STUDIES IN AGGLUTINATION USING
BACTERIUM ABORTUS AS ANTIGEN

Thesis for Degree of M. 5.
Harold Kenyon Archbold
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smtmnza IN AGGLUTINATION USING BACTERIUM ME; AS ANTIGEN.

STUDIES IN AGGLUTINATION USING MCTERIUH ABORZQfi AS ANTIGEN.

THESIS

Submitted to the Faculty of the Michigan State College of
Agriculture and Applied Science in partial fulfillment of the requirements

for the degree of Heater of Science.

Herold Xenon gchbo 1d

June 1925.

JHESIS

TABLE or commie.

1. Introduction.
11. Bevin of Literature.
111. Technic Employed.
IV. History of Strains of w m Used.
7. hperinental data.
a. Cross Agglutination.
1. Comparative Charte.
2. Average Chart.
h‘. Agglutinin Absorption.
Tables of Results.
c. The Effect of The Hydrogen Ion Concentration of the

Antigen Upon The Agglut ination Test.

VI. Discussion.
VII. Conclusions.
VI 1 I .Aclmcvlelgment .

11. Bibliography.

93788

studies in Agglutinatien Using m m as Antigen.

Int roduct ion.

i'he agglutination test has generally been accepted as a
practical means of diagnosing bovine infectious abortion by most
investigators, although a few prefer the complement fixation test.
The latter claim that the agglutination test is too variable in
its reaction, uhile the complement fixation test is more delicate
and specific, and the reagents which enter into the reaction are
more easily controlled. Recent work, comparing the results of
the two tests, show that they agree very closely.

At the present time a different technic for the agglutination
test is employed in almost every laboratory, which in all probability
causes a considerable degree of variation in the results obtained. A
study of the various factors which effect the agglutination test and
their subsequent solution will lead to a much more reliable method of
diagnosing bovine infectious abortion, and consequently make the test
more acceptable to all concerned.

Within recent years much time and effort have been devoted by
investigators to the detection and formulation of distinct imno- I
logical types among the members of a given species of bacteria, the
members of thich are culturally identical or nearly so. The purpose
of this study is to determine the existence of different imunological
types of m m, and the possibility of .1. existing difference
in agglutinability, (both of which would effect the reliability or the
test and result in a marked difference in the reading of the results

by different laboratories) by means of cross agglutination and

-2-

agglutinin absorption tests. Also to determine the agglutinability

of antigens of different hydrogen ion concentratibn.
Review of Literature.

Xolmer (1) states that, "lietchnikoff, Charin, and Rogers had
noticed peculiarities in the growth of Bacillus pyocyaneus, when cul-
tivated in imune serum, which we now believe were due to agglutinins,
Cruber and Durham and Bordet(189d - 1896) were the first to recognise
that the agglutination reaction was a separate function of imune
sem. 'hile investigating the Pfeiffer phenomenon of bacteriolysis
with Bacillus coli and the cholera vibrio, these investigators found
that if the respective immune seruns were added to bouillon cultures
of these two species, the cultures would lose their turbidity, flake-
like clumps would form and sink to the bottom of the tube, and the
supernatant fluid would become clear. Gruber at the same time called
attention to the fact that agglutinins were not absolutely specific
for their own antigen, but would agglutinate, to a lesser extent, closely
allied species of bacteria.

I'In 1896 Pfaundler drew attention to a peculiar phenomenon ob-
served when bacteria were grown in an immune serum. Long, and more
or less interlaced threads of bacteria deve10ped, tie a... regarded
as due to agglutinins. it that time considerable emphasis was laid
upon the importance of Pfanndler's reaction, but at present the
ordinary agglutination tests have superseded this reaction as a prac-
tical diagnostic procedure.

”In 1896 Widal and Drdnbaum first turned these facts to
practical use in the diagnosis of typhoid fever. These investigators

found that the serum of patients suffering from typhoid fever aegires

-3-

a ma: agglutinating power for bacillus typhosus, and since this
phenomenon generally manifests itself comparatively early in the
disease, its recognition has considerable diagnostic importance.
It is purely accidental that we speak of the 'Widal Reaction' in
typhoid fever, rather than of the 'Grlinbaum Reaction', for the
latter observer conducted similai-‘Zuiiidoegendent of Vidal, but,
owing to a lack of patients, Widal preceded him in a publication
of a more extensive work.

