
even vary within these limits. The writer sometime ago reported
results on a series of ten specimens of blood from ten healthy
adults, studied to determine the differences in opsonic power in
healthy individuals. The leucocytes were washed twice with
0.85 salt solution. The number of staphylococci taken up by
100 polynuclear leucocytes was counted in two preparations, sepa-
rately incubated. The phagocytic indices as determined by two
workers A and B are as follows:

Source. A B

N ... 9.48 7.63

J 9.16 5.20

B 8.75 6.80

McL 7.27 4.37

Z 6.63 5.12

C 6.60 5.37

S 5.89 4.82

0 5.84 4.57

H 5.49 6.03

Ns 4.91 4.37

The great error introduced by this method becomes evident
upon the determination of the opsonic indices when each serum
is used for comparison with all of the others. In the series A,
the normal opsonic index varies from 0. 52 to 1 . 93, while in series
B it varies from 0.57 to 1.75. Moreover, the indices obtained
by the two workers A and B vary considerably for the same sera.

DILUTIONS OF SERUM. 41

Bolduan has reported determinations of the opsonic index
of normal individuals and finds that as far as the bacillus of tuber-
culosis, staphylococci and streptococci are concerned, there is
marked variation not only in different individuals but also from
day to day.

Moss has found opsonic indices for staphylococci to vary
from 0.18 to 0.56 in normal rabbits.

The same serum has also been tested a number of times
with the same leucocytes and bacterial emulsion. Bolduan re-
ports opsonic index determinations for a single serum which
vary from 1 . 05 to 1 . 46. The writer has reported determinations
made by taking five specimens of blood from the five fingers
of the same hand of a healthy adult. The leucocytes used for
the determination were washed twice in 1.5 per cent potassium
citrate in normal salt solution. The phagocytic indices for
Mic. pyogenes aureus are shown in the table:

1st 100 leukocytes 2d 100 leukocytes Phagocytic

counted. counted. index.

Specimen 1 166 196 1.810

2 176 183 1.79.')

3 198 199 1.985

4 185 175 1.800

5 230 190 2.100

If the indices for each specimen of blood be compared with
all the others, the opsonic index as determined by counting the
number of cocci in two hundred cells will be found to vary from
0.85 to 1.22.

It thus seems evident that if normal serum varies as much
as has been found, the variations from the normal, unless marked,
can indicate but little. The most that would seem warranted
is to determine whether a serum is "high" or "low" in opsonic
content, as the case may be.

Dilutions of Serum.

Wright has usually used undiluted serum in the determina-
tion of the opsonic index. Simon, Lamar and Bispham have
shown that by the dilution of some sera there often is a rapid
exhaustion of phagocytic power of the leucocytes. They have
observed that undiluted pig's serum manifests a most intense
opsonizmg effect on staphylococci but that this action diminishes
markedly upon dilution of the serum. Human serum, on the
other hand, though not having as marked an initial opsonizing

42 VACCINE AND SERUM THERAPY.

power, will retain this power on much greater dilution. They
have further found that in human beings, who are supposedly
in good health, the phagocytic power in concentrated serum will,
in some cases, diminish in proportion to the degree of dilution,
wrhile with the serum of other individuals, more rapid exhaustion
takes place.

Moss has compared the opsonic index in various dilutions
of serum and has found that the index obtained in one dilution
is not proportional to that obtained in all the other dilutions.

Walker's reports on the results obtained by the dilution of
serum differ from those obtained by Moss. Walker has found
that with many bacteria, undiluted normal serum will opsonize
so many bacteria in a heavy suspension of bacteria that the leu-
cocytes will contain so many microorganisms, that counting is
impossible. If the bacterial suspension be made less heavy fewer
bacteria will be taken up by the leucocytes, but under ' this
condition the serum will not be exhausted of its opsonin and
part will be lost in the estimation of the opsonic index. A
serum containing much less opsonin may opsonize just as many
bacteria in a light bacterial suspension as a serum containing
more opsonin. "When thin suspensions of bacteria for which
much opsonin exists in the serum are used it generally happens
that both the sera sensitize all the bacteria so that the work if
accurately done will produce equal phagocytic indices for both—
in other terms, an opsonic index of unity — regardless of the real
relation of the serum. Phagocytic indices proportional to the
sera tested may readily be obtained by diluting all the sera equally
to a sufficient degree, and using with these diluted sera a thick
bacterial suspension." For some bacteria as B. typhosus, cer-
tain strains of streptococci and tubercle bacilli, Walker does not
recommend the dilution of serum, while for staphylococci,
some strains of streptococci and tubercle bacilli and for B. coli
he does.

Walker's views concerning the utilization of all the opsonin
capable of sensitizing the particular species of bacteria by the
use of heavy bacterial suspension and diluted sera, must be favor-
ably received. Before they can be accepted and followed it will,
however, be necessary to disprove the results of Moss and others
concerning the dilution of opsonin by the dilution of the serum.

i:.\< TKRIAL EMULSIONS. 43

BACTERIAL EMULSIONS.

Wright, in his technique, attempts in every case to break
ii] > all clumps of bacteria and tries to get such strength of suspen-
sion that the number of bacteria per leucocyte can be easily
determined. With certain organisms trial tests are necessary.
After this, the suspension may be diluted or bacteria added as
may be indicated.

Most investigators have experienced difficulty in obtaining
suitable suspensions and Wright himself has changed his tech-
nique for making bacterial suspensions from time to time. The
difficulties that have been experienced are of two kinds: those
dealing with the strength of the bacterial suspension, and those
concerning the condition of the microorganisms in the suspension.

Difficulties depending upon the strength of the bacterial
suspension. Numerous investigators have given definite figures
to be obtained for the phagocytic index with normal serum. In
most cases it is desirable that the phagocytic index for normal
serum be between six and fifteen. Simon has found that when
the emulsion contains between 666,000 and 2,000,000 microor-
ganisms per cubic millemeter the best results are obtained. Re-
cently Walker has emphasized certain facts that must be taken
into consideration in determining the strength of the bacterial
emulsion. If the suspension does not contain enough bacteria
to exhaust all the opsonin in the strongest serum, a certain amount
of opsonin will, according to Walker, be lost in the estimation of
the opsonic index. In order to get the right strength of bacterial
suspension, Walker tests the same with serum diluted 1 to 30
and 1 to 15. If the 1 to 15 dilution of serum shows a phagocytic
index twice as great as that obtained in the 1 to 30 dilution then
enough bacteria are present in the suspension. Too such a sus-
pension he, however, adds more of the culture, because having
more bacteria than are absolutely necessary will not change the
opsonic index but will certainly furnish enough bacteria to ex-
haust the serum of its opsonin. Only with the staphylococci
will a too heavy suspension effect the true index. This Walker
believes is due to the action of a product of bacterial growth.
To overcome the excessive phagocytosis when heavy suspensions
of bacteria are used, Walker dilutes serum. However, as has been

44 VACCINE AND SERUM THERAPY.

mentioned before, there are 'conflicting views concerning the effect
of dilution of serum for which the opsonic index is to be
determined.

DIFFICULTIES ARISING BECAUSE OF THE PARTICULAR
SPECIES OF BACTERIA IN THE SUSPENSION.

Anyone who has made opsonic index determinations has at
times had slides to examine in which the bacteria were not
well stained and in which the microorganisms were apparently
clumped. Frequently only shells of organisms remain and at
times the bacteria are completely dissolved. These difficulties
are apparently dependent upon the particular culture used but
to a greater extent upon the agglutinins and lysins present
in different sera. To overcome this difficulty young cultures
are preferred because they stain better than do old cultures.
To overcome agglutination and lysis the serum is in some cases
heated to 55° or 65°. The clumping of bacteria in opsonic
index preparations is given little attention by Wright. Other
investigators have, however, regarded clumping of bacteria
as responsible for the occasional enormous differences of the
indices determined. Walker gives tables which indicate the
objection of clumps on the uniformity of phagocytosis and has
prepared a technique by which he obtains clump-free bacterial
suspensions. According to this technique the bacteria are rub-
bed gently in a mortar with small, and gradually increasing,
quantities of salt solution until a thick suspension is obtained.
This suspension is then filtered through moistened scraped filter
paper until in a stained specimen no more -clumps are found.
Even though a technique may be devised to obtain relatively
clump-free suspensions of bacteria, this will not do away with the
clumping occasionally observed in opsonic index preparations,
because the action of agglutinins in undiluted or only slightly
diluted sera will again cause clumping of the bacteria.

LEUCOCYTES.

There -has been but little criticism of the method of collection
of leucocytes, as proposed by Wright. Simon has used 0.1 per
cent ammonium oxalate instead of 1 . 0 per cent sodium citrate
in salt solution. Some investigators have drawn the corpuscles

CRITICISM OF CALCULATION OF OPSONIC INDEX. 45

off after the first centrifugalization while others have attempted
to remove the serum of the blood and the citrate or oxalate
solution by several washings with normal salt solution.

To the source of the leucocytes Wright attaches little impor-
tance, while most other investigators have aimed to obtain leuco-
cytes from the blood of apparently healthy individuals. The
work of Peterson and Hiss and Zinsser indicates that the leuco-
cytes contain substances that are of importance in immunity
and that these substances vary in the leucocytes of normal and
immunized animals. For this and other reasons it seems advis-
able in all cases to obtain the leucocytes from the blood of normal
individuals.

Walker draws the blood from which the leucocytes are to
be obtained into citrate solution heated to 37° C. The leuco-
cytic cream is kept at this temperature up to the time of mix-
ture with serum and bacterial emulsion. He believes this to be
of great importance. In experiments performed by the writer
and others, this has not been found to be of especial value in the
production of uniform phagocytosis, nor do Walker's tables
show a marked uniformity, for the number of bacteria per
leucocyte vary from 1 to 1 7 in twenty-five leucocytes examined
on the same slide.

CALCULATION AND DETERMINATION OF THE
OPSONIC INDEX.

According to Wright's method the opsonic index is deter-
mined and calculated by dividing the degree of phagocytosis ob-
tained with patient's serum by that obtained with serum from a
normal individual.

Simon has proposed an entirely different method for the
calculation of the opsonic index. He had observed that with
many species of bacteria, due to lysis and fragmentation, great
difficulty is experienced in determining the phagocytic index and
that the strength of emulsion greatly changes the amount of
phagocytosis. To remedy this, Simon first proposed that the
percentage of phagocytic cells be determined by counting 50 cells
taken in sequence. Later Simon compared the percentage of
phagocyting leucocytes with patient's serum to the percentage
of phagocyting cells with pooled normal serum. The value ob-

46 VACCINE AND SERUM THERAPY.

tained for pooled normal serum he called 1.0, and the index de-
termined in this way he called the percentage index as contrasted
to Wright's bacillary index. These two indices Simon finds can
be made to agree to the second decimal place.

Moss and others have found that the absolute numbers of
cells phagocytosing depends in normal or slightly diluted sera on
the strength of the bacterial suspension, and that in low dilutions
practically all the cells can be made to engulf bacteria. It is
evident that by Simon's method only sera having low indices can
be investigated, unless the bacterial emulsion or serum are di-
luted. Simon has recommended that light bacterial suspensions
and at times diluted sera be used so as to be able to determine
the percentage index.

Strong, in his early experiments with anti-plague serum,
determined the index according to Wright's method. Later,
however, he substituted this method by one in which the highest
dilution of an immune serum which gives a marked phagocytosis
is compared to the same dilution of a normal serum. If, with a
certain dilution of an immune serum marked phagocytosis is ob-
served while the same dilution of a normal serum gives only slight
phagocytosis, the immune serum is regarded as having an increased
opsonin content. Even on this method Strong places little re-
liance.

Although Simon prefers to accept his index to Wright's
when there is disagreement between the two, most investigators
have preferred Wright's opsonic index.

LEUCOCYTES IN WHICH THE BACTERIA
ARE TO BE FOUND.

According to Wright's method the bacteria are to be
counted in the polynuclear neutrophiles. These cells when
Wright's technique is followed will be found along the margins
and end of the spread.

In all work on the determination of opsonic indices it has been
found that there are great differences in the number of bacteria
taken up by the individual leucocytes. Moreover, great differ-
ences have been found in the phagocytic index as determined by
different investigators examining the same slide. Even the same

LBUCOCYTB8 KXAMINED. 47

investigators have obtained markedly different phagocytic indices
on the same slide. The writer has reported work showing that
there is not always agreement between results obtained by count-
ing the bacteria in 100 and 200 polynuclear leucocytes on the
same slide. Bolduan, Moss, Cole, Jeans and Sellards, Simon and
others have published 'results showing striking differences in the
counts obtained in 50, 100, 200 and more cells. Bolduan supposes
that counting the number of bacteria ingested by 100 or 150 cells
will give a fairly correct average. Potter, Dittman, and Bradley
believe counts in 100 cells suffice in most cases. Moss, in an effort
to determine how many leucocytes must be examined, has de-
cided that after counting the bacteria in three hundred cells there
is still an error of ten per cent ; and when 50 cells only are counted
his experiments show a variation of from .8 per cent to 30 per
cent. Most investigators determine the number of bacteria taken
up by at least 100 leucocytes.

Large numbers of leucocytes must be examined because of
the variability of the number of bacteria taken up by the indi-
vidual leucocytes. Cells which are apparently normal as far as
can be determined by staining and which may contain an average
of from 5 to 8 bacteria per leucocyte, will phagocyte from 20 to
30 microorganisms at a maximum while other leucocytes may
take up none. This difference in the number of bacteria taken
up by individual leucocytes has been supposed to be due to the
leucocytes themselves and to clumps in the bacterial emulsion.
Walker has suggested that the leucocytes be kept constantly at a
temperature of 37° C., and that they be well mixed after incuba-
tion with serum and bacterial emulsion. Undoubtedly all in-
vestigators have mixed the leucocytes, bacteria and serum well
after incubation and before making the spreads to be examined.
This procedure, however, does not overcome the differences in
numbers of bacteria per leucocyte, as is shown in Walker's tables,
in which the variations in numbers of bacteria ingested range
from 1 to 15, 2 to 16, 1 to 17, and 4 to 16 in four sets of 25 leuco-
cytes examined. It cannot be concluded from this that there is
a uniformity of phagocytosis, although Walker's tables do show
a uniformity of phagocytic index in groups as small as 25 leuco-
cytes. It is evident, however, that if 25 leucocytes only were
to be examined, the opsonologist might, after he had examined

48 VACCINE AND SERUM THERAPY.

24 leucocytes, have some choice for the 25th one to he examined,
which choice would change the result from 1 to 17. Based on
this selection the opsonic index could be materially lowered or
raised as might be desired.

In one of the early contributions on the subject of opsonic
index, Simon, Lamar, and Bispham recommend that in making
the spread of the mixture after incubation, the spreader slide be
merely kept in contact with the blood without touching the lower
slide, for, "Otherwise it may happen that most of the leucocytes
containing organisms, are carried to one end, while only the empty
cells are found in the intervening space." In the laboratories
of the Johns Hopkins Hospital, Cole, Moss, and Jeans and Sellard
have made determinations of phagocytic indices determined in
different parts of the slide. It was found that the leucocytes
collected near- the edge of the smear contain decidedly more bac-
teria than those toward the center of the slide. By dividing a
slide into three zones they found that at the end of the smear the
leucocytes contain more bacteria than in the first and middle
zones. To explain these differences these men assume that the
polymorphonuclear leucocytes containing the largest number of
bacteria are so increased in size that they are drawn to the end
of the slide while the smaller ones drop out earlier. These results
indicate that, contrary to Wright's suggestion, one ought not to
determine the number of bacteria at the end of the spread.

Wright's method of the determination of the opsonic index
has yielded such marked differences in results that more work on
this subject seems justifiable. It must be remembered that the
opsonic index is of no value whatever unless carried out with the
greatest care and by some one who has had considerable exper-
ience. Modifications of Wright's technique have arisen with too
much rapidity to make it possible to lay down any definite rule
for the determinattion of the opsonic index. However there are
only few modifications that have made it possible to get more
reliable determinations of Wright's opsonic index.

CHAPTER V.

OPSONIC INDEX IN HEALTH AND

DISEASE.

The active participation and function of the white blood cells
in the protection of the body against infection was first carefully
studied by Metchnikoff. He regarded the leucocytes as cells
whose object is to remove from the body and its tissues bacteria
and foreign material. On this he based his theory of immunity.
It is not strange that Leishman, Wright, and others should have
suspected differences in phagocytosis in health and disease. In
1902 Leishman, and especially Wright, emphasized the difference
in phagocytic ratio observed in individuals with infections run-
ning favorable or unfavorable courses. While trying to find a
method by which to control the administration of vaccine, Wright
discovered opsonins. With the discovery of opsonin and the
development of a method for the determination of the same, it
was evident that the opsonic index in health and disease should
be investigated. Wright found that in individuals with localized
or chronic infections there is usually a decrease in the opsonin
content of the blood. This decreased opsonin content as deter-
mined by the opsonic index, according to Wright's theories, is
due to the fact that in local infections but little of the bacterial
substance is absorbed, and so gives rise to but a small amount of
active immunity. When this is the case the local infection be-
comes chronic.

By the method of the determination of the opsonic index,
Wright has demonstrated that following the injection of vaccines
made from the cultures of infecting organisms, there is first a
drop in the opsonic index and later a rise of the same. The drop
in the index he calls the "negative" phase and the rise the "posi-
tive" phase. The observation of negative and positive phases
was made by Ehrlich in 1892, who found that following in-

50 VACCINE AND SERUM THERAPY.

jection of tetanus toxin there is at first a slight diminution in the
anti-toxin in the milk of the animals injected while later there
is an increase in the amount of anti-toxin. Likewise Solomonson
and Madsen found that similar results are produced in the blood
when diphtheria toxin is injected. Agglutinins, precipitins, bac-
teriolysins, after injection of killed cultures of bacteria, are also
at first decreased and later increased.

Wright and Douglas did not, as has been supposed by many,
find that all pathogenic bacteria are sensible to opsonins. They
divide the pathogenic bacteria into four groups:

a. Those bacteria that are sensible to bactericidal, bacter-
iolytic and opsonic action of the normal human blood. B. ty-
phosus and Spir. cholerae belong to this group.

b. Those bacteria that are sensible to opsonic and especially
bactericidal action of normal blood serum. Examples of this
group are to be found in B. coli and B. dysenteriae.

c. Those bacteria that are definitely sensible to opsonin
but not to the bactericidal action of normal blood. B. pestis,
Mic. melitensis and Mic. pneumonias belong to this group.

d. Those bacteria that are insensible to the opsonic and bac-
tericidal action of normal human blood serum. Examples of
this group are found in the bacillus of diphtheria.

Practically all of Wright's observations on opsonic immuni-
zation were determined in man. In the human, following the
injection of killed cultures, the negative phase is usually of short
duration, lasting from 24 to 48 hours. The positive phase may
not, but usually follows the negative phase and lasts from 3 to
4 days to two weeks. In active immunization Wright reasons
the curve of protected substances may be affected in different
ways. If the dose of vaccine injected is very small, the negative
phase may be omitted, while if a very large dose is injected it will
be marked and the highest index in the positive phase may not be
as high as it was at the time of injection. Usually in active im-
munization a number of injections of vaccine must be made.
These may produce one of three effects on the opsonic index:

a. If injections are made after the index has returned to
the normal, the index curve may show a series of indices above
and below the normal. This is what happens, according to Wright,

CURVK 01 PROTECTIVE SUBSTANCES. 51

when the individual is allowed to recover completely between
injections. The curve is illustrated in Fig. 12A.

b. The injections may be given at such times that one nega-
tive phase is superimposed upon another negative phase and in
this way produce a cummulative effect in the duration and degree
of the negative phase. This result is obtained according to Wright
when active immunization is pushed rapidly or forced. The curve
of protective substance is shown in Fig. 12B.

c. The injection may be made at such times and in such
amounts that there is a summation of positive phases. This is
the result sought for in immunization. The curve representing
the amount of protective substances in this case is represented
in Fig. 12C.

FIG. 12.

Wright, in his treatment of infections by the injection of vac-
cine, tries to inject such doses at such intervals of time as to give
him a continuous "high tide phase." It is to be noted, however,
that even with the most favorable summation of positive phases
Wright has not obtained an index as high as 4 . 0 except in rare in -
stances. This, of course, is entirely out of proportion with the amount
of other immune bodies produced by active immunization.

Before the principle and methods as formulated by Wright
can be accepted it is necessary to decide (a) that opsonins are of

52 VACCINE AND SERUM THERAPY.

importance in immunity and that the method of determining the
opsonic index is accurate; (b) that in the natural recovery from
an infection the opsonic index changes, as Wright aims to change
it, by the injection of bacterial vaccine; and (c) that the injection
of vaccine produces the changes described by Wright.

IMPORTANCE OF OPSONIN IN IMMUNITY AND ACCURACY

OF THE METHOD OF THE DETERMINATION

OF THE OPSONIC INDEX.

Spontaneous phagocytosis has been described from time to time.
With the introduction of Wright's opsonin theory and the develop-
ment of the technique of opsonic index determination, phagocytosis
has been found to occur, in most cases, only when fresh serum is
present. Neufeld and many others have found that contrary to
Wright's statement phagocytosis is not entirely dependent upon
opsonins, for bacteria with little virulence may be taken up by
the leucocytes even though no serum is present. The existence
of opsonin, however, and its relation to phagocytosis can no longer
be doubted; the part it plays in immunity has not yet been
satisfactorily demonstrated. Many of the objections made to
Metchnikoff's phagocytic theory of immunity present themselves
in the opsonic theory of immunity. It has been found that highly
virulent bacteria are not nearly as readily ingested by leucocytes
as are less virulent ones. Bacteria engulfed by leucocytes are
not always dissolved and disposed of for it has been demonstrated
by various investigators that certain species of bacteria produce
or secret substances that are antogonistic to and actually kill
leucocytes. Even if it is accepted that opsonin plays an important
part in immunity, the opsonic index determined by Wright's
method, need not necessarily be an index of the patient's im-
munity to certain infectious microorganisms. Agglutinins, bac-
teriolysins, and anti-toxins, all of which are substances better
known than are opsonins, are only circumstantial evidence of
greater or less importance in the complex phenomenon of immu-
nity. The body has various means of defense against bacterial
invasion and action so that the assumption that all the protec-
tive substances and means against bacterial diseases and infec-
tions manifest themselves in such a manner that they can be de-
termined from the opsonic index seems unreasonable.

OPSONINS IN IMMUNITY. 53

In the previous chapter the accuracy of the method for the
determination of the opsonic index has been criticised. It is
certain that the method is complicated, requires a considerable
amount of experience, and with all the modifications and improve-
ments in the technique is still subject to such great error that
even in experienced hands the determinations of the opsonic
index must be regarded as of doubtful value.

CHANGES IN THE OPSONIC INDEX IN NATURAL
RECOVERY FROM AN INFECTION.

