 

THE BACTERICIDAL ACTIVITY
OI? BOVINE BLOOD FOR BRUCELLA

Tigosis for {he Degree of M. S.
MICHIGAN STATE COLLEGE

Evern Marie Wood
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THE BACTERICIDAL ACTIVITY OF BOVINE BLOOD FOR BRUCELLA

by
Evelyn Marie Wood

A THEIS

Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

MASTER OF SCIENCE
Department of Bacteriology

February, 19”.;

ACKNOWLEDGMENT

I wish to thank Dr. I. F. Huddleson for suggestions
in this project, and Dr. G. C. Richardson for
veterinary assistance.

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L331:

The Bactericidal Activity of Bovine Blood for Brucella

Introduction ............................................. 1
Experimental
materials and methods ................................h
Experiments and results
I. Determination of incubation period ......... 5

II. Comparison of bactericidal activity
or plasma and serwm eeeeeeeeeeeeeeeeeeeeee 6

III. Comparison of the bactericidal activity
of plasma and whole blood ................ 7

IV. Effect of time interval on.the
bactericidal activity of blood ........... 7

V. The effect of heat on.bactericidal

activity eeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeee 9

VI. The effect of filtration on.the bactericidal
property of bOV1nB plasma 0000000000000... 9

VII. The concentration of bactericidins .........10

VIII-IX. The influence of the presence of
Brucella agglutinins in plasma
on bactericidal ROtIVity eeeeeeeeeoeeeell

X. The effect of the ingestion of colostrum
by new born calves on.bactericidins ......11

XI. Bactericidal activity of plasma of normal
cows, those after recovery from infection
and after vaccination 00000000000000.0000012

XII. The comparative susceptibility of the three
species of Brucella to bactericidal

action COOOOOOOOOOOOOOOOOOOOOOOOOOOOOI0.0.13

XIII. The comparative susceptibility of rough

(R) and smooth (8) types of Br. abortus ‘
to the bactericidal action one-v13;- '

plasma oneeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeOCIB
Discussion eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeelh

Summary 00000000000000...0.000000000000000000000000000000025

Literature cited eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoee

TEE BACTERICIDAL ACTIVITY OF BOVINE BLOOD FOR BRUCELLA.

The bactericidal property of blood has been under extensive
study since the early work of Nuttal (11) in 1888. It is a more
complex subject than generally supposed and its significance not
easily interpreted. At present it is impossible to bring the
results of different workers, who have investigated bactericidins,
into harmony and draw general conclusions because of the differences
in the methods of study. However, certain variables in the bacteri-
cidial capacity of normal serum have been established. Mackie and
Finkelstein (6) have made a comprehensive study of the occurrence
of bactericidins in the blood serum of several species of animals,
and the susceptibility of different bacteria to their action. They
found that the bactericidal power of blood for a given micro-
Srganism varied in different species of animals and in individual
animals of the same species. Furthermore, different bacteria react

differently in their susceptibility to the bactericidins.

.This study represents a cooperative project of the Bacteriological
Section of the Michigan State Agricultural Experimental Station and
the Bureau of Animal Industry of the United States Department of
Agriculture. The work was carried out in the Brucellcsis Laboratory

of the Department of Bacteriology.

.2-
The workers just mentioned included the three species of Brucella
in their studies of serums from sheep, ox, human, rat, pig, horse,
rabbit, guinea pig and pigeon. They found tint serums from all
the species possessed bactericidal action towards Brucella.
However, the comparative bactericidal effect of the various sermon
and the difference in susceptibility of the three species of
Brucella to their action, were not accurately measured. The
method used by Mackie and Finkelstein was not one of quantitative
measurement. The bactericidal effect was determined by loop
transfers (before and after incubation) from each mixture to a
plate of culture medium. The bactericidal effect was estimated
by comparison of the end-points of growth in the test and control
series. No bactericidal action was noted until after 20 to 21.;
hours of incubation.

Mackie and Finkelstein (6) attribute the bactericidal
property of normal serum towards Gram-negative bacteria to a
"thermolabile" bactericidin consisting of complement and a sensitizing
antibody. While the existence of certain natural bactericidal anti-
bodies in normal serum has been proved by Muir and Browning (8), there
has been no clear evidence to show that the bactericidal property of
normal serum depends entirely on bactericidal antibody.

Irwin and associates (5) were the first to test the susceptibility
of the genus Brucella to the bactericidal action of bovine blood.
These workers found that bovine whole blood, serum, and plasma
exhibited slight bactericidal action for Brucella. E; abortus was

found more susceptible to the action of bactericidins than Br. suis.

.3-

Striedter and Avrukina (ILL) observed that Br. melitensis was

 

relatively more resistant to the bactericidins in serum of guinea
pigs and sheep than was 2:; abortus.

