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MI

A STUDY 9F mom ABURWS AGGLUHNATINB SERA
FOR THE”? BANERINDM AND THERAPEUTIC VALUE

The-sis for Degree of M. S.

H. W. JOHNSON
I929

 

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‘ll

A STUDY OF BRUCELLA ABORTUS AGGLUTIRATIM SERA
FOR {MEIR BAG'I'ERIOIDAL AN D TERAPFJH‘IC VALUE

A STUDY OF BRUGELLA.ABORTUS.AGGLUTINATING SERA
FOR THEIR BACTERICIDAL AND THERAPEUTIG_VALUE

Thesis

Submitted to the Faculty of’Michigan state
College of Agriculture and Applied Science in
partial fulfillment of the reqiuremente for the

Degree eijBeter of Science.

H: W. ggpnecn
June 1929

1.
2.
3.

4.
5.
6.
7.

8.
TABLE OF CONTENTS

Introduction.
Review of literature.
Experimental data.
A. Serum production.
(1). Technique (including history of strains of
Brucella abortus used).
(2). comparative charts.
B. Bactericidal effect in vitro.
(1). Technique of cultural method.
(8). Tables of results.
(2). Whole blood. ‘
(a). Heist-Lacy technique and tables of results. ,
c. Bactericidal effect in vivo.
(1). Preparation of infective material and method of
I exposure of experimental animals.
(2). Method of determining infection.
(3). Discussion of charts. .
General discussion.
Summary.
Acknowle dgement .
Bibliography.

100241

INTRODUCTION.

In response to inquiries of the medical and Veterinary
professions, an evaluation of anti-ears for Brucella abortus
infections was undertaken.

It is a Iell knoun fact that in infections with Brup
cella abortue in man or animals the manifestations of the
disease are accompanied by the appearance in.the blood serum
of agglutinins, percipitins, or complement fixing anti-
bodies, any or all of which may be present. Their impor-
tance, aside from'eerving as an indicator of infection or
previous contact with the organism, apparently has never
been studied in relation to Brucella abortus.

The role played by the above named antibodies against
infections has long been a problem.under investigation.
They appear in the blood serum and tissue extracts in
varying concentrations. In other words the body has pro-
duced antibodies, or its components have been altered in
their physical or chemical state so as to make possible the
determination that there has been contact between the ani-
mal body and the microorganism.

For a long time there has been doubt expressed among
inyestigators as to the immunological importance of anti-
bodies. Speaking of the agglutination test as used fer
standardizing anti-meningococcic serum,F.IM. Huntoon and
R. H. Hutchinson (1) say, "This test is recognized merely

as an indication that the horses have been under immuniza-

tion for a considerable time". In reference to antipneumo-
coocic sera they say (2), “The presence of agglutinins is no
indication of the protective value of the serum, and is used
only when diagnostic sera are desired". There are, however,
numerous instances of immunity without a demonstrable enmunt'
of the above named antibodies. Furthermore, with a relatively
high concentration of the antibody substance there is often
present a chronic infection. It is also evident that the
serum or tissue extracts of apparently uninfected animals
never contain the specific antibodies to any appreciable
extent.

éhe value of knowing the significance of the antibodies
present in the tissues and blood serum in Brucella abortus
infections is not only of scientific but of medical and
economic importance as well. It was not thought advisable
to determine at this time the relative importance of the
individual types of antibodies, but to make a thorough study
of their importance collectively. So, with the above idea
in mind, this paper presents a report of the study of
several Brucella abortus sera of high agglutination titre.

REVIEW OF LITERATURE

Qhe literature reveals very little research pertaining
to the standardization of antibacterial serum by either in
vitro or in vivc methods. This is not only true of Brucelle
abortus antisera, but of other antibacterial sera as well.
The presence of antibacterial antibodies has been discussed
in some instances, but there has been no method developed by
experimental work which would standardize their immune or
therapeutic properties. Certain investigators have drawn
conclusions concerning the values of agglutination, comple-
ment fixation, or precipitation tests from observations, or
from a few experiments which when extended and further inves-
tigated did not prove true.

Cole andeoore (5) say, "In spite of all that has been
written concerning the theoretical principles involved in
the preparation of anti-pneumeooccic serum, and in spite of
all the reports of its therapeutic application which have
appeared, it is very difficult to learn from.the literature
on the subJect exactly how these sera have been prepared or
standardized". This is also found true in relation to all
entisera.

In their study of the production of anti-pneumococcic
serum.they emphasize the following points, (a) immunological
specificity of the organism used for immunization, and (b)
the serum should have the power of protecting mice against

large amounts of virulent culture. Serum.for>type I pneumo-

ceccus ms the only one in which they were able to demon-
state any therapeutic value.

Veil, Richard, and Torrey (4) have shown that human
serum from pneumonia patients sensitizes guinea pigs
passively, but they were unable to conclude whether or not
such action was due to agglutinins, complement fixing bodies,
hemolysins, or precipitins. They did not attempt to demon-
strate any protective value for the serum.

Amoss and Wallstein (6) and Flexner and Amoss (6) show
that a rapid precipitation of anti-meningococcie serum has
decided benefits. The immune bodies of the horse serum were
estimated by testing its agglutinating and opscnizing power
with normal and parameningococcus strains, and by determining
its power to fix complement in the presence of antigens made
from these strains. Its anti-infectious power was deter-
mined by incubating varying amounts with one minimum lethal
dose of living meningccoccic antiserum for one hour at 37° 0.
and injecting the mixture intraperitoneally into young
guinea pigs weighing not less than 90 gm. or more than 110 gm.

Plexner and Amos (7) found that specific anti-serum
acts curatively by increasing the migration of leucocytes, by
promoting phagocytosis directly, by agglutinating the
meningococci, and by neutralizing endotoxin. Hence specific
anti-serum seems to provide the logical therapeutic agent
with which to combat epidemic meningitis.

McCoy (8), discussing the control of biological products

says, "There are but three antibacterial sera, anti-meninge-
coccic, anti-pneumonia, and anti-dysenteric, that are sub-
Jeot to official tests". In each case results secured with
any given serum are compared with a control serum distri-
buted by the Hygienic Laboratory.

ThJotta (9), Brekke (10), muir and martin (11), and
Pundit (12) demmnstrated that in the quantitative estimation
of immune bodies in bactericidal sera the "Phenomena of
Neieeer and Wechsberg" (1901) or "Complement Blocking":must
be considered and examined in an endeavor to bring about
satisfactory results with immune bodies.

Hazzi {15) treated one group of white rats with.anti-
abortus serum and another group with antiemelitensis serum.
Twenty-four hours later he injected them with lethal doses
of melitensis or abortus. He found that the anti-abortus
serum protected the rats against melitensis as well as
against abortus, and the anti-melitensis serum protected
them.against abortus also. Apparently there had been no
other experimental investigation reported showing the anti-
bacterial value of serum in relation to Brucella abortus

infections.

MODE OF PREPARATION OF SERUM

The problems presented in respect of the preparation of
an anti-abortus serum seem essentially similar to those en-
countered in the preparation of anti-pneummcoccic serum. In
each case similar factors must be dealt with; first,
numerous and varying types with very low, if any, toxin pro-
duotion; and second, antibody specificity for the several
types, as shown by agglutinin adsorption tests. Hence it
was assumed that by employing a similar method to that used
in the production of anti-pneumococcio and anti-meningococcic
serum similar results could be expected.
The cultures used were culture 238, which was isolated
July 1928 from the college Dairy Herd, and culture 811.,
isolated from.a fetus from the experimental herd in 1916.
The method consists of beginning with relatively small
doses of the organism in question.and gradually increasing
the size of the doses as rapidly as possible. Five slightly
different methods of injections into goats, cows, and a
horse were employed, namely:
1. Three intravenous injections of the growth from.one
agar slant, cultures 83L and 258, were used with a
three day interval between injections. (See Table I)

2. Subcutaneous and intravenous injections were given
alternately every second day until three injections
had been given. (See Table II. III, and 1v.)

3. Subcutaneous and intravenous injections were given

alternately every third day until three injections
had been given. (See Table VIII.) In each case
five to ten days were allowed to intervene between
series of injections.

4. Single injections were given subcutaneously every
ten days. (See Tables v and VI.)

5. Intravenous injections were given every second day
for three injections, maintaining a five day interval
between each series of injections. (see Table‘VII.)

In the methods just outlined, slight, if any, febrile
effects were noted after the first injections. The most
marked reaction as a rule followed the first or second series,
of injections. The second or third series apparently pro-
duced an anaphylactic shock, or hypersensitisation reaction
(washed culture being used) which may be due to the rapid
splitting of the bacterial proteins by the antibodies produced
as the result of previous injections. Since individual ani-
mals vary in their susceptibility and responses, slight
variations were noted.

In preparing a polyvalent serum of this type it was
thought desirable to employ an avirulent strain to build up
the titre and follow this with injections of virulent strains.
This method was employed in the injections of horse 100,
(Table I.) with slight increase in titre, following the use
of the virulent strain, but a marked emaciation end.loss of

appetite followed until it was deemed advisable to bleed the

10

horse to death. In contrast to this type of injection goat
185? (Table VIII.), receiving only an avirulent strain,
produced a much higher titre serum.

.An attempt was made to desensitiee the animals after
the method of Bull (14) (using a dilute suspension of the
organisms and injecting very slowly intravenously), who has
observed that when bacteria are injected into the blood
stream of animals immune to the same organism are clumped
and deposited in the blood vessels of the brain, lungs, and
in the spleen. Using this method,favorable results were
obtained in cows 04 and 28 (Tables II and III.).

‘11
TABLE I. SERUM PRODUCTION CHART
Cultures used, SxL and 238.
Horse 100.

 

' Date of

 

 

 

 

 

 

 

 

 

L ‘ L

 

- L

I 3
7131000 P7 Af‘er I
8/12/38 ,l:2000 r.bleedin8,

, out
I I I

' Remarks and ' Date of '
'injection ' Amounts 'cultures used'titre test' Tit!“ : Remarks,
v ; ~- T ; I T . T
. 6/29/29 ,1 agar slant , 81L , . ,1:50 ., ,
. 6/50/28 . . Temp. 105.6 , 7/ 4/28 ,l:lOO P, ,
, 7/ 1/28 ,Ho injection , , ,l:200 T. .
' 7/ 4/28 '1 agar slant ' 81L ' '1:1000 x' -'
' 7/ 5/28 '1 agar slant ' 81L ' 7/ 9/28 'l:2000 r' '
I 7/ 6/28 :1 agar slant : 8xL : :lzsooo 7: I
7 7 7 7 ‘ 7 7 7
. 7/ 9/28 .1 agar slant . 81L . .121000 x. .
. 7/10/28 71 agar slant . BxL .. 7/ 9/28 .172000 2. .
. 7/11/28 71 agar slant . 83L . 71:4000 r. .
I 7/15/28 :1 agar slant : BxL I :l:looo x: '
' 7/18/28 '1 agar slant , 83L ' 7/20/28 'l:2000 s, ‘
, 7/17/28 '1 agar slant , BxL‘ ' 'lzsooo r, :
. ; r ; 21,1000 .3 T
: 7/20/28 :3 agar slants: 238 : 7/25/28 '1:2000 r' '
. 7 . 7 7
1.4000 s

7 7 7 7 7 # 7 7
7 7 7 7 7 "h— 7 7
77 7 7 7 7132000 17 7
. 7/2l/28 .3 agar slants7 . 8/ 7/28 .l:4000 r. .
7 7 7 7 71:3000 T7 7
; ; :1 ; :1:4000 xT :
: 7/22/28 :3 agar slants: : 8/11/28 :lzeooo x: :
' . ' ' 'l:lOOOO m. '
I I I I ~ I I I
7 I 7 7 ' '- 7 , 7 3010!. 7
7 7 7 7 6/26/28 7}}3 E7 injec- 7
7 7 7 7 7 ’ 7 tion 7

' ; ' '7

I I

I I

I I

I
I
I
I

 

injections were given.intravenously.
strong reaction (complete).

partial reaction (moderate).

trace of a reaction (slight).

