A SEROLOGICAL STUDY OF STRAINS
0F PASTEURELLA MULTOCEDA ISOLATED
FROM PRlMATES

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
NINA C. RUNFOLA
1970

"ng:c

  
   

. Bx:

LIBRAR Y f
Michigan Sure

‘I-Illi‘;

, emit?! f

  
 

f; 1 , '

_ BINDING BY
HMS 8: SONS’

l soyuwuaq-

ABSTRACT

A SEROLOGICAL STUDY OF STRAINS OF PASTEURELLA MULTOCIDA
ISOLATED FROM PRIMATES

 

BY

Nina C. Runfola

An attempt has been made to differentiate twenty-

two primate strains of Pasteurella multocida into the

 

known serotypes of this species. a. multocida has been

 

separated into serological groups on the basis of cap-
sular antigens (14, 29, 46) and into different types on
the basis of somatic antigens (37). In this present work,
differentiation of capsular groups has been attempted by
indirect hemagglutination tests. Somatic antigens have
distinguished by gel diffusion analysis of lipopolysac-
charide obtained by phenol-water extraction of cells.
Since somatic antigens are part of an endotoxin or lipo-
polysaccharide complex, chicken embryo lethality tests
have been used to determine the endotoxicity of the prep-
arations. The use of the fluorescent antibody technique
was attempted in an effort to detect capsular antigens.
However, type specific fluorescein conjugates of anti-
sera could not be obtained and this technique was observed

to be effective only for species identification.

A SEROLOGICAL STUDY OF STRAINS OF PASTEURELLA MULTOCIDA

 

ISOLATED FROM PRIMATES

BY

.r 2/
‘1

Nina CiyRunfola

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1970

TABLE OF CONTENTS

LIST OF TABLES O C O O O O O O O O O O O O 0

LIST OF FIGURES . . . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . . . . .

REVIEW OF THE LITERATURE . . . . . . . . . .

Early Studies . . . . . . . . . . . . .
Capsular Studies: Colonial Variation .
Capsular Studies: Nature of Capsular
Antigens . . . . . . . . . . . . . . .
Capsular Studies: Classificatio Based
on Capsular Antigens . . . . . . . . .
Somatic Antigens: Isolation and
Characteristics . . . . . . . . . . .
Somatic Antigens: Classification Based
on O Antigens . . . . . . . . . . . .
Relationship with Other Gram-negative
Organisms . . . . . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . . .

Cultures . . . . . . . . . . . . . . . .
Media . . . . . . . . . . . . . . . . .
Preparation of Antisera . . . . . . . .
Indirect Hemagglutination Test . . . . .
Fluorescent Antibody Technique . . . . .
Capsule Stains . . . . . . . . . . . . .
Gel Diffusion Technique . . . . . . . .

RESULTS . . . . . . . . . . . . . . . . . .

DISCUSSION . . . . . . . . . . . . . . . . .

BIBLIOGRAPHY O O O O O O O O O O O C O O O 0

ii

Page

iii

iv

12
14
16
18
18
18
18
19
21
22
23
25
37

42

LIST OF TABLES
Table
l. ORIGIN, ASSOCIATED DISEASE AND COLONIAL

FEATURES OF THE PRIMATE CULTURES . . . .

2. RESULTS OF CAPSULE STAINS OF PRIMATE
CULTURES C O O O O O O C O O O O I O O O

3. RESULTS OF PRELIMINARY RAPID SLIDE
AGGLUTINATION TESTS OF SALINE EXTRACTS .

4. HEMAGGLUTINATION TESTS OF SALINE EXTRACTS
EMPLOYING VARIOUS GROUP A SERA . . . . .

5. PRECIPITATION REACTIONS OF SOMATIC
ANTIGENS OF THE PRIMATE CULTURES . . . .

6. LIPOPOLYSACCHARIDE YIELDS OF THE PRIMATE
CULTURES I O O O C O O O O O O O O O O O

7. CHICKEN EMBRYO LETHALITY TESTS OF
LIPOPOLYSACCHARIDE PREPARATIONS
OF THE PRIMATE CULTURES . . . . . . . .

8. SUMMARY OF RESULTS OF SEROLOGICAL TESTS .

iii

Page

26

27

27

29

32

33

35

36

LIST OF FIGURES

Figure Page

1. GENERAL SCHEME OF ENDOTOXIN STRUCTURE . . 12

iv

INTRODUCTION

Throughout the years many proposals for a

serological classification of Pasteurella multocida

 

have been suggested. The significance of such a class-
ification lies in the necessity for effective vaccines
for protection against several diseases of domestic
animals. It has been found that certain serotypes within
this species are responsible for certain diseases. Early
workers used host specificity as a criterion of class-
ification, while others utilized fermentation tests.

The presence of different specific capsular antigens

made possible a classification into capsular groups.

More recently, the differentiation of specific somatic
antigens has allowed a classification based on both cap-
sular and somatic antigens.

Examinations of strains of P. multocida isolated

 

from many domestic animals have previously been made.
However, only very few monkey strains have been studied
The strains studied in this present work were obtained
from the National Center for Primate Biology at Davis,
California, and most were isolated from monkeys with
clinical symptoms of disease.

1

At present, four capsular groups are recog-
nized within this species, and are designated by the
letters A, B, D and E (14). The method of choice in
the separation of these groups is indirect hemagglutina-
tion. By this method, erythrocytes are treated with
capsular extracts of bacterial cells. It is believed
that some of the underlying lipopolysaccharide is
extracted with the capsule and that this lip0polysaccharide
adsorbs to the erythrocyte, with a resulting exposure of
capsular antigens on the surface (2). In the presence of
specific antiserum, hemagglutination then occurs.

Eleven somatic antigenic types have been ident—
ified by Namioka and Bruner (34) by means of agglutina-
tion tests, and have been designated by the arabic num-
erals 1 through 11. These workers have used both capsular
and somatic designations in the serological classification
of the species. It was found that a single somatic type
might be associated with several capsular groups.

In this present work, somatic antigens have been
extracted by phenol-water treatment of cells, followed
by ethanol precipitation. Merthiolated saline solutions
of the lipopolysaccharide were placed in gel diffusion
plates for antigenic analysis. Initial studies of the
reactions in gel diffusion plates indicated the presence

of a multiple antigen-antibody system, and for this reason,

capsular extractions were performed previous to phenol—
water extraction in order to remove capsular antigens.
The endotoxicity of lip0polysaccharide preparations was
determined by a study of the biological activity, since
no single chemical criterion of endotoxin purity has
been described in the literature. Chicken embryo
lethality tests were used as indicators of biological

activity.

REVIEW OF THE LITERATURE

Early Studies

 

Pasteurella multocida is the Species name for

a group of Pasteurellae characterized by an absence of

 

hemolysis, the production of oxidase and indol, the
absence of urease, and a failure to grow on Mac Conkey's
medium (17). This group of bacteria includes the causa-
tive agents of hemorrhagic septicemia in cattle, fowl
cholera and swine pneumonia, and has been implicated as
a secondary invader in various diseases (16).

The species name Pasteurella multocida was not

 

used until 1939 (47). Initially a zoological classifica-
tion based on host specificity was employed (28). The
organisms were named according to the host species from

which they were isolated--hence the names P. boviseptica,

 

P. aviseptica, P. suiseptica and so forth. Inherent in

 

 

this system of classification were several disadvantages.
In the first place, the organisms responsible for a single
type of disease were identified by different names.
Secondly, organisms grouped under one species name might
cause several different types of disease. All of this led

to much confusion.