The irmediate objectives in this study, were (1) the development
of a controllable bactericidal system which would detect the occurrence
of bactericidins in bovine blood serum or plasma, (2) the employment
of the system in following the changes, if any, that occurred in the
blood of calves from birth to maturity, in immunised animals and in
animals infected with Brucella.

While the developnent of a satisfactory bactericidal system was
in progress, it was noted that many factors influenced the end results.
A few of these were, (1) period of incubation, (2) number of organisms
used, (1;) dissociation of the organism, (5) interval between collecting
blood and setting up the test, (6) the use of serum, plasma or whole
blood, (7) filtrationcf serum or plasma and the type of filter used,
(8) heating of serum or plasma.

A considerable portion of the Thesis is devoted to a study of

the factors which influence the results of a bactericidal system.

HPERILENTAL

Materials and Methods:

Preparation 35 culture: A single, smooth culture of Br.

 

abortus was used unless otherwise stated. The micchrganism

was grown on beef liver agar slants for 148 hours at 37°C. The
growth was rancved by means of a sterile wire-loop and sus-

pended in sterile diluting fluid which consisted of 0.05 per

cent Tryptose peptone and 0.5 per cent NaCl in distilled water.

The bacterial suspension was thoroughly mixed and then diluted

to a scale reading of 23 on the Libby Photrcnreflectometer.

The standard suspension contained from 800,000,000 to 1,300,000,000
live Brucella organisms per ml. Serial dilutions ranging frm
13100 through lsl,000.000 were made from this suspension. The
number of bacteria present was determined by floating l m1.cf the
131,000,000 dilution over the surface of a Tryptose agar plate.

The Tryptose agar contained crystal violet in a lsl,OO0.000 dilution.
The plates were incubated at 37'C for four days. The number of
colonies counted was recorded as representing the initial number

of bacteria added to the blood plasma or serum from the different
dilutions.

Prejaration .93 Ma Blood was drawn asoeptically from the
Jugular vein of the animal and collected in sterile bottles. For
the test requiring whole blood or plasma a sufficient amount of
sterile saturated sodium citrate was placed in each bottle to
prevent the blood from clotting. Whole blood, plasma, or serum

samples that were not used within a few hours after collection

-5-

were refrigerated at 14°C. When plasma or serum was used, the
blood was centrifuged for thirty minutes and the supernatant
drawn off aseptically. The serum or plasma samples were col-
lected from normal animals as regards to Brucella unless other-

wise stated.

Ebtperiments and Results:

I. Determination of incubation periods In establishing

 

a standardised procedure, the incubation period necessary to
attain the maximum bactericidal effect was sought. Five
duplicate series of plasma-bacterial suspensions and controls
were made as follows: Into each of a series of sterile, cotton
plugged, 10 ml shell vials was pipetted 1 ml of sterile diluting
fluid. To the first tube, 1 ml of plasma was added, and mixed
with a 1 ml syringe. Then, 1 ml of the mixture was transfered
to the next tube, and thus continued until the desired number
of plasma dilutions were obtained. To each vial, 1 m1 of a
l:1,000.000 dilution of the standard suspension of organisms
was added. The plasma-bacterial suspension was then thoroughly
mixed, and placed at 37'C. Each duplicate series was removed
from the incubator at intervals of 2, h, 8, 10, and 214 hours,
and plated on Tryptose agar. The results obtained are recorded
in Table I. The results show that the bactericidal action of
bovine plasma is not one of abrupt killing, but a gradual process,

dependant upon the time of incubation and the ratio of plasma

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to the number of organisms. In the trio highest dilutions of
plasma, the organisms multiply during. the first 2 hours, and
then show a slight decrease in number during the next 10 hours.
After the thh hour an increase in numbers takes place. Evidently
the bactericidins are toneak in concentration to prevent growth.
The absorption of a minute amount of bactericidins by the bacteria
apparently has a bacteriostatic action and only a slight bactericidal
action.

Later, it was observed that when a large number of bacteria
were added to undiluted plasma, reduction in the number occurred
gradually over a period of 96 hours. Thus, demonstrating that
the maximum bactericidal effect depends upon the length of time
that the plasma and bacteria are incubated together. Although
bactericidal activity continues throughout 96 hours of incubation,
the evaporation of the liquid is so great that it is difficult
to interpret the results in terms of the original dilution.
Therefore, a 1.8 hour incubation period was used in most of the

experiments that follow.

II. Comerison of bactericidal activity of plasma and serum
Several tests were mde to determine the bactericidal activity of
plasma in comparison to that of serum. In every case plasma proved
to be more active than sermn. Table II illustrates the comparative
differences when the procedure given in Experiment I, was used.
According to these data, a 1:16 dilution of plasma has the equivalent

activity of a 1:14 dilution of serum. Other comparative studies have

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been made on the bactericidal activity of plasma and serum and

will be discussed in proceeding experiments.

