THE GENERiC RELATIONSHIP OF
BRL‘CELLA, PASTEURELLA
PFEIFFERELLA AND HEMOPHILUS
THESIS FUR THE DEGREE OF M. S.
Ina Maxson

1934

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THE GENERIC RELATIONSHIP OF BRUCELLA, PASTEURELLA,
PFEIFFERELLA AND HEMOPHILUS

THE GENERIC RELATIONSHIP OF BRUCELLA, PASTEURELLA,
PFEIFFERELLA AND HEMOPHILUS

Thesis

Submitted to the Faculty of.Michigen State College
of Agriculture and Applied Science in partial
fulfillment of the requirements for the

degree of Master of Science

Ina Maxson

Dec., 1934,.

1
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1

 

THESIS

CON TEN TS

I. Introduction
11. Historical Resume
III. History of Cultures
IV. Experimental
(1) Bacteriological studies of smooth strains.
(2) Methods of dissociation used.
(3) Bacteriological studies of rough strains.
(4) Bacteriological comparison of the smooth and
rough forms.
(5) Rabbit immunization with the smooth and rough
forms of all organisms.
(6) Methods used for the agglutination tests and
the reading of titres.
(7) Results of the agglutination tests.
(8) Agglutinin absorption studies.
V. Discussion
VI. Conclusions
VII. Acknowledgement.
VIII.Literature Cited

I. INTRODUCTION

In the literature of the past 15 years, fragmentary
observations have appeared suggesting a serological re-
lationship between various species of Brucella and
Pasteurella, Brucella and Hemophilus, Brucella and
Pfeifferella*, and other combinations.

In 1950 Mallmann published a preliminary report of
the interagglutinability of the Brucella, Pasteurella,
and Pfeifferella genera. He observed quite marked cross
agglutination. Using Brucella ab ortus and B3. gigs,
immune rabbit serums, he Obtained as high titres with
the heterologous antigens as with the homologous ones.

No attention.was paid to the state of dissociation of the
cultures used.

It is well known that when a smooth strain of most
genera shifts to its rough fonm, profound antigenic
changes occur. Very frequently the rough strain is
serolognrally different fram its smooth.prototype. White
(193 ) has demonstrated that the rough forms of the species
of the genus Salmonella have a.ccmmon rough antigen that
is demonstrated serologiially by marked cross agglutination.
These same species in the smooth.phase are distinctly
different serologically.

* Although the genus Pfeifferella has been merged with
Actinobacillus in the latest edition of Bergey's "Determin-

ative Bacteriology," the name is retained in this thesis
so as not to confuse with.other members of the Actinobacillus

group.

-2-

This antigenic relationship of the rough phase
of other genera may also be true of the Brucella,
Pasteurella, Hemophilus, and Pfeifferella groups.

A review of the literature of these genera would
indicate that such might be the case. Furthermore, a
close examination of the properties and characteristics
of these genera shows a very close similarity.

For these reasons, a study of the Brucella, Pasteurella,
Hemophilus, and Pfeifferella groups has been made. The
work consists of morphological studies of both smooth and

rough phases of representative species of these genera.

II . HIS TOR ICAL RESUME

A serological and phys iOIOgical correlation of Brucella
and Alkaligenes bronchisepticus was reported by Evans in
(1918). In the same year Povitzky and Worth (1918) showed
a cross agglutination between 513;. bronchisepticus antigen.
They report no cross agglutination between g. pertussis
and g. influenzae.

Ferry and Hoble (1918) found the same serolog ical
relationship between Ag. bronchigepticus and g. pertussis;
also a cultural, morphological, and physiological similarity
which is more marked after growth for some time on an
artificial medium.

Francis and Evans (1926) found that 57 per cent of

positive tularemia serums showed cross agglutination with

 

\I:

-3-

BB. abortus and BB, melitensis. In 3 cases the titres
were equal for all 3 of these organisms, although as a
rule the heterologous titres were lower. Burnet (1127)
also reported interagglutinability 0f.§° tularensis and
Brucella in tularemia and Mediterranian fever.

Huddleson found positive cross agglutination of
Pfeifferella1mallei and BB, abortus.

In contradiction to the results obtained by Mallmann,
Priestly (1933) reported no serological relationship between
the Brucella and the Pasteurella genera. Thompson (1933)
1lsted BB. mallei ser010gically with strains of B5, abortus

and B. tularentis and found no evidence of cross agglutination.
III. HISTORY OF CULTURES

All of the following Brucella organisms used in these
studies are from.the culture collection of Dr. I. F. Huddleson.
BB, abortus 966 was isolated in 1930 from a cow from
a Michigan herd.

_B_1;. _s_u_:_1._§_ 436 was isolated in 1928 and obtained from
Crawford from Washington D. C.

BB. melitensis 316 is of human origin. It was obtained
from Dr. Burnet, Pasteur Institute, Tunis, Algeria, and
dissociated by Mallmann and Gallo (1933).

B15. melitensis 384 was isolated in 1929 frcm a mammary

 

lymph node of a goat.

BB. abortus 4a was isolated in 1924 from an aborted

 

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-4-

fetus from a cow from the Michigan State College experimental
herd. It was dissociated by Mallmann and Gallo (1933 ).
E13: ,s_1_11_§ 404 was isolated in 1922 from a premature
fetal pig frcm a naturally infected sow. It was received
from J. W. Connaway, University of Missouri. This was
also dissociated by Mallmann and Gallo (1933 ).
Br. _s_y_i_s_ 401 was isolated in 1925. The source of the
culture is not known. It was obtained from Purdue University.
B_I_‘. melitensis 319 was obtained from Dr.K. F. Meyer,

 

Hooper Foundation, San Francisco, May 10, 1921.