Several workers, however, recoqnized the pos-
sibility of a serological classification. Among the
first of these was Cornelius in 1929 (22). Employing
an agglutinin-absorption test, he separated twenty-six
strains into four groups. He also observed a lack of
correlation between serological group and animal origin
of his bacterial strains. Observing that some of
Cornelius' strains tended to be inagglutinable, Yusef (50),
in 1935, utilized a precipitin test, in which fourteen of
his twenty-one strains fell into three serological groups.
By agglutination tests, Rosenbusch and Merchant (47), in
1939, observed three serological groups, which also exhib-
ited distinct differences in their fermentation of xylose,
arabinose and dulcitol. They suggested that a serological
classification replace the earlier zoological classifica—

tion and that the name 3. multocida replace the host

 

Species names. They did observe that all of their avian
strains fell into their Group I, although their Group II
comprised strains of all origins except avian. Little

and Lyon (29) demonstrated the existence of three serolog—
ical groups by a rapid slide agglutination test. Their
passive immunization tests indicated that monovalent serums
protected mice against organisms of the homologous, but

not heterologous, type. In 1947, Roberts (46) found four

immunological groups by means of cross protection tests

in mice. He did, however, observe strains which did
not fall into any of these groups and therefore sug-

gested that other serotypes might exist.

Capsular Studies: Colonial Variation

 

By a study of colonial morphology, the existence
of several colonial variants was demonstrated within this
species. Although terminology differs throughout the
literature, three main variants have been described:
iridescent (smooth), blue (rough) and mucoid (7, 18, 20).
The most common methods of distinguishing these variants
are observation through obliquely transmitted light and
an acriflavine test (7). By means of obliquely trans-
mitted light, smooth colonies appear iridescent, usually
of a somewhat greenish nature, mucoid colonies exhibit
a slight reddish iridescence, and rough colonies appear
noniridescent (7). Mucoid colonies are usually the lar-
gest in size and are mucoid or slimy in appearance. Smooth
and rough colonies are generally smaller in size and lack
a slimelayer. In acriflavine, smooth colonies remain in
suspension, rough colonies clump and mucoid cells form
slimy precipitates (7). Capsular polysaccharides from
iridescent variants differ both biologically and chem-
ically from those of mucoid variants. Those isolated

from iridescent variants are immunOgenic and serologically

active, while those from mucoid variants are not (18).
In addition, mucoid cells contain hyaluronic acid in
their capsules (18). Rough variants have not been
found to possess any capsular polysaccharides.

Several workers (20, 23) have attempted to deter-
mine the dissociation pattern by which these variants

arise and the most probably route seems to be: (20):

 

iridescent >mucoid
rough rough

Various attempts have been made to associate the
different variants with different degrees of virulence.
Iridescent variants are most often isolated from animals
with acute disease while rough and mucoid variants are
usually recovered from chronically infected or carrier
animals (5, 20). Generally, iridescent variants possess
the highest, and rough variants the lowest, virulence for
mice (19). Mucoid cultures are usually associated with
moderate virulence for mice, although they may vary widely

in this reSpect (l9).
Capsular Studies: Nature of Capsular Antigens

The capsule of P. multocida is thought to consist

 

of both protein and polysaccharide components (1, 44).

Prince and Smith (44) detected two types of capsular

antigens, termed a and B . Both were thought to have
protein components since both stained with thiazine red.
However, trypsin did not affect the precipitation of
either, indicating the presence of other substances. The
8 antigen was resistant to boiling, which suggested a
polysaccharide component, while a was susceptible to boil-
ing and was precipitable at pH 3.8, indicating that it
was mostly protein (44). Briefman and Yaw (3) hydrolysed
the capsular polysaccharide and chromatographically
identified ribose and galactose. This was unusual in that
no other workers had ever reported ribose as a constituent
of capsular polysaccharide. Examination of capsular
polysaccharides by Knox and Bain (27) revealed nonhomo-
geneous preparations consisting of 11.2% ketose, 8.2%
nitrogen, hexoseamine, fructose, galactose, N-acetylgluco-
samine and the reducing sugars: glucose, mannose and
glucosamine. Ouchterlony tests and ethylene glycol-acetone
fractionations indicated the presence of several compon-
ents in the capsular polysaccharide (27). Because the
ratio of fructose: glucosamine varied from one prepara-
tion to another, Knox and Bain (27) suggested the possibil-
ity of a basic chain containing all of the above-mentioned
sugars with varying fructose side chains.

The protein-associated capsular substance has been
shown to be serologically active, as indicated by its

antigenicity and its ability to remove the protective power

from antisera (l). The serological activity of capsular
polysaccharides has also been demonstrated (3, 27, 43),
although immunogenicity of these substances alone has

not been observed. These polysaccharides probably act

as haptens, which are immunogenic only when associated
with protein (27). Knox and Bain (27) failed to observe
any protection in mice, rabbits or cattle subcutaneously
injected with purified trypsinized capsular polysaccharide
when challenged three weeks later. However, these tryp—
sinized polysaccharides did remove some of the protective
power for mice from rabbit and cattle antisera. The a
antigen of Prince and Smith (44), which was thought to
contain more protein than did the 8 antigen, was also

observed to be more antigenic.

Capsular Studies: Classification Based on Capsular Antigens

 

Most of the early efforts at a serological class—
ification of this species employed the use of whole organ-
isms. In 1952, Carter (4) identified three serotypes-—A,

B and C--by means of precipitin tests with soluble capsular
material. Indirect hemagglutination, a more sensitive
method, then came into use (6), in which capsular extracts
were adsorbed to erythrocytes. By this method, two more
types, D (6) and E (12), were identified. However, Carter

suggested that his group C be drOpped (14) since only very

10

few strains fell into this category. His classification,

therefore, consisted of four groups (14):

A: possessing a wide host range;
B: isolated only from cattle and buffalo;
D: possessing a wide host range;

E: isolated only from African cattle.

This classification scheme is still in use at present.

It should be noted that most of the mucoid cultures,
producing large amounts of hyaluronic acid, fall into
Carter's group A. Type E strains have only been isolated
in central Africa. Although they cross react to a small
degree with type B strains, there are sufficient sero-
logical differences to justify their placement in a sep-
arate group (12). This was confirmed by Perreau (39).
Namioka and Murata (35) confirmed Carter's groups A, B

and D by indirect hemagglutination and slide agglutina-
tion tests. They observed that the slide agglutination
was the less sensitive of the two methods. This was in
agreement with Carter (9) who observed that agglutination
tests were not suitable for these organisms due to some
spontaneous clumping in saline and to an inagglutinability
of many capsulated strains, especially if recently isolated.
Mouse pretection tests can also be used to group these
organisms into capsular groups, although they are not as

simple to perform as indirect hemagglutinations. Carter (15)

11

observed a linear relationship between the protective
capacity of sera, as measured by the logarithm of the

number of LD 3 against which 4.25 x 10“2 milliliters

50
of serum protected 50% of the mice, and the reciprocal

of the serum titer, as measured by indirect hemagglutina—
tion.

Many studies have been undertaken to determine
the capsular types associated with different diseases.
Capsular groups A and D are widely distributed with respect
to host range and disease (11). Fowl cholera is usually
associated with group A organisms (10). Hemorrhagic
septicemia of cattle is generally caused by strains of
groups B and E. Human infections have been reported,
usually as a result of dog or cat bites or entry via the
respiratory tract (13). At present, only capsular groups
A and D have been recovered from humans (13). However,
the role of endotoxin in disease cannot be ignored, since
degradation of the capsular in viyg can be expected.