I s It”

FBI-UNI!”

TABLE

II.

Culture. used, 8117.

SERUM PRODUCT ION CHART

18

 

 

 

 

 

 

 

 

 

 

Goat 1000
' Date of ' Injections ' Date of ' ' '
‘infieetien 7 Am°unt' 7 given 'titre test' T1tr° 7 R°mark°7
5 if :I 5 i.— i‘ J
: 11/1/28 :.5 agar slant:Suhcutaneous: : 1:1000 x : :
, 11/5/28 ..5 agar slant'Intravenous , 11/7/28 , 1:2000 2 , ,
, 1:4000 7
: 11/5/28 :.5 agar slant:Subcutaneous: 11/12/28 7 174000 2 : :
I I I I I 1:8000 T I I
.7 / / 7 7 7 7 7 7_
7 11 12 28 7.5 agar slant7Intravenous 7 7 1:2000 P 7 7
7 11/14/28 7.5 agar slant7subcutaneous7 11/25/28 7 1:4000 7 7 7
7 11/16/28 7.5 agar alant71ntravenous 7 11/25/28 7 1:4000 P 7 7
7 7 7 7 7 1:8000 T 7 7
.: 11/25/28 :75 agar slant:suboutaneoue: : 122000 P :' I
, 11/25/28 , 1 agar slant,1ntravenons , 12/5/28 , 1:4000 2 , ,
, 11/27/28 , 1 agar slant,subcutaneous, , 1:8000 T , ,
' 12/5/28 '.5 agar slant'lntravenous ' ' , ' '
' 12/7/28 ' 1 agar'slant'Suboutaneous' 12/18/28 ' 138383 i ' 7
: 12/9/28 : 1 agar slantzlntravenous I z ' : '
I
I I I I I I I
7 12/18/28 72 agar slants73ubcutaneous7 7 1.4000 P 7 7
7-12/20/28 72 agar slants7lntra7enous 7 12/29/28 7 1:8000 r 7 7
7 12/22/28 72 agar slants7Suboutaneous7 -7 ° 7 7
' 12/29/28 '2 agar slants'lntravenous ' ' ' . ' '
' 12/51/28 '2 agar slants'Suboutansous' 1/9/29 ' izgggg‘; ' 7
' 1/ 2/29 ’2 agar slants' ' ' ‘ ' '
I I I I I I I
I I I I - I I I
77 1/ 9/29 73 agar slants7suboutaneous7 1/20/29 7 1:4000 P 7 7
7 1/11/29 73 agar slants7Intravenous 7 7 1:8000 T 7 7
I I I I I . I Before I
' ’ ' ' 11/1/28 ' i223 ; ' injeo- '
' ' ' ' ' 7’ ‘ tion '
I I I I I I I
x - Strong reaction
P =2M088rate rsactimn
T “ Slight reaction

13

 

 

 

 

 

 

 

 

 

TABLE III. SERUM PRODUCTION CHART
Culture used, 831,.
00' C4.
’ Date of 7
'inJeotion 7 Amounts : Injection. ' Date °f ' ' 7
2:: 7. 7 Sivan 'titre test' Titre 7 Remarks,
7 l ‘
11/ 3/28 ..5 a a. ' L I
I 11/ 5/25 ;.a 7.3.: 33:71777777777777. 7 I 171000 17 i '
7 11/ 7/28 '55 agar elant' 77 ' 11/12/28 7 132000 T 7’ '
' ’ I I ' 1:4000 T ' :
' 11/13/23 .5 a 317 a ' ' 7 .
7 11/14/28 :75 agar 8%::::Intr8:enoua ' ' 1‘2000 P : I
’ 11/16/28 7.5 8881‘ slant 77 ' 11/23/28 ’ 1:4000 P I
r A ' ' I 1:80 '
_: L 00 T I '
7 11 1
11/23/28 '55 8 I j ; 2
7 11/25/28 1.5 732: 2%zfifi71ntr83°n°nfi I 12/ I 1:2000 r ' 7
7 11/27/23 7 1 agar slant'Suhcutaneoum' 4/28 7 134000 T I '
' ' . 7 7 7 1.8000 7 7 7
7 12/ 4/28 7 5 agar slant. ' ' '
7 travenous . ' 7
7 12/ 5/28 7 1 agar slant'gn ' 7 1.1000 2
2 boutaneous 12 - ' '
7 12/ 8/23 3 a a, ' u 7 /15/28 7 1.2000 2
. 7 g slantszlntravenous 7 w 7 174000 7 7 Slight I
I . ' I I .hOOk I
7 12/15 28 72 agar slants' ' '
Subcutaneous ' '
7 12/17 28 72 3831- Ilanta'ln ' 7 1:1000 P
travenous 1 - ' '
712 19 2 7 7 2/25 28 1.200
7’ / / 8 :2 agar slante:Subcutaneous7 / : 174008 5 I 7
7 12/25/28 72 agar slant 7 ' ' 7 *7
travenous ' 7
' 12/23/7‘3a ‘2 agar 818771787m 3 3 3 3 ' '
s Subcutaneous' .
112/50/23 :5 agar slantai1ntravenous 7 1/‘7/29 : 1.1000 T : I
I ' ‘ L L L I
7 1/7/ 29 73 agar slants. ' '
Subcutaneo 7 '
7 1/9/ 29 73 agar slants71ntravenouga' 1/17/29 ' 121000 P ' :
. . 7 7 1.2000 T 7
7 ' r 7 '
' . I I ' 1085 1' f —r
7 7 3 Be ore 7
7 7 : : 11/13/28 7 1:50 P 7 injeo- 7
' ' 7 7 ' 13100 I ' tion 7
I ' 1e500 ' I
I ' ' '

1/20/29

 

1" Strong reaction
P 77 Moderate Mftion
T ' Slight reaction

TABLE IV.

Culture used, 83L.
Goat 490.

SERUM PRODUCTION CHART

14

 

’ Date or

:injection 1

7:722
9/25/23

27727::

10/25/28
11/ 1/28

3? 2523

r

Amounts

agar
agar
agar
agar

agar
agar

agar

7 Injections
' given

-
j

‘Intravenous

II
II
II

slant
slant
slant '
slants:

I
II

slants’
slants:

. ‘ C C ‘ fl ‘ ¢ ‘ ‘

slantszsubcutaneous
I

' Date
:Titre test'

8/15/28
:7 :72

10/19/28
10/50/28

of '

Titre

'l:500
~'l:10000
'l:10000
'1:8000
'l:8000
'l:8000
'l=BOOO
'1:8000
:1:8000

agar
agar
agar

slant7subcutaneous7 7
slant7Intravenous 7 11/ 7/28 71:8000
slant7suboutaneous7 11/12/28 71:8000

WNWBHBHBN

N"d

Remarks:

 

11/12/28
11/14/28
11/15/28

11/25/28
11/25/28
11/27/28

12/ 7/28
12773733

FHJUI

agar
agar
agar

agar
agar
agar

agar
agar
agar

U f

slant:Intravenous
slant78ubcutaneous' 11/19/28
slant.Intravenous : ll/23/28

._‘_

'l:8000
:l:8000

I I I
slant7subcutaneous7 7
slant7Intravenous 7 12/ 7/28 7
slant7auboutaneous7

I I;

1:8000
I
slantIIntravenous

slant78uboumaneous
slant7 "

12/17/28 1:8000

0":-

"d'a‘

4---.---..-J7

 

‘Cdd‘C‘CCCCPC-..C.‘.1P..CQ.C.UCOC.‘C

12/18/28
12727::

C‘QC‘O‘CO

FHJFJ

agar

agar
agar

slant78ubcutaneous7
slant7lntravenons 7 12/29/28 1:8000
slant'Subcutaneous7 7

.7 7 8/25/28 71:25

7 7 71:50

I I I

I
I
I
I
I
I
I

NH

....fleogdgqonrooaee...0dlpflsoCC‘CCC-OCCC..Pg¢

Before
injec-
tion

.““C'CCO‘.‘-.“.‘-

 

x - Strong reaction
P I"Moderate reaction
T = Slight reaction

TABLE-V. SERUM PRODUCTION CHART

Culture used, 82L.

15

 

 

 

 

 

Goat 101.
7 33:. of 7 Injections ' Date of ' ' I
Iinjection I ANDunt. 7 given "1‘r. test' Titre ' Remarks.
: 11/25/28 :.5 agar slant:8uboutaneous: 12/ 5/28 : 1:200 .x : :
1:500 P
' 12/ 5/28 '.5 agar slant'Subcutaneous' 12/18/28 ' 1:1000 7 ' '
I I I I I I I
7 12/18/28 .2 agar slants.Suboutaneous' 12/29/28 ‘ 1:1000 7 7 7
' 12/28/28 .2 agar slants'Suboutaneous. 1/ 8/29 . i:%ggg 7 7 7
= 7
I 1/ 8/29 :5 agar slants:8ubcutaneous: 1/18/29 : 1:2000 r : :
1:4000 T
‘ 1/18/29 '5 agar slants'Suboutaneous' 1/28/29 ' 1:1000 7 ' '
' ' ' ' 11/23/28 ' 1:100 x ' Before '
' ' ' ' ' 1:200 P ' injeo- 7
' ' ' ‘ ' 1:500 7 ' tion '
I I I I I I I
71212 V1. SERUM 2702007107 05227
Culture used, 81L.
00‘ Bee
7 Date of 7 Injections ' Date of ' ' '
'injection 7 Amounts 7 given 7titre test' Titre ' Remarks,
' 11/25/28 '2 agar slante'subcutaneous' 12/ 5/28 ' 1:500 x ' '
I I I I I I I
' 12/ 5/28 '2 agar slants'suboutaneous. 12/18/28 ‘ 1:1000 7 ' '
12/18/28 2 agar slants Subcutaneous 12/28/28 1:1000 7
: 12/28/28 :2 agar slants:Subcutaneous: 1/ 8/29 : 13:000 7 7 z
1: 000 7
' 1/ 8/29 '2 agar slants'Subcutaneous' 1/18/29 ' 1:1000 7 ' '
I I I I I e I I
' 1/18/29 '2 agar slants'suboutaneous' 1/25/29 ' 1:1000 7 ' 37fore 7
7 7 7 7 11/25/28 7 «1123‘; ; 7 injec- 7
7 7 7 7 7 ° 7 tion 7

 

x - Strong reaction
P 7' Moderate reaction
T ' Slight reaction

TABIE VII. SERUM PRODUCTION CHART

Culture used, 831..