Wright, at various times, has reported observations on
the phagocytic reaction and opsonjc index in infections running
favorable and unfavorable courses. He has found that recovery
and increased opsonic index accompany each other. Hektoen
has reported that in pneumonia the opsonic index is at first low
and rises as the patient's condition improves so that at the crisis
the index is above normal. In patients that have a persistently
low opsonic index in this disease death usually follows. Tunni-
cliff has found that in scarlet fever the opsonic index for strepto-
cocci is below normal early in the disease and, as the acute symp-
toms subside, it rises above normal. Later on it again becomes
normal. Ruediger has found in erysipelas a sharp rise in the
index for streptococci as the temperature begins to fall. Hamilton
isolated pseudo-diphtheria bacilli in 75 per cent of cases of acute
otitis media and found wide variations in the index for this organ-
ism in these cases. Clark has found that the typhoid opsonic
index drops before a relapse in typhoid fever.

The writer in a study on the opsonic index in erysipelas made
observations on the changes in the opsonic index in unvaccinated
patients to determine the relation between the opsonic index
for Streptococcus erysipelatos and recovery from, migration, re-
currence and desquamation in erysipelas. The most instructive
of these cases are the two which follow:

Case I. — S., a man, aged thirty-eight years, who had a migra-
tory, recurrent erysipelas of the face, ears, scalp, and neck, was
admitted to the hospital on the fourth day of his disease. When
his face was involved, his index was 0.7; when it was desquamat-
ing, the index had risen to 1.3; when his face was again involved

54

VACCINE AND SERUM THERAPY.

seven days later, the index was 1.4; and when it was desquamat-
ing again, the index had dropped to 0.8. When his neck became
involved, one day after his face began to desquamate for the
second time his index was 1.0; two days later, with an index of
1.1, his back became involved, and three days later, with an
index of 3 . 2, his shoulders and neck were again involved, and two
days later, with index of 1.1, the back and shoulders began to
desquamate. (Chart I.)

T OP'
T. INO.

105° 0.4
10t°
103"
102°
101° 2.0
100°
99° 4.0
• 0.5

DAY OF
DISEASE

4 5 6 7 8 » 1ft 11 12 13 14 15 16 1? 18 19 20 21 22 23 24 25 26 27 28 2!) 30 31

CHART I. — Temperatrue (unbroken line) and opsonic index (broken line) in a case of

erysipelas (Cnse I).

Case II. — A., a man, aged twenty-four years, suffering with
erysipelas of the face and ears, was admitted on the eighth day of
the disease with an index of 0.9. The next day his temperature
dropped and desquamation began ; his index was found to be 1.1.
Subsequently the index, though the patient was perfectly well,
remained below unity. (Chart II.)

TEMPERATURE

13 14 (•

UPSOMIC INDEX

CHART II. — Temperature (unbroken line) and opsonic index (broken line) in a case of

erysipelas (Case II).

In these two cases there has been no constant change of index
corresponding with desquamation and recovery.

OPSONIC INDEX IN RECOVERY FOR INFECTION

55

To further study the relation of the opsonic index to the
course of the disease, the indices of all patients who had received
no vaccine were tabulated with regard to the day of the disease,
and an average opsonic index for each day was determined.

TABLE SHOWING THE: OPSONIC INDEX AT THE TIME OF ADMISSION, BEFORE KILLED
CULTURES WERE INJECTED, AND THE DAY OF THE DISEASE.

Day of Disease.

1

2

3

4

5

6

7

8

9

10

11

0.6

1.2

0.6

0.7

0.7

0.4

0.9

0.8

1.1

0.6

1.0

0.3

1.1

1.1

1.1

1.0

1.1

0.8

0.9

0.7

1.0

2.4

1.4

0.9

0.6

1.3

1.7

1.0

1.7

1.5

0.6

1.6

0.7

1.1

1.0

1.2

0.45

1.1

1.45

1.1

0.86

0.4

1.05

0.96

1.23

0.6

0.9

Average.

The composite chart indicates that erysipelas causes an in-
crease of the opsonic index which reaches its maximum about the
third day of the disease, and is followed by a gradual fall. The
subsequent course of the chart represents, in very large part, ob-
servations made upon recurrent, migratory, and complicated
cases. Ruediger found that the index was high during an attack
of erysipelas.

l.o

I."

OLA

10 - n

CHART III. — To show the average opsonic index and the day of disease before killed
cultures of streptococci were injected.

It is to be noted that the determinations made and repre-
sented in the last curve do not represent the index in any one case.

56 VACCINE AND SERUM THERAPY.

The indices in individual cases are so variable and show such great
irregularity that determinations of the opsonic index in any case
give little indication of the severity of the disease and is of no
value in prognosis.

Most investigators have found that in normal individuals
the curve of opsonic index is very irregular. Moss in thirteen
observations on one individual determined indices which varied
from 0.52 to 1.95 and in another from 0.42 to 1.66. Bolduan
reports observations in which one patient's serum varied in opsonin
content as determined by Wright's opsonic index from 0.82 to
2 . 50 in seventeen days. Inasmuch as the opsonic index during
infections shows no greater variation than during health it can
hardly be accepted that determinations of opsonic indices are of
much assistance in diagnosis and prognosis in disease.

Up to the present, most investigators have found that in the
natural course of infection there is no regular curve of the opsonic
index and that in the natural recovery from an infection the
changes in opsonic index are not those which Wright attempts
to obtain by injection of killed cultures of bacteria.

CHANGES IN OPSONIC INDEX PRODUCED BY INJEC-
TIONS OF KILLED CULTURES.

Wright has observed that the injection of killed cultures is
usually followed by a change in opsonic index for the organ-
isms injected. If the amount of killed cultures injected is large,
he states there is a marked negative phase, after which there
may be either a marked or slight increase in the opsonic index.
If the amount of killed cultures injected is small, the negative
phase may be small or absent entirely and following this there
may or may not be a positive phase. Wright has attempted to
regulate injection of killed cultures so as to get as small a nega-
tive phase and as large a positive phase as is possible. To accom-
plish this, he has determined the number of killed cultures in emul-
sions and has injected certain numbers of bacteria at a dose, the
number varying with the species of the organism and the opsonic
index changes obtained by their injection. In some cases Wright
has resorted to autoinoculation, which is accomplished by mas-
sage or manipulation of the infected part. This partly removes

OPSONIC INDEX FOLLOWING VACCINE INJECTION'S. 57

the toxins from the diseased tissues to healthy tissues where im-
mune substances are actively produced. The amount of mas-
sage and manipulation Wright regulates by the changes produced
in the opsonic index.

While numerous investigators have reported results of vac-
cination based on opsonic index determinations and have ob-
tained, after injections, almost always a uniform rise in the opsonic
index, most investigators have not found this to be the case.
Even though Wright claims to have obtained changes in opsonic
index showing considerable regularity, yet on examination of
his charts one sees curves of most marked irregularity; the index
is not constant in normal individuals ; shows no consistent increase
or decrease in different stages of infection, and is* influenced by
many non-specific processes as menstruation, exercise, food, etc.
Numerous investigators have found that even though there may
be first an increase in opsonic index following vaccination, yet
this is soon followed by a fall of the index and still improvement
of the condition will be observed.

The results obtained on determinations of opsonic index in
health and disease are so variable that its importance as a means
of diagnosis, prognosis and indication of actual immunity
must be doubted. It is definitely certain that the index is of
no value whatever unless carried out carefully by one having had
considerable experience in its determination.

CHAPTER VI.

THE NATURE OF OPSONINS

The theories of Wright concerning opsonins have received
attention chiefly because of the apparent value of their quanti-
tative determination in health and disease and in the regulation of
dosage and interspacing of injections of bacterial- vaccines. Nu-
merous investigators, however, have attempted to determine their
importance in immunity, their structure, and the conditions
which influence their action.

Wright has found opsonins, the substances which prepare bac-
teria for phagocytosis, present in the serum of normal, diseased
and vaccinated individuals. Neufeld and Rimpau, on the other
hand discovered substances which prepare bacteria for ingestion
by leucocytes, present in certain immune serum. Inasmuch as
both the opsonin of Wright and the bacteriotropin of Neufeld and
Rimpau perpare bacteria for phagocytosis, they are by many
considered as identical substances. Various investigators, while
admitting that these substances may be identical in some sera,
do not regard them as the same substances in all sera. Wright
has regarded opsonin as an important factor in immunity, but
has made no distinction between the opsonin present in normal
and immune sera. Numerous investigators, however, have dem-
onstrated and emphasized certain differences in the bacteriotropic
substances in normal and immune sera.

SPECIFICITY.

While Wright and Douglas did not discuss the question of
specificity of opsonins, still it is evident that they consider opso-
nins as specific. The opsonic index in disease, according to Wright,
shows certain deviations from the normal for certain microor-
ganisms, while for other organisms it deviates but little from the
opsonic index obtained with normal serum. On thisjDbservation

BPFECT OF HEAT ON OPSONIN. ."/»

Wright has based a method for the diagnosis of certain microor-
ganisms in infections. This can, however, only be regarded as
logical if opsonins are specific. Bulloch and Western found that
in normal serum, by absorption tests, the specificity of opsonin
for B. pyocyaneus, B. tuberculosis and staphylococci could be
determined. Muir and Martin, Simon, Russell, Potter, Dittman
and Bradley, and others, have found that normal opsonins are
not specific. Muir and Martin, and Russell, however, by absorp-
tion tests have determined that immune opsonins are quite spe-
cific. vSimon, however, has not been able by investigation to
prove the existence of specific immune opsonin.

EFFECT OF HEAT ON OPSONIN.

Wright has consistently assumed that normal and immune
opsonins are identical and has preferred to regard all opsonins
as thermolabile. There are, however, on the other hand, a large
group of observations which show that while opsonins in immune
serum resist a temperature of 55° C. for one hour, opsonins in
normal serum will no longer be able to prepare bacteria for phag-
ocytosis by the leucocytes after such an exposure to heat. These
observations show plainly that immune opsonin is thermostable,
while normal opsonin is thermolabile. The basis for Wright's
assumption that they are all thermolabile is not evident, inas-
much as Wright and Reid have shown that in the serum of cer-
tain patients suffering from tuberculosis there is a thermostable
opsonin. On this observation Wright has based a method for
the diagnosis of tuberculosis, which can only have for a founda-
tion the assumption that in tuberculosis specific immune bodies
which are heat resisting, are produced. From the various inves-
tigations reported, normal and immune opsonins manifest a marked
difference in ability to withstand heat.

STRUCTURE OF OPSONINS.

Opsonins have, by numerous investigators, Savtchenko,
Besredka, Loehlein and Dean, been regarded as identical with
amboceptors (fixateur). Muir and Martin have shown that not
every immune body produces an opsonizing effect. Hektoen has,
from a series of experiments, decided that opsonins are distinct
substances or anti-bodies. Neufeld and Rimpau, Neufeld and

60 VACCINE AND SERUM THERAPY.

Toepfer, Keith, Bulloch and Atkin, agree with Hektoen and Wright
and Douglas that opsonjc action is due to the presence of hitherto
unknown distinct bodies.

Before this can be accepted, however, it will be necessary to
repeat many of the experiments that have been made, inasmuch
as in England and America especially, these investigations were
made at a time when no distinction was made between normal
and immune opsonins.

The existence of normal and immune opsonins is now quite
generally accepted. Neufeld in a consideration of the causes of
phagocytosis, states that he believes bacteria and foreign bodies
are only taken up by the leucocytes when the latter are stimu-
lated. He bases this assumption on the phagocytosis of red blood
cells by leucocytes, which occurs only when a special haemotropic
serum is present. In the haemotropic serum, according to Neu-
feld, the physico-chemical condition is so changed that a part of
the body is modified so as to serve as a stimulus or appetizer for
the phagocytes. Virulent organisms dissolve with greater diffi-
culty and give off less appetizer, and because of this there is less
phagocytosis of virulent organisms than of organisms with de-
creased virulence. Spontaneous phagocytosis, according to Neu-
feld, is due to changes in the cell, one of these changes accident-
ally stimulating the leucocytes to phagocytosis. In immuniza-
tion Neufeld believes a specific immune substance is produced,
which substance modifies bacterial or other cells so that they will
serve to stimulate the leucocytes to phagocytosis. Immune opso-
nin or bacteriotropin, as he prefers to call it, is a thermostable
substances which does not require complement. Normal opsonin,
on the other hand, is believed by Neufeld to produce its action
because of normal amboceptor and complement, which gently
dissolves bacteria and in this way stimulates the leucocytes to
phagocytosis. The assumptions of Neufeld are borne out by
numerous investigators.

Immune opsonins resist temperatures up to 55° for one hour,
65° C. at times not being sufficient to destroy their action. If
the opsonizing action of immune serum is once lost it cannot be
regained by the addition of fresh complement. Muir and Martin
have found that inactivated immune opsonin absorbs little or no
complement. Because of the properties of immune opsonins, they

STRICTURE OF OPSONINS. 61'

generally regarded as belonging to the anti- bodies of
the second order of Ehrlich. They apparently possess two groups,
the opsonophore and the haptophore. Of these the opsonophore
L-nmp is destroyed by heat, age, acids, etc. It is thus seen that
immune opsonins resemble the agglutinins and precipitins in
structure and by some investigators bave been thought to be
identical with agglutinins.

Normal opsonins, though not acknowledged by Wright to
be different from immune opsonins, have characteristics by which
they differ from immune opsonins. Normal opsonins resemble
complement in that they are absorbed or fixed by sensitized and
non-sensitized bacteria, blood corpuscles, specific precipitates and
indifferent bodies, and exhibit thermolability and susceptibility
to deterioration by age. Noguchi has found that normal opsonins
resemble complement in that they are highly labile bodies, loose
their action on standing several days, are preserved for a long
time when present in blood in a dry state, and in this condition
can be heated to 135° C. without destruction of their functions.
Recently Muir and Martin, Levaditi and Inman, and Hiihne and
Neufeld have ascribed the action of normal opsonins to comple-
ment. Cowie and Chapin have found that normal guinea pig
serum restores the opsonic power to normal serum which has been
heated to 55° C. They believe from their experiments that opso-
nins (normal?) exert their action because of an amboceptor-com-
plement group. Hektoen has recently published results of experi-
ments from which he concludes that the activating element is free
from the opsonin and therefore he believes that opsonins belong to
the third order of anti-bodies of Ehrlich.

It now seems quite definitely established that the action of
normal and immune opsonins is due to entirely different factors
in immunity, and that immune opsonins are distinct anti-bodies
probably belonging to those of the second order of Ehrlich.

RELATIONS OF OPSONINS TO LEUCOCYTOSIS.

No consistent relation has been found to exist between changes
in opsonic index and general or localized increase in the number
of leucocytes. Simonds and Baldauf have recently found that fol-
lowing an injection of heated bacteria there is a decrease in leuco-
cytes after which there is a marked increase in leucocytes with an

62 VACCINE AND SERUM THERAPY.

ultimate return to the normal. These changes in the number of
leococytes they find precede the changes in the opsonic index.
When the index is at its height the number of leucocytes has re-
turned to the normal. Various methods designed to increase the
number of leucocytes have been resorted to but have all been of little
avail in increasing the opsonin content in the blood as determined
by Wright's opsonic index. If, however, the leucocytes can be
stimulated to phagocytosis an increase in the number of leucocytes
in the infected region must certainly be of value. Opsonins are
by some supposed to originate in the leucocytes. The importance
and possibility of this becomes more evident from the work of
Peterson and Hiss and Zinsser. Potter believes that the opsonic
index varies with the source of leucocytes.

RELATION OF OPSONINS TO STIMULINS.

Metchnikoff has described a series of experiments in which
the introduction of serum, either from normal or immunized ani-
mals, greatly increased phagocytosis. This action, he supposes,
is exerted on the leucocytes and is of great importance in phago-
cytic immunity. The substance in the serum which he regards as
stimulating leucocytes he calls "stimulin." Stimulins are prob-
ably the same as opsonins, even though up to the present time,
in the observations reported, their action is on the leucocytes and
not on the bacteria.

OPSONINS AND AGGRESSINS.

Endotoxins or the aggressins of Bail, have by some been sup-
posed to account for decreased phagocytosis or low opsonic in-
dices obtained with some sera. Aggressins are supposed to injure
the leucocytes and inhibit their action. The part the endotoxins
play in preventing phagocytosis has not yet been determined.
Dorr, Sauerbek and others have shown that aggressins are not
difinitely specific. Neufeld from experiments has decided that
the lack of phagocytosis with virulent organisms does not depend
upon the injury to leucocytes and for this reason does not believe
that decreased phagocytosis is due to the action of aggressins.

OPSONINS, STIMULINS, AGGRESSINS. 03

INFLUENCE OF CHEMICALS AND REACTION ON

OPSONINS.

Various salts, even in small amounts, influence opsonic action
markedly. Noguchi has shown that in normal serum, reaction
exerts considerable influence on phagocytosis, it being most marked
when the serum to be tested is neutral in reaction.

NON-BACTERIAL OPSONINS.

Opsonins have been found to be produced for other cells than
bacteria. Hektoen has reported opsonins for blastomycetes and
red blood cells. Likewise it is possible to get marked phagocytosis
of inert particles of charcoal, stains, etc., upon the addition of
certain sera.

CHAPTER VII.

VACCINE THERAPY

For centuries it has been known that following an attack of
certain acute infectious diseases there remains a certain loss of
susceptibility to the contraction of a second attack of the same
disease. Early in the eighteenth century this experience was
utilized by vaccination for small-pox. When it was found, about
the middle of the nineteenth century, that many acute and in-
fectious diseases have for their etiological factor certain micro-
organisms known as bacteria, attempts were made to induce im-
munity by inoculation of killed or attenuated cultures of these
organisms. Pasteur in this way successfully immunized against
chicken cholera, but while a certain amount of success by the
injection of killed cultures was obtained, still it was found imprac-
ticable for reasons that are not understood nor can be considered
here. Recently, however, Haffkine has successfully immunized or
vaccinated against cholera and Pfeiffer and Wright against ty-
phoid fever. It would prove impracticable to employ active im-
munization against all organisms with which man may be infected,
for this would require frequent injections of all kinds of organisms.
Active immunization to be applicable generally must be such that
it can be instituted after infection has occured. In this Pasteur
was successful in the disease of hydrophobia. It was found, how-
ever, that when the method of active immunization is begun after
infection has occured, there is a super imposition of a mild form
of the disease upon a severer form, thus accentuating the disease.
This observation lead to a relatively long period of cessation of
this method of treatment.

In recent times active immunization after contraction of the
disease, has again become important. This has been due espec-

BACTERIAL VACCINES. 65

ially to the efforts of Wright. It is important to note that the
method of vaccination was used by Wright before he had dis-
covered opsonins. From his later observations Wright has decid-
ed that opsonins are of the utmost importance in immunity to
some organisms, and are decreased in amount during most infect-
ions but that they can be increased by the injection of bacterial
vaccine.

BACTERIAL VACCINES.

It was stated in a former chapter that in bacterial infections
the infected body absorbs bacterial substances and products.
According to the amount of substance and products absorbed
immune substances are produced. When much is absorbed suf-
ficient immune substance is usually produced to cure the disease,
but when little is absorbed not enough immune body is produced
to cure the infection which then becomes chronic. To produce
enough immune substances to affect a cure from an infection
Wright and Douglas have employed the well known principle of
active immunization. The method they have suggested is to
actively immunize by injection at the proper time, of proper doses
of killed Cultures of the organisms causing the infection. The
effects of injection of killed cultures or vaccines according to these
investigators is to produce certain changes in the opsonin content.
Wright and Douglas attempt to immunize by giving injections
of such numbers of bacteria as will produce only a slight negative
phase which lasts only a short time and is followed by a rise in
opsonic index which lasts for some days. When the index begins
to fall after the subsidence of the positive phase, another injec-
tion is made and the effect on the opsonic index is determined.
An attempt is made to inject such doses at the second and sub-
sequent injection that there shall again be only a slight negative
phase and that during the positive phase the opsonic index shall
be higher than following the first injection. An effort is made to
keep the opsonic index of the individual above normal. If the
index shows a marked drop after any injection, the dose has been
too large. By means of vaccines Wright claims that the anti-
bacterial power of the blood can be increased for any microbe
invading the body. In 1906 Wright was able to say that he had
by this method achieved uniform success.

66 VACCINE AND SERUM THERAPY.

DOSAGE.

Wright has, by means of the opsonic index determined the
proper dosage for the different bacterial vaccines and while he as
not stated that definite amounts must be used, still he has sug-
gested certain doses. His principle in dosage is to use the smallest
dose that will give a good rise in the opsonic index and never to
increase the amount injected until it has been ascertained that
the doses which have been injected have been too small to produce
a good rise in the opsonic index.

The doses of vaccine recommended by Wright are as follows:

Bacillus coli 5 to 50 million bacteria

Micrococcus gonorrhoeas 5 to 50 million bacteria

Micrococcus pneumonise 10 to 50 million bacteria

Bacillus pyocyaneus 5 to 5 thousand million bacteria

Micrococcus pyogenes 50 to 1 thousand million bacteria

Streptococcus pyogenes 10 to 25 million bacteria

Bacillus tyhosus 5 to 50 million bacteria

Koch's New Tuberculin 1-1,000 to 1-400 milligrams

These doses are by some considered purely arbitrary, because
the personal characteristics of the individual to be immunized,
the duration, extent and severity of the infection, must all be con-
sidered in active immunization. While these doses may be arbi-
trary they at least offer some guide as to the number of organisms
to be injected at a time. At times in active immunization the in-
jection of vaccine is accompanied by injection of specific immune
serum. This will pave the way for larger doses and more powerful
vaccine injections.

Injection of the doses recommended by Wright usually pro-
duces no constitutional symptoms. Wright has stated that at
times malaise, slight general muscular pain and headache are
observed on the day following the injection but as the positive
phase comes on there is a marked buoyancy of spirits and stimu-
lation. Most investigators on this subject have never observed
untoward symptoms when the small doses recommended by Wright
are injected, while others have at times observed a rise of tem-
perature, nausea, vomiting, etc. According to the principle of
active immunization probably the greatest amount of immunity
results when there is some reaction for in active immunization the
individual or animal undergoes a modified or less severe form of
the disease.

BACTERIAL VACCINE. 07

The point of injection of bacterial vaccines is considered by
Wright to be of importance in the success of vaccination. Those
regions of the body are selected where there is rapid absorption
by the lymph and blood. It has been found that on subcutan-
eous injection greater amounts of immune body are formed than
upon intravenous injection. This is especially illustrated in
the production of certain haemolytic sera.

If Wright's method of vaccine treatment of bacterial diseases
is to be judged it must be remembered that the injection of killed
cultures did not originate with Wright, but that Wright has added
opsonic index determinations to control the time and doses of
these injections. Opsonic index determinations have, however,
been found to be open to so many sources of error, of such great
variability in health, disease and after injections of vaccines, that
many clinicians have decided upon the dosage and inter-spacing
of injections by clinical symptoms rather than based upon opsonic
index determinations. Most clinicians have, in the first injection,
used the dosage recommended by Wright but in subsequent in-
jections have used such amounts as seemed warranted and indi-
cated. Hektoen, adhering to the reliability of opsonic index de-
terminations, holds that the index is of diagnostic and prognostic
importance, while Park, Cole, Bolduan and others regard the index
as unreliable for such purposes.