The main purpose of such a study of capsular
groups is its applicability to vaccination procedures.
Type B organisms are employed for bacterin production
against hemorrhagic septicemia (8). Bacterins against
secondary pneumonia of swine are prepared with groups A
and D organisms (8). Heddleston (24) observed that Little

and Lyon's (29) capsular types 1 and 3 were capable of

12

infecting chickens and that a bivalent vaccine employ-

ing both types was necessary for effective protection.

Somatic Antigens: Isolation and Characteristics

 

Endotoxin, consisting of lipopolysaccharide with
O antigenic side chains, can be isolated by a variety of
methods, including phenol-water, aqueous ether, acetic
acid and trichloroacetic acid extractions. Endotoxin is
part of the cell wall of gram-negative bacteria, probably
forming a layer surrounding the mucopeptide of the cell
wall. Although no one definite structure for endotoxin
has been determined, it is generally thought to consist
of a lipid moiety, a backbone of 2-keto—3-deoxyoctanoic
acid (KDO), heptose and phosphate (21) and O antigenic
side chains composed of repeating sugar components (30).

A general scheme of the structure is shown in Figure l.

 

 

KDO-hep-hep-glu-gal-glu-NAcGlu-[j:}—4 “}~4“}.u

 

 

 

Lipid --
———KDO-hep-hep-glu-gal-glu-NAcGlu-L::k“{::}~4;;}v~
moiety
w._* F-KDO- -hep- hep- glu-gal- glu-NAc Glu-E::F—{ __f-{::}
L.____Y__ “I! L.--_,If “V”— -1-
Backbone Core 0 side chains

Figure 1. General scheme of endotoxin structure. KDO:
2-keto-3-deoxyoctanoic acid; hep: heptose;
glu: D-glucose; gal: D-galactose; NAcGlu:
N-acetylglucosamine.

 

13

The lipid moiety is thought to consist of two glucosamine
units, substituted with fatty acids. The number of KDO
molecules in the backbone is uncertain. O antigenic
groups, consisting of specific sugar sequences, are
repeated to form 0 side chains. Individual endotoxin
molecules are thought to aggregate to form larger struc-
tures.

Since no chemical assay has been defined by which
endotoxin can be identified, biological assays must be
employed. Among these are chicken embryo lethality, pyro-
genicity and the elicitation of the Schwartzman phenomenon
(30). Controversy exists as to the exact location of the
toxic portion of the molecule.

One of the earliest reports of lipopolysaccharides

isolated from P. multocida was by Pirosky (40, 41, 42).

 

These Boivin-like antigens were isolated from both smooth
and rough variants. He was able to demonstrate sero-
logical differences between various 0 antigens. Type
Specific lip0polysaccharides were also isolated by
MacLennan and Rondle (31) in 1957. Some of the more recent
work in this area has indicated that a serological class-
ification of this species on the basis of capsular groups
alone is not sufficient. Bain and Knox (2) isolated
lipopolysaccharide from Roberts' type I cells by phenol—

water extraction. They observed that Asian type I strains

l4-

possessed a different kind of lipopolysaccharide than

did the Australian type I cells. Heddleston, Rebers

and Ritchie (25) isolated a particulate lipopolysac-
charide by successive extractions with cold form-

alinized saline. They demonstrated that the lipopoly-
saccharide from an avirulent, noncapsulated avian strain
induced active immunity against virulent capsulated organ-
isms in mice, rabbits and chickens. Namioka and Murata's

(36) studies of P. multocida indicated the presence of

 

both common and specific 0 antigens.

Somatic Antigens: Classification Based on O Antigens

 

Among the first to separate organisms of this
species into serotypes on the basis of O antigens were
Namioka and Murata (37). Namioka and Bruner (34) were
able to separate the Species into ten 0 groups, each
designated by an arabic numeral. When 0 and capsular

groups were correlated, eleven serotypes resulted:

 

 

Carter's Namioka and Bruner's
capsular group 0 groups
A 1, 3, 5, 7, 9
B 6
D l, 2, 3, 4, 10

They acknowledged the difficulty in classification accord—

ing to O antigens due to the occurrence of multiple cross

reactions.

1.

15

They also observed the following:

Almost all 5:A cultures were isolated from
cases of fowl cholera;

Most 1:A, 3:A, 1:D and 4:D cultures were
recovered from cases of sheep or swine
pneumonia;

Only groups 5:A and 9:A killed three-
month old chickens within 24 hours;

All 0 group 6 cultures were obtained

from cattle with hemorrhagic septicemia;
Several 0 groups could be subdivided into

subgroups.

Further studies of the 0 groups of P, multocida by

 

Namioka and Murata (38) indicated the following:

1.

2.

A new O group, 11, was observed;

Capsular group B strains do not cause
hemorrhagic septicemia of cattle unless

0 group 6 is present; ll:B strains do not
cause the disease.

Fowl cholera is caused by types 5:A,

8:A and 9:A;

Types 1:A, 3:A and 7:A are responsible for
pneumonia and secondary infections of man

and various animals.

16

Murata, Horiuchi and Namioka (33) observed that host
age affected pathological changes in chickens, even
with serotypes that were known to cause acute fowl

cholera.

Relationship with Other Gram-negative Organisms

 

Several workers have attempted an examination of

the relationship between P. multocida and other Pasteu-

 

rellae by means of taxonomic methods. Talbot and Sneath
(49) analysed many characteristics by means of a computer.
With the basic assumption that each characteristic carried
equal taxonomic significance they observed that Pasteu-

rella pestis and Pasteurella pseudotuberculosis were more

 

 

closely related to each other than they were to P. mul-
tocida. However, a distant relationship did exist. Also
using Adansonian taxonomic methods, Smith and Thal (48)

divided the genus Pasteurella into two groups:

 

l. Oxidase positive strains, including

P. multocida, P. hemolytica and

 

 

P. pneumotropica;

 

2. Oxidase negative strains, including

3. pestis and g. pseudotuberculosis.

 

They suggested that the first group be given the genus

name Pasteurella and the second group Yersinia. They also

 

17

observed that P. multocida strains were somewhat

 

heterogeneous, although they did show 85% similarity
with one another.
One other group of workers has tried to study

the relationship between P. multocida and other gram-

 

negative organisms. Employing sonic disintegrates and
immunodiffusion tests, Prince and Smith (45) studied

cross reactions between P. multocida strain 925 and an

 

other gram-negatives: Actinobacillus lignieresi,

 

Brucella suis, Hemophilus canis, Escherischia coli,

 

 

Neisseria catarrhalis, P. hemolytica, P. pseudo-

 

 

tuberculosis and Hemophilus influenza. Cross reactions

 

 

were observed between P. multocida and all of these strains

 

except B. suis and H. influenza. Since sonic disintegrates

 

were used, many internal antigens, and possibly enzymes,
were involved. However, one must consider the possibility
that these reactions may have been due to common compon-
ents of all gram-negatives, such as enzymes and cell wall
constituents. The failure to observe cross reactions with
two of the other species may have been due to differences

in disintegration rates between species.

MATERIALS AND METHODS

Cultures

Twenty-two strains isolated from monkeys were
obtained from the National Center for Primate Biology
in Davis, California. Cultures were maintained on Difco
stock culture medium. All cultures were transferred to
fresh stock culture medium at four to five month inter-

vals.

