A study of the mechanisms of immunity to pasteurella haemolytica infection

A study

of

the

mechanisms

of

haemolytica

immunity

to

Pasteu. rel. la

infection.

I

HELEN BARBARA EVANS

A thesis

for

submitted

University

the

degree
of

1979

ZG

dý8N10ý

of

Edinburgh

Doctor

of

Philosophy


DECLARATION

The

work

project

concerned

diseases

of

findings

were

at

the

sheep,

and

Research

work

presented

and

where

conjoint

was

played

in

design

of

the

results.

thesis

was

in

the

a large±:

of

respiratory

with

Institute.

experiments

part

some of

collaboration

this

of

of

consequently
in

obtained

Moredun

this

investigations

with

the

tation

in

reported

the

experimental

my colleagues

Nevertheless,

thesis

carried

out

necessary,

a

was
were

experiments

most

and

the

by

of

myself

full

role

interpre-

W O1 C-/aAAs
HELEN
October

EVANS
1979


CONTENTS

Pa qe No

DECLARATION

Summary

1-

Acknowledgements

4

Abbreviations
GENERAL

in

used

text

INTRODUCTION

Characterisation

of

The distribution
healthy
amongst
Diseases
of

P. haemolytica

Septicaemia

2.

Pneumonia

P.

of P.
sheep

sheep

1.

for

Requirements
P. haemolytica

haemolytica

haemolytica

associated

Experimental
in sheep

Septicaemia

2.

Pneumonia

Models
of
laboratory

7-

38

10-

17

18-

19

20-

26

vaccine
for
study

against
of

27

infection

with

of

P.

pasteurellosis

haemolytic

animals

GENERAL MATERIALS

AND METHODS

Bacteriological

techniques

Strains

of

P. haemolytica

Strain

of

Escherichia

Enumeration
Laboratory

6

with

a suitable
and methods

reproduction

1.

5-

types

efficacy

vaccine

3

of

animal

Mice
Inoculations
1.

Subcutaneous

2.

Intraperitoneal

3.

Intranasal

28-

31

32-

38

39-

56

39-

41

42-

43

in

coli

bacteria
techniques


Vaccines

page

No

44-

48

49-

56

57-

88

58-

59

Antigens
1.

Preparation

of

P.

2.

Preparation
P. haemolytica

of

heat-killed

Preparation

of

Absr rption
hydroxide

of

Emulsification
oil
mineral

of

Bovine

vaccine

Techniques

collaborators

for

sheep

P.

in

antibodies

lambs

with

P13 virus

and

bacteriology

and

Quantitative
P.. haemolytica
enzyme-linked

P.

lungs

mouse

sera

Pathology

1-

of

haemolytica

Challenge
of SPF
P. haemolytica

Chapter

to

responses

examination

test

with

antigen

transformation
Lymphocyte
P. haemolytica
antigens

IHA

aluminium

onto

absorbed

by

employed

Histological

SSE

vaccines

antigens

albumin

haemotica

to
estimation
of antibody
in sheep sera by a micro(ELISA)
immunosorbent
assay

Experimental

infection

of

mice

with

haemolytica
Introduction
Materials

and

Experiment
varying
with

1-

Methods
Intranasal
numbers
of

2Viable
Experiment
in the lungs
of mice
Experiment

thiopentone
intranasal
Experiment
live
and

infection
P. haemolytica

of

mice
60-62

counts
of P. haemo]_ytica
infection
after

3-

Comparison
of halothane
for
sodium
as anaesthetics
infection

4Comparison
of IN ii_ection

heat-killed
P. haemolytica

63-

64

65-

67

68-

70

and

with


Page

No

71-

72

73-

74

75-

77

8The effect
Experiment
of
of vaccination
P. haemolytica
type Al vaccine
mice with
in the liver
of organisms
upon the growth
following
IP challenge
P. haemolyti.
ca
with
type Al 3 weeks later

78-

80

Discussion

81-

88

5-

Experiment
procedure;
challenge

Intraperitoneal
challenge
for
preparation
of inocula
of mice and determination
of

Experiment
type

Al

in

6-

Growth

of

the

livers

and

P.

