STUDIES ON THE BACTERICIDAL AND AGGLUTINATIVE
POWER OF SERUM AND PLASMA OF NORMAL
AND PULLORUM INFECTED TURKEYS

By
WONG Y* WAI

A THESIS
Submitted to the School of Graduate Studies of
Michigan State College of Agriculture and
Applied Science in partial fulfillment
of the requirements for the
degree of
DOCTOR OF PHILOSOPHY
Department of Bacteriology and Public Health
June, 1949

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ACKNOWLEDGEMENTS
The author is grateful for the generous
help, guidance and instruction given to him
by Dr. H. J. Stafseth, Professor and Head of
the Department of Bacteriology and Public
Health, Michigan State College.

STUDIES ON THE BACTERICIDAL AND AGGLUTINATIVE
POWER OF SERUM AND PLASMA OF NORMAL
AND PULLORUM INFECTED TURKEYS

TABLE OF CONTENTS
Page
Introduction..........

.........

0

Review of Literature .................... • 1
Materials and Methods
Discussion and Results
Summary

......

........... .....17
............... .22
•...... *32

Bibliography ............................. 34
Tables ........... ............... ... ..38-60
Figures

....

*...... .61-76

Introduction

There have been numerous reports in the literature
regarding the bactericidal property and the inhibitory
zone of serum.

Much work has been done by others on

normal animal serum using various species of organisms.
The bactericidal action of each serum varies with each
species and strain of organisms.

Since the bactericidal

action varies toward each species of organisms, it Is
necessary to test the specific serum and plasma on spe­
cific organisms.
The object of this experiment was to Investigate
the bactericidal action of turkey serum and plasma on
Salmonella pullorum.

First, observations were made on

the killing power of normal turkey serum and plasma and
then on the killing power of infected turkey serum and
plasma.

The agglutinating titers, and blood cell counts

were also observed to see what influence they have on
bactericidal action.

The results explain why the

plasma of infected birds had less bactericidal action
than normal plasma, and why prozones occurred in the
lower agglutinating dilutions.

-1-

Revlew of Literature
The observations of Nuttall (1888) and many others
first contributed to our knowledge of serum reactions. It
has been recognized that the bactericidal property of serum
is a variable one, differing according to the animal spe­
cies and the type of micro-organism concerned.

There has

been some uncertainty regarding the specificity or nonspecificity of natural bactericidal effects.

Muir and

Browning (1908) reviewed the literature on this subject
and studied the specificity of these reactions by absorp­
tion methods.

They found that treatment of a normal serum

with increasing amounts of bacterial suspension produced
first a diminution of the bactericidal action towards the
homologous bacterium, and also a decrease in the effect
of natural complement-fixing and agglutinating antibodies.
This suggested the likelihood that the bactericidal ef­
fects of normal serum may be due to multiple specific
antibodies sensitizing bacteria to the lytic action of
complement.
Thjotta (1919) showed that during immunization there
is produced along with the antibodies, a complement-inhibi­
ting substance which he believes to be separate and dis­
tinct from agglutinins, precipitins and bactericidal am­
boceptor.

If sufficient dilution of the serum Is made

-2and if extra complement is added, the immune serum will
show bactericidal action, whereas undiluted, fresh Immune
serum, mixed with the homologous organisms, exhibits
little if any bactericidal effect.
It was shown by Georgevitch (1926) that killed or­
ganisms incubated with serum neutralized non-3pecifically
the bactericidal action of the serum.

It is noteworthy

that the neutralizing substance acts at 0°C. and is more
active at 37°C*

This agent, irrespective of the organism

from which It is produced or the serum used for the bac­
tericidal test, affects strongly the bactericidal anti­
bodies for certain organisms, and is less active towards
others*
The complements of different animal species were
interchangeable in these bactericidal reactions and cer­
tain observations recorded led him to assume that dif­
ferences in the bactericidal properties of sera towards
particular organisms depend on variation in the anti­
body rather than in the complement.
Gordon and Wormall (1928) have shown how bacteriolysis
of Shigella dysenterjae by normal guinea pig serum depends
on the combined action of complement and a thermostable
factor removed from the serum by absorption with organisms.
The question is further complicated by the fact that dif­
ferent mechanisms may be concerned in the bactericidal

-tr­
action of normal sera and that the factors Involved may
vary with different organisms.
Pettersson (1928) classified the bactericidal agents
of serum into alpha lysins and beta lysins.

The former

apparently represent the complement acting with a sensi­
tizing agent analogous to an immune body*

The latter,

according to Pettersson consist of a stable “activating11
agent (resisting a temperature of 65°C. for a half hour)
and an activable principle which unites with the bacteria
in the presence of the activating agent.
Knarr (1929) showed how leucocyte extracts kill such
organisms as streptococci, staphylococci, pneumococci,
and Bacillus anthracis, whereas the alexin of serum acts
on Salmonella typhosa, Escherichia coli, and Vibrio
cholerae.
Pinkelstein (1951) has done much work on the bacteri­
cidal action of serum, and he summarized his results as
follows s
1. An analytical study has been made of the mechanism
of natural bactericidal action by the serum of various
animals towards certain organisms exhibiting the maximum
reactivity to this effect*
2. The serum-complement has no bactericidal action by
itself.

An antibody-like agent invariably acts as an in­

termediary agent, “sensitizing” the particular organism
to the action of the complement and is capable of being
“absorbed” by it from serum at 0°C.

-43* This sensitizing agent is stable at 55° C. but
labile at 60°-65°C*

In this respect it resembles natural

hemolysins and agglutinins, but contrasts with the more
stable immune antibodies and the more labile nature of
the complement-fixing antibodies,
4# The absorption tests demonstrate the high degree
of specificity of these natural bactericidal antibodies
for particular bacteria*
5. A non-specific extracellular substance occurs in
bacterial cultures which may neutralize or inhibit these
antibodies, and interferes with their sensitizing action
even at 0°0.

This substance is liberated in large amounts

in cultures heated to 12Q°C,
In 1932, FInkelstein demonstrated that the bacteri­
cidal property of normal serum towards gram-negative
bacteria is labile at 55°C. for 30 minutes; the factors
responsible for the corresponding effect on gram-positive
bacteria are stabile at this temperature.

Thus the gram-

negative and gram-positive organisms are acted on by se­
parate agents, the 11thermo-labilerf and "thermostabile”
bactericidins respectively.

Bactericidal effects are

more frequent and pronounced towards the gram-negative
than toward the gram-positive bacteria*

The ”thermolabile”

bactericidin consists of complement and a sensitizing anti­
body*

The lability of the bactericidin is due to the

-5lability of the complement*

The antibody is stable at

60°C. and specific for the particular organism acted on.
The “thermo-stabile” bactericidin in undiluted serum with­
stands a temperature of 57.7°G. though labile at 60°C; its
lability is considerably increased in diluted serum and in
slightly alkalinized serum though unaltered by slight
acidity.
The work of Gordon (195b) demonstrated that the ab­
sorption of both normal unheated and heated sera by dead
bacteria fails to yield any evidence of the existence of
a series of specific antibodies in serum.

The loss of

bactericidal power consequent upon absorption is never
specific for the absorbing organism but is always general.
Mudd (1953) showed that sensitization by serum renders
various dissimilar bacteria similar with respect to their
surface properties.

This convergence of surface proper­

ties is carried further by homologous immune than by
heterologous or normal sera, and the homologous immune
sera are effective in higher dilutions.
The work of Gordon and Johnstone (1940) also showed
that the absorption of a normal serum by a series of
strains of one organism causes a general diminution in
bactericidal power for all the strains, but there is a
more striking diminution for the strain with whibh the
serum was absorbed.

Three strains of Micrococcus catar­

rhal! s were used to absorb a guinea pig serum.

They

-6demonstrated that the complement titer of guinea pig
serum was high, that of human serum low and that of
rabbit serum still lower.

The results show low bacteri­

cidal action of human serum on the gonococcus, whereas
guinea pig serum with a higher and rabbit serum with a
lower complement titer were both markedly bactericidal.
In this experiment one human serum had no bactericidal
action on Vibrio cholerae but another human serum killed
Vibrio cholerae in one hour.