"At the present time this agnostic reaction is known as
the Gruber-Widal reaction. It has proven of great value to a
large nuber of different investigators, not only in making the
seam diegnosis of typhoid fever, but in other infections as well“.

!he agglutination test for the diagnosis of infectious abor-
tien in cattle was probabh first used in 1907-1908, by a Danish
veterinarian, Grinstead (2). His results, haever, were not
published until near the end of 1909. Grinstead showed that the blood
of cows which had aborted, agglutinated the abortion bacilli in
higher dilution than the blood of normal animals.

lcradyean and Stochman (3) were the first to demonstrate
that the agglutination test was of considerable diagnostic value
in the abortion disease, Very shortly afterwards Holth published
similar preliminary observations upon this test. The former investi-
gators concluded from their rather meager data that, "having regard
to what has been the experience in other diseases for which the test
has been extensively used, the difference between the agglutinating
power of a serum of a cow affected with abortion, and that of some
normal animals, is too slight to inspire confidence in the test".
Later these men reported favorably upon the agglutination reaction

and its value as a diagnostic test in infectious abortion. In this

report (4) they state, "In conclusion it may be said that the
agglutination test has now passed beyond the probationary stage,

and deserves to be adopted as the method of diagnosis which is

most generally applicable in suspected cases of contagious abortion.
The discovew ef such a reliable method of diagnosis removes what

has hitherto been the greatest obstacle in grappling successfully

with the disease either by private effort or under state regulation.
It my also be noted that it provides an easy means of ascertaining whether
there is any real foundation for the view that other organisms tlnn the
abortion bacillus, discovered by Bang, are responsible for abortion

on an episootic scale”.

Iohler and from (5) made a comparative study of the value of
the two serological tests and recommended the agglutination test, due
to its simplicity and the saving of time, except in doubtful cases,
when the complement fixation test may be employed to advantage.

Seddon (6) pointed out several very important factors in
connect ion with the agglutination test for infectious abortien. Re is
of the opinion that this-test is not simply a matter of dilution, but
a quantitative reaction. This same fact has been reported en by
several other investigators.

Connaway (7) states, "i'he serological tests, agglutination and
complement fixation, when properly applied, are as reliable for the
diagnosis of infectious abortion in cattle, as tuberculin is for the
detection of tuberculosis".

billions (8) expresses himself as follows, ”The frequency of
abortion in a herd usually corresponds fairly well with the intensity

of infection with Main My. In those herds where abortion

and other interferences with repeoduction.occur, the agglutination

and complement fixation test usually show intense infection with W
am. But such reactions can only show that M93212 9.3%

is somewhere active or has been active, and cannot showrthat it caused

a given abortionP.

Fitch (9) after making single agglutination tests on all animals
in several different herds and checking the histories of tin animals
‘with his tests, reaches the following conclusion , "At present the
results of the agglutination test cannot be utilised as a basis for
control measures for abortion disease".

Birch and Gilman (10) commenting on the three statements above
state, “If these views represent , as we believe they do, a fairly
reliable cross-section of opinion as it exists in regards to the value of
the serological tests in the practical handling of infectious abortion
it is evident that there are yet fundamental considerations on which
all are not agreed, nevertheless there are many well established facts
which are quite universally accepted and.to which additions are being
made”.

In a later paper on further studies with.the agglutination test,
Fitch.(ll) states that it is the most reliable of the serological
methods of diagnosing infectious abortion. He says further, "It'will
indicate the amount of hemJinfection, although it'will not pick out
individual aborters. A positive reaction to this test means past or
present infectionP.

Rettger (12) believes that the agglutination and complement
fixation tests may be satisfactorily applied in.the.diagnosis of
infectious abortion. their constancy and reliability make them.inp

dispensable in the study and control of this disease.

-7-

According to'Williams (13) "In the diagnosis of contagious abortion
the most practical method without sacrificing animals is presumably by
the agglutination or complement fixation tests. In the application
of‘these tests to abortions occuning in.clinics such as the ambulatory
clinic of the low York State Veterinary College and others, combined
with cultural searches and guinea pig inoculations from the abort and
the membranes, the presence of m m is recognisable in
about 60% of cases. But in many of these, other bacteria are also
present, Ihat relation, if any, they have to the abortion is not
known”.