16

 

 

 

G03‘ 1850
7 Date of 7 7 Injections ' Date of 7 7 7
'injection 7 Amounts 7 given 7titre teat' Titre 7 Remarke7
_I_ l L L I L _I_
7 7 7 7 7 7 7 7
7 10/15/28 7.5 agar elant71ntravenoue 7 7 7 7
7 10/15/28 7.5 agar elant71ntraven0ue 7 10/20/28 7 1:1000 7, 7
7 10/17/88 7.5 agar elant7Intravenoue 7 7 1:2000 7, 7
7 ' 7 7 . 7 7 7 7
7 10/25/88 7 1 agar elant7Intravenoue 7 7 7 7
7 10/25/88 7 1 agar elant7Intravenoue 7 7 7 7
7 10/27/28 7 1 agar elant71ntravenoue 7 7 7Died 7
7 7 7 7 10/ 3/23 7 1:200 P710/88/287
7 7 7 7 1:500 7

I
L

I
L

 

x 7' Strong reaction
P .7 Moderate reaction

'1‘ =7 Slight reaction

TABLE VIII.

Culturi need, 81L.

SERUM PRODUCTION CHART

17

 

 

 

 

 

 

 

Goat 185E.
' Date of 7.1n1ectione 7 Date of 7 7 7
7injection.7 Amount: 7 given 7titre teat7 Titre 7 Remarke7
7 7 1 1 r
7 11/ 1/23 ..5 agar alant7Subcutaneoue7 11/ 8/28 :1:2000 x : 7
7 11/ 4/28 ..5 agar elant7Intravenoue 7 .1=4000 7 . .
7 11/ 7/38 ..5 agar elant7Subcutaneoue: 11/12/23 .izaoog 7 . .
7 7 I . 7 3300 T 7 7
4 L 7 _ A
7 'r— r 7 *_" *
I
7 11/12/28 '.5 agar elant' 7 11/19/28 71:4000 r ' 7
7 11/15/28 '.5 agar elant' 7 - '1:8000 7 ’ '
7 11/18/28 7.5 agar alant7811ght shock7 11/25/28 71:4000 r 7 7
7 v 1:8000 7
L I I L I
7 ' ’ 7 f : 7
7 11/25/29 ..5 agar elant7 7 12/ 5/28 71:8000 x 7 ,
7 11/26/28 ..5 agar alant7811ght1y 171-, 71:10000 7 , .
7 , .creaeed shock. 71320000 1' . 7
7 11/29/28 . 1 agar elant7- . , . 7
' :v— 3 3 I v #7
7 12/ 6/28 7 1 agar a1antjMarkea shock: 12/14/28 71:8000 x 7 '
7 12/ 9/28 7 no injection7 7 71:10000 P ' '
. 12/12/28 7 no injection7 7 71:20000 7 ' '
l 7 7 7 71:30000 7 7 I
7 I I T 7 I I
, 12/29/28 7 1 agar elant73ubcutaneoue' 12/29/28 71:1000 x 7777: an
, 12/31/23 7 1 agar alantvxntravanona 7 71:2000 7 77.7:- large.
, 1/ 2/29 7 1 agar elant7subcutaneoue' 71:4000 7 vbleedingv
7 7 7 : 71:8000 T I 7
I ' ' ‘
7 1/ 9/29 :5 agar slantehntravenoue 7 11/ 1/2e '1:25 7 737“" '
7 7 .1350 T injec- '
7 ' 7 7 7
7 a , tion
f 7 ' L_ I I

 

z - Strong reaction
P '7 Moderate reaction
T ' Slight reaction

18

TECHNIC 0F METHOD FOR DETERMINING TE! BACTERIOIDAL
ACTIVITY OF THE SERA

Pour methods were employed to determine the relative
potency of the various sera prepared. Three cultural
methods eere used and one in.vivo.

The agglutinating sera from the human and animal
sources were tested from time to time by the rapid agglup
tination method developed by Huddlescn and Carlson (15).
mhe results are shown in Tables I to VIII inclusive. The
agglutination test was employed for the purpose of demon-
strating its value as an indicator of the badtericidel or
therapeutic properties of the serum.in question then can»
pared with in vivo bactericidal tests.

The method used to demonstrate the presence of bacteri-
cidal substances in the sera is as folloes: a standard
suspension of living organisms was prepared from.a 48 hour
growth on liver infusion agar slants so that a turbidity or
one, two, and five centimeters by the Gate's nepholometer
was obtained. Dilutions of 1:25, 1:50, 1:100, 1:200, 1:600,
1:1000, 1:2000, 1:4000, 1:8000, 1:16.000, 1:32,000, 1:64,000,
1:128,000, l:856,000, l:512,000, and l:l,024,000 of the high
agglutinating serum were prepared with sterile physiological
salt solution (PH‘678). To one cubic centimeter of each or
the above dilutions was added one drop of the standard bac-
terial suspension. The drops Iere delivered 1rom.capillary

pipettes of 12, 16, and 20 gauge, standardized by the use of

19

the united States wire gauge. Dilutions of the serum and
suspensions of like turbidities of the living organisms eere
used as controls. The tubes of serum dilutions and bacterial
suspmnsions were incubated at 37 degrees Centigrade and
seeded with a d mm. wire loop after 2, 10, and 24 hours
incubation in portions of gentian violet liver agar plates

as described by Huddleson (16). The results were noted at
varying intervals, and final reading taken 72 hours after
the 24 hour plating.

The final readings were tabulated in accordance with
the amounts of growth, as shown in Tables IX to XIX inclu-
sive. An even, dense growth was charted as three plus, a
seeded area with moderate but not dense growth was charted
as two plus, one with but scattered grewth.was charted as
one plus, and no growth was charted as negative. Contami-
nation and modifications of the above discussed method of
charting are explained on the individual charts.

This so-called 77quantitative technie77 was.employed in
place of the plate counting method in an endeavor to avoid
such errors as the clumping of organisms by the high agglu-
tinin content of the serum, and dead organisms present in

the standard suspension.

2

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. I a I . I . I . I . I . I . I . I . fl . + . 7¥ . ++ .. .
. a I . I 7+7 I . I r I . I . I b M“ . M . +++PI+H+P +++7PH£ flm.
. ++ . I . I a I . I . I . I . # . . . fl . . fl .0 O .
. a I a I u I p I P I p I F I . W» H _ +W+ p H . M - MS H. mm
. +++ . I . I r I . I . I . I . # . . . . . I .o .
a a I717 I p H . I . I . I . I . # .?7 fl . +++ a * . # . H: N .
. + . I . I a I . I . I . I . I . I a + a I . + . ++ .e .
. L- I a I a I . I P|7I . I p I a I . * . I . * . ++ 75 HS dN.
. + . I . I . I . I . I . I . I . I . + . I . xfl . I .ohfl OH. 0H
. b I 7; I 7% I p I . I p I . I a I a * . M a * . I .a .
. +++ . I . I e I . J . I . fl . I a I . + a . * . I ..H .
P F I F I L I _ I u I F # L # .— # p * p I” . ... a I P A N F
. + . I .. I a I .. I . I . I . I . I . I . I . + . I .enfi #N.
. a I p I p I a I _ I . I 7+7 I P I .P7 I ..IHW. + . I _ .
omwd . +++ . I . I . I . I . I . I . I . I . I . I . +++ . I .ehfi OH. NH
Ednom . a I r I P I . I fiL“ . I 7% I P7 I 7? I a m . +++7P7 I . .
mohm . +++ . I . I . I . I . H a I . I . I . 7I . . +++ . I ..H .
n . a I P7 I . I . I . . fl . I p I . I _ I . +++ . I P Q N P mhi
. . .oooeo.ooom7.cooafi.ooom .oooe .ooom .ooOH . com . com . ooH . o . . mane .opao am
mMHdBmm.Moono. H . H N.. H . H . H . H . H . IMW7. IAI7. IAW7. Iflm.. Imm.—QOHPQD. Ho
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21

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mason ma nopms me.> mg npeonm assessed 7 +++
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”71.7mm? 7” 7 ”77 71m ”starring ”7 ”as”
n H +++“ W H +++ H N H +++ H +H+ H +++ H +++ H +++ W +++ h +m+ H +++ ”.Hn OH” mm
mm - +++ - m — +++ — + . +++ - .7“ . +++ — +++ — +++ — +++ - +++ - +++ .eHfl N p
h h? b h p h r b h I IP b b I?
”$7. - H a; 7. 7 Km “rm .Hm Tm ”7:7me577”
dons . +++ . ++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ ..nn on. 0H
sauce amonm. 7r, 7 . ++ r . o .e . o r, 7n» L . vat .
. +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ ..n: m .
.P pl 0 I r b p r h p P h h o b h
. +++ . ++ . +++ . ++ . +++ . ++ . +++ . +++ . w++ . +++ . +++ . +++ ..Hn wm.
QOHPQQSOHH. p p P . - -I p a a . u r P —
.mnn Nb Hound. +++ . ++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ ..hn OH. NH
06.68 mwflfiddQM- p h p r L F p L _ p u h .
. +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ ..Hn m .
u p u u p u p — u p . a a a
. .ooomm.oooafi.ooom .oooe .ooom .OOOH . com . oom . 00H . on . mm . mean .uuauqfim
uMHdSom .Mooflo. - . . . . . II... . IIII . 7|ll . ..III . Ill .doapdn. HO
. . H u H . H . H . H . H . H . H p H . H . H .IfiodH _ owddw

 

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22

TABTE XI

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seesaw soaaapaaa asmHHm 7 a
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Ansonw oz 7 7
neeonm assessed a +++
:paonw openeooa 7 ++
nHOHpepsqu mason me apaonm pnMHHm a +
poems and seamen o.> mm cepquampnoo 7 e
. +++ . ++ 1 ++ . +++ . +++ . +++ . ++ ..+++ . +++ . +++ . +++ . *7 . + ..Hn fim.
. — ++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . + . + . .
. +++ . ++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . ++ . +++ ..nn OH. mm
a . ++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . ++ . +++ . u
. +++ . +++. +++ . +++ . +++ . +++ . +++ a +++ . +++ . +++ . +++ . ++ . +++ .ehfl N .
. . +++. +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . .
. +++. +++ . +++ . ++$ . ++ . +++ a +++ . +++ . +++ . +++ . +++ . ++ . * pond fim.
. . +++. +++ . +++ . ++ . +++ . +++ . +++ . +++ . +++ . +++ . ++ . ++ . .
. +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . ++ .. ++ —eh£ OH. OH
. . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . ++ . ++ . .
. +++ . +++ . +++ . +++ a +++ . +++ . +++ . +++ . +++ . +++ . +++.. +++ . +++ ..An N .
. . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . .
. ++ - ++ . +. . ++ . +++ . +++ . + . +++ . ++ . ++ . +++ . ++ . ++ .eHS #N—
. . +++ . +++ . +++ . +++ . +++ . ++ . +++ a ++ . +++ . +++ . ++ . ++ . .
BMMMM_ +++ - +++ . +++ . +++ . +++ _ +++ . ++ . +++ . +++ — +++ . +++ . ++ . ++ .eofi."H OH. NH
monm. . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . ++ . +++ . .
n . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ ...HH.H N .
. . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . .
mthamm.Moono.©oo¢o.000mm.ooomH.ooom .OOO¢ .ooom .ooon . mmm_. com . mmm.. on a 7mm..dwmmw .mummmnm
. . H . H . H . H . H H H . H u H . H . H . H . H . a a. a
_ a u — -