The rabbit serum, which

had a lower complement titer than the guinea pig serum,
was again the more bactericidal, and Inactivation of
complement completely destroyed the bactericidal action
of both sera.

They showed that in many species specific

antibodies can be individually absorbed or that there
is a general bactericidal antibody which can be so modi­
fied by contact with a large excess of any particular
organism or strain as to render It specifically inactive
for that organism or strain.
Agglutination and Inhibiting Zone
The older hypothesis, suggested by Eisenberg and
Volk (1902) accepts Ehrlich1s conception of the agglu­
tinin as being made up of an antlbody-bacteria binding
portion (haptophore) and a flocculating portion
(zymophore), and assumes that by heating or aging, some
of the agglutinin is so modified (agglutinoid) that the

-7
clumping component Is destroyed without, however, affect­
ing the binding portions*

As a result, the agglutinoid

may still unite with the bacteria but does not produce
flocculation.

In order to explain the inhibition effect

in high serum concentrations, it is assumed that the ag­
glutinoid in these concentrations has a greater affinity
for bacteria and is therefore bound to them to the ex­
clusion of effective agglutinins*
The second hypothesis is put forward by Zln&ser
(1923) as follows:
Agglutinoid zones are analogous to zone phenomena
of other antibody reactions, notably the precipitin re­
action, and are definitely dependent upon quantitative
union between antigen and antibody and have nothing to
do with deterioration of antibody by heat or otherwise*
In various colloid precipitations in which serum is
involved, moderate heating of the serum will strongly
reduce its ability to precipitate a suspension.

When

normal serum is heated it is likely that there is a
change in Its colloidal state producing a certain amount
of colloidal protective property In the serum.

In re­

actions between bacteria and anti-serum, it Is likely
that the antibody carries Into union a not inconsiderable
amount of active serum constituents.

The so-Galled

specific action of the agglutinoid is probably due to
the fact that the antibody carries into union with the

-8bacteria inactive protein which is colloidally protective
by virtue of the heating.
Shibley (1984) summarized his results as follows:
1« Immune agglutinating serum possesses a specific
charge-reducing effect which is quantitatively related to
the agglutination titer of the serum.

This effect is

lost when the serum loses its agglutinating power; that
is, after adsorption of agglutinin by homologous bacteria.
Adsorption by heterologous organisms does not affect this
property.
8. A highly protective non-agglutinating serum did
not show this specific charge reducing effect.
A few years later he worked on the mechanism of the
agglutination of bacteria.

He concluded that the process

of sensitization by agglutinating serum consists of a se­
lective coating of the bacteria by the globulin of the
antibody.

This film formation causes the bacteria to

take on the characteristics of particles of denatured
globulin.

Subsequent agglutination of the coated bacteria

follows the laws governing the flocculation of particles
of denatured protein by electrolytes.
Shibley (1989) heated serum from 55°G. to 76°C. for
10 minutes, then made agglutination tests.

He found that

the inhibition zone (prozone) begins at 64°c.

The zone

then widens to reach a peak at 66®C. to 69°C.

Above this

the zone narrows and disappears at 7S°C.

Coincident with

-9this narrowing and. its loss, there is a corresponding
drop in the agglutinative titer.

At 76°C. all aggluti­

nation disappears.
He proposed the hypothesis that the inhibition zone
is caused by a modification of the agglutinin; (a) it
still retains its binding power although when union has
taken place the agglutinin-bacteria complex fails to
clump, and (b) it has a greater affinity for the bacteria
than the unchanged agglutinin.
How, when the heating level is further raised, it
will be seen that the inhibition zone is reduced and then
disappears.

He explains this on the assumption that this

higher heating further modifies the modified agglutinin
so that It now loses its binding power.
When serum was heated to 70°C. for 10 minutes and
titrated at pH 7, prozones were wider than those of lower
pH.

At pH 5.4 no prozones were present (serum heated at

74°C.)
With 16 billions of organisms per cc in the titrating
mixture, no prozone appeared.

Titrating serum antigen

mixture containing 8 billions of organisms gave some pro­
zone while tubes containing 2 billions of organism gave
wider prozone.

He also absorbed serum with 64 billions

organisms per cc and produced no prozone but absorbing
with 2 billions of organisms per cc gave a prozone.

-10
Eagle (1930) suggested that agglutinating and
precipitating antibodies are a specifically altered
fraction of the serum globulin.

The antigen-antibody

complex, whether it be sensitized red cells, agglutinated
bacteria or precipitate, formed by a soluble protein and
the corresponding antiserum, contains this antibody glo­
bulin, demonstrable chemically, immunologically and by
a change in the cataphoretic, flocculating, interfacial
and complement-fixing properties of the antigen towards
those of the protein with which It has combined.
In the case of the cellular antigen, this antibody
is present as an invisible film of specifically adsorbed
protein, while in the precipitation reaction, it may
constitute the bulk of the material formed.

In both

cases the originally hydrophilic globulin has become waterinsoluble (denatured) upon combination with antigen.
This change in properties is not a phenomenon peculiar
to the Immune reactions but is a commonly observed and
as yet unexplained property of adsorbed proteins, res­
ponsible for their sensitizing effect upon other-wise
stable colloidal suspensions.

It is suggested that in

the case of the immune reactions, this denaturation of
the antibody globulin is due to the fact that its speci­
ficity is determined by hydrophilic groups.

When these

combine with antigen, hydrophobic groups necessarily
face the water phase, determining the surface properties
of the antigen-antibody complex.

But when normal serum

-11protein is adsorbed, since there are no groups with a
specific affinity for the antigen, the molecules na­
turally orient themselves at the interface so that the
hydrophilic groups face the water, and the adsorbed
protein acts as a protective film*
There are therefore three factors which determine
specific flocculation:

(1) the hydrophilic antigen is

covered, with (2) a film of immune globulin, denatured
by its combination with antigen*

In the absence of

electrolytes the charge due to the ionization of this
protein suffices to prevent aggregation*

Minute con­

centrations of (3) electrolytes, however, depress this
surface charge below the critical value necessary for
stability*

The resultant aggregation is therefore

primarily of the immune globulin surfaces, and only
incidentally of the associated antigen*
With insufficient immune-serum, only a very small
portion of the cell surface is covered with antibody
globulin; most of the impacts are between hydrophilic
antigen surfaces, Ineffective in producing cohesion.
The more immune serum, the greater Is the proportion
of antigen surface covered with the sensitizing de­
natured protein, and the correspondingly greater the
proportion of effective impacts*
The optimum hydrogen ion concentration for floc­
culation is intermediate between that of the original

-12
cell and that of the antibody globulin, shifting towards
the latter as the degree of sensitization Is increased
(more extensive antibody film).

At the optimum reaction,

ionization takes place and therefore the surface charge
is minimal.

No added electrolytes are necessary to pro­

duce aggregation.

In a more acid or a more basic reaction,

the surface charge, due to the ionization of the adsorbed
protein, causes a mutual repulsion of the particles; but
traces of electrolytes depress this charge and allow the
cohesion of the denatured antibody films.

The floccula­

ting ion is always the one opposite in charge to the ion­
ized protein, and its flocculating efficiency increases
enormously with Increasing valence.

The further from the

isoelectric zone, the greater is the degree of ionization
and the more electrolytes are necessary to depress the
surface charge below the critical value.
Jones and Orcutt (1934) reported that when two agg­
lutination inhibitory sera specific for Brucella abortus
were added to a strong B. abortus agglutinin, agglutination
was inhibited and a prozone developed.

Bacteria not ag­

glutinated in the prozone serum can be centrifuged and
re suspended in the same mixture and remain in suspension.
When the original supernatant is replaced with salt so­
lution, agglutination usually occurs promptly although,
where the concentration of prozone serum is considerable,
an additional washing with saline solution may be required

-13to induce clumping.

They think the failure to agglu­

tinate may be attributed to the deposition on the sur­
face of the deposited globulin film of a substance
which reduces the cohesive properties of specifically
sensitized organism.
Duncan (1937) states that the salt optimum deter­
mines the maximum combination of antibody with antigen
and thus influences the quantity of agglutination mea­
sured by the highest effective serum titer.