BB. abortus 5a was in the laboratory prior to 1916.

Bpsteurella avicida was obtained from the Michigan State
College stock culture collection.

Pasteurella tularensis 38 was a non pathogenic strain
from Dr. Francis, obtained. from the Michigan State Department
of Health culture collection.

B. bovisepticus was isolated from a buffalo with
hemorrhagic septicemia. It was obtained from the Bureau
of Animal Industry, Washington, D. C.

B. pertussis was obtained from the Michigan State
Department of Health culture collection.

B. influenzae 1601 was obtained from the Michigan State
Deparmnent of Health culture collect ion. It was rough when
obtained.

BB. mallei was obtained from the Michigan State

College stock culture collection. It was rough when obtained.

-5-
BB. mallei F87 was obtained from the Bureau of

Animal Industry, Washington, D. C.
IV. EXPERIMENTAL

1. Bacteriological Studies of Smooth Strains.

Bergey (1930) classifies the organisms of the
Brucella, Pasteurella, Hemophilus, and Pfeifferella groups
as minute Gram negative bacteria with a tendency to bi-
polar staining in the Hemophilus and Pasteurella genera.
All the organisms used in the following studies are non-
motile. With the exception of B. avicida all, are inactive
in carbohydrate media, none of them producing acid or gas.

B.inf1uenzae, B, pertussis, B. tularensis, B. _e_g__u_.’_i_-
septicus, and the Brucella group are aerobes. B. avicida
and Bf.mallei are facultative aerobes.

All organisms used produce small grayish colonies.
BB. mallei liquefies gelatin very slowly. No other organisms
of these groups liquefy gelatin.

There is, thus among all of these genera a close
morphological and physiological similarity of organisms.

Table I on the following page gives the characteristics
of these bacteria as found in Bergey‘s manual of Determin-

ative Bacteriology, (1933 ).

-6-

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-7-

At the beginning of these studies all cultures were
plated and S colonies fished. A chicken infusion medium
was used for culturing all organisms throughout this work.
For about 3 months stock cultures of.B._p§rtussis and B.

influenzae were held upon blood chicken infusion agar, and

 

B, tularensis upon chicken infusion agar containing 0.1
percent cystine. However, the colonial appearance of the
cultures remained the same upon chicken infusion agar
without the blood or cystine and these ingredients were
omitted from the stock medium.

All organisms were checked morphologically and
physiologically with Bergey's classification of these genera.
The size of the rods of the same culture varied. Sometimes
they appeared as cocci and at other examinations the organisms
were definitely bacilli. However, they were never long
rods when in the S colonial state.

Bipolar staining was not noted in the organisms with
an S colonial appearance. All of the colonies were small,
gray, glistening and moist with smooth edges and surface.

,B. avicida although tested several times, at no time
produced acid in 0.5 percent sucrose chicken infusion broth
with Andrade's indicator.

‘B. tularensis grew in milk. This is contrary to Bergey.
This may have been.due to the fadt that this was an old culture
accustomed to growing upon artificial media. The reactions

of all of the other cultures in litmus milk were the same as

Bergey's

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‘8-

.BB. mallei S was lost before it was checked for
its ability to liquefy gelatin in a stab culture. .BB.
mallei R did not liquefy gelatin. No other organism of these
groups attacked gelatin which was also true according to
Bergey.

The following table II shows the morphological and

physiological characteristics of the cultures used.

-9-

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-10-

2. Methods of Dissociation Used.

BB. melitensis 384, BB. abortus 4a, BB. suis 404

 

and BB. melitensis were dissociated to their R strains by

 

repeated 48 hour transfer in 10 percent homologous serum broth
at 37°C. .§£°.§El§ 404 R and BB. melitensis 316 R were
dissociated back to their S prototypes by mailmann and Gallo,
(1933). None of the other dissociants have been reverted.

,B. avicida R was dissociated by holding the culture in
the ice-box for several months. During this time the culture
became a mixed R and S foruL Typical S colonies were fished
several times, but after being held for sometime as stock,
during which time the culture was transferred only occasionally,
the R type appeared when plates were streaked.

Attempts were made to dissociate B. tularensis and B.
_pertussis by repeated 48 hour transfer at 37°C., but each
time the organisms died before typical R colonies were obtained.
It had been.noted that when the S organisms were groWn.upon
1 per cent dextrose chicken infusion agar, which had been
used for holding stock strains, the cultures tended toward the
R type. When.grown upon chicken agar without dextrose, the
culture remained more typically S. Concluding from this
that it was perhaps the carbohydrate producing this change
from.S toward R, these organisms were grown in 1 per cent
sucrose broth and dissociated to their R types by repeated

48 hour transfers at 37°C.

-11...

.B. influenzae was an.R form when received. Attempts

 

to revert this organism to the 3 type by animal passage
were unsuccessful. A mouse was given.an intraperitoneal
injection of 1 cc. ole. influenzae culture, containing the
organisms from 1 slant. Within 24 hours the mouse died. The
culture was not recovered. In second and third atteumts the
mice did not die.. At 72 hours they were killed,but the
injected culture was not recovered.