IP
LD50

haemolytica
of

spleens

normal

mice
Experiment
SC routes
type
tica

7prior
Al

Vaccination
to challenge

2Mechanisms
Chapter
in sheep
ytica

of

of

immunity

mice by IP and
P. haemoly
with

to

P.

haemol89-108

Introduction
Materials

and

Methods

]Growth
Experiment

of
in the presence
in vitro
and serum

P.
of

haemolyti
ca.
lung wash cells

2Experiment
The roles
of antibody
immunity
(CMS) in combined
mediated
P13 virus
of SPF lambs with
and P.
type Al

91-

92

93-

95

and cellinfection
haemolytica
97-103
104-108

Discussion
3Non-specific
Chapter
P. haemolytica

resistance

to
109-129

Introduction
Materials

and

Methods

1The effect
Experiment
given
of Carovax
24 hours
to mice 48 hours,
and 6 hours

to challenge
prior
upon the LD
of P. haemol. y50
tica

110-111

112


Page

treatment
to
prior
of
growth

2Experiment
Effect
of vaccine
intervals
time
mice at different
P. haemolytica
on
with
challenge
in the liver

bacteria
Effect
of dilution
of challenge
with

4Experiment
dual
components

Effect
of treatment
on
of the vaccine

5-

Experiment

B188
with

of Carovax
P. haemolytica
116-117

P.

with

challenge

of

113-115

3Experiment
on the outcome
type Al

of

No

Al

type

haemolytica

Determination

indiviwith
the outcome

LD5

of

of

'treated

.
E.

co"

untreated
mice and in mice
24 hours
before
challenge
vaccine
in

118-119

120-121

6Effect
with
Experiment
of treatment
24 hours
before
Carovax
with
challenge
B188 upon the growth

E. coli
of the organism
in the livers
of mice

122-123

Discussion

124-129

4Chapter
and challenge

into
investigations
Further
P. haemolytica
of mice with

vaccination
130-1153

Introduction
Materials

and

1Experiment
of P. haemolytica

mice

and

and challenge

135

A2,

Al,

types

with

A6
136-138

A9

Experiment
procedures

3upon

P. haemolytica

Effect

of
challenge

type

4-16
Experiments
types
different
of
by the model
gated
P. haemolytica
with
Experiment
P.

of LD Q values
Zack
for
C57

Determination
strains

2Vaccination
a
of mice with
(types
Al,

A2 and A6) P. haemo3ytica_

Experiment
trivalent

vaccine

132-134

Methods

haemolytica

challenge

Discussion

17 -

139-142

A2
Cross-protection
P. haemolytica,
of IP challenge

P.

between
investiof

mice
143-144

Vaccination
type

with

vaccination
various
of mice with

A9

SSE

haemolytica

of

mice

with

and

HKO,

and

typt-

A9

145-147

148-153


Page

Chapter

5-

for
tica

detection
from
sera

the
in

Development

IHA

of

and

to
of antibodies
vaccinated
mice

No

tests

ELISA

haemoly-

P.

154-121

Introduction
Materials

and
1-

Experiment

for

dilutions

157-159

Methods
Determination
mouse

of
in

sera

IHA

end-paint
tests

homologous
and heterologous
using
SSE extracts
P. haemolytica

160

2Preliminary
titration
Experiment

of
P. haemolytica
type Al SSE in the presence
to
of immune mouse serum and titrations
dilutions
determine
optimal
of conjugate
3-

Experiment

Titrations

of

sera

types
of P. haemolytica
antigens
dilution
conjugate
using
at 1/200
comparable
achieve
at
absorbance

4Experiment
of cross-reaction
P. haemolytica

and

161-163

SSE

Al-A12
to
400 nm

164-165

degree

Determination
of the
between
the types
in the. ELISA test

of
166-167
168-171

Discussion
DISCUSSION

CONCLUDING
Mechanisms

of

172-190

immunity

to

P.

in

haemolytica

174-1.81

sheep
Models

of

P.

haemolytica

infection

in

mice

182-190
191-212

Bibliography
Appendix

1

213-214

Appendix

2

215-217

Publications

arising

from

this

thesis.