Antibody-

antigen combination reaches its maximum at the salt
optimum and it diminishes progressively as the salt
concentration deviates in either direction from this
optimum.
Wiener and Herman (1939) believe that the precipitative and agglutinative reactions take place in two
separate stages: (a) a specific combination between the
antigen and its antibody and (b) a non-specific stage
of aggregation of the sensitized particles, in which
electrolytes play a role.
The work of Pauling, Campbell and Pressman (1934)
indicates that the forces responsible for combination
and attraction of antigen and antibody molecules may be
classified as electronic van der Waal*s attraction,
Coulomb attraction, attraction of electric dipoles or
multiples, formation of hydrogen bonds, etc.

The spe­

cificity of interaction of antigen antibody molecules

-14
arises from their structural complementariness, which
permits close contact of the molecules over sufficient
area for these weak forces to cooperate in forming a
strong antigen-antibody bond*
The weight of evidence indicates that further com­
bination of the initial antigen-antibody complexes to
form a precipitate is a specific rather than a non­
specific reaction and is due to a continuation of the
primary combination step to form a framework structure
of alternate antigen and antibody molecules.

Further­

more, it appears that both precipitating antigen and
precipitating antibody must be multivalent or at least
bivalent*
Wiener (1944) showed that the prozone phenomenon
may be due to the presence in sera of a mixture of
blocking and agglutinating antibodies.
Jenkins (1946) found that sensitized bacilli can
release antibody at high temperature and take up more
at low temperature.

The released antibody is a globulin

and is accompanied by another globulin which is not spe­
cific*

The released antibody presents some characteristics

which differentiate it from serum antibody*
Hayes (1947) favors the idea of the altered
(by heating) physical properties of the cell*

This al­

teration of the bacterial cell causes the colloid ad­
sorbed from the serum to protect the organism from
antibody action.

He classified the prozones into 3

-15
groups as follows:
serum*
heated*

Prozone "A", occurs with unheated

Prozone “B", occurs with sera which have been
Prozone “C", occurs when heated S* typhi 0

suspension is mixed with unheated serum.

On the basis

of the results of his experiment, he proceeded to ex­
plain such interference as follows:
1* A primary non-specific adsorption of “albumin
fraction1* by the cell will prevent the fixation of ag­
glutinin on the cell surface*
2* A secondary adsorption of “albumin fraction" by
the agglutinin-cell complexes which inhibits aggregation*
Blood Cells and Their Kesponse to Infection
Chandhuri (1927) found that the number of erythrocy­
tes in a unit volume of blood Is significantly higher in
the sexually normal adult male than in the normal adult
female of the fowl,
Hofmeister (1934) reported that leghorns with acute
infection showed a sharp rise in the number of pseudoeosinophlle leucocytes, reaching a peak in 1 to 3 days*
At the same time small lymphocytes decreased In numbers
and returned to normal within 8 days.

A moderate in­

crease in large lymphocytes occurred 2 to 4 days after
the increase in pseudo-eosinophils and returned to nor­
mal after 7 days.

The percentage of neutrophils

in

inoculated resistant birds was somewhat higher than in
non-inoculated resistant ones.

-16Biely, Jacob and Palmer (1935) found that the range
of red blood cell count of birds was 1,805,000 to
3,845,000 per cu.mm.

The mean red cell count of the male

was significantly higher than that of the female but
there was no significant difference between the mean
leucocyte counts of males and females.
At the World's Poultry Congress in 1939, Roberts,
Severens and Card reported that the number of lymphocytes
is greater in resistant than in susceptible fowls.
The work of Scholes (1942) Indicates that resistance
more probably depends upon temperature differences than
upon differences in the number of lymphocytes in the
blood.

-17Materials and Methods
Blood cell counts.

The method of Wiseman (from

Olson, 1935) was used for the routine study of turkey
blood.

The diluting fluid consisted of 50 mg. of

phloxine, 5 ml. of neutral formalin, and 95 ml. of
Ringer1s solution.

The ordinary red blood cell dilu­

ting pipette was used.

The blood was diluted 200 times.

The filled pipettes were allowed to stand for several
hours to allow the cells to take up the dye before the
count was made in the hemocytometer.

The ends of the

pipettes were closed by stretching a heavy rubber band
lengthwise around the pipette during the interval to
avoid loss of fluid.

The cells were counted in the

usual manner; that is, the erythrocytes In 80 of the
smallest squares were counted, and the result multi­
plied by 10,000 which represents the number of ery­
throcytes per cubic millimeter of blood.
The number of leucocytes was obtained by counting
the acidophilic granulocytes which were specifically
stained by phloxine in the entire ruled area of the
hemocytometer (9 mm^).
The differential count of leucocytes was made from
the stained blood smears.

Thin blood smears were

stained with Wright's stain.

A daily cell count was

made on normal and infected turkeys.

-18Freparation of culture.

A smooth strain of S.

pullorum (Isolated from a turkey) was used in this
experiment.

The organism was grown on nutrient agar

slants for 24 hours at 37°C.

The growth was removed

by means of a sterile wire loop and suspended in ste­
rile diluting fluid, which consisted of 0.05$ tryptos©
peptone and 0.5$ sodium chloride in distilled water.
Ten ml. of this diluting fluid was usually used for
each agar slant.

The bacterial suspension was trans­

ferred into a sterile test tube, then thoroughly mixed
and diluted to

turbidity Ho. 2 of McFarland's nephelo-

meter (For standard turbidity, refer to “Laboratory
Diagnostic Methods" by Kolmer).

Serial dilutions ran­

ging from 8x10-^ to 8x10^ were made from this suspen­
sion in the same diluting fluid.

The number of organisms

present was determined by plating 0.25 ml. or 0*1 ml.
of 10"*^ and 10“^ dilutions.
used.

The pour plate method was

The nutrient agar was melted, cooled to 45°C.

and poured Into the Fetri dish which was rotated to mix
the content well before the agar solidified.

When the

agar was solidified the plates were incubated for 3 to
4 days at 37° C. and then the colonies were counted.
The initial number of bacteria added to the blood plasma
or serum from dilutions can be calculated by multiplying
the number counted by the dilution factor.

-19Preparation of blood.

Each bird was tested for

§.• pullorum infection by using the stained antigen rapid

whole-blood test.
Blood for the bactericidal test was drawn aseptically from the wing vein of the bird and placed in a
sterile bottle.

For the tests requiring plasma, 0*1 ml.

of sterile saturated sodium citrate solution for each
10 ml. of blood was placed in the bottles*
The plasma was separated from the whole blood by
centrifuging at 1,800 r.p.m. for 25 minutes.

The super­

natant was poured off aseptically into another sterile
tube.

Serum was obtained by allowing the blood to clot

after which it was centrifuged if necessary.
All bactericidal tests were made within 24 hours
after collection of blood unless otherwise stated.
During this period the blood specimens were kept in an
ice box (4°C.).
Turkeys were immunized by injecting 1 ml., 2 ml.,
3 ml*, or 4 ml. of dead S. pullorum (8xl010 permit
suspended in 0.85$ saline intravenously.

Later, live

S* pullornm was injected into the same turkeys intrave­
nously.

Repeated injections were given until the turkeys

showed a high agglutination titer.
The bactericidal test.

In order to show the maxi­

mum bactericidal activity of serum or plasma on S. pullorum, two methods of setting up the tests were used:

-201. One ml. or 0.5 ml, of undiluted serum or plasma
was added to a series of tubes to which was added
the same volume of diluting fluid containing live
organisms in varying numbers.

2. Serial dilutions

of serum or plasma, in 1.0 ml. or 0.5 ml. amounts,
were placed in the sterile tubes to which were added
the same volume of diluting fluid containing a con­
stant number of live S. pullorum.

The tubes were

shaken and incubated at 37°C.; the length of time
varied with the experiment.

At the end of the period

of incubation, 0.25 ml. or 0.1 ml. of the mixture was
taken from each tube and pJa ced in a sterile Petri
dish.
10

Melted nutrient agar was cooled to 45°C. and

ml. amounts were then poured into each Petri dish.