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spawn ego no apeonm oz 7 o
npaonm oz 7 7
Apaonm assessed 7 +++
ataonm mumsmeoa 7 ++
GOHPmpdonH mason ma apaonm panHm 7 +
page Home seamen oi. mg cmpenHampsoo .... ...
. +++ . +++ . e . ++ . +++ . ++ . ++ . ++ . +++ . ++ . o . o . + ..An em.
. r +++ a +++7. +++ r +++ r ++m8. +++ . +++7r,+++ . +++ . +++.h + 7» + 7» .
. +++ . +++ _ + . +++ _ +++ . +++ . +++ . +++ . +++ . +++ . + . + . +++ ..nn 0H. mm
. . +++r1+ . +++ . +++ T ++++ +++ T +++ . +++ a +++ . +++; +++L +++L .
. +++ . +++ _ +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . ++ . ++ . +++ ..nn m .
. . ++wt. +++7r +++ a +++ . +++.r +++ rt+++ . +++ . +++ r +++.e +++ r +++.r »
. +++ . +++ . ++ . ++ . ++ . +++ . + . +++ . + . +++ . o . m . + ..nn em.
. 7r +++ _ +++ r ++ 7. ++ 7. +++ . + . +++.. +++7. +++.. +++ . + . + ,. .
. +++ . ++ . ++ . ++ . ++ . +++ . +++ . +++ . ++ . +++ . +++ . +++ . ++ ..nn OH. oH
. . +++ r +++ P ++ 7r ++ _ +++ur +++ . +++ _ +++ . +++7r +++ . +++ . +++ . .
. +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ ..ng m .
. .P7+++ . +++ . +++7. +++a +++ . +++._ +++ . +++.r +++ . +++7. +++ r +++ . .
. +++ . +++ . ++ . +++ . ++ . +++ . +++ . +++ . + . 7+ . + . o . + ..nn em.
Home . 7r. +++ . +++ . ++ . ++ . +++. +++ . ++ . + 7. ++ . + . + .r +++ . .
aspen . +++ . +++ . +++ a +++ . +++ . +++. +++ . +++ . +++ . +++ . +++ . +++ . ++ ..nn OH. NH
. 7,7 r14++ . ++ . +++ . +++ . +++. +++ . +++ . +++ . +++ . +++7r ++ . +++ . .
Sachem. +++ . +++ . +++; +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ ...H .
. . +++ . +++ . +++ _ +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . n m .
. .oooea_ooomm.oooaH.eoom .oooe .ooom .OOOH . com . oom . ooH . om . mm . asap .aaquqflm
mammaomgoono. H . H . H . H . H . H . H . 7|H.l . I..H...7 . IHII . I7Hl. .. Iml .nogmp. mo
. . . . . . . . _ . . . . .IdodHL omgc

 

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24

who data in Tables 1x to XII inclusive show the in
vitro bactericidal effect of horse serum containing specific
agglutinins. The agglutination test was employed to evaluate
the height of antibody production in all sera used in this
test. Several normal sera were used with no bactericidal
effect demonstrated. Inconsistencies may be observed in
Tables IX and X which.sers believed to be due to the change
in the pH of the salt solution. It may also be observed as
shown in Table IX that with a marked change in pH’a decrease
in.the growth is evident in both.serum.dilutions and con-
trols. While in Table X only a slight change in the pH of
the salt solution was evident, no decrease in the controls
and only a slight decrease in the higher dilutions could be
noticed.

The results charted in Tables XI and XII shes the only
positive bactericidal results obtained by the use of
specific horse serum. It may be observed that bactericidal
effect is exemplified to only a very limited degree in the
twenty-four hour seeding of the lower dilutions. The fee

slight variations can not be accounted for.

TABLE XII I

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mesonm oz 7 7
row mesonm unsung; 7 +++
museum canneeoa 7 +7
seesaw asmarm 7 +
+++. +++ . + . + a 7 . 7 . + . ++ .+++ .+++ .+++ .+++ .+++.+++.+++.+++.+++..Hn «N.
. . p a . . - — . . p a — . a u u .
+++a +++ . + . 7 . 7 . I . ++ . 7 .+++ .+++ .+++ .+++ .+++.+++.+++.+++.+++..nn em.
. . . . . . . . . . _ . . . . .65? . mm
+++. +++ . +++ . ++ . ++ . + . +++ . +++ .+++ .+++ .+++ .+++ .+++.+++.+++.+++.+++..A£ em.
. . . . . . . . . . . . . . . . . .
+++. +++ . s . ++ . 7 . ++ . +++ . +++ .+++ .+++ .+++ .+++ . s . r a I .+++.+++..H: em.
. . . . . . . . . . . . . . . . H %. .
. . .
+++. +++ +++ . ++ . + . ++ . ++ . +++ .+++ .+++ .+++ .+++ .+++.+++.+++.+++.+++..n£ or.
. . . . _ . . . . . _ . . . . . . .
+++. +++ . ++ . ++ . ++ . ++ . ++ . 7 .+++ .+++ .+++ .+++ .+++.+++.+++.+++.+++..Hn or.
. . . . . . . . . . . . . . . .65? .
. . . . . . . . a . . . . . u — ++H. e . NN
+++ +++ ++ +++ +++ . ++ . +++ . +++ .+++ .+++ .+++ .+++ +++ +++ +++ +++ an or
p — u _ . - u . u a -
+++. +++ . s . +++ . ++ . +++ . +++ . +++ .+++ .+++ .+++ .+++ . s . r . 7 .+++.+++..nn or.
. . . . . . . . . . . . . . . . .7 um. .
+++. +++ . +++ H +++ H ++ ” a++ H +++ U +++ ”+++ ”+++ Ha++ ”+++ ”+++.+++.+++.+++.+++..Hn m .
u u - a u u n —
+++. +++ . +++ . +++ . ++ . +++ . +++ _ 7 .+++ .a++ .+++_.+++ .+++.+++_+++.+++.+++..As m .
. . . . . . . . . . . . . . . .A ”F L
+++. +++ . . . . . . . . . . ... . . . . “++- e . mm
. . +++ . +++ . +++ . +++ ..+++ . +++ .+++ .+++ .+++ .+ + .+++.+++.+++.+++. . A: m
. -
+++. +++ . s . .r . +++ . +++ . +++ . +++ .+++ .+++ .+++ .+++ . a . e . 7 .+++.+++..Hn m .
. . . . . . . . . . . . . . . . H %a a
O _ . . . . . . . . . . . . . . . . . as
U. . . . . . . . .. . . . . . . . . . 955 .mm
M . 000¢NOT 000m Hm.000©nm. 0000M... 000$? 000mm. 00005 0000. 000$. 000m. 000 r. 000. 00m. 00 r. on . mm .nOHPdn. “Noam.
3.. ... . H. . .... . H . H _. H _ r . ... . H. . .... . ... .H. .7. H. H.H..I§oflH.Me+0
. . . . . . . . . . . . . . . . . L I.

 

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mm\mr\m

26

TABLE XIV

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maHeoHoa oH eaaosa on sea 7 a
nOHpsaHaspuoo s s
museum oz 7 7
Agoaw pangfla 7 +++
gpaoam openeeQfl 7 ++
seaoam paerm 7 7
+++ . I _ I . I . a . ++ . ++ . +++ . +++ . ++ . +++ . +++ . +++ ..an em.
P F r b F b h h I? L P L h -
+++ . 7 _ 7 . + . + . ++ . ++ . ++ . +++ . a . +++ . +++ . +++ ..93 em.
h P Ir L r h h Fl h {F P F up - NN
+++ . I . I . I . + . ++ . ++ . ++ . +++ . +++ . +++ . +++ . * ..HS em.
I h r r h B! h h — h b in p h -
+++.. I . I . I . + . ++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ ..nn Hm.
b h P n in, u p n L F b h L B
+++ . I . I . I . ++ _ + . ++ . +++ . +++ . ++ . +++ . +++ . +++ ..93 OH.
. . . _ . F H r T . F . . .
+++ . 7 . 7 . 7 . ++ . ++ _ ++ . +++ . +++ . +++ . +++ . +++ . +++ ..nn Or. .
b! n h u l- L n h L a p — |—’ u mm
++ . I . 7 . I . ++ . ++ . ++ . +++ . +++ . + . +++ . +++ . a ..HH 0H.
_ >1 L h r — bl — u u u u a —
+++ . 7 . I . + . ++ . +++ . +++ . +++ . +++ . +++ . +++ . ++ . +++ ..as OH.
+ r r F . . _ L r . . . . F
++ . I . I . I . + . ++ . +++ _ +++ . +++ . +++ . +++ . +++ . +++ ..an m .
P P L n L P L n L h — lml - —
+++ . 7 . I . + . ++ . ++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ ..nn m .
n h n L a - r pl - p p [—I uli - NW
+++ . I . I . + . ++ . +++ . +++ . +++ . +++ . +++ . +++ . +++ . * ..an m .
F —I P + u — a . u n u p u -
+++ . 7 . I . + . +++ . +++ . +++ . +++ . ++ . +++ . +++ . +++ . +++ ..n: m r
. .. . . . . . . . a . . . J .
.oooeo_ooomm_oooer.000m .oooe .000 .000 . oom _ oom . OOH . on . mm . asap .aepaHHm
Mooflo. H . H . H7 . ... . H. . ... . .r . IIHWI . 77.7.7.7 . IIHII . lurll . Ilml. .QOHPGQ. Ho
. . . . . . . . a . . . .ISOHH . mmwdw
mQOdeer_asamm
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B7

TABTE XV

hpfidfipnsa .mu m a nomfipqa anacqmpm
apaonm ownmpuwom vnmarm 7 «

copaaadpnoo 7 *
Avgonm 02 I 7
«339% 3322.54 ... +++
npsonm mpmnodoflvl ++
npsonm pnmflnm a +

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 +++ . I . I . I r I . +++ . +++ . +++ . +++ ..hfi #m.
.mfir iL, . F LI L. .r .7 IL L .
. +$+ . I a I m I . I . +++ . +++ . +++ . +++ ..HS dw—
. . . _ E; . _ . . b . mm
. +++ — I u I .- ..l p .I - +++ — +++ - +++ - +++ - OB fiN—
. L . _ . L . . . . .
. +++ 1 I . I . I . I . ¢++ . +++ . +++ . +++ ..Hfl #N.
r‘ I. V . p . . . a . b
. +++ - I n .l — .I - I - +++ - +++ - +++ - +++ .03 0F.
. P . p F . u . . . III.
. +++ . I . I I II . I . +++ . +++ . +++ . +++ shfl On.
. _ P \b . . P7 . . . . mm
. +++ . I . I . II . I . +++ . +++ . +++ . +++ ..H£ OH.
. . . . P . . . . . .
- +++ - II a II n I- n It - +++ - +++ — +++ - +++ .0B or.
r. . . . . . . . . . .
. +++ . + . + 1 fl . fl . ¢++ . +++ . +++ . +++ ..HQ N .
. . . . . . . . . . .
. +++ . ... . + . a" . n." . +++ . +++ . +++ . +++ ..E m .
flohcfigam - a n p a u — lb - a — NN
gnwm - +++ - ... — 8n — + - ... - 1+ - * - +++ - +2....- 10ng N —
Hmonm . a b a u u p a — _ p
. +++ . + . + . + . +++ . +++ . +++ . +++ . +++ ..H: m .
. . . . . . . . . . .
. . . . . . . . . .Hwafldldddddqflm
mMHdHBm .MOOQO.OOM¢ .OOMN .OOMH . WWW. _ WIMIW . mam-W . ...-WWW . Ilmlm... .fioapwn. Ho
. . . . . . . . _ r .IdodH . omddw

 

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noapaaadpqoo 7 LL .