ORGANISMS USED IN VACCINATION.

Many of the species of pathogenic bacteria have been used in
active immunization. Two kinds of vaccine treatment have been
employed, one consists of the injection of killed cultures of the
causal organisms and the other of the autoinoculation from the
infected focus by means of massage and manipulation.

In the injection of vaccines, so called "stock culture" vac-
cines and "personal" or "autogeneous" vaccines have been used.
It has been found that for some organisms, vaccines made from
stock cultures will give as good results as will personal or auto-
geneous vaccines. Stock vaccines have the advantage that no
time need be lost in the preparation of the vaccine, and for nearly
all of the species of bacteria causing infections amenable to treat-
ment by active immunization, stock vaccines are equally as good
as autogeneous vaccines. The principle exceptions are the vac-

68 VACCINE AND SERUM THERAPY.

cines for Streptococcus pyogenes, Mic. pneumoniae and, accord-
ing to some, for the Mic. gonorrhoeae.

Some investigators have used vaccines containing several
species of bacteria. Serum and vaccine manufacturers have pre-
pared vaccines suitable for active immunization against one or
many different sepcies. Wright at one time made no particular
effort to identify the species of the causal organism, simply mak-
ing a vaccine from the cultures obtained by inoculation with
material from the lesion. This is open to many objections, for
at times the causal organisms may be such that it will not grow
on ordinary media or else the causal organisms may not grow as
well as secondary invaders and saprophytic species. Because of
the mixed and undetermined vaccines used in immunization it
may frequently happen that those organisms, for which immuni-
zation is necessary or beneficial, are not injected.

In all cases it ought clearly to be remembered that if bene-
fits are to be derived from active immunization the vaccine must
consist of a suspension of the causal organism. To determine
these, microscopic examinations of the material from the lesion,
and isolation of the organisms when possible ought to be made.
This cannot be too strongly emphasized, especially to the practi-
tioner who can buy from the druggist vaccines said to be good
for boils, acne or whatever the condition may be, while as a mat-
ter of fact the vaccine may not contain the organism causing the
particular lesion which is to be recovered from as a result of active
immunization. This has undoubtedly been the cause of some of
the failures observed by some clinicians and has, as was the case
with tuberculin, decided the practitioner against the use of bac-
terial vaccines in certain cases of infection in which vaccine might
have been of value.

PREPARATION OF BACTERIAL VACCINES.

While killed cultures of bacteria have for some time been
used in active immunization, the amounts injected had been only
indefinitely determined. Wright and Douglas originated and de-
scribed a method for the preparation and standardization of bac-
terial vaccines to be used in immunization. The causal organisms
according to this method are grown on artifical culture media
after which they are suspended in sterile salt solution. The num-

CONTROL OF INJECTION OF VACCINE. 09

her of bacteria per cubic centimeter of the suspension is deter-
mined by drawing into a capillary pipette one volume of the
bacterial suspension, and one or more volumes of fresh blood
from a puncture in the finger. These volumes are then mixed
well and a drop of the mixture placed on a clean slide. With a
spreader which is placed in front of the drop, a smear is made.
This is then allowed to dry, is fixed and stained with a suitable
stain, usually Loeffler's methylene blue. The slide is then counted
by determining the number of red blood cells and bacteria in
from ten to twenty-five fields of the high power of the microscope.
Normal human blood contains five million red blood cells per
cubic millimeter. After having established the ratio of red blood
cells to the bacteria in the suspension, it is easy to determine the
number of bacteria per cubic millimeter or cubic centimeter.
The tube containing the bacterial suspension is now sealed and
heated in a water bath, to 60 or 65° C. for one hour. After kill-
ing the bacteria in the suspension, a dilution, suitable for injection,
is made in a bottle containing 50 c. c. of sterile salt solution.
The bottle is closed with a rubber cap and sufficient carbolic acid
or lysol is added to make an ultimate dilution of O.o per cent.
After this the vaccine is tested for sterility on suitable culture
media. To obtain vaccine from the bottle a drop of pure lysol
is placed on the rubber cap. This is done to sterilize the outer
surface of the rubber cap. Then the sterile hypodermic needle
is inserted through the cap, the bottle turned up and the piston
of the syringe is withdrawn until the desired amount of vaccine
has been taken into the barrel of the syringe.

CONTROL OF INJECTION.

While Wright and his pupils have claimed to regulate the
dosage and inter-spacing of injections entirely by the opsonic
index, others have found the opsonic index too unreliable to serve
as a guide in the administration of bacterial vaccines. Wright
and his pupils have not always based their decisions on dosage
and time for injection, on determinations of the opsonic index.
It frequently happens that at the time of the first injection of
vaccine their patients have an opsonic index well above normal.
In most cases the treatment is based on determinations made by
serum drawn twenty-four hours earlier. The time for the second

70 VACCINE AND SERUM THERAPY.

and subsequent injection is not always controlled, even by Wright
and his pupils, by the opsonic index.

In accepting opsonins as important factors in immunity other
immune substances must not be lost sight of. Various investi-
gators have designed methods by which the action of agglutinins
and bacteriolysins is' to be inhibited, so that opsonic indices may
be determined and used as a guide for the dosage and inter-spac-
ing of injections. The justifications for such regulation of dosage
and determination of the proper time for injection is not evident.
In a patient suffering with cerebro-spinal meningitis, from the
spinal fluid of which Mic. meningitidis was cultivated, the writer
tried to govern the dosage and inter-spacing of injections of
meningococcus vaccine by the opsonic index. After three to
four minutes of incubation of the mixture of leucocytes, men-
ingococcus suspension and patient's serum, all of the organisms
were found to be dissolved. It is evident that lysins in this case
were probably of more importance in immunity than were opso-
nins. Clark has proposed that in determining the opsonic index
of serum of typhoid patients, the serum be heated to 56° C. in
order to destroy the lysins for the typhoid bacillus. By follow-
ing out this method Clark finds that relapses follow upon a drop
in the opsonic index. How much more important would it prob-
ably be to determine the curve for lysins in a case where lysins
are present in such amount that it is necessary to destroy their
action before opsonic index deternr nations can be made? This
would seem more rational, especially because our knowledge con-
cerning lysins in immunity is greater than it is concerning opsonins,
which may or may not play any part in immunity. .

According to the determinations of the opsonin content of
the blood, Wright and his pupils have found that the opsonic
index begins to rise two to five days after injection, and usually
reaches its height after about five to eight days. In most cases
adherents of the opsonin theory make injections every four to
eight days. The appearance in the blood, however, of the better
known anti-bodies comes somewhat later. Bacteriolysins are
found to be present at times in the spleen twenty-four hours after
injection of culture. They do not, however, appear in quantities
in the blood until five to nine or fourteen days later. Agglutinins
are found to be present in the blood from eight to twelve days

ANTI-BODIES AFTER VACCINE INJECTIONS. 71

after injection of cultures. Typhoid agglut in ins 'usually do not
appear in a typhoid patient's blood until after ten' days of the
disease have elapsed. Furthermore, it is noted that anti-toxins,
bactericidins and agglutinins can be enormously increased by
correct inter-spacing of injections and proper dosage. Wright
himself has called attention to the value of determining these
different substances in immunity before injections are repeated.
If injections are made entirely according to the opsonic index, they
may be made before the bactericidal and other protective sub-
stances have increased in amount, several negative phases may
thus be superimposed on each other, and the protective substances
be much decreased. With the role of opsonins in immunity in
doubt, the difference in sensibility of different species of microor-
ganisms to opsonins established, and the presence of marked
bacteriolytic action of certian sera for certain species of infecting
organisms observed, the control of the process of active immuni-
zation by the opsonic index alone does not seem justifiable. The
immune bodies of importance in immunity to the particular species
of bacteria ought to be established so that the dosage and inter-
spacing of injections could be regulated by determinations of the
presence of these substances. Meakins has, on this basis, studied
immunity to dysentery bacilli, Streptococcus pyogenes, Micro-
coccus pyogenes and Bacillus tuberculosis. Such methods would
undoubtedly result in a better accomplishment of a higher grade
of active immunity.

It is necessary, however, to refer to the great success obtained
by Wright, and others following Wright's teachings, in the treat-
ment of bacterial infections and diseases according to the deter-
minations of the opsonic index. Wright has stated that he has
not achieved success only in a certain percentage of cases, but that
he has had uniform success by following his method of active im-
munization. Ross has given a list of diseases successfully treated
based on the determinations of the opsonic index. This list in-
cludes furunculosis, pustular acne, sycosis, cystitis, otitis media,
empyema, gleet, gonorrhoeal rheumatism, acute gonorrhoea, local-
ized tuberculosis, lupus, tuberculosis of the joints, bladder, kid-
ney, epididymis, glands, peritoneum, iris and lungs. Surely a
method that will give uniform success for as large a list of dis-

72 VACCINE AND SERUM THERAPY.

eases as this, is one that must be of the utmost importance in
clinical medicine.

Because the methods for the determination of immune bodies
are still so inaccurate and in many cases unreliable, and because
the defensive agencies of the animal body against bacterial in-
vasion and multiplication are so manifold and complex, numer-
ous investigators have relied upon the clinical control of dosage
and inter-spacing of injections of bacterial vaccine. At all events,,
the value of active immunization by injection of bacterial vaccines
will ultimately rest upon clinical results. While it is relatively
easy in most cases of infection to tell when complete recovery
has taken place, still it is difficult to tell what clinical symptoms
and signs are associated with the best method of immunization.
The conditions ultimately desired are definitely known, but the
conditions in the diseased body which will produce these ulti-
mately desired effects, are little understood, Probably the best
illustration of this is seen in the use of tuberculin in the treatment of
tuberculosis. Tuberculin has been known and used in treatment
for nearly twenty years, and still today it is an open question
whether the dosage shall be regulated to give an anti-toxic or an
anti -bacterial immunity. Clinical evidence and experience is
deceiving as is well demonstrated in the literature on tuberculin.

With some organisms, and in certain cases treated by the
vaccine method, excellent results have been obtained when the
dosage and interval between injections has been determined by
clinical signs and symptoms. The number of cases in which
the treatment is controlled clinically is probably larger than the
number of those treated according to Wright's method. Many
investigators, although determining the opsonic index during the
process of immunization, have found that it is quite as safe to
administer vaccine when controlled by clinical results as by op-
sonic index determinations. In clinical control of vaccine treat-
ment, it is aimed to have the dose so small and the injections so
far apart that immunization is accomplished without the pro-
duction of clinical symptoms. The doses that are usually given
are those recommended by Wright, though some have used larger
doses without the production of any reaction.

In most clinics the doses injected are gradually increased.
This is also the procedure recommended by Wright. If this

STAPHYLOCOCCUS VACCINE. 73

method is followed, it frequently happens that there may be an
exacerbation in the pathological condition. This is especially
observed in furunculosis produced by the staphylococci. Fre-
quently following the second injection of a vaccine of this organ-
ism a new furuncle will appear. The writer has obtained better
results by first injecting the largest dose and then at subsequent
injections to give constantly decreasing doses. The reasons for
following this method are based entirely upon clinical experience
and may be due to the fact that if the second and subsequent
injections are larger than the preceding one, too much of the im-
mune substance is used up in combining with the bacterial vac-
cine injected or there may be an actual hypersusceptibility to
these organisms.

The interval between injections when the opsonic index is
not determined, lies usually between seven and ten days, which
is about the time required for the production of known immune
substances. Whenever a reaction follows an injection the in-
terval between injections is lengthened so as to allow sufficient
time for the disappearance of all symptoms.

Staphylococcus Infections. The various infections caused by
the Micrococcus pyogenes group have been considered most satis-
factory for vaccine treatment. In the list of Staphylococcus infec-
tions treated by bacterial vaccines, Ross includes boils, carbuncles,
acne, sycosis, felons, styes, and septic wounds. The results ob-
tained in the treatment of furuncles and carbuncles by killed
cultures of the infecting organisms have varied. Probably the
best results have been obtained in localized surface infections.
In the treatment of these cases it has been found that autogen-
ous vaccines are not necessary. It is, however, important in all
cases to determine the species and variety of organisms causing
the infection and to use this variety in the treatment of the path-
logical conditions. The Staphylococcus vaccines with which the
writer has obtained the best results are those made from different
isolation of the same variety of organisms. Thus a Mic. pyogenes
aureus vaccine which has given good results is one made from

74 VACCINE AND SERUM THERAPY.

three isolations, the organisms being isolated from the lesions of
chronic marked furunculosis, from an acute boil on the face, and
from a patient having multiple pustules on the back of the neck.
Most patients suffering with furnucles and carbuncles show a
marked improvement within twenty-four hours after injection
and some of these patients remain well after that. Everyone
who has treated a number of patients suffering with furuncles or
carbuncles must have observed that there occasionally is recur-
rence of these conditions after apparent recovery following upon
the injection of suitable vaccines. If Wright's method of vacci-
nation according to the opsonic index is followed out, there are
apparently more recurrences than when the injections are given
at intervals not less than seven days nor greater than ten days.
In all cases the best results will be obtained if a small incision is
made in the carbuncle or furuncle twenty-four hours after vacci-
nation. By this method the collection of leucocytes or pus is
drawn off, the pressure is relieved and the bacteria ingested by
the leucocytes are removed. The advantage of this becomes
evident when it is realized that staphylococci secrete a leucocidin,
which destroys leucocytes. If the leucocytes containing bacteria
can be removed before they are killed the value of phagocytosis
in immunity is evident. After this the pus is evacuated daily
from the furuncle or carbuncle and the wound is not allowed to
heal until it has been found that for several days no more pus is
being formed. In addition to draining the furuncle or carbuncle
through a slight incision, thorough cleansing of the skin with
benzine is of great advantage in preventing the formation of new
foci. The doses used in most clinics for these conditions are gradu-
ally increased, the first injections usually consisting of three
hundred million cocci. As was mentioned before, apparently
better results are obtained when at the first injection six hundred
million cocci constitute the dose, while the later doses contain
fewer cocci, at times only one hundred million. While this method
of vaccination does not depend for its results only on the effects
of vaccines, still it is a method which gives good results without
the formation of large scars.

Acne vulgaris has been successfully treated by vaccination
only in certain cases. The value of bacterial injections in this
condition was so emphasized by Wright that for a time it was

STREPTOCOCCUS VACCINE. 75

the common belief among clinicians that vaccinations with killed
cultures of Micrococcus pyogenes albus were applicable in all cases
of acne. This, however, is not the case. In the lesions of many
patients suffering with acne vulgaris, the white skin coccus cannot
be found. In such cases either the species of organisms causing
the pustules must be injected or else vaccine treatment should
not be applied. A number of the cases of acne which have been
treated by the writer have failed to show microorganisms that
could be cultivated. When the vaccine treatment is used other
means of treatment must not be discontinued. Incisions and
evacuation of the pustules, cleansing of the skin with benzine,
expression of comedones or black heads, the bathing of the infected
part in hot water, application of sulphur or other suitable lotions
and the regulation of the diet are all of assistance to overcome
this disease. Even under these measures the treatment is not
always successful.

The treatment of sycosis barbae or barber's itch with staphy-
lococcus vaccines has not always been as successful as has been
reported. Two patients with this disease in the City Hospital
in New York City, were treated by Ross with injections of staphy-
lococcus vaccine. No satisfactory results, however, were obtained.
The unsatisfactory results obtained by the injection of staphy-
lococcus vaccine is easily explained when it is realized that sta-
phylococci cause secondary infections in only some of the cases
of sycosis barbae. In these cases only can staphylococcus vaccine
be of benefit.

Streptococcus Infections. Various kinds of streptococcus in-
fections have been treated by vaccines. Usually these infections
are acute and severe and it often is a question whether sufficient
time for the production of immune bodies as a result of vaccina-
tion will elapse before death or spontaneous recovery terminates
the disease. Arthrites, pneumonia, pericarditis, erysipelas, local •
infections and endocarditis, etc., have been treated by the use of
vaccine. At times striking results have been obtained, while
in other cases not treated by vaccination there has been equally
striking recovery. One needs only to remember the cases of !
streptococcus infections of the uterus, of septicaemia and ery- ;
sipelas that recover in a few days without any specific
treatment, to realize that it is easy to attribute beneficial results

76 VACCINE AND SERUM THERAPY.

to the use of streptococcus vaccines in cases in which the same
results might have been obtained without them. In 1907, the
author treated 3 7 patients suffering with erysipelas by injections of
killed cultures of streptococci isolated from the lesions of ery-
sipelas. From the results obtained it was impossible to determine
the value of the injection of the killed cultures of streptococci.
While the injection of from twenty-five million to one hundred
million streptococci did not prevent migration and recurrence,
the apparent shortening of the duration of the disease suggests
that streptococcus vaccines are of some value. The effect upon
the duration of the disease w^as found to be uncertain because,
during the time of the investigation, no suitable cases remained
untreated with which comparison could be made. It is known that
the duration of the disease varies in different years. Ross and
Johnson have recently reported on the treatment of erysipelas
with a vaccine made from streptococcus erysipelatos, and conclude
that such vaccine exercises a specific and controlling influence on
the course of the disease. These investigators make initial injec-
tions of from ten to twenty million killed streptococci, and repeat
these injections every day or two according to the clinical results
obtained. Criticism of the results obtained is not possible because
of the meagre reports of the cases treated by these investigators.

Wright and others have reported successful treatment of
streptococcus septicaemia by the injection of streptococcus vac-
cine. It must, however, be remembered that streptococcus septi-
caemia is at times recovered from even when no specific treatment
is given.

In streptococcus immunization autogenous vaccines have
usually been preferred, though Gabritschewsky and others have
gotten good results with stock vaccines.

Gonococcus Infections. Probably the best results of the use
of gonococcus vaccines have been obtained in cases of gonorrhceal
arthritis. Cole and Meakins obtained encouraging results in
about twenty cases of acute and chronic gonorrhceal arthritides
when treated by injections of stock vaccines of Mic.gonorrhoeae.
These investigators treated their cases according to the opsonin
content of the blood as determined by the opsonic index, but
feel that probably as good results might have been obtained had
the injection been given every ten days, which was about the

GONOCOCCUS AND PNEUMOCOCCUS VACCINE. 77

time determined upon by the opsonic index. The doses injected
in these cases varied from five hundred million to one thousand
million cocci.

Treatment of acute and chronic urethritis by vaccines has
given some, but relatively few, beneficial results, and is by no
means to be regarded as a sufficient measure to obtain a cure from
this condition.

Micrococci pneumoniae Infections. Apparently some good re-
sults have been obtained in the treatment of chronic infections
with this organism. The dose injected varies from ten million to
one hundred million. In acute pneumonia the course of the
disease is usually too short to derive beneficial results by means
of active immunization.

Tuberculosis. The bacillus of tuberculosis may produce its
lesions in almost any part of the body. Bacteriological examina-
tion of the lesions produced will vary. In some lesions, especially
those localized in the interior of the body such as the glands,
joints and bones, the tubercle bacillus alone is responsible for the
condition found. In other lesions, as in pulmonary and intestinal
tuberculosis, there are present in addition to tubercle bacilli other
bacterial species. Active immunization, by the injection of killed
tubercle bacilli theroetically will be of benefit in lesions due to
the tubercle bacillus alone, while in lesions produced by tubercle
bacilli and other species it will not be very efficacious.

In the specific treatment of tuberculous conditions, various
modifications of so-called tuberculin have been used. Old tuber-
culin is the concentrated germ-free glycerine bouillon on which
tubercle bacilli have been grown for several weeks. Before fil-
tration the tubercle bacilli are killed by heat. Deny's bouillon
filtrate, "B. F." differ from old tuberculin in that filtration is done
before killing the bacteria by heat. Tuberculin R, "T. R. "con-
tains the powdered tubercle bacilli suspended in salt solution.
Usually 1 c. c. contains 2 m. g. of powdered bacilli. Tuberculin
0, "T. 0." is the supernatent opalescent liquid obtained after
emulsifications of ground up tubercle bacilli in distilled water,
are centrifuged. Bacillen Emulsion, "B. E." is a suspension of
ground up tubercle bacilli in fifty per cent glycerine. It contains
5 m. g. in 1 c. c. of suspension.

78 VACCINE AND SERUM THERAPY.

Our knowledge concerning the effect of tuberculin on the
bacillus of tuberculosis and the tissues of the body in which the
bacillus is found, is still indefinite. Before tuberculin can be scien-
tifically applied as a therapeutic measure, it is necessary to deter-
mine the part played by the moderate reaction in the tissues and
what is to be accomplished by immunization with tuberculin.

The reaction in diseased tissues following the injection of
tuberculin may be either mild, in which case, there is redness due
tohyperasmia and congestion, or it may be marked, producing
necrosis and sloughing of the diseased tissues. In all attempts
at immunization with tuberculin the mild reaction is preferred
because it does not lead to the liberation of tubercle bacilli as a
result of the necrosis of tissues, but does furnish to the lesions
an increased blood supply.

The conception of the immunization accomplished by tuber-
culin injections differs, there being two important theories. Koch,
Wright and others believe that anti-body, specific to the action
of the tubercle bacillus, is produced. This, according to their
views, is accomplished through stimulation of all the defenses
of the body. In conformity to their views on the objects of
tuberculin immunization, tubercle bacilli are injected in the form
of "T. R." or "B. E."

Maragliano, Sahli, Denys, Trudeau, and others adhere to the
toxin immunization theory, according to which tuberculin injec-
tions lead only to tolerance or immunization to the toxin liberated
by the tubercle bacillus. In accordance to this theory, old tuber-
culin as well as other tuberculins are injected, the doses being
gradually increased to the highest point of tolerance. The lesion
according to this theory is only secondarily effected, there being
no acquisition of immunity to the tubercle bacillus.

Tuberculin treatment is usually only undertaken in tuber-
culosis of the glands, joints and bones, though in some clinics it
has also been used in pulmonary and other forms of tuberculosis.
Koch would limit its application to such cases as are still
curable — that is, have not advanced too far nor are complicated
by other bacteria. In deciding upon cases amenable to tuber-
culin treatment Koch relies upon the body temperature as the indi-
cator. In uncomplicated and curable cases the temperature does not
rise much above normal. Various measures have been taken as

TUBERCULINE, COLON AND TYPHOID BACILLUS VACCINE. 79

guides in tuberculin treatment of tuberculosis. Wright injects
only amounts large enough to produce a positive phase in the
opsonic index and in some cases of tuberculous glands and joints
has accomplished good results by autoinoculation. Trudeau,
Denys and others have regulated the dosage and inter-spacing of
injections by clinical signs and symptoms. They usually inject
1-1000 m. g. of old or 1-10,000 m. g. of "Bacillen Emulsion."
After this, by a long continued treatment they gradually increase
the dosage. The doses injected are not increased nor the interval
between injections shortened if there are any general or local re-
actions, as evidenced by fever, local redness and edema. In this
method no stress is laid on the absolute amount of tuberculin
injected. When the patient has no tolerance to tuberculin, Tru-
dean has usually observed chronic toxaemia and progression of
the disease.