The contents were mixed by rotation and then allowed
to harden after which the plates were incubated at
37°C. for 3 days.

Colony counts were made and compared

with those of the control tubes.

The same procedure

was repeated at the end of 4 hours, 8 hours, 24 hours,
and 48 hours.
Table No. 1 shows how the dilutions were prepared
and the amounts of S. pullorum suspension used.
The S. pullorum antigen was made by growing the
organisms in large bottles.

After several days of

growth in nutrient agar at 37°C.
moved with 1% phenol water.
by centrifugation.

the organism was re­

The bacteria were washed

The supernatant was poured off and

-21-

the organisms resuspended in diluent containing 0.25$
phenol.

The bacterial suspension was diluted to tur­

bidity No. 1 of McFarland1s nephelometer.

This bac­

terial antigen was used for agglutination tests.

The

pH of this antigen was adjusted with a Beckman pH
meter.
A serial dilution for the agglutination test was
set up as follows:

1.9 ml. of the S. pullorum antigen

was placed in tube No. 1 and 1.0 ml. in all the follo­
wing 10 tubes, then 0.1 ml. of plasma was placed in
the first tube.

The content was mixed well and 1.0

ml. was transferred into the second tube, etc.
Table No. 2 for agglutination dilutions.

See

-2 2 -

Resuits and Discussion
All conditions must be taken into consideration
in order to obtain a true blood picture.

Factors such

as species, age, sex, temperature, food, and various
abnormal conditions have direct effect on blood compo­
sition and cell count.

So, in order to obtain accu­

rate results, all turkeys should be of the same age,
sex, etc.

After taking all these important factors

into consideration, a daily cell count was made on
all turkeys to be used in these experiments.

It was

found that the average cell counts of 18 three-month
old normal turkeys was as follows:
Total leukocyte count

- 12,000 per cubic mm.

Total erythrocyte count

-2,600,000 per cubic mm.

Total heterophil

count

- 43$ of the

total leucocyte

Total lymphocyte

count

- 51$ of the

total leucocyte

Total eosinophil count

- 1$

of the

total leucocyte

Total basophil count

- 2$

of the

total leucocyte

Total monocyte count

- 3$ of the total leucocyte

This agrees quite closely with the differential leu­
kocyte count of Johnson and Lange (1939).
After a sufficient number of blood cell counts
were made on normal turkeys, one half of these birds
were infected with S. pullorum and daily cell counts
were continued.

It was found that 24 hours after in­

-25jection of* S, pullorum there was a sudden increase of*
heterophils which remained at a high level for about
three days, then began to drop quite rapidly.

The

percent increase of heterophils apparently depends on
the amount of bacterial suspension used and the suscep­
tibility of the turkey.

The heterophil count usually

returned to normal about six days after the administra­
tion of S, pullorum.

The lymphocytes in this experi­

ment, however, showed only a slight although rather
irregular increase in most cases.

The differential

counts showed more young, large lymphocytes after the
rise of the agglutinating titer.

This may indicate a

relationship to antibody formation.

The eosinophils,

basophils and monocytes were found to be increased
somewhat, but not to any significant degree in this
case.

The erythrocyte count decreased somewhat after

the administering of S. pullorum.
The total leucocyte count usually increased gra­
dually after each successive oral administration of
organisms as shown in Figures 2, 5 and 4, whereas there
was a very sudden increase in total leucocyte count
after the first intravenous injection of organisms.
There was less variability following the later injec­
tions as shown in Figures 6 and 7,

It stands to rea­

son that infections occur more readily as a result of
intravenous injection.

The birds which were Infected

-24orally generally showed low agglutinating titers whereas
those which were Infected intravenously showed very high
agglutinating titers when the same dosage of organisms
was used.
Bactericidal tests.

The results obtained in this

experiment indicate that the bactericidal action of
plasma is somewhat greater than that of the serum.
why that is the case is not yet known.

Just

However, plasma

may have more antibody present than the serum.

It is

possible that during the process of coagulation some of
the antibody may have been removed with the fibrin,
thus causing a decrease in bactericidal action.

Plasma

is more like normal blood than serum and reacts more ef­
fectively.

Therefore, all subsequent tests were made

with plasma.
Tables 3 and 4 show that the serum and plasma of
different normal turkeys vary with respect to bacteri­
cidal power.

Tables 5 and 6 show that the plasma is

more bactericidal than the serum.

Plasma from the in­

fected turkeys also have greater bactericidal action than
the serum of the infected turkeys (Tables 7 and 8)•

Plas-

mae from the orally infected turkeys (Tables 9, 12, 14, 15,
and 16) were more bactericidal than those of Intravenous­
ly infected birds.

The normal plasma was the best of

the three when compared with the plasma of low aggluti­
nating titer.

When the agglutinating titer was sufficient­

ly high, plasma of the orally infected turkeys showed

-25greater bactericidal action than the normal plasma.
Tables 17 and 18 show that the plasma of the turkeys
which had been injected with dead S. pullorum antigen
possessed better bactericidal action than the plasma
of the birds which had been injected with living S.
pullorum antigen.
Plasma lost its bactericidal property after ab­
sorption with S. pullorum antigen.

The amount of

bactericidal action lost depends on the amount of
bacterial antigen used for the absorption.

Plasma

lost its bactericidal action also when a Salmonella
choleraesuis antigen was used for absorption which
shows non-specificity of bactericidal action.

The

results were obtained through many repeated experi­
ments.
When plasma was diluted four times, the immune
plasma showed better bactericidal action than the
normal plasma.

After 16 hours of incubation, the

mixture of plasma and bacteria was pipetted out and
plated in nutrient agar.

The normal plasma showed

numerous colonies in 1:16 dilutions,whereas the immune
plasma showed numerous colonies in 1:64 dilutions*
These two plasmae showed little or no growth of bac­
terial colonies at lower dilutions which indicates
that the plasma is bactericidal or bacteristatic at
the above dilutions.

-26In the presence of excess complement, normal plas­
ma showed numerous colonies in 1:52 plasma (saline) di­
lutions whereas there were about the same number of
colonies in the 1:128 immune plasma dilutions.

This

indicates that the bactericidal action increases some
when sufficient complement is present.
An excess amount of guinea pig complement (0.1 ml)
was added to each tube to see what influence it would
have on bactericidal action.

Table 19 shows that the

bactericidal action improved slightly in the unheated
normal and immune plasma, but it (guinea pig complement)
had absolutely no influence on the plasma which had been
heated at 56°C. for one hour.

Heating at 56°C. may have

destroyed the antibodies as well as the complement or
guinea pig complement may not be an adequate substitute
for turkey complement.
Agglutination tests.

Ordinarily agglutination

titration mixtures of plasma and antigen, incubated
at 57°C., showed no prozones except sometimes with
old plasma.

No noticeable difference in agglutination

or titer was observed when the pH ranged from 6 to 8.5.
However, when incubated at 56°G., all the agglutinations
at three different pH levels showed prozones in the
first three dilutions and showed less Inhibitory zone
agglutination at a lower pH than at a higher pH.

-27When the immune plasma was heated at 56°C. for
one hour, the precipitate filtered off, and the ag­
glutination test was run at 56°C., the test showed
no inhibitory zone at all (Table 20).
When the plasma was filtered through No. 05
Selas filters immediately after the blood was drawn
from the birds, it showed an inhibitory zone as had the
untreated plasma.

If this plasma was allowed to stand

for several days in the ice box and was then refiltered
through a filter of the same porosity, the plasma
showed little or no inhibitory zone.

When plasma was

frozen at -55°C. for two days and the sediment re­
moved after thawing, it showed very little inhibitory
zone .
When 0.1 ml. of fresh pullorum negative plasma was
added to the immune plasma (0.1 ml.) which had been
heated at 56°C. for one hour and filtered, it showed
no inhibitory zone; but if the above immune plasma was
mixed with 0.1 ml of old pullorum negative plasma, It
showed some Inhibitory zone (Table 21).
Table 22, shows results of absorption of plasma
with S. pullorum and 3. choleraesuis antigen.

Both

showed inhibitory zones before absorption but none
afterward and both showed decrease in agglutinating
titer after absorption.