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

aw Agonm oz 7 I
museum 33354 ... +++
nekouw opmnouoz 7 ++
55% 23.8 .. +
L
L +++ L 7 L 7. L I L 4+ L ++ L ++ L ++ L +++ L * L +++ L +++ L +++ L.nn «m.
L L L L L L T L I p L r L L L L
L +++ L 7 L 7 L I L + L ++ L ++ L +++ L +++ L +++ L +++ L ++¢ L +++ L.nn #m.
L L L L Li P L L L L L L L b L mm
.L +++ L I L 7 L + L + L ++ L ++ L ++ L +++ L +++ L +++ L +++ L +++ L.Hn fiNL
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I80 L LI L L L L L L L L L L L L L mm
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mourn L L L L L L L L L L L F L L L
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TABLE XVII

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XVIII

TABIE

.30

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31

It may be noted from results shown in Table XIII when
72 hour old specific human serum inactivated with.guinea pig
homplement was employed there appeared a zone in the 1:16.000
to l:512,000 dilutions of the serum; and from results in
Tables XIV.and,XV when fresh specific human serum was emr
ployed a zone is seen in the 1:500 to l:64,000 dilutions of
the serum. While in 72 hour old specific human serum and
normal human serum no bactericidal action was observed. The
above results were checked and found to be constant with
only a few slight variations.

It may also be observed from results shown in Tables XVI.
XVII, and XVIII When specific cow serum was employed there
appeared a zone in the 1:400 to 1:64.000 dilutions of the
serum. while no bactericidal action was observed when
specific cow sera inactiVated with guinea pig complement
were tested. Three day old specific cow sera were also
observed to show no bactericidal effect when the in vitro
method was employed. Her was the bactericidal effect in-'
hanced by defibrinating or citrating the blood.

It was also observed that with an increase in the con-
centration of the bacterial suspension there was no bacteri-
cidal effect. The same was true when the specific nor: were
inactivated with guinea pig complement and employed with an

increase in the bacterial suspension.

32

SLIDE CELL DETERMINATION OF THE BACTERICIDAL
POXER OF WHOLE BLOOD

iaitland {17), wright, (18), and wright, Colebrook, and
Stores (19) describe a technic in which whole blood mixed
with a known quantity of bacteria was incubated in slide
cells. as an experimental method for the investigation of
certain phases of immunity. In the above cited experiments
the expression of bactericidal value was obtained in terms
of the number of colonies which developed in a given volume
of blood (the number of implanted organisms being known) and
represents the combined action of the cells and serum over a
period of 24 to 48 hours at 57° C. The results of which
were compared with those of serum alone.

The technic described by Heist and Solis-Cohen (20) and
later applied to experinerms on several organisms by Parks
(21) and Kolmer and Borow (22) was somewhat similar in pur-
pose and results to the method mentioned above, but employed
capillary tubes in place of slide cells. ‘

The following observations were made using a modifica-
tion of the technic described by Kaitland. A standard tur-
bidity of living organisms was prepared by the use of the
Gate's nephelometer; three complete slide cell groups‘were
prepared and incubated for varying intervals, namely 2, 10,
and 24 hours, at 57° C. and the growth was charted in re-

ference to the amount, three plus indicating abundant growth,

33

two plus moderate growth, and one plus only slight growth.
No growth at all was charted as negative.
No bactericidal effect was observed when specific

horse, cow, and goat sera were employed.

TABLE XIX. SET UP FOR WHOLE BLOOD EXERILENT. e/12/2e.
Cow # c4.

 

' Whole Blood ' organisms ' Serum dilutions
1 ' 12 guage ' none ' 12 guage 1:50
2 ' none ' 12 guage ' 12 guage 1:50
3 ' none ' 12 guage ' none
4 ' 12 guage ' 12 guage ' none
1:25 ' 12 guage ' 12 guage ' 1:25
1:50 ' 12 guage ' l2 guage ' . 1:50
1:100 ' 12 guage ' 12 guage ' 1:100
1:200 ' 12 guage ' l2 guage ' 1:200
1:500 ' l2 guage ' 12 guage ' 1:500
1:1000 ‘ 12 guage ' 12 guage ' 1:1000
1:2000 ' 12 guage ' 12 guage ' 1:2000
1:4000 ' 12 guage ' l2 guage ' 1:4000
1:8000 ' 12 guage ' 12 guage ' 1:8000
I i 8

 

Standard turbidity 5 cm. (organisms)
Serum from Cow c4, e/7/2e

PREEaRATION OF INFECTIOUS MATERIAL Ah METHOD
OF EXPOSURE

Four virulent organisms from different sources were
used in the in vitro portion of this experiment. Culture
238 was isolated July 1928 from the College Dairy Herd.
Culture 251 was isolated from the college Dairy Herd.
Culture 196 was isolated from the fetal.nembranes from a
cow in the college Dairy fierd on January 14, 1929. Culture
12 was obtained from Mathew's, Purdue University.

The virulent strains of Brucella abortus were grown on
liver infusion agar slants for 48 hours, in an atmosphere of
10 z 002 and aerobically. The growth was removed with
physiological salt solution, and sprinkled over the feed of
the guinea pigs, or injected intraperitoneally in standard
doses. when administered by feeding one third to one agar
slant per guinea pig Was used. The varying amounts given
intraperitoneally are shown in each table. In each case

the exposures were several times the infecting dose.

METHOD OF DETERMINING INFECTION

The exposed guinea pigs were under observation for a
period Of six weeks after exposure to infection. This time
is sufficient for lesions to develop. it the end of this
period they were bled for an agglutination test. The

agglutination test was run in accordance with the method

35

develOped by Huddleson (15).

The guinea pigs were then killed and autopsied and a
search Was made for any anatomical changes. An attempt was
made to culture Brucella abortus from.the lungs, liver,
spleen, kidneys, and testicles. The organs were smeared
on gentian violet liver infusion agar plates as described
by Huddleson (16). The plates were placed in sealed Jars
containing 10 m 002 and incubated at 37° C. for three or
four days. At this time the plates were examined for the

presence of Brucella abortus.

DISCUSSION AND CHARTS OF IN VIVO METHOD

At the end of the six weeks period following the ex-
posure to infection the guinea pigs were normal in external
appearance and had gained weight in a majority of cases.
at autopsy the spleen appeared to be the organ most commonly
showing infection, as may be observed in both anatomical
changes and cultural findings. The spleen was usually but
not always several times enlarged and showed numerous grayish
white nodules on the surface. The liver was the next most
common fecus of infection and was usually congested and con-
tained few or numerous grayish white foci, varying from the
size of a pin point to about two millimeters in diameter.
These foci were usually raised and glistening. The lungs in

several cases were pneumonic or hemorrhagic and showed small

36

grayish foci. In the kidneys in a few cases there were small
pin-point hemorrhages and cysts one to three millimeters in
diameter filled with sanguinous fluid. One or both testicles
sometimes were abscessed. The epididymus was more often
involved. The lymph glands were involved to some extent in
several pigs. The tables which follow will be discussed by

groups somewhat in detail.

3v

GROUP a 1.

TaBLE XX

 

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38

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I 7 dga I , I ea 3 I
— M I acflr . ex
14 zéOOszI : swant I I I I I :DIargea. . I , <0
I I I I i ' d hemorrflag%%h I I .
1' I ' I I '«cat'tel°e_ areas VI"!- 1 '
I I I D - n18) . 1 n08.
I umO . uIc I
I I I I (One _;I 1 111 3-1 I I
I I ' *<-v°8h ‘LOC n I; norm?” I- ‘7- rtuSI1:500
I I I ' IQngl b ut A i c 5 Boabq d
I I . ' .S.Ieen a 9 - shout v" . I t fcun .
I I I , o? 38W 1001 a %epbtlc no
I I ' I 8 1/2 , Iwith f aiameter. ‘I r1 I
I I , I ' 28 - 1n “acflred I I
i i I r af-' r m [9/5/ ‘Infli. 1 m7 and-S 9 D JP. 184-!
’ ' ' I In ° - . 4505‘° vam n 5 a I 83*10 ' I
I ' I ' tel? 1n ' I «J 9,8 and. Sip .‘v
' I 5/28 IHCC."eCtionI 'g‘an9 ’13 SWigflt a ' I
' I '1 agar '8 I '3 . ' ringulnaa -' I
l - Ir ~ I ea. fiOnlq
I t I I war euh I
75 I405gm.' I Swan I 1 I I ' :en gshowed few4p§ normgn I
I I I Iulo ”een .
I I I I S I : 18h I I
I I I i I t ' areas.bu5dant grdy o’andsi I
' I ' ' I ' I I 'with fiepatic Wymphsaper' B abortus 1:500
' I I V ' 0010 1 _ edo‘ ' ’ d? N
' . . . . . . . .fI Int-II enarg gm. I .1101; few .
» ' I ' ' 3 ' 2 I ' ’giéiaw ingulgax) with I I
I I 1 _ .
3 I ' I .' I 8 / ' '9/5/28, warged (5 01 and I ,
t I I ' I ' Ihr. af-'4 Ogm" ,sgfge cheesy fie Sa‘ I I
l I 130. in I rhag ° -
I I Iter mOr nor I I
‘ I ' 17 338% ;8/5/28 ' ' jection I :very Eegwands 5'4 X . '
I t I ' I I I Lma . '17 areas 52a IB°abO d .
. ' ' I I . w e sma heumon t foun ' I
; ' I I f’ ' '1 4“) areds a1? gray ‘
' I : 2 1/2 ' I / .1 Few Sm I . I
I ' l fig , 8 f I9/5/28,in ungi in ”Hugs. I ' I
I _ s ' 1 hr. a ”'5508m’I ish foo ottered‘ bortus 73500
I I8/5/28 Icc. I§e 'OnI ' ,fiungs Sn . areas. Inot fou ,
I ~ . '1 agai I ' Laectl ' .hemorrhaglgw (2-4 mm‘) .
I m — I 1 2 ' Sma '
17 '590g ., I swan ' I 8 /f_,q85gm.I9/5/28.k1anay 0 area-
' I , '0.1 'hr. g 'u I Inecrotl
I '1 a ar '8/55/338 ICC. 'ter }n-I '
I . I g 1° ctlon
I 5 1 ' Swant I :18 ML
m. I I
18 I42 g ' I
I

 

 
 

 

 

40

'III GROUP 7,41.

‘1

7‘ 7

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—
.dflSOH HOG. owWQDr” o m o I .o m
a:unu. . . a“ mmmpn camagauoewg. mm\m\m_ a mpm. mm\N\m 559mm 00 m . a m¢¢. om
.mfivhopw m. Aoaa mlrv rrdam 30h. u u . .
p P . bl - P r bfl 4L

 

mm... 35 on . .. . m m on ._ .
.o m p . aa m rv m mar ad a m.mm\m\m”.ammmnu mm\m\m

3:59.353 pad. oflmwcnnoamg Sham hum?

nan-I-L-puo-u-

 

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n
—
b
—
anamm .00 m .. .. .SMmbm” mr
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n
n
u
h

p . _ _ P _i|J

'unmmOP— MWAHHUHHHH - — humour uOMMB @- .u ORPH'WMWWH — .02..

dd Rm. 58de . mmngnflu humanism ...dw mo. .3 w.§momxo. @33an "$803 lap .mnowop.m.fim
OHPHH.lOdH0POdm. . mvmfl . 9%.. Ho mumm— owm¢. m _

ommd . . . .pgwfimrh. . .vflrwma J

 

nogomnnH rammofinmmwnpam

“coo.om .r onpfip mOHVaqusrmmwv .Ar % omaomv asnmm :pflg.dmpmona.dnd goapommnH Op womogxm mmflm

41

DISCUSSION OF RESULTS IN
TABLES XX, XXI, XXII, AND XXIII.