Tuberculin treatment has proven especially beneficial in
lesions in the joints, bones, glands, skin and urinary bladder,
although good results have been obtained in pulmonary and other
forms of tuberculosis.

Bacillus coli Infections. Different infections produced by
Bacillus coli have been treated by injections of vaccines made with
this organism. The vaccine method of treatment has been found
to be most successful in cases of chronic cystitis due to B. coli com-
munis, although cholecystitis, appendix abscesses, endometritis
and other local infections with this organism, have at times re-
sponded favorably to this method of treatment. The number of
bacteria injected is seldom more than fifty million, usually smaller
doses than this being used. Vaccines made from cultures isolated
from the patient usually give the best results. The injections are
repeated about every ten days.

Bacillus typhosus and Bacillus paratyphosus Infections. The
typhoid bacillus group consist of several important members.
Recognition of this fact has led to the use of vaccine made from
cultures isolated from the patient to be treated. Vaccines made
from killed cultures of typhoid bacilli have been used for immuniz-
ation as well as for treatment of pathological conditions due to
members of this group.

Pfeifler and Kolle have made vaccines for immunization
against typhoid fever from agar cultures, while in England the

80 VACCINE AND SERUM THERAPY.

vaccine usually is made from bouillon cultures. The organisms
are killed by one hour's exposure to 53° C. to 60° C. Pfeiffer and
Kolle inject sufficient amounts of vaccines to produce a local reac-
tion lasting several days, and a general reaction, as is demon-
strated by fever and malaise, lasting one to two days. Following
the first injection, two to three injections of increasing amounts
are made at intervals of eight days. The English method also
produces marked disturbances at times. The duration of immunity
conferred by this method of treatment varies, and is usually re-
garded as lasting from one to three years. The results of such im-
munization are still doubtful but statistics indicate that immunized
individuals are less likely to contract the disease, and that when
the disease is contracted it is less severe and less often fatal.

Typhoid bacillus vaccine has been tried in the treatment of
typhoid fever. Here it has met with but little success. The best
results with the vaccine have been obtained in cystitis, cholecysti-
tis, and the local abscesses following attacks of typhoid fever. The
doses injected range usually from five million to fifty million bacilli,
the injections being repeated at intervals from eight to ten days.
Autogenous vaccines usually give the best results.

Bacillus dysenteriae Infections. Vaccines made from dysen-
tery bacilli have been used in prophylaxis as well as treatment of
bacillary dysentery. Inasmuch as there are different strains of
these bacilli, it is advisable to use vaccines made from the cultures
isolated from the patient or with all of the different strains of B .
dysenteriae.

Shiga has tried various methods of active immunization and
has found that the best results are obtained either, by injecting
subcutaneously anti-dysenteric serum and killed cultures of dysen-
tery bacilli, or, by taking by mouth the killed cultures of this organ-
ism. By Shiga's method of simultaneous injection of anti-dysen-
teric serum and dysentery vaccine the death rate in Japan due to
bacillary dysentery has been much reduced. The duration of im-
munity, according to Shiga, is about two months.

Vaccine treatment of dysentery can only be applied in cer-
tain cases. By the method of active immunization, immunity can-
not be established in much less than eight days, and for this reason
only subacute or chronic cases are amenable to treatment by vac-
cines. The number of cases treated by this method is still too

I. \IMf\IZATION AGAINST HYDROPHOBIA. 81

small to warrant a conclusion. It seems quite certain, however,
that vaccine therapy may prove of value in this disease. It must
l.c lionu- in mind that there are different strains which can produce
the disease and that an autogenous vaccine will most likely give
the best results. Usually fifty million bacilli are injected, the inter-
val between injections being from eight to ten days.

Cholera Vaccines. Vaccines made from cholera vibrions have
been used in protective immunization. Statistics on this method
are still too meagre to warrant definite conclusions on its value.

Less common Infections. Vaccine treatment has been ap-
plied in most all infections produced by bacteria. The results
obtained' have varied, though Wright believes that the method
is applicable in the treatment of all infections caused by bacteria.

SPECIFIC VACCINES FOR DISEASES OF
UNKNOWN ETIOLOGY.

Rabies, Hydrophobia or Lyssa. This is a disease whose eti-
ology is only indefinitely established. The organism responsible
for the condition has never been grown. By some investigators,
certain peculiar bodies, found in 1903 by Negri in the large nerve
cells in the central nervous system, are regarded as the causal factors
in the disease. Although these bodies are not universally accepted
as the etiological factors, they are quite generally accepted as spe-
cific to this disease.

The inability to establish the etiology has not prevented the
establishment of methods of immunization to this disease. The
principle of the method of immunization most generally practised
is" based on the establishment of an active immunity during the
period of incubation of the disease. In man the period of incuba-
tion usually lasts from four to six weeks, though it has been found
to be as short as fifteen days and as long as one year. The rela-
tively long period of incubation makes it possible to establish an
active immunity before the symptoms of the disease develop.

Immunization is accomplished by repeated injections of so-
called "fixed virus." Pasteur found that tissues and fluids from
rabid animal vary considerably in virulence, and that the viru-
lence of virus can be changed considerably. If successive re-inocu-
lations into rabbits be made from the virus of a rabid dog the virus

82 VACCINE AND SERUM THERAPY.

is increased in virulence for rabbits. After a number of passages
through rabbits the virus finally can no longer be exalted in viru-
lence. This is then called the fixed virus, is usually obtained from
the cord and kills a rabbit in six to seven days. . This fixed virus
can, by drying, be gradually decreased in virulence so that after
fourteen days of drying it has lost all virulence for rabbits. To
immunize the individual, virus of low virulence is first injected,
after which inject ions are made with virus of greater virulence until
at the end of the treatment injections of cord which has dried only
from two to three days, are made.

At the present time there are in the United States many so-
called ' 'Pasteur Departments" for immunization against rabies. The
particular technique employed in such departments varies in some
details, still the fundamental and essential principles are the same.

In every case treated, an attempt is made to establish definitely
the diagnosis of rabies. Two methods are employed to accomplish
this: one is to examine for Negri bodies and the other is to make
inoculations into rabbits. For both of these methods it is es-
sential that the head of the rabid animal be preserved intact and
sent to the laboratory for diagnosis. It is therefore recommended
that rabid animals be killed by other methods than those which
mutilate the head.

As soon as the diagnosis is established, or if this is impossible
because the animal cannot be found, the patient should submit to
treatment. This requires from two to four weeks and. consists
usually in daily injections of cord of hydrophobia rabbits, which
has been aseptically dried over caustic potash at a temperature of
about 23 ° C. for varying periods of time. After drying, a part of
the cord is suspended in bouillon to which either 0.25 per cent car-
bolic acid or glycerine has been added as a preservative. The in-
jections are made into the subcutaneous tissue and are usually not
followed by much disturbance. If there is an inflammatory reac-
tion, it is to be treated as any other cellulitis. During the treatment,
the patient may go about his work, his bowels should be kept open
and alcoholic excess avoided. Immunization is not fully attained
until about two weeks after the last injection of virus.

Immunization is usually and preferably practiced at the hos-
pital of Pasteur departments. The New York City Department of
Health has also practised immunization by sending virus to physi-

SMALL-POX VACCINATION. 83

cians outside of the City of New York. In such cases a stronger
course of treatment is followed and an effort is made to limit this
practice to places not more than twenty-four to thirty-six hours'
distance from the laboratory.

The results of Pasteur treatment or active immunization
against hydrophobia are most satisfactory. Without specific treat-
ment the mortality in patients bitten by rabid dogs varies from 50
to 80 per cent, while in patients who have undergone a course of
active immunization the death rate is about 0.5 per cent. To get
good results, early treatment is essential, Bites on the face and
head, and bites by mad wolves are not immunized against as suc-
cessfully as bites by dogs in other parts of the body. After the
symptoms of the disease have developed, active immunization can
no longer be expected to be of value.

Immunization against rabies has also been attempted by the
injection of anti-rabic serum. Babes and Lepp in 1889 found that
they could protect dogs against rabies by injection of serum from
dogs actively immunized to rabies. Tizzoni and Centanni have
reported favorable results with anti-rabic serum. Marie has re-
cently advocated the simultaneous injection of virus and anti-rabic
serum. By this method it is possible to immunize against hydro-
phobia more rapidly. The value of these newer methods has not
been proven so that at the present time conclusions as to their
value are impossible.

Small-pox. Small-pox is a disease for which the etiological
factor- has not been determined, although Guanieri, Councilman,
Calkins and others have found protozoan parasites in the lesions
of the disease.

For a long time it has been known that one attack of small-
pox will protect against subsequent contraction of the disease.
This has led to the practice of acquiring immunity against severe
forms of the disease by intentional inoculation and contraction of
mild forms of the disease.

In 1796, Jenner inoculated a boy with virus from cow-pox on
a dairy-maid's hand, and found that in this way cowr-pox can be
transmitted and immunity to small-pox be produced. Since then
vaccination against small-pox by inoculation of cow-pox has been
quite universally adopted. It is now accepted that cow-pox is a
modified form of small-pox and that small -pox vaccination is a

84 VACCINE AND SERUM THERAPY.

process of active immunization by means of living organisms at-
tenuated by passage through cows.

The methods of obtaining virus for vaccination vary. Up to
1870, virus was usually obtained from the pustule of the vacci-
nated individual. Now, however, vaccine is usually obtained from
young calves which have been inoculated on the abdomen and
thighs with vaccine from the pustules of healthy children or calves.
The pustules on inoculated calves are cleaned and opened on about
the third or fourth day and from the material contained in them
either so-called "vaccine points" or "glycerinated virus" is made.
The animals from which the vaccine is obtained are kept as clean
as possible, asepsis and anti-sepsis being employed as much as is
possible. When all precautions are taken the vaccine still con-
tains many organisms.

Vaccine points are made by dipping sterile bone slips into the
material from the pustule. The vaccine is then dried, kept under
aseptic conditions and is ready for use. Glycerinated virus is made
by softening the pustular material with glycerine. Glycerine has
the additional advantage of killing many bacteria in the virus and
makes it possible to put the virus up in tubes. Small-pox vaccine
is seldom entirely sterile, the organisms present, however, in a well-
prepared vaccine, consist of only few species and are usually harm-
less. After two to three weeks action of glycerine, vaccine is
frequently free from bacteria.

The durability of potency and efficiency of small-pox vaccine
varies. Some vaccines lose their value in one month while others
will remain efficient for three to four months. This emphasizes
the importance of using fresh virus for vaccination against small-
pox.

The method of vaccination varies. It is the custom now to
produce scarifications of one-eighth to one-fourth of an inch in
diameter on the well-cleaned arm or thigh. When vaccination is
made on the arm, the area selected is about the point of insertion
of the deltoid muscle. In cleaning the part where vaccination is
to be made it is to be remembered that all antiseptics must be re-
moved with sterile water before inoculation is made. Scarifica-
tion is only made severe enough to produce an exudation of serum,
bleeding being avoided as much as possible. After scarification,

FUTURE OF OPSONINS AND VACCINES. So

v.-ux Miit' on the point or from the tube is well rubbed in, the serum
is allowed to dry and a dry sterile dressing is put on.

The reaction produced by successful vaccination usually ap-
pears after about three days. Locally, there is at first a papule,
wh idi becomes a pustule on the eighth or tenth day. About the
end of the second week the vesicle changes to a scab, which comes
off and leaves a scar. Constitutional symptoms usually appear
about the third day and last until the end of the first week after
vaccination. When there is infection, cellulitis and sloughing fol-
low; these conditions are treated as any other infection, but usually
ought not occur.

The duration of immunity varies and is uncertain. The
longest time that immunity can be certainly relied upon is two
years. The rule for vaccination which is generally adopted and
advised consists in vaccination within the first or second year,
certainly before entering school, re-vaccination within the tenth to
fifteenth year and after that whenever there is an epidemic of
small-pox or possibility of exposure to the disease.

The efficiency of vaccination against small-pox cannot be
doubted. The mortality in vaccinated individuals is between five
and eight per cent, while in the unvaccinated thirty-five to forty
per cent of the cases terminate fatally. The disease thus is milder
in vaccinated than in unvaccinated individuals. Moreover fewer
cases of small-pox occur in the vaccinated than in those not vac-
cinated against small-pox.

FUTURE OF OPSONINS AND VACCINES.

As the method of determining the opsonic index stands today
it is of questionable value in diagnosis and treatment of bacterial
diseases. Opsonins may play no part in immunity; on the other
hand, they may be quite important, and we be lacking in compre-
hension of their value and their determination. Further efforts
should be made to study them.

The treatment of the bacterial diseases by killed cultures
ought not be cast aside because of the unreliability of the present
method of determining the opsonic index, inasmuch as opsonins
are not the only immunizing substances produced by the injection

86 VACCINE AND SERUM THERAPY.

of bacterial vaccines. It seems that the injections of killed cul-
tures have been both of prophylactic and curative value. The
curative value is probably limited to the treatment of chronic
diseases, because time is required to actively immunize. More-
over this method may be of especial value in the treatment of
infections due to species that have many varieties, for here the
individual may be immunized to the special organisms causing
the disease. Injections of killed cultures will not cure all infec-
tions and bacterial diseases and in some cases many injections
may be required to obtain the desired result.

Attention is again called to the fact there is no vaccine for
boils, acne or any other condition, but there are vaccines for im-
munization against infections by the Mic. pyogenes aureus, albus
or citreus, streptococci, typhoid bacilli, pneumococci, gonococci,
etc. Vaccine therapy, to give good results, requires that in all
infections treated the causal microorganisms or virus must be
determined, before vaccines are resorted to.

Credit is to be given to Wright, not for the discovery of the
method of immunization by injections of killed cultures, but for
its revival. The employment of this method of inducing immunity
and treating bacterial infections and diseases need not necessarily
be associated with opsonins and opsonic immunity.

CHAPTER VIII.

SERUM THERAPY

In the discussion of theories of immunity, mention was made
of the fact that in 1887 Fodor showed that the juices of the nor-
mal Hying body, especially the blood, possess bacteria-destroying
property. Buchner, Behring, and Nuttall were among the first
to coroborate the results of Fodor. It was later recognized that
the cell-free blood serum of normal animals possesses properties
of destroying bacteria. It is to be noted, however, that the blood
of every species of animals will not destroy all species of bacteria.
Furthermore, while in certain cases the degree of natural immunity
corresponds to the amount of the bacteria-destroying substance
present, still these two conditions are not always found in the
same case. The substance which destroys bacteria, Buchner
called "alexine."

In 1888 Hericourt and Richet made the observation that
blood serum from an animal immunized to Staphylococcus pyo-
septicus, confers when injected intraperitoneally into rabbits,
an immunity to this organism. Babes and Lepp in 1889 reported
the possibility of protecting animals against rabies by the injection
of body fluid from animals immunized to rabies. However, these
results were given but little attention until Behring and his
pupils systematically investigated the subject.

Behring and Kitasato, in 1890, reported that mice can be im-
munized to tetanus by the injection of blood serum obtained
from rabbits artificially immunized to tetanus. These investi-
gators further reported that serum from tetanus-immune animals
protects against the primary intoxication produced by the tetanus
bacillus.

In 1888 Roux and Yersin discovered diphtheria toxin. Beh-
ring and Wernicke, in 1891, showed that blood serum from diph-

88 VACCINE AND SERUM THERAPY.

theria immune guinea pigs or rabbits possesses the ability, when
injected intraperitoneally, to immunize normal guinea pigs against
diphtheria and a diphtheria infection already in progress.

In 1892 these investigators further found that it is possible
to immunize larger animals to diphtheria, and demonstrated in
the blood of these animals protective and curative substances
which can be transferred by the serum to other animals. The
doses necessary to cure the disease were found to be larger than
those required to immunize against the same. The amount of
protective and curative substance produced was found to vary,
to some extent at least, with the degree of active immunization
of the animal from which the serum was obtained. It was fur-
ther found by Behring that serum from animals immunized to
diphtheria will protect against the toxin of Roux and Yersin.

Based on these observations, Behring has formulated a law
which is generally known as "Behring's Law." According to
this dictum, the blood or blood serum from an animal possessing
acquired immunity is able to transmit this immunity to a sus-
ceptible individual or animal when this blood or blood serum is
injected in the right amounts into the susceptible animal or in-
dividual. Individuals naturally immune to a particular infec-
tious organism, do not possess in their blood or blood serum im-
munizing substances which can be transmitted to another in-
dividual. Immune substances which can be transmitted thus
are not present naturally but must be produced by a process
of immunization which consists either of undergoing the
natural course of the disease or submitting to some method
of artifical immunization. As a result of these investigations,
there has arisen the practice of passive immunization, or, as
it is more frequently called, serum therapy. The development
of methods and success obtained by passive immunization
against the action of tetanus and diphtheria bacilli, led to
the belief that it would be a relatively easy matter to make
specific sera against all disease-producing bacteria. It was
soon found, however, that this is not possible. Acquired
anti -toxic immunity has only a relatively limited application.
Most pathogenic bacteria hold their special poison so firmly
within the cell that the poison is only freed when the cell
disintegrates, or when certain stimulating conditions as are found

ANTI-TOXIC AND ANTI-BACTERIAL IMMUNITY. 89

in the body exist. Conditions favorable to the liberation of
toxins have up to the present time been produced for a few
bacterial species, so that most bacteria only liberate their
toxin in the tissues of the body invaded. As a result of
immunization to two widely different substances, toxin and
bacterial cells, two different classes of immune sera have been
manufactured. Of these two classes, the anti-toxic sera neutralize
certain morbific bacterial products, while the ant i -bacterial sera
destroy the bacterial cells. It is not to be inferred from this
that the same serum may not be both anti-toxic and anti-
bacterial, for diphtheria toxin and cells, for example, may be
used in immunization. Under such conditions the blood serum
obtained will have both anti-toxic and anti-bacterial value.

Acquired anti-toxic immunity is an immunity due to a neu-
tralization of toxin by a specific anti-toxin. The substance that
is formed as a result of the combination of toxin and anti-toxin
acts like a harmless and stable compound. Ehrlich, basing his
observations on studies of the union of toxin and anti-toxin,
classes the anti-toxic bodies formed with the anti-bodies of the
first order, that is, the labile susbtance or complement takes no
part in the reaction. In natural immunity to certain toxins the
presence of specific anti -bodies cannot be demonstrated and un-
der these conditions immunity is rather a non-susceptibility of
the cells to the toxin. Metchnikoff has shown that tetanus toxin
circulates freely in the blood of naturally immune animals. In
certain other cases of natural immunity toxin combines with cells
that are of little importance to the body or which are but little
affected by the particular toxin. Acquired anti-toxic immunity
thus differs from natural immunity in the formation of immune
bodies. These immune bodies are found in the body fluids,
especially the blood serum and can be injected into other animals
or individuals and in this way confer upon them a passive
immunity.

Acquired anti-bacterial immunity is an immunity depend-
ent upon the rapid destruction of bacteria. The process
is one of solution or lysis. The anti-bodies which produce lysis
belong to Ehrlich's third order of immune bodies, that is, com-
plement, which is easily destroyed by age, heat, etc., is necessary
to bring about the solution of bacteria. Natural immunity to

90 VACCINE AND SERUM THERAPY.

certain bacteria is associated with the presence of lytic bodies.
Behring found that blood serum from the white rat which is nat-
urally immune to the action of anthrax bacilli, exerts a marked
destructive effect on anthrax bacilli, while blood from animals
not immune to anthrax bacilli exert no such destructive effects
on these organisms.

On the other hand blood serum from the dog and the
pigeon, both of which species are practically immune to the
action of anthrax bacilli, posses no demonstrable bactericidal
power. It is evident that natural immunity is not in all cases
due to bactericidal action of blood serum of non-susceptible
animals. Acquired anti-bacterial immunity differs from natural
immunity to the action of bacteria, in that definite specific
immune bodies are present. These' immune bodies are found
in the body fluids, especially the blood serum, and dissolve
bacteria.

UNTOWARD EFFECTS OF SERUM INJECTIONS.

In the conference of passive immunity, blood and blood serum
injections are made almost exclusively. Blood transfusions were
made by Denis in 1667. While some good results were obtained,
fever, embolli, bleeding, heamoglobinuria, and urticaria were
sometimes produced by these blood transfusions. Up to 1894,
before diphtheria anti-toxin was first generally used, injections
of blood and blood serum were rather rare. Following the wide-
spread practice of injection of diphtheria anti- toxin in
blood, exanthemata followed in approximately twenty -two
per cent of the cases. With the injection of diphtheria anti-
toxin in blood serum, exanthemata were produced in only six or
seven per cent of the cases. From time to time after 1894 cases
have been reported in which injections of diphtheria anti-toxin were
followed by skin manifestations. While the sequellas of serum
injections are usually not serious, still at times serious symptoms
and even death have been reported as following injections of
serum.

In 1905, v. Pirquet and Schick originated the term "Serum
Krankheit," or serum disease. These investigators found that
serum disease varies, two rather definite types of the disease
being recognized; one type which results from the first injection of

SERUM DISEASE. 91

serum and another type which follows the second and subsequent
injections of serum.

Serum disease following the first injection of serum mani-
fests itself after an incubation period of eight to twelve days,
and is largely independent of the amount of serum injected. Of
the symptoms of this disease, fever is the most constant, lasting
usually during the entire course of the disease. The height of
fever, however, is not an indicator of the disease, but from the
curve of the temperature a prognosis can be made, because the
temperature drops by lysis. Together with fever there is usually
an exanthema, appearing most frequently as an urticaria. This
appears first about the point of injection, later it is distributed
symmetrically and usually itches severely. At times there is swell-
ing of the glands, especially of the glands found in the region in-
jected. The symptoms of pain in the glands are of prognostic
value, in as much as the pain usually disappears or abates before
the size of the gland decreases. During the period of incuba-
tion the leucocytes are increased in number, but during the height
of the disease the number of leucocytes is markedly diminished.
Joint pains are found in a small percentage of the cases of serum
disease. The metacarpal, hand and knee joints, are most fre-
quently effected. Usually these pains do not last long. In cer-
tain cases there is edema, but albuminuria is seldom or never
present. The mucous membrane is seldom effected, which dis-
tinguishes the condition from scarlet fever and measles. The
disease is further distinguished from measles by the absence of
Koplick spots, coryza, conjunctivitis and increased efflores-
cence about the point of injection. From scarlet fever the con-
dition is distinguished by its non-communicability by contact, and
absence of scaling, nephritis and angina.