218


1

SUMMARY

to

Immunity
using

was

studied

in

infection

haemolytica

P.

of

models

haemolytica

Pasteurella

both

lambs

and

mice.

Immunisation
.vaccine

of

specific

a sodium

containing

P.

haemolytica

type

Al

P.

haemolytica

type

Al,

type

parainfluenza
of

responses

increased

lambs
transfer

this

although

by

haemolytica

alveolar

from

by

from

to

P.

vaccinated

The

passive

repeated
did

säure vaccine,

upon

failed

serum

leucocytes

Serum

effect

with

transformation

produced

challenge.

with

Lymphocyte

serum,

not

protect
lambs

vaccinated
in

haemolytica

facilitate

vitro,

phagocytosis

in

macrophages

a

of

infection

upon

challenge.

the

with

challenge

against

after

with

a bacteriostatic

had

P.

blood

lambs

from

recipients

3 virus.

markedly

of

vaccination

extract

superimposed

hyperimmune

of

salicylate

protected

peripheral

lambs

pathogen-free

of

vitro.

w

Two models

were

caused
cleared

because

similar

haemolytica

Intranasal

investigated.
llytica

P.

of

lesions
from

in

the'lung
lesions

infection
the
by
were

in

infection

lung

of

mice

3 days

48 hours
produced

later.

after
bj

the

were

mice

with

P. haemoBacteria

infection,
introduction

and
of


2

heat-killed

haemolytica,

P.

this

model

was

investigated

not

further.

P. haemolytica
gastric

vaccination

of

tica

sodium

containing
types

various

The
to

type

Al,

commercial

24 hours

or

14 days
no

such

before

detection
vaccinated

of

mice.

vaccine

was

immunosorbent

There

to

P.

was

of

between

mice

the

P. haemolytica
that

was

given

with.

P.

was

used

Carovax.

vaccine,

challenge,

indicating

challenge

antibodies

challenge

cross-protection

with

protection

An enzyme-linked

haemolytica

challenge

before

coli,
the

Vaccines

against

protected

Pasteurella

challenge

When

non-specific.

haemoly-"

P.

observed.

the

Escherichia

also

The

of

P.

of

in

spleen.

investigated.

little

intraperitoneal

against

protected

T10

of

12 or

and
extracts

was

-and

was

liver

extracts

and

strains,

investigate

7 days

All

strains

When given

and

A8,

model

liver

the

intraperitoneally

mice

various

salicylate

homologous

with
the

A6,

Al,

the

with

in

growth

upon

to

in

multiplied

mucin

effect

introduced

vaccine
type

Al,

protection
1 hour,

was
48 hours,

haemolytica

observed.

assay

haemolytica
considerable

was
in

developed
the

sera

cross-reaction

for
of


between

sera

extracts

of

from
the

mice
different

immunised
types

with
of

P.

sodium

salicylate

haemolytica.


4
Acknowledgements

am deeply

I
Dr.

G. H.

out

this

grateful

to

for

their

Lawson

K.

project,

thanks

My sincere

Sharp,

M.

Dr.

J.

for

many

and

for

are

also

Dr.

N.

Burrells

for

advising

aspects,

Dr.

B Rushton

on

Mr.

pathology,

house,
Mr.
pbs,

Mr.

B.

would
librarians

reading.
Research
Council.

A.

like

also

typing

and

Mr.

at
this

L.

to

McVittie

Mr.

A.

Suthe-rland

to
the

Moredun

manuscript
Throughout

Studentship

this
awarded

Mr.

I was
the

and

the

advice

animal

of

photograI

assistance.

Evans

project
by

help
of

and

Miss

A.

technical

many

for

C.

statistics,

Skeoch

J':

Donachie

Mr.

preparation

Institute,

and

and

technical

Miss

W.

to

staff

on

for

for

thank

the

advice

Inglis

Mr.

and

W. Angus

and

for

Aitken,

D.

tests

Mr K.

through-

encouragement.

am endebted

me on ELISA

C.

I.

Dr.

Gilmour

I

and

and

advice

due

J.

M. McLauchlan

Easter

and

their

and

supervision

excellent

discussions.

useful

P. W. Wells

Dr.

G.
for

Miss

A.