Schroeder (14) states that ”If the agglutination test for bovine
infectious abortion is oxjcan be made serviceable to distinguish with
certainty between safe cows which, though' they have been exposed
to infection, and may have aborted, and.may show'some reaction, and
dangerous carriers of abortion.bacilli, it would tend greatly to reduce
the difficulties that must be overcome in attempts to suppress the
abortion evil through the use of sanitary measures".

It appears from the foregoing review of literature that the present
day investigators are gradually accepting the agglutination.test as a
valuable aid in the diagnosis of bovine infectious abortion and they‘have
come to look upon it as almost indispensable.

Of recent years many inyestigators have atmmpted to separate
strains of an organism into different addefinite groups by means of
the agglutination and agglutinin.absorption tests. Some of these
investigationdhave given valuable information.on the subject while
the results obtained by others have been fruitless. 'Iithout a doubt
these efforts, in some instances, have served a very useful purpose, but
Torrey (15) thinks that we may extend this method of grouping in such

a manner as to depart from a natural system of classification. He states

-3-

that a type or group, if established upon a rational basis, should
represent a distinct serological entity.

By the use of the agglutinin absorption test, Hermanies (16)
showed that gonococcus could be separated into six distinct hetero-
logous types. The agglutinins produced by strains of one type could not
be absorbed by strains of another, In five of these types, the agglu-
tinins were always bound by the strains belonging to the same type.
The antigenic complex of the strains forming a type seemed to have
a similar constitution, Strains forming type 3, however, varied in
their agglutinogerb and absorptive capacity, for, while a number of
strains bound the agglutinins produced by some strains completely,

- the binding capacity in others was more or less limited and variable,
and in d few entirely absent.

Warren (17) reported that the agglutination test does not serve
to differentiate strains of gonococcus into serological groups.

Torrey (18) reports, ”An analysis by agglutinin absorption
methods of the serological relationships of 7'! gonococcus strains,
isolated from cases of acute and chronic gonorrhea and its complications,
indicated that they may not be distributed among a number of clear-cut
immunological types. Straight. agglutination tests have yielded almost
no evidence as to specific serological relationships".

Ilaning and Clemenger's (19) results indicate that the B.
influensa of Pfeiffer is really an associated group of bacteria without
the marked unity in its immunity reaction as is seen in other epidemic
causing bacteria. Their results "show that almoet every strain as
regards to agglutination is a law unto itself!,

mall and Dickson (20) were able to separate nine strains of

B.inf1uensa into four distinct groups. Their work was based upon

-9-

cross-agglutinatien and checked by the agglutinin absorption test.

Bell(21) maintains that the influensa bacillus represents
a heterologous group of organisms as shown by cross-agglutination
and agglutinin absorption tests and tint identical strains do occur.

Using cross agglutination, agglutinin absorption, and
protective tests with strains isolated from different localities,

Boos (2:) reports that the various strains of B. influensa apparently
do not differ in kind.
from laitland and Cameron (23) studied 38 strains of B, influensa isolated
/_hospital patients during a period when no cases of epidemic influensa
were seen and found that nearly all the strains possess a serological
individuality as determined by agglutination and agglutinin absorption
tests".

Rivers and Kohn (24), working with 13 meningitis strains of
influensa bacillus, found that eleven were culturally alike and fell into
two groups by the agglutinin absorption test. Seven strains fell into
group 1, Three strains fell into group 2, with one intermediate group.
The other two strains stood alone culturally and serologically.

Peasier and liar (25) perfonied a series of absorption tests
with formalinised suspensions of Ms m and Baot. melitensis
which led to a fourfold grouping of 14 mg, m and Bact. melitensis
strains. The grouping revealed these principals: .

l. "in antiserum cannot be exhausted by strains of another group".

8. " A strain acts in a uniform manner(qualitatively) on all

strains in another group under the same absorbing conditions."

3, "Strains within the same gr-"rup do not necessarily act in a

uniform manner on one another when absorbed from the same antiserum".