The disease following the second and subsequent injections
of serum varies somewhat with the interval between injections.
If an injection of serum is made from twelve to forty days
after a preceding one, the incubation period is very short, the
disease appearing at times in less than one hour after injec-
tion. If the interval between injections is from forty days to
six months, there may be an immediate reaction, or else an im-
mediate reaction with another reaction six to eight days later.
If the interval between injections is over six months there is usu-

92 VACCINE AND SERUM THERAPY.

ally no immediate reaction, the symptoms appearing six to twelve
days after injection. When the interval between injections is
six days or less, the disease is not produced. Serum disease
follows re-injections more frequently than it does in the first in-
jection and is usually produced by smaller amounts of serum.

The "immediate reaction" occuring when re-injection is made
within an interval of twelve to forty days, manifests itself in one
to six hours after injection and usually reaches its maximum
within twenty-four hours after injection. The "second reaction"
is not seen after the first injection of serum but occurs most fre-
quently when, the interval between injections is between forty
days and six months. The incubation period for the second
reaction is somewhat shorter than it is for serum disease produced
by first injections. The symptoms of serum disease produced by
re-injection are usually more acute and general but of shorter
duration. In many cases there is vomiting.

When serum disease was first recognized it was supposed to
be caused by toxin in the immune serum. However, as early as
1894, Heubner expressed doubt as to the importance of specific
immune sera in the production of the disease. Later the mani-
festations of serum disease were produced by the injection of nor-
mal serum. In 1906 Rosenau and Anderson of the Hygienic
Laboratory of the Public Health and Marine Hospital Service
of the United States, published a work in which experiments were
reported on "sudden death" of guinea pigs following serum in-
jections. These investigators found that re-injections of normal
horse serum into guinea pigs are very poisonous if the interval
between injections is more than ten days. The length of time for
which this hypersusceptibility persists has not been definitely
determined but lasts at least as long as two years and two days.
On the other hand when the interval between injections is less
than ten days, normal horse serum will produce no such effect.

The nature and causes of the reaction have been matters of
considerable investigation and contention. The reaction was at
first regarded as one resulting from the injection of toxin or poison.
The long period of incubation, however, is explained with diffi-
culty by such a conception of the phenomenon. While it is true
that to produce disease by certain toxins, a period of incubation
is necessary, this period is seldom as long as eight to twelve days.

ANAPHYLOXIS. 93

It can hardly be conceived that horse serum has the ability to
increase its amount of poison as can bacteria and other organisms
capable of self reproduction. Rosenau and Anderson have de-
termined quite definitely that the reaction is due to a difference
in the susceptibility of individuals and not the toxicity of the
serum.

v. Pirquet and Schick conclude that the phenomenon is due
to the presence of specific anti-bodies. According to these in-
vestigators the anti-bodies are not precipitins, for man does not
produce the precipitins readily with horse serum — in children
three weeks are required before the precipitins can be detected
and are^ found in the blood only from four to nine weeks. The
"inline li ate reaction" which occurs when the second injection of
horse serum is made twelve to forty days after the first, Pirquet
and Schick explain as being due to the already present serum
anti-bodies which combine with the immunizing substance in serum
and produce poisonous substances, which in turn produce the
disease.

Gay and Southard, in 1907, reported experiments which in-
dicate that the theory of Pirquet and Schick is untenable. These
investigators believe that "sudden death" in guinea pigs "sensi-
tized to horse serum" is due to a substance they call "anaphylac-
tine." This substance is found in normal horse serum, is not
absorbed by the tissues of the guinea pig and is eliminated slowly.
The anaphylactine in the guinea pig increases the avidity in the
cells of the guinea pig, so that when more serum is injected the
cells are "overwhelmed in the exercise of their eliminating func-
tions, and functional equilibrium is so disturbed that local or
general death may follow." Rosenau and Anderson had shown
that the hypersusceptibility of the guinea pig for a certain protein
is manifested upon a second injection of the corresponding protein.
Later they showed that the substance anaphylactine is specific
in the same sense. These investigators could not demonstrate

o

this substance in the blood of the sensitized guinea pig during the
incubation period, nor at any time in the blood serum of man,
monkeys and cats.

The investigations on the phenomenon of hypersusceptibility
of guinea pigs to horse serum in connection with serum disease
in man, has lead to a considerable advancement in the methods

94 VACCINE AND SERUM THERAPY.

of administration of specific immune sera. While anaphylactine
has not been found in the blood serum of man, later investigation
may show that the phenomenon of serum disease in man corresponds
to sudden death and anaphylaxis in guinea pigs.

The markedly beneficial results of injections of specific im-
mune sera in the prevention and cure of certain diseases, have
so far outweighed the undesirable and serious results which have
followed serum therapy, that most physicians have given little
attention to the contra-indications of serum injection. As the
indications and contra-indications for injection of immune serum
are better understood by physicians fewer objections to serum
therapy, scientifically applied, will be made. That serious ob-
jections to the injection of immune sera have some foundation,
is evidenced by the fact that Rosenau and Anderson have been
able to collect a considerable number of cases of sudden deaths
in man after the injection of immune serum. It has been noticed
from the first that in the cases of sudden death following the in-
jection of diphtheria anti-toxin there is marked respiratory em-
barrassment. Rosenau and Anderson believe the essential lesion
of serum anaphylaxis is localized in the respiratory centers.
In collecting statistics on this subject these investigators have
found two cases, "and also others have come to our notice," in
which sudden death followed the injection of anti-toxin into
asthmatics. From these observations Rosenau and Anderson
conclude that "the knowledge of the fact that injection of horse
serum into asthmatics may be attended with danger, should be
considered in the use of anti-toxins."

While there are some cases in which the injection of horse
serum has been followed by sudden death due to a hypersuscepti-
bility to horse serum, most of the deaths have undoubtedly been
due to other causes. Frequently physicians and laymen wrongly
attribute undesirable conditions to the use of diphtheria anti-
toxin and other immune sera. In the use of some immune sera,
especially diphtheria anti-toxin, recovery from the disease is so
rapid and early, that the physician and patient overestimate the
physical condition of the patient, and as a result the patient is
frequently allowed a certain amount of exercise. This at times is
followed by unfavorable symptoms. The condition in these cases is
probably due to the action of diphtheria toxin rather than

INDICATIONS OF SERUM INJECTIONS. 05

of diphtheria anti-toxin. The heart paralysis which develops
in some cases is probably only observed because by the use of
diphtheria anti-toxin the patient remains alive long enough so
that the heart paralysis may be observed. The action of diph-
theria toxin on the heart and other organs has been known for
some time and in serum laboratories it is not an unusual occurence
to have an apparently well horse, which is undergoing immuni-
zation to the diphtheria bacillus and its toxin, die very suddenly
after exercise. Post diphtheritic paralyses have been observed
long before the introduction of the use of diphtheria anti-toxin,
and to attribute this condition to anti-toxic serum is to attrib-
ute a symptom to a remedy, where the symptom is really one
found in the disease treated by the remedy, diphtheria anti-toxin.
In all cases treated with immune serum the patient ought to be
kept quiet for some time after injection of serum, even though
he has apparently recovered from the disease.

Following the good results obtained in the treatment of
diphtheria by diphtheria anti-toxin and the protection against
the disease conferred by the so-called immunizing dose of
diphtheria anti-toxin, the serum has been used very extensively.
In many cases, undoubtedly, serum has been used without proper
indications. Many physicians have used diphtheria anti-toxin
even though the diagnosis of diphtheria was not definitely made,
because it was assumed that no harm could come from the use of
the serum while great benefits might result if the condition were
one of diphtheria. Some physicians have injected the immuniz-
ing dose fully as indiscriminately ; whole families, attending physi-
cian and nurses have received immunizing doses of diphtheria
anti-toxin. The indications for protective and curative injec-
tions of immune serum ought to rest on a better foundation.
This is especially true because hypersusceptibility to serum, or
serum disease, is more frequent following re-injections of serum.
It is evident that serum ought only to be injected into the indi-
vidual when certain indications are present.

Specific treatment with immune serum is only indicated when
the diagnosis, and, in certain cases, the etiological factor has been
determined. It frequently happens that a bacterialogical diag-
nosis cannot be made immediately. Under such conditions the
clinician is warranted in using specific serum if there is such

96 VACCINE AND SERUM THERAPY.

clinical evidence as is usually found in infections caused by the
corresponding organisms, or if the patient has been exposed to
infection from a previously diagnosed case of the communicable
disease.

When injections of immune sera are to be made frequently,
and over long periods of time, as is the case with most anti-bac-
terial sera, the injections should be made at intervals of less than
ten days, because, if the interval is longer than this, serum disease
is more likely to develop.

The indications for injection of immunizing doses are not
clear nor definite. Nurses and doctors, however, now rarely re-
ceive immunizing doses of immune serum. This is especially
true of diphtheria anti-toxin. The reasons for the abandonment
of the practice of immunizing doctors and nurses, are based on
the fact that doctors and nurses are better able to avoid infec-
tion and so usually do not contract the diseases of their patients,
and because the protection conferred by passive immunization
is only of short duration. Doctors and nurses, because so
frequently exposed to infectious diseases, would have to be
injected frequently and at different intervals of time to be
immune to the various infections of their patients. Serum
injections at intervals of more than ten days, however, are more
likely to give rise to serum disease.

The custom concerning immunization of individuals against
possible infection because of exposure, varies. It is the rule of
some physicians, upon the diagnosis of diphtheria in one member
of the family, to give all members of the family an immunizing
dose of diphtheria anti-toxin. In some of the large children's
hospitals and clinics diphtheria anti-toxin is given to all patients
on admission, this being done to avoid the outbreak of diph-
theria epidemics. Such preventative measures hardly seem
warranted. The custom of most physicians now is to give im-
munizing doses of some specific ant i -sera to such members of a
family or household as are most likely to contract the disease;
thus for example, diphtheria anti-toxin is given only to the
children in a home in which there is diphtheria. On this basis
administration of anti-sera is justifiable, while indiscriminate
immunization by the injection of serum is costly and may
also sensitize individuals to serum, which might later make the

CONCENTRATION OF IMMUNE SERA. 97

use of serum difficult or impossible even though injection of
immune serum were definitely indicated. While it is true that
some sera are ordinarily injected at intervals of two or three
weeks without the production of serious results, still it frequently
happens that such a degree of hypersensitization to serum de-
velops that passive immunization must be given up entirely.

Since the introduction of serum therapy, large numbers of
persons have been injected with immune serum and great bene-
fits have been derived from the use of different sera. The cases
of serum sickness, especially the rashes, have been relatively fre-
quent, yet are so far outweighed by the beneficial results that
they must not prevent the physician from the use of immune
sera when they are indicated. The cases of sudden death fol-
lowing injection of serum fortunately have been very rare. As
further knowledge concerning the untoward effects of the injec-
tion of horse serum is gained, serum disease and sudden death
will become less frequent. The essential points, which have so
far been developed, indicate, that specific sera are to be used only
after a definite diagnosis of the disease, that there is some danger
attending the injection of horse serum into asthmatics, and that
untoward effects of serum injections are more frequent when the
interval between injections ranges from ten days to several years.

CONCENTRATION AND PURIFICATION OF SERUM.

Frequently large amounts of specific immune substances are
to be injected in the treatment of infections. Hypodermic in-
jection of large amounts of serum, however, is painful and more
likely to produce serum disease, especially the rashes. These
objectionable features of serum therapy have resulted in an
attempt to produce serum of greater concentration and purity.
It has been found that bacteria and toxins from certain cultures
are more potent and cause animals to produce immune sera of
higher protective value. As a result virulent cultures, or toxins
produced by virulent cultures, have been used in active immuni-
zation of the animal that is to furnish the immune serum.

Attempts have been made to obtain an increased potency by
separating the immune substance from the non-immune sub-
stance of specific serum. Dieudonne, in 1897, showed that the
protieds precipitated from diphtheria anti-toxin by acetic and

98 VACCINE AND SERUM THERAPY.

carbonic acid, contain none of the anti-toxins. In this same
year Belfanti and Carbone found that with the globulins precipi-
tated by magnesium sulphate, diphtheria anti-toxin is thrown
out. Atkinson, in 1901, and others since then, have shown that
during immunization the serum-globulin content of serum in-
creases. Gibson and Banzhaf, however, have found that the
increase in serum-globulin is not necessarily associated with the
accumulation of anti-toxin in the blood. Early in 1906 Gibson,
working in the Research Laboratories of the Department of
Health of New York City, reported practical methods for con-
centrating and purifying diphtheria anti-toxins. According to
this method, by half saturation of anti-toxic serum with ammon-
nium sulphate, the globulins, nucleo-proteids, and similar sub-
stances are thrown out. These contain the immune bodies. The
precipitate is again dissolved in a saturated solution of sodium
chloride. Now only the anti-toxin fraction and some of fhe
globulins are in solution. After filtration, acetic acid is added
to the filtrate to precipitate the anti-toxic globulins which are
then filtered off, dried with paper, dialyzed, neutralized and again
dialized for several days. After dialysis the solution is made
isotonic by the addition of sodium chloride. The sulution is then
filtered through a Berkefeld filter to remove any bacteria that
may be present and chloroform added as an antiseptic. This
method has been attempted for the various immune sera, but up
to the present time it is profitable only for the concentration of
diphtheria and tetanus anti-toxins. Recently Banzhaf has found
that more concentrated anti -toxin may be obtained by heating
the serum to 57° for some hours before separating the anti-toxic
globulins. By Gibson's method the concentration is increased
from three to five times, while by Banzhaf 's from eight to ten times.
By the use of concentrated and refined serum the consti-
tutional disturbances and rashes are somewhat less frequent and
not as severe. The principle advantage of the product obtained
by these methods, however, lies in the fact that a large amount
of immune substance can be injected in small amounts of material.

DRIED IMMUNE SERUM.

Relatively recently, immune sera, especially anti-diphtheritic
and anti-tetanic sera, have been dried after concentration and
refinement. It was mentioned earlier that when serum is dried

ORAL ADMINISTRATION OF IMMUNE SERA. 99

soon after being drawn, the immune body and especially the
complement are preserved longer. Among the advantages of
dried immune sera may be mentioned: the lack of deterioration,
the cheapness of manufacture; the convenience of carrying about
in the medicine case; and the possible administration of immune
substances by the mouth. Undoubtedly these products will
prove to be of considerable value and are not to be lost sight of.

ORAL ADMINISTRATION OF IMMUNE SERUM.

In 1SU2 P^hrlich discovered that ricin anti-toxins can be ab-
sorbed by the intestinal canal. In 1893 Wernicke, working with
Behring, established the fact that anti -toxic substances in the
body fluids of diphtheria-immune animals are absorbed by the
digestive tract; thus, dogs fed on the meat of diphtheria-immune
sheep obtain some immunity to diphtheria. The possibility of
oral administration of anti-toxic sera has been recently advanced
by McClintock and King. These investigators have found that
the best results by oral administration of diphtheria anti -toxin
are obtained by the following method: "One half -hour before
administering the serum the child is given one glass of one per
cent sodium bicarbonate solution. When the anti -toxin is given
there is added one minim Fl. Ext. Opii and from four to ten
minims of saturated solution of salol in chloroform. When pos-
sible no food should be given for at least four hours before ad-
ministering the serum." This method is adopted to inhibit the
digestion of the anti-toxin so as to make the absorption of the
unchanged anti-toxin possible. Dried diphtheria anti-toxic globu-
lins were found to give satisfactory results. By oral adminis-
tration of diphtheria anti-toxin neither serum sickness nor
anaphylaxis was observed. At present this method is still of
questionable value and requires further investigation.

ANTI-TOXIC SERA.

It was stated earlier that sera containing bodies which neu-
tralized toxin are called anti-toxic sera. The immune bodies
are receptors of the first order of Ehrlich (see p. 16), which have
been thrown off as a result of overproduction of certain receptors.

100 VACCINE AND SERUM THERAPY.

The overproduction of these receptors occurs as a result of the
presence of specific toxins, found in infections with specific or-
ganisms, or after experimental injection of certain toxins. As is the
case with other anti-bodies, anti-toxins are present to some de-
gree in many normal individuals, thus diphtheria anti-toxin was
found by Bolt on to be present in the blood serum of 30 per cent
of the horses examined, and in 50 per cent of the children and 81
per cent of the adults examined by Wasserman. Various sub-
stances have been used to produce an increase of specific anti-
toxins and as a result a considerable number of anti-toxins have
been made. Among these may be mentioned the anti-toxins
which combine with the toxins of the organisms of diphtheria,
tetanus, pyocyaneaus, symptomatic anthrax, and botulism, with
snake and spider venoms, with ricin, aprin and robin, and
with the glucosids of toad stools and poison ivy. Of the anti-
toxic sera, the anti-diphtheritic and anti-tetanic have been of
most importance.

In the treatment of diseases for which anti-toxic sera have
been made, it must always be remembered that anti-toxin neu-
tralizes prepared toxin and combination can take place only when
the receptors of the toxins and anti-toxins are free. As soon as
the cells and toxins have combined and the body cells have been
injured as a consequence, no amount of anti-toxin can protect
the cell from the action of the poison molecule. Anti-toxic
bodies can only anchor and render inert or harmless such toxins
as are free or have uncombined receptors. This emphasizes the
importance of the early administration of anti-toxic serum be-
cause at this time the poison molecules can be anchored by the
anti-toxic substances instead of combining with the cells of the
body.

DIPHTHERIA ANTI-TOXIN.

Ferran, early in 1890, and Fraenckel and Behring later in the
same year, published methods by which experimental animals
can be immunized to diphtheria. Behring and Kitashima, in
1891, published methods for the immunization of guinea pigs to
diphtheria toxin. In 1892 Behring and Wernicke emphasized
the presence of protective substances in serum of diphtheria-im-
mune animals. Serum therapy as applied to the treatment of

DIPHTHERIA ANTI-TOXIN. 101

diphtheria in man began in 1891 and 1892. The first diphtheria
anti-toxin was put on the market in August, 1894. Since then
diphtheria anti-toxin has been used extensively.

The method for making diphtheria anti-toxin has gone
through various stages of development. All diphtheria anti-
toxin used in the United States is made practically after the same
method, which is as follows: Young, vigorous and absolutely
healthy horses are immunized, although other species of animals
have also been used. During immunization the animal receives
repeated injections of diphtheria toxin. This toxin is obtained
by growing a virulent culture of B. diphtheria on the surface of
beef bouillon in an Ehrlenmeyer flask. After one week's growth
at a temperature of 35° to 36° C., the culture is rendered sterile
by adding carbolic acid in amounts to make a 0 . .5 per cent car-
bolic acid solution. After 48 hours the diphtheria bacilli have
settled to the bottom, when the bouillon is filtered through a
Berkefeld filter. The germ-free bouillon contains the diphtheria
toxin and ought to be potent enough so that 0.01 c. c. will kill
a 250 gram guinea pig on or before the fourth day after
subcutaneous injection. The horse or animal to be immunized
receives enough of this toxin in bouillon to kill five thousand
guinea pigs of 250 grams each. At the time this amount
of toxin is injected the horse also receives an injection of ten
thousand units of diphtheria anti-toxin. After three to five days,
when the fever has subsided, a somewhat larger dose of toxin and
the same amount of anti-toxin are injected. A third injection is
made after another interval of from three to five days. After
this usually no more anti-toxin is injected with the toxin, the
doses of which are constantly increased and injected at intervals
of from five to eight days. After about two months of treatment,
if the immunization has been successful, the horse will usually
tolerate enough toxin at one injection to kill one hundred thousand
guinea pigs of 250 grams each.

At the end of six weeks to two months, samples of blood are
drawn and tested as to protective value. If the anti-toxic value
is high, the horse is usually bled to death by tapping the jugular
vein. The withdrawal of serum is made under aseptic conditions.
The bottles containing the blood are slanted and after four to
five days the serum is drawn off.

102 VACCINE AND SERUM THERAPY.

By means of Gibson's or Banzhaf's method the serum is now
concentrated and refined. After this it is standardized, i. e., its
strength is determined. The United States government deter-
mines the unit of diphtheria anti-toxin in the United States, and
the Government requires that all diphtheria anti -toxin made by
manufacturers, having a United States government license, must
conform to this standard. This emphasizes the importance of
using only diphtheria anti-toxin made by manufacturers having a
United States Government license. Because the toxic value of
diphtheria toxin changes with ageing and other conditions, the
Hygienic Laboratory of the U. S. Public Health and Marine Hos-
pital Service, issues from time to time standard anti-toxic serum.
With this serum the strength of the toxin used in determining
the anti-toxic value of serum is gauged. The "immunity unit,"
or unit of anti-toxin, is the amount of diphtheria anti-toxic serum
which will just neutralize one hundred times the smallest amount
of toxin necessary to kill a 250 gram guinea pig in four days.
The anti-toxic value of serum is determined by mixing one hun-
dred times the smallest fatal dose of fresh diphtheria toxin with
varying amounts of the diphtheria anti-toxin, allowing the mix-
tures to stand for fifteen minutes and then injecting them into
250 gram guinea pigs. The smallest amount of serum which will
protect a 250 gram guinea pig for more than four days against one
hundred times the smallest amount of diphtheria toxin necessary
to kill a 250 gram guinea pig, contains one unit of diphtheria anti-
toxin.

At the time of bleeding immunized horses, that is after six
to eight weeks of immunization, the serum may contain from
one hundred to one thousand units of diphtheria anti-toxin per c. c.
The concentrated and refined diphtheria anti-toxin contains usually
from three hundred to two thousand units of anti-toxin per c. c.
The various manufacturers of diphtheria anti-toxin now furnish
their product in syringes containing a determined number of
units of anti-toxin. These packages are stamped so as to indicate
the date after which they can be exchanged, free of charge, for
more recently tested serum. Because of the degeneration in
potency of diphtheria anti-toxin in the fluid condition, it is desir-
able that no sera be used after the date when they are to be ex-
changed for new sera.

DII'HTHKklA ANTI-TOXIN. 103

The therapeutic application of diphtheria anti-toxin has given
most satisfactory results, and is now recognized and regarded as
the specific for diphtheria. The statistics on the death rate from
diphtheria have been collected at many different times, and all
indicate that, while before diphtheria anti-toxin was used over
fifty per cent of the cases resulted fatally, since the application
of this method of treatment, the death rate from diphtheria has
fallen below twenty per cent, and according to some statistics to
five per cent. In Boston the death rate of diphtheria from 1888
to 1894 was 43.2 per cent, while from 1895, after which diphtheria
anti-toxin was used extensively, to 1904, inclusive, the death
rate has fallen to 11.84 per cent. Still better results will prob-
ably be, obtained as this method of treatment becomes more uni-
versal and is better understood.

The method of treatment of diphtheria with diphtheria anti-
toxin varies. However, some definite principles of treatment
have been established. It is very essential that all cases be treated
as early as possible. The reasons for this are clear when it is
understood that as toxin is liberated by diphtheria bacilli, it tends
to combine with the cells of the body. After this union has once
taken place the cell is rapidly injured. In order that diphtheria
anti-toxin combine with these toxins it is essential that the immune
substances be present at the time of the liberation of the toxin.
Most statistics show that in ordinary cases of diptheria the death
rate is nil when diphtheria anti-toxin is injected on the first day
of the disease. Even when treated with diphtheria anti-toxin on
the second day, the death rate is below five per cent; however,
when specific treatment is not instituted until after the fourth
day, twenty per cent of the patients do not recover.