In one instance, plasma

-28which had been kept in an ice box (4°G.) for 6 days, and
was then filtered, and subjected to an agglutinating test,
showed no inhibitory zone.
All the results mentioned indicate that the inhibi­
tory substance is In the colloidal state and that it
can be removed from the plasma by various physical treat­
ments such as aging, freezing, heating, centrifugation,
and filtration.

The plasma must be altered in some

way before the inhibiting colloidal substance can be
formed.

Before going into further discussion, let us

review the work of Shibley.

He said that raising the

temperature increases the quantity of agglutinin changed
into agglutinoid.

Now, if this is true, why is there no

change of agglutinating titer after heating?

In my experi­

ments, the plasma which was heated at 56°C. for one hour
showed a large amount of sediment after heating.

When

the sediment was removed it showed no decrease in agglu­
tinating titer, which indicates that none of the agglutinin
was lost after heating at 56°G. for one hour.

In fact,

in most cases it showed a stronger agglutination than the
unheated plasma.

So it is unlikely that It is due to the

alteration of agglutinin.

Another fact which shows that

there is no alteration in the agglutinin is that the
filtered heated plasma which gives no prozone at 56°C.
incubation, will give prozone when old, normal plasma
is added to it.

We know that there is no pullorum

-29agglutinating antibody in normal plasma, yet the old,
altered pullorum negative plasma causes prozone when
added to the pullorum positive plasma which had been
heated at 56°C. for one hour and centrifuged (Table 21).
This suggests that a non-specific colloidal substance
interferes with the agglutination.

When plasma was

raised to a higher pH, (8.5) a wider range of orozone
was produced.
was present.

At lower pH (5.4) little or no prozone
The above may have something to do with the

iso-electric point of protein.

Since pH 5.4 is almost

the iso-electric point at which most of the globulin may
be precipitated this pH may cause the flocculation of
the colloidal particles, and thereby remove the substance
which interferes with agglutination.

The prozone can also

be removed when a sufficient quantity of bacterial anti­
gen may absorb most of the colloidal particles; therefore,
no prozone will occur in the absorbed plasma (Jamil and
Stafseth 1949).

The above results are unlikely to be due

to degraded agglutinin, since prozone "A,f occurs without
heating serum.
Plasma from infected birds shows larger colloidal
particles present more often than the plasma from normal
birds, and it has been found that the rate of sedimenta­
tion of infected blood is greater than that of nonnal
blood.

The cause of this phenomenon is not clear.

It

is apparently connected with the ratio of albumin, glo-

-30bulin, and fibrinogen in the plasma, which influences
the formation of larger colloidal particles and the
rate of sedimentation; or there may be some degree of
protein alteration In the infected blood which causes
the protein to become precipitated more readily than in
normal blood.

This may lead to an explanation of the

reasons why diseased blood of pullorum Infected turkeys
is altered more easily than normal blood, as shown by
the formation of larger colloidal particles In plasma
after removal from the blood stream*

Very old plasma

which shows heavy precipitate does not give a prozon©
when the precipitate is filtered off before titrating
agglutinating sera.

It is usually the intermediate

alteration of plasma that causes a prozone (Table 23).
It appears that a certain degree of alteration of
plasma protein produces the proper size of colloidal
particles which are the cause of prozones.

When the

colloidal particles have gone beyond this size, the
Inhibitory effect Is lost.

It is possible that the

very old plasma loses its inhibitory effect because
the plasma-protein has been aggregated into larger
particles which no longer can be adsorbed on the sur­
face of the bacteria cell, and therefore, does not
prevent the adsorption of agglutinating antibody.
The lower bactericidal action of the undiluted
Infected blood plasma may also be due to the

-51col loi dal particles which are adsorbed on the surface
of the bacterial cell preventing absorption of anti­
body.

The fact that diluted infected blood plasma shows

greater bactericidal action than normal diluted plasma,
indicates the possibility that the diluted plasma does
not have a sufficient quantity of colloidal particles to
prevent the absorption of antibodies.

Plasma from the

orally infected turkeys with a high agglutinating titer
showed greater bactericidal property than plasma of the
intravenously infected turkeys.

This may indicate that

little or no protein alteration took place in the former
whereas more protein alteration occurred in the latter or
it may have to do with the contacts which the antigen
makes with immunologically active tissues in the intestinal
wall (Tables 10 and 12).
The results of this experiment indicate that plasma
is apt to deteriorate after it has been removed from the
circulatory system.

During the process of deterioration,

certain inhibitory colloidal particles are formed.

These

colloidal particles when adsorbed on the surface of bac­
terial cells, interfere with the absorption of antibody,
thereby producing prozone.

Since such colloidal protein

particles appear to be formed by the alteration (denaturation) of protein after it has been drawn from the blood
stream, the bactericidal action of the infected turkey
plasma may be better in vivo than in vitro.

-32Summary
1. The average number of blood cells per cubic mm. of
blood In normal turkeys was: 2,600,000 erythrocytes
and 12,000 leucocytes.

The percentage of the various

types of leucocytes was:
heterophils

43$

lymphocytes

51$

eosinophils

1$

basophils

2$

monocytes

3$

2. The erythrocytes decreased after oral or Intravenous
administration of S. pullorum.
3. There was a sharp rise in heterophils after intra­
venous administration of S. pullorum.
4. Lymphocytes did not Increase much during the first
part of the infection, but gradually increased
during the later stages of the infection.
5. The agglutinating titer rose before there was any
significant Increase in lymphocytes.
6. Plasma showed greater bactericidal property than
serum.
7. Normal undiluted plasma showed greater bactericidal
property than plasma from infected turkeys.

-358. Normal plasma diluted to

1:16 showed less bacteri­

cidal action than plasma from the infected turkeys
in the same dilution.
9. Plasma

from the orally infected turkeys showed

greater bactericidal power than plasma from the
Intravenously Infected ones.
10. Plasma with a high agglutinating titer showed the
best bactericidal action.
11. The bactericidal property was destroyed by heating
at 56°C. for one hour.
12. When the immune plasma was heated at 56°C. for one
hour, the precipitate filtered off, and the agglu­
tination test showed no inhibitory zone.

Freezing

and aging the immune plasma will remove the factor
which causes prozones.
13. Old, deteriorated normal plasma added to the prozonefree plasma caused prozone which suggests that the
prozone is due to non-specific colloidal particles.

-34BIBLIOGRAPHY
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-3 5 -

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-36—
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Table 1
Dilutions of Salmonella pullorum
Suspension UsecT in this Experiment

8

Tubes
Saline

9 ml

Pullorum
Suspen­
sion

1 ml

9 ml

9 ml

9 ml

9 ml

9 ml

9 ml 9 ml 9 ml

A m t . of

Sal.pul.
Susp.

'?=“

1 m l — >•

—■-'Jss.

Transfer­
red

Dilutions l o -1

io

~2

io

-3

io

~4

io

“5

i o -6 l o

-7

ic r 8 io

~9

Table 2

Agglutination Test Mixtures

Dilutions
;-Tubes
Dilutions

10

8

1
£0

1
TO

1

1

1

1 : 1

1

1

Antigen
added

1 .9ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml

Plasma
added

0.1ml

Mixture
trans­
ferred

1

TO! TTO 3TO BTOjlSTO.'TOTOjSITO .KSTO

1 ml 1 ml II ml ll ml 1 mljl ml 1 ml 1 ml
1-

The Salmonella pullorum antigen was adjusted to the
desired plT, (8.4) 1.9 ml. of the antigen was placed in
tube No. 1 and 1 ml in the following tubes; 0.1 ml of
the testing plasma was pipetted into tube No.l and mixed
well; then 1 ml was transferred into tube No. 2 etc*

1 ml

1 ml|

Table 3

The Bactericidal Activities of 2 Normal Sera
Serum 1
Normal
serum
dilution
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2