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Evans (26) reports, "The agglutinin absorption test with
4’ strains of Brucella meletensis has shown:
"1. This species may be differentiated into at least seven
serological groups. Four of these groups include only one
or two strains each, and are relatively unimportant.
"3. The majority of bovine and percine strains fell into one
large group (30strains) which is designated variety abortus.
Two strains of human origin were in this variety. Twocf the
mall serological groups are so closely related to this one
that they should be considered as subvarieties.
'8. Simple agglutination tests can not differentiate between
varieties, abortus and melitensis”.
Iliger (27) found a aiture of a typical 3. coli and B. dysentery
shiga, which yielded immune sera possessing agglutinating properties
of practical equal quantity for each other. Absorption experiments made
with their respectibe sera indicated that two distinct agglutinins were
produeed in about equal amounts in the process of imnisation of rabbits.
The agglutinins were specific ones, each for its culture, and accessory
' agglutinins left the specific agglutinins quantitatively unaffected.
Avery (28) found that the biological classification of pnemococcus
distinguishes four distinct groups. He based these types upon well
defined imunological differences.
Hooker (29) found that strains of B. typhosus could be placed
into three fairly well defined groups. He based his classificat ion upon

the complement fixation and agglutinin absorption tests.

-11-

Technic Deployed.

We The sera were obtained from thirty rabbits
each of which had previously been injected with a suspension of

a different strain of living m abozgul. Each strain was
grown for twenty-four hours at 37°C on a slant of beef liver agar.
The growth was suspended in sterile physiological salt solution and
diluted until turbidity corresponded with tube 5 of McFarland's
hephelometer. Immediately after preparation one cubic centimeter
of the suspension of the organisms was injected into the marginal
ear vein of the rabbit. Each of the rabbits used had previously
reacted negative to the agglutination test using a strain of M.
m as antigen. Three weeks after injection,the rabbits were
bled from the ear in order to determine the titre of the serial.
The titre in each case proved sufficiently high enough for the
study. The rabbits were then bled from the heart, the blood
allowed to clot at room temperature, placed in the ice box over
night, centrifuged on the following day, and the clear serum re-
moved and placed in the ice box until used.

The same procedure was followed in building up sera for
the agglutinin absorption tests. Tn this case only eight strains
of m M were used.

c utinat T 8 Ten tubes were used in the cross,
agglutination test. Two c.c. of salt solution was placed in the
first tube and one c.c in the remaining nine tubes. Two-tenths
c.c. of serum was added to the first tube, giving a dilution of

1-10. One c.c. of this mixture was transferred to the second

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tube, giving a dilution of 1-20. This process was carried through
the remaining tubes and one c.c. of the mixture in the last tube was
discarded,thus diluting the original serum one-half on each transfer.
To each of the ten tubes one c.c. of the antigen was added, which
again resulted in diluting the serum one-half in each tube and de-
creasing the turbidity of the antigen one-half. The final dilutions
were: 1-20, 1-40, 1-80, 1-160, 1-320, 1-640, 1-1280, 1-2560, 1-5120,
and 1-10240, The tubes were incubated at 37°C and readings taken at
the end of 24 and 48 hours. The thirty sera were run against each of

the thirty antigens thus giving cross-agglutination of homologous

and heterologous st rains.

 

W Thirty strains 01' seam spams vero arm
for 24 hours at 37°C on liver agar slants as recomended by Huddleson(50).

The growth of four slants was washed off with phenolised salt solution,
pH 6.0, and the suspension made up to correspond with tube number one
of the lcParland's nephelometer, and stored in dark brown bottles in

a cool place.

W The bacterial growth from 25 liver agar
slants was washed off with 20 c.c. of phenolised salt solution. To
the suspension, 20 c.c. of homologous serum (diluted 1-!) was added

thus giving a dilution of 1-10. This was incubated at 37°C for two
hours and placed in the ice box over night, The suspension was centri-

fuged the next day and the clear supernatant fluid, which in reality
is the serum diluted 1)“ 1o, pipetted off. no agglutination test
he run on the series, using the homologous antigen, to see if all the

homologous agglutinins had been removed. If there was an degree of

~13-

agglutination, the serum was reabsorbed in the following manner. The
growth from five slants was washed off with the phenolised salt solution,
and the suspension concentrated by centrifugation. The supernatant
fluid was discarded and the sedmented organisms added to the 1 to 10 serum.
This as incubated as before and centrifuged. This method of reabsorp-
tion was carried on until the agglutination test showed that the
homologous agglutinins had all been removed. Then the absorbed serum
was run against 26 heterologous antigens and the results recorded.