Group one shows the results obtained from guinea pigs
injected intraperitoneally with varying amoumts of hyper-
immune horse serum and microorganisms (strain 238). The
agglutination titre was determined at the end of a six weeks
period after injection. Guinea pigs one to eighteen inclu-
sive showed an agglutination titre of 1:500, while guinea
pigs % 19 and 20 showed only a slight agglutination reaction.
The last two were treated with two cubic centimeters of serum
only. This group of guinea pigs gained weight in the
majority of cases. TYPical Brucella abortus lesions were
found in all excepting guinea pigs % 6, 19, and 20. Although
typical lesions were found in all but three of the pigs, there
is a noticeable decrease in the apparent systemic reaction as
the amount of serum was decreased. In every case in which the
administered serum was decreased Brucella abortus colonies
were not obtained from seedings of the organs, although there
were macroscopic changes which were characteristic of Brucella
abortus infection. It was also noted that in Table XX where
the largest infecting dose was given and one tenth cubic
centimeter of serum.was given simultaneously, one guinea pig
showed foci in.the liver while the organs of the other

guinea pig were normal. Brucella abortus was not cultured

 

from either guinea pig.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

#____-----‘
'I a 10130 . A
A . . . ’I’e . ”a: ... ”t (311:).
+ ' nat 1011 tlt _ ,— 0 armil #J- u l ‘-
. - agg"u“l ~98 aflu ”V 'hggo
I — In'S serum. I a Cu"ture , ‘titre
BTOV v hrs.) 0* qupt :gfter
”fill-to (4d ' + in-Yoii lbw w
fl “/5 ‘;-.gal” 3 cu ' 3&0 L9 r_ ”(Io-S I all...
iVea I :ihfllec 'tODSV
Pip‘ 7.2808 I a . ~. il/ll‘fjrs A. _3z_.
Each 0 I I , Mate ' Utousy 11m“ U s ' ROD
l“r . {yht U9 ‘ Ill- 4 ,7 '1 :41
- I'el‘o I . a - w'tfl
. I I . ante . of mu . n are»
. ago Di". a“ Iafter I - we 0 a
I “'0 I um ' - , ’ S d ' e
Weight'ti‘ sera with '. 1n d'5 WKS'ItOP J “MI set-“ere . W a foam“ '- 500
i I ' use ' 30139 I I . v ‘1 88V L . f1." ““ bot-tub . '7 a I 1 :
Piglbefore tre I Cu‘tu-re IJ 1 ’ I.“ 8.1. £0010 «so 345' a. are SP'E’en’
- . f s "3. 1 II ”I I
N00 ex‘podtbe' I I I4/w/29‘ Flag-girls“ _ _.'~" fool, (...:- h: I in +1.]: mix.“ '
' are Ifore' '5; 1 ’29'550gfn.’ 30 Q OJ?‘ :y/lbli [— 'T. ell... argeCL. 5.311;}. iilulLLu/o ’d'
I S I I 1 .25 IV/ / . I “Tu 1“? GOD-1 Gen 30/.) Iv.» ‘ .bol"tu~8 10111; ~'_7 .500
I _ I ' 2 I ' /, ’n 9 "' iv 8'5" 0‘ ~ - ‘11-4 II- a . 9nd .
1 i565gmoI , '79Ong'4 ‘/¢ ' H 3001 and 9* , in Spweefl “ I
. ¥ 2' ( --a f" 7‘." . 81* J. J 4 1 _ .. I
r ' ‘L I '5/1/89' I ! ‘ C‘Q .‘ g‘LQ-g‘l‘ 1 ‘31. I‘Lvel I -!-riaxle:"7. '
r I 7:25 I , 'Tuflb; hemorrflaée“ I I» ~ ' “Q
i - I ‘ c ir‘ 1'. ' I ‘ LG 1 ‘ 50
2 I785gm°' I I ' O m '4/1/39’Pgagish ioclo A uerfrflagefiy, abortus iour I -
6 r' . ‘0‘ , - . lb “ ..- , 43’ -.
I I I I’2 I ag'ég g I Q’ U min 90}1. ' Spleen'- steen' '
U — (‘10 . I T
ad I '- . - I" ~ ~ 1}" .30, ' a ‘ '_ ..
5 1450gm. ' I I i I .r "a“ 9 ‘Iflvceir 71" nge“ 8110. a b %_ , b0 “Ht 118 foulifl [-1 . 500
I ' ' I a , '545gm.u4/”/6 '50 P e‘ I- er - .5. ? i; wivera I ‘
' 5/7/29' I 'fOCl’ . Ironic. ”iv :qrf .ih u¢fi7.(ajkidn€y‘
I - I I I I’Inigs . In Cio Spve . « fOPio ISp d ‘30
4: 495gm. I I I c" wI“ViSh £0 . ' (V‘I‘aylsn J g ‘- rtuS fOu-FL "I, :DL
I , I I , 4/1/2J,gergym&w witfl e n “i numer- 3, ago m and kld-'
I j I ‘ " .L H...'Q“. ..L ’ ' - m l "-
I “/1/39'4703911. I 1.5 1’]. + ”ray I" 1"}. - O 3.1 : r:"'€‘o~ . I 11* -I ve '
r I . 'I 00 la 1 'I" ' er ' 5'5 .._ p, ‘I 91* C” I e}?- 43 (1
- .1 0 3.1V UL} / n - 111,1
I I ' o '1 Gen “Q80- “ 4'0 I ’
I ‘ - I l I 3 DP" '9 WSCE‘DH gfl ab ortu.“ 1:500
5 4808m‘ I I 41’ 1/2 9 'OU‘ ° utiCe—e c" ‘0' -I ell and I
I i L I 1 20,555,911. ' / ,Ieft t6 ” . 413198113111 Sp 8
.1 j, . up " -. ‘5‘
l I ' 'I 5‘00 '5/ / I I I grayisll £0010 'Kidfley' '
' . - ' . . ' .rivsr 71Eraea. L ,wzeoo
6 '65531110 I I '4/7/29,F)O (f3 811 a c: . 8.019811! _ I
I 1 l 1 '1/29 '400gln. I L I . af';‘ 1811 IO 01..” . éh I
I I 5 I I .1 gr 0 .. 'I- {firth \v l I
3 ' I 7 1500 I ‘g ITiVer owp‘xa" Wit“ I, c3762. d ‘300 '
5 i "' I r ‘ I)- ‘l’ I n ‘ “ - Q 4‘ m ' '1 :9
i 7 '4208m°. ' ‘ ' x4/I/29'5‘biX R”in“ in cub £201. '3 abortu” EOFIIan
I r7 r I ‘4 ‘ I O s.
' l I n 435g5~1° fOC ' oriiy ' -I (3": all
1 I 112‘ '1 (49' iero‘us c: ‘ 7 ~ th irl 11111.7“ 1'
F f ' I 12500 '“/ / ' Iyiver n9“8 x 1101mm:1 ”1 '. . q found .1;500 ES
' '4 (I. O i. T' I‘ ~ I. w'ver.
, . . aJ a .v: . live 1 l
I I64 'I ,29I55ng I / Iglf‘ 4 1101110‘ . I Al. +11 _ "‘ found a . O
, / \ new - , iov ” tuo ‘ w 50
I ' 1 1 . 'I 000 1 ‘ ' I ' IUIJ-gs I) . ‘ (Ora-318 ‘1 . - b. ab 03? - Q 116 ' '
v 5 m ' ' I . ' .4/‘/29'lar89& Wltn If ray f0¢1'~ Iin KidneJ V I
9 I52 g 0' ' 3/1/29: vofigm' !;iver nul’fler ouioima-I . fiOtn isms eel’lo f uIld I
I I A % - ' _ I .l 0 -
I '1 .7009 : I I 0-8 X -. .- rtus '7 “000
o L 0 N ' een SSGLIO .2 abo 1 en I
I 5 H1. I - ’ ' ‘ ‘ . '4/1/ggsz .' leS abSCS. river ._ 11:. .1 .Ver’ 8p 8 9'
10 172 g L 1 , 29 505gfl.r / test 10 _, ”nOnlC' —‘ ,11’1 1. - v
L ' I O '5/1/ ! I 1 {rs "' pilewm '1 gen 4"5 X and. Klane" . , CI ' O
~r . 1:400 , Iluna poci. sp~ . 01 I tus foun ,w;50
' 800m.: ' ' ' ' egrgray * . h ray i0 ' 'B. abor
‘7 I6 b I I I Oogm, 14/1/5 Orma'I Wlt g “ tered gray "1’1 Sg'Ieeno '
' ' 7‘4000 ' I 'IiVeI - Lleen thce 1 'Bo abortu:iver, :1:d0 Q;
I a. o I '
8203111.. I I I 4/1/29‘focl. 8.1) . er .- ‘in 1mg, _‘ kidrley" H
12 I ' I lz/q/Zg '615gm. I ' umOnj-c’ L163 ,q ., Gen, and t1)
' , I 1:8000 I‘ I ' .Iungs ' ?nespleen 55 p e*P I E4
- J“ l O
I. ' I '7 :8000 g . ‘ : Liver _ 8 X nborflflav1 YJlfi in- I . n SID-1 Gen. '
- I ' g ' lean 6' m rficla ' l I ' q)
14 '595gm‘r ' ' x ,Sp foci. Dupe conseSted" I 1U
' 1 C '”‘
I I r I I '4: 1/291graJ 1 warge and E86» I + found 0
, . , . 1/29 seam-I W S t as W B m I '1 =500 s
S ' . s. . ,. , en‘ “
' I ' 1.16000 'U/ I ' IBOth t8 f 01 Spleen 'in lver Q ' as
' " I . I I «217’ O ’ I 'I 88110 ' i
m. I 0 II— {110.} .- 'Sp 6.
15 705g I . I 1V€r 7 , gun I m
' . ' L . ' I/za'goes en“arge”' ’B. abortwaiver. ."=5OO
' L I I 1 29 [6'15ng ’4/' y I . I‘ arabr Iin 'Iul’Lg'S, 1' 1
l I ' " OO '5/ / ' I 1‘10 0 lee b '7 ' , qp-‘een'
I I 1 :1 b0 I ' l Twigs U113 111110 L twice norma ' and ... I
' _ I L I I o '3 eeI’l ' 1v
6 Egm. / a . 0p“ '
15 1 . ' I n / /29,5q0gm.:4/‘/29If001 oint hemorrhagam bortu“ found .‘z500
‘ '54 1 U ‘ o ’ " 1‘ . . k.
I a r - nln P . ~ lean In. a
' I ' 7352000 I ' ' ‘IungS Bray foclo DP foci-I°n SUIeen‘ '
17 '7658‘31'I _ I ~T I: I lLiver logma‘ with gfzyen- ,1 I '
X 1 - ‘na I
I I I ,I 1 29 4-5 . - w ingul j I
. I . wag .mgm. W :superflai Canasta“. ,L s .- fi .
I 5 m. " ' ' ' 1v fire-S pin p 01183: 1001. B. a and I .
18 65 g ' *’ 'Iuror numerouscD 2 math 'in SPIBen :
I I f I "‘ ' e ‘ - .8. . -.
. ‘ - . ' I "é‘fieen 6-7 X $23210 1a" 111' “ kidney I
- I t y I 4. _ '. Su- . -18IO‘80-
I ‘ I I ' 750m. 14/” /29 ,gray’ £0 Gina hepaft 10 all C I fl
' I ' 'E/I/2915 O ' 'guinajs ted. ' ' I q fourld '
' I 7:54:000 I I ' Iand cOQgeS morrhages° B. abortu“p d Ilz500
' 10 m. I - ' I . .- Oin‘b he 5-6 I . 9- I1 een ctr]— I
-I 9 6 8 , I ' I ,Tungs pln P i Spleen ,1n _I P
3l 1 I I ' IIiVér grills”. £0011'._L’:°L(3j.‘6‘~.1 in- IKj-dney. r
..—' " "u e - d
I ' ' I '4/1/39'X norma7- D Etly Gujarged° . abortus foun ‘w.5oo
_ _ I l ,r‘ 4900‘fi10 I / . 'IS 81 igl _ 817 BC arid I .
, ' I ' ,5/1/39, 6: ' .gulna cattered' gr .1 Iin Spween L
20 .5858m" ' ' ' I ' ' ocI. Spleenw "”ve. "“ '1-500
I ' ' I ' '4/1/29If . d inrignt eJ eSoI '
. . . WI /29 595em° . a" 1“ om hemorrhag , - .
' ' .‘I 28 000 IU ' T‘ ' S Pin-p - " leen ,
I I . L Lung - f 01- DP I
_ '_ I . dy O f and 7 ‘500
I 5 m I a I er gr 1: S O ‘ o
. . 1.128.000 ' . ‘ Iiiver “ grag ’B abortus £9“ 'Ioeoo
- I ' g I . rm 0 ' . I
m I 1 29 e no 001. . . an
22 .5403 . I g 29.570811“ I4/ / 'thc 08.11118er grayfth !21.11 I lveI' I
‘ ' I 'I 5'72 000 35/1/ ‘ ' 'T'iver - Stwice norffla’.Y W l ’81).1 Gen. I
I : ’ I” - 1
25 '475gm., . ' ' I m '4/7/29Ispigggonso - r I“ abortus found ,1-500
' I ' I5/1/29'4708 . ‘ Lad . 01110. lee.‘ 1eenté. Ween. '
' . I '1 .572,000 I r tlmgs Pmu‘m a £00110 bpj‘ ‘ln Sp ,
I470gm. - , . ?7 r t I Scattered gr yr ~ra7 fool. I
24: g 4 *1/ng 'I Wltx ton—s I ma
I r g 25 m. I / 5 X norma _ rhageSo tuS £011 , 5 O
' 1/29 :4 g I . t nemor . IB abor ,. w I: O
F I ‘0 ()0 5 . g ' —p01n .7 00 l. ' unQ
4 O I In . 0 f . - '1 en I
I ' - ”31’0" ’ ' 'Iungs P umerous'glt'd (3 '11’1 Sp 8
25 Iéaogm.' 1 L ' 1 'Liver -sgightwy enwarggn. 'kidney. I
l ' '\ Ween . ° 8,.1 S - '
I l . n I
‘ I ' ' 9:500gm.'4/I/ZQIEEPerf101a1 lnggeted° I '7'500
' ' '1 .1’024,00 ' I ' '1a’rge oint hemlorrhag ‘I‘TO cu‘ItuI'eS
I - ‘ n- ’ i J
26 ,445gm. ' i I ' 'Lungs p1 pouS gray food. a '
' ' ' I , I I iICIV er nuinfr ht“ 1? 8n" arge ° = d I
I - . u‘ ~1
. . . 1/29 ' Dead . spleen S g . abortus .fw .1 too
I I ' rum 5/ I I £0010 'fi' ~ " lVer’ '
' " ' NO se " ' I 1er0LlS gray. h Iin " 131168" I
27 14:05ng ' I ,1/2 dose 1 , 'I‘iVer ~tngge norma-I Wlt ,and SP" 9811.
1 ' Y 'enrned. '4/1/29.Sp1ee§oc? '
' C
I ' I um : thus .650gm': .sray
I m ' "' I NO - 361' 11101; in.‘ I '
28 .5303 " , .jectedI
I
. I I '
'~' .; '