'BB. mallei was also an R strain when received. It had

been held as a stock culture for many years.

3. Bacteriological Studies of R strains.

As with the S forms of these organisms, morphologiral
and physiological studies were made of the R types. At
scme time during the period in which they were being observed
for the following studies all R forms were Gram positive.
This was not contamination for the same culture later after
several transfers decolorized and took the counter stain,
then again returned to Gram positive. Also Gram positive
and Gram negative organisms were often present in the same
slide preparation. All R cultures at scme time during their
use in agglutinin absorption studies showed barred and bipolar
staining with methylene blue.

The R colonies were more Opaque, larger, duller and

flatter than the S type, with a granular and wrinkled appearance.

-12-

An exception was found in the B. avicida colonies, in which
case the S and R colonies were much the same in size. The
I forms were not flattened,but possessed the typical R surface
and opaqueness.

Some of the organisms acquired motility in their

change to the R type. 'B. avicida did not change its reaction

 

toward litmus milk. 'B. pertussis lost its ability to produce
an alkaline reaction in litmus milk.

The R forms of the Brucella genus retained their inactivity
toward carbohydrates. ‘B. avicida in its R form still did not
produce acid in 0.5 per cent sucrose chicken.broth and the
reaction of this organism for the other sugars remained the

same as in the 3 form. .§° pertussis R'by dissociation had

 

gained the ability to produce acid in all the sugar media
used, although in its 3 form no acid was produced in any
sugar medium.

The ability to liquefy gelatin was not acquired by any
organism by dissociation.

Table III, on the following page gives the morphological
and physiological properties of the R forms of these groups

of organisms.

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-14-
4. Bacteriological Comparison of the S and R Forms.

From the studies of the morphological and the cultural
properties of the S and R types, a varying degree of change
in the different organisms is seen. The R type in each case
was determined only by colonial appearance. From the data
shown in the preceding tables there appeared to be an increase
in the ability of all R organisms to become Gram positive.
All R organisms had increased in size, also there was an
increased tendency to barred or bipolar staining. Motility
in several cases was found in the R form of the bacteria.

,B. pertussis,R, an.organism non-active in carbohydrate

 

media, had acquired by dissociation the ability to produce
acid in all the sugars used, dextrose, sucrose, mannite,
levulose, and maltose. No other organism showed any change
in activity toward the sugars used.

There appeared to be no correlation between the R and
S types in their reaction in litmus milk. Some had become
more active, others less active. .EE- abortus 966 S, BB.

suis 436 S and BB. melitensis 384 S gave an alkaline reaction

 

in litmus milk. .§£~ suis 404 R, BB. melitensis 316 R and Br.
:melitensis 384 R had become more active toward litmus milk
by dissociation, reducing the litmus as well as first showing

the alkaline production characteristic of the S prototypes.

-15-

5. Rabbit Immunization with the S and R Forms of All

Organisms Studied.

Rabbits were immunized with both the smooth and rough
dissociants of each organism.in which both forms were available.
The immune serums for BB. abortus 966 8, BB. gggg 436 S and
B3, melitensis 384 S were produced by l intravenous injection
of the unkilled cultures. One cc of a suspension with a
density corresponding to tube No. 3, McFarland's nephelometer,
was used. About 3 weeks later the animals were bled aseptically
from the heart.

Antigens of all other cultures were killed by heating
at 60°C for 1 hour and with l exception all were injected
intravenously. An intraperitoneal injection ole. influenzae R
was made because spontaneous agglutination produced clumps
in the bacterin too large for safe intravenous injection.

The bacterins were given every 3 to 4 days in 1 cc
amounts of a suspension, with a density corresponding to
tube No. 3, McFarland's nephelometer. At this density about
1,000,000,000 organisms per cc were present. An average of
about 7 injections were made before the titre was sufficiently
high to bleed the rabbits. The blood was collected under
sterile conditions from the heart and all serums were preserved

with 0.5 per cent phenol.

-16-

6. methods Used for Agglutination Tests and the Reading

of Titres.

All antigen suspensions used in the agglutination tests
were made in 0.1 per cent salt solution containing 0.1 per
cent phenol. A density comparable to tube number 2 McFarland's
nephelometer standards was used.

The serum serial dilutions were also made in 0.1
per cent salt solution containing 0.1 per cent phenol. Each
tube contained 0.5 cc. of the serum dilution. To each was
added from a dropper 1 drop of‘the antigen.suspension. This
gave a density correSponding to about 3/4 the density of
mcFarland's standard tube number 1. A control tube was made
for each test by omitting the serum, using only the saline
solution plus antigent After adding the antigen suspension
the tubes were shaken and incubated for 48 hours at 37°C.

At the end of this period the titres were read over a student
reading lamp, held against a dark back ground. The shade was
turned in such.a way that the bottom.edge was perpendicular

to the table, and the shade between the reader and the light.
The tube containing the gglutinated suspension was held aganst
this perpendicular edge above the light. All readings were
made with a hand lens, for the clumps in many cases were too
mnall to be seen with the unaided eye. After this imtial
reading the tests were placed in the ice box over night and

read again.
The titres of the immune serums for their homologous

antigens are given in the following table 4.

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-18..

After determining the titre of each antiserum with
its homologous antigen, the titres of all immune serums
with all heterologous antigens were tested with the following

results. Table V.