The agglutination test used in this work is the same as that
used 'above except that eachbf the tubes contained one c.c. of salt
solution and one c.c. of the absorbed serum was placed in the first tube.
The method of making progressive dilutions was the same. The resulting
dilutions were: 1-40, 1-80, 1-160, 1-320, 1-040, Only five tubes were
used at it was thought that this would give a satisfactory end point,
which proved to be true in each case. The tubes were incubated, at 37°C
and readings taken at the end of 24 and 48 hours. mly the 48 hour
readings are recorded here as they were approximately the same.

0 f 00 in It was necessary to prepare a calorimeter

which would have a range of pH from 4.0 to 8.8 with a difference of a
.4 p2 between each tube. A prepared tablet for each pl requied was placed
in a tube containing 10 c.c. of distilled water and dissolved. The
following indicators were added to the corresponding tubes: Tubes pH 4.0
and 4.4 eight drops of Brom Phenol Blue were added, tubes of pH 4.8,.
5.2, 5.6 eight drops of Methyl Red were added, tubes of pH 6.0, 6.4, 6.8,
7.2 and 7.6 eight drape of Bram Tumol Blue were added, and for tubes of
pl 8.0, 8.4, and 8.8 eight drops of Cresel Red were added. A crystal

of Thymol was added to each tube as a preservative. This calorimeter)

~14-

is used as a standard to prepare solutions of corresponding hydrogen ion
concentration.
t f Anti e o Differen ‘: Strain 2a was selected for this
study, Antigens were prepared with the following pH values: 4.0, 4.6,
4.8, 5.2, 5.6, 6.0, 6.4, 6.8, 7.2, 7.6, 8.0, 8.4, and 8.8. Preliminary
tests had been carried out on 10 c.c. of distilled water to determine
the number of drape of [/1 £01 and 1/1 Naon it’took to obtain a buffered
solution which would correspond to each of the above pH values on the
colorimeter. A capillary pipette was used throughout the work for the HCl
and one for the NaOH, in order that the drops would be uniform. 200 c.c.
of each buffered solution was made up using the above data as a bale, and
each solution was checked with the colorimeter. A 24 hour beef liver
agar slant growth of strain 2a was washed off with each of the buffered
solutions. The turbidity of each was made to correspond with tube
nuisber one of llcFarland's nephelometer. The suspensions were checked
to determine their value and stored until ready for use.
W m Eight tubes were used in the test. 2 c.c. of the
antigen was added to each tube. The serum was added to each tube in
the following amounts:
1st tube............... .08 c.c. of serum, giving the dilution 1 to 25
2nd tube............... .04 c.c. of serum, giving the dilution 1 to 50
3rd tube............... .02 c.c. of serum, giving the dilution 1 to 100
4th tube............... .01 c.c. of serum, giving the dilution 1 to 200
5th tube............... .08 c.c. of l to 20 sort, giving the dilution 1 to 500
6th tube............... .04 c.c. of 1 to 20 serum, giving the dilution 1 to 1000
7th tube.................02 c.c. of 1 to 20 serum, giving the dilution 1 to 2000
8th tube............... .01 c.c. of 1 to 20 serum, giving the dilution 1 to 4000
Seven sera was used, including two negative and five positive. The tubes
were incubated at 37°C and readings taken at the end of 24 and 48 hours.

0
The lattems recorded here as both readings were approximately the same.

TABLE I.

History of Strains of Bat-M91231 anloyed in these Studies.

COCOOOOOOOOOOOO...0.0.0.....00..0.00000000000000000000000000000000000000000

Strain

Date of Isolation

Origin

COCCOOOOCOOOOOOOOOO0.0.0.0000...0.0.0....OOOOOOOOOOOOOOOOOO0......00......O

“0.2.H

16
30

28

60

100
200
300
600
808

1639
2010
Berlin
C

D1

3
higland
G. B. Swine
H16
3221

J

King

I.
Boadard
I

Unknown
1915

1910

1916

1915
Unknown
June, 1917
1919

Sept., 1919
Feb. 1920
June, 1920
Jan., 1920
1922

1922

May, 1919

1923

Rec'd 1920
Unknown
March 1917
1923

Prior to 1916
unknown

1920

1922

1924

1916

Unknown
December 1920
Unknown
Unknown

Received from B.A.& prior to 1915

Aborted fetus (herd A, Mich.)

0311‘s EXP. State AbOl'tihg '0'.