 

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I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cow Serum f 04. ngg‘utinetion titre 7:” do
4 r7 . «x . ~ I 4‘ n a " ”I“ i; 1 '~" ' In; Li" 1'11 ' "‘18
Each Pig Received “/0 Agar S“ent (48 are.) of up idle be ‘10 SeraI thL .
. 2 T F ’ 'ngg.
Weight: 4880 . v ,. TV I 'titre
h f re It1_ I Serum Di‘.' gate .'ei3ht Mate n.:., n I geeterio‘oeioa‘ ca.
Pig, 6 O z . . a I - ft f an ' autonsv findinwe a. 1. ~ eiter
No. expo- 'tre used Witn , in— 1: er I: uw- n i u - S , findings , eu-
' ' o . , .I, NBS-‘7 L.
1 sure I 132:8- ! Cu” ture I Jected' U WAS. I O: a ' 13013st
ore _ , I
x I I ‘ ' ‘ je‘i: of do so evi’"ed. Fiver:3. «bortue found ,
,3 --,".' - , ‘,"‘,.1 '73,“ ... ~‘I',r'_ffl 1‘: -_ l 7 (Sr '7 z E} K‘:
29 '620gm.' __ ' 1:25 :'Z/'I /g9: ofiOgm. :4/; 32/ 9:1".umer @113 .231 5;; fggl, T4 ”Jami ill 0453:, , V v , ' CO
I I t;vice EEOlfimf' Sizza. 1ft 8 I calm ,
3 4+: I i j I :qun‘ nin—noint ”6 UixLGEeSII '
‘ " I ' ' An'it 7'- hr“ \ ¢'.‘ wlm‘x.‘ ":471‘1 \ £3113 T’t’ic‘ j?“ Til/Nd ‘ 3 h,
. ivei CicN« .ooi. Try Lei. Iuo e¢~) w 3’ * I"-SID
o I": 2/, )9 ‘4 _ : . .3 . . '1 k J
50 '6203m°' ' ' 1'25 ! fi/ /29: 6803m.:4/M :tiioe nJrL1a‘. night teS£l~ . in Sp' 1
I I I n. i:
C' 8 $0808 8:89. 7 I
I l I 1_ I ' L L
I a in - pneumonic Liver few‘ A 44,04um a '
‘ ' ‘ . 'n/ l: LY n 1 (j ’c'j 1:3:fiSJP/W e . __ flH-Vfilein- I '1). a U EV!" L L C (:01; 1‘3 :500
51'450gffl. I - ' 1 :50 '0/ .1/ 89 140081110 a45/0/ A} ‘51. L4,)" .L.\..ch. a .L..—L\LJ::. I” . 11.94; 4. £0110: 1
I I I I I I 'OLlS "Large STE}: 1033... '
q _ 4 I. I
I I I v I I I ifllllgS _ ”0111"“0 11-4,, 316;,103’311345/ ,§ .15. L. L‘CJ. U) S ECUIAL 7 ,7 3
52 'BTOgm I _ I 1:50 ‘5/ /Z9: 54Og1m’4/2/BSIZiVer - gi*°ey fooi. Spieen 1 in:“ ”e:, Sp sen, :oU
I I I ' 4-5 X norme . Ldfit WEST -
' ' ‘ ‘ ' ' '“unge - pin goo int fittemori eé n. Joust “ ‘ mu. 0 ,
r I I .. ~ 'e ’ ': : ert ’ 'V‘ "xi “'x 9w ‘74'3' Sen} 212:7 ‘Rizi 111 :If‘eei: e111 .93i130
53 '500gm. ' ~ ' '7 o '00 '0/1/fii9 'UL/ubl L. 14/6/529 Inf-1:176]: A v {31.0%, :3; ;_,q:"_;m;f, O Y or: T, ..
3: LD 1) .2881). Li“(/ Jx ink/‘4.qu . I 1-1‘.‘..J_-t; AL
9: 9 ; ; ‘T ; .Ilver - vex: ieI gre. lelo :3. obortue found I
i ‘ - 'n ..., [P ‘ fl -..‘I H , T, -r {1'2 ,c 'I A 11') .. ° .-. (-.., .:fi.--; 7,, :43" if,“ (I u; z i"; -
54 ,585gm., - , .100 ,5/1/59,450gm.,4/4/n9,agieen ffb X nonlu ”Le 'Efi—ffi:“fl “A“ I 00
are? fool. Inione~. I
‘L 1 L I I I 1t: c, , y
I I I I I I 17.11.1138 - pill 1:: ‘il’it HE, LOL‘I’fiELgGSJ '
z I x x x LLiVEJ‘ few Eilel‘ ,gre oei . ID. e3bortu.3 rerun] '
f-‘r ’- «r 1', 1". r a: . 17f}, V." .1 -.J, '1 ., - 3 1 7’}: , 0 fr; '3
55 1570013. ‘ - ‘ 7 3500 ‘5/1/29'4’ égm. 14/2/29Io [3 6811 U‘4 3“ ”0431197“ .3121.)- 3 ' 4.1.; .LV El Van-Mu ' 7 .uOU
,. r l I I I I ,3:l“£;.'T fOCi. SUJEB rilclu" 21.11- Is‘p" {3611 1
I I' I I I I Ibai1‘7‘2 COLLAWLLIL-u I 7 '
i-‘ I "P "- '
' ' ' ' ' Tungs oxeulorio Live r few N aoortue iound r-
. r; r: ... I? ‘ ' " ‘4.” '7 '300
56 '400gm.' - ‘ 1'500 :°/"/29:”705m-‘4/ /29'~rie fooi. .ln Spieen. I
I l I
I I I I I I I '3. abortus: few '
I I I l I I I 'Coionies from ' '
57 '4603m.‘ - ' 137000 '3/“/29'550gm.'4/2/29' river few grey fooi. '“unms wivcr, :7-50u
| I I I I I I ' ST) €6n,&';1’ld kid-
I I I I I I I 1118:;- I
I.. ,, . I
' ' ' Tungs pin noint hemorrl ages. 5. abortus found
, . - i , , I"! 50
38 .425gm. I - I '1 .7000 I5/ /29: 4503111. '47/2/29:T 1.3781» few gray £001.. :11”; spleen. , 0
9T : l i j I ,Iunee pneumonic. liver grey ,B. abortus found ,
59 '525gm,I - I 1:4000 ,5/1/29,665gm.,4/2/29,foci. Spleen twice norne". ,in.Sp"een and ,"z500
: L A, L ,_ , ,Kidney grey fooi (severeT). Lkioney. L
c: ’ T ' ‘ _ .,
, . ' . ' an“ 4:52:32: 5 B. foam:
I J D - ,
4o '6408‘m.' - ' I $4000 '5/‘/29 '600gm. ‘4/2/29 § . th f in Sp" sen, MIMIC-3y.1 500-
' ' ' 3 . ' ' 11X norfila ‘V‘an gray. 001.121.1161. teSticje
L z I I u I .Right teetic‘e absoeesed. ’ .
. . z I x I ILungs pneumonic. Liver nu- I I
u u s l x : Imerous grey fooi. Spleen 4-5I I
I u u z x l IX norma‘ — gray foci. KidneyIB. abortus found I
41 .520gm.. - . 1:8000 .3/1/29.mogm.:4/2/29:petechia-I hemorrhages. BothIin SpIeen and I1 500
I n I x x Itestic”es absdessed and se— Itestio‘e. ‘
u u x I x x :mina'I vesic‘es fiTTed with I I
I I I I I I Igray f"? uid, I I
I ' ' ' ' ' 'Lungs grey fiooi. Liver grey'B. abortus found '
42 I4655m.‘ - I ":8000 '5/ /29'620gm.'4/2/29'fooi. Spleen twice normei. 'in ”iver and 'T;5oo
I I ' 'ep’een.
I x I I ' I _M1un s pin-point hemorrhages. B. abortus found ;
45 '555gm.‘ - ' T:16,OOO '5/1/29'520gm.'4/2/29' Liver grey foci. Spleen 5- ~4Iin "iver and ,I;500
' ' ' ' ' 'X norma? with grey fooi. ,sp"een. ,
I I I I l r - ,
, , , , , , ,Lunge — pneumonic. Liver - , ,
44 ,520gm., _ , 1:16,000 ,5/1/29I610gm.,4/2/29,gray fool. Spleen grey fool ,B. abortus found '7‘ROO
, , , . , , , ,W1th 5—4 X norma‘. Kidneys win spieen , '“
, 3 , , , 1 pin-point hemorrhages. , .
u u I ' x u I IIiver - grey foci (few). 'B. abortus found '
45 :445gm., ~ . T:52,000 :5/1/29I4953m.I4/2/29ISpleen 50% eniarged with 2 Pin kidney and '7z500
: z I I It I ITarge (3 mm.) gray foci. "iver '
I ' I l l l I l . T
46 ,445gm., - , 1:32,000 ,5/1/29,520gm.,4/2/29, Liver scattered gray foci. IB' enortus found 11:500
~ .in Kidney.
I‘ I I l I I I ' I p ‘
u. abortus found '
I | c I r o - ,
47 3325gm,' _- :_T,64,000 15/1/391565gm.:4/2/29' {idney emaTT gray fool 'in ‘iver, sp een, '1 500
I y 1 , Y , I Iand kidney. I
I I I I I . B. abortus found
48 ~ 1 O l .. ' . . '
. L . , , , , :and sp'I een. ,
' ' . 0 o
49 '35Ggm.: - ' 7.‘28,OOO :5/"/29:45Bgm.:4/2/29:Irver 5 light gray areas. 'Eh.323:§FS gognd '1 500
I an ungI ' .
I . I I .
50 575 - 1.1 ' 1 ' ' , 'L s neumonic nleen 2 X I I
, gnu, , . 28,000 5/ /29 555gm. 4/2/29,n13§nelawp . s; ' __ ,., ,500
l l l
. , a 3 ,
51 ,580gm., - , 1:512,000 ,5/ /29 ,400gm.,4/2/29,Liver 1 gray fooi on margin.,B. abortus found 1,
l I l Spween 50d; [0 81’1" arged. in 'Yiver. ' .500
,7 ' ‘
_52 I455gm.: - , 1.513 000 ,5/ /29:5.Ogm.,4/2/29;qungs pneumonic. Spleen 50%,
5 - 3 enlarged. , ‘ '13500
I
5 .565gm.. . ,1; 1 ,024 000 5/1/29 540gm.,4/2/2ei:ungs pneumgnic, , _ ,1,500
54 '5603m.' - '1.1 024 000 '5 2 ILiver - gray fooi. Spleen I I *—
' ' ' O , ’ ' /1/ 9:450gm. :4/2/29'2 X noun-a1. 3 ~ '1:500
' ' ' i l I IL . —"'
ungs pneumonic. Liver gray'
I I _ I . q ,. a
55 '4503m.' 'No serum :5/3/29'49ng.'4/2/29'fool. Spleen 5-b X norme‘I fig wggrtzidfognd '7.500
I . , ' ' 'with gray foci. x g C ver,..
I I I l l ‘—
._ , , , :Iung s pneumonic. Liver grey,
55 I39Ogm.: _ INo serum ,5/ /29,460gm.,4/2/29:foci. Spleen 5-4 X normaw -,B. abortus found ,7
I I I gray foci. Iymph gland ,in "ung and ”iver .500
I I a . , , :3- 4 X normei. I '
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44