The dose to be given in the different conditions varies, and
no hard and firm rules can be laid down. There are, however,
some quite generally accepted rules as to doses.

The ordinary case of diphtheria, treated on the first day of
the disease, receives injections of from two to three thousand
units of anti-toxin. When the serum is not given until the second
day, usually three to four thousand units are injected.

In the pharyngeal and laryngeal types of diphtheria larger
doses are usually given — seldom less than five thousand units.

104 VACCINE AND SERUM THERAPY.

When the disease comes under observation late or is very
severe twenty thousand to one hundred thousand units may be
necessary. Evidence has been obtained that toxin may be taken
from the cells with which it has combined when very large doses
of anti-toxin are injected. No case of diphtheria should be re-
garded as being too severe or too far advanced to be treated by
diphtheria anti-toxin, but in such cases large doses should be given,
as small doses are of no avail, because they do not neutralize all
the toxin present. The age of the patient, unless very young,
should have no influence on the amount of anti-toxin injected.

Diphtheria anti-toxin, as any other therapeutic agent, should
be given in sufficient quantities to accomplish the full therapeutic
action. To determine this the effects of diphtheria anti-toxin
must be recognized. The results of injections of sufficient amounts
of anti-toxin are: general improvement of the patient's condi-
tion, reduction of fever, improvement of the pulse, but most
noticeable of all is the shriveling of the membrane, decrease of
discharge, and less fetid odor of the breath. When these effects
do not appear from six to eight hours after injection, more diph-
theria anti-toxin should be injected. Re-injections usually are
larger than first injections, and are indicated at any time when
the patient's condition becomes more grave, or when improve-
ment does not come in six to eight hours after injection. At
times diphtheria anti-toxin does not give satisfactory results in
the treatment of diphtheria. In most of these cases, however, the
treatment is begun too late or there is infection with other organ-
isms. Of these organisms streptococci are most important and
are responsible for many of the fatal cases of diphtheria. Such
cases are at times treated with both diphtheria anti-toxin and
anti-streptococci serum.

Various attempts have been made to administer diphtheria
anti -toxin by the mouth. The work of McClintock and King on
this method has already been referred to. Before oral adminis-
tration of diphtheria anti-toxin can be generally employed, more
experimentation will be necessary.

The' use of dried diphtheria anti-toxic globulins, which are
dissolved in salt solution before injection, is relatively recent.
If the results obtained by their use are as favorable as has been
reported, it may be expected that they will be readily taken up

IMMUNIZATION~~AGAINST DIPHTHERIA. 105

by the medical profession. Serum, however, has the advantage
of always being ready for use and not requiring sterilization
before administration.

In some of the severer cases of nasal diphtheria, anti-toxin
has been sprayed^on the membrane with some beneficial results.
Frequently too, it is observed that diphtheria bacilli remains viable
in the nose and throat after an apparent recovery from the dis-
ease. Such persons frequently are the cause of the spread of
diphtheria. Various attempts have been made to remove the
diphtheria bacilli from the nose and throat of these apparently
well individuals. In ' addition to the antiseptic methods em-
ployed in the treatment of this condition, diphtheria anti-toxin
has been applied locally. Recently it has been proposed to ac-
tively immunize such individuals by the injection of bacterial
vaccines made from the cultures of diphtheria bacilli isolated
from the patient.

Immunization of well individuals against possible diphtheria
infection has been practiced for some time. It has been quite
definitely proven that injection of a relatively small number of
anti-toxic units will protect from four to six weeks against in-
fection with the diphtheria bacillus. The doses generally recom-
mended are from three hundred to five hundred units in small
children and one thousand units for older children and adults.
While the custom concerning the immunization of well persons
varies, it is quite generally accepted that children who have been
exposed to diphtheria should receive at once immunizing doses of
diphtheria, while adults seldom are treated in this way.

The definitely beneficial results which have been obtained
by the use of diphtheria anti-toxin in the treatment of diphtheria,
ought to be sufficient to convince the practitioner that diphtheria
anti-toxin should be used in practically all cases of diphtheria.
Moreover the practioner ought to know enough concerning the
symptoms and complications of diphtheria, and the untoward
effects of serum injection to distinguish the conditions dependent
upon the disease and those dependent upon the serum injected.
It is not unusual for the practioner to diagnose the transient serum
rashes as erysiplas, and the edema, following serum injections,

106 VACCINE AND SERUM THERAPY.

as those due to Bright 's disease. Practioners are undoubtedly
largely responsible for the misconceptions of laymen concerning
the effects of diphtheria anti-toxin injections. As a result of these
misconceptions frequently consent to use diphtheria anti-toxin,
when it is definitely indicated, cannot be obtained, and yearly
numbers of children whose lives could undoubtedly have been
saved are carried to the grave.

TETANUS ANTI-TOXIN.

In 1890, Behring and Kitasato immunized mice and rabbits
to tetanus by injecting cultures of the bacillus of tetanus. These
investigators found that blood from rabbits immunized to tetanus
bacilli, is able to protect mice against tetanus. The first reliable
anti -tetanic serum to be used in man, was put onto the market
in 1896.

The process of producing anti-tetanic serum, is similar to that
employed to produce anti-diphtheria serum— blood is drawn from
horses that have received injection of increasing amounts of tetanus
toxin. The toxin injected is produced by growing the tetanus bacillus
on bouillon. After ten to fifteen days of incubation under anaerobic
conditions, the bouillon culture is filtered through a Berkefeld filter.
The germ free filtrate contains the toxin, which is present usually in
large amounts. At the first injection into the horse usually one-half
c. c. of toxic bouillon, together with anti-tetanic serum are in-
jected. The amount of 'toxin is increased at each injection, until
finally seven hundred to eight hundred c. c. of toxin are tolerated
when given in one injection. After the third injection the anti-
tetanic serum is usually omitted. After several months of treat-
ment, and complete recovery from the last injection, the horses
are bled and the serum is collected. The serum is then concen-
trated and refined after the same methods that ar^e used for diph-
theria anti -toxin.

The standardization of tetanus anti-toxin until very recently
has been indefinite and unsatisfactory. At the present time
different standards exist in the various countries. The unit of
anti -toxin for tetanus for the United States has been fixed as that
amount of tetanus anti -toxin that will protect a 350 gram guinea
pig for ninety-six hours against one thousand times the smallest
fatal dose of tetanus toxin. In order that the standard may be

TETANUS ANTI-TOXIN. 107

tin.- same throughout the United States the Hygienic Laboratory
of the United States Public Health and Marine Hospital Service
sends out at regular intervals a stable precipitated tetanus toxin
\vliich is called the "test dose" and contains one hundred times
the smallest fatal dose. It will be observed that a unit of
tetanus anti-toxin contains more than ten times as much neu-
tralizing value as does a unit of diphtheria anti-toxin. The var-
ious anti-tetanic serum producers furnish their product in suitable
syringes, and, as is the case with diphtheria anti-toxin, state the
date after which it is desirable that.the serum be returned if not
used.

The value of tetanus anti-toxin and the methods for adminis-
tration can only be judged after study of the action of the tetanus
bacillus and its toxin. Tetanus in man is usually the result of
the subcutaneous introduction of tetanus bacilli, some foreign
matter and usually saprophytic organisms. Thus the disease
follows most frequently upon puncture and contused wounds
as the result of injuries made by machinery, pistol shots, trauma-
tism especially in stablemen, infection of the naval during or
after birth, and sometimes as a result of the injection of con-
taminated vaccines and sera. The distribution of the organism
is very wide, being found practically everywhere where there is
animal habitation.

After the entrance of the tetanus bacillus, usually there is
little change produced in the tissues infected. Suppuration occurs
practically only as a result of other organisms. Usually the or-
ganisms remain localized. The disease and its symptoms are
entirely dependent on the absorption of the toxins produced by
the tetanus bacilli. Tetanus toxin circulates in the blood and
lymph, but shows no symptoms until it unites with and is ab-
sorbed by the end organs of the motor nerves and the central
nervous system. To reach the end organs the toxin is^Mwried
through the axis cylinders. It is thus seen that when the symp-
toms of the disease appear, the toxins have already combined with
the cells, are exerting their toxic effects on them and can no longer
combine with the anti -toxin which is not taken up by the central
nervous system. In cases not treated with tetanus anti-toxin, Rose
has found that ninety-one per cent of the cases in which the incuba-
tion period is short, terminate fatally; 81.3 per cent of those

108 VACCINE AND SERUM THERAPY.

having a medium incubation period die, while of the cases with a
long incubation period, 52.9 per cent of the patients die.

Tetanus anti -toxin can only bind tetanus toxin before it is
taken up by the nerve cells and axis cylinder. Frequently, how-
ever, the disease is not suspected until after the symptoms appear.
Because of this the value of tetanus anti-toxin lies mainly in its pro-
phylactic application. As a preventative of tetanus, tetanus anti-
toxin has proven to be of great value. However to obtain bene-
ficial results injections should be made early, as soon after in-
fection as is possible, and inasmuch as immunity lasts only two
to three weeks, the injections should be repeated as long as danger
of infection exists. The immunizing dose which is generally
used^ consists of fifteen hundred immunity units. The wounds
after which immunizing doses of tetanus anti-toxin are to be
used are those made by burns with toy pistols, fire-works, pistol
shot wounds, injuries occuring while working about the stable
or infected with fertilized garden earth or manure. The value of
this method cannot be definitely determined, for if tetanus anti-
toxin is used successfully, it is difficult to decide whether or not
there has been infection with the tetanus bacillus. It is the be-
lief of some, however, that tetanus can be prevented entirely if
after all injuries of the kind likely to be infected with tetanus
bacilli immunizing doses of tetanus anti-toxin are injected early.
Jordan states that in the United States in 1903, there were 4,449
Fourth of July injuries, of which 406 cases died of tetanus ; while
in 1907, when tetanus anti-toxin was used quite universally for
such wounds, there were only sixty-two deaths caused by tetanus,
following 4,413 Fourth of July injuries.

The use of tetanus anti -toxin as a curative measure has not
given universal success. This is undoubtedly due to the fact
that after the symptoms appear the tetanus toxin has already
combined with and injured the nerve cells, and is no longer amen-
able to neutralization with tetanus anti -toxin. It is usually
conceded that if tetanus anti-toxin is not given within thirty hours
after the appearance of 'symptoms, subcutaneous injections of
anti-toxins is of little avail. When the disease has advanced,
the large nerves in the vicinity of the infection and the spinal
canal have been injected. The therapeutic dose of tetanus anti-
toxin administered varies. If it is a case of short incubation, that

TETANUS ANTI-TOXIN. 1 0<)

is eight days or less, injections of from 15,000 to 20,000 units are
made every two to eight hours until there is abatement of the
symptoms. These cases usually terminate fatally, even when
tetanus anti-toxin is given. The cases where the incubation period
is longer are more frequently recovered from. In all cases early
injections in large doses are indicated when the symptoms have
appeared, and radical methods of injection are justifiable, because
most cases without anti -toxin terminate fatally. As a result of
tetanus anti-toxin, the death rate in animals as determined by
Behring has been found to be reduced from 88 per cent to 40 or
45 per cent. The curative value of tetanus anti -toxin in man
cannot be well demonstrated by statistics. While the results are
not nearly as favorable as those obtained by treating diphtheria
with diphtheria anti-toxin, still tetanus anti-toxin ought to be
used in all cases of tetanus.

Relatively recently a dried tetanus anti-toxin has been made.
This powder is used principally as a dressing for wounds which
are likely to be injected, but it may also be used for injection
after dissolving in salt solution. For the latter use it has the ob-
jection that it dissolves with difficulty. While relatively little
is known concerning it, it has advantages similar to those of dried
diphtheria anti-toxin and may prove of considerable value to the
medical profession.

As a result of the decreased number of cases of tetanus fol-
lowing Fourth of July wounds, when immunizing doses of tetanus
anti-toxin are given, the physician should in all cases of such injury
administer to the patient an immunizing dose of tetanus anti-
toxin. Moreover all wounds in which tetanus infection might
occur, should be thoroughly cleansed and the patient immunized
with tetanus anti-toxin. When symptoms of tetanus have de-
veloped the patient ought to have every advantage of any bene-
fits that may be derived from the use of large and repeated in-
jections of tetanus anti -toxin.

ANTI-BACTERIAL SERA.

Sera containing substances which result in the death and
destruction of bacteria are called anti -bacterial sera. Acquired
anti -bacterial immunity depends on destruction of bacteria be-

110 VACCINE AND SERUM THERAPY.

fore they have had sufficient chance for multiplication and pro-
duct ion of poisons which kill the cells in the body. For this reason
anti-bacterial sera are frequently called bactericidal sera. In
the serum of individuals and animals that have acquired anti-
bacterial immunity, various kinds of anti -bodies are found. The
best known of these bodies are bacteriolysins, opsonins, precipi-
tins and agglutinins. These prepared anti-bodies are introduced
into the body in passive immunization. The importance and
value in immunity of all of these anti-bodies is not known, but it
is generally accepted that agglutinins and precipitins are of little
or no value, while opsonins and bacteriolysins are considered of
importance in anti -bacterial immunity. Bacteriolytic immune sera
depend for their action on specific substances called ' 'bacteriolysins, ' '
which dissolve bacteria. The value of opsonins in bactericidal
sera to be used in injections for the purposes of conferring passive
immunity, is still indefinitely understood, although some investi-
gators have attached considerable importance to their presence
in sera.

The anti -bodies in anti -sera called agglutinins and precipi-
tins belong to the second order of receptors, the bacteriolysins
to the third order, while in regard to opsonins, no statement can
be made at the present time — some regarding them as belonging
to the second and others to the third order of receptors.

Anti-sera containing receptors of either the second or third
order are easily inactivated by heat, age, acids, etc. After inacti-
vation they are no longer able to produce agglutination, precipi-
tation, lysis, or opsonification. The loss of power to produce these
effects depends on the destruction of ferment -like substances, which
are a part of the agglutinin and precipitin receptors and are fur-
nished to the receptors of the third order by the fresh blood serum.
(See page 18.) Inactivated bacteriolytic serum differs from in-
activated or aged agglutinating or precipitating serum in that
on the addition of fresh serum the anti -bodies producing lysis
can be re-activated, while the agglutinating and precipitating
properties cannot be restored in this way. Because the different
specific bacteriolytic sera used in passive immunization are usually
not freshly drawn and therefore have been inactivated by age,
the individual or animal immunized must furnish the complement
or ferment-like substance so as to make the destruction of bacteria

ANTI-BACTERIAL SERA. Ill

possible. The normal individual in order to utilize all the lytic
and possibly the opsonic anti-body in a specific serum, therefore
must possess large amounts of complement. Numerous investi-
gators, among which may be mentioned Ehrlich, Morgenroth and
Metchnikoff, have found that in certain diseased conditions there
is an actual decrease in complement. Moreover, as far as can be
determined, complement is not increased during disease and im-
munization. Richardson has shown that the serum of typhoid
patients is not usually able to destroy typhoid bacilli, but that
upon addition of fresh serum from a normal individual the typhoid
immune serum is able to bring about complete lysis of typhoid
bacilli. Numerous similar observations have been made in natural
infections in man. In the efficiency of ant i -bacterial sera the
presence of sufficient amounts of complement undoubtedly plays
a large part and in certain cases the lack of complement probably
is the cause of failure of beneficial action of certain specific anti-
bacterial serum. Various attempts have been made to increase
the amount of complement. Ant i -complement has been used
to immunize in order to excite the over-production of complement.
Injection of fresh complement with the immune serum has been
tried by some investigators. Efforts have been made to preserve
the complement in immune sera by freezing the serum and keeping
it cold until it is injected. Recently dried immune sera have
been advocated because it is known that complement is preserved
for a long time in serum dried down soon after it is drawn. It is
questionable, however, whether by any of these methods enough
complement will be present to activate the amount of immune
bodies injected. Hiss recently has published results on the cura-
tive action of subcutaneous and intraperitoneal injection of ex-
tracts of leucocytes from normal rabbits. Of this leucotic extract
Hiss says, "The action of the leucocytic extract may be due to
the enhancement of the bacteriolytic action of the animal's plasma
by the introduction of complement — but is most likely chiefly due
to the poison neutralizing or destroying bodies."

The practical application of specific ant i -bacterial sera in
the treatment of the various infections in man has been tried on
a large scale. It has been found that by the early injection of
serum from animals actively immunized to various organisms,
animals can be rendered non-susceptible to infections with pneu-

112 VACCINE AND SERUM THERAPY.

mococci, gonococci, streptococci, typhoid and dysentery bacilli,
cholera spirillae and many other microorganisms. Based on these
results, many different anti-bacterial sera have been used in passive
immunization in man. The results of anti-bacterial sera in the
treatment of diseases of man have, however, not been as satis-
factory as those obtained with specific anti -toxic sera. The im-
portance of complement has already been emphasized. There are,
however, other reasons why anti -bacterial sera have not yielded
as good results as anti-toxic sera.

Anti -bacterial substances, with the aid of complement, have
the power of destroying bacteria but not of neutralizing bacterial
toxins. For this reason it is essential that anti-bacterial sera be
used early enough in the disease, before the bacteria have had a
chance to multiply v sufficiently and to set free enough poison to
injure and kill the cells. Early injection of specific sera unfortu-
nately is not possible in many of the diseases for which specific
anti -bacterial sera have been used, but as our methods of early
diagnosis are improved earlier administration of these sera will
be made. It is to be remembered that it is of the utmost impor-
tance that these sera be used before the infecting organisms
have multiplied greatly.

Another reason of the failure of anti -bacterial sera to cure
disease is dependent upon the fact that as the bacteria are dis-
solved and destroyed by the action of immune substance and com-
plement, the intracellular toxins are liberated. If the body cells
are not able to cope with the increased amount of toxin liberated
upon the solution of the bacteria, they will die and the gravity of
the disease be increased. Hiss has produced evidence to show
that the leucocytes contain substances neutralizing these poisons.
To have liberated the minimum amount of toxin, anti-bacterial
sera should be administered when the number of invading bacteria
is relatively small, which will be early in the disease.

Anti -bacterial sera are standardized with more difficulty and
less reliably than are the anti-toxic sera. Attempts have been
made to determine the number of smallest fatal doses of bacteria
a given amount of serum will 'protect against. It is to be remem-
bered, however, that with living microorganisms, a multiple of
the fatal dose is not as much more severe than a single dose as

ANTI-STREPTOCOCCIC SERUM. 113

the multiple would indicate. Attempts have also been made
to determine the protective value of specific anti -bacterial serum
by injecting varying amounts of sera, and bacteria, which have
been mixed in vitrio and allowed to act for some time before in-
jection. The value of some of the lytic serum, as determined by
this method, is high. Frequently it is not possible to test the bac-
tericidal power when, as a result of the difficulties of determining
the amounts of specific bactericidal substances in the serum, the
agglutinating and precipitating values are determined. This
method of standardization is not followed because agglutinins
and precipitins are regarded as bactericidal substances but because
these anti-bodies, to some extent at least, accompany bactericidal
power.

As was stated earlier, specific anti-bacterial sera have been
made by injections of practically all the disease producing bacteria.
The most important of these are the sera which contain specific
substances by which they destroy streptococci, meningococci,
pneunococci, typhoid and dysentary bacilli, and staphlycocci.

ANTI-STREPTOCOCCIC SERUM.

Anti-streptococcic serum was first made bv Marmorek, in
1895. This investigator immunized horses by injections of in-
creasing doses of living virulent streptococci. With serum from
these animals he was able to confer passive immunity to rabbits
and also made attempts to treat erysipelas and pueperal fever in
the human. Denys and LeClef have immunized animals with
bouillon cultures of streptococci and claim to get favorable results
upon injection of serum from the immunized animals. Van der
Velde, realizing that there are different strains of streptococci, and
that the anti-sera produced are specific for the especial strain
used in immunization, immunized animals with the different
strains of streptococcic, thus making what is called a "polyvalent"
anti-streptococcic serum.

The sera which are now on the market are almost universally
made by immunizing horses with repeated injections of increasing

114 VACCINE AND SERUM THERAPY.

doses of killed and later living bouillon cultures of numerous
strains of streptococci recently isolated from patients. Several
months are required to immunize horses from which the immune
serum is to be obtained. After testing the serum for sterility it
is usually tubed in suitable syringes.

The method of action of anti-streptococcic serum is little
understood. The serum contains agglutinins but these have not
been regarded of value in immunization. With the discovery of
bacteriotropins by Neufeld and Rimpau and opsonins by Wright
and Douglas new light has been thrown on the action of these
sera, for it has been found that in anti-streptococcic serum there
is present a specific substance which makes streptococci vulnerable
to ingestion by leucocytes.

Standardization and determination of the amount of pro-
tective substances in anti-streptococcic serum has not been suc-
cessful, because the susceptibility of animals to streptococci varies
and because the method of protective action of the serum is little
known. Usually the dose injected is regulated according to cubic
centimeters of polyvalent anti-streptococcic sera. The potency
of this serum decreases relatively rapidly so that it ought not to
be used later than six months after the bleeding of the immunized
horse.

Anti-streptococcic serum has been used especially in septi-
caemia, local infections following traumatism, streptococcic pneu-
monia, meningitis, rheumatism, erysipelas, pueperal sepsis, scarlet
fever, secondary infection complicating tuberculosis, and in fact,
in all streptococcus infections.

The therapeutic and prophylactic results obtained by the use
of anti-streptococcic serum vary considerably and are apparently
dependent on various conditions, such as grade and kind of im-
munizing serum, day of disease on which injections are made,
amount of serum injected, virulence of infecting organism, and
determination of the invading organism. From the various re-
ports which have been received from clinicians who have used
these sera, there can be no doubt as to the beneficial results which
may at times be obtained from its use. In order that good results
may be obtained, however, it is essential that it be definitely
determined that the streptococcus is responsible for the diseased
condition, that a high grade of polyvalent serum be given early

ANTI-MENINGOCOCCIC SERUM. 115

in doses varying from 10 to 40 c. c., and that these injections he
repeated at intervals of from four to eight hours.

After injection of anti-streptococcic serum the patient usually
rests more quietly for several hours. This, however, need not
indicate that recovery from the disease will follow. The thera-
peutic effects of the serum are manifested by the relief of symp-
toms, decline in fever, improvement of the pulse, and subsidence
of the nervous symptoms. These effects usually appear within
twenty-four hours after injection if the serum is to be of value,
and if no relief comes within twenty-four hours after two injec-
tions of from 20 to 40 c. c. of serum, no beneficial results from the
use of the serum are to be hoped for. No untoward effects of the
serum are met with except the occasional skin rashes which have
already been discussed elsewhere.