Aborted fetus (herd A, Mich.)

Udder of cow 995 (Abortion Exp.herd)

In laboratory prior to 1916.

Aborted fetus (near Owosso, Mich)

Placenta of cow following abortion.
(near Ubly, Niche)

Aborted fetus (herd A, Mich)

Udder of cow (herd A, Mich)

Udder of cow (abortion exp.herd)

Udder of cow (abortion exp. herd)

Aborted fetus, Michigan.

Aborted fetus, Michigan.

Placenta of cow following abortion
(abortion exp. herd)

Udder of cow (abortion exp. herd)

Dr. Graham, Illinois University.

Obtained in German 1919.

Aborted fetus (herd A, Mich.)

Aborted fetus near Detroit.

Unknown.

Royal Veterinary College April 1919

Aborted fetus of sow(near Lansing,Mich)

Aborted fetus, Kasas Exp.Stat ion.

Aborted fetus (nutrition Exp.herd).

Aborted fetII (1161‘. B, M1011)

Obtained 1923 from Dr. Stockinan,England.

Aborted fetus (herd A, Mich.)

Purdue University, 1924.

Wisconsin University 1916

kperimental Data.

Win; During the last decade many investigators

have attempted to group strains of an organism by the use of
cross agglutination. A review of the literature shows that some
very encouraging results have been obtained, but failures also
have been recorded.

In this work, thirty strains of M11122 gbortg. were
used in the production of thirty sera. Thirty different antigens
were prepared from the same strains, and an agglutination test was
perfomed with each serum against each of the antigens. Charts
from 1 to 60 inclusive show the results of these tests, each chart
representzhe results of agglutination of one serum against the
thirty antigens. The black columns show the end point of agglutina-
tion at the end of 24 hours, while the red calms denote the end
point of agglutination at 48 hours.

It may be seen that antigen £221 is agglutinated only by its
homologous serum and by heterologous serum 2a. There were seven sera
whose agglutinability was variable. These seven were H221, 806, .1, D1.
7, 1, and 100. The variation was quite marked in several instances.

The agglutinability of the antigen was not constant for each
sera. lo line could be drawn to setcff any one group of strains,
unless it be antigen E221. Taking the results of agglutination of 29
of the antigens, excluding B221, one will note that the average end point
of agglutination is about 1 to 1280. There were some antigens which were '
agglutinated by a serum in a dilution as high as 1 to 10240 and as low
as l to 40. (Chart 31 denotes the average agglutinability of each of the

antigens against all of the sera. Two antigens differ from the remain-

-17-

inc antigens very distinctly. H 221 has the lowest average of all.
The average agglutinability of Roadard is much lower than that of
the remaining 28 strains.
W: In addition to the use of cross agglutination
in attempting to classify strains of a certain organism, much work has been
done with the agglutinin absorption test in this respect. Better
results have been attained with the latter, although failure has been
omen.

In this work eight of the onginal strains were used to produce
sera. They were, 3221, 806, 400, 2a, 2010, Boadard, Swine l, and I.
Each strain was absorbed .by its homologous strain, and to make sure
that all of the homologous agglutinins were taken out, an agglutination
test was run on the absorbed serum using its homologous antigen. A
negative test showed complete absorption of the homologous agglutinins.
If a positive test was obtained the corn was reabsorbed until a
negative test was obtained. When the serum was proved to be absorbed
agglutination tests were run against 26 heterologous antigens. Serum
400 was absorbed also by the heterologous antigen 2a, and then the
absorbed serum was run against the same antigens.

Tables 2 and 3 give the results of these tests. The results
were recorded as P(partia1), + (positivq, -(negative), and 0 (no test).

Strain 400 is the only antigen which absorbed completely all the
agglutinins from its homologous serum. Swinl left agglutinins in for
one strain. 2010 absorbed all agglutinins except for two strains. 806
and I left agglutinins for three strains. The remaining tests show that
after absorption of the homologous agglutinins, there remained agglutinins

for a majority of the heterologous strains.

 

W Many laboratories vary as to the pH of the

antigen used in their agglutination test. Table 4 gives the results

of the agglutination tests using antigens of different pH values.