Number of
bacteria
added
7
7
7
7
7
7
7
7
7

x
x
x
x
x
x
x
x
x

109
108
10*7
10®
10®
104
103
10~
101

Colony Count
Incubation period in hours
4
8
24
48
N
N
698
84
16
9
2
4
0

N
338
144
21
7
2
0
0
0

29
2
1
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0

31
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0

Serum 2
1
1
1
1
1
1
1
1
1

2
2
2
2
2
2
2
2
2

7
7
7
7
7
7
7
7
7

10^

X IO9
X
X
10l
X 10 8
X 10®
X 10t
X 103
X 10^
X IO1

N
N
N- 231
35
428
6
27
3
18
0
7
0
0
0
0
0
0

N - too numerous to count

Table 4

The Bactericidal Activities of 2 Normal Plasma©
Plasma 1
Normal
plasma
dilution
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2

Number of
bacteria
added
8
8
8
8
8
8
8
8

x
x
x
x
x
x
x
x

109
10®
107
10®
10®
104
10®
102

Colony Count
Incubation period in hours
24
4
8
48
N
N
N
N
634
131
36
15

N
N
N
N491
110
25
3

N
N
1000
493
7
1
0
0

N
N
201
29
0
0
0
0

Plasma 2
Normal
plasma
dilution
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2

Number of
bacteria
added
8
8
8
8
8
8
8
8

x
x
x
x
x
x
x
x

N -

109
10®
10'
10®
lOf
104
10®
102

Colony Count
Incubation period in hours
24
4
8
48
N
N
N
N630
138
38
12

N
N
N
1144
358
93
18
4

Too numerous to count

N
N
952
247
49
6
0
0

N
N
338
35
3
0
0
0

Table 5
Rate of Bactericidal Action of Serum and Plasma

Normal
Serum
Dilution
1
1
1
1
1
1
1
1

2
2
2
2
2
2
2
2

Normal
Plasma
Dilution
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2

Number of
Bacteria
added
15
15
15
15
15
15
15
15

X
X
X
X
X
X
X
X

10®
10®
10l

10®
101

104
10®
102

Number of
Bacteria
added
15
15
15
15
15
15
15
15

x
x
x
x
x
x
x
x

102
10®
107
10®
10®
104
10~
102

4
N
N
N
1480
265
73
30
19

Colony Count
Incubation period in hours
8
24
48
N
N
N
619
153
41
6
6

N
208
35
2
0
0
0
0

N
N
0
0
0
0
0
0

Colony count
Incubation period in hours
24
48
4
8
N
1110
21
3
0
1
0
0

N
26
7
0
0
0
0
0

18
12
2
0
0
0
0
0

0
0
0
0
0
0
0
0

Table 6
Rate of Bactericidal Action of Serum and Plasma

Normal
serum
dilution
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2

Normal
plasma
dilution
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2

Number of
bacteria
added
15
15
15
15
15
15
15
15

x
x
x
x
x
x
x
x

10®
10®
101

10®
10"
10’
10"
10^

Number of
bacteria
added
15
15
15
15
15
15
15
15

x
x
x
x
x
x
x
x

109
10®
10?
10°
10°
10’
10®
10

Colony Count
Incubation period in hours
4
6
24
48
N
N
684
93
9
2
3
1

782
446
64
8
1
0
0
0

19
1
0
0
0
0
0
0

1
0
0
0
0
0
0
0

Colony Count
Incubation period in hours
24
48
8
4
N
1254
322
39
5
1
1
1

414
47
19
2
0
0
0
0

18
3
0
2
0
0
0
0

4
0
0
0
0
0
0
0

Table 7
Hate of* Bactericidal Action of Serum and Plasma
From Infected Turkey

Infected
serum
dilution
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2

Infected
plasma
dilution
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2

Number of
bacteria
added
7
7
7
7
7
7
7
7

x
x
x
x
x
x
x
x

10*
10®
10^
10®
10~
10’
10£
102

Number of
bacteria
added
7
7
7
7
7
7
7
7

X
x
x
x
x
x
x
x

109
10s
10?
10°
10°
10*
10°
102

Colony Count
Incubation period in hours
24
48
8
4
N
N
N
1522
164
76
17
18

N
N
N
480
182
27
20
21

N
N
N223
47
2
1
1

N
181
4
0
0
0
0
0

Colony Count
Incubation period in hours
24
8
48
4
N
N
N
890
62
34
13
8

N
N
N
464
75
14
9
2

N - too numerous to count

N
N
N219
5
1
0
0

N
N
682
6
2
1
0
0

Table 8
Rate of Bactericidal Action of Serum and Plasma
Prom Infected Turkey

Infected
serum
dilution
1:2
1:2
1:2
1;2
1:2
1:2
1:2
1:2
1:2

Infected
pi asma
dilution
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2

Number of
bacteria
added
6
6
6
6
6
6
6
6
6

x
x
x
x
x
x
x
x
x

109
10®
1C>X
10°
10°
lOf
10°
10®
101

Number of
bacteria
added
6
6
6
6
6
6
6
6
6

x
x
x
x
x
x
x
x
x

10^
10®
10?
10®
log
log
lOg
lOf
101

Colony Count
Incubation period in hours
4
8
24
48
N
1750
207
29
2
1
1
0
0

682
584
39
4
0
0
0
0
0

8
3
0
0
0
0
0
0
0

1
0
0
0
0
0
0
0
0

Colony Count
Incubation period in hours
24
4
8
48
N
1044
44
7
2
0
0
0
0

336
57
3
1
0
0
0
0
0

0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0

Table 9
Hate of Bactericidal Action of Plasma
Orally Infected Turkey

No. of
bacteria
added
Plasma
diluted
to 1:2

8
8
8
8
8
8
8

x
x
x
x
x
x
x

10^
10°
lo£
10b
10$
10*
10°

Colony Count
Period of incubation in hours
4
8
24
48
N
N
N1221
304
105
50

N
N
N
305
84
0
0

912
121
59
0
0
0
0

49
9
3
1
0
0
0

Intravenously Infected Turkey

Number of
bacteria
added
Plasma
diluted
to 1:2

8
8
8
8
8
8
8

x
x
x
x
x
x
x

10^
10°
10^
10°
10°
10^
10°

Colony count
Period of incubation. In hours
4
8
24
48
N
N
N
1339
435
176
117

N
N
N
422
109
48
39

N
382
52
8
0
0
0

N
23
1
1
2
0
0

Table 10
Rate of Bactericidal Action of Plasma
Orally Infected Turkey

Number of
bacteria
added
Plasma
diluted
to 1; 2

8
8
8
8
8
8
8

x
x
x
x
x
x
x

10?
10°
10l

10°
10°
10^
10°

Colony Count
Period of incubation in hours
4
8
24
48
N
N
N
278
58
15
4

N1868
411
102
9
0
0

524
12
5
2
0
0
0

14
2
1
0
0
0
0

Intravenously Infected Turkey

Number of
bacteria
added
Plasma
diluted
to 1:2

8
8
8
8
8
8
8

x
x
x
x
x
x
x

10®
10°
io;
lot
10A
10*
10°

Colony count
Period of incubation in hours
4
8
24
48
N
N
N
1460
336
86
57

N
N
N
1208
254
54
48

N
N
N
N
N
N
N

N
N
N
N
N
N
N

Table 11
Hate of Bactericidal Action of Plasma
Both Turkeys Were Infected Intravenously

Plasma
diluted
to 1:2

Humber of
bacteria
added

Colony Count
Period of incubation in hours
4
8
24
48

8
8
8
8
8

N
H
N
N
N- 1360
332
281
108
42

x
x
x
x
x

10^
10°
10*
10°
10°

Agglutinating Titer

Plasma
diluted
to 1:2

-

258
107
15
0
0

1/640

Number of
bacteria
added

Colony Count
Period of incubation in hours
4
8
24
48

8
8
8
8
8

N
N
N437
120

x
x
x
x
x

10^
10°
loZ

10,10°

Agglutinating Titer =

H
H
1400
310
50
1/320

N
N
1380
36
0

Table 12
Rate of Bactericidal Action of Plasma
Orally Infected Turkey

Humber of
bacteria
added
Plasma
dilution
1:2

8
8

X
10a
X 108
8 X
10l
8 X 10%
8 X 105
8 X 10%
8 X lc r

Colony Count
Period of incubation in hours
4
8
24
48
H
N
H
820
288
21
11

H
H
761
51
22
3

0

242
11
0
0
0
0
0

Intravenously Infected Turkey

Humber of
bacteria
added
Plasma
dilution
1:2

8
8
8
8
8
8
8

X 1°9
X 10®
X
X
10t
X 10®
X 104
X 103

10I

Colony Count
Period of incubation in hours
4
8
24
48
H
N
H
H
1064
373
73

H
H
H
221
141
40
4

960
74
21
22
0
0
0

Table 13
Rate of Bactericidal Action of Plasma
Plasma of an orally infected turkey