Anti-streptococcic serum, even though its curative results are
uncertain, ought to be used in every case of acute streptococcus
infection. The use of the serum in scarlet fever is not universal
but probably should be resorted to in the severer cases. In the
chronic cases of discharging sinuses, etc., streptococcuc vaccines
are at times used to greater advantage than anti-streptococcic
sera.

ANTI-MENINGOCOCCIC SERUM.

Agglutinating substances were discovered in 1903 by Jaeger,
in rabbits experimentally immunized to meningococci. Since
then numerous attempts have been made to produce specific
anti-sera to be used in passive immunization of the human. It
was not until 1906, however, that injection of such anti-meningo-
coccic serum was followed with any degree of success. In 1906
Kolle and Wasserman reported results on the use of specific
meningococcus serum which they had been able to produce in
animals either by intravenous or subcutaneous injections of
cultures of meningococci killed by heating to 60° C., or by in-
jections of extracts of meningococci, obtained by shaking these
organisms for four or five days in suspension in distilled water.
Jochman in this same year reported results on a specific anti-
meningococcic serum prepared after similar methods. The effects
obtained by the use of these sera have, however, not been as

116 VACCINE AND SERUM THERAPY.

favorable as those obtained by the injection of a serum prepared
by Flexner and Jobling.

The method of preparation of the curative meningococcic
serum of Flexner and Jobling, is to inject into the horse first in-
creasing doses of killed meningococci, later increasing doses of
living meningococci and finally increasing doses of an extract of
meningococci. The extract injected contains the endotoxin liber-
ated by the action of a meningococcus autolytic enzyme which as
Flexner has found, is able to destroy the cell substance of the
meningococcus.

The action of Flexner and Jobling's serum, according to the
originators, is three fold. It exerts an anti-toxic action, is bac-
tericidal and is bacteriotropic or opsonifying. Of these effects-
clinicians who have used the serum emphasize especially the ap-
parent anti-toxic action.

Flexner and Jobling have summarized the results of the treat-
ment of 400 cases of epidemic cerebrospinal meningitis. Accord-
ing to this summary it has been found by the various clinicians
using this serum, that the period of illness can be shortened, the
chronic lesions largely prevented and the percentage of mortality
markedly reduced. Probably the principle reason for the differ-
ence in efficiency of Flexner and Jobling's serum as compared to
Kolle and Wasserman's and Jochman's specific sera lies in the
method of application. Flexner and Jobling have emphasized the
importance of reaching the causal organisms with the serum. For
this reason lumbar puncture and withdrawal of a certain amount
of cerebrospinal fluid is first made, after which, without removing
the needle, injection of the serum is made directly into the sub-
arachnoid space of the spinal cord. Recently the persist ance of
Diplococcus intracellularis in the lateral ventricles, after apparent
death of the organism in the spinal sub-arachnoid space, has been
emphasized by Knox and Sladen. For the treatment of this con-
dition, Gushing and Sladen have suggested the advisability of ven-
tricular puncture and injection of ant i -meningococcus serum.

The results which have been obtained by the use of anti-
meningococcus serum as made by Flexner and Jobling, have been
so beneficial in acute and chronic cases of meningitis due to the
Diplococcus intracellularis that its use is indicated as a specific
therapeutic measure.

ANTI-GONOCOCCIC SERUM. 117

ANTI-GONOCOCCIC SERUM.

Various attempts have been made to produce an anti-gono-
coccic serum for passive immunization of man. It is a well known
fact that in the human no immunity results from an attack of
gonorrhea, and for this reason, together with the lack of favorable
results from the use of anti-gonococcic serum, most investigators
have concluded that a specific immune serum for Mic. gonorrhoeae
cannot be produced. In most of the attempts at the production
of anti-gonococcic serum old cultures of the organism have been
used.

In 1906,Torrey described an anti-gonococcic serum with which
he had attained beneficial results in the treatment of some cases
of gonorrheal arthritis. In an attempt to make anti-gonococcic
serum, Torrey made the observation that the toxin of the Mic.
gonorrhoeae, which is present in old fluid cultures, is toxic for
the small laboratory animals. The toxin for the gonococcus has
been studied at various times, and has been generally regarded
as being derived from the dead and disintigrated bodies of the
organism, though by some it has been claimed to be a true ex-
tracellular toxin. Torrey found that it is not possible to im-
munize the small laboratory animals to this toxin, but that in
fact at times a true hypersusceptibility may develop as a result of
injections of this toxin. This investigator observed, however, that
animals can be immunized to the living and dead organisms of
gonorrhea, and that in the blood of animals so immunized agglu-
tinins and lysins are present. Based on these observations, Tor-
rey holds that the efficiency of the serum depends upon specific
bactericidal substances which act because of lytic rather than of
opsonifying power. Phagocytosis, according to this investigator,
is of little importance in the destruction of gonococci.

Torrey and Rogers have prepared an anti-gonococcic serum
for use in treatment of gonorrheal infections in man. The method
of preparation of this serum, as perfected and recommended by
these investigators, and used by the different serum producers is
as follows: Strong healthy rams receive intraperitoneal injections
of increasing amounts of twenty-four hour old ascitic agar cultures
of various, recently isolated, virulent strains of Mic. gonorrhoeae.
The culture is suspended in salt solution, and for the first two

118 VACCINE AND SERUM THERAPY.

injections, this suspension is heated to 65° C. for one-half hour
before the injection is made. Nine or ten injections are usually
necessary to produce serum of high value. After immunization
is completed, the animal is bled from the carotid arteries, and the
serum allowed to separate out. After this the serum is collected,
filtered and tested for sterility. Immunization is made in rams
because blood from these animals is apparently little toxic for the
human. Polyvalent serum is used because the immune bodies for
one strain are specific for that strain, and inasmuch as the strains
of gonococci vary in the different infections, immune bodies for
all strains must be present in the serum.

Standardization of this serum has not been effectual. It
seems quite definite that this serum possesses no anti-toxic nor
opsonic value, but is dependent for its efficiency on its lytic action.
Because of a lack of any better method of determining the immun-
izing value of this serum, determinations have usually been made
of its agglutinating value. The amount of serum, injected is based
on cubic centimeters rather than units of immune substance.

Torrey and Rogers' anti-gonococcic serum has been tried
in the treatment of the various gonorrheal infections. From time
to time favorable and unfavorable results have been reported.
The greatest value of the serum apparently is manifested in the
treatment of the complications of gonorrheal urethritis, such as
prostatitis, epididymitis, orchitis, cystitis, salpingitis, endocarditis,
pleuritis, meningitis and especially arthritis. The acute condi-
tions, urethritis, vaginitis, and conjunctivitis, are little effected
by this treatment.

The method of treatment with anti-gonococcic serum consists
of injections of 2 c. c. of serum at intervals of from one to four
days, as may be indicated clinically. The injections of serum are
made in the loose subcutaneous tissue in convenient parts of the
body. In every case treated with anti-gonococcic serum all the
other methods known to be of value in the treatment of the par-
ticular condition should be employed. Anti-gonococcic serum seems
to cause serum disease more frequently than most of the other
immune sera. In all cases it is to be remembered that, because
this method of treatment frequently must be carried over a long
period of time, serum injections must be made at intervals of not
more than seven to eight days.

ANTI-PNEUMOCOCCIC SERUM. 119

While anti-gonococcic serum, as prepared by Torrey and Rogers'
method, has produced beneficial results in many conditions,
it is to be remembered that improvement and recovery have not
constantly followed its injection. Moreover injection of this
serum ought not to be the only method of treatment instituted.

ANTI-PNEUMOCOCCIC SERUM.

Man after an attack of pneumonia possesses a certain degree
of immunity. Immunity, however, does n^;t always follow pneu-
monia, nor is it lasting. Early in the history of the diplococcus of
pneumonia, attempts were made to artificially immunize animals,
with the result that it was found that a certain degree of immunity
can be produced. In as much as the pneumonococcus does not
produce an extracellular toxin, ant i -toxic immunity cannot be
produced in animals as a result of artificial immunization with this
organism.

The anti-pneumococcic serum now used is usually taken from
horses that have received increasing injections of dead and later
of living cultures of recently isolated pneumococci. Most of the
sera are polyvalent, the best known being those made by Roemer
and by Pane. The method of action of anti-pneumococcic sera
is little understood. It is quite definitely established that it
possesses no anti-toxic value. The curative action of the serum
then, must depend on its ant i -bacterial power. Lysins, until
recent times, were supposed to 'account for the immunizing value
of the serum. Neufeld and Rimpau and Wright and others have
shown that anti-pneumoccic serum contains bacteriotropic or
opsonifying substances which prepare the cocci for ingestion by
the leucocytes.

In the treatment of pneumonia with anti-pneumococcic serum
usually relatively large amounts of serum are injected. The
treatment as recommended by clinicians who have had most ex-
perience, is to inject 20 c. c. into the subcutaneous tissue twice
a day until the symptoms are relieved. None of the other known
methods of treatment are suspended during serum treatment.

The results which have been attained by the use of the serum
have varied a great deal. Some observers have lauded the use

120 VACCINE AND SERUM THERAPY.

of this serum, but when all cases are considered the results are not
convincing as far as the curative effects of the serum are con-
cerned. The reasons for the failure of a specific anti-pneumococcic
serum are probably dependent on the absence of an anti-toxin
in the serum, injection after the disease is far advanced, and the
difficulty in determining the species of organism causing the pneu-
monia. The latter reason must be evident to the clinician, because
the disease of lobar pneumonia may be caused by various differ-
ent species of organisms. Although in general, the therapeutic
action of anti-pneumococcic sera has not been marked, still
physicians who have used it in pneumonia have been impressed
with the general improvement of the patient, the lowering of the
temperature and the absence of complications, following its use.
Important untoward symptoms seldom have developed from its use.
While anti-pneumococcic serum is not definitely a curative
measure, the physician is warranted in using it whenever the
patient's condition becomes serious. In all cases it is to be
remembered that if beneficial results are to be obtained with
this serum it must be injected early.

ANTI-TYPHOID SERUM.

Before the discovery of the organism causing typhoid fever,
it was known that an attack of typhoid fever, usually gives some
protection against a second attack of the disease. After the
discovery of the typhoid bacillus it was found that animals repeat-
edly injected with non-fatal doses of this organism will ultimately
be protected against otherwise fatal doses. It was also found
that by injecting blood serum from immunized animals, a passive
immunity against typhoid bacilli can be conferred to other animals.
Based on these results various anti-typhoid sera have been made
for the specific treatment of the disease of typhoid fever in man.

Anti-typhoid sera are usually obtained from horses which
have been immunized either by repeated increasing injections of
dead and living cultures of typhoid bacilli or typhoid bacillus
toxins. Most of the sera prepared have, however, not been anti-
toxic but anti-bacterial. Chantemesse has prepared a so-called
anti-toxic serum by injecting horses with increasing doses of pure
cultures of typhoid bacilli grown for some time on a macerated

ANTI-TYPHOID SERUM. 121

splenic pulp and defibrinated human blood medium. Tavel by
repeated injections of two weeks old bouillon cultures, sterilized by
the addition of 0.5 per cent of carbolic acid has made a serum
which is supposed to possess anti-toxic substances. Most of the
sera are, however, made by injecting into horses and other animals,
increasing doses of dead and living typhoid bacilli of various
strains. For these sera little or no anti-toxic value is claimed,
the immunizing properties being dependent on the lytic power of
tne serum.

In the treatment of typhoid fever with these sera, 10 c. c. and
frequently larger quantities are injected daily until improvement
occurs.

The results following the use of anti -typhoid serum have been
disappointing. In most cases no effects on the course of the
disease have been observed following the use of this serum.
Chantemesse in 1902, reported, that by the use of his serum the
death rate in children due to typhoid fever was 3 per cent, whereas
the death rate in all the children of Paris who received no such serum
injections, was 19 per cent. Occasionally observers have obtained
and reported cases in which improvement is rapid after the in-
jection of anti-typhoid serum. As is the case with many of the
anti -bacterial sera, there is in general no influence on the course
of the disease. In some cases, however, a drop in the fever and
improvement in the pulse and general conditions have been re-
ported as resulting from the use of this serum. Various reasons
have been assigned for the failure of the curative action of the serum,
the most important of which are, the lack of anti-toxic substances
which can combine with the toxin liberated after solution and
disintegration of the bacilli due to the specific lytic substances in
the serum, and, the failure of the body to supply a sufficient
amount of complement so as to get the destruction of the invading
typhoid bacilli.

Jez has prepared an extract from the spleen, bone marrow
and lymph glands of animals immunized to typhoid bacilli. This
extract is usually administered by the mouth, and is supposed to
possess anti-toxic properties. The value of this extract is doubtful.

Anti-typhoid sera are still in the experimental stage. With
the sera now produced no marked beneficial results can be hoped
for, and are only used in certain severe cases of the disease.

122 VACCINE AND SERUM THERAPY.

ANTI-DYSENTERIC SERUM.

Soon after the discovery of the bacillus of dysentery by
Shiga in 1892, the treatment of bacillary dysentery by the use of
immune sera was undertaken. It was found by Shiga, that the
organism which is now regarded as the etiological factor in bacillary
dysentery is readily agglutinated with blood from patients suffer-
ing with this disease. Following the discovery of the etiological
importance of this organism in the dysenteries in Japan this or-
ganism was found by Flexner in the dysenteries in the Philippine
Islands, and in an epidemic of dysentery in Germany by Kruse.
In 1903, Duval and Vedder found this organism in the discharges
of dysentery patients in United States, and in 1903, Duval and
Bassett, working at the Thomas Wilson Sanitarium near Balti-
more, Maryland, isolated this organism from the stools of children
suffering with summer diarrhoea. Since Shiga's discovery of the
organism, B. dysenteriae has been found in association with dysen-
tery in almost all parts of the world, and the etiology of bacillary
dysentery now seems well established. While at first it was sup-
posed that the different organisms isolated from stools in these
cases were the same, later investigation has shown that there are
two principal varieties of the species of Bacillus dysenteriae. Of
these the original Shiga isolation represents a strain which does
not ferment mannite, while the organism isolated by Flexner
represents a type which produces acid on mannite. The mannite
fermenting type has now been found to include at least two and
probably three different strains. The original Shiga type has
been found especially in the cases of epidemic dysentery in adults,
while the mannite fermenting types are most frequently found,
although not exclusively or as the only type, in the stools of chil-
dren suffering with summer diarrhoeas. The latter fact empha-
sizes the especial importance of the mannite fermenting types of
dysentery bacilli to the dysenteries in the United States.

A further difference has been found between the two main
types; the mannite fermenting types do not produce an extra-
cellular toxin, while the type, not producing acid on mannite,
produces an extracellular toxin. Because of these differences
in the ability to produce extracellular toxin, antitoxic immunity
can be produced by experimental immunization for the Shiga
type, while for the mannite fermenting types the production of

ANTI-DYSENTERIC SERUM. 123

anti-toxic immunity is not possible experimentally. These differ-
ences have been recognized recently and only after much work
had been done on the treatment of bacillary dysentery with specific
immune sera. Because of this the value of serum therapy in
dysentery can be determined only from the relatively recent
literature on this subject.

The results obtained by the use of ant i -dysenteric serum in
the treatment of dysentery have differed markedly. In Japan,
by the use of Shiga's ant i -dysenteric serum, the mortality of
dysentery has been reduced from 22-26 per cent to 9-12 per cent.
Vaillard and Dopter have collected statistics on two hundred
cases treated with anti -dysenteric serum, in which cases there was
a mortality of only 2 per cent. In the United States on the
other hand, no great beneficial results have been attained by the
use of the serum as is evidenced from the extensive investigations
made in 1903 under the direction of Dr. Flexner.

There are various reasons for the differences in results which
have been obtained by the use of anti -dysenteric serum. The
patients treated in the United States were largely children under
three years of age, while a large percentage of the cases for which
favorable results are obtained with serum treatment, occurred
in adults. The day of the disease on which serum injections are
made is probably earlier in such locations where epidemic dysen-
tery is regarded as a serious disease. In the United States many
cases of frequency of stool are only simple diarrhoeas, because of
this the patient usually receives no medical attention until the
disease is well advanced. Probably the most important reason
for the difference of the curative value of the different sera is
dependent on the differences in the specific substances in the
serum. Evidently those sera which have produced the most
beneficial results possess anti-toxic properties, while those used
in the United States are principally or wholly anti -bacterial.
Shiga says of his serum that it is "bactericidal as well as anti-
toxic and — therefore is more effective than anti-typhoid serum."
Practically all of the anti -dysenteric sera have been polyvalent,
i. e., the animals furnishing the serum have been immunized to the
various strains and toxins of dysentery bacilli, as the case may be.

The method of treatment with anti-dysenteric serum varies
with the severity of the disease. Shiga suggests that in mild

124 VACCINE AND SERUM THERAPY.

cases one dose of 10 c. c. of the serum be injected. In cases of
medium severity two injections of ten c. c. each, the interval be-
tween injections ranging from six to ten hours, are recommended.
In the severer cases, 40 to 60 c. c. in all are to be injected, but
never more than 20 c. c. daily. The serum used in the United
States has been injected in larger amounts, sometimes as much
as 100 c. c. being injected in one day.

The effect of treatment with Shiga's serum has been to
decrease the number of stools, cause the blood and pus to disap-
pear from the stools, restore the temperature to normal, and to
lessen the pain and tenesmus. When this serum is used late in
the disease the beneficial effects manifest themselves much later.
The serum used in the United States has not influenced the course
of the disease to any extent, nor is it possible to determine any
particular improvement in the condition of patients so treated.

Coyne and Auche have reported eleven cases of dysentery
produced by the Flexrier, or mannite fermenting, type of dysentery
bacillus, which were treated very successfully with a polyvalent
serum. The curative effects of this serum were probably due to the
action of the anti-toxin to the Shiga type of bacillus dysenterine.

It is to be hoped that specific anti-toxic sera can be made
for the mannite fermenting group of dysentery bacilli, or that
Shiga type anti-toxin will be found to be efficient in combatting
infections with the mannite fermenting group of dysentery bacilli.
This is especially desirable because the mannite fermenting
organisms are largely responsible for the childrens' summer
diarrhoeas in the .United States.

ANTI-STAPHYLOCOCCIC SERUM.

Various anti-staphyloccic sera have been made. They are
usually obtained from horses and other animals that have received
repeated injections of dead and living cultures of these organisms.
The method of its action is not clearly understood although any
protective value it may have is probably due to lytic and opsoni-
fying power. The value of the serum, as it can now be obtained,
is inconsiderable, and its injection in the treatment of staphy-
lococcus infections is seldom or never warranted.

INDEX OF NAMES.

Page

Atkinson 98

Babes and Lepp 83, 87

Baldwin 37

Banzhaf 98, 102, 106

Barber 38

Behring 100

Brhnnn and Kitasato 15, 87, 106

Brhring and Kitashima 100

Bfhring and Knorr 15

BfliriiiK and Wernicke 87, 100

Belfanti and Carbonne 98

Besredka 60

Bolduan 37, 41, 47, 67

Bolton 100

Bordet 20

Buchner 14, 87

Bulloch and Atkin 61

Calkins 84

Chamberland 14

Chantemesse 120, 121

Chauveau 14

Clark 54, 71

Cole 38, 47, 48, 67

Cole and Meakins 28, 76

Coyne and Auche 124

Dean 60

Denis 90

Denys • 79

Denys and LeClef 20, 113

Dieudonne , 97

Dorr 63

Duval and Bassett 122

Duval and Vedder 122

Khrlich 15, 50, 111

Ferrari 100

Field 20

Flfxner 122, 123

l-'lc.Mier and Jobling 116

Foa and Bonome 14

Fodor 14, 87

Fraenckel 100

Gabritschewsky 77

(lay and Southard 93

Gibson 98, 102, 106

Guerini 84

Haflkine 65

Hamilton 54

Hektoen 60, 64, 67

Herricourt and Richet S7

Heubner 92

IIN> and Zinsser 45, 63, 111, 112

Huhiu- and Neufeld 62

Jaeger 115

.!<•;, us and Sellards 30, 47, 48

126 INDEX OF NAMES — CONTINUED.

Jenner 84

Jez 121

Jochman 115

Keith 61

Klebs 14

Kolle and Wasserman 115

Koch 79

Kruse 122

Leishman 23, 49

Levaditi and Inman 62

Loehlein 60

Madsen 50

Maragliano 79

Marie 83

Marmorek 113

McClintock and King 99, 104

Meakins 72

Metchnikoff 14, 20, 53, 63, 110

Moss 38, 41, 42, 46, 47, 48, 57

Morgenroth Ill

Muir and Martin 60

Neufeld 61, 63

Neufeld and Rimpau 21,59,60, 114, 119

Neufeld and Toepfer 61

Noguchi 62

Nuttall 14, 89

Pane 119

Park 37, 67

Pasteur 14, 65, 82

Peter-sen 45, 63

Pfeifler 65

Pfeiffer and Kolle 80

Pirquet and Schick 90, 93

Potter 37

Potter, Dittman and Bradley 47, 60

Richardson Ill

Roemer 119

Rosenau and Anderson 92, 94

Ross 34, 72, 76

Ross and Johnson ". 77

Roux 14

Roux and Yersin 87

Ruediger 54

Russell 60

Sahli 63

Salmon and Smith 14

Sauerbek 63

Savtchenko 60

Shiga 81, 122, 123

Simon 37, 41, 43, 44, 45, 46, 47, 48, 60

Simonds and Baldauf 62

Solomonson 50

Strong 46

Tavels 121

Tizzoni and Centanni 83

Torrey and Rogers 117

Trudeau 79

Vaillard and Dopter 123

v. der Velde 113

Walker 31, 32, 37, 38, 39, 42, 43, 44, 45, 47

Wasserman 100

Wright 27, 30, 31, 35, 37, 38, 40, 44, 49, 51, 57, 59, 60, 65, 67, 72, 72, 75, 79, 119

Wright and Douglas 21, 23, 27, 30, 33, 50, 59, 60, 66, 69, 70, 71, 114

Wright and Ried 60

Wright, T. H 29

INDEX.