Media

 

Blood (ox) agar; serum tryptose agar; nutrient

broth.

Preparation of Antisera

 

Smooth strains were grown on blood agar and
mucoid strains on tryptose agar. Growth was washed off
with saline, the bacterial cells collected by centrifuga—
tion and resuspended in 0.5% formalinized saline. After
overnight incubation at 37°C a sterility test was per—
formed by placing a drOp of the suspension into a tube of
beef heart infusion semi-solid medium, followed by 24
hour incubation at 37°C.

18

19

Immunization schedules consisted of the follow-
ing: Two rabbits were inoculated with each vaccine.
Each rabbit was injected subcutaneously with 0.25 cc of
the vaccine with Freund's adjuvant in each of four sites.
One month later, 1.25 cc was again inoculated subcutan—
eously into each of four locations. One month after
this, 1.0 cc was injected intravenously (without Freund's
adjuvant), followed in four days by 2.0 cc intravenously
and four days after this by 3.0 cc intravenously. Four
days after the last injection the rabbits were bled out
by cardiac puncture. All sera were inactivated at 56°C
for 30 minutes and then frozen.

Antisera were prepared against five primate
strains: MMU 291, MRA 245, ATR 1407, ATR 1403 and MMU 5791.

Sera to other P. multocida strains were made

 

available by Dr. G. R. Carter.
Indirect Hemagglutination Test

The procedure used was a modification of the
one described by Carter (6).

Saline extraction of P, multocida: 18-24 hour

 

confluent growth from a blood agar or serum tryptose
agar plate was washed off with 5.0 cc of physiological
saline. This was heated at 56°C for 30 minutes and cen-
trifuged. The supernatant was transferred to another

tube for incubation with red blood cells.

20

Incubation of red blood cells with extract:
Red blood cells were obtained from either chickens or
humans and washed three times with 10 cc amounts of
physiological saline. All hemagglutination tests
described in the Results were performed with chicken
erythrocytes unless otherwise stated. 0.1 cc of packed
red blood cells were added to each saline extract and
incubated at 37°C for 2 hours. The red blood cells were
centrifuged out and washed three times with physioloqical
saline. Physiological saline was then added to the blood
cells to yield a 0.5% suspension.

Indirect hemagglutination test: 1:10, 1:20,
1:40 and 1:80 dilutions of sera were made in physiological
saline. 0.25 cc of the treated red blood cell suspensions
were added to 0.25 cc of the serum dilutions. The tubes
were shaken and allowed to stand at room temperature for
approximately two hours. A positive reaction was con-
sidered to be one in which agglutination was observable
in the bottom of the tube, blood cells failed to "run"
when the tubes were tilted, and clumping of the hood
cells was observable when the tubes were tapped lightly
enough to dislodge the cells from the bottom. Control
tubes consisted of blood cell suSpensions in which the
blood cells had been treated with physiological saline

instead of with a saline extract.

21

Fluorescent Antibody Technique

 

Fractionation of serum: An amount of satur—
ated ammonium sulfate solution was added to rabbit
serum so as to produce a final concentration of 45%.
After incubation at 25°C for four hours, collection
of the precipitate by centrifugation, and redissolv-
ing of the precipitate in distilled water, the same
procedure was repeated on the globulin solution. 'The
globulin solution was dialysed for 24 hours at 4°C to
remove ammonium sulfate.

Protein determination: A biuret test was per-
formed on the globulin solution to determine the pro-
tein concentration.

Conjugation of the globulin with fluoroscein
isothiocyanate (FITC): FITC (0.025 mg/mg of protein
to be labelled) was dissolved in a volume of pH 9,

0.1 M Na

HPO which was half that of the globulin to be

2 4

conjugated. A volume of 0.2 M Na2HP04

the volume of the globulin was added dropwise to the

equal to one-fourth

globulin, followed by dropwise addition of the FITC solu-
tion. The pH was adjusted to 9.5 and the volume adjusted
so as to achieve a concentration of 0.05 M NaZHPO4.

This mixture was incubated for 2-1/2 hours at 25°C and
then centrifuged. The conjugated globulin was dialysed

for several days at 5°C against pH 7.5 buffered saline

22

until no fluorescence was observed in the dialysate
under Wood's light. Borated merthiolate was added
to a concentration of l:10,000 and the conjugates were
frozen in small quantities.

Staining of slides: Chicken red blood cell
suspensions treated with bacterial saline extracts
were prepared and smears were made on slides, air dried
and fixed with light heat. In some cases, heat fixed
smears of bacteria themselves were used. The fixed
smears were covered with the conjugates and incubated
for 15 minutes in a moist chamber at room temperature.
The slides were dipped in pH 7.5 buffered saline, washed
in pH 7.5 buffered saline for 10 minutes, rinsed in dis—
tilled water and air dried. Buffered glycerol saline
was added prior to addition of a coverslip. The slides
were observed with American Optical fluorescence micro-

sc0pe.

Capsule Stains

 

Smears from blood agar growth were made in saline
with 5% horse serum and fixed in methanol. The slides
were covered with crystal violet for one minute, washed

and dried, according to the method of Jasmin (26).

23

Gel Diffusion Technique

 

Phenol-water extraction of P. multocida:

 

18-24 hour confluent growth from a blood agar plate
was washed off with 5.0 cc of distilled water. An
equal volume of 88% phenol was added and this mixture
was heated at 68°C for 20 minutes with frequent mix-
ing. After centrifugation, the aqueous phase was trans-
ferred to another tube and to this 10 volumes of cold
0.4 gram % sodium acetate in 95% ethanol was added. This
mixture was incubated 1-2 days at 4°C after which the
lipopolysaccharide precipitate was centrifuged out and
washed twice in cold 0.4 gram % sodium acetate in 95%
ethanol. The precipitate was dried, dissolved in 1.0 cc
of 1:10,000 merthiolated saline and stored at 4°C.
Preparation of Noble agar: Difco Noble agar
was dissolved in physiological saline (1 gram/100 cc)
by bringing the saline to a boil. The hot agar was immedi-
ately filtered through a millipore microfiber glass disc
prefilter, Type AP20, and then autoclaved at 121°C for
15 minutes. 1.0 cc of 1% borated merthiolate per 100 cc
of agar was added. The agar was distributed in 5.0 cc
amounts into 50 x 12 mm diSposable petri dishes with
tight fitting lids. Six wells (0.5 cm diameter) arranged
circularly around one center well (also 0.5 cm diameter)

were made in the agar. All wells were one centimeter apart.

24

Gel diffusion tests: The lipOpolysaccharide
obtained from the phenol-water extraction was placed
in the center well and the antiserum in the surround—
ing wells, or vice versa. The plates were allowed to
stand at room temperature, and the wells were refilled
twice at three day intervals.

One or three saline extractions of the bacterial
cells at 56°C were, in some cases, performed prior to
the phenol-water extraction in order to remove capsular
polysaccharide impurities from the final lipopolysaccharide
preparations. The procedure was identical to that des-
cribed for the saline extraction used preparatory to the
indirect hemagglutination test, except that the super-
natant was discarded and the sedimented cells subjected

to phenol-water extraction.

RESULTS

Table 1 consists of a description of the sources
of the cultures which were studied and of the type of
colonial variants found. Except for cultures received
prior to the start of this project, an attempt was made
to determine the major colonial variants present, since
only iridescent and mucoid cultures possess enough cap—
sular material to study by means of serological tech-
niques.