III. Comparison of the bactericidal activity of plasma

and whole blood: The procedure used in determining this

 

difference is as follows: The bacterial dilutions were pre-
pared as mentioned previously. A standard suspension of _B_r_=
abortus was diluted serially from 13100 to 131,000,000. A
uniform quantity (1 ml) of plasma or whole blood was added
to each of a series of sterile vials in which had been placed
1 ml of the bacterial dilution. The contents were mixed, and
then incubated at 37‘C for LLB hours.

In Table III is set forth the differences between the
bactericidal action of whole blood and that of plasma. Bovine
whole blood was found to exhibit far less bactericidal power
than plasma or serum. The method used in this experiment proved
to be a more reliable measure of the bactericidal effect of blood
than the one used in preceding experiments. Thus, this method

was adopted as the standard procedure.

IV. Effect of time interval on the bactericidal activitx

 

of blood: It was noted during the progress of the study that
the results on the plasma from the same animal often varied
from day to day. Since no particular attention was paid to
the time that elapsed between the collection of the blood fans
the animal and its examination, it was thought that the time

interval might be an important factor in influencing the results.

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A series of samples were-collected from several animals and
permitted to stand at different. lengths of time and in different
states before pssforming the tests. The results of one time
interval experiment are shown in Table IV. It is obvious from
the results that variations in bactericidal activity can be
attributed to the time factor and also the state in which the
plasma was held before its examination. The results of the
examinations show that bovine plasma from different animals
may show either an increase or decrease in bactericidal
activity upon standing. Plasma separated from the blood cells
or aged in the presents of cells was found to undergo a change
in its activity. Similar experiments were conducted using serum
in place of plasma. The same changes in activity were noted.

Numerous experiments were conducted to seek a plausible
explanation for the changes which took place in the bactericidal
activity of plasma when it remained for several hours before
examination. Several test tubes containing plasma were divided
into two groups. One group was plugged with cotton, and the
other was sealed with rubber stOppers. Bactericidal tests
were run at weekly intervals. The results showed that the
plasma taken from the rubber stoppered tubes did not undergo
the changes in bactericidal activity to the same extent as
the plasma from the cotton plugged tubes. The tubes plugged
with cotton would allow more aeration of the plasma. Thus,
it was thought that by passing oxygen or carbon dioxide through

the plasma one might attain the same alteration in bactericidal

.9...

activity that took place when the tubes of plasma remained for
several hours at ice box temperature. Oxygen and carbon dioxide
were passed separately through the samples of plasma, after which
samples were sealed and placed at h‘C for 2 days. The treated
samples were not significantly different in their bactericidal
activity than the control plasma sample held for the same length
of time.

Since the refrigeration of plasma samples does not prevent
changes in their bactericidal activity, more accurate results
might be expected if the samples are tested immediately after

collection from the animal. '

V. The effect of heat on bactericidal activity: Samples
of plasma and serum were divided into three equal portions.
One portion was heated at 56'C for one hour, another at 58'!)
for one hour, and the remaining left as a control. The method
used in Experiment III was employed for comparing the bactericidal
activity of these samples. Table V illustrates that heating at
these mentioned temperatures markedly destroyed the bactericidal
effect of both plasma and serum. However, even after heating,
the activity of plasma was still greater than that of semen.
The bactericidal activity of samples heated at 56'C, was not

markedly different from that of the samples heated at 58.0.

VI. The effect of filtration on the bactericidal property

 

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the bactericidal property of bovine plasma, three different

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filters of different porosities were choosen, (l) a Berkefeld
Cl)‘which is known.to retain.complement, (2) a Seits, and (3)
a fine porosity Jena fritted glass filter. The filters were
sterilized and the plasma drawn through into a sterile container
by means of a vacuum.pump.

.The procedure used in determining the bactericidal property
was the same as described in.Experiment III. The results shown
in Table VI brought forth a new aspect to the subject of
bactericidins. ‘While the Berkefeld filter completely removed
all bactericidins, the more permeable Seits and Jena fritted
glass filter apparently retained an "antibactericidal" substance.
As a result the bactericidal activity of the plasma was increased.
The validity of these results were ascertained by repeating the

procedure.

‘VII. The concentration of bactericidins: An effort was
made to concentrate the bactericidins in'bovine plasma by
preforming the following experiment: Thirty mls of sterile
bovine plasma were placed in a sterile cellophane tubing and
dialysed against C.P. glycerine; the sample being agitated
constantly during the period of dialysis. The water was dialyzed
rapidly out from the plasma, and within two hours the volume of
plasma was reduced to 7.5 mls. Although highly concentrated,
the syrup-like plasma.remained in a perfectly clear state. The
concentrated plasma, and an original sample were serially diluted

and tested as in Experiment III. The results in Table VII show

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that the highly viscous plasma was not as effective in killing
the organism in the first two dilutions as was the control
plasma. However, the data indicate that it is more bactericidal

in the higher dilutions.