Sclater,
for

Armstrong
help

supported

Agricultural

C.

with
by

proof.
a

Research

-

5
Abbreviations

AM

= alveolar

BA

= bovine

C.

=

control

CFA

=

complete

cfu

=

colony

CMI

=

cell-mediated

DW

=

distilled

E=

in

used

text

macrophage
albumin

Freund's

forming

adjuvant

unit

immunity

water

absorbance

ELISA

=

gelatin-Hanks

= Hank's

immunosorbent

enzyme-linked

0.1%

balanced

salt

solution,

assay
containing

gelatin

HBSS

= Hank's

H. E.

= haematoxylin

HKO

= heat-killed

HuRBC

= human

IFA

= incomplete

IHA

indirect
=

IN

= intranasal

IP

intraperitoneal
=

IT

= intratracheal

balanced

solution

salt
and

eosin

organisms
blood

red

cells

Freundes

adjuvant

haemagglutination

intravenous

IV

=

MIF

= macrophage

MV

missing

migrätion

value

inhibition

factor


6

broth

NB

=

nutrient

ND

=

not

done

n. t.

=

not

treated
density

OD

optical

PBS

phosphate-buffered

saline

PBS/Tween

phosphate-buffered

saline

Tween

P13

=

containing

20

type

parainfluenza

PVP

polyvinyl

RBC

red

SC

subcutaneous

SE

standard

error

SED.

standard

error

3

pyrrolidone

blood

S. I.

=

stimulation

SPF

=

specific

SSE

=

sodium

V

=

vaccinate

cells

of

difference

index
pathogen

salicylate

free
extract

of

means

0.05%


INTRODUCTION

GENERAL

is

Pneumonia
of

sheep

necropsied

it

cause,

considerable
in

least

its

bacteria,
lungs

of

the

the

organism

is

1954).

with

a variety

also
of

studied

predominantly

basis

of

important

not

cattle

in

of
and

Particular
associated

the

certain
types
with

is

to

is

c

and

have

organism

pneumon'. a and

Europe

been
into

subdivided
and

and

or

America

biochemical
the

may

sheep,

pneumonia

P. haemolytica

of

the

a septicaem

North

species

from

conditions

restricted

both

with

characteristics

which

with

at

Pasteurella

clinical

other

associated

of

is

similar

of
is

The characteristics

features.

which

isolated

may be

frequency

greatest

most

(Carter,

the

some

of

associated

fever"

on

is

commonly

"transit

types

disease

is

P. haemolytica

extensively

the

from

stages,

the

organism

to

terminal

from

disease.

industry

the

losses

pneumonia,

Organisms

sheep,

may not

report

The

the

isolated

the

consequence.

haemölytica.
be

that

likely

1977).

economic

and
with

total

the
is

in

quoted

reflect

accurately
this

figures

the

proportion

(Veterinary

Report,

II

Analysis

a high

Kingdom

United

the

Diagnosis.

Investigation
Although

in

in

lesion

important

an

ý

antigenic
are

other

types

8

with

in

septicaemia

reviewed
the

points

salient

1967;
be

will.

been

extensively
1973)

Thompson,

a later

at

considered

only

and
point

Vaccines

still

aimed

in

available

that

inadequate,
vaccines

this

at

are

in

that

many

the

insufficient

The

general

unsatisfactory

pasteurellosis

use

immunising

nature

of

vaccine

field

the
by

supported
trials

vaccine

of

This

current

against
is

possibility

the

of

powers

protect

haemolytica

disease.

of

uptake

or

are

P.

nevertheless

outbreaks

to

latter

disease.

of

but

the

either

or

prevention

country*,

implicated

indicates

the

(Pyke,

1966).

Development

hampered
to

in

introduction.

this

is

(Carter,

recently

has

This

sheep.

in

reproduce

the

of

past

the

*Carovax,

Wellcome

Ovivac-P,

Pasticidin,

Middlesex,

England.

haemolytica

P.

by the

disease

absence

either

Foundation
Hoechst

has

vaccines

in

Ltd.,

of

a suitable

sheep

or

Beckenham,

Pharmaceuticals,

in

been

system
laboratory

England;
Hounslow,

is


9

This

animals.

development

These

not

immunisation,

satisfactory

mechanisms

has

of

a full

involved

aspects

will

in

also

only

hindered
but

it

progress

has
of

protection

against

be discusscý

prevented

also

understanding

in

towards

immune

the

the

this

organism.

introduction.