Table 2 presents the results of capsular stain-
ing. By the Jasmin method of staining, the capsule
appears colorless against a light purple background.
The bacterial cells stain dark purple. All of the cul-
tures possessed capsules, although the capsules of five
strains appeared to be very thin. None of these five
strains was typable by the serological methods employed.

Preliminary rapid slide agglutination tests
were performed, employing erythrocytes treated with
saline extracts of the cultures. The results, presented
in Table 3, indicate that three of the ten strains
examined fell into Carter's capsular group A.

25

 

26

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.pmcflEumump Doc "0
“waanHm>m no: coflumEu0msa "n
“pm>Hmowu mumB mmusuaso on» mEau may um pmcaaumump um
msam pooHn uummm nonhumfip cacouno “cane mapmmufipwuu waved mom me<
on pooan pummm mfluwcouflumm msummnfl>wuu msuom Hmn me¢
msam woods unmwm pmumupwnop ucfisu mum> msgmmuw>whu msuo¢ Nev med
msam ocean uummm mfluflpumoflumm amfiuwcouwumm mapmmufi>mmu msuow omm mam
ucmommpflufl meow pooHQ msocm> mcowmma HMHSUHHU HHMEM MDDMHDE momom: mmom DEE
mafia 203m Hmmmz a mspmmufi>mmu msuo< man med
enam vocab ummmm nu msummwM>HHu msuo< mom mam
unmommpHuH vocab pummm mflcoadocm muumaze monomz Hmhm Dz:
pfloosz vocab pummm mmmwmombm uw>fla HHmEm “gamma msummufl»wuu mouom nova med
pfloosz OOOHQ pummm mmmmmomnm um>fla HHmEm “Spawn msummnwpmuu m5u0< move maé
pfioosz vocab uummm mommmomnm nw>wa HHmEm “sumac msuwmuw>muu msuo¢ mmma m9<
pfioosE cam coflmma mo nmzm :oflmma 850 muumHDE momomZ «cam :22
unmommpHuH paoosz
pfloosz nm3m Hommz mmcunocflnm msummuflpmmD msuo< need ma4
on nmzm Hammz mmSHHOCHnm mumflpmu monomz mvm «m:
on c0amma mo QMBm mmmn HHmu um mmmomnd muumHse monomz Hmmv Dz:
UHOUDE £03m Hmmmz mmmwmflp xuoumuflmmmu momma msummum>wuu msuod mvm ma<
on nm3m Hammz mmmmmflp muoumuammmu momma msummhm>fiuu msuo¢ mum mad
pfioose mcofiuwuomm Hmmmz nu moummnfl>fluu msuofi wvm ma¢
cam madm.
ucmomwpflufi mcoflumuomm HMmmz nu msummufl>wuu mauo¢ omm mad
paw paces:
0| pooHn uumwm numwo muumasfi monomz Ham 322
on pooHn pummm sumac muumeE momomz comm DEE
on mcoflumuomm Hommz mmfluHsUAMMHU huoumnwmmmm msmaoeocwm monomz Hmm M02
ammuspmmm cmEHommm mmmmmao meMAUOmmd chHHO mumEflnm musuasu
HMHCOHOU mo condom

 

 

monsuHsU wumEflnm mo mmusummm HMHGOHOU paw mmmmmflo Umumw00mm¢ .camfluo

H mqmdB

27

TABLE 2

Results of Capsule Stains of Primate Cultures

 

 

 

Presence of Presence of
Culture Capsule Culture Capsule
MCY 681 +a ATR 1335 +
MMU 3866 + ATR 1403 +
MMU 291 + ATR 1405 +
ATR 820 + MMU 5791 +
ATR 848 + ATR 863 +
ATR 843 + ATR 789 +
ATR 849 + MMU 6059 +
MMU 4531 + ATR 256 +2
MRA 245 + ATR 442 +
ATR 1407 + ATR 751 +a
MMU 5162 + ATR 906 +a

 

a: Only a very thin capsule was detected.

TABLE 3

Results of Preliminary Rapid Slid Agglutination Tests
of Saline Extracts

 

 

Erythrocytes treated Serum
with saline extracts of: Type A Type B Type D Type E

 

 

MCY 681 - - - -
MMU 3866 - - - -
ATR 820 - - - -
ATR 848 - - - -
ATR 843

ATR 849

ATR 1335
ATR 1403
ATR 1405
MMU 5791

u +-+-+|
z
o
I
z
o

 

ND: Not determined
Sera used in these tests were: Hull (Type A), 100 (Type B),
37 (Type D), and 33 (Type E).

28

Erythrocytes treated with saline extracts of
all of the cultures were employed in indirect
hemagglutination tests against group specific sera.

Table 4 shows the results obtained when several group A
sera were employed. Eleven of the cultures gave
hemagglutination with group A sera. Serum dilutions

were carried out only as far as 1:80, with the excep-
tion of Hull group A serum which was diluted only as

far as 1:40. Serum dilutions were generally not carried
out further than 1:80 because the actual HA titer of

the serum was not of as much interest as was the fact
that hemagglutination was observed in the lower dilutions.
The large number of tests performed also made further
dilutions impractical. The other group A sera (MMU 5791,
MRA 245, MMU 291, P8, P1059, VA3, 9A) were tested and no
hemagglutination was observed.

Six of the untypable cultures were passed through
seven—day old chicken embryos in an attempt to recover
organisms with a higher degree of virulence and possibly
more capsular material. One tenth of a suspension of
cells washed off from a blood agar plate with 5 milliliters
of physiological saline was inoculated via the yolk sac.
Allantoic fluid was harvested 24 hours later and used to
treat human type 0 erythrocytes for use in the indirect

hemagglutination tests. Allantoic fluid from embryos

29

inoculated with physiological saline served as controls.
Five of the six cultures killed the embryos within 24
hours, while the saline—inoculated embryos remained
alive.

However, no hemagglutination could be demonstrated

against any of the group A, B, D or E sera tested.

TABLE 4

Hemagglutination Tests of Saline Extracts
Employing Various Group A Sera

 

 

Chicken erythrocytes
treated with saline

Reciprocals of Serum Dilutions
Showing Hemagglutination

 

 

extracts of: Sera: Hull 1403 3397 X73
MCY 681 ND - - -
MMU 3866 ND - - -
MMU 291 40 - - -
ATR 820 ND - - -
ATR 848 ND - - -
ATR 843 ND - - -
ATR 849 ND 40 — -
MMU 4531 40 — - -
MRA 245 40 - - -
ATR 1407 40 10 10 —
MMU 5162 ND 10 10 -
ATR 1335 ND — 20 -
ATR 1403 ND 10 - -
ATR 1405 ND 10 10 —
MMU 5791 ND 80 40 -
ATR 863 ND - 10 -
ATR 789 ND - — —
MMU 6059 ND - - -
ATR 256 ND - - -
ATR 442 ND - - -
ATR 751 ND - - -
ATR 906 ND - - -
Untreated - - - -

 

ND: Not determined

30

Four group-specific fluorescent antibody con-
jugates were prepared using the following sera:
1403, 100, 2121, 1243, representing groups A, B, D,
and B, respectively. One milliliter of each conjugate
was adsorbed with the confluent growth from 5 plates
each of the three other groups of organisms. The adsorp-
tion was carried out for two and one half hours at 37°C.
No group-specific reactions could be demonstrated by
this method, since all of the organisms employed fluores—
ced with all four of the conjugates. The use of rhoda—
mine B as a counterstain did not eliminate the non-
specificity, nor did dilution of the conjugates. However,
the conjugates were species-specific, since neither

Pasteurella hemolytica nor a Cogynebacterium fluoresced

 

when tested against any of the conjugates.