- 19 -

TABLE V. .
CROSS AGGLUTINATION OF ALL R AND S ARTISERUMS WITH ALL
R AND S HOMOLOGOUS AND HETEROLOGOUS ANTIGENS

(D
H
S
(D

 
 
  

   
      

   
     
  

   

IR

   

     

     

        

         

    
      

Antigens

melitensis 516 R

melitensis 319 S
equisepticus S

   

   

abortus 5a S
suis 456 S
suis 401 S
abortus 4a R
tularensis S

   

     

avicida R

    

    

    

   

   
 

- influenzae R

.L o

    

  
      
  
 

 

Br. melitensis 384 8

Br.
P. bovisepticus 3

Positive abortus
Br. suis 404 R
P. avicida 8

Br.
Br.
P.
P.
P.

     

  

Br.

 

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Br.

     

      
   
 
  
   
   
   
  
  
 

    
  
   
 
    
   
   

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ens
a
a

    
 

   
   
 
  
 
   
   

 
       
 

    
   
 

The figures represent the number of the tube of’greatest dilution
showing an agglutination of a turbidity of 2 or greater.

The dilutions are designated as (1) 1-20, (2) 1-40, (5) 1-80,
(4) 1-160, (5) 1-320, (6) 1-640, (7) 1-1280, (8) l-2560.

' Positive serum.from a naturally infected cow.

- 20 -

There is marked cross agglutination between the Brucella,
Pasteurella, Hemophilus and Pfeifferella genera as shown in
the preceding table. The Brucella immune serums agglutinated
all antigens, usually giving a slightly higher titre for their
Specific antigens.

‘Pi. mallei S antigen.did not give a positive titre in
a dilution greater than 1 to 80 with smooth immune serwm.
With rough immune serums a slightly higher titre was produced
and positive abortus serum from naturally infected cow agglu-
tinated 2:. mallei S in as high dilutions as it did its
homologous antigens.

Some of the species antiserum of each of the other genera
used agglutinated some Species of all genera in dilutions
as high as l to 1,280.

The agglutinating titres of B3. abortus and g. pertussis
S antigens, with all serums were more nearly parallel,than
either antigen showed with other members of its own group,
although @2- abortus and g. pertussis S antiserums did not
agglutinate equally with all antigens. g, pertussis S serum
agglutinated g. avicida S and R strains in dilutions equal to
its homologous antigens. ,2. avicida S serum showed only
slightly less agglutination for g. pgrtussis S but only
one half this titre for g._pertussis R.

Pf. mallei R serum gave a low titre for g. avicida S
and R, the titre being approximately equal for both strains.

2. avicida 3 serum gave a low titre for g§.:mallei S and R

- 21 -

antigens. .g. avicida R serum agglutinated both in a slightly
higher dilution.

Tables VI and VII contain the agglutination data for the
rough immune serums. The R serums gave a higher titre for
the specific R antigens than for the homologous S antigens.
However for the heterologous agglutination, sometimes the
titre was higher for an R serum and an R antigen, sometimes
for an R serum and S antigen, and in.some cases the same
titre for both 3 and R was shown. In general the Brucella
R serums gave a rather higher titre for heterologous S
antigens than homologous S antigens. Likewise the S Brucella
antigens seemed to show more agglutination for the heterologous

R serums than those of their own group.

' 22 -
TABLISVI.

CROSS AGGIDTINATION OF R IMMUNE SERUMS WITH HOMOLOGOUS
AND HETEROLOGOUS R ANTIGENS.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Br. abortus 4a R 6 4L5 5
Br. suis 404 R 4 6 2 8 5
Br. melitensis 516 R 5 7 5 - 4
'Br. melitensis 584 R 4 4 5a 2 5
.2. avicida Ry! 5 5 5 7 7 2
H._pertussis R 4 4 2 4 6 4
Pf. mallei R 5 5 4 5 7

 

 

TABLE VII.

CROSS AGGIUTINATION OF R IMMUNE SERUMS WITH HOMOLOGOUS
AND.HETEROLOGOUS S ANTIGENS.

 

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Br. melitensis 584 S’ 2 l l 5 4 5
P. avicida S 4 5 4 5 5 2
P. bovisepticus S 4 4 4
Pi tularensis S 4-5 4 6'7
H. effusiss_3 4 4 4 5
. maIlei S 5 2 4 1“5

 

-25..

The picture for the interactions of the S antiserums
with homologous and heterologous antigens was much the
same as for the R antiserums. Tables VIII and IX show
these data. Higher titres for specific antigens and
antiserums were shown with a varying relationship of the
heterologous serums and antigens. The g, pertussis S
immune serum with Ba. abortus 966 S antigen gave a higher
titre than with the B3. abortus 4a R antigen. §.pertussis
S immune serum with P. tularensis S and R antigen gave
a higher titre for the R antigen, practically none being
shown for the S strain, ,g. avicida S and R cultures have
a large and equal amount of agglutination for their Specific
antiserums and for §;_pertussis S immune serums

The data for positive abortus serum from a naturally
infected cow were interesting. For some cultures demon-
strating twice as high a titre for the S strain as for
the R and for others twice as high a titre for the R as
the S strain.

In the Pasteurella genera, aside from the specific
agglutination of g. avicida S serum and 3. tularensis S
serum.with their specific antigens, the agglutination titres

of the S Pasteurella serums were higher for the R antigens.

-24..
TABLE VIII.

CROSS AGGLUTINATION OF S INWMNE SERUKS WITH HOMOLOGOUS
AND HETEROLOGOUS R ANTIGENS.