10

Characterisation

taxon,

The

(1932)

Cross

and

in

disease

In

of

a study

by

the

isolated

of
reactions.

non-motile,

gram-negative

in

diameter

haemolytic.

48 hours

Cultures
and

organisms,

and

pneumonic

(Spray,

bacilli
(1932)
"typical"

biochemical
were

and

and

"typical"
was

similar

The

"atypical"

were

which

organisms,

and
to

an

cocco-

Newsom

haemolytic

II

as

týicum

ovise

"atypical"

Groups

to

isolated

organisms,

cattle,

were

referred

were

gram-negative

and

mm

These

of

sheep

short,

3-5

indole-negative.
III

and

translucent,

a study

from

(1921).

organisms

named

isolated

group

of

were

In

a

groups

Pasteurella

1923).

defined

flat

Similar

lambs

bovine

cocco-bacilli.

non. -haemolytic,

bovisepticus.

Bacillus

Jones

and

II

1921).

of

incubation,

were

Group

indole-positive

from

were

the

of

cases

organisms

bipolar

agar

after

I

with

(Jones,

morphology,
Group

serological

on blood

three

colonial

Newsom

associated

earlier

from

distinguished

criteria

Colonies

years

by

description

first

the

eleven

bacteria

Jones

pneumonia

an organism

although

appeared

introduced

was

designate

to

cattle,

had

organism

haemolytica

P.

haemoiytica

P.

of

and

group.
and

III

group

Cross
The

of
resembled


11

Group

Jones'

I

and

(The "typical"
Pasteurella

was

has

group

it

is

but

does

not

produce

reactions

of

species

as

classified

and

and

attacks

certain

were

adopted
are

(Cowan

P.

facultatively

fermentation,

1974).

Steel,

and

anaerobic.
by

sugars

haemolytica

diverse,

are

and

identification

criteria-for

by Biberstein,
listed

non-

a gram-negative,

as

aerobic

gas

of

this,

These

(1960).

been

since

defined

oxidase-positive

Cultural
view

is

which

rod,

is

haemolytica.

Pasteurella

multocida).

P. haemolytica
motile

designated

in

the

and Knight

Gills

here,

of

in

diminishing

order

The

of

of

importance.

1.

Haemolysis

on blood

haemolysis
strain,

considerably

varies
and

directly

P. haemolytica
distinguished,

is

sometimes

underneath

haemolysis

agar.

is

the

a useful
and
as

from

However,

colonies.

by which

feature

latter

strain

detectable

only

P. multocida

the

extent

can
is

be

non-haemolytic.

to


,2
i

2.

Absence

of

uniform

characteristic

in

indole

to

contrast

P.

a

baemolytica,

P.

of

is

This

production.

multecida,

is

which

indole-positive.

3.

Growth

on

grows

well

P.

4.

In

McConkey's

does

multocida

Failure

to

is"oxidase-positive,
it

from

most

the

milk.

haemolytica

P.

characteristics,
feature

a useful
of

blue

methylene

these

wbereas

agar,

not.

reduce

to

addition

McConkey's

on

haemolytica

P.

agar.

distinguishes

which

Enterobacteriaceae,

which

are

oxidase-

negative.

Satisfactory
bacteria

are

fermentation
often

fermentation
difficult

media

inconclusive
strains

biotypes,

designated

be associated

is

achieve,

poor,

of

with

P.

as

1973).

haemolytica

A and

T.

growth

pneumonia,

and

on many

obtained
Smith
from

Biotype

these

with

and results

(Thompson,

classified

to

to

reactions

(1959

sheep

A strains
biotype

are
B)

into

two

appeared

T strains

13

with

lambs.

of

septicaemia

characterised

further

fermentation

reactions,

to

colonies

grey

small

arabinose

biotype

while
brownish
but

not

lost

blood

but

in

to

penicillin

of

methods

sensitivities

larger

by

10 days,

colonies

trehalose

with
2 days,

within

A strains

Biotype
broth

ageing

formed

fermented

and

trehalose

not

morphology,

A strains

agar,

10 days.

within

and

Biotype

sheep

later

were

colonial

curves

formed

rapidly

viability

of

fermented

and

arabinose

more sensitive

and

cultures

were-

than

and tetracycline

T strains.

biotype

A variety

haemolytica

P.

subdivide

that

established
from

on

T strains

centres

basis

1961).