Gel diffusion analysis of the somatic antigens
was attempted, employing lipopolysaccharide (LPS) obtained
by means of phenol-water extraction as the source of
antigenic material. Precipitation due to the reactions
between these preparations and the standard antisera
against the eleven Namioka O antigenic groups was tested.
The results are summarized in Table 5. Three groups of
lipopolysaccharide preparations were used. One group of
preparations consisted of LPS from phenol-water extraction

of the cells. Several of these preparations precipitated

31

with many of the antisera. Double and triple lines of
precipitation were noticed in gel diffusion plates con—
taining these preparations. Since there existed a pos-
sibility that capsular polysaccharides might be con-
taminating these preparations, thereby causing extra
lines of precipitation, it was decided to perform one

or several saline extractions of the cells at 56°C
prior to the phenol—water extraction. It was assumed
that these procedures would reduce the amount of capsular
polysaccharide which might be present in the phenol-
water extracts and also might expose some of the 0 anti-
genic sites covered by the capsular material. From
Table 5 it can be observed that the number of precipita-
tion lines was reduced by saline pretreatment. In most
cases there was little difference in the number of pre-
cipitation lines observed after one or three saline pre-
treatments. However, often different 0 groups were dem-
onstrated in the three LPS preparations of a single cul-
ture.

The yield of material obtained by each of the
three extraction procedures was determined and is pre-
sented in Table 6. The yields of most cultures which had
been subjected to three saline extractions prior to phenol-
water treatment generally ranged from 0.8 to 1.9 milli-

grams per two plates. The yields per two plates of growth

32

TABLE 5

Precipitation Reactions of Somatic Antigens
of the Primate Cultures

 

 

Precipitation
with Antisera

Precipitation
with Antisera

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LPS to the follow- LPS to the follow-
Preparation ing 0 Groups Preparation ing 0 Groups
MCY 681: a 1,4 ATR 1335: a 4,5,7,8,9,11
b - b 8
c 1,8,11 c 8,11
MMU 3866: a 5,7,9 ATR 1403: a 4,5,7,8,9,1l
b 1 b 8,11
c - c -
MMU 291: a 7,11 ATR 1405: a 4,5,7,8,9,1l
b - b 8,11
c 1,11 c 7,8,11
ATR 820: a 4,9 MMU 5791: a 1,2,3,4,7,8
b 7,8 b 1,3
c 7 c 1,3,4,ll
ATR 848: a 4,9 ATR 863: a 7,8
b 7,8 b 7,8,11
c 7 c 1,7,8
ATR 843: a 7,8 ATR 789: a 4,5,7;8,9,11
b 7,8,11 b 7,8,11
c - c 4,8,11
ATR 849: a 7,8 MMU 6059: a 5,7
b - b l
c 8 c -
MMU 4531: a 2,3,4,5,7,8,9,11 ATR 256: a 3,4,5,7,8
b — b -
c — c 1,8
MRA 245: a l,3,4,8 ATR 442: a 4,5,7,8,9
b l b 7,8,11
c 11 c 4,8,11
ATR 1407: a 4,7,8,9,11 ATR 751: a 4,5,7,8,9
b 7,8,11 b 7,8,11
0 ~ c -
MMU 5162: a 5,7 ATR 906: a 4,5,7,8,9
b 1,2 b 8
c - c -

 

U'DJ

LPS from phenol-water extraction;
LPS from phenol-water extraction preceded by

extraction;

LPS from phenol-water extraction preceded by

saline extractions.

one saline

three

33

were determined since these were the amounts dissolved
in one milliliter of merthiolated saline for use in the
gel diffusion tests. No reduction of yields was observed

as the number of saline pretreatments was increased.

TABLE 6

Lipopolysaccharide Yields
of the Primate Cultures

 

 

LPS Yield (mg/2 plates)

 

 

 

No previOus One previous Two previous
LPS saline saline saline
Preparation extraction extraction extractions
MCY 681 1.7 1.3 1.9
MMU 3866 1.2 1.1 1.7
MMU 291 0.7 0.8 1.2
ATR 820 2.1 1.1 1.7
ATR 848 ND ND 0.9
ATR 843 ND ND ND
ATR 849 ND ND 0.2
MMU 4531 ND ND 0.8
MRA 245 ND ND 1.1
ATR 1407 ND ND 1.0
MMU 5162 ND ND 1.0
ATR 1335 ND ND 0.9
ATR 1403 ND ND 0.8
ATR 1405 ND ND 1.2
MMU 5791 ND ND 1.4
ATR 863 ND ND 0.3
ATR 789 ND ND 1.1
MMU 6059 ND ND 1.1
ATR 256 ND ND ND
ATR 442 ND ND 0.2
ATR 751 ND ND ND
ATR 906 ND ND 1.0

 

ND: Not determined.

34

The lipOpolysaccharide preparations in which three
saline extractions had preceded phenol~water treatment
were employed in chicken embryo lethality tests to deter-
mine if one of the biological effects of endotoxin
(lipopolysaccharide) could be demonstrated. It is known

that salmonella gallinarium endotoxin administered intra-

 

venously in the range of 0.007 to 0.008 ug is the LD50
dose for eleven day old chicken embryos (32). Eleven
day old embryos were inoculated intravenously with 0.02
pg doses contained in 0.05 cc of 0.2% formalinized saline.
Control embryos were inoculated with 0.05 cc of 0.2%
formalinized saline. Death within 24 hours was considered
to be a criterion of endotoxicity. The results are
shown in Table 7. Due to the limited number of fertile
eggs available at the time these tests were performed,
only two embryos were inoculated with each LPS prep-
aration. Six of the eight LPS samples tested killed both
embryos within 24 hours. The failure of the two samples
to kill both embryos may have been due to an insufficient
dosage of lipopolysaccharide. No death was observed in
the control embryos.

A summary of the findings with respect to the
capsular and somatic groups is presented in Table 8. Only
the somatic groups observed in preparations which under—

went three saline extractions prior to phenol-water are

35

included in this summary, since it was felt that these
samples would contain the least amount of contaminating

capsular polysaccharide.

TABLE 7

Chicken Embryo Lethality Tests of Lipopolysacharide
Preparations of the Primate Cultures

 

 

Number of deaths

 

 

LPS Preparation Number of embryos inoculated
MMU 5162 1/2
ATR 1335 2/2
ATR 1403 2/2
MMU 5791 2/2
ATR 863 2/2
ATR 442 2/2
ATR 789 2/2

ATR 906 1/2

 

0.2% formalinized saline 0/2

 

36

TABLE 8

Summary of Results of Serologic Tests

 

 

Culture Capsular Group . Somatic Groupa

 

MCY 681
MMU 3866
MMU 291
ATR 820
ATR 848
ATR 843
ATR 849
MMU 4531
MRA 245
ATR 1407
MMU 5162
ATR 1335
ATR 1403
ATR 1405
MMU 5791
ATR 863
ATR 789
MMU 6059
ATR 256
ATR 442
ATR 751
ATR 906

Ital

| >35>Svki>fiibfiab I

,8,ll

lml\l\l)—'||—"
V
1.;
f—l

H
H

Q
1.;
H

I bkdl bkdhhdl m I
~ ‘ ““
mcn m~4uam
‘ ‘ - ‘
H Idaabra
H H ~ H
H
H

 

a: Corresponding to Namioka's somatic groups.