VIII. The influence of the presence of Brucella agglutinins
in plasma on bactericidal activity: In order to determine if any
correlation existed between the agglutination titer and bacteri-
cidal activity the following experiments were performed. A normal
adult animal whose plasma showed no agglutinins in a 1:25 dilution
or above was given an intravenous injection of a suspension of
virulent 31;. abortus organisms. The agglutination titer and
bactericidal activity were recorded at weekly intervals for the
first month after injection and thereafter at two week intervals.
As shown in Table VIII, the bactericidal activity varied even
when the agglutination titer remained constant. The bactericidal
activity became progressively less in each succeeding test.

In a similar experiment conducted on a calf which had been
vaccinated with a killed suspension of 2.1:. :23. The rise and
fall of the agglutination titer could in no way be corrolated with
the flucuation taking place in the bactericidal activity of the plasma.

The data on this animal is shown in Table II.

I. The effect of the ingestion of colostrum by new born

calves on bactericidins: The bactericidal activity of plasma

 

from five different calves was determined before they ingested.

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colostrum and at various intervals up to several weeks afterwards.
The results of this study on one animal are set forth in‘Table I,
and are representive of all that were studied. The remarkable
increase in bactericidal activity shortly after the ingestion of
colostrum, and the progressive increase thereafter, was typical

for all calves tested.

XI. Bactericidal activity of plasma of normal cows, those
after recovery from infection and after vaccination: The results
in Table II are representative of a comparative study of the
bactericidal activity of 15 normal cows, one adult cow known
to have developed resistance to Brucella infection following
recovery, 10 animls which were immunized by vaccination, 8
infected cows, and 3 cows that recovered from brucellosis in-
fection as indicated by a marked decrease in their agglutination
titers from a previous high level. The procedure used was the
same as that of Experiment III. The number of bacteria added
(as shown in Table II) represents an average of the number of
bacteria used in all the samples examined. All plasma samples
examined from infected animals show little or no bactericidal
power. Plasma of individual animals often vary considerably
in bactericidal action but mrlced contrast between the colony
counts from normal plasma-bacterial suspensions and those from
infected animals is unusual and must have some significance.

The activity of plasma from recovered animals was not equivalent
to that of most normal animals. Little, if any difference was
noted between the bactericidal action of normal, resistante, and

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-13-

III. The comparative susceptibilitLof the three species

 

of Brucella to bactericidal action: One smooth strain of each

 

of the species of Brucella was selected for the purpose of
demonstrating their comparative susceptibility to the bactericidins
in bovine plasma. The bacterial suspensions were prepared in the
usual manner. The plasma was that of an normal animal and the
procedure described in Experimnt III was used. The results are
tabulated in Table III. 21;.- 33.13. was found to be the most
resistant of the three species to the action of bactericidins,

while Br. abortus was the most suspectible.

XIII. The comparative susceptibility of rough LR) and

 

smooth (8) types of Br. abortus to the bactericidal action

 

of bovine plasma: A smooth culture of Br. abortus was

 

dissociated by multiple transfers in sterile broth consisting
of 1.0 per cent Tryptose peptone, 3.0 per cent dextrose, and
0.5 per cent NaCl. Standard suspensions of the R and S types
were prepared as previously described. The plasma samples were
from a normal, vaccinated, and infected cow. The procedure .
described in Experimnt III was used. The data are tabulated
in Table XIII.

The results show that the rough type of organism is far
more susceptible to the bactericidal action of bovine plasma
than is the smooth type. The contrast is especially pronounced
in the plasma from the infected car. As shown previously, plasma

from infected animls has relatively no bactericidal activity towards

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'11;-
smooth strains of £2 abortuso In the case of R type, the plasma
is as bactericidal as plasma from a normal or vaccinated animal.
In View of the differences in susceptibility to bactericidins
shown by a rough strain, it would seem important to use a culture a

in a bactericidal system that is free from dissociated «113;.

DISCUSSION

Topley and Wilson (15) define complement as ---" a thermolabile
substance, or complex of substances , present in varying concentrations
of blood serum of most normal snimls, which has the property of bringing
about lysis of certain cells and bacteria in conjunction wiﬁ: certain
antibodies which render the cells or bacteria sensitive to its action."
They consider their definition incomplete because it is still an open
question as to the exact role of complement and antibody in the anti-
bacterial property of normal senm. llany theories have been proposed
to explain the combined activity of "bacteriophilic complement” and
”specitk antibody.” In 1909 Muir and Browning (8) began a series of
”harmonize designed to determine whether or not complacent could be
absorbed out by homologous bacteria and thereby diminish the bactericidal
activity on both homologous and heterogenous organisms. They found that
the treatment of a normal serum with increasing amounts of a killed
suspension of a bacterium, usually produced effects in the following
orders (a) diminution of the bactericidal action on the same
bacterium; (b) diminution of tho bactericidal action on other
bacterium; (c) diminution of hemolytic complement. The latter