 

S serums

  
  

  

abortus 5a S

Antigens

   
    
 

Br.
P tularensi

  

  

a or us
s
Hm ens S
me itensis
vi
u arens
er uSS
ma e

  
   
  

a.

  

 
     
    
    

  
  

  

R

     

TABLE IX.

CROSS AGGIIJTINATION OF S INEJUNE SERUMS WITH HOMOLOGOUS AND
HETEROLOGOUS S ANTIGENS

  

serums

  
    
 

      
     

   

    
        

  

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-25-

All the antigens of’each.organism studied have been made
with cultures with the same colonial appearance, either typical
S or R colonies. But these antigens made up on different dates,
although apparently in the same stage of dissociation, will
give varying titres with the same serum. Not only is the agglu-
tinating antigen a cause of varied results, but the state of
dissociation of the cultures used for immunization will, of
course, control the amount of’S or R agglutinin in the antiserum
produced. Some of the cultures used in these studies have been
held in stock for several years as evidenced by the culture
histories. Thus they are likely of different serological
ccmposition than recently isolated organisms. McKenzie,
Fitzgerald and Irons (1955) in studies of S and R variants
of‘g schotmulleri, §_morgani and Shigella paragygenteriae
(Flexner), state that their "results appear to demonstrate
that serological, morphological and physiological characters
are capable of independent variation during the dissociative
processes.“ A dissociation apparent only serologizally may
have taken place.

While there can.be no doubt of a marked serologizal relation
between these groups of organisms the state of dissociation
influences this interagglutinability. This may'explain the
inability of some to find cross agglutination of these groups
or organisms. For this reason agglutinin absorption studies
have been made to determine whether the S agglutinogens of
one organism are the same as the R agglutinogens of another
or whether some apparently S organisms are more R than S or

so called R strains are more S than R.

-26-

8. Agglutinin Absorption Studies.

Agglutinin absorption studies of eadh immune serum
were made of all antigens showing a positive cross agglutination
in dilutions of 1 to 40 or over. For agglutinin absorption
all serums were diluted 1 to 50 with 0.1 per cent salt solution
containing 0.1 per cent phenol. To avoid dilution the growth
was washed from 1 slant culture with 5.5 cc. of diluted
serum. This was absorbed at 57°C for 1.5 hours. This super-
natant solution was decanted to another Slant, absorbed the
same length of time at 57°C., again centrifuged, and finally
titrated against the absorbing antigen. This was sufficient
absorption for some of the serums. Those which were not
completely absorbed were incubated with another dose of antigen,
for 1.5 hours and held over night in the ice box to completely
remove the Specific agglutinins.

For still other serums it was necessary to dilute
the immunt serum with physiological saline. This made the
gglutinin absorption less Specific. Otherwise it was im-
possible to completely absorb the agglutinins with one antigen,
for another antigen of the same organism, but grown at a
different time. That is, unless the absorbing antigen.and the
antigen used for testing complete absorption for specific
agglutinins were not made up at the same time the result was
the same as though two different organisms had been used.

For the absorption in physiological saline a dilution
of 0.1 cc. of serum in 0.6 cc. of 0.86 per cent saline was
used. After absorption sufficient distilled water was added

(diluted 8 times) to make an approximately 1 to 50 dilution

-27-

which at the same time produced a 0.1 per cent salt solution
for titrations, the same serum and salt dilution which had
been used in the other titrations. The absorptions with the
R Brucella antigens were made in this way.

After the immune serums had been absorbed the agglutination
titre of each was tested again with each antigen which had
previously Shown a positive cross agglutination in dilutions
of l to 40 or over.

The following tables Show the results of agglutinin
absorption and the cross agglutination titres after absorption
compared with cross agglutination results before absorption.

The first column for each serum in these tables are the
original titres of the unabsorbed serums with the homologous
and heterologous R and S antigens. These titres are represented
by the number of the1nbe of greatest dilution showing an
agglutination of a turbidity of 2 or greater. The dilutions
are designated as (l) l-20, (2) 1-40, (5) 1-80, (4) 1-160,

(5) 1-520, (5) 1-640, (7) 1-1280, (84 1-2560, (9) 1-5120.

The second column for each serum presents the titres
of the absorbed serums with the homologous and heterologous
S and R antigens. The dilutions are designated as (1) 1-50,
(2) 1-100, (3) 1-200, (4) 1-400, (5) 1-800, (6) 1-1600, (7)
1-5200.

CROSS AGGLUT INATION OF 8 AND R IMMUNE SERUMS, ABSORBED BY BR. ABORTUS
966 S ANTIGEN, WITH HOMOLOGOUS AND HETEROLOGOUS S AND R ANTIGENS.

-28..

 

 

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WITH.HOMOLOGOUS AND HETEROLOGOUS S AND R.ANTIGENS.