The only marked difference shown is on the antigens of a pH
4.0, 4.4, and 4.6. In these, there is a tendency for the acid antigen
to cause the formation of a flocculent precipitate in the presence of
serial, which settles to the bottom of the tube. It has the appearance
of EB“! to vhich alum has been added. The occurrence of this
phenomenon was constant in tubes of pH 4.0, and 4.4,and in two sera
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-1’-

Discussion.

From the data obtained in csess agglutination it may be
seen that no two antigens were agglutinated to the same degree
by each of the thirty sera. However, there were many antigens
which were agglutinated to the same degree by s single heterol-
ogous serum. Chart number (8) shows that antigen 100 produced
agglutinins to a markedly low degree for antigens 2a, 400, and
316. This occunsnce is difficult to explain as no other serun
,sgglutinated thse three antigens to such a low degree. In many
cases the agglutinins produced by an antigen gave a much higher
titre for heterologous antigens than for the homologous antigen.
The charts show that strain H221 differed markedly in its agglutin-
ability from the other strains, or the agglutinins produced by the
other strains with the exception of fl 2a were of a different type
than those which are necessary to cause a clumping of this anti-
gen. If £221 is to be considered alone, some reason must be given
for such an individual inagglutinability. Kmiede, Cooper, and
Provost (31) report the possibility of existence of degraded
variants. They den-ibe such strains as having lost their agglutin-
sbility and a marked loss in antigenic power. In this case 3221
scene to have a very low agglutinability but possesses an average
antigenic power. Therefore, one of its prOperties function, whiflle
another has degraded or has been lost almost entirely. Krmnwieds (31)
and his co-workers also state that a degraded variant may stimulate

the production of agglutinins which it is relatively unable to find,

One may notice that 3221 antigen is agglutinated in relatively low

dilutions by its homologous serum, as compared to heterologous strains.

-20-

All of the other antigens stimulated the production of agglutinins of
a fairly high titre for both the homologous and heterologous. It is
possible to conclude from this reasoning that strain H221 is a degraded
variant.

The fact that serum 2a agglutinated antigen H221, however, cannot
be explained by the above reasoning, 'But let us refer to the agglutinin
absorption test for a little information. When serms 3221 was absorbed
by its homologous strain it removed the agglutinins for a Zn also.

When serum 2a was absorbed by its homologous strain it removed the
agglutinins for 3221 as well. Therefore, we may be led to believe that
these two strains are closely related.

In considering the final results of cross agglutination it
is readily apparent that no group separation of the thirty strains
used is possible, except, possibly in the case of strain 3221 which
might be placed in a distinct group or the torn degraded variant applied
according to Krumwiede, Cooper, and Provost (31).

It IUOUC that the agglutinability of a given strain is of
considerable importance, especially from the standpoint of accurate
diagnosis and the obtaining of comparable results by different labor-
stories engaged in the performance of the agglutination test. The
charts reveal that antigensnrepared from different strains are not
agglutinable to the same degree by a given seru. This finding in
all probability explains the great degree of variation of the
agglutination titre obtained by different labOratories on the same
801'“.

In the agglutinin absorption test, after absorbing the sera
with their homologous strain, agglutinins remained to a low degree
for heterologous strains in every instance but one. Strain 400

removed from its homologous serum its homologous agglutinins and

-21..
those for 26 heterologous strains. In this case strain 400 was able to
remove all agglutinins, the production of which was due to the stimulus
of this strain. Ira-wilds (31) and his co-workers believe that such
a statement can safely be made, but they report two exceptions to this.
First, normal agglutinins may be present; second, agglutinins whose
production was stimulated by degraded variants may be present. They
state that all of the latter agglutinins are not always absorbed by the
degraded variants which stimulated their production. We will consider
these exceptions under those cases where agglutinins for heterologous
strains were present after absorption.

When serum 400 was absorbed by heterologous strain 2a both
homologous and heterologous agglutinins were removed. This tends to
show that strains 400 and 2a may be very closely related.

The seven sera which contained heterologous agglutinins, after
homologous agglutinin? absorption, present a different problem. we
might’explain their presence by calling than normal agglutinins, but the
sera of the rabbitsvere tested before injection for agglutinins, and 1
all gave negative results. The presence of heterologous agglutinins
might be explained by refering to the strains which stimulated their
production, as degraded variants. Isit logical to say that out of eight
strains. only one could be classed as being normal, and the remaining
seven as degraded variants? The presence of heterologous agglutinins
in the absorbed serum, might be explained by calling them non-specific
agglutinins. It seems possible for a strain to stimulate the production
of two kinds of agglutinins, namely specific and non-specific. The
specific agglutinins wouldbe absorbed by the homologous strain only,
while the non-speci fic agglutinins would remain in the com. If a
homologous strain removed its homologous agglut ininn and also removed

others which were thought to be heterologous, all would have to be

-21.
those for 25 heterologous strains. In this case strain 400 was able to
remove all agglutinins, the production of which was due to the stimulus
of this strain. Kruwiede (31) and his co-workers believe that such
a statement can safely be made, but they report two exceptions to this.
First, normal agglutinins may be present; second, agglutinins whose
production was stimulated by degraded variants may be present. They
state that all of the latter agglutinins are not always absorbed by the
degraded variants which stimulated their production. We will consider
these exceptions under those cases where agglutinins for heterologous
strains were present after absorption.

When serum 400 was absorbed by heterologous strain 2a both
homologous and heterologous agglutinins were removed. This tends to
show that strains 400 and 2a may be very closely related.

The seven sera which contained heterologous agglutinins, after
homologous agglutinin? absorption, present a different problem. we
mightlexplain their presence by calling than normal agglutinins, but the
sera of the rabbitsvere tested before injection for agglutinins, and I
all gave negative results. The presence of heterologous agglutinins
might be explained by refering to the strains which stimulated their
production, as degraded variants. Is it logical to say that out of eight
strains, only one could be classed as being normal, and the remaining
seven as degraded variants? The presence of heterologous agglutinins
in the absorbed serum, might be explained by calling them non-specific
agglutinins. It seems possible for a strain to stimulate the production
of two kinds of agglutinins, namely specific and non-specific. The
specific agglutinins would be absorbed by the homologous strain only,
while the non-specific agglutinins would remain in the serum. If a
homologous strain removed its homologous agglutinins and also removed

others which were thought to be heterologous, all would have to be

 

-22-
classed as specific. This occurred when serum goadard was absorbed
by its homomous strain, for the agglutinins for Berlin, King, and
2a were also removed. Thus the remaining agglutinins were non-specific,
and also the homologous strain was unable to remove them. This also
occurred in the other six sera. Therefore, it seems possible to assume
that a strain may stimulate the production of both specific and non-
specific agglutinins. Variations have been noticed in the agglutination
tests of various laboratories on the same sera. Investigators are de-
sirous of eliminating all possible variations in this test with the
ultimate view of standardisation. It was thought that the pH of the
antigen might be one of the causes ofvariation in sgglutinability, so a
series of tests were carried out using antigens of different pH. The
only variations noticed in these tests were recorded in antigens with
a p! of 4.0, 4.4, and 4.8. It seemed that in these antigens, the con-
centration of the acid was so strong, that it had the prOperty of
causing the albumin of the serum to be precipitated to the bottom of
the tubes in a flocculant mass. This interfered with the reading of the
tests, so it was thought advisable to use an antigen whose pH was some-
what higher than 4.8. The antigens used in the cross agglutination
and agglutinin absorption work had a pH of 6.8 and all of the reactions were
easily read and no precipitation was noticed. to differences were
noted in sgglutinating power in, antigens from pH 5.2 to pH 8.8. The
alkaline antigens did not appear to interfere with agglutination. So, if an av—
erage is taken between these two extremes it should give an antigen
of a pH which would give satifactory results. This numberical average

is p! 7.0. The literature shows that agglutination test is more
efficient‘if the antigen is slightly acid. So, if the p! of the antigen
to be used in the test is 6.8. it is believed that the results of the

test would be much more constant.

-25-.

Conclusions.

Gross agglutination does not serve to group the thirty
strains of W m used in this study, although one
strain appeared to be of a different group.

The agglutinin absorption test as used shows no specific
differences between the twenty-six strains of MILE! abortgl
used. This test, however, has possibilities and should be given
further study.

The hydrogen ion concentration of an ant igin does not
effect its agglutinability unless the antigin has a m of 4.0 or

below.

The writer wishes to acknowledge his indebtedness to
lir I. Forrest Huddleson, Hr. W.L.lallman, and Dr. Ward Giltner
for the assistance and helpful suggestions received during

the course of these studies.

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(a)

(9)
(10)

(ll)

(12)

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‘ Not verified.

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