effect brings forth an important questions "are bacteriophilic

-15-
complement and hemolytic complement analogous?” A corrolary
existing between the findings of Noguchi and Bronfenbrenner
(10) and the data tabulated in Table IV, may be of significance.
In their study of the variation in complement activity of guinea
pigs, they noted that a large number of serums gained in complementary
activity when remaining in contact with the clot for 146 hours, while
some serums become weakened during the same length of time. Their
observation coincides with the finding that the bactericidal activity
of plasma or serum may increase or weaken upon standing. The interval
of time elapsing between the withdrawal of the blood and the testing
of its activity has proved to have an important bearing on the
quantitative measurement of bactericidins. Of further interest is
the fact that plasma or serum does not have to be in contact with
blood cells or clot in order to accomplish a rise in bactericidal
power. Therefore, one can exclude the possibility of lecocytic
extracts (leukins), or other cellular components, as being responsible
for the increase in activity. Upon standing, either a substance
which inhibits bactericidal activity is diminished, or a bactericidal
agent is set free from a plasma constituent. If the increase in
bactericidal activity was found to be proportional to the increase
in complement activity, one could say that ”hemolytic complement"
and "bacteriophilic complement” were analogue. However, even
though the supporting evidence is a prologue to this theory, it
would require further investigation before such a conclusion
could be reached. In contrast to this proposal, Dunlap (1) claims

that unlike hemolytic complement, the complement concerned in

-16..
bactericidal action is absorbed by charcoal. It is generally
supposed that there are more than one component involved in the
bactericidal phenomenon. Therefore it would not necessarily
have to be the complement absorbed by charcoal, but possibly
the so-called "sensitizing antibody."

The length of time that blood-bacterial suspensions are
incubated together is of importance in determining the anti-
bacterial effect of‘whole blood, serum, or plasma. In low
dilutions of plasma (1:2 to 1:16) the killing of Brucella
organisms is a gradual process, and the diminition of
bacteria is directly proportional to the time of incubation.
In higher dilutions of plasma (1:32 and above), the number of .
organisms shows a gradual decline for the first few hours and
then a sudden rise upon further incubation. One explanation
of this may be the proportion of antibody to antigen which
is not sufficient to inhibit the growth over an extended period
of'thme. 'When antibody is present in sufficient quantity to
sensitize all the bacteria to the lytic action of complement,
the decline in bacteria depends upon the incubation period.

By using a constant anmunt of plasma, and varying the number
of bacteria, the degree of killing obtained depends on the
proportion of bacteria to bactericidins and the incubation
period. For comparative purposes, a hB hour incubation period
proved satisfactory for the species of Brucella.

The incubation period for in m bactericidal tests is
dependent upon the bacterium employed. In an experiment by

Mackie and Finkelstein (7) it was noted that after an incubation

-17-

period of three hours, for E. typhosa and certain other organisms,
maximum.effect was obtained and furﬂher incubation did not elicit
appreciably greater killing.

No explanation can be given for the greater bactericidal
activity displayed by plasma in comparison with serum. The only
known difference between plasma and serum.is the presence of
fibrinogen in the fomer. Yet fibrinogen, when separated from
plasma, displays no bactericidal effect. Fibrinogen is precipitated
when.plasma is heated at 56’C for one hour. Plasma retains more
bactericidal activity than does serum when treated at the same
temperature. This is an indication that a bactericidal agent exists
in plasma which is not present in serum.

Irwin.and associates (5) compared the bactericidal activity
of a plasma cell mixture with that of whole blood. Their findings
‘were that the activity of this mixture was never more pronounced,
and occasionally less than that of whole blood. However,.the blood
cells present in the plasma may have lowered its activity. Citrated
‘whole blood has little, if any bactericidal action against E5. abortus.
The difference in.the activity of plasma and whole blood is especially
pronounced in normal animals. ‘Where complete killing of 1200 organisms
by 1 ml of whole blood has not been noted, 1 ml of plasma from the
same animal may kill as high as 60,000 31;. abortus organisms. This
finding is representative of the difficulties encountered when trying
to correlate 211.29. results with the bactericidal phenomenon that

occurs in vivo.

 

It has been recognised generally that hemolytic and bacteriOphilic

complement are destroyed by heating normal serum at 55'0 for 30 min.

-18-
The bacteriophilic complement is considered by'Nackie and
Finkelstein (6) to be the active principle in the killing of
Gramnnegative organisms, and'when it is destroyed by heating
the bactericidal activity is lost. They assert that Gramp
positive organisms are acted upon by a separate mechanism,
because of the thermostahility of the bactericidal agent. The
occurrence of heat stable bactericidal properties in.serum.has
also been studied by Salter (13),'who believes that different
organisms are affected by a common agent and not by multiple
specific agents. Although the bactericidal activities of both
bovine plasma and serum were markedly weakened by heating at
56'C for 1 hour, it was noted that their activity towards .35.:
abortus was not totally destroyed. The thermostable bactericidins,
as suggested by Selter, may be a common agent to both Grampnegative
and Gramppositive bacteria, while the labile complement may be
needed as a specific agent in killing Gram-negative organisms.