CROSS AGGLUTINATION or 3 AND a IMMUNE SERUMS, ABSORBED BY BR. ABORTUS
966 s ANTIGEN,

-28a-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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-29-

 

 

CROSS AGGIUTINATION OF 8 AND R IMMUNE SERUMS, ABSORBED BY BR. SUIS 456 S
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2,560. fig, melitensis 319 S absorbed by g;. abortus 966 D

 

antigen, agglutinated.§§. abortus 4a R antigen in a higher
dilution than before absorption, but showed no reroval of
agglutinin for its specific antigen. .gg. abortus 966 S
antigen removed none of the agglutinin from 3:. mallei immune
serum for £3. abortus 4a R antigen or 2:. mallei R antigen.
However, 3;. mallei R immune serum, absorbed bY.§£- abortus
4a R antigen showed no agglutination for its specific antigen,

3:. mallei R.

fig. melitensis 316 R immune serum showed no absorption

 

by RE. abortus 966 S antigen for its specific antigen. The
agglutinin which 3. avicida R immune serum contained for
,2. avicida S antigen was not removed by absorption with 23.
abortus 966 S antigen, but much of the agglutinin for g.
avicida R was removed. 23, abortus 966 S immune serum un-
absorbed by any antigen gave a slightly higher titre for 2.
avicida S than R.

§£,.ggi§ 404 R immune serum agglutinin for g. tularensis
S was not absorbed by 2;. abortus 966 S antigen. ,g. pggtussis
S agglutinin was absorbed and g. pertussis R agglutinin was

Iaot. The §._gertussis R agglutinin was absorbed from 23.

 

znelitensis 319 S immune serum. ,gg. suis 404 R immune serum

 

 

.agglutinin for its Specific antigen was removed. The agglu-

tinins for g; tularensis S and H. Eertnssis R were removed

 

-45...

 

from 23. melitensis immune serum by absaption with 33.
abortus 966 S antigen.

In the cross agglutination studies with unabsorbed serums,
the agglutinating titres 0f.§£° abortus and g. pertussis S
antigens with.all serums were more nearly parallel than
either antigen showed with other members of its own group.
After agglutinin absorption of all serums the titres with
these two antigens are the sane in the majority of cases,

although.not as nearly parallel as before absorption. fig,

suis 401 S immune serum absorbed by g. pertussis S antngen

 

showed no decrease in agglutination titre for Br. abortus

966 S antigen. Br. suis 404 R immune serum absorbed by

~

 

2. tularensis S antigen.showed no decrease in titre for

 

.3. pertussis S, but the titre of 23. suis 404 R immune serum

 

absorbed by g. tularensis S antigen was negative for g;.

 

abortus 966 S antigen (page ).

The immune serumscf the Brucella organisms, g3, abortus

 

5a S, fig. suis 401 S, 25. melitensis 519 S, and fig. meliten-
sis 516 R, which were absorbed by fig. suis 404 R antigen,
showed no removal of agglutinin for Br. abortus 966 S antigen.

The absorbed serums agglutinated g. pgrtussis S antigen

 

in a l to 50 dilution, vith this exception, fig. melitensis

q

516 R absorbed immune serum, agglutinated g. pertussis D

 

antigen with the same titre as the unabsorbed g3. melitensis

 

R serum. 23. suis 404 R unabsorbed immune serum agglutinated

both 23. abortus 966 S antigen.and g. per ussis S antigen

 

-44-

in l to 160 dilution. 2;. abortus 5a S abscrbed by'gg.

melitensis 316 R antigen rennved much of the agglutinin for

 

Br. abortus 966 S, The titre of this serum for H. pertussis
S antigen was higher than before absorption. .3. tularensis S
immune serum absorbed by H. pertussis R antigen did not agglu-
tinate H3. abortus 966 S antigen. No removal of agglutinin

for H. pertussis S was shown.

 

-fi.
_

The R pertussis S unabsorbed serum agglutinated g.

 

avicida S and R altigens in dilutions equal to its homologous

antigen, g. pertussis S immune serum absorbed by H. pertussis

 

S antigen showed but slight removal of 2. avicida S agglutinin.
2. avicida R agglutinin was canpletely removed. H. pertussis
S immune serum absorbed by g. avicida R antigen agglutinated
2. avicida S antigen.in a slightly lower titre than before
absorption, but H. pertussis S antigen was agglutinated in

l to 50 dilution as compared to l to 1280 dilution, the
original titre of the unabsorbed serun.

Every attempt to show an absolute serological simiharity
between any two organisms has resulted in just such data as
shown above, complete absorption or the absence of absorption
when the opposite might be eXpected. Many of the tests which
gave varying results which could be questioned as error, have
been repeated with the same absorbed serum and the same antigen.

The same titre was found each.time.

I-.I,V

-45..

The cross agglutination studies of these 4 genera
have shown apparently inconsistentrssults. The results of
the agglutinin absorption studies were doubly varied, for
instead of one variable factor there were two, the agglutin-
ating antigen and th€,absorhing antigen. The antigens for
titration of absorbed senrms were made up in quantities
sufficient to last several weeks. Thezabsorbing organisms
were freshly transferred and cultured each time that they were
used. This probably in some cases eXplains the difficulty
found in completely absorbing the specific agglutinins.
Although bacteriologically apparently the same antigens
were used for absorbing and for the agglutinating check on
suificient absorption of the specific agglutinins, two

serologically different organisms may have develOped.

-46-
V. DISCUSSION

The cross agglutination studies have shown that there
is interagglutinability of these genera and the agglutinin
absorption results have emphasized this fact. It has also
emphasized the fact that agglutination titres are influenced
by the state of dissociation of the cultures used as antigens.
In many instances the lad; of correlation between members
of’tke sane genera in the quantity of agglutinin produced
and the agglutinogen is as great as the variation between
members of different genera.

Previous reports of dissociation and serological

classification of each of these groups demonstrate this

same irreguhirity of results. Evans (1918) found fig.