7 days,

within

biotypes

growth

(Smith,

antibiotics

the

on

These

whereas
revealed

one

1950),

Godbille,
Bosworth

and

haemolytica

concerned

mainly

or

Glover,

and

in

three

Biberstein,

Gills

to

the

an

species

with

by

distinct

Cross,

1932),

haemolytica
two

(Florent

and

(Montgomerie,

means
and

the

P.

1930),

serotypes

1938),

studies

antigenically
and

to

attempts

Early

Newsom

Edington,

reactions.

subdivide

used

was

1921;

(Tweed

been

serologically.

(Jones,

da

P. multoci.
studies

P.

have

of

Knight

basis

of

agglutination
(1960)
somatic

attempted
antigens
6


14

by

direct

and

arthritis

types
II)

and

ion

be

basis

the

on

to

expected

of

septicaemia,
of

nasopharynx

by

this

into

these

and

These

method.
a wide

cover

fell

a

two

spectrum
groups

main

fermentat-

additional

tests.

P. haemolytica

the

subdivide

into

species

(IHA)

developed

was

antigen
(1960).
blood

(bovine

cells

haemolytica
in

raised
reaction

by

and

rabbits

their

with

the

of

species

other

and

into
between

IAA

tests

types

have

This

were

IRA
defined

sharply

also

red

antisera

strains.
eleven

Knight

from

antigens
with

as

bovine

of

agglutination

cross-reactions

A variety

Gills

soluble

selected

this

using

sensitisation

against

divided
and

RBC)

An

serotypes.
test,

to

successfully

used

Biberstein,

involved

test

The

been

several

haemagglutination

indirect

types,

has

which

heat-stable

a soluble,

possesses

component,

capsular

P.

the

examined

haemolytica,

P.

of

were

would

which

strains,

from

from

derived

cattle

pneumonia,

and

mastitis,

individuals,

healthy

and

sheep

bacterial

autoclaved

using

conditions,

clinical

of

variety

(I

from

Strains

cells.

of

tests,

agglutination

minimal.

been

developed
4


15

to

51

classify
of

fever

these

strains

(HuRBC)

cells

dilutions

test

rabbits.

All

strains

were

the

found

red
that

if

the

study,

the

of

sera

haemolytica,

the

found

be

to

from
by

instead

used

were

agglutination

treatment

after

cells,

IHÄ test

presence

was modified

dilute

from

the

distinct

cells

red

red

against

were

clearer

in

suspended

titres
P.

pig

0 human
in

test

cases

rabbit

normal

was employed
immunised

sheep
rather

than

to

Gills

and

Knight

to
with

a

type

strains.

unknown

The
been

typed,

were

of

strain

was

this

This

to

polysaccharide

raised

totally

and

study.

obtained

antigen,

determine

it

IHA

test

widely

of

be

Biberstein,
As

used.

became
prepared

antigen
would

it

from

agglutinated

in

Guinea

isolated

type

onto

sera

and

In

has

then

were

this

attempted

soluble

immune

in

plasma.

T'e

adsorbed

homogeneous

HuRBC,

known

was

(1966).

patterns

Canada.

specific

multocida
Cameron

haemoiytica

of

strains

with

in

(1956)

Carter

P.

of

which

serologically

of

haemolytica.

strains

shipping

from

of

P.

subdivide

agglutinated

apparent
from

larger
that

a particular
by

2 or

numbers

of

3 antisera

strains
a batch

occasionally
strain

(1960)

of

P.

were

of

haemolytica

(Biberstein,

1965).


16

In

this

some cases

original

different

were

due

the

typing

to

trays

(Shreeve,

strains

P.

divisions

of
and

classified
tation

tests

degree

of

representing

as

either
(Biberstein

correlation

In

to

(1960)

biotype

biotype

was
A by

Group

found

and

is

which

to
by

to
was

the

the

basis

was
fermen-

of

A reasonable
two

methods

s biotype
and

Group

IT.