It has long been known that hemoiyiic complement could be
removed from serum by means of a filter of exceedingly fine
porosity. The Berkefeld filter has proved most satisfactory.

By filtration through a Berkefeld (I), plasma looses all of its
bactericidal properties. If one attributes this loss to
bacteriophilic complement retention, then this again brings it

in close relationship to hemolytic complement. In comparing the
activity of filtrates from Berkefeld, Seits, and Jena fritted glass
filters, a very interesting resultant was noted. “While Berkefeld

filters deprived the plasma of its activity, the Seits and fritted

-19..
glass increased the bactericidal effect. This increase may be
explained by the retention of either ant" anticomplementary
substance or an inhibitor of bactericidins. In either case the
size of this substance must be larger than that of the bactericidins
which passed through.

No appreciable increase in bactericidal activity can be
obtained by concentrating plasma. The change in viscosity, or
the altering of the combining action of the bactericidins in a
high concentrated plasma appears to retard bactericidal action.

By diluting the plasma back up to its original volume, it regains
its original activity.

Gengou (h) in 1899 drew attention to the fact that bacteria are
agglutinated as well as lysed by a specific antiserum and that this
effect may greatly reduce the number of colonies in a plate count;
the clumping of the bacteria producing single colonies. For this
reason, lackie and Finkelstein (7) assert that bactericidal tests
which have been based on colony counts give an unreliable index of
bactericidal activity. It is acknowledged here that the agglutination
titer of plasma may have an indirect bearing on the number of colonies
counted. However, as a comparative index of the bactericidal activity
of plasma from normal or animals showing high agglutimtion titers,
this method has proved very reliable. As shown in Table VIII and II,
when the agglutinins are not demonstrable in plasma in a dilution of
1:25 or above the bactericidal action of the plasma is markedly
higher before the same animal has developed specific agglutinins. If
the bacteria placed in the presence of a high titered plasma were
agglutimted, a lower colony count should be obtained than when the

plasma was negative. When plasma samples from a single animal show

-20-
a constant high agglutination titer over a period of weeks, the
colony counts in a standard bactericidal test should remain
approximtely the same, if the titer influenced the resultant
bacterial counts. This was not observed.
If the resistance of an animal to brucellosis was in direct
relationship to the bacteric idal action of its plasma towards

Br. abortus _i_n_ vitro, then upon immunization of the animal an

 

increase in bactericidal activity should occur. However, the
reverse is often the case. Calves which were immunized by
vaccination, exhibited more bactericidal activity before imnnnisation
then after. The bactericidal antibodies are undoubtedly of different
character then those responsible for resistance to infection. As yet,
bactericidal tests have not proved to be a measure of immnity or
susceptibility of an animal to any specific infection.

The plasma from new born calves, before they have ingested
colostrum, has little, or no bactericidal effect. Forty-eight
hours after the ingestion of colostrum there is a sudden increase
in bactericidal action, and continues to rise for several weeks.
The age at which calves show maximum bactericidal activity cannot
be definitely stated, as it varies with the animal. In certain
animals studied, the bactericidal activity of the plasma from
calves 5 days old were within the range of that of an adult cow.
is a collorary to this study, it is of interest to cite the
findings of San Clemente and Huddleson (12) in mm ”Electrophoretic
studies of the proteins of bovine serums with respect to Brucella.”

From the electrophoretic studies upon the serum of a calf at birth,

-21-

before the ingestion of colostrum, it was observed that, (a)
calf serum obtained before the ingestion of colostrum showed
a high concentration of a. -globulin which might almost equal or
even exceed the concentration of albumin: (b) 7’ -globulin was
extremely low or absent. Six days after birth and after it had
ingested colostrum the following changes had taken place: (a)
the high a peak had decreased from 1414.2 to 30.); per cent of
the total area, while the 7’ peak had risen from 2.3 to 20.7
per cent. Twenty-two days later the electrophoretic pattern
began to approach that of an adult cow. is 1 -globulin is
associated with the antibody content of serum, one might assume
that the sudden rise in 7' ~globulin was responsible for the rise
in bactericidal activity. However, the fact that infected cow's
serum has a much higher 7 -globulin content then does that of
an normal animl, but a lower bactericidal capacity, would tend
to disprove any such assmnption. Nevertheless, colostrum either
provides bactericidins or stimulatesthe production of these agents.

