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

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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 milliliters50

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 be2 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

TABLE

1

Origin,

Associated

Disease

and

Colonial

Features

of

Primate

Cultures

Culture

Primate

Origin

Associated

Disease

Source

of

Specimen

Coloniala

Features

MCY

MMU

MMU

ATR

ATR

ATR

ATR

MMU

MRA

ATR

MMU

ATR

ATR

ATR

MMU

ATR

ATR

MMU

ATR

ATR

ATR

681

3866

291

820

848

843

849

4531

245

1407

5162

1335

1403

1405

5791

863

789

6059

256

442

751

906

Macaca

gynomolgus

MacacamuIatta

Macacamulatta

Aotus

trivirgatus

Aotus

trivirgatus Aotus

trivirgatus Aotus

trivirgatus Macaca

mulatta

Macaca

radiata

Aotus

tfivirgatus Macacamulatta

Aotus

trivirgatus

Aotus

trIVirgatus Aotus

trivirgatus Macacamulatta

Aotus

trIVirgatus

Aotus

trivirgatus

Macacamulatta

Aotus

tfivirgatus

Aotus

trivirgatus Aotus

trivirgatus Aotus

trivirgatus

Respiratory

difficulties

Death

Death

_b

_b

Upper

respiratory

disease

Upper

respiratory

disease

Abscess

at

tail

base

Rhinorrhea

Rhinorrhea

Gum

lesion

Death;

small

liver

abscesses

Death;

small

liver

abscesses

Death;

small

liver

abscesses

Pneumonia

_b

_b

Small

circular

lesions

Peritonitis;

pericarditis

Very

thin;

dehydrated

Peritonitis

Thin;

chronic

diarrhea

determined

at

the

time

the

cultures

were

received;

information

not

available;

not

determined.

secretions

blood

blood

Nasal

Heart

Heart

Nasal

secretions

Nasal

secretions

Nasal

swab

Nasal

swab

Swab

of

lesion

Nasal

swab

Nasal

swab

Swab

of

lesion

Heart

blood

Heart

blood

Heart

blood

Heart

blood

Heart

blood

Nasal

swab

Venous

blood

Heart

blood

Heart

blood

Heart

blood

Heart

blood

c

Mucoid

and

iridescent

(Blue

and

mucoid

_c

Mucoid

_c

_c

Mucoid

Mucoid

Iridescent

andmucoid

Mucoid

Mucoid

Mucoid

Iridescent

Blue

Blue

Some

Blue

Blue

_c

Blue

iridescent

26

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

I-"

f—l

H HQ

H H

Ibkdlbkdhhdl

mI

~‘““

mcn

m~4uam

‘‘-

‘

HIdaabra

HH

~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.

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