Page

Acne vulgaris 75

Agglutinins 17, 19, 50, 53, 71,110

Aggressins 7, 63

Alexines 14, 87

Amboceptor 18, 60

Anaphylactine 93

Anaphylaxis 93

Anti-bacterial sera 106

Anti-bodies 16, 1"

Anti-dysenteric serum 122

historical 122

differences due to strains of bacilli .' 122

preparation of 123

met hod of treatment with 123

results obtained with 124

Anti-gonococcic serum 117

preparation of . ... 117

method of treatment with 118

results obtained with 118

Anti-meningococcic serum 115

Flexner and Jobling serum 116

administration of 1 16

results of treatment with 116

Anti-pneumococcic serum 119

preparation of 119

method of treatment with 119

results obtained with 120

Anti-staphylococcic serum : 124

Anti-streptococcic serum 113

preparation of 113

actien of 114

method of treatment with 114

results obtained with and value of 115

Anti-typhoid serum 120

preparation of 1 20

method of treatment with 120

results obtained with 121

Jez's immune extract 121

Anti-toxic sera 99

diphtheria anti-toxin 100

tetanus anti-toxin 106

Anti-toxic globulins 97. 102, 106

Anti-toxin 17, 19, 53, 89, 99

Appendix abscesses MI

Arthritis, streptococcus 76

gonococcus 77

Bacillus coli, sensibility of 50

suspensions 28

vaccine 80

Bacillus diphtheriae, anti-toxin 88, 100

sensibility of 50

toxin " 87, 101

vaccine 105

Bacillus dysenteriae anti-serum, sensibility of 50, 122

vaccine. . 81

128 INDEX.

Bacillus, pestis sensibility of 50

Bacillus pyocyaneus, suspension of [[-\\ 28

vaccine gg

Bacillus tuberculosis, immunization to with tuberculin 78

suspension of 29

staining of 35

vaccine (tuberculin) 78

Bacillus typhosus, anti-serum 120

opsonic index determination for 71

sensibility of 50

vaccine 80

Bacterial agglutinins 17, 19, 50, 53, 71, 110

Bacterial emulsions ' _ 27 31

clumping in. . 3Q, 40

Bacterial precipitins 17, 19, 50, 110

Bacterial vaccines 64

autogenous 68

colon bacillus 80

cholera spirillum • 81

control of injections of 70, 73

dosage of • ' 66

dysentery bacillus 81

gonococcus 77

paratyphoid bacillus 80

preparation of 69

point of injection of 67

staphylococcus 74

streptococcus 76

stock culture 68

f tuberculosis (tuberculin) 78

typhoid bacillus 80

Bacteriotropins 21, 59

Bacteriolysins 17, 19, 50, 53, 71, 89, 110, 112, 117, 119, 120, 123

Barber's itch 75

Behring's law 88

Calculation of opsonic index 35, 45

Carbuncles 74

Chemicals, effect on opsonic index 64

Chicken cholera 35

Cholecystitis 80, 81

Cholera, immunization against 65, 81

sensibility of spirillum of 50

vaccine against 81

Clumping of bacteria in bacterial suspensions 30, 44

Complement 18, 98, 110, 112

Complementophore group 18

Concentration of serum 97, 102, 106

Cystitis 79, 80, 81, 118

Cytophylic group 8

Diphtheria anti-toxin 89, 100

dried 104

dosage 103

oral administration of 104

preparation of 101

standardization of 102

therapeutic action of 104

treatment with 103

Diphtheria immunization, active 105

with anti-toxin 105

Diphtheria toxin 87, 89, 101

Dysentery, bacillary, immunization against 81

vaccine treatment in 81

serum treatment in 122

Ehrlich's side chain theory 14

Emulsions, bacterial 27, 31

Endocarditis, gonococcus 118

streptococcus 76, 115

INI. 1 29

Kndometrilis. colon bacillus infections 80

foaococcuA infection! 118

54, 70

Kixinii of smears (Or opsonic index determination^ :: 1

Fixed \ i ru s. hydrophobia 82

Furtinculosjs 74, 86

(ilohulins in iinniuiic sera 98

precipitation of 98, 102, 106

diphtheria 102

tetanus 106

( Minocorciis infection, serum therapy 117

vaccine therapy 77

Haptophorc »Toup 16

Hydrophobia 65, 81

diagnosis of v_'

1'asieiir treat nt 81, 83

llypcisiisceptibility to serum 90, 105

to vaccine 73

Immune sera 87

anti-l>acten:d seia 109

anti-dysenteric serum 122

anti-^onococcic serum 117

anti-meningococcic serum 115

anti-pneumococcic serum 119

anti-staphylococcic serum 124

anti-streptococcic serum 113

anti-typhoid serum 120

anti-toxic sera 99

diphtheria anti-toxin 100

tetanus anti-toxin iL 106

dried 104, 109, 1 1 1

polyvalent \ \'.'>

Immunity, acquired 12, 65, 89

actice 13, 65

definition and classification of 12

passive 13, 89

theories of 14, 15, 20

Immunizing substances 13, 88, 89

elimination and loss of 20

Infections 7

course of 8, 11

elimination of causal factors 11

general react ion in 10

incubation period in 9

local reaction in . : 10

Leistungakern i.">

Leucocytes, accumulation of 10

in immunity !.'>, 20, 21, 45, 63, 111, 112

in opsonic index determinations, examination of 35, 46

source of 26, 44

Leueocytosj.s. relation to opsonic index 62, 111, 112

Lydl 17, 19, 50, 52, 71, 89, 110, 112, 117, 119, 120, 123

Meningitis, opsonic index in 70

serum treatment of 115

vaccine t reat ment of 70

Micrococciis meliteiisis. sensibility of 50

Micrococcus meningitidis, anti-serum 115

suspension of 28

Microcoecus pneiimoniae. anti-serum 119

sensibility of oO

suspension of 28

vaccine (>(>. 77

Micrococcus pyo^enes. anti-serum U I

infection, treatment with anti-serum 124

treatment with vaccine 74

suspension of, for opsonic index determination 28

\ accine 74

Negative phase 50, 51

130 INDEX.

Opsonic index 21, 23, 24

after injection of bacterial vaccines 57

bacterial emulsions for 27, 31, 43, 44

calculation of 35, 45

dilution of serum for 41

in acute otitis media 34

in erysipelas 54

in health and disease 50, 53, 54

in tuberculosis 60, 79

in typhoid fever 54, 70

leucocytic emulsion for 26, 44, 46

mixture of bacteria, leucocytes and serum for 31, 39

normal serum for 26, 39

smears for 33

staining of smears for 35

serum for ' 24, 39

technique for determination of '. . . 24, 38

Opsonin 20, 49, 59, 110

effect of chemicals on 64

effect of heat on 60, 61, 62

effect of reaction on 64

immune and normal 20, 21, 59, 60, 61, 114, 119, 124

nature of 58

non-bacterial 64

specificity of 20, 59, 61, 62

structure of 60, 61

importance in immunity 20, 50, 53

Oral administration of anti-sera 99, 104

Pericarditis, gonococcus 118

streptococcus . 76, 114

Phagocytic index 24, 39, 45

Phagocytosis 20, 21, 45

causes of 14, 20, 61

spontaneous 30, 61

Pneumonia. 76, 119

Pneumococcus infections, serum treatment 119

vaccine treatment 77

Pooled serum 26

Polyvalent immune serum 113, 123

Positive phase 50, 51, 71

Precipitins 17, 19, 50, 93, 110

Pus ' 10

Rabies 81

Reaction, influence on opsonic index 64

Receptors 15

first order 16

second order 17

third order . 18

Septicaemia 9

anti-streptococcic serum 114

streptococcus vaccine 76

Serum, anti-bacterial 106

anti-toxic 99

concentration 97, 102, 106

disease 90, 105

dried immune 98

dried diphtheria anti-toxin 104

dried tetanus anti-toxin 109

oral administration of , 99, 104

pooled normal 26

purification of ,97, 102, 106

therapy 87

Small-pox vaccination , 84

duration of immunity from 85

preparation of vaccine for 85

Smears for determination of opsonic index 33, 48

examination of 35, 46

staining of 35

i\Di:\.

Spirillum cholerae. immuni/ation against 81

sensibility of 51

Staphylococci, suspension for opsonic index determination 28

Staphylocoecus infect ions, serum t rent ment of 124

vaecine t reat ment of 74

Stimiilins 20, 63

St rcptococci, suspension for opsonir index determination 29

Streptococcus Infection*, eenim treatment of 113

\ ace i ne treat ment of 76

Substance sensibilit rice 18

Sycosis barbae 75

, nus antitoxin l()(i

• Iried 109

prepurution of 106

staiidardi/ation of 106

t reat ment with 107

Toxin 13, 17, 87, Si'. 101. 106

produced by diphtheria bacillus 87, 101

produced by tetanus bacillus 106

Tuberculin 7 ^

Tuberculosis, treatment with tuberculin 7'.'

Typhoid bacillus infections, immunization against 65, 80

serum treatment of 120

vaccine treatment of .so

Typhoid bacillus, sensibility of 50

suspensions of for opsonic index determination 71

Urethritis, gonococcus; serum treatment of 1 18

vaccine treatment of 77

Vaccine therapy 65

bacterial vaccines 65

Bacillus coli 79

Bacillus dysenteriae 81

Bacillus paratyphosus 80

Bacillus typhosus 80

control of injections 70

dosage of 66

gonococcus 76

pneumococcus 77

preparation of ti'.t

Sprillum cholera 81

staphylococcus 73

streptococcus ^ . 75

tuberculin 77

of unknown etiology, attenuated virus. . 81

rabies, virus 81

immunization with 83

preparation of 82

results obtained with s:<

small-pox virus 84

immunization with s;>

preparation of 84

results obtained with s.">

Diagnosis and Treatment of
Diseases of Women

... By ...

H. S. Crossen, M. D.

Clinical Professor of Gynecology, Medical Department Washington University;

Gynecologist to the Washington University Hospital, and Chief of the

Gynecological Clinic; Consulting Gynecologist to the Bethesda

Hospital, St. Louis Female Hospital, and

St. Louis City Hospital.

816 Pages. 700 Illustrations. Price : Cloth $6.00. One-half Morocco, $7.50.
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CONTENTS.

Chapter I. Gynecologic Examination Methods.

Chapter II. Gynecologic Diagonsis.

Chapter III. Gynecologic Treatment.

Chapter IV. Diseases of External Genitals and Vagina.

Chapter V. Lacerations and Fistula of Pelvic Floor, Perineum, External Geni-
tals and Vagina.

Chapter VI. Inflammatory and Nutritive Diseases of the Uterus.

Chapter VII. Displacements of the Uterus.

Chapter VIII. Fibromyoma of the Uterus.

Chapter IX. Malignant Diseases of the Uterus.

Chapter X. Pelvic Inflammation.

Chapter XI. Other Affections of Fallopian Tubes, Peritoneum and Connective
Tissue.

Chapter XII. Diseases of the Ovary and Parovarium.

Chapter XIII. Malformations.

Chapter XIV. Disturbance of Functions.

Chapter XV. Invasion of the Peritoneal Cavity for the Treatment of Gyneco-
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Chapter XVI. After-Treatment of Operative Cases.

Chapter XVII. Medico-Legal Points in Gynecology.
Appendix — Formulae — Index.

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Cjolden rvules of Surgery

Aphorisms Observations Reflections

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Science and Art of Surgery

A Guide for Surgeons and Those Who Would Become Surgeons

By

Augustus Charles Bernays, A. M., M. D.
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and Anatomy, St. Louis, Mo., U. S. A.

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Tin- Education of a Surgeon. About Fees.

On Scientific Communications to the Litera- Off with the Cloak of Superstition.

ture of Medicine and Surgery. Inflammation and the Confusion It Has
Science and Surgery. Caused.

GOLDEN RULES OF SURGERY:

A-'psis. Genito-Urinary. Nose.

Anesthesia. Operations. Goitre.

AMSCI Joints. Shock.

Abdomen. Kar. Oesophagus.

Appendicitis. Krysipelas. Pelvis.

Aneiirysm. Gangrene. Rectum.

Artery Hlccding. Hand and Foot. Spine.

l'»urns. Mojst Dressing. Throat.

Breast. Mouth. Veins.

Can Minor Surgical Operations Be Done in Office?

Death Following Miner Surgical Operations.
Fractures and Dislocations. Therapeutic Hints.

Irrigation Dreinage cf Abdominal Cavity.
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(jolden JKules of .Pediatrics

Aphorisms, Observations and Precepts on the Science and Art of Pediatrics:
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ing, and the Principles of Scientific Treatment.

By
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Professor of Diseases of Children and Clinical Midwifery, Washington University,

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CONTENTS.

Introduction.

Part I.
General Rules of Diagnosis.

General Rules.
Loss in Weight. Appetite.
Convulsions.
Physical Examination.
Head and Neck.
Some Deformities.
Teeth and Gums. Dentition.
The Enanthemata.
Vomiting. Hcmatemcsis.
Diarrhea.

Distended Abdomen.
Abdominal Pain.
Abdominal Swellings.
The Nose and Nasopharynx.
The Larynx.

Anomalies of Breathing. Cough.
The Lungs.

The Heart and Circulation.
The Urine. The Eruptions.
The Nervous System.
Paralysis.

Tremor, Choreiform Movements. Head-
ache, etc.

Changes About the Eyes.
Changes About the Ear.
Clinical Syndromes.
Fever. Chronic Fever.
Status Gastricus.
The Typhoid State.
Infantile Atrophy.
Gastroenteric Infection, Diarrhea.
Chronic Indigestion in Older Children.
Chronic Constipation.
Peritoneal Irritation.
Severe Anemia. Edena.

Ascites. The Adenoid Face.

Acute Pneumonic Consolidation.

Intestinal Obstruction.

Nervous State. Scrofula.

Tuberculosis.

Impending Heart Failure.

The Syndrome of Cerebral Irritations.

Golden Rules of Prognosis.

Part II.

Golden Rules of Hygiene and Infant
Feeding.

The Nursing Mother.
The Wet Nurse.
Artificial Feeding.
Feeding the Sick.

Golden Rules of Treatment.
General Therapeutics.
The Newly Born.
Diseases of the Mouth.
The Neck and Scalp.
The Throat.

The Respiratory Organs.
Gastroenteric Diseases.
Rickets and Scurvy.
Heart and Circulation.
The Blood.

The Genito-Urinary Organs.
The Nervous System.
Specific Infectious Diseases.
Malaria. Cerebro-Spinal Fever.
Diphtheria. Intubation.
Tuberculosis. Pertussis.
Mumps. Septicemia.
Rheumatism and Endocarditis.
Syphilis. The Exanthemata.
The Severe Infectious Fevers.
The Skin.
Formulary.

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Golden Rules of Dietetics

By A. L. BENEDICT, A. M., M. D.

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same; Merey Hospital, Buffalo; Member of the Academy of Medicine and

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Physiologic Chemistry.

Daily Requirements of the Human Body.

Standard Diet in Health.

Quantative Estimate of Diet.

Approximate Methods of Checking Diet Weight and the Excretions.

Transmutability and Reservation of Food.

Waste of Food.

Predigestion of Food.

KmerKeney -Methods of Introducing Nourishment.

Preserved Foods.

Methods of Cooking.

Compositions of Natural and Commercial Foodstuffs.

Food Adjuncts.

Purine Bodies.

Important Constituents of Foodstuffs.

Distinctly Deleterious Foodstuffs.

General Hygiene of Eating.

Diet Tests.

Condensation of Atwator & Bryant's Analysis of Foodstuffs.

Part II.

Principles of Dietetics According to General Pathologic Conditions.

Infant Feeding.

Diet in Critical Physiologic Periods.

Diabetis, Glycosuria.

Obesity and Leanness.

Chronic Diseases of Nitrogeneous Metabolism.

Diseases of the Urinary Organs.

Diseases of the Ductless Glands.

Diseases of the Liver.

Diseases of the Pancreas.

Diseases of the Digestive Organs.

General Preversion of Digestive Functions.

Functional Intestinal Diseases.

Organic Intestinal Diseases.

Diseases of the Heart and Blood Vessels.

Blood Diseases.

Hemorrhagic Diseases.

Bone Diseases.

General Principles of Feeding in Fevers.

Infectious and Parasite Diseases.

Respiratory Diseases.

Skin Diseases.

Diseases of the Nervous System.

Surgical Emergencies and Operations.

450 Pages, Octavo. Price $3.00

Chapter

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11.

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III.

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IV.

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Chapter

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Chapter

VII.

Chapter

VIII.

Chapter

IV

Chapter

X.

Chapter

XI.

Chapter

XII.

Chapter

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Chapter

XIV.

Chapter

XV.

Chapter

XVI.

Chapter

XVII.

Chapter

XVIII.

Chapter

XIX.

Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter
Chapter

I.

II.

III.

IV.

V.

VI.

VII.

VIII.

IX.

X.

XI.

XII.

XIII.

XIV.

XV.

\\ I

XVII.

XVIII.

XIX.

XX.

XXI .

XXII.

XXIII.

XXIV.

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A VERY
YOUNG OVUM IN SITU

By

G. Leopold,

Dresden
Authorized Translation By

W. H. VOGT, A. M., M. D.

Obstetrician and Gynecologist, Lutheran Hospital, St. Louis, Mo.

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Examination of h

ar

By

Selden Spencer, A. B., M. D.

Instructor of Otology in the Washington University Medical Department.

St. Louis, Mo.

With an Introduction

By
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Professor of Otology, Medical Department Washington University,

St. Louis, Mo.

67 PAGES OF TEXT
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CONTENTS.

Chapter I. Methods of Procedure (General Consideration).

Chapter II. The Kxternal Ear.

Chapter III. Diseases of the Canal.

Chapter IV. The Middle Ear.

Chapter V. The Middle Ear (Continued), Non-Suppurative Condition-.

Chapter VI. The Middle Mar (Continued), Post-Suppurative Conditions.

Chapter VII. The Middle Ear (Continued), Suppurative Conditions.

Chapter VIII. The Middle Ear (Continued), Purulent Otitis Media.

Chapter XI. The Middle Ear (Continued), Purulent Otitis Media.

Chapter X. Tlie Middle Mar (Continued), Operations in Chronic Purulent Otitis

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Chapter XI. The Internal Ear.

Chapter XII. Hearing Tests.

Chapter XIII. Intra-Cranial Complications.

Chapter XIV. Kxercises in the Surgical Anatomy of the Temporal Hone.

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Suggestive Therapeutics,
Applied Hypnotism

and Psychic Science

By H. S. MUWRO, A. M., M. D.

Americus, Ga.
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in Europe has been alive to the importance of this subject for many years and much
has been written on it by such men as Schofield, Bernheim, Forell and Duboise. These
writings have been in the nature of research work and have not been devoted to the
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Chapter I. Introduction.

Chapter II. Suggestion: Its Uses and Abuses.

Chapter III. Hypnotism: A Demonstration of the Efficiency of Suggestion.

Technique of Inducing the Hypnotic State.

Chapter IV. Theory and Practice of Suggestive Therapeutics.

Chapter V. Simple Suggestions, or Suggestion Without Hypnotism.

Cahpter VI. Hypnotic Suggestive Therapeutics Applied in Medicine, Surgery.

Chapter VII. The Psychological Factor in Obstetrics.

Chapter VIII. Training the Subconscious Self for Health and Strength.

Chapter IX. Correct Diagonsis a Safeguard Against Blunders.

Chapter X. Philosophy and Religion and Their Relation to Health.

Chapter XI. Conservation of Energy, Education and Control of Emotions.

Breathing, Relaxation, Dietetics, Exercise, etc.

Chapter XII. Roughing It as a Means of Health.

Chapter XIII. Are All Specialists Egotists?

Chapter XIV. Personality as a Factor in Therapeutics.

Chapter XV. Environment: Its Influence in Therapeutics.

Chapter XVI. Brutality of Frankness: Honesty Imperative.

Chapter XVII. Physical and Mental Hygiene; Character as a Resource of Health.

Chapter XVIII. Suggestion in Education, Character Building, etc.

Chapter XIX. Moral Stamina a Therapeutic Power; The Higher Art in Therapeutics,

and the True Physician.

Chapter XX. Self-Mastery as a Fine Art.

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Arteriosclerosis

ETIOLOGY, DIAGNOSIS, PROGNOSIS, PROPHYLAXIS AND

TREATMENT

By

L. M. Warfield, A. M., M. D.

With an Introduction by
H. S. Thayer, Baltimore, Md.
8 ORIGINAL ILLUSTRATIONS.

150 PAGES.

PRICE $2.00

PUBLISHER'S ANNOUNCEMENT

This book is particularly opportune. The rapid pace which Americans are living,
the worry and mental strain under which the majority of their time is spent, has made
this a nation of arterio-sclerotics. The author has laid stress upon prophylaxis as well
as given the most rational treatment known to modern times. The text is embellished
with instructive original illustrations. Sent anywhere on receipt of price.

Diseases of The Skin

By

A. H. Ohmann-Dumesnil, A. M., M. E., M. D., Ph. D., etc.

Formerly Professor of Dermatology and Syphilology in the St. Louis College for .Medical
Practioners; the St. Louis College of Physicians and Surgeons; the Marion-
Sims College of Medicine; Member of the St. Louis Medical Soeietj',
of the Missouri State Medical Association, of the American
Medical Association, of the 1st, 2d, 3d, 4th, 5th
and 6th International Dermatological
Congress, etc.

THIRD EDITION

THOROUGHLY REVISED AND ENLARGED

150 ORIGINAL ILLUSTRATIONS
600 PAGES. PRICE: CLOTH $4.00. MOROCCO, $5.50

PREFACE

Tin's book is not a treatise. The intention has been to make of it a practical guide
to the easy recognition of skin diseases, as well as to their successful treatment. The
remedies which have been recommended are such as may be found in every practitian's
armamentarium medicinorum. No attempt has been made to write an elaborate work,
but rather to furnish, in a clear, concise manner, just that information most desired by
medical students and general practitioners.

TABLE OF CONTENTS.

Chapter
Chapter
Chapter
Chapter
Chapter
Chapter

I. The Skin.

1 1 . Anatomy.

III. Physiology.

IV. Diagn<>-K
V. Etiology.

VI. Pathology.

Chapter VII. Therapeutics.

Chapter VIII. Prognosis.

Chapter IX. Symptomatology.

Chapter X. Classifications.

Chapter XI. Diet in Skin Di>ea>r>.

Chapter XII. Food Eruptions.

Chapter XIII. Appendix.

C. V. MOSBY MEDICAL BOOK AND PUBLISHING CO.

Grand Avenue and Olive Street, :: :: St. Louis, Missouri.

Office Treatment of Rectal Diseases

By

R. D. Mason, M. D.

Professor of Rectal Diseases in the Creighton University,
Omaha, Nebraska.

New 4th Edition. 367 Pages. 87 Illustrations. Price $2.50

Tuberculosis of the Nose and Throat

By

Lorenzo B. Lockord, A. B., M. D.
Denver, Colorado

Consulting Laryngologist to the Agnes Memorial Hospital, Denver.
504 Pages. 85 Illustrations. 64 of which are colored. Price $5.00

THE C. V. MOSBY MEDICAL BOOK AND PUBLISHING CO.

St. Louis, Mo.

Hand Book of Rectal Diseases

By
L. J. Hirschman, M. D.

Professor of Clinical Proctology Detroit College of Medic -ine and Surgery.

150 Illustrations
400 Pages. Including 2 colored Plates. Price $4.00

Gonorrhea in Women

By

Palmer Findley, M. D.

Professor of Gynecology in the Medical Department of the University of Nebra-ka.

Omaha, Nebraska.

128 Pages. Royal Octavo. Price $2.00

Chronic Constipation

By
J. A. McMillian, M. D.

Professor of Therapeutics in the Detroit College of Medicine and Sursrery,

Detroit, Michigan.

257 Pages. Price $2.00.

THE C. V. MOSBY MEDICAL BOOK AND PUBLISHING CO.

St. Louis, Mo.

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