The bactericidal property of calf plasma is developed at an
early age. Within three days after birth the bactericidal
capacity my have reached the extent of an adult animal, or even
exceed it. It is a well established fact that calves are highly
resistant to Brucella infection. Whether high resistance and
high bactericidal activity on the part of plasma are related in
any way is problematical. Since plasma from adult animals that
have never knowingly been exposed to Brucella infection, in many

instances, show just as high bactericidal activity it would appear

.22-
that such a question cannot be answered satisfactorily until
such adult animals have been exposed to infection to determine
their susceptibility. ‘

The changes in the bactericidal activity of bovine plasma
cannot be correlated with the age of the animal. Occasionally,
plasma from a normal adult animal may have greater bactericidal
activity than that of an month old calf. It is evident that the
factors governing the bactericidins in. 11.19. are multiple. The
diet of the animal may play a part in this role. Ecker and
associates (2) have shown that a correlation exists between the
concentration of ascorbic acid in the blood serum of guinea pigs
and the, complementary activity of the serum. It is possible
that a relationship may also exist between ascorbic acid and
bactericidal activity, as complement undoubtedly plays a part
in the bactericidal mechanism.

The antibacterial activity of plasma from acquired immune,
and animals immunised by vaccination lies within the range of
the variable activity of that of normal animals. Although
immune individuals may be resistant to Brucella infection, the
resistance cannot be foretold by means of a bactericidal system.
The absence of bactericidal activity in plasma from infected
animals, and the low activity of plasma from those having recovered
from infection must have immunological significance. Fiessinger
and Cattan (3) report a disappearance of the bactericidal action
of whole blood in typhoid fever patients, a phenomenon similar

to that noted in bovine plasma from infected animals. Irwin

-23--
and associates (5) had likewise observed that certain cows,
recovered from a previous infection of _B_1:. abortus, showed a
loss in bactericidal properties of whole blood as compared
to that of presumably normal animals.

Striedter and Avrukina (114) were unable to demonstrate
any differences in the bactericidal activity of serums fran
healthy guinea pigs (highly susceptible) or those infected
with or recovered from brucellosis. In view of these results,
the differences noted in the bactericidal activity of plasma
from normal and infected cows may not hold true for guinea
pigs.

It has been generally assumed that a sanitizing antibody
takes part in bactericidal phenomena. Maokie and Finkelstein
(7) regard antibacterial antibodies as being highly specific.
These specific antibodies, if related to antibodies produced
by immunisation, should be greatly increased during infection.
However, the antibacterial effect is lessened. If an optimum
preportion of combining power exists between antibody and
complement, or antibody and antigen to obtain bactericidal
activity, then an increase in antibody content may decrease
the action of complement. It was noted by Neisser and
Weeksberg (9) that a marked pro-zone often occured in bactericidal
tests. A particular dilution of serum might exert no bactericidal
action, while a much higher dilution resulted in the complete
killing of the bacteria. In accordance with these findings, a

pro-zone phenomenon was observed whenever extremely high dilutions

.214-
of plasma were tested for bactericidal effect upon a constant
number of organisms. The high activity shown in the pro-zones
may be due to the existence of the proper ratio of antibody to
antigen and not the proportion of complement to antibody. If
the pro-zone cf bactericidal action depends upon the presence
of an optimum amount of antigen to antibody then by serial
dilution one may arrive at a dilution in which the optimum
combining proportion obtains for bactericidal activity. However,
since it was found in this study that more than one pro-zone
occurred, this would hardly serve as an explanation.

The literature is incomplete on the susceptibility of
the species of Brucella to bactericidal action of bovine
plasma. Irwin and associates (5) have found 25.. £13 to be
more resistant to the bactericidins of bovine blood than P5.
abortus. While Striedter and Avrukina (114) have noted 2:.

melitensis relatively more resistant to the bactericidins in

 

serum of guinea-pigs and sheep than Eubortus. The arrangement
of the three species in regards to their reactivity to normal
bovine plasma is as follows: (a) 25. abortus is markedly more

susceptible than the other two; (b) _B_l_'_. melitensis is less

 

resistant than _B_1_'.. £545.

The rough type of _B_r.. abortus is far more susceptible
to the bactericidal activity of bovine plasma than the smooth
type. Plasma from an infected animal is as highly bactericidal
to the rough type as that of a normal animal. In accordance

with these findings is the work of Mackie and Finkelstein (6).

.25-
They noted the R variant of E typhosa proved more susceptible

to serum bacteriolysis than the 8 form.

SUMMARY

1. The interval of time elapsing between the withdrawal
of blood from the animal and the testing of its bactericidal
activity has a direct bearing on the bactericidal action of
whole blood, serum, or plasma. The majority of plasma samples
increased in activity upon standing at h'c for h8 hours or
more, while a few samples diminished in activity. Because of
this unpredictable change in activity it was found that more
accurate results could be obtained when the samples were examined
immediately after withdrawal from the animals.

2. The bactericidal action of bovine plasma _i_n_ m is
not one of abrupt killing, but a gradual process. The effectiveness
of this action is dependent upon the time of incubation, and the
ratio of plasma to the number of organisms used. As a comparative
method, a constant amount of plasma added to varying numbers of
organisms proved more satisfactory.