The data in Tables XXIV and XXV show results from
guinea pigs injected intraperitoneally with Varying amounts
of serum simultaneously with one third of an agar slant of
microorganisms, culture 198. Serum from human and bovine
sources were employed. It may be observed that each guinea
pig had an agglutination titre of l to 500 at the end of a
five weeks period following injection. It is also apparent
that there was a slight loss in weight in the guinea pigs
treated with human serum, and an increase when the bovine
serum.was employed. This may have been due to the fact that
older pigs were employed when.the human serum was used. In
all excepting one pig (number 48) gross anatomical changes
were observed, although pigs 46 to 54 inclusive showed a
marked decrease in the extent of the gross anatomical changes.
It may also be observed in the higher dilutions of bovine
(pigs 50, 52, 55, and 54) serum no growth was obtained from
cultures of the organs. One may also observe a zone in the
gross pathological findings from the guinea pigs treated
with a dilution of l to 64,000 to 1 to 1,024,000 of bovine
serum {Table XXV). As both the decrease in gross pathologi-
cal findings and the failure to culture the organism from
the organs of the infected guinea pigs are overlaping, it
would indicate that there is a dilution factor necessary in
obtaining beneficial results. When human serum was employed,

the results were uniformly negative

45
DISCUSSION

It is interesting if not instructive to compare the
results of the "in vitro" and.the "in vivo" methods. The
results presented in this paper, based upon the "in vitro"
bactericidal study of 11 sera from human, bovine, equine,
and caprine sources indicate that the hyperimmune bovine
sera and the immune human.sera show a slightly varying
bactericidal zone in dilutions between 1 to 500 and 1 to
64,000. While the "in vivo" study of 5 sera, employing 255
guinea pigs, indicates that the bovine sera has a therapeu-
tic value in dilutions between 1 to 52,000 and l to 1,028,000.

From these observations the inference may be drawn that
in a specific serum for Brucella abortus the bactericidal
effect may exhibit itself in a zone brought about by dilu-
tion. It is not yet apparent how the dilutions of the sera
and the bactericidal action are related to one another.

It is clear that the agglutinin.production and the
agglutinin titre are not a measure of the serum's lytic
power. Whatever be the chemical nature of the various pro-
ducts derived from the body tissues and fluids, which bring
about the bactericidal action under experimental conditions,
their concentration in the serum is certainly not in direct
proportion to the agglutination titre; nor is the concen-
tration of the lytic substance of the sera in.direct rela-
tion with the therapeutic value, as was shown by the pre-
sence of the lytic action in very high dilutions of the sera

following exposure to infection, are not quantitatively

related.

1.

2.

3.

4.

46

SUMMARY

Observations on hyperimmune bovine, equine, and
caprine sera and immune human sera show a zone
of bactericidal action.

This zone appeared in the hyperimmune bovine
sera and the immmne human sera, and was inde-
pendent of the agglutination titre.

The zones in the "in vitro" and the "in vivo"
tests do not coincide.

Cow sera appeared to give the more satisfactory

re 8111138 0

4?.

ACKNOWLEDGEMENT

The writer wishes to acknowledge his
indebtedness to Dr. 1. Forest Huddleson,
Dr. Ward Giltner, and also L. T. Clark, and
Dr. A. S. Schlingman of Parke, Davis and Co.
for assistance and suggestions received during
this investigation. The writer also wishes to
acknowledge the financial aid received from
Parke, Davis and Co. which made this work

possible.

48

BIBLIOGRAPHY

(1) Jordan, E. O. and Falk, I. S. Newer Knowledge of Bac-
teriology and Immunology. Chapter 70, p. 926.

(2) Ibid. p. 929.

(3). Cole, H. and Moore, H. F. The Production of Antie
pneumococcic Serum. Vol. 26, 1917, p. 557-565.

(4) Weil, R. and Torrey, J. c. Immunological Studies in
Pneumonia. Jan. 1, 1916, p. 1-11.

(5) Amoss, H. L. and Wollstein, H. A method for the Rapid
Preparation of Anti-Meningitis Serum. march 1, 1916,
p. 405-419.

(6) Flexner, S. and Amoss, H. L. The Rapid Production of
Anti-Dysentery Serum. Vol. 21, 1915, p. 515.

(7) Flexner, s. and Amoss, H. L. Chemical Versus Serum
Treatment of Epidemic Meningitis. Vol. 25, 1916, p. 685.

(8) Jordan, E. O. and Falk, I. S. Newer Knowledge of'Bac-
teriology and Immunology. Chapter 72, p. 958.

(9) ThJotta. on These So-called Neisser and Wechsberg
Phenomena. Journal of Immunology, Vol. 5.

(10) Brekke. Quoted by Thjotta, 1916.

(11) Journal of Hygiene. 0n the Deviation of Complement by

the Serum and Its Antiserum.and Its Relation to the

Percipitin Test. Vol. 6, p. 282.

(12) Pondit, c. G. An.Experimenta1 study of the Neisser -

Wechberg Phenomena. Journal of Hygiene, Vol. 21, 1923,

p. 406.

(15)

(14)

(15)

(16)

(17)

(18)
(19)

(20)

(21)

(23)

49

Mazzi, Renato. Sui termini di raffronto fro 1'aborto
epizootico e la febbre di Halta. Biochimica e Terapia
sperimentale, Vol. 9, 1922, p. 71-80.

Bull, 0. G. .. method of Serum.Treatment of Pheumococcic
Septicemia in Rabbits. Journal of Experimental Hedicine,
Vol. 22, 1915, p. 466.

Huddleson, I. P. and Carlson, E. R. Journal of American
Veterinary Medical Association, Vol. 70, n.s. vol. 25,
Ho. 2, Nov., 1926, p. 229.

Huddleson, I. F. Tech. Bul. Ho. 49, High. Agr. Exp. Sta.
Part 4.

maitland, H. B. Bactericidal Power of the Blood in
Slide 0611. British Journal of Experimental Medicine,
Vol. 7, 1926, p. 161-167.

Wright, Sir Almonth E. Lancet, V01. 1, 1919, p. 485.
Wright, Colebrook, and stores. New Principles in
Therapeutic Incubation. Lancet 1, Ibid, Vol. 1, 1925,

p. 565.

Eeist, G. D. and Solis-Cohen, S. Bactericidal Action of
the Whole Blood uith New Technique for the Determination.
Journal of Immunology, Vol. 5, July, 1918, p. 261.

Parks, B. S. Bactericidal Actian of the Whole Blood as
Determined by the Heist-Lacy Method. Journal Lab. and
Clin. Zed. Vol. 11, Dec., 1925, p..269-274.

Kolmer, A. and Borow. Adoption of the Heist-Lacy method
for Determining the Bactericidal Activity of the Whole

Blood for the Chemotherapeutic Investigations: studies

in the Chemotherapy of Bacterial Infections.

of Infectious Diseases Vol. 51, p. 116-124.

Journal

~50

Sep28'45
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