DISCUSSION

Of the eleven cultures whose capsular groups
were determined, all were observed to be group A strains.
Since strains of groups B and E have only been isolated
from cattle, it was suSpected that the primate strains
would possess either A or D capsular antigens.

The remaining eleven strains could not be typed
by the serological methods employed. Several possibili-
ties exist which may explain this failure to detect cap-
sular antigens. Of the eleven group A cultures, most
consisted of mucoid or iridescent variants at the time
these studies were initiated, indicating an amply supply
of capsular material. In contrast, many of the remaining
cultures were composed of rough variants, possessing
relatively small amounts of capsular substances. There-
fore, the absence of adequate amounts of capsular antigens
would prevent their detection. Most of the eleven cul—
tures classified as group A lost their ability to cause
indirect hemagglutination after maintainance and trans-
fer in stock culture medium for one year, indicating that
dissociation to the rough variants had probably occurred.
The presence of other unknown capsular groups within the
species may also have been responsible for the inability

37

38

to type these cultures on the basis of their capsular
antigens, although this possibility seems to be less
likely.

Many attempts were made to study the untypable
cultures. Passage of these cultures through chicken
embryos was performed in an effort to increase the num-
bers of virulent, capsulated organisms. Both chicken
and group 0 human erythrocytes were employed in indirect
hemagglutination tests. However, none of these efforts
were successful. Treatment of the saline extracts of
mucoid cultures with hyaluronidase was also attempted
in order to remove the nonantigenic hyaluronic acid from
the material and possibly expose more of the capsular
antigens, but these efforts were also unsuccessful. Tan-
ning of the erythrocytes was employed in order to deter-
mine whether indirect hemagglutination could be demon-
strated by allowing some of the protein of the capsular
material to adsorb to the erythrocytes. However, this
procedure was also ineffective in producing hemagglutina—
tion with the untypable cultures.

It was hOped that the fluorescent antibody tech-
nique would present a rapid means of identification of
the different capsular groups. The inability to obtain
type specific fluorescein conjugates within this species,

even after adsorption, seems to indicate the presence of

39

many common surface antigens. NonSpecific staining due
to technical reasons does not seem likely since organ—
isms of other species, including the closely related
Pasteurella hemolytica, did not fluoresce after treat-
ment with the conjugates.

The complexity of the antigenic makeup of

Pasteurella multocida was demonstrated by analysis of

 

the somatic antigens. Antisera representing Namioka's
eleven somatic types were used. Each of these antisera
was prepared against intact formalinized organisms, since
purified lipOpolysaccharide is poorly immunogenic. It
was assumed that degradation of some of the surface com-
ponents, including the capsule, occurred within the host,
thereby exposing some of the somatic antigens. Namioka
(36) regarded his somatic classifications as groupings
based on antigenic complexes composed of several factors.
In this case, each somatic designation may represent the
presence of several 0 factors. The antisera used in this
study were not adsorbed with organisms of the ten heter-
ologous types and therefore the reaction of a single
lipOpolysaccharide preparation with several antisera, as
is observed in Table 5, may be due to cross reactions.
The chemical composition of the 0 factors corresponding
to each somatic designation has not as yet been examined

and thus the antigenic determinants involved are not known.

40

Table 8 indicates that the primate strains
reacted mainly with antisera to four of the somatic
groups: 1, 7, 8 and 11. In some cases the lipopolysac-
charide from a single strain reacted with several of
these antisera, possibly due to cross-relatedness of
the antigens of these groups. Adsorption of each of the
four antisera with organisms of the three heterologous
types might clarify these results somewhat. Lipopolysac-
charide from several strains gave no precipitation with
any of the antisera. Mutations resulting in loss of O
antigens might be responsible. It should also be noted
that the number of saline extractions performed prior
to phenol—water treatment affected the kinds of 0 groups
observed. For cultures receiving no saline pretreatments,
the presence of capsular antigens may have been reSpon-
sible for some of the large number of precipitation lines.
Each saline pretreatment prior to the phenol-water extrac-
tion probably removed more of the surface components from
the cell.

Further study is necessary to understand the

nature of the capsular and somatic antigens of P. multocida

 

and their role in infection. The numbers of these antigens
remains unclear. Prince and Smith (44) have found two

types of capsular material in all P. multocida organisms

 

they have studied. Working with a single strain of organ—

isms they demonstrated the presence of sixteen other

41

soluble antigens. Since these were detected in sonic
disintegrates of cells, it is not known how many of these
are 0 antigens. The number and kinds of common antigens
also remain unknown.

In summary, eleven of twenty-two monkey strains
were found to possess group A capsular antigens by means
of indirect hemagglutination tests. The four main somatic
antigenic types observed by precipitation reactions in
gel agar were 1, 7, 8 and 11. The fluorescent antibody
technique was ineffective in this serological typing since
type specific fluorescein conjugates of antisera could

not be prepared.

BIBLIOGRAPHY

BIBLIOGRAPHY

Bain, R.V.S. 1955. British Veterinary Journal 111:
492-498. Studies on haemorrhagic septicemia of
cattle IV. A preliminary examination of the anti-
gens of Pasteurella multocida type I.

 

 

Bain, R.V.S., and K. W. Knox. 1961. Immunology 4:
122-129. The antigens of Pasteurella multocida
type I. II. Lipopolysaccharides.

 

Briefman, L., and K. E. Yaw. 1958. Journal of
Bacterioloqy 75: 236. Monosaccharides isolated
from the capsule of Pasteurella multocida.

 

 

 

Carter, G. R. 1952. Canadian Journal of Medical
Sciences 30: 48-53. The type specific capsular
antigen of Pasteurella multocida.

 

 

 

Carter, G. R. 1952. Canadian Journal of Comparative
Medicine 16: 150-152. Some comments on pasteurel-
losis.

 

 

Carter, G. R. 1955. American Journal of Veterinary
Research 16: 4813484. Studies on Pasteurella
multoEida. I. A hemagglutination test for the
identifiEation of serological types.

 

 

 

 

Carter, G. R. 1957. American Journal of Veterinary
Research 18: 210—213. Studies on Pasteurella
multocida. II. Identification of antigenic
characteristics and colonial variants.

 

 

 

 

Carter, G. R. 1957. American Journal of Veterinary
Research 18: 437:440. Studies on Pasteurella
multocida. III. A serological survey of bovine
and porCine strains from various parts of the
world.

 

 

 

 

Carter, G. R. 1958. Nature 181: 1138. Failure of
the agglutination test to identify types of
Pasteurella multocida.

 

 

42

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

43

Carter, G. R. 1958. British Veterinary Journal
114: 356-357. Some characteristics of type A
strains of Pasteurella multocida.

 

 

Carter, G. R. 1959. American Journal of Veterinary
Research'20: 173-175. Studies on Pasteurella
multocida. IV. Serological types from Species
other than cattle and swine.

 

 

 

 

Carter, G. R. 1961. The Veterinary Record 73: 1052.
A new serological type of Pasteurella multocida
from Central Africa.

 

 

Carter, G. R. 1962. Canadian Journal of Public Health
52; 158-161. Animal serotypes of Pasteurella
multocida from human infections.

 

 

Carter, G. R. 1963. The Veterinary Record 75:
1264-1265. Proposed modification of the sero-
logical classification of Pasteurella multocida.