 

abortus and g3. melitensis to be alike morphologically,
culturally, biochemicallyzand serologically. She found
that g3, abortus antiserums with.§3. abortus or garmelitensis

antigens gave the same titre, but g3. melitensis antigen

 

with g;. abortus and 23. melitensis serums gave a different
titre for each. She concludes that gg.eabortus and fig.

melitensis antiserums contain more than one agglutinin, that

 

the agglutinin in the two antiserums are alike in kind

but differ in proportion and that the correSponding
agglutinogens are present in the antigens in corresponding
variation.

Feusier amn heyer (1920) divided the abortus and

IQIW In _

-‘17-

melitensis strains into 4 groups by agglutinin absorption
tests. Evans (1925) divided the Brucella organisms into
7 groups by agglutinin absorption tests and Burnet (1925)
also by agglutination and agglutinin absorption tests
divided them into 2 types.

rcutt (1926) wonking with 6 strains found no serological
distinction. Two melitensis strains were found to be identical
with the bovine and swine strains by means of agglutination
and agglutinin absorption studies. Carpenter and Beak
(1929) because of the serological difference shown between
the R variants suggested a division of the H3. abortus
strains into 2 groups.

The reason for this kaleidoscopic appearance of tfle
cross agglutination and agglutinin absorption results of the
Brucella group seems to be given by Zdrodowski, Brenn and
Voskressinski (1950) who state that the R and S forms
showed distinct serologhzal differentiation. The organisms
charged their serological and bacteriological positions
according to their state of dissociation.

Also Blastridge and.chlpine (1932) divided the abortus
and melitensis organisms into 5 serological groups. Evidence
of variation in serological properties of the genus Brucella,

especially g3. melitensis under odinary'laboratory con-

 

ditions has been noted and discussed from the standpoint
of explaining the lack of agreement in results reported by
various investigators. The strains are not correlated

into the same groups serologically and biologically.

-48-

These reports would seem.to show a serolOgical differ-
ence when a cultural, morphological, and physiological
variation was not visible. In the present attempt to
correlate the Brucella, Pasteurella, Hemophilus, and Pfeiffer-
ella groups the only characteristic used to determine the
R or 8 type of organism has been its colonial appearance.
hackenzie, Eitderald and Irons (1933) in a study of the
independent variation of the biological characteristics
of bacteria as a result of dissociation, state that the
"agglutinative, agglutogenic absorptive qualities" vary
independently of each other. In the reversion of the
R cultures back to the 8 types not once vas complete
reversion of all characteristics observed. Thus an organism
may be smooth according to more concepts, but rough to
others. A serological examination of organisms will
detect character changes not biochemically or morphologically
noted, thus the irregularity in serological classification.

A review of work done upon the Pasteurella genera
shows the same picture, Varied serological titres from
different states ef'dissociation, varied classification
of apparently identical cultures.

LeKruif (1921) found the coexistence of organisms
of different degrees of virulence in cultures of the
bacillus of rabbit septicemia. as called the virulent
bacteria the D form and the less virulent the G fornt

Agglutination tests showed a marked serological difference

-49-

in these two types of culture. They were morphologically
indistinguishable in form and possessed like fermentation
reactions. The G form flocculated rapidly in fluid media
and formed translucent bluish colonies of low virulence.

Lal (1927) has summarized previous work in the correlation
of the Pasteurella genera, in part as follows:

Gochenour (1924) was unable to distinguish the strain
isolated from the tissues of a buffalo from other Peateur-
ella organisms by employing cultural, serological and
animal protection methods. Tanaka (1926) stated that there
is not sulficient difference between the members of the
group in agglutination, fermentation, or complement
fixation properties to be of diagnostic value. Some
investigators divided the group into two or three distinct
types, each consisting of a few organisms indistinguishp
able from each other, although derived from different
animal hosts. Koske (1904) reports 3. avicida indis-
tinguishable from 33. spis_by agglutination reactions.
Roderick (1922) studied the morpholgocal, cultural,
biochemical and immunological prOperties of the Rasteurella
genera and divided it into 2 groups, (1) a bovine swine
type and (2) an ovine avian-rabbit-cavia type. Vassar-
mann and Ostertag (1904), Hutyra and harsh (1912) found
that the immune sera against one organism was protective
against the homologous infection, but not against the

heterologous bacteria of this group. Joest (1906) reports

-Sg-

that blood serum strongly ii'nmunized against one member
of this group will be found protective. Lal concluded
from his work that the Pasteurella organisms tested by

ic

F);

complement fixation showed a certain amount of group Speci

reaction, an exception being 3. lepisepticum. Homologous

 

stra';s showed a high degree of specific fixation. Although
not easily distingiishable the organisms were a distinct
Species.

Hughes ( 950) by reciprocal agglutination tests with
several Stains of g. avicida showed a close serological
relationship. Jebster (1850) on the basis of colony
formamion divided 2. avicida into three groups. A
"fluorescent" colony of high virulence, stable in suSpension

found in severe epidemics, a “blue" colony unstable in

m

uspensions associated with endemics and an "intermediate"

23

3;:-

The Hemophilus genera, probably because of its
greater instability on artificial media, more than any of
the others showed varying serOIOgical results. Particularly
was this true of the H. influenzae organisms, a culture
which very readily becomes dissociated when.he1d in stock
and is very difficult to revert to its original form.