Plasma
dilution
1:2

Number of
bacteria
added

Colony Count
Period of incubation in hours
4
8
24
48

8
8
8
8
8
8
8

N 1190
N- 824
504
95
7
11
4
0
0
2
0
1

X
x
x
x
x
x
x

10Q
10®
10?
108
10®
104
105

13
3
1
0
0
0
0

Plasma of an intravenously infected turkey

Plasma
dilution
1:2

Number of
bacteria
added

Colony Count
Period. of incubation in hours
8
24
48
4

8
8
8
8
8
8
8

N
N
N
760
213
51
18

x
x
x
x
x
x
x

10^
108
10?
10°
10®
104
103

N
N
N
712
161
54
13

N
N
N
N
N
N
N

Table 14
Rate of Bactericidal Action of Plasma
Plasma of an orally infected turkey with a high
agglutinating titer.

Reacting
substance

Number of
bacteria
added

Colony Count
Period of incubation in hours
4
8
24
48

Plasma
dilution
1:2

9
9
9
9
9
9
9

N
N
N
N420
106
67

x
x
x
x
x
x
x

109
log
107
10®
10®
10^
10®

N
N
814
92
35
12
3

N
N536
11
0
0
0

N
95
1
0
0
0
0

Plasma of an intravenously infected turkey.

Plasma
dilution
1:2

9
9
9
9
9
9
9

X lo g
X 10
X IQ7
X 10%
X
10A
X i°i
X 105

N
N
N
N
N344
140

N
N
N
N442
162
92

N
N
N1426
256
112
52

N
N
776
115
55
11
8

Plasma of a normal turkey •

Plasma
dilution
1:2

9
9
9
9
9
9
9

X
X
X
X
X
X
X

109
1°8
10l

X°!
10&
10«
10s

N
N
N
N
822
235
111

N
N
N
N
N
914
141

N
N
N
N
N
N
N

N
N
N
N
N
N
N

Table 15
Rate of* Bactericidal Action of* Plasma
Plasma from an orally infected turkey with a high
agglutinating t i t e r . ___________________
Reacting
substance

Number of
bacteria
added

Plasma
dilution
1:2

6
6
6
6
6
6
6

x
x
x
x
x
x
x

10®
10®
lo'
10*
10®
104
105

Colony Count
Period of incubation in hours
4
8
24
48
N
N
N
N382
66
20

N
N
N
472
101
5
0

N
241
3
0
0
0
0

Plasma from an intravenously infected turkey with a
high agglutinating titer.

Reacting
substance

Number of
bacteria
added

Plasma
dilution
1:2

6
6
6
6
6
6

x
x
x
x
x
x

10®
10®
10'
10®
10?
104

Colony Count
Period of incubation in hours
4
8
24
48
N
N
N
N528
61

N
N
N
451
301
6

N
N
N
360
61
8

Pi asm a from a normal turkey

Reacting
substance

Number of
bacteria
added

Plasma
dilution
1:2

6
6
6
6
6
6

x
x
x
x
x
x

10®
10®
10'
10®
10®
104

Colony Count
Period of incubation in hours
8
24
48
4
N
N
N
N
360
70

N
N
N
N
801
147

N
N
N
N
N
N

Table 16
Rate of Bactericidal Action of Plasma
Plasma from an orally infected turkey with a high
agglutinating titer.

Reacting
substance

Number of
bacteria
added

Plasma
dilution
1:2

6
6
6
6
6
6
6

x
x
x
x
x
x
x

10®
10°
10^
10^
lof
lof
10°

Colony Count
Period of incubation in hours
4
8
24
48
N
1101
295
47
6
3
1

N
1022
101
21
5
0
0

N
242
2
0
0
0
0

Plasma from an intravenously infected turkey with a
high agglutinating titer.

Reacting
substance

Number of
bacteria
added

Plasma
dilution
1:2

6
6
6
6
6
6
6

x
x
x
x
x
x
x

1°9
10®
107
10f
1°5
104
103

Colony Count
Period of incubation in hours
4
8
24
48
N
N
N
1110
207
63
5

N
N
310
550
102
7
1

N
N6
1
0
0
0

Plasma from a normal turkey

Reacting
substance

Number of
bacteria
added

Plasma
dilution
1:2

6
6
6
6
6
6
6

x
x
x
x
x
x
x

109
102
10
10c
10°
104
10^

Colony Count
Period of incubation in hours
4
8
24
48
N
N
N
891
431
71
9

N
N
N
N
1120
220
26

N
N
N
N
N
N
N

Table 17
Rate of Bactericidal Action of Plasma

Tube
No.

Dilutionl Number of
bacteria
of
plasma
added

1
2
3
4
5

1 s2
1:2
1:2
1:2
1:2

15
15
15
15
15

x
x
x
x
x

10*?
10^
10J
lOg
10^

Colony count
4 hour incubation
IDA
I
IA
ID
N
N
175
33
7

N
N
N
869
123

N
N
N
675
87

N
N
N
1007
102

N*
N
N
92
19
2

20 hour incubation
1:2
1:2
1:2
1:2
1:2

1

2
3
4
5

15
15
15
15
15

X
X
X
X
X

10K
10&
104
iof
102

10

4
3
2
2

N
NN 226
N
25
N- 13
215
2

N
N
N
188
115

9
6
4
0
0

ID - Plasma of a turkey immunized with dead
S. pullorum.
IDA Plasma of "ID*1 adsorbed with 3, pullorum.
I s
Plasma of
a turkey immunized with live S. pullorum.
IA *
Plasma of
"I* adsorbed with 3* pullorum.
N*«

Plasma of

a normal turkey.

Table 18
Rate of* Bactericidal Action of Plasma
5 hour incubation
Tube
No.

Dilution
of
plasma

1

1:2
1:2
1:2
1:2
1:2
1:2

2

3
4

5
6

Number of
bacteria
added

N«

7
7
7
7
7
7

N
N
N6
5
0

X
X
X
X
X
X

10l

10®
10®
101
ioi

102

Colony Count
ID
N
N
N
840
120
16

I
N
N
N
N
N
521

24 hour incubation
1
2
3
4
5
6

1:2
1:2
1:2
1:2
1:2
1:2
N1
ID
I

-

7
7
7
7
7
7

X
X
X
X
X
X

10o
10*>
10s
10*5
102

170
0
0
0
0
0

N
140
25
0

0
0

N
N
102
95
3
0

Normal plasma
Plasma of the infected turkey immunized
with dead S. pullorum organisms.
= Plasma of the infected turkey immunized
with live S. pullorum organisms.

Table 19
Rate of Bactericidal Action of Plasma

Tube
No.

1
2
3
4
5
6
7
8

Di lu ti on
of
plasma
1
1
1
1
1
1
1
1

4
8
16
32
64
128
256
512

Number of
bacteria
added
7
7
7
7
7
7
7
7

X 10^
X icr
X io 'i
X 10?
X 10?
X 10?
X 10?
X 106

N*
74
790
N
N
N
N
N
N

I

N fC
16
64
360
N
N
N
N
N

12
95
186
N
N
N
N
N

IC
2
12
45
120
1500
N
N
N

NHC

3HC

N
N
N
N
N
N
N
N

N
N
N
N
N
N
N
N

16 hour incubation
1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512

1

2
3
4
5
6
7
8
N«
X
N*C
IC

“

N'HC

=

IHC

=

-

7
7
7
7
7
7
7
7

X
X
X
X
X
X
X
X

10^
10?
106

io3
103
103
103
103

0
154
N
N
N
N
N
N

0
0
4
998
N
N
N
N

0
3
320
N
N
N
N
N

0
0
0
0
701
N
N
N

N
N
N
N
N
N
N
N

Normal plasma
Plasma of the infected turkey

Normal plasma with excess complementadded
Plasma of the infected turkey withexcess com*
plement added.
Normal plasma heated at 56 C. for one.hour.
with excess complement added.
Plasma of the infected turkey heated to 56 C.
for 1 hour with excess complement added.