(1959

A
Knight
An
as

classified
Group

of

serotype

Gills

in

two

a selection

each
the

T

Biberstein,

this,

Smith'.

included

biotype

1962).

Biberstein,

2,

study.

system

relate

between

T corresponded

by

previously

this

A and

T on

Gills,

general,

I. of

serotype
Smith,

A or

and
was

tubes

suggested

haemolytica,

correspond

exception

to

P.

biotype

with

1972).

To confirm

classification.

and

later

these

Perspex

a microtitre

seemed

species

modified

in

biotype

the

of

was

Wasserman

Thompson,

and

interfere

substituted

(1960).

Knight

of

strains

to

that

not

discovered

adapted

haemolytica

the

who

the

was

discovery

of

technique

the

cross-reactions

were

did

in

types

of

demonstrated

was

(1966)
for

Biberstein

Smith's

Gills

was

there

which
The

serotype

test

the

Later,

antigens,

capacity

A 12th

used.

It

Thompson

1 ml

a mixture

others

procedure.
and

of

in

to

types.

minor

Biberstein

of

but

culture,

between

due

was

II

of

"

B)


17

Biberstein,

Gills

6,7,8,9,11
biotype

A and

biotypes

A and

homology

between

between

a biotype

of

used

to

1975).
of

vision
one,

and

regarding

haemolytica

P.

the

to
The
P.

the

biotypes

available
haemolytica

into
feel

biotypes

as

1965).

This

brevity

strains

Al" and

has

not
of

"Type

A and

evidence

been

etc.

these

separate

which

(Thompson

and

that

there

is

groups

grounds

species

(Smith

will

for
be

the

be

Mould,
subdia valid
for
and

sake

referred

each

broadly

the

are

so

of

could

groups

two

resolved,

P. haemolytica
T3 ",

two
T

was

hybridi-

which

suggests

that

homology

the

proteins

patterns

into

of

Electrophoresis

constituent

species

degree

(30-50%)

1968).

gave

some workers
the

the

between

interspecies

Francis,
of

but

T strain

for

obtained

gels

subdivide

corresponded

A strains,

to

T.

a high

showed

a biotype

and

polyacrylamide

serotype

'type

often

biotype

a^-. d homologies

nucleic

haemolytica

A and

1,2,5,

belong

to

?.U to

and

biotype

(Biberstein

sations
in

P.

two

that

than

of

T of

Serotypes

now considered

3,4

serotypes

investigation

An

less

12 are

and

(1960).

Knight

and

Thal,

of
to

as

18

distribution

The

haemolytica

P.

of

types

healthy

amongst

sheep

The
of

study
of

P.

the

upper

has

respiratory

tract

1970).

Thompson,

that

the

same types

with

the

possible

Type

A2

whereas
5,8

and

some

healthy

types

9 were

well

the

tonsils
being

Sixty-four

of
P.

contrast,

of

95 of

type

100

T

100

(Gilmour,

adult

survey

and

only

showed

the

that

the

as

Type

was

isolated

65% of
and

from
these

these
1974).

Fraser,
these
were

proportion

A

of

nasopharynx

sheep,

6% of

such

Britain.

Great

swabs

1966).

USA,

the

Thompson

not

was

Britain,

haemolytica

P.

showed

Britain,

which

Great

in

nasopharyngeal

haemolytica

A 12 month

in

T strains

In

flocks

Thompson,

in

represented

type

A12,

in

rare

Shreeve

Biberstein,

and

predominant

apparently

sheep.

isolates

yielded

were

healthy

USA and Great

type

flocks

frequently

normal

of

the
of

healthy

apparently

1966;

(Biberstein

USA

outnumbered

strains

from

strains

in

exception

in'the

identified

of

A survey

occur

in

the

assisted

isolated

been

Thompson,

and

has

types

haemolytica

P.

of

haemolytica

(Biberstein

sheep

different

of

epidemiology

sheep.

from

and

recognition

sheep
of

of

type

T.

sheep