Pittman (1951) reported that spontaneous conversion of g.

influenzae from its S to R type readily took place in

 

artificial culture media. It may be delayed by certain
cultural procedures or hastened by growth.in type specific
antiserum, The artificia1.conversion of'R strains to the S
form has been Observed, but this change is carried out only

with difficulty.

Bordet and Sleeswyck (1910) found in freshly isolated

g. pertussis organisms grown.on chocolate agar and

 

plain agar, a serological difference. Old stock strains
did not show this difference. Krumwiede and Mishulow (1925)

in studies of a series of strains of g. pertussis of typical

 

morphological and cultural characteristics, divided them
into two groups by agglutinin absorption tests.

Leslie and Gardner (1951) by cross agglutination and
agglutinin absorption divided 52 strains of g. pertussis
into four serological groups. All freshly isolated organisms
fell into one group. By growth on various media all
cultures have been changed tozall into all four types.

Roos (1919) concluded that the various strains of
influenzae do not differ in Kind as indicated by cross
agglutination and agglutinin absorption tests. Valentine
and COOper (1919) reported that their results indicated
that under the term "B. influenzae" they were dealing with
a group oiiorganisms which were heterOgenous in character
as determined by immunological reactions, cross agglutination,
and agglutinin absorption tests.

Povitzky and Denny (1921) found 4 to 7 influenzal
meningitis strains, isolated years apart, proved by agglutinin
absorption, to be of one type. Fran the respiratory cases
5 strains from different individuals were obtained which
were of the same type. No other groups of more 2 members
were obtained. In general they found that strains of the

same immunological characteristics had the same cultural

-52-

properties, but there were some exceptions.

Jordan and Sharp (1922) found that as a rule each

strain of g, influenzae possessed a serolOgical individuality.

 

Occasionally strains from different sources were serologically
identical. Repetition of agglutination tests with.the same
strain antigen and serum did not give identical results.

At scme time almost every serum a glutinated every antigen,
although at other times the results were negative. They

found cross agglutination between 3. pertussis and g.

 

influenza . Five strains of g. pertussis agglutinated in

 

 

dilutions of 1 to 50 or 1 u; 100 with serums obtained from
two different rabbits immunized against an influenzae
strain. The serums of two other influenzae immunized
rabbits did not show agglutination. These antiserums

against 6 strains of.Alk. bronchisepticus gave negative

 

 

titres. 2ittman (1951) by means of cross precipitation
and agglutination reactions divided 15 8 strains into 2
serological groups.

Dovitzky (1918) showed that g;. mallei gave variable
serologi:a1 reactions under different conditions of growth.
Only 1 strain out of 15 in the stock collection p:oved
constant in serological reactions, and that only when great

care was exercised in the preparation of the culture medium.

.:.s~‘.

VI . SULLJLRY.

To Show the relationship between the Brucella, Pasteur-
ella, Hanophilns and the Pfeiiferella.genera, bacteriological,
morphological, cultural, physiological.ard serological
(cross gglutination and agglutinin absorption) studies have
been made 01' these organisms in their smooth and rough
forms. The dissociation state of these bacteria has been
determined by colonial appearance alone.

Cultures which have been held in stock for many years
have been used as well as more recently isolated Strains.
These organisns are alike morphologically and physiologically.

Immune serum was prouuced by rabbit injection with
both smooth and rough strains. Cross agglutination tests
showed interagglutinability of all genera used. In ahhaat
every case immune serums showed the highest titres with their
specific antigens. Rough serums of the Brucella genus have
shown higher titres for a smooth heterologous antigens than
the smooth types of their own genus.

No closer relation was found among the rough forms
than among the smooth, either bacteriologically or serologi-
cally. an S immune serum often demonstrated higher
agglutinability for some S antigens, some R antigens, and
sometimes the same titre for both 3 and R heterologous
antigens. The reciprocal agglutination and agglutinin
absorption tests often varied. This has been explained
by the intermediate states of dissociation of cultures used.
Although the colonial appearance of the cultures in these

tests were checked constantly for deiinite S or R types,

'5‘“ I

.II I ..vrufis‘.

.u.

.I.Iu

-54..

changes brought about by slight difference in medium
composition, temperature of incubator and frequency of
transfer over a period of several months, can.be detected
only through ser010gical examination and have brought
about the results shown. The organisms can not be divided
into the same groups by biological and serological
classification.

The literature shows a marked lack of agreement of
serological classifications of all these groups individually.
These studies have not only shown the irregularity of
serological results within each group of organisms, but tn;
the same dissimilarity exists between groups as within
groups. This dissimilarity does not detract from the

proof of relationship of these genera but rather adds to it.

-55-

VII. CCLJlLSIOHS

(l) The Brucella, Pasteurella, demophilus and Pfeifferella
genera are close 3 related morphologically, physiolOQically

and serologically.

(2) The degree or’their interagglutinability deperds upon
the state of'dissocrition of the cultures used for the

serologiazlstudies.

(5) Agglutinin absorption studies desbnstrate the same
irregular serological similarity between genera which is

shown within genera.

(4) Cultures apparently identical b; morpholOgical and
physiological properties are found to be variants by

serological examination.

 

I .1

: titsflifi.

VI I . A CKN 017L123 cm: ‘1“ .

The writer wishes to express appreciation to those
who aided in this work; to Dr. 11’. L. Llallmann, under
whose supervision these studies were planned and carried;
and to others of the Bacteriology Department, who assisted

with animal immunizations and the preparation of media.

. 4'4. t,‘

 

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