N
N
N
N
N
N
N
N

Table 20
Agglutination Titrations

Dilutions
;
-

A

lncuba+
tion : 1
1
1 1 1 1
1
1
1
1
1
temp. 20 ’ 35 ’ 80 160 520 64o 1280 2560 5i£o 10240,
0 !
6 37 C • ++* +++ ++ + +++ + ++ + ++ +++ + 4
+ [

B

7

pH
■

C 8.5

+++

+++

+4

+4

+

37 °C. ++± ++ + + + + +++ ++ + +++

+44

++

++

4

37°G• + +* +++ +++ +++

D

6

56°C. ' +i

++

+ + ++* + 4+ +++

++4 ++4

++

4

E

7

56°0.

+i

++ f*++ 4 4+ + 44

+ ++ 4 + f

++

4

++ + +* + ++ +++

4+4 + +3f

++

f

F 8,5

t

56 °C. !
i +

G

6

56°C. *+<• +++ + + + + 4+ 4+ + + ++

+ ++

+4

H

6

56°C. :

+ ++ + + ++ +4+ +++ + + +

++ +

++

.

56 °C. ■»+* ++i + ++ 4++ ++ + +++

++ +

4+

4
+±

+
4

A )
B ) = Plasma of different pH with 37°C. incubation
C )
E ) - Plasma of different pH with 56°C. incubation

F )
G
H
I

= Plasma heated at 56°C. for 1 hour then filtered
before running the agglutination test.
= Plasma heated as in 11G" with 0.1 ml. of egg
albumin added to it.
= Plasma heated as in “G11 with 0.1 ml. of normal
plasma added to it.

Table 21
Agglutination Titrations

Dilutions

U
V

00

w

X

•

• • •
00 €0 00

1
1Incubation
pH ! temp*
| 1
20
L.
f
56 °C.
i:
56°C.

i
40

i
—

1

1
1 1
1
1
1
1
1
80 160 320 640 1280 2S60 5120 10240
+++ +++

¥

++± + *¥

56°G •
56 °C •

|

4+*fj

+ ++

¥ +¥

++

+

¥+y

++

f

+++± f ++ + ++ + +¥: +*+ + +*
i
;

ff

++

yy-f

•“

!
i
1
1

1

I

±
1

**"¥

¥ ++

++

¥

-

i

i

U

«

V

=

W

=

X

=

Plasma filtered through Selas filter #03 immediately
after blood was drawn from turkey•
Plasma which was refiltered through S e l a s filter #03
after it has been standing in ice box for 2 days*
Plasma heated at 56°C* for an hour, centrifuged and
the supernatant,used.
The supernatant of "W11 plus 0*1 ml* of old stored
normal plasma*

Table 22
Agglutination Titrations

Dilut ions
-----1
1

Incuba­

1
1
20 " ¥ o “
+±

CD

tion
temp.

H O

PH

i
1
1
1
1
l
160 520 640 128b 25$0 5120 10240

1
p

8.4

37 °C.

Q

8.4

37 °C.

R

8.4

37 °C.

S

8.4 i 37 °C.
8.4 37 °C.

T

+++
;++f± + ++

++ +

+++

**

+*

«•»

+ + * + ++
■f*+

++t

f

«•

-M-** ***-1 ***

+++

+1

+

+ *+

+ 4 + +++ + ■*+

1

f

f

P -

Plasma untreated*

Q r

Plasma absorbed with S. pullorum antigen,

R -

Plasma absorbed with S. choleraesuis

S =

Plasma left standing in ice box (4°C) for 6
days before tested.

T =

+

Fresh plasma not stored.

antigen.

Table 23

Agglutination Titrations

Tempera* 1
ture
20

J

•

CD

56 °C.

±

K

8.4

56 °C.

444

8.4

56 °C.

M

8.4

56 °C.

4

N

00

56°0.

444

O

56 °C.

4

•

L

•

pH

00

Dilutions

1
“10

1 1
80 TBO

1

1
1
1
1 _ 1 _
"SIU 1260 2560 5120 10240

4 4 4 44 + 44

4-4

4

444 444444 44 +

44

4

444i 444± 4+ 4 + 44 4 44

44

4

4

44 444 4-

4

MM

4+ 4 444 444 4

4

-

4

-

4

4

44 44* *4

4

-

±

-

J » Plasma frozen for 3 days at -35°C. (not filtered).
K s plasma of J, but filtered before testing.
L - Plasma heated at 56°C for 1 hour and filtered
before testing.
M - Plasma not heated nor filtered.
N - Plasma heated to 56°C. for 1 hour then centrifuged,
supernatant used.
0 = Plasma heated as in N, but not centrifuged nor
filtered.

Figure 1

Percent of Heterophils
in normal birds

Heterophils

10

50

20
Days

40

50

Figure 2

Total number of leukocytes per MM5
in turkey

thousand

10

20

30

40

Days
Organisms
Given(lccJ
orally

Given same
amount of
organisms

Given same
amount of
organisms

Figure 3

Percent of heterophils
in turkey

70#

60#

50#

40#

10

20

30

Days
Organ!sms
given (lcc)
orally

Given same
amount of
organ!sms

40

Figure 4

Percent ot jieterophils
in turkey

70#

50#

40#

LO

20

30

Days
Organisms
atisull8®1
)
orally

Given same
amount of
organisms

40

Figure 5
Number of red blood cells per cubic mm
in turkeys
4M

s Million

3M
Turkey
2,5M
Turkey B
2M

10
Days
lcc pullorum
given orally

Same
amount
given

Figure 6

Total leukocytes per

in turkey

30T
1,000

25 T —

20T

15T

10T
Days
Organisms given
1 C C \ j n f

a

Same
amount
given

Same
amount
given

Figure 7
Percent of* heterophils
in turkey

20

30

Days
Organisms
given(lc c )
intravenously

Same
amount
given

Same
amount
given

Figure 8
Number of red blood cells
per cubic mm*
in turkey

Million
3M

2M

10

20

Days
Organisms
given (lcc)
Intravenously

Same
amount
given

30

40

50

Figure 9

Number of lymphocytes
per cubic mm.
in turkey

T ‘ 1,000

15T

10T
lymphocytes
5T

agglu tinating
titer

Organisms
given (lcc)
intravenously

Same
amount
given

Figure 10
Total leukocytes
per cubic mm
in turkey

30T

T = 1,000

20T

1ST

10T

Days
Organisms
given (lcc)
intravenously

Same
amount
given

Same
amount
given

Figure 11
Percent of heterophils
in turkey

70#

60#

50#

40#

10
Days
Organisms
given & cc )
intravenously

Same
amount
given

Figure 12
Humber of red blood cells
per cubic mm,
in turkey

4M

= Million

3M —

2.5M —

10

20

Days
Organisms
given (lc c )
intravenously

Same
amount
given

30

40

50

Figure 13
Number of lymphocytes
per cubic mm
in turkey
1,000

Agglutinating
.titer

20T

15T

10T

5T

20

l cc organisms
given intra­
venously

Same amount
Given

30

40

Same amount
Given

50

Figure 14
Total leukocytes
per cubic hub
in turkey

1,000
25T

20T

1ST

10T

10

20

30

40

Days
Organisms
Same
Same
given(lcc)
amount amount
intravenously given given

Same
Same
amount amount
given given

50

Figure 15
Number of red blood cells
per cubic mm
in turkey

s Million

3M

2*5 M

2M
10

30

20
Days

Organisms
given(lcc)
intravenously

Same
amount
given

40

50

Figure 16
Humber of lymphocytes
per cubic mm
in turkey

1,000
25T
Agglutinating
Titer ^
20T

1ST

10T

5T

Number of
lymphocytes

10

lcc organisms
given intravenously

20

Same
amount
given

30

Same
amount
given

40

50