3. Plasma has a greater bactericidal action then does serum.
Whole blood shows only a slight bactericidal activity.

h. The activity of plasma and serum is markedly lowered by
heating at 56°-58’c for one hour but is not totally destroyed.
Fibrinogen is precipitated at this temperature, but plasma still
retains a higher activity than does heated scrim. Therefore, the

difference in bactericidal activity of serum and plasma cannot be

.26..
accounted for by the presence of fibrinogen.

5. The bactericidal property of plasma can be removed
by filtration through a Berkefeld (W). The more porous filters
such as Seits and Jens fritted glass retained an 'antibactericidal"
substance, and thus the filtrates show a greater bactericidal
effect than does the unfiltered control sample.

6. There was an appreciable loss in bactericidal activity
when plasma was concentrated to a highly viscous state. The
activity was regained upon diluting the concentrate back to its
original volume.

7. The agglutination titer of bovine plasma cannot be
corralated with its bactericidal activity. The presence of
agglutinins in the plasma proved incidental to the comparative
index of the bactericidal activity of normal plasma to those
which showed high titers.

8. Plasma of new born calves, before they have ingested
colostrum, has little, or no bactericidal activity. Shortly
after the ingestion of colostrum the activity increases and
become progressively greater as the calf matures.

9. Plasma from cows infected with brucellosis has little
or no bactericidal effect upon 35. abortus. Plasma from animals
that have recovered from infection show a lower bactericidal
activity than do normal animals. No apparent differences were
shown in the bactericidal activity of normal animls, acquired

immune, and vaccinated cows.

~27-
10. The species of Brucella vary in their susceptibility
to the action of bactericidins in bovine plasma; 135. abortus

being the most susceptible, 31;. melitensis and _B_r_. suis the

 

least.
11. The rough type of 2:. abortus is far more susceptible

to the bactericidal action of bovine plasma than is the smooth

type.

1.

2.

3.

h.

5.

7.

-28-
LITERATURE CITED

mnlop, E. 11., Fixation of complement by g. typhosa and
normal guinea pigs scrim; A contribution to the
study of natural antibodies, J. Path. and Bact.,
2: 769-795. 1928.

Ecker, E. 3., Pillemer, 1..., Wertheimer, D. The effect
of ascorbic acid on the constitution of complacent.
J. of Imunol. _3_h_a 16-50. 1933.

Fiessinger, 11., Cattan, R., Le pouvoir bacte’ricide du
sérum et du plasma a) 1'e’gard du bacille d'Eberth,
Presse méd. 29: 1185-1188, 1928.

Gengou, 0., E'tude sur les rapports entre les agglutinines
et les lysines, Annalee Inst. Pasteur, 3.2: 6142-656,
1899.

Irwin, u. 11., Beach, B. 1.. Bell, r. 3., Studies on the
bactericidal action of bovine whole blood and serum
towards Brucella abortus and Brucella suis, J.
Infect. Dis., 58-59. iii-22, 1936.

Beckie, T. J., Finkelstein, M. H., The bactericidins of
normal some; their character, occurrence in
various animals and the susceptibility of different
bacteria to their action. J. Hyg., 23 1-24, 1932.

Mackie, T. J., Finkelstein, 1!. H., Natural bactericidal
antibodies: Observations of the bactericidal

mechanism of normal serum, J. Hyg.. 28 35-55. 1931.

8.

9.

10.

11'.

12.

15.

15.

.29-

Muir, R., Browning, C. H., On the bactericidal action of
normal serum. J. Path. and Bact., Es 76-91, 1909.

Neisser, H., Wechsberg, F., Mllnoh. Med. Wschr., £58., 697, 1901.

Noguchi, H. , Bronfenbrenner, J., Variations in the complement
activity and fixability of guinea-pig serum. J. hper.
Med.. _1_§_: 69-77. 1911'.

Nuttall, G., Experimente ﬁber die bacterienfeindlichen
Einflﬂsse des thierischen Kﬂrpers, Ztschr. f. Hyg.

11. Infektionskr, g: 353, 1888.

San Clemente, C. L., Huddleson, I. F., ElectrOphoretic
studies of the protein of bovine serums with respect
to Brucella. Studies in brucellosis II., Mich. Agr.
Exp. Sta., Bul. 182, 14.1., 191.3.

Belter, H., Thermostabile bakterienfeindliche serumstoffe,
Ztsohr. f. Hyg. u. Infektionskr., 22: 313-323, 1918.

Striedter, V., Avrukina, 8., The bactericide capacity of
blood 32.11.29. experiments and its significance for
iinmunity in brucellosis, Dept. of Especially Dangerous
Infections, Viem, Moscow, 1940. I

Topley, W. W. 0., Wilson, G. 8., Principles of bacteriology
and immnology, 2nd Ed. 139, 1938.

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