 

 

Carter, G. R. 1964. Canadian Journal of Micro-
biology 10: 753-756. Correlation between
hemagglutifiating antibody and mouse protection
in antipasteurella (Pasteurella multocida) sera.

 

 

 

Carter, G. R. 1967. Advances in Veterinary Science
11: 321-379. Pasteurellosis: Pasteurella
multocida and Pasteurella hemolytica.

 

 

 

 

Carter, G. R. 1968. Outline of Veterinary Micro-
biology. Michigan State University Bookstore.

Carter, G. R. and E. Annau. 1953. American Journal
of Veterinary Research 14: 475-478. Isolation
of capsular polysaccharides from colonial variants
of Pasteurella multocida.

 

 

 

Carter, G. R. and R. V. S. Bain. 1960. Veterinary
Reviews and Annotations 6: 105-128. Pasteurellosis
(Pasteurella multocida). A review stressing recent
developments.

 

 

 

Carter, G. R. and C. H. Bigland. 1953. Canadian
Journal of Comparative Medicine 17: 473—479.
DiSsociation and virulence in strains of
Pasteurella multocida isolated from a variety of
lesiOns.

 

 

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

44

Cherniak, R. and M. J. Osborn. 1966. Federation
Proceedings 25: 410. The structure of the
heptose-containing backbone of Salmonella
typhimurium.

 

 

 

 

Cornelius, J. T. 1929. Journal of Pathology and
Bacteriology 32: 355-364: An investigation of
the seroIOgicaI relationships of twenty-six
strains of Pasteurella.

 

 

Elberg, S. S. and Cheng—Lee Ho. 1950. Journal of
Comparative Pathology and Therapeutics 60: 41-50.
Studies on dissodiEtion in Pasteurella multocida.

 

 

 

Heddleston, K. L. 1962. Avian Diseases 6: 315-321.
Studies on pasteurellosis. V. Two immunogenic
types of Pasteurella multocida associated with
fowl cholera.

 

 

Heddleston, K. L., Rebers, P. G. and A. E. Ritchie.
1966. Journal of Immunology 96: 124-133.
Immunizifig and toxic properties of particulate
antigens from two immunogenic types of Pasteurella
multocida of avian origin.

 

 

 

Jasmin, A. M. 1945. Journal of Bacteriology 50:
361-363. An improvéd staining method for demon-
strating bacterial capsules, with particular
reference to Pasteurella.

 

 

Knox, K. W. and R. V. S. Bain. 1960. Immunology 3:
352-362. The antigens of Pasteurella multocida
type 1. I. Capsular polysaccharides.

 

 

Lignieres, J. 1901. Annales de l'Institut Pasteur
15: 734-736. Contfibution a 1‘etude et la
classification des septicemie hemorrhagiques les
"Pasteurelloses."

 

Little, P. A. and B. M. Lyon. 1943. American Journal
of Veterinary Research 4: 110-112. Demonstra-
tion of serological types within the nonhemolytic
Pasteurella.

 

 

 

Luderitz, 0., Staub, A. M. and O. Westphal. Bacterio-
logical Review 30: 192-255. Immunochemistry of
O and R antigens of Salmonella and related entero-
bacteriaceae.

 

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

45

MacLennan, A. P. and C. J. M. Rondle. 1957.
Nature 180: 1045-1046. ’Pasteurella septica:
tHe occurrence of type-Specific polysaccharides
containing aldoheptose sugars.

 

 

Milner, K. C. and R. A. Finkelstein. 1966.
Journal of Infectious Diseases 116: 529-536.

 

Murata, M., Horiuchi, T. and S. Namioka. 1964.
Cornell Veterinarian 54. 293-307. Studies
on the pathogenicity of Pasteurella multocida
for mice and chickens on the basis of’O groups.

 

Namioka, S. and D. W. Bruner. 1963. Cornell
Veterinarian 53: 41-53. Serological studies
on Pasteurella multocida. IV. Type distribution
of the organiSma on the basis of their capsules
and 0 groups.

 

Namioka, S. and M. Murata. 1961. Cornell Veterinarian

 

51: 498-507. Serological studies on Pasteurella
multocida. I. A simplified method for capsule
typing of the organism.

 

 

Namioka, S. and M. Murata. 1961. Cornell Veterin-
arian 51: 507-521. Serological studies on
Pasteurella multocida. II. Characteristics of
somatic (0) antigen of the organism.

 

 

Namioka, S. and M. Murata. 1961. Cornell Veterin—
arian 51: 522-528. Serological studies on
Pasteurella multocida. III. 0 antigenic analysis
of cultures isolated from various animals.

 

Namioka, S. and M. Murata. 1964. Cornell Veterin-
arian 54: 520-534. Serological studies on
Pasteurella multocida. V. Some epizootiological
findings resuIting from O antigenic analysis.

 

 

Perreau, P. 1961. Revue d'Elevage et de Medecine
Veterinaire des Pays Trgpicaux 14: 245-256.
Contribution a ITetude immunologique de Pasteurella
multocida.

 

 

 

Pirosky, I. 1938. Comptes Rendus des Seances de la
Societe de Biologié 128: 346-347. Sur l'existence,
chez les bariants smooth et rough d'une souche de
Pasteurella aviseptica de deux antigenes glucido—
lipidiques serologiquement distincts.

 

 

 

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

46

Pirosky, I. 1938. .Comptes Rendus des Seances de la
Societe de Biologie 127: 98-100. Sur l'antigene
glucido-lipidique des Pasteurella.

 

 

Pirosky, I. 1938. Comptes Rendus des Seances de la
Societe de Biologie. 127: 966-969. Sur les
propietes immunisantes antitoxiques et anti-
infectieuses de 1' antigene glucidolipidique de
Pasteurella aviseptica.

 

 

 

Prince, G. H. 1969. Comparative Pathology 79:
179. Production of antigens of Pasteureila
multocida and detection of antibodies against
them in’EOVine sera.

 

 

 

Prince, G. H. and J. E. Smith, 1966. Journal of
Comparative Pathology 76: 303-314. Antigenic
studies on Pasteurella multocida using immunodif-
fusion techniques. I. Identification of the
soluble antigens of a bovine hemorrhagic septicemic
strain.

 

 

 

Prince, G. H. and J. E. Smith, 1966. Journal of
Comparative Pathology 76: 315-320. Antigenic
studies on Pasteurella multocida using immunodif-
fusion techniques. II. Relationships with other
Gram-negative species.

 

 

 

Roberts, R. S. 1947. Journal of Comparative Pathology
and Therapeutics 57: 261-278. An immunSIOgiCal
study of Pasteurelia septica.

 

 

 

Rosenbusch, C. T. and I. A. Merchant. 1939.
Journal of Bacteriology 37: 69-89. A study of
the hemorrhagic septicemie Pasteurellae.

 

Smith, J. E. and E. Thal. 1965. Acta Pathologica et
Microbiologica Scandinavica 64: 213-223. A
taxomonic study Of the genus Pasteurella using a
numerical technique.

 

 

Talbot, J. M. and P. H. A. Sneath. 1960. Journal of
General Microbiology 22: 303-311. A taxomonic
study of Pasteurella septica, especially of strains
isolated from human sources.

 

 

 

Yusef, H. S. 1935. Journal of Pathology and Bacterio-
lo 41: 203-206. A contiibution to the serolog-
ical classification of Pasteurella strains.

 

 

”IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIEIIIT“