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 Pasteurel l a mu l t ocida

A S tudy on the I solation, Ident i f ication and

Charact e r ization of New Zealand S t rains .

A The s i s P r e s e nted in P a rt i a l F u l f i lme nt o f t h e

Re qu i rement s f o r t he Degree of Ma s t e r o f S c ie n c e

i n Microbio l ogy at Ma s s ey Un ive r s i t y , N e w Zeal and .

RhysJohnJon

1988
 ii

ABSTRACT

P a s t e urella mul t ocida is a sma l l Gram negative b a c i l lus which causes

di s e a s e in man y anima l s pe c i e s . I t s pat hogeni c it y i s a t t r ibuted t o
t w o mecha n i sms : inva s i vene s s a n d t o x i n product ion . T h e propo rt ion o f

s t r a i n s w h i c h p r oduce t o x i n i s l o w but s u c h s t r a ins c a u s e s evere

a t r o ph i c rhinit is in s w ine . I n v a s i ve n e s s is t h e p redomi n a n t

me c h a n i sm of pathoge n i c i t y a nd is la rge l y dependant on the

po s s e s s ion o f a capsule .

P. m u l t o cida i s divided i n t o f ive s e rotype s ( A , B , D , E and F ) ba sed

o n t he antigenic s t ructure of the capsule a nd a c o r relat i on exi s t s

between serotype , di sease a nd t h e anima l species a f fected . Howeve r ,

ma n y i s o l a t e s a r e n o n - t yp a b l e d u e t o t h e p o s s e s s i o n o f a non
a n t i gen i c ( hy a l u ro n i c a c i d ) caps u l e . The presence o f a n ant igenic
capsule and a hyaluronic a c id capsule a re independant . ie one , both
o r n e ither ma y be present .

To facilitate the i s o l a t ion of P . mul t o ci da we a s s e s sed the

s e l e c t ive medium of Smit h and Ba s ke rv i l le ( l 9 8 3 ) a n d found that many
New Zea la nd i s olates of P . mul t o c i da grew poorly in the absence o f
b l o od ( not p re s ent i n Smi t h and Ba s ke rville ' s medium) . Furt hermore ,

m a n y i s o l a t e s were inhib i t e d by P o l ymixin and t h e high a l kal inity

(pH 8 . 6 ) o f t he medium .

On t he b a s i s of these o b s e rv a t i o n s we f o rmu l a t e d a m o d i f i e d

s e le c t ive medium which omit ted P o l ymixin and w a s c ompo sed o f a blood
a g a r b a s e at neut r a l pH . H o weve r , i t cont a ined t h ree ant ibi o t i c s
( Ge n t a myc i n , Bacitracin a n d Myco s t a t i n ) present in the o riginal
med i um o f Smi t h and Baske rvi l le . T h i s modi f ied medium propagated a l l
our test i s o l a t e s o f P . m u l t o c i da w h i c h w e re d e r ived f r om s even
 iii

animal spe c ie s ( f owl , sheep, goat , c at t l e , r abbit , dog , c at ) . It

al s o supp re s sed the backg round f l o ra present in swabs t aken f rom the
nas al cavity o f rabbit s .

B o t h t h e t radit io nal is o l at ion t e c hnique o f p l at in g specime n s on

b l o od agar and t he modi f ied s e l e c t ive medium were used t o obt ain
is o l at e s o f P . mu l t o c i da f rom s e v e r al specie s o f dome s t ic animal .
T h e s e were : pigs ( 1 5 is o l ate s ) , rabbits (25) , cat s ( 1 0) , dogs (21)
and deer ( 1) . I s o l ates we re examined in some det ail .

I s o l ates were serotyped u s ing the indirect haemagglutination as s ay

( I HA ) . They were found to be T ype A (7%) , B (1%) , D ( 9%) or

u n t ypable ( 8 3 % ) . This is s imilar t o overseas f indings .

T h e I HA as s ay is a l aborious technique s o two po s s ible alternat ive s

w e r e e x amine d . vi z s o d ium d o de c y l s u l p h a t e po l y ac r y l amide - ge l

a n a l y s is ( S D S - P AGE) of p r o t e in s and r e s t r ic t io n e n d o n u c l e as e

an alys is ( REA) o f DNA . Both technique s f ailed t o dis t inguish bet ween
P. mul t o c i da s e r otyp e s in t he s e n s e t h at is o l at e s w e r e e xt reme l y
h e t e rogeneous and mo s t g ave a unique pat t e rn both with SDS-PAGE and

REA .

T h is hete rogeneit y allowed t he ident ification o f individual s t r ains

of P . m u l t o c i da and c o n s e qu e n t l y S D S - P AG E w as used in an

e pidemio l ogical inve s t ig ation which t raced t he o rigin o f an outbreak

of respirat o ry dis e as e in r abbit s and provided evide nce t h at t h e

outbre ak w a s n o t ( as was ear l ie r believed) due t o t he int roduction

of r abbit s f rom ove r s e as int o a New Z e a l and c o lony . F u rt h e rmo r e ,

u s ing t his app roac h we were ab le t o show t h at s t r ains wh ich c ause
s eve re pneumonia were not c o n f ined t o t he lowe r re s pirat o ry t ract
but could al s o be c a r r ied in t he nasal c avit y . This is c o n s is tent
w it h the accepted hypothesis t h at P . mul t oci da is an oppo rtunis t ic
pathogen .
 iv

Severe a t r oph ic rh i n i t i s i s a d i s e a s e o f p igs w h i c h i s c aused by

t oxin-producing s t r a i n s of P . mul t ocida . The di s e a s e is respons ible
f o r muc h e c onomi c l o s s ove r s e a s but h a s not been rep o r t e d i n New
Z e a l and . We obt a ined a toxigenic s t rain of P . mul t oc i da and used it
to va l i d a t e an in vi t ro ( ma mma l i a n ce l l cu l t u r e ) me t h o d for
de t e ct i n g t oxin - p r o du c i ng s t r a i n s . T h i s me t h od w a s t he n u s e d t o
examine New Zea land i s o lates o f P . mul t ocida . None o f these were
s h own t o be t ox i gen i c . T h i s s u g ge s t s t h a t t ox i g e n i c s t ra in s , if

present i n New Zea land, a re not c ommon .

D i seases due to P . mu l t o ci da a re commonly t reated with antimicrob i a l

agent s such a s t h e P e n i c i l l ins , Aminog l yc o s ides and Su lphonamide s .

St rains which a re re s i s t ant to these agents a r e p re s ent in ove r s e a s

c o unt r i e s a nd re s i s t a n ce is associated w it h the pre sence of

pla smids .

We det e rmined the min imum inhib i t o ry concent rat ions (MIC) , o f four
a n t im i c r o b i a l s ( Penici llin G , S t r e p t o my c i n , Tet r a c y c l ine and
S u lphadi a z i ne ) f o r New Z e a l and i s o l ates . Con s iderable variat ion wa s

f o u nd b e t w e e n i s o l a t e s but no i s o late wa s o f s u f f i c i e n t l y h igh

re s i s t ance t o be cons ide red " re s i s t a nt " . This was de spite the fact

t h at 1 2 of 73 (16%) of t he f i e l d i s o l a t e s p o s s e s s ed p l a smids of a
s imi l a r s i z e ( lMda l to SMda l ) to p l a smids known to c a rry ant ibio t i c

re s ist a nce markers .

D e spit e it s import ance a s a pat hogen of swine , cat t le , s heep , fowl

and r abbit s , P. mul t o c i da has been l it t le s t u died in New Zealand .

T h is t he s i s repre sents a pre l iminary s t age t o a f u l l e r unde rst anding
of the import ance of P . mul t o c i da in t his coun t r y a nd t he best means
of c ont ro l . The p o s s i b i l it y of outbre a k s of d is e a s e due t o
P. mul t o ci da , eg at rophic rhinit i s , may we l l s t imu l a t e f u rther work
on this organism .
 V .

ACKNOWLEDGMENT S

I am inde b t e d t o the D epa rtment o f Mi c r ob i o l ogy and Genet i c s f o r

p roviding t h e f a c i l i t i e s f o r t h i s pro j ect.

I wish t o thank my supe rvi s o r , John Cla rke , f o r h i s cont r ibut ion t o
t h i s work . H i s z e a l and gramma t i c a l dext e r i t y were mo s t apprec iated .

T ha n k s go t o Ron T u c ke r and P a u l H o cqu a r d , f o r t he i r e f f o r t s in

s upplying mat e r i a l s , a n d t o t he f o l lowing pe ople a nd o rg an i z at ions
f o r donat ing mat e r i a l s a nd providing f inanc i a l a s s i s t ance :

The Leona rd Conde l l Trust

Min i s t ry of Ag r i c u l t ure and F i sheries

Rex P a t t e rson ( G ibco NZ Ltd)
Drs J.M . Rutt e r and P . D . Luther ( ARC I n s t i t ute, United Kingdom)

I would l ike to t h a n k Geo rge I onas for g e ne r o u s l y p r o v i d i n g

t e chni c a l a dvice . I a m a l s o indebted t o h im f o r h i s ph i l o s oph i c a l

t e ach ings , espe c i a l l y h i s balanced concept o f a utopian s o c iety .

F inally I thank my c l a s smat e s Mike , Nicky, S a l l y and St eve f o r t he i r

f r iends hip and t o t he t e chnic a l s t a f f f o r ente rtain ing u s a l l .
 vi

TABLE OF CONTENTS

Page

T I T LE P AGE . . i
ABS TRACT ii
ACKNOWLEDGEMENTS . . V

TABLE OF CONTENTS vi
L I S T OF F IGURES xi
L I S T OF TABLES . . xiii
INTRODUC T I ON . . 1

CHAPTER 1 : Historical Review

1.1 Clas s i f i cation a nd Nomenc l a t u re 4

1.2 Serological Cha racterisat ion o f P . mu l t o c i da
1.2.1 E a rly Attempts at Group i ng . . 9
1 .2.2 D i scove ry o f Caps u l a r T ypes 12

1.2.3 D i sc overy o f Soma t i c Ant igen ic Groups 16

1.2.4 Agreement between Typ i n g Met hods . . 19
1.2.5 Bacteriophage Typ ing . . 20
1.3 Ant igen i c St ruct u re o f P . mul t ocida 20
1.4 Toxige n i c S t r a i n s o f P . mul t oc i da . . 26
1.5 D i s e a s e As s o c i a t ions and Ep idemiology o f
P. mul t o c i da

1.5.1 Gene r a l 29
1.5.2 Catt le 31

1.5.3 S heep 33
1.5.4 Goa t s . . 35

1.5.5 Rabbi t s 37
1.5.6 S wine . . 38
1.5.7 Dee r 39
1.5.8 Cat s a nd Dogs 39
1.5.9 Man . . . . . . 40
 vii

1.6 The Re l a t ionsh ip o f Type o f P . mul t oc i da t o

Hosts a n d D iseases . . . . 40
1.7 Immun i t y and Va c c ines . . 45
1.8 Ant ibiotic Resist ance and the P resence o f
P lasmids . . . . . . . . . . . . . . 50
1.9 Iso l a t i on and Ident i f i c a t i o n o f P . mu l t o ci da 52

CHAPTER 2 : I s olation of P. mu l t ocida from t h e Na s a l Cavity:

Eva luat ion and Modi fication of a S e lective Medium

2.1 Int rodu c t ion . . . . . . . . . . .. . . . . . . . . 54

2.2 Mate r i a ls and Met hods
2 . 2 . 1 P repa ration o f Media . . . . . . . . 56
2 . 2 . 2 P repa rat ion and S t anda rdizat ion of

Cu ltures . . . . . . . . . . . 58
2 . 2 . 3 Eva luat ion o f 8HPG Medium 59
2 . 2 . 4 The Effe ct o f Ant ibiot i cs on the

G rowth of P . mul t o c i da a nd the

Suppression of Othe r Organisms . . 59

2 . 2 . 5 I noculation o f Modi f ied Select ive

Medium ( SM ) . . . . . . . . . . . . . . 60
2.3 Resu l t s

2 . 3 . 1 Evaluat ion of 8 HP G Medium . . . . . . 61

2 . 3 . 2 The Ef fect of Ant ibiotics on the

Growth o f P . mu l t o c i da o n BA . . . . . . 63

2 . 3 . 3 Evaluat ion o f Modi f ied S e lect ive

Medium ( SM ) 66

2.4 D iscussion 69
 viii

CHAP TER 3 : I s olat i o n of P . mu l t ocida, S e r o typinq o f

I s olates and Compa r i s on of The s e by SDS-PAGE

and Re s t r iction Endonuclease Ana lys is (REA)

3.1 Introduct ion . . . . . . . . 72

3.2 Mat e r i a l s and Methods . . 72

3 . 2 . 1 I s o l a t i o n and Ident i f ic a t ion o f
P. mul t o c i da . . . . 73
3 . 2 . 1 1 Mat e r i a l s 74
3 . 2 . 1 2 Met hods 73
3 . 2 . 2 P rocedures f o r the IHA A s s ay
3 . 2 . 2 1 Ma t e r i a l s 74
3 . 2 . 2 2 Me thods 76
3 . 2 . 3 P rocedu res for SDS-PAGE Analys i s

3 . 2 . 3 1 Ma t e r i a l s 84
3 . 2 . 3 2 Methods 88
3 . 2 . 4 P rocedures f o r Re s t r i c t i on Endonuclease

Ana lys i s ( REA)

3 . 2 . 4 1 Mate r i a l s 92
3 . 2 . 4 2 Methods . . 95
3.3 Re s u l t s
3 . 3 . 1 I s olat ion o f P . mul t oc i da and S ome
Epidemi o logical Aspect s . . . . 99
3 . 3 . 2 S e rotyp ing : Va l idat ion o f IHA As s a y
a nd i t s Application t o t he Typing o f

F ield S t rains

3 . 3 . 2 1 Va l idat ing the !HA As s a y 103

3 . 3 . 2 2 S e r ot yping o f F i e ld I s o l a t e s 104

3 . 3 . 3 SDS -PAGE Ana lys i s o f S t r a ins o f

P. mul t oci da . . . . . . .. . . 105

3 . 3 . 4 Epidemio logical Trac ing o f
P. mul t o c i da by SDS-PAGE . . .. . . . . . . . . 107
 ix

3 . 3 . 5 T h e Source o f a P . mul t o c i da Iso l a t e

Assoc i ated w i t h an Epidemic o f

Snuff les . . . . . . . . . . . . . . 113
3 . 3 . 6 Compa r ison o f P . mul t o ci da Serotypes
with SOS-PAGE and REA . . 116
3.4 D iscussion 119

CHAPTER 4 : The Detect ion of Toxigenic S t ra i n s of P . mu l t ocida

Derived f rom New Zealand Animals

4.1 Int r o duct ion . . . . . . . . . . . . . . . . . . . . 120

4.2 Mat e r i als and Met hods
4 . 2 . 1 P repa rat ion of Rea gents and Medi a . . 121
4 . 2 . 2 Me thods . . . . . . . . . . . . . . 124

4.3 Resu l ts

4 . 3 . 1 Va lidat ion of the EBL Assay . . 128

4 . 3 . 2 Assay o f F ield Iso l ates 130
4.4 D iscussion 131

CHAP TE R 5 : Compa r i s on of New Zealand I s o l ates of P . mul t ocida

With P a rt icular Reference to P l a smids

5.1 I nt roduction . . . . . . . . 132

5.2 Ma t e r i a ls and Met hods . . 1 32

5 . 2 . 1 Examination of P . mul t ocida for

P lasmid C a r riage

5 . 2.1 1 Mate rials 133

5 . 2 . 1 2 Met hods . . 133

5 . 2 . 2 P . mul t o c i da : Det e rminat ion o f the

Minimum I nhibi t o ry Concent rat i ons o f

Ant ibiot ics

5 . 2 . 2 1 Mat e r i a ls 134

5 . 2 . 2 2 Methods 135
5 . 2 . 3 P rocedu res for SOS -P AGE Analysis 136
 X

5 .3 Resu l ts . . 136

5 .3.1 The Ant ibiotic Sens i t ivity o f S t rains

o f P. mul t ocida.. . . .. . . 139

5 .3.2 Comparison of P rote ins of P . mu l t o ci da .

P l asmid C a r rying S t r a i ns Versus Non-
P lasmid C a r rying S t r a i ns . . 147

5.4 D iscussion 147

CHAP TER 6: Gene r a l D i s cus s io n . . . . . . . . . . . . . . . . . . . . . . 151

6 .1 The Deve l opment o f a Select i ve Medium f o r

the I solat ion o f P . mult o c i da i n New Zeal and . . 152

6.2 Iso l a t i on and Se rotyping o f P. mul t o c i da

f rom New Zealand Animals . . . . . . 152

6 .3 S D S - P AGE Ana lysis of Field Isolates o f

P. m u l t o c i da.. . . .. . . . . . . . . 154

6.4 Compa rison o f P. mu l t ocida S e rotypes by

Rest r ict ion Endonuclease Ana lysis ( REA) . . . . . . 155

6 .5 Assay f o r Exot oxigenic S t r a i ns o f

P. m u l t o c i da .. .. .. .. .. . . . . 156

6 .6 Ant ib iotic Resist ance and i t s Re lat ionsh ip

to P l asmids i n New Zea land S t rains o f

P . m u l t o c i da .. 157

6.7 Out look 158

AP P END IX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

B I B L IOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
 xi

L I S T OF F IGURES

F igure P a ge

1.1 G ram S t a i n o f P . mul t o c i da 5

1 .2 Ant igen ic Cha racte r i s t i c s of P . mu l t o c i da 18

1.3 Type A Colon ies of P . mult ocida . . . . . . . . . . . . . . . . . . 22

1 . 4 Type D Colonies o f P . mu l t ocida . . . . 22

2. 1 The Suppre s s ion of Na s a l Bacte r i a l F l ora by a

S e lect ive Medium ( SM ) . . . . . . . . . . . . . . 67

3.1 SDS-PAGE P ro t e i n Ana lys i s of P . mu l t o ci da I s o lated

f rom Seve r a l Anima l Species . . . . . . . . . . . . . . . . . . . . 10 6

3.2 SDS -PAGE Ana l y s i s o f P . mult ocida I s o lates f rom Swine . . 108

3 .3 SDS-PAGE Ana l y s i s o f P . mul t o c i da I s o l ates f rom Dogs . . . . 109

3.4 SDS - PAGE Ana lys is o f P . m u l t o c i da I s o l a t e s f rom Cat s . . . . 110

3.5 SDS -PAGE Ana l y s i s o f P . m u l t o c i da I s o lates f rom Rabb i t s . . 111

3 . 6 SDS-PAGE Ana lys is of P . m u l t o c i da f rom Rabb i t s . . . . . . . . 1 12

3 .7 SDS-PAGE Ana ly s i s o f P . mul t oc i da I s o lates f rom Rabb i t s

:A compa r i s on o f lung a n d na s a l t ra c t i s o lates . . . . . . . . 114

 xii

3.8 SDS -PAGE Ana l ysis o f P . mul t o c i da I s o lates f rom Rabb i t s

: A compa r i s o n o f i s o l a t e s obt a ined b e f o re and a f t e r

t he int rodu c t ion o f anima l s , f rom o ve rsea s , t o a
rabbit c o l on y . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

3.9 SDS -PAGE Ana l y s i s o f P . mul t o c i da s e rotype s A, B and D . . 1 17

3 . 10 Re st rict ion Endonuclease Ana lys i s o f P . mul t o ci da

: A compa r i s on of serotypes A and D . . . . . . 118

4.1 Cell Culture Monolaye r o f Embryo n i c Bovine Lung Cells . . 129

4.2 Cell Culture Monolaye r o f Embryo n i c Bovine Lung Cells

: The cyt opa thic e f f ect o f P . mu l t o c i da exot oxin . . 12 9

5.1 Agarose -Ge l E lect rophore s i s o f DNA f rom P lasmid-

Cont a ining F e l ine I s o la t e s o f P . mu l t o c i da . . . . . . . . . . 137

5.2 Aga rose-Ge l E lect ropho re s i s o f DNA f rom P l asmid-

Cont a in ing Canine I s o l a t e s of P . m u l t o c i da . . . . . . . . . . 138

5.3 Variat ion o f MICs Between S t ra i n s o f P . mul t oc i da . . . . . . 142

5.4 SDS-PAGE Ana lysis o f P . mul t ocida I s o la t e s f rom Dogs

:A compa r i s on of pla smid-cont a i ning i s o lates with

i s o l a t e s wh i ch do not contain p l a smids . . . . . . . . . . . . 148

5.5 SDS-PAGE Ana lys i s o f P . mul t ocida I s o la t e s f rom Cats

: A comp a r i s o n o f pla smid-cont a in in g i s o lates with

i s o l a t e s w h i ch do not cont a i n p l a smids . . . . . . . . . . . . 149

 xiii

L I S T OF TABLE S

T ab l e P age

1.1 P. mul t o c i da Soma t i c Groups a nd the i r Re lation t o

Capsu l a r Type . . . . . . . . . . . . .. .. . . .. .. .. .. 18

1 .2 Host Range o f P. mul t oci da 29

1 .3 Re l a t i onsh ip o f P. mul t ocida Capsu l a r Type to D isease 41

1.4 Re l a t i onsh ip of P . mu l t ocida S e r ot ype t o Host and

D isease . . 44

1.5 D i f f e rent i a l Charact e r ist ics o f Pa s t eure l l a Spec ies . . . . 53

2.1 The Ab i l it y of 8HPG Medium t o P ropagate P. mul t o c i da . . . . 62

2.2 The E f fects o f D i f f e rent Leve ls o f a Combinat ion o f

Ant ibiot ics on P. mul t ocida . . . . . . 64

2.3 The E f fects o f Low Leve ls o f a Comb inat ion o f

Ant ibiot ics o n P . mu l t ocida . . 65

2.4 The E f fects o f Individual Ant ib i ot i cs a t D i f f e rent

Concent rat i o ns on the Growth o f P . mul t ocida 68

2 .5 The Abi l it y o f SM Medium to Recover P. mul t o c i da . . .. . . 69

3.1 P. mul t oc i da Stra i ns Iso lated f rom Rabbits . . . . . . . . . . 101

3 .2 The P roport i ons o f Pneumonic Lesions f rom whi c h

P. mul t oc i da was Iso l a t ed in Winte r a n d S umme r . . . . . . . . 102

xiv

3.3 S e rotyping P rototype S t r a ins o f P . m ul t o c i da by I HA

Assay . . . . 103

3.4 Serotypes o f Field I s o l ates a s Dete rmined by the I HA

Assay . . 104

4.1 As s ay o f P ro t otype S t rains of P . mu l t o c i da f o r

Exotoxin P roduction . . . . . . . . . . . . . . . . . . . . . . 130

4.2 Assay o f F i e ld I s o l a t e s o f P . m u l t o c i da f o r Exotoxin

P roduction . . 131

5.1 Assay o f S t r a ins o f P . mul t oc i da f o r P la smids . . . . . . . . 136

5.2 An Evaluat i o n o f P l a smids Carried by St rains o f

P . mult ocida . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

5.3 Minimum I n h ibit o ry Concent rations o f Ant ibiot i c s For

P. mul t o c i da . . . . . . . . . . . . . . . . . . . . . . . . 140
 1

INTRODUCTION

P a s t e u re l l a m u l t o c i da is asso c i a t e d w i t h many spe c ies o f a n ima ls

usua l ly as a c ommensa l o f the uppe r resp i ra t o ry t ract but it is a lso
a potent i a l pathogen . As a pathogen it causes a range of dise ases .
These inc lude pneumonia o f seve r a l a n ima l spec ies, shipping fever o f
c a t t le and s h e e p , fowl cholera of domest i c h e ns a n d a t r o ph i c
r h i n i t is o f swine .

T h ese dise ases h a ve resu l t e d i n c o n s i de r a b l e e c o n omi c l o ss and

therefore it i s not surprising t h at P. m u l t o c i da has been
i n t ens ive l y s t u d i e d ove rse as . T he st udies h oweve r we re f o r man y
ye a rs less p r o d u c t i ve t h a n t h e y c o u l d h a ve b e e n f o r t h re e ma i n

re asons . The w i de h ost r a n ge o f P. m u l t o c i da was not r e c o gn i zed

wh i c h led t o a mu l t ipl i c i t y o f n ames for what we now recogn i z e as

one bact e r i a l spe c ies . The o rgan ism was not adequately dist i ngu ished
f rom e it h e r Ye r s i n i a spp or P . h a emo lyt i ca and the a n t i genic

c o mp l e x i t y o f P . mul t ocida w a s c o n t i n u a l l y unde rest imate d . Even

t o da y our u n de r s t a n d i n g of P . mu l t ocida l e a v e s mu c h to be
e s t a b l ish e d b u t t he fact that it c o n t i n ues t o b e a n i mp o r t a n t

p a t hogen o f a n ima ls ensures that i t w i l l cont inue t o b e intens ive ly

st udied .

T w o gene r a l me c h a n isms , i n vasiven ess a n d t o x i n p r o du c t i o n , a re

r e sponsib le f o r the p a t h o ge n i c i t y o f P . m u l t o c i da . The f o rme r is

p r obably t h e mo r e imp o r t a n t and depends t o a l a rge extent o n t he

possession o f a capsule . The re a re t h re e common c apsu l a r t ypes , vi z

A, B a nd D . These det e rmine the s e r o t ype a nd a c o r re l a t i on e x ists

b e t w e e n t h e s e r o t ype , t h e d i s e a s e c a us e d a nd t h e h ost spe c i e s

a f f ected .

T o x i n produ c i n g s t r a i ns o f P . m u l t o c i da h ave been recogn i z ed only

recent ly ( De Jon g , Oei a nd Tetenburg , 1 9 8 0 ) , a re appa ren t l y r a re and

so f a r have been l imited to pigs whe re t hey cause a seve re f o rm o f

 2

a t rophic r h i n i t is . T h is dise ase is c ommon in Europe and, i f media

repo rts a re r e l i able ( " The Dominion " D ecembe r 2 2 , 1 9 8 7 ) a lso a f fects

p i gs i n Aust r a l i a . As yet seve r e a t r op h i c r h i n i t is h a s not been

r e p o r t ed i n New Z e a l a n d a l though a m i ld f o rm o f t he disease , of
undete rmined a e t iology , has been reported ( Hedges, 1981) i n p igs in
the Wa ikat o .

T re a tment o f disease due to P. mul t oc i da usua l l y involves one o r a

c o mb i n a t i o n o f the f o l l o w i n g a n t imi c r ob i a l a ge n t s , Penicillin,
Te t r acyc l i ne , S t reptomy c i n and S u lphonamides . The cont inual use o f
t h ese d r ugs h a s l e d t o t h e de v e l o pm e n t o f resist a n t st r a i ns o f
P. m u l t o c i da a n d th is resist ance i s assoc iated with plasmids .

A ma j o r pract ical di f f i c u l t y i n s t u dy i n g the p revalence of

P. m u l t o ci d a i n a n ima ls is t h a t s i n c e i t is c a r r ie d i n t he nasa l
t ra c t in r e l a t ively low numbers it may frequent l y go undetected when
n a s a l swabs a re examined by the st a n d a r d t e c h n ique of p l a t ing on
b l ood aga r .

A f u r t h e r d i f f i c u l t y i n studying t h e o r g a n i sm is t h a t t h e o n l y
a v a i lable t e c hn ique t o dete rmine t h e c apsu l a r type i s t h e indirect

h a emagg l u t i n a t i on a ss a y ( I H A) . T h is is l a bo r i o us , is often
inte rfe red w i t h by the p resence o f n o n -ant ige n i c hya luronic a c id on

t h e bact e r i a l surface and f requent ly g ives negat ive resu lts because

m a n y i s o l a t es o f P . m u l t o c i da do n o t poss e ss a c a psu l e . T h ese

d i f f icult ies h a ve to some extent i n h i b i ted e p i demiolog i c a l studies

on t he different antigenic t ypes .

The a b o ve c o ns i de r a t i o ns resu l t ed in the f o rmu l a t i o n of the

f o l l owing a ims f o r this resea rch p roje c t :

1. T o de v e l o p a s e l e c t i v e me d i u m w h i c h w o u l d f a c i l i t a t e t h e
iso l at ion o f P . mul t o c i da f rom nasal swabs .
 3

2. To obt a i n and examine i s o l a t e s o f P . m u l t o c i da f r om s eve r a l

spe c i e s o f anima l i n New Zea land .

3. To establish t he I HA a s s a y a nd u s e i t t o d e t e rm i n e t h e
se rotype s o f New Zealand i s o l a t e s o f P. mul t o c i da .

4. To examine t he po s s ib i l ity o f u s ing a lt e rnat ive approaches f o r

d i f f e r e n t i a t i n g b e t we e n s e r o t ype s of P. m u l t o c i da . This

involved t he examinat ion o f prote ins ( by SDS-PAGE) and D NA ( by

REA) t o de t e rmine i f a n y c o r r e l a t i o n b e t we e n s e r o t ype a n d

prote i n s ( o r DNA) c ou ld b e e s t ab l i s hed which might a c t a s a n

a lterna t i ve method f o r det e rmin i ng t he s e r otype .

5. To e s t ab l i sh an in vi t ro mamma l i a n c e l l culture method f o r the

det e c t i o n o f exotoxin-produc ing s t ra ins o f P . mul t o c i da and t o
use t h i s me t h od t o de t e rmine i f New Z e a l and i s o l a t e s p roduce
exotoxin .

6. To s c reen New Zeal and i s o la t e s o f P. mu l t o ci da f o r re s i s t ance

to ant ib i o t i c s ( Penic illin, S t r ep t o myc i n , T e t r a c y c l i n e and

S u lphonamide) . T o e xamine t he s e isolates f o r the p r e s ence o f

pla smids a nd t o de t e rmi ne if a c o r re l a t ion exi s t s between
ant ibiot ic re s i s t ance and t he p re s ence o f plasmids .
 4

CHAPTER 1

H i storical Review.

1 . 1 Cl a s s i f i c a t ion and Nomenclature

T h e P a s t e u re l l a c e a e a re a f ami l y o f G r a m nega t i ve , f a c u l t a t ive l y

a n a e r o b i c c o c c o ba c i l l i w h i c h a r e n o nmo t i le , nonspo r e f o rming and
exhibit pleomo rph ism ( F igure 1 . 1 ) . They a re dist ingui shable f rom t he

En t er ob a c t er i a c e a e and Vibri on a c e a e be c a u s e t h ey a re o x ida s e a n d

c a t a l a s e po s i t i ve , d o n o t p r oduce appre c i able amount s o f hydrogen
s u lphide and in the c a s e o f mo st spec i e s do not produce gas f r om the
f e rme n t a t i o n o f c a rb o h y d r a t e . The f ami l y i s d i v ided i n t o t h ree
gene ra : P a s t e u rel l a , 6 spec ie s ; H a em oph i l u s , 19 s pe c i e s and

A c t in oba ci l l u s , 5 spe c i e s (Mannhe im, 1 9 8 4 ) . U s ing biochemi c a l t e s t s

P a s t eurel l a c a n b e di f f e rentiated f rom Ha em oph i l us and

A c t i n oba ci l l us . Furthermo re Ha em oph i l u s s pp requ i r e c e rt a in growth

f a ct o r s whe r e a s Pa s t e u re l l a s pp a r e n on- f a s t idious organi sms which

grow on s i m p l e me d i a ( Ma n n h e i m , 1 984) . M e mbe r s of t he ge n u s
P a s t e u re l l a , i n pa r t i c u l a r P . h a e m ol y t i c a , a re re l a t e d t o t h e

a c t i noba c i l l i ( Smi t h , 1 97 4 ) and i t c a n b e d i f f i cu lt t o di s t ingu i s h

bet ween t he t w o . Howeve r t he a c t i n ob a c i l l i po s s e s s a unique st icky
c o l oni a l texture which i s useful in d i f fe rent iat ing between t he two
genera ( Mannhe im, 1984) .

The first r e p o rt of the isolat ion of a b a c t e r i um f i t t i n g t h e

de s c r ipt ion o f t h e o rg a n i sm n o w k n o wn a s P . mul t oc i da w a s made by

B o l l inge r in 1878 ( s ee C a rte r , 1 9 7 6 ) . At t h i s t ime Lou i s P a s t eur

d e mo n s t r a t e d t h a t this organism c auses fowl chole ra . He t h e n

p r o duced t h e w o r ld ' s f i r s t l abo r a t o ry made v a c c ine t o c omba t t h e

disease ( s e e Mannheim, 1984) . I n 1 8 8 3 Bu r r i l ( s e e Nami o k a , 1 97 8 )
i s o l a t e d P . m u l t oc i da f rom t he b l o o d o f a dome s t i c f ow l s u f fe r ing
f rom f o w l c h o le ra . He de s c r i b e d t he o r g a n i sm and c a l led i t
Mi c r oc oc c us gal l i cidus . This name did not a c h ieve gene r a l a c c eptance
5

F igure 1 . 1 Gram Stain of P . mu l t ocida .

The ce l ls a re sma l l , Gram negat ive , ple omo rph i c rods .

1 0 0 0 x Magni f icat ion .

 6

and it was still known a s ' the bacillus of fowl cholera ' when
i s o l a t e d a n d de s c r ibed b y P e r o n c i t o in 1 8 8 7 ( see S m i t h and W i l s o n ,
1 985) . I t w a s i n t h a t ye a r t h a t T r e v i s a n c o ined t he gene r ic name
Pasteurel l a a s a t r ibut e to P a s t e u r f o r h i s e a r l i e r w o r k on fowl
cholera ( s ee Ca rte r , 1 9 8 4 ; Smith and W i l s o n , 1985) .

D e s p i t e t he above report s Lehmann a nd N e umann [ 1 8 8 9 ] ( s ee Namioka ,

1978 ) a re g i ven c redit for the first a t t empt at c omp rehen s i ve l y

de sc ribing P . mul t oc i da wh ich t h e y de s ignated Ba ct e ri um mul t oci dum .
Howeve r i n 1 8 9 3 S t e rnberg named t he organ i sm
Ba c t eri um sep t i ca em i a e haem orrh agi ca e a f t e r i s o lat ing it f rom a c a s e
of h a emo r r h a g i c s e pt i c a emi a , a di s e a s e of cattle r e a r e d in hot
c o unt r i e s ( N a mi o k a , 197 8 ) . This pro l i f e ration o f n a me s is not
s u rp r i s i n g because at the end o f the 1 9 t h century it w a s cons ide red
that i s o l ates de r i ved f r om d i f f e r e n t ho s t spec i e s rep r e s ented

di f fe rent species o f the same genus . T h i s view w a s de r ived f rom the

c ommo n o b s e r v a t i o n t h a t du r i n g n a t u r a l l y o c c u r r i n g o u t b r e a k s o f
disease , t ransmi s s ion of P . m u l t oc i da r a re l y o c c u r red between

di f f e re n t host s pe c i e s ( S mi t h , 1 97 4 ) . T h i s phe n ome n o n w i l l b e

discus sed later . F o l l o w i n g t h i s inaccurate line of r e a s oning

Lign i e r e s [ 1 9 0 0 ] ( see Smith a n d W i l s o n , 1 9 8 3 ; C a rte r , 1 9 67 ) added a

spe c i f i c name for each o rganism based upon the an ima l which it was

is olated . F o r examp l e , P a s t e u re l l a s pp c a u s ing f o w l c h o l e r a wa s

de s i g n a t e d P . a vi s ep t i ca . That c a u s ing sept ic aemia and ha emo r rhage

in c a t t l e was de s ignated P . b ovi sep t i ca whereas if it wa s i s o l ated

f r om s h e e p L igni e re s c a l led i t P. ovi s ep t i c a ( Sm i t h a nd W i l s o n ,

1 9 8 3 ; Carter, 1967) .

I n p r ep a r i ng T op l e y and Wi l s o n s f i r s t e d i t i o n o f ' P r in c ipals o f

B a cte r i o l o gy , V i r o l o gy a nd Immu n i t y ' [ 1 9 2 9 ] ( s ee Smit h and Wil son ,

1983) t he authors rec ogn i zed t h a t on mo rpho logical a nd b i o chemi c a l
g rounds t he s t ra i ns of L i gn i e r e s c o u l d be grouped a s a s ingle
specie s . T o encomp a s s t h i s new g rouping Topley and W i l s o n ( s ee Smi th
and Wi l s o n , 1983) co ined the n ame P a s t e urel l a sept i ca i n 1 9 2 9 . T h i s
n a me g a i n e d wide but not u n i ve r s a l a c cept a nc e and h a s been u s e d
 7

unt i l qu i t e rece n t l y , e s pe c i a l l y in the B r i t i s h C ommo n w e a l t h

( C a rt e r , 1 9 6 7 ) . Notwithst anding t he conclus ion that P . s ep t i ca could

infect s eve ral spe c i e s of an ima l , i s o lates of the o rg a n i sm tended to

be mo re p a t hogenic for t he spe c i e s o f a n ima l f rom w h i ch they were
i s o l ated t han for other anima l specie s . Topley and W i l s on [ 1 9 2 9 ] ( s ee
Smi t h , 1 9 7 4 ) t he re f o re concluded that P . s ep t i ca could be subdivided
into ' b i o t yp e s ' . H o weve r t h e nature of t h e d i f f e r e n c e s b e t ween
' b iotype s ' , except for differences in host range , wa s not spe c i f ied
( Smit h , 1 97 4 ) .

D e s p i t e t he p r e s t ige a s s o c i a t e d w i t h Topley a nd W i l s o n a nd t he i r
f i r s t edit ion o f ' P rincipa l s o f Bacterio logy, Virology a nd Immun i t y '
t he n ame P . s ep t i c a d i d n o t a c h ieve unive r s a l approva l be cause it
did not c omp ly with the rules o f nomenc l ature ( see Smith and Wi l s on ,

1983) . C o n sequent l y i n 1 9 3 9 Ro s e nbusch a n d Me rchant ( s ee Smith and

Wi l s on , 1 983) propo s ed the a l t e rnat ive n ame Pa s t e u re l l a mu l t o c i da .

T h i s n ame w a s a c c epted by t h e a u t h o r s o f Be rge y ' s Ma n u a l in 1 9 4 8

( s ee Smi t h and Wi l s on , 1 9 8 3 ; Namioka , 1 9 7 8 ) . Howeve r t he picture was

c o mp l i c a t e d by t he t e mp t a t i o n to clas sify st ra ins caus ing

haemo r rhagic sept i c aemia o f c a t t le and bu f f a loes unde r the sepa rate
spe c i e s n ame Pas t e u re l l a bovi s . This temptat ion wa s f in a l ly re s i s t ed

s i n c e p o s it ive di f f e rent iat i o n f r om othe r membe rs of P . mul t o c i da

cou l d b e made o n l y by immuno l og i c a l o r s e r o l ogic a l me a n s . ie . It

rep r e s en t s a d i f f e rent se rotype rather than a diffe rent biotype of

t he s ame o rganism ( Ca rte r , 1967) .

At t h i s p o int the re ade r may deduce , i f only from the t it le of t h i s

the s i s , t h a t t h e n ame P . m u l t oc i da h a s gene ra l l y b e e n a c c e pted

f o l l o w i n g its a c c ep t a nce by Be rge y ' s Ma n u a l i n 1 9 4 8 . Howeve r f o r

ma n y y e a r s a f t e r t h e f i rs t edit ion of t h e man u a l w a s pub l i s hed

s t r a i n s o f P . mul t ocida have been re fe rred to a s ' t he haemorrhagic

sept i c aemia pasteu r e l l ae ' . This loose de s ignat ion w a s used by s ome
aut h o r s t o include both P . mul t oc i da a nd P . haemol y t i ca ( Ca rt e r and
Bain , 1 960) . This t e rm is u n s a t i s f a c t o r y e ve n if l imited t o
P. m u l t oc i d a because in p r a c t ice ma n y s t rains do not cause
 8

h a e m o r r h a g i c ,s e p t i c a e m i a a nd n o w a d a y s the t e rm h a e mo r r h a g i c
s ept i c a emia i s re s e rved t o de s c ribe acute sys temic pa s teure l l o s i s in

cattle ( Ca rt e r and Bain , 1 9 6 0 ; Carte r , 1 9 6 7) .

T a lbot a nd Sne a t h ( 1 9 6 0) examin ed many s t r a in s of P a s t eurel l a us ing

c l a s s i c a l morph o l og i c a l and b i ochemic a l t e s t s . They conc luded that

P. mul t oci da was only distant l y re lated to
P a s t e u re l l a ps e u d ot ubercul os i s and the plague baci l lu s
P a s t e u re l l a pest i s . Howeve r t h e s e l a t t e r t w o o rgani sms were found t o
b e c l o s e l y r e l a t e d t o e a c h o t he r . Th i s imp l i e d t h a t the genus
Paste urella should b e s p l i t ( T a lbot and S n e a t h , .1 9 6 0) .
I n ve s t i g at i o n s b y Smi t h and T h a l [ 1 9 6 5) ( s ee Cart e r , 1 9 67) support
T a lbot a nd Sne a t h s f i ndings . F u rthe rmore Smith and T h a l ( s ee Cart e r ,
1 9 67) demon st rated that membe r s of t h e genus P a s t e u re l l a f e l l into

t w o g r o up i ngs . One gr oup c o n t a ined the o x i da s e po s i t i ve spe c i e s

P . m u l t oc i d a , P. pn e u m ot r op i c a , P. ureae, and P . h a em ol y t i c a

[P. a e r ogen e s a nd P . g a l l i n a ru m have s i nce b e e n a dded t o t h i s

g roup ing] . The s e cond group conta ined the oxida se negat ive o rgani sms
P. pes t i s a nd P . pseud ot uberc u l osi s . With t h i s in f o rmat i on in mind
t h i s s e cond gr ouping wa s removed f r om the genus P a s t e u re l l a and is
now c l a s s i f ied i n t he genus Ye rsin i a a s Y. pe s t i s and

Y. p s e u d ot uberc u l os i s ( C a rt e r , 1 9 6 7) .

The s pe c ie s name ' mu l t o c ida ' i s de r i ved f rom the Lat in adject ives

' mu l t u s ' [ ma n y ] and ' c idus ' [ k i l l i n g) . Hence t he n ame me a n s ' many
killing' which is appropriate because P . mul t oc i da c a u s e s mo rt a l ity

i n a w i de range o f hos t s . Pedant ica l ly speaking it s hould be spe lt

mu l t i c i da , h oweve r the G r e e k ' mu l t o c ida ' has bec ome t he a c cepted

ve r s i o n . The l o g i c a l pronounc iat ion is t he r e f o re mu l / t o / c i / da and

not mu l / t oc / i / da as is commonly used .

 9

Not e : The a b o ve sect ion illustrates the ma in historical

l a ndma r k s in t h e n omenc l a t u re o f P . mul t o c i da . Between

1878 and 1 9 2 9 a vast range of names we re gene rated f o r

t he m a n y b i o t ype s of this o rgan i sm . T h e s e n a me s a nd
t he i r re f e r e n c e s h a ve been repo rted by H u s s a i n i [ 1 9 6 6 ]
( see Smith, 1 97 4 ) .

1.2 S e r o logical Charact e r i s a t i o n o f P . mu l t ocida

1 . 2 . 1 E a rly At tempt s at Grouping

The e l u c idat ion o f P . mu l t o c i da s e r o l ogy has been a s l ow proce s s .

T h i s h a s been due in pa rt t o a cont in u a l unde re s t imat i on o f the
o r g a n i s m ' s c omp l e x i t y . Lign i e res [ 1 9 0 1 ] ( s ee C a r t e r , 19 6 7 ) n amed

s t ra i n s a f t e r the anima l spe c ie s fr om which t hey w e r e i s o lated . By

do i n g t h i s he m a y h a ve su spected that s e r o l o g i c a l di f f e r e n c e s
e x i s t e d be tween i s o l a t e s f r om d i f f e rent h o s t s . Howeve r , mo st ea rly
i n ve s t i g a t i o n s s e em to have a s s umed that the spe c ie s was
s e ro l o g i c a l ly homogeneou s . T h i s did not appa re nt ly i n h ibit t hem f rom
sugge s t ing a mu l t itude of di f f e rent names f o r t he o rg a n ism .

U s i n g s e r o l o g i c a l t e c h n i qu e s t he exi s t e n c e o f di f f e rences between

i s o l a t e s of P . m u l t ocida was s h own independent ly by s eve ral wo rke r s .
Co rne l iu s [ 1 929 ] , Yu sef [ 1 9 3 5 ] and Ochi [ 1 9 3 3 ] ( s ee Cart e r , 1 9 6 7 ) us ing

' a g g l u t i n i n a b s o rp t i o n ' , p r e c ip i t a t i o n a n d a gg l u t i n a t ion a s s a y s

r e s p e c t i v e l y w e re able to d i s t ingu i s h f ou r s e r o l og i c a l t ype s .

Ochi [ 1 9 3 3 ] ( see Nami o k a a n d Mu r a t a , 19612) n amed his four

s e ro l o g i c a l groups A , B, C and D . He obs e r ve d that dome s t i c f o w l s

w e re not a t t a c ked by P . mu l t ocida strains of b o v i ne or igin .

Rec ip r oc a l ly , c a t t le and bu f f a lo were not a t t a c ked by fowl i s o l ates .

Ochi ' s t ype A c on s i s ted o f c u lt u r e s i s o l a t e d f r om c a s e s o f f o w l
c h o l e r a a n d w e r e f ound t o po s s e s s pathoge n i c i t y f o r f o wl a n d sma l l
l a bo r a t o ry an ima l s only . Type B s t ra i n s were i s o lated f rom c a s e s of
h a emo r r h a g i c s ept i c a emi a o f c a t t l e a n d f r om s imi l a r d i s e a s e s o f
s heep a nd swine . T h e s e s t r a i n s did n o t p r oduce f owl chole ra in
 10

dome s t i c b i rds . T h i s repres ent s t he f i r s t clear ind i c a t ion that t he

h o s t r a nge o f P . m ul t oc i da i s related to t he serotype . Och i ' s Type C

p o s s e s s ed no p a t hogen i c i t y f o r f o w l s a n d fa i led t o p roduce e ither

haemo r r h agic sept icaemia i n c a t t le or a s imi l a r condit i o n i n sheep

a nd s wine . Ochi ' s t ype D was d i s s imi l a r to types A, B and C . Howeve r
we w i l l not cons ide r it furthe r be cause it has s ince been shown to
e quate with the c u r rent spec i e s P . haemol y t i ca ( C a rt e r , 1967) .

Note : St rictly spe a k ing systemi c pasteure llosis is a

b a c t e r a emia a n d n o t a s e pt i c aemia s i n c e P . m u l t oc i da
does n o t mu l t i p l y i n t h e b l o od s t r e a m . H o we ve r t h i s

the s i s follows t he gene r a l practice and uses the

de s i g n a t i o n s e pt i c aemia when this is the a c cepted

t e rm i n o l ogy f o r a part i c u l a r disease . The us age o f t he

term pa steure l l o s i s i s di s c u s sed in Sect ion 1 . 5 . 1 .

Unt i l t he late 1 9 3 0's who le o rganisms we re used a s both immunis ing

a n t i ge n s and a s react ing ant igen in s e r o logica l t e s t s . Howeve r in
1 9 3 8 P i ro s ky ( s e e Car te r , 1967) , us ing an agar-ge l immunodi f fus ion
procedure ( AG I D ) , s t u d i e d p o l y s a c c h a r i de ex t r a c t s f r om s mo o t h
c u l t u r e s o f P . m u l t oc i da . He f o und t h a t ant i s erum p repa red u s ing
w h o l e o rg a n i sms r e a c t ed w i t h t he pol ys a c c h a r ide e x t r a c t s of s ome
i s o l a t e s only . T h a t is , he s h owed that s e ro logi c a l s peci f i c i t y was
at least in p a r t de t e rmi n e d by t h e p o l y s a c c h a r i de c a p s u l e ( s ee

Ca r t e r , 1 9 67 ) . T h i s led him to s ugge s t t h at P . mul t oc i da i s o l ates

c o u l d p o s s i b l y be d i v i de d i n t o s e r o l o g i c a l g r o u p s b a s ed o n t he

ant ige n i c i t y o f t he po lysaccha ride ( Ca rt e r , 1 9 6 7) . Howeve r he found

that s o me strains did n o t react with any ant i s e ra , inc luding

a nt i s e r um a ga i n s t t he s t r a i n unde r t e s t . T h i s i s a p r ob lem which
pers i s t ed even when other t yp i ng methods were deve l oped . As a result
many i s o l a t e s a re untypable by the va r i o u s typing me t hods empl oyed
a nd t h i s r ema i n s t r ue up to t h e p r e s e nt day . T he p r o b l em o f
untypable s t ra i n s i s ret urned t o late r .
 11
; 0

F o l lowing P i ro s ky ' s sugge s t i o n that P . mul t oc i da could be c l a s s i f ied

serologically on the ba s i s of it ' s capsular p o l y s a c c h a r i de

Ro senbu s c h and Me rchant [ 1 9 3 9 ] ( s ee Carte r , 1 9 6 7 ; Robe rt s , 1 9 4 7 ) used

t ube a g g l u t i n a t i o n rea c t i o n s augmented with b i o c hemi c a l t e s t s t o

d i v i de P. mul t o c i da int o t h re e g r oups . The b i ochemi c a l t e s t s were
based o n differences in t h e f e rment a t i o n of xylo s e , a rabinose and
du lc i t o l , howeve r t h i s approach has not appa rent ly been cont inued by
other worke r s and i t s usefulne s s is unce rtain . Also t he s e ro l ogi c a l
t e s t s w e re n o t de f i nit ive b e c a u s e they de tected many c ro s s react ions
be tween P . m u l t o c i da i s o l a t e s . The s e c r o s s reactions were p robably

due at least in p a r t to t h e u s e of w h o l e o r g a n i sms rather than

ext r a c t s . Neve r t h e l e s s L i t t l e and Lyon [ 1 9 4 3 ] ( s e e Ca rte r , 1967)

ide n t i f i e d t h r e e s e r o l o g i c a l g r o ups o f P . m u l t o c i da u s i ng s l ide

aggl u t i n a t ion t e s t s . They f ound that mo re i s o l ates were typable by

t h i s m e t h o d t h a n by u s ing t h e t ub e agglut inat i on procedure of

Ro s e n b u s c h a n d Me r c h a n t ( s e e C a r te r , 19 67 ) . H o w e v e r t h e s l ide
agg l u t i n a t i o n me t h od gave even mo re c r o s s react ions than the tube
agglut inat ion me t hod of Ro senbu sch and Me rchant ( s ee C a rt e r , 1 9 67 ) .

In h i nd s ight the c r o s s re act i o n s were pr obably due t o the ant i s e rum

c a u s i n g agglut i n a t ion o f o r g a n isms on t he b a s is o f t he i r caps u l a r

p o l y s a c c h a r i de a n d / o r t h e i r s oma t i c ant igens . T h e s e two ant igen

systems a re known t o exi st a nd va ry more or less i ndependant ly . It

is t he re f o re not s u rp r i s ing that compl i c a t ed c ro s s r e a c t ions occur

when r e a c t ing c omp lex a n t i g e n p r e p a r a t i o ns with e qu a l l y c omp lex

ant i s e rum . Somat ic ant igens a re di scus sed late r .

A d i f f e rent approach to t he serological cl a s s i f icat ion of

P. mu l t o c i da was t a ken by Robe rts ( 1 9 4 7 ) . He pa s s ive ly immuni zed mice

with a n t i s e r a r a i sed t o P . mu l t oc i da s t r a ins f rom di f fe rent anima l s

and f r o m wide g e og raph i c a l s ou r c e s . When cha l lenged t he immunized
a n im a l s were f o und t o be p r o t e c t e d a g a i n s t some s t r a i n s but not
othe r s . These ' mouse protect i o n ' t e s t s s ugge s t ed t h a t s ome isolates
we r e c l o s e l y r e l ated s e ro lo gica l l y wh i l s t o t he r s were r e a d i ly
di s t i n g u i s h a b l e b e c a u s e o f a lack o f c ro s s prote c t ion . F rom h i s
 12

r e s u l t s Robe rt s spe c u l a ted t h a t P . m u l t oc i da was comp o s e d o f f o u r

diffe rent s e r o l ogical groups . The s e h e named with t he Roman nume r a l s

I, II, I I I a n d IV . H e observed t h at typable s t rains o f P . m ul t oc i da

i s o l ated f rom cases o f haemo r rhagic sept icaemia in c a t t l e f e l l into
g r oup I ( Ro b e r t s , 1947 ) T he s e b o v i ne g r o up I s t r a i n s p r ob a b l y
equate w i t h O c h i s typ e B grouping ( s ee Nami oka and Mu r at a , 1 9 6 12 ) .
I n t e re s t i n g l y Robe r t s a l s o i s o l a t ed many ' unt ypable ' st rains . That
is, ant i s e rum t o any of the four g r oups f a i led to protect pas s ive ly
immuni zed mice against s ome f ie ld i s olates .

1 . 2 . 2 D i s cove ry of Capsular Type s

I . A S e r o l o g i c a l Approach

C a rt e r ( 1 9 5 2 ) a t t emp t e d t o g r oup s t r a i n s of P . mul t oc i da us ing two

me t h o d s . These were t he t ube a g g l u t i n a t i o n t yp i n g m e t h o d of

Ochi [ 1 9 3 3 ] ( s ee C a rt e r , 1967) a nd L i t t le & Lyo n ' s [ 1 9 4 3 ] ( see
C a rt e r , 1967) s l ide agglut i n a t i o n t e s t . H e w a s un s u c ce s s f u l i n the

sense that he c ou l d not r e p r o du c e t h e e a r l i e r pu b l i s h e d w o r k .

C a rt e r ( 1 9 5 2 ) t h en a t t empted t o g r o up s t r a i n s u s i ng a n AG I D t e s t
employing a s a l i ne e xt r a c t a s a n t i gen . T h i s app r o a c h i s s ome w h a t

s imi l a r t o t h a t o f P i r o s k y ( s ee above ) , however P i r o s ky used what

was then c a l led " Boivin'' ant igen which in pract ice probably cont a ins

b o t h cap s u l a r po l y s a c c h a ride and h e a t s t a b l e soma t i c ant igens . In

c ont r a s t Carter ' s s a l i ne ext r a c t s p r obab l y c o n s i s t e d mo s t l y o f

capsu l a r polys acchar ide . Using t h i s method Ca rter was succe s s ful in

demo n s t r a t i n g t h ree s e r o l og i c a l g r oups (Carter, 1 9 52 ) . H oweve r i t

was unsat i s f act ory f o r s c reen ing l a rge numbers of i s o l a t e s s ince it
i nvo lved b o t h t h e c o n s umpt i o n o f l a rge amount s of r e a g ent s and

s u f fered f rom t he problem of c ro s s react ions when f ield i s o lates , a s

opposed t o ' p rototype s t rain s ' were examined .

The f a i l u re o f t radit i onal t e s t s t o c l e a r l y de f i n e se ro logical

d i f feren c e s in isolates led Ca rte r ( 1 9 5 5 ) t o u s e w h a t w a s then a
relat ive l y new typing procedure . T h i s wa s the I ndi rect
 13

H a emaggl ut ina t i o n A s s ay ( I HA ) . Th i s me thod invo lved seve r a l steps .

B a c t e r i a l c a p s u l e w a s f i r s t removed f rom c e l l s by s a l ine and heat
extract ion . This ext r a c t wa s then mixed with h uma n t ype 0

e r yt h r o c yt e s . The ext r acted c ap s u l a r mat e r ia l st icks to the

e rythrocytes a n d rema ins even a f t e r t h e c e l l s h ave been wa s he d . The

' s e n s i t i zed' ( c apsular polysacchar ide c oated) e rythrocytes w e r e t hen
mixed with s e r i a l dilut i ons of P a s t e u rel l a a n t i s e rum and i n c ubated
f o r severa l h o u r s . By t h i s t ime the c oated e rythrocyt e s ma y or may

not be a gglu t i n a t ed . Agg l ut inat ion indic a t e s t hat the P . m u l t oc i da

s t rain being t e s t ed is o f the same se rolog i c a l group a s the s t r a i n
u s ed t o produ c e t he a n t i s e rum a nd vi ce vers a . T h i s me t h o d h a d a

f u rthe r bene f i t in that i t c o u l d b e u s e d t o a s s a y t h e t i t re o f

ant ibody to t he cap sula r polys accha r ide .

The I HA assay is supe rior to p re vious me t h ods , e s pe c i a l ly

agglutination t e s t s , for seve r a l reasons .

i. I t requires only sma l l qu ant i t ie s o f a nt igen .

ii . Fewe r s t ra i ns a re found to be untypab le .

i i i . The r e s u l t s showed gre ate r rep roducibilit y .

iv . Fewe r iso lates showed cro s s react ions .

Mo s t of the s e a dv a n t a ge s can be a t t r ibuted to the g reater

s e n s it ivity of the I HA assay . H oweve r the me t h o d has the

d i s advantage o f be ing ve ry laborious .

C a rt e r ( 1 9 5 5 ) f o und t h a t h uman Type 0 e ryth rocyt e s w e r e t h e mo s t

s u it able f o r h i s IHA a s s ay because t hey did not a gglut inate in the

p re sence of n o rma l rabbit or bovine s e r um . S h e e p a nd c h i c ke n

e rythrocytes were unsat i s factory bec a u s e they cont a i n ant ige n s which

c ro s s -react w i t h ant igens o f P . mul t oc i da ( C a rt e r ( 1 9 5 5 ) .

U s ing the IHA a s say Carter ( 1 9 5 5 ) ident i fied four serolog i c a l g roups .
These he de s i g n a t e d T y p e s A, B, C, and D . They equ a t e d w i t h
Robe rt s ' s mo u s e p rot e c t i on groups II, I, III and I V r e s pe c t i ve l y
( Carte r , 1955) .
 14

A f u r t he r ca p s u l a r t ype w a s ident i f ie d in 1 9 6 1 ( Ca rte r , 1 9 6 1) . It

wa s i s o la t ed f rom seve r a l c a s es o f bovine haemorrhagic s ept i c aemia
in cent r a l A f r i ca . This new g r o u p w a s de s i g n a t e d Type E . It is
s e r o l og i c a l l y r e l a t e d t o T ype B b u t s u f f i c i e n t l y di s s im i l a r t o
wa r rant the sepa r ate serological cl a s s i f i cat ion ( C a rt e r , 1 9 6 1) .

Re cent ly a new capsular t ype , de s ignated Type F , w a s i s o l a t ed f rom

t u r keys ( Riml e r and Rh o a de s , 1 9 8 7) . It s howed some s imi l a r i t y t o
Type D by the acrif lavin tes t . To date l it t le i n f o rma t i o n is
ava i l able on t h i s Type .

The a bove i n f o r ma t i o n s u gge s t s that t he re a re s i x t ype s of

P. mul t oc i da . Ho wever C a r t e r ( 1 9 6 3) s ugge s t ed t hat Type C should be

dropped f rom the c l a s s i f i c a t ion a s " it does not rep resent e i the r an

imp o rt ant type o r group" . He con s i s t a n t l y found that ant ibody t it re s

t o Type C were l o w a n d cons idered t ha t it was " not a cap s u l a r type

in the s a me s ense that the others were" ( Carter, 1 9 6 7) .
Nami o k a ( 1 9 7 8) ree xamined t he e x i s t e n c e o f Type C P . mul t oc i da . He
conc luded that t h e Type C reference s t r a in had become ant ige n i c a l ly

rough . This imp l i e s that a Type C grouping may exi s t but , if so , no

s t a nda rd f u l ly encapsulated prototype i s ava i l able .

S e v e r a l imp o r t a n t modi f i c a t i on s o f t he IHA a s s a y h a ve b e e n made

s ince its int r o d u c t i o n by C a r t e r ( 1 9 5 5) . C a r t e r and Rappa y ( 1 9 6 1)

mod i f ied the a s s a y by us ing f o rma l inised human t ype 0 eryt h rocyt e s .

This p r o c e du r e ext ended t he she l f life of the s en s i t i zed

e rythrocytes and a l so re s u l t ed i n ant ibody react ions o f highe r t i t re

wh i c h f u r t h e r i n c rea s e d t he s e n s i t i v i t y o f t he t e s t ( C a r t e r and

Rappay , 1 9 6 1) . Another modi f icat ion w a s the pret rea tment o f muc o id,

hya l u ronic-ac id-c o nta in ing st rains with hyaluronidase . This resu lted

i n an increase i n t he numbers of s t r a i n s which we re typable (Carter,

1 9 7 2 ) . T h i s is d i s cus sed lat e r .

 15

I n f u rther deve loping the IHA a s s ay S a wada , Rimler and Rhoade s ( 1 9 8 2 )

s ub s t i t u t e d g l u t a r a lde h y de f i xed s h e e p e r y t h r o c yt e s f o r h um a n

e ryth rocyt e s . T h i s in i t s e l f i s an adva nt age be cause s heep b l o o d i s

read i ly ava i l able i n Ve t e r i n a ry labo r a t o r ie s . Furthe rmo re f ixat ion
with g lut a r a ldehyde ove rcome s the problem o f hete roph i l ic ant ibodies

and f u rt h e r e x t e nds the s he l f life of t he ce l l s . I HA a s s a ys

p e r f o rmed with Human type 0 e rythrocytes o r f ixed sheep e rythrocytes
e s s e n t i a l l y g i ve t h e s ame result s ( S a wada , Riml e r and Rh o a de s ,

1982) .

I I . A Non Serolog i c a l App roach

P re ceeding the s e r o l og i c a l ident i f ic a t i on o f caps u l a r type s by I HA

assay, va r i o u s a t t empt s were made at g r oup ing st ra ins of
P. m u l t o c i d a by o t h e r c r i t e r i a ( C a r te r , 1967) . T h i s app r o a c h w a s

b a s e d on c o l o n i a l morph o l ogy a nd t he host spe c i e s f r om w h i c h

dif fe rent colon i a l types were i s o lated . T h e colonia l t ypes f e l l into

th ree groups : muc o id, smooth and rough ( C a rte r , 1 9 67 ) . B r aun ( 1 9 5 3 ]

( s ee Ca rt e r , 1 9571 ) f o und t h a t the a c r i f l a v i n t e s t ( see below ) ,

f i r s t int roduced b y Alles s a ndrini and S ab a t u c c i ( 1 9 3 3 ] ( s ee C a rt e r ,
1 957 ) w a s a mo re r e l iable indi c a t o r of v a r iant type than w a s s imp le
obs e rvat ion of c o l o n i a l mo rpho logy (Carter , 1 9 5 7 1 ) . Mu coid colonies
c ont a i n l a rge amo u n t s o f c a p s u le wh i c h r e a c t s w i t h a c r i f l a v i n t o

g i ve a s l imy p r e c i p i t a te . Rough c o l o n i e s po s s e s s l it t le , if any,

capsule . T h e y f l o c c u l a t e i n a c r i f l a v i n whe r e a s t h e i nt e rmed i a t e

' smo o t h ' variety r e a c t s t o g i ve a homo g e n e o u s s u s pe n s i o n . Rough

s t ra in s tend not t o be pat hogen ic ( C a rt e r , 1957 1 ; Smith and W i l s o n ,

1983) . T h i s me t h o d o f grouping s t r a in s w a s o f cons ide rable v a l u e

init ially, howeve r it was l a rge l y s u p e r s e d e d by t h e s up e r i o r

di s c r iminat i ng powe r o f t he IHA a s s ay . Neve rthe l e s s rough va riant s

a re untypable by t he I HA method due to t he i r l a c k o f capsule s o t he
a c r i f l a vi n t e s t i s s t i l l a u s e fu l t o o l f o r s c reening i s o l a t e s t o
e s t a b l i s h i f t hey a re rough ( C a rte r , 1 957 1 ) .
 16

1 . 2 . 3 D i s c overy of S oma t ic Ant igenic Groups

Many of t he earl ier s ero logi c a l stud ies o n P . mul t oc i da resulted i n

t he p u b l i c a t i o n of res u l t s which were not c o n f i rmed by o t he r

w o r ke r s . T h i s wa s i n p a r t due t o a l a c k o f apprec i a t ion o f t he
ant igen i c complex i t y of t he o rg a n i s m ( Na m i o ka and Mu rat a , 19 6 1 2 ) .
Ca rter ( 1952 ) repo r ted that P . mul t oc i da p o s ses sed a s oma t i c antigen

common t o all member s o f the spec ies . This v iew changed in 195 6 when
Yaw a n d c owo rkers ( see Nami o k a and B r une r , 1963) s t ated t h a t t he
wide d i f ferences o b s e rved in h o s t range c o u ld not be expla ined by

va riat i on in caps u l a r type a l one . Consequent ly t hey s uggested t h a t

o rgani sms w i t h dif ferent somat i c ant igens might exi s t t o account f o r
t h i s p h en omen o n . C a r t e r s upp o r ted t h i s c o n cept ( Ca rter , 19 6 1 ;
Carte r , 19 6 3 ) . He h a d typed many s t r a ins by their c aps u l a r ant igen

and suggested that o t her ( s u r f a ce ) ant igen s p robably exi s ted .

I t might be a s s umed t h a t P . m u l t oc i da s oma t i c a nt i gen s c o u l d be

i n ve s t i g a t ed u s i n g o ne o f t he t e c h n i q u e s p rev i o u s l y u sed f o r

st udying t he caps u l a r ant igen . Ho wever di f f i c u l t ies were exper ienced

when c o nvent i o n a l a gg l ut in a t i o n pro cedu res were used to s t udy t he
soma t i c ant igen . F o r example, s ome i s o lates were not agglut inated by
homo l o g o u s ant i se r a whi le o t he r s showed mu l t iple c r o s s rea c t i o n s
( Ca rte r , 196 7 ) . T h i s was probably due t o t h e test interact ing w i t h

the capsular polys a c c h a r ide

A breakthrough occur red with t he work of Namioka and Murat a ( 19 6 1 2 ) .

They found that agg lut ination tes ts yielded more c o n s i stent res u l t s
i f cultures , whet her u sed f o r ant igen or ant i serum prepa rat ion , were

f i rst t re a t ed with Mo l a r HCl wh i c h r em o v e d the c aps u l a r

p o l y s a c c h a r i de . C ro s s rea c t i o n s i f t he y s t i l l o c c u r red c o u l d be
minimi sed by absorbing the rabb i t sera w i t h cross rea c t i ng s t r a in s
t h u s res u l t ing i n w h a t t hey c a l led ' spec i f i c f a ct o r ' s e r a spec i f i c
f o r soma t i c antigen s . This approach of inc rea s i ng t he speci f i c i t y o f
s e r o l o g i c a l rea c t i o n s b y ab s o rb t i o n i s s im i l a r t o t h a t u sed f o r
ent e r i c b a c te r i a such as t he s a lmonel l ae . For this rea s o n t h e
 17

s oma t i c a n t i gen o f P. m u l t o ci da i s o f ten r e f e r red t o a s t he 'O'

a nt igen ( C a rter , 1967) .

Namioka ( 1 9 6 1 2 , 19613) , u sed c r o s s abs orbtion methods t o divide the

s oma t i c a n t i gen s o f P . mu l t o c i da i n t o t w o g r ou p s : ' c ommo n ' and
' spec i f i c ' ( see F igure 1 . 2 ) . He fu rt her demo n s t ra ted the exi s tence
o f s ix spec i f ic soma t i c a nt igens t h ree of wh ich were a s s o c i ated with
s t rains p o s se s s ing Carte r ' s Type A capsule and t hree with Type D . By
c omb i n i n g c a p s u l a r t yp e nomenc l a t u re w i t h t he s oma t i c a n t igens
A r a b i c numb e r t he s t r a i n s were a s s igned t o a ' Serotype ' ( Namioka ,

1 9 613) .

L a ter Nami oka ( 1 9 6 3 ) i dent i f ied a ddi t i o n a l s oma t i c a n t i gen s a n d

demo n s t r a t ed t he re l a t i ve i n dependence o f s oma t i c a n d c a p s u l a r

a n t i gens . i e He had ea r l ier shown t hat a s t ra in o f P . mu l t o ci da with

a s pec i f i c c a p s u l a r t ype c o u l d p o s s e s s one of s e ve r a l s oma t i c

a n t i gens . H e now showed t hat a s t ra in po s ses s i ng a spec i f i c soma t i c

g r oup c o u l d p o s s e s s one o f seve r a l c a p s u l a r a nt igens . F o r example

( T able 1 . 1 ) , a s t ra i n o f P . mul t o c i da po s ses s ing a Group 3 s oma t i c

a n t igen c o u l d po s se s s e i ther a Type A o r a T ype D c a ps u le . Other
s oma t i c a n t igens we re f o und o n l y i n a s s o c i a t i on with one spec i f i c

t ype o f c a p s u l a r po l y s a c c h a r i de . F o r ex a mp l e , Group 9 s omat i c

a n t i gen h a s only been f o und a s s o c i a ted w i t h a Type A c a p s ule . To
date t w e l ve s o ma t i c g r o up s h a ve been i de n t i f i e d a nd t he i r

r e l a t i o n s h ip with the capsu l a r type has been determined ( Table 1 . 1 ) .

 18

Table 1 . 1 P . mu l t ocida Soma t ic Groups and the i r Rel a t i o n t o

*
Caps u l a r Type .

Capsu l ar Type Sornat i c Group Serotype

A 1 lA
3 3A
5 5A
7 6A
8 BA
9 9A
8 6 68
11 1 18
D 1 10
2 20
3 3D
4 40
10 1 00
E 6 6E
1 1F
3 3F
7 7F
12 1 2F
From Cart erC 1 96 7)
Based on R i mier and R h o a d es ( 1 98 7 )

The exi stence o f somat i c s ubgroups w a s shown b y Namioka ( 1 9 6 1 3 ) and

Namioka and Mu rata ( 1 9 6 3 ) .

F igure 1 . 2 An t igen ic Cha rac ter i s t i c s o f P . mu l t oc i da .a

Cap sule <Kl

1
2
WHOLE CELL 3

:m -----------
4
5
Somat i ( OJ S p ec i f i c 6'
Ant i g en 7
1: 8'

Cornmon 9
Ant i g en 10

I 1 1
12

B a s e d on Nam i o k a ( 1 97 8 >
C o m p osed o f sever a l subg rou ps
 19

1 . 2 . 4 Agreement between Typing Methods

S h i g i d i a n d Mu s t a f a ( 1 9 8 0 ) f o und t h a t there was g o o d a g r e ement

between the tube agglut inat i o n a s s ay o f Namioka and t he AG ID method
in i de n t i fying t he soma t i c ant igens . T h i s implies t h a t both methods
i n v o l v e t h e s a me ant i g e n s . H o w e v e r s e r o t ype s de t e rmi ned by one
typ ing method do not a lways c o r relate w it h result s u s ing a dif fe rent
me t h o d . B r o gden and P a c k e r ( 1 9 7 9 ) c o mp a r e d s i x d i f fe r e n t t yp i n g
me thods . These w e re :

i. The s l i de a g g l u t i n a t i o n t e s t of Litt le and Lyon [ s ee

Section 1 . 2 . 1 ] .

ii . The I ndirect Haemagglut inat ion A s s a y of Carter [ s ee

Sect ion 1 . 2 . 2 ] .

i i i . The AG ID test of Heddle ston [ see Section 1 . 2 . 3 ] .

iv . The s l ide and t ub e agglut i n a t i o n a s s a y s o f N amioka [ s ee
Sect ion 1 . 2 . 1 ] .

v. The mo use protect ion test of Robe rts [ see Sect ion 1 . 2 . 1 ] .
vi . The a c ri f la vin/ hya l u r onida se dec apsulation test o f Carter
[ s ee Sect ion 1 . 2 . 2 ] .

Brogde n and P a c k e r ( 1 9 7 9 ) c o n c l uded that a c o r re la t i on between typing

me thods could not be shown . Often i s o l a t e s be longing t o one s e rotype

would f a l l into several s e r o t ypes when a di f fe rent t yping met hod was

used . This phenomenon i s probably due t o the complex nature o f the

P. m u l t oc i da capsule . I s o l a t e s with one o r two s e r o t yping ant igens

in c ommon may d i f fe r in othe r ant igens which anothe r typing method

recogn i s e s ( B rogden and P a c ke r , 1979) .

D i f f e rences between resu l t s o f IHA and AG I D t yping c an po s s ibly be

exp l a i ned by t he in vo lveme n t of d i f fe rent a n t ige ns . Origin a l l y it
was t h o ught that the IHA ant i gen was l ipopolysacch a r ide ( C a r t e r and
Rappa y , 1 9 6 2 ) . Howeve r it has s ince been shown that
l ipopo lysacchar ide is the ma j o r ant igen re spons ible for t yping ba sed
on t he AG ID t e s t whe reas the capsula r polysaccharide det e rmines the
re s u l t s obt a ined by IHA (Manning, 1984) .
 20

1 . 2 . 5 B a c teriophage Typing

The e x i s t ence o f P . mu l t ocida - s pe c i f i c b a c t e r iophage s h a s been known

for many yea r s . N o succe s s h a s been ach ieved in u s ing bacter iophage
as a t yping tool ( Ca rte r , 1967)

1.3 Ant igenic St ructure o f P . m u l t ocida

The o c c u r r e n c e o f ant i ge n i c p o l ys a c c h a r i de s in P . m u l t o c i da w a s
first reported over fifty ye a r s ago by Hoffe n r e i c h [ 1 92 8 ] and
D ingle [ 1 9 3 4 ] ( s ee Cart e r a n d B a i n , 1960) . S ince t h e n t he ant igenic
s t ructure of the o rganism has been shown t o be complex .

Carte r ( 1 9 52 ) used t ube agglut ina t i o n and caps u l a r s we l l ing

technique s to show that P . m u l t oci da p o s s e s sed a caps u l a r ant igen .

He p r e s umed t h i s t o be c ompo s e d o f p o l y s a c c h a r ide a n d p r oduced
evide n c e which s ugge s t e d t hat strains also h ad a c ommon
(polys a c c h a r ide conta ining) s omat i c ant igen . Carter a n d Annau ( 1 9 5 3 )
late r a na lysed c a p s u l a r ext r a c t s f rom s eve ral P . mul t ocida s t r a ins

and c o n f i rmed t h a t the c a p s u l a r a n t i g e n w a s i n d e e d c ompo s e d of

poly s a c c h a r ide . I nt e rest ingly a non- immunogenic muc o i d

p o l y s a c c h a r i de w a s also r e c o ve red and u s ing a h y a l u r o n i da s e

depolyme r i s a t io n t e s t i t wa s ident i f i ed a s hya luron i c a c id ( Ca r t e r

a n d Ann au , 1 9 5 3 ) .

Many f re s h l y i s o l a ted s t r a in s o f P . mu l t o c i da do n o t agglut i n a t e

when mixed w i t h h o mo l o g o u s a nt i s e rum ( Ca rte r , 1972) . This is
surp r i s ing be cause t he po s s e s s i on o f a capsule can b e demon s t r ated

in ma ny o f t he s e s t ra ins by us ing s imple c apsule - s t a i n ing me t hods .

F u r t h e rmo re t he b i nding o f T ype - spe c i f i c ant ibody t o t h i s capsule
has been shown by capsular swe l l ing techn iques ( The c a p s u le enla rges
when a n t ibody b inds t o it ) ( C a rt e r , 1 9 52 ) . With the knowl edge that
many e n c ap s u l a t e d s t r a i n s o f P . m u l t o c i da do i n d e e d b i nd T ype
s pe c i f i c a n t i b od y it is now t h ought t h a t t he n o n - i mmu n o g e n i c
 21

hyaluronic a c id on the surface f o rms a l a t t ice framework t h roughout

t he caps u le , rathe r t h a n a d i s c r e t e laye r . T h i s latt ice i nte r f e re s

with agglut inat ion react ions b y s t e r i c a l l y h inde ring t h e b inding o f

o n e ant ibody mo l e c u l e t o mo re t h a n one b a c t e r i um . B i nding o f the
a n t i b o dy t o mo re t h a n one s ite on a s i ngle host is u n imp a i r e d
( Ca rte r , 1 9 6 7 ; Carter a n d Bain , 1 9 6 0) .

The problems a s s oc i a t ed with the inhibition o f agg lut inat ion due t o
h y a l u r o n i c a c i d e x t e nd s t o t h e i n d i r e c t h a emagg lut i n a t i o n a s s a y
( I HA) . This led C a r t e r ( 1 9 7 2) t o modi f y t h e IHA a s say . This was
discus sed i n Section 1 . 2 . 2 .

The product ion o f hya luronic acid i s mo s t p r o l i f ic i n C a r t e r Type A

s t ra ins . T h i s endows the c o l o n i e s w i t h a mu c o i d appe a r a n c e ( see

F igure 1 . 3) a n d t h u s they can b e dist ingu i s hed f rom Type D st r a ins

( s ee F i g u r e 1 . 4) w h i c h p r o du c e l it t le hyaluronic a c i d and f o rm
smooth c o l o n i e s . I t i s not present in Type B s t r a ins and at tempted
de c a p s u l a t i o n o f t hese s t ra in s by h y a l u r o n i d a s e l e ave s t hem

una f fected ( Ca rt e r , 1 9 6 7 ; Carter a n d Bain , 1 9 6 0) .

F rom the w o r k done by Carter and Annau ( s ee above) it ma y have been

dedu c e d t h a t c a p s u l e e xt r a c t s of P. m u l t o c i da c o n t a i n o n l y one

a n t i ge n i c s pe c i e s ie p o l y s a c c h a r i de . H o w e v e r B a i n ( l 9 5 5) u s ed a

s a l ine /pot a s s ium t h i ocyanate ext r a c t ion method followed by a polar

o rgan i c - s o l vent s epa r a t i on method to i s o l a t e both a prot e i n f r act ion

and a p o l y s a c c h a r ide f r a c t i o n f ro m t he c a p s u l e o f Robe r t s Type I

s t ra ins . T h i s type o f of P . mul t oc i da is equiva lent to C a r t e r Type B

s t r a ins ( s e e Section 1 . 2 . 2) . He t hen demo n s t ra ted that t he protein

comp l e x w a s a p r o t e c t i ve a n t i g e n bec a u s e r a bb i t a n t i s e r um when

absorbed w i t h the protein complex had a dimin i shed protect ive powe r
a s shown by the mou s e protection t e s t . T h i s c learly showed that the
p o l y s a c c h a r i de wa s not the o n l y p r o t e c t i ve ant igen in c a p s u l e
ext ract s . B a i n ( 1 9 5 5) sugges ted t ha t this protein component could be
used in f ut u re P . mu l t ocida vaccine s .
22

F igure 1 . 3 Type A Colonies of P . mu l t o cida .

The colonies a re app roxima tely 2 . 0mm in diamet e r a ft e r

2 4 h r s incuba t i on a t 3 7 C o n b l ood aga r .

N o t e that the colonies a re mucoid . This i s in cont rast

t o Type D c o l onies ( see F igure 1 . 4 ) wh ich a re smooth .

1 0 x Magn i f i c a t ion

F i gure 1 . 4 Type D Colonies of P . mul t o cida .

The colonies a re app roximately 2 . 0mm in diamet e r a f ter

2 4 h r s incuba t ion at 3 7 C o n blood agar .

N o t e the smooth appea rance o f colon ies a s i l l u s t rated

by t he dema r c a t ion o f a d j acent c o l o ni e s . This is in
c o n t rast t o Type A c o l o n i e s ( s ee F igure 1 . 3 ) which a re

muc o id .

1 0 x Magn i f icat ion

1 3
 23

Bain [1955] ( s ee Cart e r , 1 9 6 7 ; C a r t e r and B a i n , 1960) cont inued h i s

a n t ige n i c a na ly s i s of P. m u l t o c i da and u s i ng AGID p r o c e du re s

revea led twe lve antigens in s a line extract s . P r i nce and Smi t h ( 1 9 6 6 1 )

u s e d AG I D a nd immunoe l e c t ropho r e t i c [ IEP ] t e c hniqu e s t o ident i f y

e i ghteen s o l uble ant i gens o f P . mul t ocida . S i xteen o f t h e s e ant igens
we r e shown to be common t o Type A and B s t ra in s . Fu rthe rmo r e seve ra l
o f these s ixteen ant igens we re found t o b e s h a red by s ome othe r Gram
negat ive o rg a n i sms . The rema i n i ng t wo a n t i gen s , den o t e d a and p ,
we r e found t o be s e r o t ype spe c i f i c . That i s , found o n l y i n Type A
and Type B s t rains o f P . mul t oc i da respect ively ( P r ince a nd Smi t h ,

19661) . It s e ems rea s onable t o s uppose t h a t t hese t w o ant igens (a

and Pl r e p r e sent the T ype - sp e c i f i c ant igens . C l e a r l y t he t ype

s pe c i f i c a n t igen e x i s t s aga i n s t a c omp lex background of sha red o r
p a r t i a l ly s h a red ant i gens .

S i n c e t he w o rk of P r ince a nd S m i t h ( 1 9 6 6 1 ) t he n umbe r of known

s u r f a c e a n t igens h a s been f u r t h e r i n c re a s e d . Bha s i n and Lapo i n t e
Shaw ( 1 9 8 0 ) u sed cros sed immunoe lec t ropho re s i s t o a n a l y s e a st r a i n o f

P. mul t oc i d a be l onging t o C a r t e r ' s Se r o t ype S : A . T h e y de s c r ibed

n i neteen c e l l a s s o c i a ted ant igen s .

P e nn and N a gy ( 1 9 7 4 ) used AG I D a nd immunoe l e c t roph o re t i c techniques

t o s t udy e x t r a c t e d c e l l u l a r ma t e r i a l f r om C a r t e r T yp e B a n d E

s t rains of P . mu l t ocida . They repo r t ed s e ve r a l l ines of

p r e c ip i t a t i o n b y A G I D . Howeve r , t he y o b s e rved t h a t two l ines

r e p re s e n t e d t he mo s t c o mmo n surface a n t i ge n s . U s ing sodium

deoxycho l a t e depolyme r i sation a n a l y s i s t hey demonst r a t ed t ha t one o f

t he s e l in e s was a non-prot e i n a c e o u s Type - s pe c i fic caps u l a r ant igen

( Penn and Nagy , 1 9 7 4 ) . By the s ame method t hey showed t h a t the other

l ine cont a i ned prote in. Interest ingly Type B prot e in a c e o u s ant igens
were p r e c ipit ated by a nt i s e ra prepa red by u s ing who l e Type E c e l l s
as immu n i z i n g a n t i g e n a n d vi c e v e rs a . T h i s i nd i c a t e d t h a t the
p ro t e i n a c e ou s ant i g e n s ext r a c t ed f iom t he capsu le o f Type B and E
s t ra in s of P . mu l t o c i da were s im i l a r ( P e nn a n d N a g y , 1974) .

F u r t h e rm o r e T ype B a n d E p o s s e s s o t he r c o mmon a n t i g e n s be c a u s e
 24

Namioka and Mu rata ( 1 9 6 4) found t h a t t hey both bel onged t o the same
s oma t i c g r o up i ng [ vi z g r o up 6] , w i t h t he e xcept i o n o f a s i ngle
isolate o f a Se r o t y pe 1 1 : B strain ( N ami o k a a nd Mu r a t a [ 1 9 6 4 ] see
Ca r t e r , 1 9 6 7) .

It c o u l d t he re f o re be conc luded t hat T ype B a n d E s t rains of

P. m u l t o c i da repre s e nt s imi l a r o rg a n i s ms d i f f e r i n g o n l y i n t he i r
cap s u le . T h i s s imi la r ity i s r e i n f o rced by t he knowledge t ha t Type B
and E s t r a i n s c a u s e s imi l a r d i s e a s e s . T h i s w i l l be r e t u rned t o
lat e r .

T h e wo r k o f P e n n a nd Nagy ( 1 9 7 4) ( s ee a b o ve) s h o we d t ha t s a l i ne
ext r a c t i o n o f P . mu l t o c i da yie lded a c omp l e x mixt u re o f ant igens .
Clea rly i f one wished to st udy c a p s u l a r polys accha r ide a l one a much

mo r e s pe c i f i c e x t r a c t i on a n d p u r i f i c a t i o n t e c h n i qu e h a d t o be

deve loped . A suitable method wa s e s tabli shed two ye a r s l a t e r by Penn

and Nagy ( 1 9 7 6) . S t a r t ing with s a l ine ext ra c t s of P . mu l t o c i da Types

B a nd E t hey used a po l a r - o r g a n i c - s o l v e n t b a s e d f ractional

p r e c ipit a t i o n me t ho d t o s ep a r a t e c a p s u l a r poly s a c c h a r i de ant igen

f rom t he e ndotoxin . They then u s e d bio logica l tests t o show that t he
cap s u l a r p o l ys a c c h a r ide ( no n -pyrogenic) h a d been c ompl e t e l y f reed

f r om t he e ndot o x i n ( pyroge n i c) f ract ion . The pu r i f i e d c a p s u l a r

ant igen w a s shown t o be a h igh mo l e c u l a r we ight ac idic
po l y s a c c h a r ide wh i c h was po o r l y. irnrnunogen i c in rabbit s . Howeve r a
do s e dependent ant ibody response was ach ieved in c a t t l e a s s hown by
I
t h e mou s e p r o t e c t i o n t e s t ( P enn and Nagy, 1 9 7 6) . This clearly

demo n s t r a t e s t h a t t h e c a p s u l a r p o l y s a c c h a r i de is an i mp o r t a n t
p r o t e c t i v e a n t ige n . Mu l t i c ompo nent who l e - ce l l va c c i n e s a re o f t e n

toxic b e c a u s e o f t h e presence o f endotoxin s o i t i s a dv a nt ageous t o

u s e a p u r i f i e d c a p s u l a r a n t i g e n s o t h e s e w o r k e r s e x p l o ited the
f i nd i n g o f a do s e depende n t response in c a t t l e . U s i n g p u r i f ied

c a p s u l e f r om both T ype B and T ype E P. m u l t o c i d a they p roduced a

c omme r c i a l h a e mo r r h a g i c s ep t i c a em i a v a c c ine ( Wa l k e r a n d F o s t e r ,
1 9 8 1) .
 25

T h e a b o ve di s c u s s i o n i l lust rates t h a t t he a n t i g e n i c ma k e up o f
P. m u l t oc i da i s complex and t h a t t he c a p s u l a r ant igen a nd s oma t i c
ant i ge n v a ry more o r l e s s independa ntly f r om s t r a i n t o st r a i n ( s ee
Sect ion 1 . 2 . 3 ) . Howeve r t he two ant igens a re not e a s i ly s eparable in
the l a b o r a t o ry .

It is n o w g e ne r a l l y a c c e p t e d t h a t t h e " c apsule " , t hat is, the

f ra c t ion removed f rom intact c e l l s b y s a l i ne extra c t i o n , i s made up
of f o u r b a s ic component s ( C a rt e r , 1967 ) :

i. Polysaccharide
ii . Hya luron i c Ac id
i i i . Lipopo lys accharides
iv . P roteins

The p o l y s a cchar ide i s respons i b l e for Type s pec i f i c immunity .

Hya l u r o n i c acid is non-antigenic and probab l y augment s t he o rgani sms

v i r u lence .

Lipopo l y s accharide i s a " cont aminant " of the caps u l a r extract . That

is , it represent s t he s oma t i c ant ige n , of wh ich t he r e a re twe lve

t ypes known ( s ee Sect ion 1 . 2 . 3 ) . Soma t i c ant igens a re n o rma lly f ound
a s s oc i a t ed with the intact ce l l , howeve r a sma l l prop o r t ion of these

a n t i ge n s a re f ound i n s a l i n e e x t r a c t e d capsule prep a r a t i o n s . The

s omat i c a nt igens represent protect ive ant igens although they a re of

l e s s e r import ance than the capsular polys a c cha ride [ s ee be low ] .

P ro t e i n comp l exe s ext r a c t e d f rom c ap s u l a r ma t e r i a l h ave not been

widely s t udied . Howeve r , those a s s o c i a ted with Type B a nd E st r a ins
o f P . m ul t oc i da a re s imi l a r , if not ident i c a l [ see above ] . Although
p r o t e i n c omponen t s de r ived f ro m t he capsule have been s ho wn to be
protect i ve ( Ba i n , 1955) a s ingle homogeneous protein which protects

has yet to be i s o lated . Howeve r pa rt i a l ly purif l ed p r o t e i n f ra c t ions

have been succe s s f u l l y used t o immunize mic e , cattle ( Carte r , 1967)
 26

a n d r a bb i t s (Bain, 1 955) . Re c e n t l y K a j i ka wa a nd M a t s umot o ( 1 9 8 4 )

demo n s t r a t e d that a protein component was a l s o prot e c t i ve i n b i rds .

T h e y p r e p a red a s a l ine e xt r a c t o f P . m u l t o c i da S e r o t ype 5 : A and

u s i n g ge l f i l t ra t i o n i s o l at e d a ( p o l y s a c c h a r i de f re e ) capsule
a s s o c i a t e d p r o t e i n c ompo n e n t w h i c h w a s p r o t e c t i ve for t u rkeys

( Ka j i ka w a a n d Ma t s umot o , 1984) .

*
1.4 Toxigenic S t ra ins o f P . mu l t ocida

The above discu s s i on implies t h a t P . mul t oc i da is a p a t hogen solely

be c a u s e of i t s i n va s ivene s s . H o wever w i t h i n the l a s t de c a de t he
importance of a s e cond virulence f a c t o r , t oxin product i o n , h a s been
e s t ab l i s hed .

Smit h [ 1 9 5 7 ] ( s ee Ca rter and B a i n , 1 9 6 0 ) demonst rated t h e presence o f

a heat l a b i l e t o x i n in t h e s upernatant o f s e ve r a l P . m u l t o c i da

s t r a i n s a f t e r cel l lys is . He f ound t hat s t e r i l e f i lt ra t e s o f wee k

old b r o t h cultures cont a i n e d 2 0 t o 1 6 0 mo u s e l e t h a l dos e s . This

t o x i n c o u l d be i n a c t i v a t e d b y f o rma l i n but ( s u r p r i s ing l y ) was
re s i s t a nt t o proteo lytic enzyme s ( s ee C a r t e r and B a i n , 1960) .

T h e s i g n i f i c a nc e o f Smi t h s ' [ 1 9 5 7 ] w o r k w a s not a pp r e c i a t e d unt i l

1 9 7 5 when I l ' ina and Za sukhin ( s ee D e Jong , Oe i and Tetenburg , 1980)

s ho w e d t h a t s t r a i n s o f P . m u l t o c i da wh i c h cont a ined a he a t labile

t ox i n , a s det e rmined by t h e mou s e let h a l i ty te s t o f Smi t h [ 1 9 5 7 ] ,

c o u l d b e i s o l a t e d f rom t he n a s a l c a v i t y o f s w i ne s u f f e r i ng f rom

p rogre s s ive a t rophic rhin i t i s [ AR ] ( a de s c ript ion o f t h i s di sease i s

g i ven i n Sect ion 1 . 5 . 6 ) . They demonst rated that t oxigenic f i lt rates

f rom t he s e s t ra i n s wou ld produce AR- l i ke l e s i ons i n t he nasal t ract

o f s pe c i f i c p a t hogen f ree [ SP F ] piglet s . This s t r o n g l y s ugge s t e d

t h a t t oxige n i c s t rains o f P . mul t o c i da we re t he c a u s e o f AR . It is
temp t i n g t o specul ate t ha t t h e toxige n i c s t r a i n s i s o l a t ed b y Smith
ove r a decade e a r l ier [ see above ] could a l so produc e AR- l ike les ions
in S P F p i g l et s . H oweve r t h i s mu s t rema i n specu l a t i o n b e c a u s e the
 27

o r i g i n s o f Smiths ' s s t ra ins a re not c l e a r a nd furt h e rmo re they a re

not now a v a i lable .

The u s e o f SPF piglets in surveys to demo n s t rate AR- le s ion-p roduc ing
st r a i n s i s expen s i ve and cumb e r s ome . T h i s p r o b l e m w a s pa rt i a l l y
ove r c ome when De Jong , Oei a n d Tetenb u r g ( 1 9 8 0 ) demo n s t r a t e d t h a t
t ox i n - c o nt a in ing ext ract s o f P . mul t o ci da , a s determined by t h e S P F
p i g l e t me t h o d , c a u s ed dermo n e c r ot i c l e s i o n s i n g u i n e a p i g s when
i n j e c t e d int raderma l ly . This d i s cove ry led t o the t e rm De rmonec rotic
Toxin ( DNT ) be ing used as a n ame for this t oxin .

The u s e of guinea pigs t o t e s t f o r P . m u l t o c i da t ox i n w a s mo re

c o n ve n i e n t t h a n intrana s a l inoculat ion of SPF piglet s . H o we v e r
in vi v o tests , inc luding the gu i n e a pig s k in test, a re both

e xp e n s i ve a n d c umb e r s ome w h e n u s e d t o s c r e e n l a r g e n u mb e r s of

i solate s . Thi s p roblem was s u rmounted whe n Ru t t e r a n d Luther ( 1 9 8 3 )

s howed t hat a cytopathic e f fe c t i s produced in embryo n i c bovine lung
( EB L ) c e l l cul t u r e s when they we re exposed to ste r i l e f i l t rates of

D N T - p r o duc i n g s t r a i n s of P . m u l t o c i da . P e nn ings and S t o rm ( 1 9 8 4 )

s howed that a s i mi l a r cytopathic ef fect c o u l d b e p r o du c e d i n
p re s e n s i t i zed [ see below ] Vero cells . The s e o b s e r v a t i o n s we re
imp o r t ant bec a u s e many i s o l a te s could now be conven i ent ly s c reened
f o r t o x i n p roduct ion .

M a n y P . m u l t o c i da i s o l a t e s h a ve b e e n e x amined a n d w o r k e r s ha ve

looke d f o r a c o r re lation bet ween capsule t ype and t oxin product ion .

Toxigenic strains of P . m u l t o c i da have been found to be

p redomi n a nt ly T ype D ( Na k a i , S a w a t a a nd Kume , 1 98 5 ; S awata e t a l ,

1984) . The r ema i nde r a re Type A ( P i j oan et a l , 1984) . An equ a l ly

imp o r t ant que s t i o n i s the d i s t r ibut i o n o f t oxin p roducing st rains

among host spe c i e s . This has appa rent l y not recieved much attent ion

and a t p resent a l l known t o xi genic s t ra ins have been i s o l ated f rom

swine .
 28

The N a t u re o f P . mul t oc i da De rmonecrotic Toxin

P. m u l t o c i da de rmon e c r o t i c t ox i n is a heat labi le i rnrnu n o ge n i c

1 6 0 0 0 0 M r p r o t e i n wh i c h c o n t a i n s s e venteen diffe rent amino ac ids

( Na k a i et al, 19841; Nakai e t a l , 1 9 8 42 ; Rut t e r and Luthe r , 1983;

Rut t e r e t a l , 1984; De Jong , O e i and Tet enburg , 1980) . The t oxin is
synthe s i z ed i n the cytop l a smic spa ce du r i ng t he l oga r it hmic growth
pha s e of P. mu l t o c i da in vi t ro . Re l e a s e of toxin into t he
s u r rounding medium occurs du ring t he s t a t i onary pha s e o f the growth
cu rve ( S awata and Kume , 1 9 8 5 ; Rutter a nd Ma cken z i e , 1984) .

The mode of act i on of P . mul t o ci da t oxin i s unknow n . Toxic e f f e c t s

a re s im i l a r to those p roduced by a d e rmo n e c r o t i c t oxin of
B o r de t e l l a b r o n c h i s ep t i c a howeve r the two mo l e c u l e s a re

s e r o l o g i c a l l y d i s t inct (Nakai et al, 19841 ) . T o x i n - c o n t a i n ing

f i l t r a t e s ha ve no s ignific ant haemo l yt i c o r phospho l ipa s e act ivity

( Rut t e r and Luthe r , 1984) .

P e nnings and S t o rm ( l 9 8 0 ) found that an enhanced toxic e f fect in Ve ro

ce l l s c o u l d be p r oduced by p r e t r e a t me n t of t he cells with a
pho sphodie s t e r a s e inh i b i t o r . P h o s ph o d i e s t e ra s e s cata lyse t he
c o n ve r s i o n o f c AMP t o 5 ' AMP . This s u g g e s t s t h a t t h e c y t opathic
e f f e c t may be c a u s ed by int r a ce l l u l a r a c c umu l a t i o n of cAMP . This

a c t i o n i s s imi l a r t o t he e n t e rotoxins produced by s ome members o f

En t e r oba c t e r i a c e a e and Vi b r i o n a c e a e ( Spei r s , Stavric and

Konow a l chuk, 1 9 7 7 ; Konowa lchuk , Spe i r s and Stavrik, 1 9 7 7 ; Guinee and

Jansen, 1 97 9 ) .
*
Note : a l s o see S e c t 1 . 5 . 6
 29

1 . 5 D i s e a s e As s oc iat ions and Epidemi ology of P . mu l t oclda

1.5 . 1 Gene r a l

T h e p ri ncipal di s e a s e s caused by P . mul t o c i da a re f o w l wh i c h a f fe c t s

dome s t i c a n d w i l d b i rds , h a e mo r r h a g i c s ept i c a em i a of c a t t l e and

buf f a l o e s , s h ipp ing feve r o f s e ve r a l h o s t spe c i e s e s pec i a l ly s heep

and c a t t l e , pr ima ry or seconda ry pneumo n i a and ab s ce s s e s in a wide

range o f a n i ma l s . The s e d i s e a s e s often invo lve o t he r a ge n t s in

concert with P . m u l t ocida . The mo s t f requent coinfect ing a g e n t s a re

P. h a em o l y t i ca and P a ra i n f l uen z a t ype 3 v i rus [PI3) .

The f o l low ing t ab l e , which i s not e xh a u s t i ve , i l l u s t ra t e s t h e w ide

range o f a n ima l s f r om wh ich P . mu l t oc i da h a s been i s o l a t e d , e i ther

a s a d i s e a s e agent o r a s a commen s a l . Anima l s for which t h e o rganism

is a ma j o r p a t h ogen a r e i n d i c a t e d , as a re a n ima l s o f c o mme r c i a l

imp o r t a nce t o New Zealand .

*
T a b l e 1 . 2 H o s t range o f P . m u l t oc i da .

eo ... . Turkey M i nk
Deer B u f fa l o Mo l e
Do g Car i bo u Mouse
D uck Cat Monkey
Goat Ch i p m u n k Mus krat
Horse Dove Peacoc k
Domest i c Hen Gerb i l Pe l i ca n
R a bb i t G u i ne a P i g Rat
s... i ne . Kang aroo Vo l e
Sheep Man V u l t ure

: From Cart er and B a i n ( 1 960 ) and Cart er < 1 9 67 ) .

: P. mul t oc i da i s a m a j o r d i sease-c a u s i n g a g ent .
Bo l d Pr i nt : An i m a l o f commerc i a l i m port ance t o New Z e a l and .

As t h e above t able i l l u s t r a t e s P . m u l t ocida c a u s e s many d i s e a s e s o f

ma n y a n ima l s . Ho weve r , the o rg a n i s m i s n o rma l l y a s s oc i a t e d w i t h

a n ima l s a s a c ommen s a l o f t h e uppe r r e s p i ra t o ry t ra c t ( URT ) but i t

c a nn o t usua l l y b e i s o l ated f rom he a lthy lungs ( Ca r t e r , 1 9 67 ; C a rte r

 30

and Bain, 1960; Hamdy , P ou nde r a nd Fe rguson , 1 95 9 ; Nga t i a e t a l ,

1 9 8 5 ; Magwood Barnum and Thomp s o n , 1 9 7 0 ; Smit h , 1955) .

C o mme n s a l s t ra in s of P . m u l t o c i da a re imp o r t a n t opp o r t u n i s t i c

pathogens and phys iologic a l " s t re s s e s " a re u s u a l ly g i ven the c redit

[if that is the r i ght w o rd ! ] for i n i t i a t ing mo s t outbreaks of

pa s t eu r e l l o s i s ( Ca rte r , 1 9 6 7 ) . Such p redispos ing fact o rs a s " s t re s s "
c a u s e t he comme n s a l t o become di s s emina ted . This wa s demons t rated by
C a v e l l e ro and S a 1 a [ 1 9 5 1 ] ( s e e C a r t e r a nd Bain , 1960) who i n j ected
c o r t i s o ne ( wh i c h i s re l e a s e d in l a rge amount s by t he b ody du r ing
pe r i o d s of st re s s ) into rats wh i c h had an i n a pp a rent na s a l

P. m u l t o c i da i n fect ion . T h e y o b s e r v e d t h a t a f t e r t r e a t me n t the

inappa rent infect ion r a p i d l y p r o g r e s s e d to an a cute s y s t emic

pa s t eu r e l l o s i s a nd many o f the anima l s died f rom the bacte raemia .

The p a t h ogen i c me chani sms by which P . m u l t o c i da c a u s e disease a re

not k n own , however it is t hought t h a t b a c t e r i a l endot oxin i s
re spons ible for the clinical effects and death in acute
bac t e r aemi a s . The numbers o f organi sms present in a n ima l s dy ing of
acute pasteure l losis is e n o rm o u s ( Carter , 1 9 67 ) so a h i gh
concent rat ion o f endot oxin would b e present in t h e b loods t ream .

I t i s gene r a l l y a ccepted that natural infect ions o f P . mu l t o ci da a re

a c q u i r e d by inha lat ion o r inge s t i o n o f t h e o r g a n i s ms . H o w e ve r ,

Da ubney a nd Hudson ( 1 9 3 4 ) s uggested that a rth ropods may b e vec t o r s o f

h ae mo r rhagic sept icaemi a . T h i s view w a s s uppo rted b y MacAdam ( 1 9 6 2 )

w h o demo n s t r a t e d t h e t r a n s mi s s i o n o f a c u t e pa s t e u r e l l o s i s f r om
c a t t l e to rabb i t s us ing t ic k s a s a vect o r . This i s int e re s t ing but

d i s e a s e due t o P . m u l t o c i da in rabb i t s i s not u s u a l l y a s s oc i a t e d

with t he s ame t yp e s of s t rains as those wh i c h cause a cute
p a s t e u re l l o s i s i n c a t t le s o i t i s p robably not important i n f ield
c o nd i t ions .
 31

1 . 5 . 2 Cat t le

The mo s t impo r t a n t di s e a s e s o f catt le a s s o c i a t ed w i t h P . mul t o c i da

a re s h ipp ing feve r , haemo r rhagic s ept ic aemia and pneumonia . S h ipping
f e ve r is t he mo s t impo r t a n t o f t h e s e and hence h a s been the mos t
s t u d i ed . T h i s i s an a c u t e s y s t emic p a s t eu re l l o s i s which t yp i c a l ly
o c c u r s a f t e r an a n ima l h a s s pent long pe riods in a s t re s sed s t a t e .
eg i n t ra n s i t . It is f o u n d w o r l d - w i de a n d i s o n e o f t h e mo s t
e c o n om i c a l l y imp o r t a n t d i s e a s e s o f feedlot c a t t l e i n the United
States ( Ca r t e r a nd Ba i n , 1 9 60) . Howeve r , de s p i t e t h e wide - s p r e a d
occu r rence o f t he di sease i t has n o t b e e n reported i n N e w Zea l and .

S h i pp ing feve r i s u s u a l l y due t o P . m u l t o c i da in a s s oc i a t ion with

o t h e r bacteria and vi rus e s . It i s thought t o be i n i t iated by a lung

i n f e ct ion wh i c h progre s s e s rapidly to a bacteraemi a r e s u l t i n g in a

h i g h g rade feve r and o ften death . T h i s view i s suppo rted by the wo rk

o f P a nc ie r a and Cors tvet ( 1 9 8 4 1 ) . They inocu l a t ed both P . mul t o c i da
and P . h a emoly t i c a into the lungs of c a lve s . This i nduced pneumonic

le s i ons . All of t he anima l s deve loped acute pneumo n i c pasteure l l o s i s

a n d s ome a n ima l s subseque n t l y died o f a b a c t e raemia w h i c h c a n be
r e g a rded as expe riment a l s h ipping feve r .

T h e conc lus ion that two o r more orga n i sms comb ine to cause s hipping

f e v e r doe s not at a l l imp l y that they play equa l r o l e s t h roughout

t h e disea s e . C l e a r ly the one wh ich become s mo st w ide ly di s s eminated
t h roughout the body i s u l t imatly the one which k i l l s the a n ima l . If

so, t he n t he mo s t c r it ical orga n i sm is P. m u l t o c i da be c a u s e

Col lier ( 1 9 6 8 ) said t hat t h i s organism i s the princ ipa l cause o f the
advanced c l in i c a l st age s of shipping feve r .

Hae morrhagic s ept icaemia i s a c l i n i c a l l y s imilar d i s e a s e t o sh ipping

f e v e r but it contra s t s i n that i t is a n acute s y s t emic
p a s t e u re l l o s i s c a u s ed by s ome s t r a in s of P . m u l t oc i da o n l y ( see
S e c t ion 1 . 6 ) a nd i s f ound p redominant l y in t ropic a l c ount r ies .
P ne umon i a in c a t t le can be caused by P. mul t oc i da e it h e r a l one or in
a s s o c iat ion w i t h other o rgani sms . This was s h o w n b y C a rt e r and
Rou s e l l ( l 9 5 8 ) who is olated both P . m u l t ocida a nd P. h a emolyt i ca f rom
t he pneumo n i c les ions o f l ungs obt a ined f r om c a lves which had died
f ro m pneumon i a . It is now gene r a l ly accept ed t h a t P. mu l t o c i da and
o t h e r agent s , e s pe c i a l l y P. h a emolyt i c a and P a r a in f l ue n z a Type 3
(PI 3 ) virus , c omb i n e w i t h p h y s i o l o g i c a l stress t o prec ipitate
pneumon i a .

T h e cont r i b u t i o n to t h e i n i t i a t i o n o f pneumonia in ca t t le , due t o

P. mul t ocida , b y non-F . mu l t oc i da o rgani sms h a s r a i sed t he quest ion

of whether t he pres ence o f the d i s e a s e in a herd can be co rrelated

w i t h t he t ype o f na s a l f lo r a c a r r ied by the anima l . Magwood, Bar rum

a n d Thompson ( 1 9 7 0 ) su rveyed the b a c t e r i a l f l ora o f the n a s a l cavity
of c l i n i c a l l y no rma l c a l v e s f r om both he a l t h y and pneumonia-prone

he rds . T h e y f o und t h a t P. m u l t o c i da was o n e o f t h e mo s t wide ly

d i s t r ibuted o rgani sms i n both popu l a t i on s . H o weve r , the pneumonia

p r o n e herd w a s not a s s o c i a t e d w i t h a na s a l pop u l a t i o n d i f f e r i ng

m a r k e d l y f r om t h e n o r m . But it w a s o b s e r ve d t h a t P . mu l t o c i da

domina ted t he nas a l f l o r a o f t he pneumon ia prone herd, in a state of

a c t ive c o l o n i z a t ion for s e ve r a l days a t a t ime a nd t ha t b a c t e ria

we r e shed i n la rge numbe r s du r ing t h i s proces s . T h i s might sugge st

that pneumon i a prone anima ls a re phys iologica l ly mo re sus ceptible t o

P. mul t oc i da dominat ing t h e i r n a s a l f lo ra . Howeve r i n t he i r report ,

Magwood, B a r r um a n d T h o mp s o n ( 1 9 7 0 ) did n o t s a y i f t h e t ype s o f
P. mul t o c i da s t ra ins wh i c h were i s o l ated f rom t he two he rds di f fe red

f r om e a c h o t h e r so i t c a n be s p e c u l a t e d t h a t t he pneumo n i a prone

h e r d ma y h a ve b e e n a s s oc i a t e d with a strain of P. m u l t o c i da

c o mme n s a l wh i ch wa s m o r e v i r u l e n t ( t hat is , it had a h i ghe r

p r open s i t y f o r being a n oppo r t un i s t i c p a t h o gen ) than the st rains

f o und in t he norma l herd . Furthermo re , t he dominance o f P . mul t o c i da
i n the n a s a l f lora i s a cyc l i c event so t he l a c k o f l a rge numbe rs o f
P. mul t o c i da i n the n a s a l pa s s age s o f pneumonic a n ima l s may b e due
t o the t ime lag between h igh numb e r s of t he orga n i sm a nd t he onset

o f clinical s ymp t om s . A t the c l i n i c a l s t a ge the n umbe r s of t h e

 33

o rgani sm i n the n a s a l c a vity wo u l d h ave r e t u rned t o a low p a rt o f

t h e cycle .

C a s e s o f P . m u l t o c i da a s s o c i a t e d ma s t i t i s h a v e b e e n r e p o r t e d i n
c a t t le b o t h ove r s e a s ( C a r t e r a nd B a i n , 1960 ; Carte r , 1976) and i n
New Zealand ( Ca rt e r [ 1 9 7 2 ] ( s e e W o o dgye r , 1976) . Howeve r , u n l i ke
s he ep [ see Sect ion 1 . 5 . 3 ] ma s t i t i s o f t h i s ae t i o logy i s not a ma j o r
p r ob lem .

I n gene ra l , de spite the ove r s e a s s it u a t i o n pas teure l l o s i s i s not a

ma j o r problem in t he New Zea l and c a t t le indu s t ry . Thi s might be due
( Cu rt i s , 1972) t o t h e e xtens ive pa s t o r i a l rea ring methods used in
this count ry . I t h a s b e e n s pe c u l a t e d t h a t w o u l d c h ange i f mo re
i nt e n s i f ie d methods of bee f p r odu c t ion a re embarked upon ( Cu rt i s ,

1 972) .

1 . 5 . 3 Sheep

T h e ma i n d i s e a s e s o f s heep wh i c h a re c a u s e d by P . m u l t o c i da a re
s h ipping f e ve r , pneumo n i a and ma s t i t i s ( C a rt e r , 1967) . The rema rks
c oncerning sh ipping feve r o f c a t t le also apply to sheep s o will not

b e di s c u s s e d f u r t h e r e xcept to s a y t h a t de a t h s due to s y s t emic

p a steure l l o s i s (a categ o ry wh ich inc lude s sh ipp ing feve r ) which a re

n o t comp l i c a t ed by pneumon i a a re ra re . Th i s impl i e s that t he ma j o r

route of i n fect ion is vi a the lungs . U n l i k e s h i p p i n g f e ve r of

c a t t le , P . mu l t o c i d a and P. h a emo l y t i ca play roles of e qu a l

impo rtance i n c a u s ing s ystemi c p a s teure l lo s i s of sheep ( B ibe r s t e i n

a nd Kennedy , 1959) .

P a s t e u re l l a pne umon i a of sheep i s mo r e i mp o r t a n t i n t empe r a t e

c l imates t ha n i n the t ropics . Howeve r , P. h a emolyt i ca i s cons ide red

t o be a more c ommon c a u se of t h i s di s e a s e t h a n P . mu l t o c i da . Th i s
c omme nt e s pe c i a l l y a p p l i e s t o l ambs w h e r e P a s t e u r e l l a pneumo n i a
o ccurs p r i ncipa l ly w i t h in t h e f i r s t month o f l i fe a n d i s t he c a u s e
of c o n s i de r a b l e loss of a n ima l s ( Ma r s h , 1 953 ; C a rt e r , 1976) .
 34

N e v e r t h e l e s s P . m u l t o c i da do e s p l a y a n o t - i n s ign i f i c ant role in

c a u s ing pneumonia in l amb s . This was demon s t rated by Stevenson ( 1 9 6 9 )
who u s ed P . m u l t o c i da i n c omb i n a t i o n w i t h myc o p l a s m a s t o p roduce

p n e umo n i a in s t r e s s e d l amb s . Howeve r , h e c o u l d n o t p r oduce t he

di s e a s e in s t re s s e d l amb s u s ing P . m u l t o c i da a l one n o r in n o rma l
l ambs us ing e ither comb in a t ion ( S tevenson, 1 9 6 9 ) . This s ugge s t s that
o t he r o rgani sms and s t re s s a re import ant i n p redi spo s ing t he anima l
t o d i s e a s e c a u s ed by P . m u l t o c i da .

The a bove di scus sion does not i mp l y that P. m u l t o c i da and

P. h a em o l y t i c a a re n o t i s o l a t e d t o ge t h e r f r om s heep pne umo n i a s .
P. m u l t o c i da is often i s o l a t e d f r om s h e e p p u lmo n a r y l e s i o n s i n
c o n j u n c t i o n w i t h P . h a emolyt i c a , h oweve r in l ine w i t h t he gene ral
r u l e with s heep pneumonia P . h a emolyt i ca u s u a l l y predominates ( Hamdy ,
P ounde r and Fe rgu son, 1959) .

Be l s c hn e r [ 1 9 5 1 ] ( s ee Ma r s h , 1953) i s o l a t e d P . mu l t o c i d a f r om t h e

l un g s o f s heep w h i c h h a d d i e d f r om pleuropne umon i a . H e conc luded

( in c o r rect l y ) that the o rganism was t he aet i o l ogic a l agent o f t h i s

d i se a se . S i nce t h i s w o r k it h a s b e e n e s t a b l i s hed t h a t Myc op l a sma
myc o i de s i s t he a g e n t o f p l e u r o p n e umo n i a . H o weve r , Be l s c h ne r ' s
conc lus ions do i l l us t r at e t he impo r t a n c e of P. m u l t o c i da as a
s e c ondary disease caus ing agent . T h i s is enhanced by St Geo rge ( 1 9 7 2 )

who demo n s t ra ted the pre s ence of P . mul t o c i da in pneumonic lungs o f

Au s t r a l i a n s h eep w h i c h had d i e d o f ' S umme r P neumon i a ' . I t i s now

gene r a l l y a ccepted that P . h a emolyt i ca is the a gent o f t h i s disease .

N o t w i t h s t a nding t h i s , T adaya n , Cheema and Muh amme d ( 1 9 8 0 ) i s o lated

P. m u l t oc i da f rom lung abce s s e s in I ranian sheep . T h i s s ugge s t s that

P. m u l t oc i da nonethe l e s s can c a u s e pr ima ry l ung di s e a s e i n t he s e

a n ima l s .

F rom the above discu s s i o n it can be appre c i a t e d that P . m u l t o c i da i s

a s s o c i a t e d w i th sheep d i s e a s e s i n many count r i e s . However the role
of P . mul t o c i da i n New Z e a la nd f l o ck s , a l t hough suspected, i s poo r l y
unde rstood . Al ley ( 1 9 7 5 ) s urveyed t he re spira t o ry t ra c t f lo r a o f b o t h
 35

n o rma l and pneumonic s heep . Despite demons t r a t ing t he presence o f a

hete rogeneous bacte r i a l population he did not repo rt the pre sence o f
P . mul t oc i da . S a l i s bury ( 1 9 5 7 ) and Downey ( 1 9 5 7 ) isolated a
P. mu l t o c i da - l i ke o r g a n i s m f r om s h e ep s u f f e r i n g f r om e n z o o t i c

pneumo n i a ( ie chron i c non-progre s s ive pneumo n i a [ CNP ] ) . Howeve r it

i s more p robable that the i r i s o lates were P . h a emolyt i ca s ince t h i s

organism i s a lmo st ubiquitous i n CNP l e s i o n s ( P rince , 1985) .

Note : A problem in interpret ing early l it e rature on P . mul t o c i da

i s that in many accounts the ident i f i cat i on o f t he spe c i e s
i s n o t s u f f i c ient l y c le a r t o di f f e rent i a t e t he o rgan i sm
f r om other P a s t e u rel l a species, in particular
P. h a em o l y t i c a . For this r e a s o n many o f t h e e a r l i e r
repo rts o f P . mul t o ci da should b e t reated w i t h a degree o f
s cept ic i sm .

1 . 5 . 4 Goats

T h e ma i n d i s e a s e s o f goats caused b y P . mul t o c i da a re pneumonias and

s y s t emi c p a s t e u re l l o s i s . I n f e c t i ons o c c u r wo rld-w ide a l t h ough it
w o u l d s eem f r om the l it e ra t u re that a h igher preva lence occurs in
t ropic a l count rie s . P a nde ( 1 9 4 1 ) i s o l a t e d a n organism which f it t ed
t he de s c ript ion of P . mul t o ci da f rom the l ungs o f many goats w h i c h

had died f rom pleuropneumonia in Indi a . He c ited e a r l ie r outb reaks

w h i c h h a d de c i ma t e d goat he r d s i n the a r e a and s u gge s t e d t h a t

P. m u l t o c i da w a s t h e c a u s a l agent . . Howeve r s i nce 1 9 4 1 Myc opl a sm a

myc o i d e s h a s b e e n a c cepted a s t he c a u s e o f pleuropneumonia . It is

t h e re f o re l i ke l y t ha t the o r g a n i s ms d e s c r i b e d b y P a n de ( 1 9 4 1 )

repre s ent seconda ry i nvade rs a s s oc i ated w i t h the p r ima ry mycop l a sma l

d i s ea s e .

B o rgman and Wilson ( 1 9 5 5 ) showed that P . mul t ocida could be i s o l ated

f rom a l a rge p r opo r t ion of l u ngs f rom g o a t s wh i c h h a d d i e d f rom
p n e umo n i a . The d i s e a s e p i c t u r e i s s imi l a r to t h a t wh i c h a f fe c t s
s heep ( Bo rgman and W i l s on , 1 9 5 5 ) . Usually a few a n ima l s die s udde n l y
 36

due t o a bacte raemi a . T h i s i s f o l lowed by c l in i c a l evidence a l ung

i n f e c t i o n w i t h i n o t h e r memb e r s of t h e h e r d . The ma i n s igns a re

letha rgy , anorexi a , a mucopu rulent di scha rge f rom the eyes and n o s e ,
cough , dyspnoea a n d a n e levated tempe ra t u re . The d i s e a s e t a kes one
t o two weeks t o run its course . Deaths o c c u r f rom the o n s e t o f

c l i n i c a l s igns w i t h mort a l i ty rates ave r a g i n g approximat e l y 1 0 % o f

the infected he rd ( Bo rgman a n d Wi l s on , 1955) .

The f i nding that P . m u l t o c i da can be i s o l at ed f rom a l a r ge

propo r t ion of goats wh i c h die f rom p n e umo n i a or s y s t emic
pa s t e u r e l l o s i s s ugge s t s that , l i ke c a t t l e and sheep , P . mul t ocida is
a c o mme n s a l of t he uppe r respi rat o ry t ract and is also an

oppo rt u n i s t i c pathoge n . This would imp ly t h a t a la rge propo rtion o f

norma l a n ima l s w o u l d ca rry the o rganism i n t he i r n a s a l cavit y . This
i s s upp o rted by Ngat i a e t a 1 ( 1 9 8 5 ) who s u r veyed t he n a s a l f lora o f

c l i n i c a l l y n o rm a l goat s in t he U n i t e d St ates . They i s o l a t ed

P. m u l t o c i da f r o m a p p r o x i ma t e l y h a l f of the a n ima l s s t udied .
F u r t h e rmo re , t h e y s h owed t h a t P. m u l t o c i da is one o f t h e mo s t
p r e domi n ant b a c t e r i a f ound i n t he norma l n a s a l f l o r a i n the sense
that i t can be rec ove red f rom a h igh propo r t i on of anima l s .

The above di s c u s s ion s ugge s t s that the t ype s of di s e a s e s a s s oc i ated

w i t h P . m u l t o c i da in go a t s a re s imi l a r t o t h o s e f o und i n s h eep .

Howeve r , much o f t he l it e ra t u re has been t a ken at f a c e value and a s

w i t h s heep many e a r l i e r repo r t s o f P . m u l t o ci da a s a cause of

di s e a s e have confused t he role o f t h i s o rgan ism ( u sua l ly a s econdary

invade r ) with that of the p r ima ry pat hogen . eg pleuropneumonia i s

c a u s e d b y Myc opl a sma myc o i de s . F u r t h e r mo re , e a r l y ident i f i c a t i o n

p r o ce d u r e s did not t a ke into account t h e t h e l a r g e n umbe r s of

P a s t e u re l l a and related s pe c i e s wh i c h a re now r e c ogn i s e d .
Con sequently s ome reports o f a P . mul t oc i da - l i ke o rgan ism may h ave
a c t ua l ly refe r re d t o P . h aemolyt i ca or a Yers i n i a spp . Nonethe l e s s
P. mul t o c i da i s c l e a r ly an impo rtant pat hogen o f goat s espe c i a l l y i n
t ropic a l count rie s . T h e organism has n o t b e e n reported a s a problem
 37

i n New Zealand but with t he g rowing emph a s i s o n f a rming goat s here

t h i s c ould change .

1 .5.5 Rabb its

Rabb i t s a re ext reme l y s u s cept ible t o P . m u l t oc i da infect ions and t he

o rg a n i sm i s r e s p o n s ib l e f o r e c onomic l o s s in product ion unit s a nd
c omp l i c a t i ons o f l ab o r a t o r y r e s e a rch p r o j e c t s ( Percy, B h a s in a n d
Ro s e n da l e , 1986) . A n umbe r of c l i n i c a l ma n i f e s t a t i o n s occur
i n c l ud i n g ' s n u f f l e s ' , p n e umon i a , o t i t i s med i a , con j un c t i v i t i s ,

pyome t ra , o r c h it i s , s ubcut a neous abc e s s e s and sept i c aemia ( F l at t ,

1 97 4 ; Ca rte r , 197 6 ; D iGiacomo e t a l , 1987 ; Hagen, Go rham and F l at t ,
1 97 6 ) .

The mo st common f o rm of pasteure l lo s i s in rabbits i s s nu f f le s . This

di s e a s e is cha r a c t e r i s ed b y a serous or mucopurulent n a s a l exuda t e .

Rh i n i t i s deve lops a fter two wee ks f rom t he initia l n a s a l infect ion .
It i s a common b u t s u rp r i s i n g f i nd i n g t ha t p rewe a n l i n g s a re not
affected, even i f t he doe is i n f e c t e d ( G i a commo , Ga r l inghouse a nd
Van Hoo s ie r , 1983) . This appa rent re s i s t a nce of p rewe a n l ings s e ems
to be un ique to rabbit s .

P. m u l t o c i da e x i s t s a s an upp e r r e s p i r a t o ry t r a c t c omme n s a l i n a

h i g h p r o po r t i o n o f c l i n i c a l l y n o rma l r abbit s . If r e s i s t a n ce is

lowe red, f o r e x a mp l e due to s t re s s r e l a t e d e xp e r im e n t a l u s e ,

i n f e c t i on can s p re a d t o o t h e r p a r t s of t h e b o dy r e s u l t i n g i n

c l i n ic a l man i f e s t a t ions ( F latt , 1974) .

F ro m the uppe r r e s pi ra t o ry t ract P . mu l t o ci d a infect ion c a n sp read

to t he lungs and cause a c u t e pneumon i a . T h i s often le ads to de ath

due t o sept icaem i a which i s re spon s ib l e for betwe en f ive a nd f i f ty
pe r cent o f dea t h s i n ove r s e a s rabbit c o l o n i e s ( Flatt , 1 97 4 ; Mann ing
et al, 1986) .
 38

Spread o f P . mul t o ci da t o the middle e a r leads t o o t i t i s medi a . T h i s

can be c omp l i c a t e d b y f u r t he r s p r e a d t o t he inner ear [ ot i t i s
i nt e rn a l . This i n fe c t i o n i s cha ract e r i sed b y head t i lt with an
a s s oc i ated l o s s o f bal ance and is c a l led t o r t i c o l l i s o r c o l loqu i a l ly
a s w ry-neck ) . An a n ima l in t h i s condit ion i s d i f f i c u l t t o t re a t w it h
a n t i b i ot i c s d u e t o t h e i n a c c e s s ib i l i t y o f t h e s it e o f i n f e c t ion
( Flatt , 1 9 7 4 ) . Unt reated cases often lead t o death due t o s t a rvat ion
because t he an ima l i s unable to o r ient a t e it s e l f to eat .

1 . 5 . 6 S wine

P. m u l t o c i da causes a variety of dis ea s e s in s wine . The more

imp o r t ant o f t h e s e i s a t rophic rhin i t i s [ AR ] r se conda ry pneumonia

a nd s wine plague . Next t o t he mycopla sma s , P . mul t o c i da i s the mo s t

common o rgani sm i s ola ted f r om pneumo n i c p i g lungs ( Go i s , Kukea and
S ie a k , 1980) . In New Z e a l a nd P . mul t o c i da h a s been i s o l a t e d f r om

c a s e s of s wine abo rt i on C a r t e r [ 1 9 7 2 ] ( s e e Woodgye r , 1 97 5 ) .

AR in s wine i s c h a racteri sed by degene rat ion of the n a s a l t u rbinates

r e s u l t ing in s nout de f o rm i t y . It is of c o n s i de r a b l e e c o n o m i c
i mp o r t a nc e ove r s e a s , e s pe c i a l l y in E u r ope ( P e de r s o n and N i e l s en ,

1983) . A mild f o rm of AR h a s been de t e c t e d ( H edge s , 1 98 1 ) in New

Z e a land, howeve r t here is no report o f t he i s o l a t i o n o f P . m u l t o c i da

f rom pigs with t h i s condi t i o n .

Ove r s e a s re s e a r c h h a s s hown t hat the pres ence o f AR i n a herd

c o r r e l a t e s w i t h t he p re s e n c e o f B or de t el l a bron ch i s ep t i c a a nd / o r
t ox i g e n i c s t r a i n s o f P . m u l t oc i da [ t h e t o x i n o f P . m u l t o c i da wa s

d i s cus sed in Sect ion 1.4] ( D e J o n g a n d A k k e rma n s , 1986; Rhode s

et al, 1 9 8 7 ; S a w a t a et a l , 1 9 8 4 ; Lugtenburg, Van Boxt e l and D e Jong ,
1984) . B. bron c h i s ept i ca a l one , o r i n c o n c e rt w i t h n o n - t o x i g e n i c
P. m u l t o c i da , c a u s e s a mi l d , t ra n s i e n t t u rb inate a t rophy known a s
' re g r e s s ive AR' . T o x i ge n i c P . mu l t o c i da causes a mo r e s e ve re
progre s s ive a t r ophy of the t urbinate s . T h i s s ugge s t s that t h e t oxin
is r e s pon s ib l e f o r t he mo re severe l e s i o n s . Thi s is s uppo r t e d by
 39

Pede r s on and B a r f o d ( 1 9 8 2 ) who ins t i l led p u r i f ied P . m ul t o c i da t oxin

i n t o the n a s a l c a v i t i e s o f pigs a n d p r o du c e d l e s i o n s w h i c h were

s im i l a r t o t h o s e of p r o g re s s ive AR . The s eve r i t y of t he l e s i o n s

produced was prop o r t iona l t o t he amount o f t o x i n g iven ( D e J o n g a nd
Akke rma ns , 1986; P e de r s o n and B a r f od , 1 9 82 ; P ede r s o n a nd B a r f o d ,
1 9 8 1 ; De Jong, Oe i and Tetenbu rg, 1980) .

Chemi c a l irritat ion of the n a s a l mu c o s a o f pigs p r i o r t o t he

i n t r oduct ion of t o xigenic P . mul t o c i da re s u l t s in a mo re rapid onset
o f c l in i c a l AR . T h i s r a i s e s t he po s s ib i l it y that B . bron ch i s ep t i ca

may cause a mi ld i r ritat ion which prog res s e s to progre s s ive c l i n i c a l

A R a f t e r inf ec t i o n b y t o xigenic P . mu l t o c i da . Wh i l e the importance

of c o infect ing agents rema ins to be c l a r i f ied in de t a i l , t he mode of

a c t i on of P . m u l t o c i da toxin is t o s t imu late bone reabso rpt ion and
s u pp r e s s o s t e o i d s ynthe s i s (Goi s , John-Ba rnes a nd Ro s s , 1983;

Pede r s on and E l l i ng , 1 9 8 4 ) . This expl a i n s the changes in g r o s s s nout

mo rpho logy obse rved in t h i s disease .

1 . 5 . 7 Dee r

L i t t l e i s known about p a s t e u re l l o s i s i n de e r but a h aemo r r h a g i c

s e p t i c aemi a - l i k e disease due t o P . mu l t o c i da h a s been de s c r ibed in

r e i nde e r ( No rd k v i s t a n d Ka r l s s o n [ 1 9 6 2 ] , see C a r t e r , 1967 ) . The

s t r a i n s invo lved were not typed but f rom the nat u re o f t he di s e a s e

i t would s eem t h a t dee r may b e sus cept ible to t h e s ame P . mu l t oci da

s e rotypes s c a t t le and t hese s t r a i n s may cause s imi l a r di s e a s e s in

b o t h spec ies .

1 . 5 . 8 Cat s and D ogs

P. m u l t o c i da d i s ease in c a t s and dogs i s not as common a s i t i s in

t he anima l s me n t i oned e a r l i e r in t h i s sect ion . Neve rthe l e s s it has
been i s o l a t ed a s a c omme n s a l of t he mo u t h and t h ro a t of a l a rge
p roport ion of c a t s and dogs ove r s e a s ( C a rt e r and B a i n , 1 9 6 0 ; C a rte r ,
1 9 67 ) . T h i s w o u ld appe a r t o b e a l s o t he c a s e i n N e w Z e a land c a t s
 I

40

bec a u s e Woodgye r ( 1 9 7 5 ) h a s s hown that 4 2 % o f a s ample o f New Z e a l a nd

c a t s c a r ry P . mu l t o ci da in t he i r mouth s .

As a disease agent of c a t s a nd dog s P . mu l t o c i d a h a s b e e n f o und

a s s o c iated with abcesses o f both spec i e s , abo r t i on in dogs a nd
pneumon ia in c a t s ( Ca rter and Bain , 1 9 6 0 ; Namioka and Brune r , 1 9 62 ;
Cart e r , 1967) . L ittle work has been done in New Zea l a nd t o
inve s t igate di s e a s e c a u s ed b y P . m u l t o c i da in these a n i ma l s .
Howe ve r , Ca rt e r [ 1 9 7 2 ] ( s e e Wo odgye r , 1976) i s o l a t e d the o rg a n i s m

f rom a c a t w i t h pneumo n i a . T h i s i s a s o l it a r y i s o l a t ion but it i s

none t he l e s s cons i s t ent w i t h overseas f i nd ings .

1 . 5 . 9 Man

Al t h ough n o t c ommo n , human i n f e c t i on s due to P. mul t o c i da a re

bec oming mo re widely recogn i sed . I n fect ion does oc c a s s i ona lly r e s u l t
f r om dog a nd c a t b ites s o it is n o t s u rp r i s ing t h a t b i o chemic a l ly

and mo rpholog i c a l ly human s t rains tend t o be ve ry s imi l a r to f e l ine

s t r a in s ( T a lbot and Sneath , 1960) howeve r the respi r a t o ry t ra c t can

become infected vi a othe r s ou rces . The s t rains invo lved appe a r t o be

opp o rt un i s t i c p a t ho ge n s p r oduc ing l ocal le s i ons only . Wh i l e

i n f e c t ion a s a re s u lt o f a n imal bite s c le a r l y rep res ent a z oono s i s

pat ients s u f f e r i ng f rom respirato ry t ract infections are
p r e do m i n a n t l y f r om f a rm i n g b a c k g r o u n d s . This also s ugge s t s a

z o o n o t i c relat ionsh ip ( Ca r t e r , 1 9 6 7 ; Rut t e r a nd Luthe r , 1984) .

P. m u l t ocida h a s been i s o l a ted f rom one human spu t um s amp le in New

Z e a l and . Inte r e s t ingly the pat ient had s ympt oms s imi l a r t o t h o s e of
tube rculos i s ( Mi l le r , 1 9 63 )

1.6 The Re lat ionship of Type o f P . mu l t o cida t o Ho s t s and D i s e a s e s

T h i s sect ion c o n s ide r s the c ap s u l a r Type ( ie A, B, D, E, F) , which

a l l u de s only to t he c apsular p o l y s a c c h a r i de . It is to be
 41

d i s t ingu i s hed f r om t he S e rotype , which refers t o t he comb i na t i on o f

c ap s u l a r type and t he soma t i c ant igen ( eg 1 : A , 3 : A, 3 :F, 6 : B, 6 :E) .

I . Re lat ionship of Capsu l a r Type to D i s e a s e .

D i s e a s e s due t o P . mu l t o cida can o f t e n be c o r related w i t h t h e h o s t

a n ima l a f fected and the s t rain Type ( i e A, B, D, E o r F) a s s o c iated
w i t h the di s e a s e ( see Table 1 . 3 ) .

*
T a b l e 1 . 3 Re l a t i onship o f P . mul t ocida Caps u l a r Type t o D i s e a s e .

Type D i sease Assoc i at i on

A Fow l cho l era.

Pr i mary and second a ry i n fect i ons in a w i d e
ran g e o f spec i es .

B Haemorrh a g i c sept i c aem i a o f cat t l e and

b u f fa l oes.
Sw i ne p l a g ue .

D Pr i mary and second a ry i n fect i ons i n a w i d e range

o f an i m a l spe c i e s .

E Haemorrh ag i c se p t i caem i a o f cat t l e i n Cent ra l

A fr i c a .

F Pr i m a r y d i sease o f turkeys.

B a sed on Cart er ( 1 976 ) .

See Ta b l e 1 . 2 for a l i st
of spec i es i nvo l ved .

As i l l u s t r a t e d above P . m u l t o c i d a i s a n imp o r t a n t di s e a s e - caus ing

a g e nt . Neve rt he l e s s , t he o rg a n i s m e x i s t s p r ima r i ly as pa rt of the

n o rma l f l o r a a nd can be recove red f r om the nasal cavity of healthy

a n ima l s . Fo r e xample Types A, B a nd D st rains are recove r a b l e f rom

t h e n a s a l c a v i t i e s of h e a l t h y c a t t l e . H oweve r the d i s t r i b u t i on o f
s e r o t ype s i n t he n o s e i s n o t e x a c t l y pa r a l l e l ed b y t he r e l a t ive

p re va lence of these t ypes as disease caus ing agent s . Thus i n c a t t le

 42

Type A i s b y f a r the mo s t common t ype i s o l ated f rom pneumo n i c lungs .

T h i s d i s e a s e i s p redominant ly f ou n d in t empe rate c l imat e s . On t he

o t he r h a nd h a e mo r r h a g i c sept i c aemia i s a d i s e a s e o f c a t t l e wh i c h

we re re a red i n t ropi c a l c l imat e s a n d i s a s s o c i a ted with s t r a in s o f

P. m u l t o c i da Type B . T h e reason f o r t h i s i s u n c l e a r but P . mul t oc i da

i s a n oppo r t u n i s t i c p a thogen and l i ke many such pathoge n s i t tends

to c au s e d i s e a s f o l l o w ing sub j e c t i on of the host to " s t re s s " . It
might p l a u s i b l y b e s ugge sted that t he s t re s s a s sociated w i t h c o l d ,
w e t we a t h e r p redi spo s e s the a n ima l t o the Type A related disea s e ,
wh i c h i s p n e umo n i a . Hot , d r y w e a t h e r o n t he o t h e r h a n d , might
p r e d i s p o s e t h e anima l t o t he Type B re l a t ed disea s e , h aemo r rhagic
sept i c aemi a .

I I . Re lat ions h ip of Serotype to D i s e a se .

" S e r ot ype " i s de f i ned a s a the c omb i n a t i o n o f c a p s u l a r t ype w it h

s oma t i c ant igen .

P. m u l t o c i da s t rains which cause f o w l c h o l e r a a re a l l t he s ame

c a p s u l a r t yp e . ie Type A . Howeve r Robe r t s ( 1 9 4 7 ) , u s ing t he mou s e
protection t e s t , e s t a b l i s hed t h e e x i s t e nce o f t h ree immuno logical
" t ype s " a s s o c iated with fowl cho l e r a . The ide a that s eve r a l " t ype s "

of P . m u l t o c i da could cause fowl cho l e r a was r e i n f o rced by

Heddleston [ 1 9 6 2 ] ( see C a rte r , 1967) who s t udied i s o l a t e s us ing both
an immun i z a t i on-chal lenge test in c h i ckens and a complement fixat ion

tes t . His r e s u l t s demo n s t r a t e d t he e x i s t a n c e of two " t ype s " o f

P. m u l t o c i da f r om c a s e s o f f o w l c h o l e r a . T h e s e t w o " t ype s " also

di f f e r ed b i o c hemi c a l l y i n t he i r a b i l i t ie s t o f e rme n t xy l o s e and

dul c it o l .

Thus Robe rt s [ 1 9 4 7 ] f ound t h a t t h ree s e ro logic a l " t ype s " of

P . m u l t o c i da we r e respon s ib l e for fowl cholera , whe r e a s
Heddl e s t o n [ l 9 6 2 ] had p r oposed t h a t only t wo " type s " w e r e invo l ve d .

Howeve r a l l i s olates were Type A s t r a ins .

 43

Namioka ( se e Sect ion 1 . 2 . 3 ) d i s cove red diffe rent soma t i c ant igens o f
P. m u l t o c i da and g a ve them a n umb e r to d i s t i n g u i s h t hem f r om

( cap su l a r ) Types . It i s rea s on a b l e t o sugg e s t that t he d i f ferences

in the a v i a n is olat e s of Robe rt s [ 1 9 4 7 ] a nd Heddle s t on [ 1 9 6 2 ] lay i n

the i r s om a t i c a n t i ge n s . This was s u pp o r t ed w h e n Namioka and
Bruner ( 1 9 6 3 ) f ou n d t ha t S e r o t ypes S:A and B :A were t h e mo s t
prevalent i s olates f rom fowl cho le ra . They f u rther showed that t he s e
t wo s e r o t yp e s e qu a t e d t o t he two immu n o g e n i c t yp e s i s o lated by
Heddle s t o n [ 1 9 6 2 ] ( s ee Carte r , 1967) N a m i o k a a n d Mu r a t a ( 1 9 6 4 )
re i t e r a t e d t h e r o l e of S e rot ype S :A as a caus a l a ge n t o f f o w l

cho lera a n d i n addi t i on re fined S e rotype B : A t o Sub s e rotype B a : A ( S a

being a s u bgroup o f somatic group 8 ) . They a l s o added a n addi t ional

S e r ot ype [ 9 : A] t o c ompile a f i n a l l i s t o f three Se rotypes caus ing

fowl c h o l e ra . This conc l u s i o n is c o n s i s t ent w i t h t he e a r l ie r

f indings o f Robe rts [ 1 9 4 7 ] ( see above ) who s a id that t h ree dif fe rent

st rains we re invo lve d .

I t should b e c l e a r f rom t he above d i s c u s s i o n that f o w l chole ra i s

caused by c e r t a i n S e ro type s of P. m u l t o c i da only . I n t e re s t ingly

c o r re l a t i o n s h a ve a l s o been seen between p a rt i cu l a r P . m u l t o c i da
S e rotype s and t he age and spe c i e s o f b i rd ( dome s t i c h e n , t u rkey,

duck e t c ) a f fected . For example Heddleston [ 1 9 6 2 ] ( s ee Ca rte r , 1967)

obse rved t hat h i s t ype 1 s t ra in [ equ iva lent t o Carter S e rotype S : A ]

was pathogenic for 1 6 - wee k - o l d a nd 4 5 - we e k - old c h i c ke n s . Type 3

[ equ iv a l e n t t o C a r t e r Se rotype B : A] wa s p a t hogen ic f o r 1 6 -wee k - o ld

t u rkeys a n d 4 5 -wee k - o ld c h i c k e n s but w a s o f low pathogenicity f o r

1 6 -wee k - o l d chicken s .

S ince t h e w o r k ( s e e above ) wh i c h demon s t r a ted t h e i n v o l vement o f

cert a i n S e ro t ypes o f P . m u l t o c i da i n c a u s i ng f o w l c h o l e r a , other

worke rs h a ve shown t h a t such a r e l a t i on s h ip occurs w i t h d i s e a s e s i n

s ome o t h e r a nima l species ( see T a b le 1 . 4 ) .

 4'4

T able 1 . 4 Re lat ionship of P . mul t oc i da Se rotype to Host and D i s e a s e .

An i ma l Host D i sease Serct y pe R e fetence

Fow l Fow l Ch o l era 5:R Cad er < 1 '367 > ,

8a : R Nam i o k a and M urat a ( 1 '364J
'3 : R

Cat t l e Haemorr h a g i c 6:8 Sh i g i d i and M u s t a f a ( 1 '3 7'3J

-Sept i caem i a 6:E

Sw i ne Pneumon i a 1 : A Cart e r ( 1 '367 > ,

3:A Cart e r and B a i n ( 1 '360 ) ,
1 :D Nam i o k a and 8runer ( 1 '363J
2:0
4:0

Rabb i t s Res p i ra t ory 1 2 : A. G i acommo, Gar l i n g h o use

Tract 3:A ar1d Van Hoos i er ( 1 '383J ,
12:0 Mann i n g et a l ( 1 '386 >
3:0

Predom i na n t d i sease-caus i n g Serot y p e .

I n s umma r y t he ide n t i f i cat i o n of " S erotype s " on the b a s i s o f both

the cap s u l e and soma t ic ant i gens has a l l owed a mo re re f ined ana lys i s

o f t he r e l a t ionship between t h i s a nd di s e a s e produced i n a variety

of host s . The above discu s s i o n i n d i c a t e s t h at s ome c o r re l a t i o n
exi s t s b e t ween t h e S e rotype o f P . mul t oc i da , the species of host

infected and t he d i s e a s e caused .

 .
I

45

1.7 Immunity and Vaccines

Immunity to P . mul t oc i da is prima r i ly humo r a l ( Cart e r , 1967) a nd t he

ant ibodies p roduced a re bacteric ida l ( Wa l ke r and Fos t e r , 1981) .

T h e f i r s t P . m u l t o c i da v a c c ine w a s a c c ident a l ly produced by Loui s

P a steur i n 1 8 7 8 . This wa s i n the f o rm o f a broth culture o f ( a lmo s t
c e r t a i n l y T ype A ) orga n i sms w h i c h h a d been le ft t o s t a n d a t room
t empe r a t u re f o r s uch a length of t ime as to rende r t he o rg an i sms
non-infect ious . This was d i s cove red by Pasteur a fter he
uns ucces s fu l ly t r ied t o produce fowl cholera i n chi ckens u s i ng the
o l d c u l t u re . H o weve r , t he s i gn i f i c a n ce o f t h e d i s c o ve r y w a s not
a pp r e c i a t e d u n t i l he r e i noculated t he s a me fowl with a f re s h
v i r u lent s t r a i n . The bi rds did not cont ract the disease a nd P a steur
c o r rect ly c o n c l uded concluded that t h e y had s omehow been ' p ro t ected'

b y t he o ld c u l t ure .

A c t i ve immun i z a t i on o f f o w l w i t h a v i rulent vacc ines was cont inued

f o r s e v e r a l d e c a de s a f t e r P a s t e u r ' s d i s c o ve r i e s . H o w e ve r t h e i r
popu l a r i t y d i mi n i s h e d be c a u s e vaccines p repa red, at le a s t
o stensibly, b y P a s t e u r s me t h o d w e r e n o t c o n s i s t a n t l y e f f e c t ive

( C a rter a n d B a i n , 1960) .

E a r l y a t temp t s we re made a t pa s s i ve l y immun i z i ng a n ima l s a g a i n s t

s e ve r a l P . m u l t o c i da diseases e s p e c i a l l y s h i p p i n g f e ve r which

r e qu i r e s only short t e rm p r o t e c t i o n ( Ca r t e r and Ba i n , 1960) .

Howeve r , t h i s was di scont inued due t o a gene r a l lack o f e f f ic a cy . I n

h i nds i ght this is not s u rp r i s i n g be c a u s e only one t yp e of
P. mul t o c i da w a s used t o p roduce t he a n t i s e rum whe rea s t he di s e a s e

c a n invo lve a n ot h e r t ype o f P . mul t o ci da o r e v e n be d u e t o othe r

o r gani sms , eg P . h aemolyt i ca . T he re f o re the immun i z ing s e ra , which

was se rotype spec i f i c , was not protect ing against di s e a s e s c aused by

other organi sms .
 46

B a in [ 1 9 5 2 ] ( s ee C a rt e r , 1967) prepa red a v a c c i ne f o r haemorrhagic

s ep t i caemia (a di sease of cattle i n t ropi c a l count r ie s ) by blending
o rganisms w i t h mine ral o i l and lanolin . Good protect ion was obt a ined
w i t h a min imum of t i s s u e damage . B a i n s t a te d t hat an o i l adj uvant
c ombined with a high number of o rgani sms were both import ant f a c t o r s
i n prepa ring vaccines t o P . mul t o c i da ( see C a r t e r , 1 9 6 7 ) .

T h e demon s t ra t ion by B a i n [ 1 9 5 2 ] ( s ee above ) o f t he imp o r t a n c e o f

u s ing a n a d j uvant in P . m u l t o c i da vac c i ne s led H e dd l e s t o n a n d
Hall [ 1958] ( s ee C a r t e r , 1967 ) t o s t udy seve r a l a d j uvant s f o r u s e
w i t h fowl cho lera vacc ine s . They f ound that a n t i gen emu l s i f ied in a n

o i ly adj uvant e l i c ited g reater protect ion t h a t aque ous s uspensions .

This f i nd i ng w a s s i mi l a r t o t h a t o f B a i n ' s . La t e r Hedd l e s t on i n
a s s o c i a t i o n w i t h Re i s inge r [ 1 9 5 9 ] ( s ee Cart e r , 1 9 6 7 ) u s e d mine ral o i l
and a r l a c e l a s an adj uvant f o r a f o w l cho l e r a va c c ine conta ining a

s ingle ( S e rotype 5 : A ) s t rain o f P . m u l t o c i da . This vacc ine was found

t o be e f f e c t ive aga i n s t cha l lenge f r om the same s t r a i n of o rgan i sm .
H oweve r , when used in t he f ie ld it was found t o be ine f fect ive due

t o the occurrence of " va c c ine breaks " . In hind s ight , these " vacc ine
b rea ks " we re not surp r i s ing bec ause later Nami o ka and
c owo rke rs [ 1 9 6 2 - 1 9 6 4 ] ( s e e Section 1 . 4 ) s howed t hat fowl cholera was

c aused by t h ree Se rot ype s o f P . mu l t ocida . A l t hough this informat ion

was not k n own to H e dd l e s t o n and Re i s i n g e r t hey did n o n e t he l e s s

s uspect t he invo lveme nt o f mo re t han one s t r a i n i n t he a e t i o logy o f

t he disea s e . In 1960 ( s ee Ca rte r , 1 967) t hey modi f ied t he i r vacc ine

b y adding a n ext r a s t r a i n of P. m u l t o c i da ( S e ro t ype 8a : A) . This

v a c c ine was found to be e f f e c t i ve in the field . They further

modif ied t h i s vacc ine b y subst itut i ng the o i l - a rl a c e l adj uvant with

a luminium hydroxide . They f ound that t h i s e l l icited protect ion f o r

t he s ame length o f t ime a s o i l - a r l a ce l ( approximately o ne ye a r ) but

did cause a s mu c h t i s s ue damage ( C a rt e r , 1967) C o n s e que n t l y

a lumin ium hydroxide h a s s ince bec ome the adj uvant o f cho i ce f o r mo s t
P. m u l t o c i da vacc ine s whe t h e r f o r f o w l c h o l e r a o r o t he r di s e a s e s
( Wa lke r a n d Foste r , 1981) .
 47

Heddl e s t on and Re i s i nge r ' s t w o - s e rotype -conta ining v a c c i ne a f fo rded

protect i o n in the f ield a lt hough it was l a t e r shown t h a t t h ree type s
o f P . m u l t o c i da a r e respons ible for fowl cholera . That i t wa s
e f fect ive could suggest that t he t h i rd t ype ( Se rotype 9 : A ) was not
present in the a re a s whe re f l o c k s were immuni zed . Ho weve r Serotype
9 : A was t he l a s t f ow l cho l e r a s t rain to be de sc ribed ( Nami oka a nd
Mu r a t a , 1964) wh i c h sugge s t s that it is not as i mp o r t a n t as
Serotype s S : A o r 8 a : A in caus i ng fowl cho l e r a .

S ince 1 9 6 0 P . mul t oc i da vacc ines have predominant ly been ' ba c t e r i n '

based . That is , made f r om k i l l e d c u l t ures or component s o f k i l led

cult ure s ( Ca rte r , 1 9 6 7 ) . The d i s e a s e s protected aga i n s t inc lude fowl

cholera , sh ipping f ever and s wine pneumonia . There a re many repo rt s
of succe s s or f a i l u re in immu n i z ing a n i ma l s aga i n s t P . m u l t o c i da
u s i ng b a c t e r i n s . Howeve r , a s a gene r a l r u l e it c a n be s a id t h a t
bact e r i n s a re not u s u a l ly e f f e c t ive un l e s s used w i t h a n adj uvant . I f
succe s s fu l , prot e c t i o n i s usua l l y only short term .

Unt i l t he last de c a de wh o l e - ce l l P. m u l t o c i da vacc ines were

re lat ively unde f i ned . That i s , t hey cons i s t ed of a n inact ivated c e l l

s u s pe n s i on s t anda rdi zed on t h e ba s i s o f c e l l numb e r s and b l e nded

w i t h a n a d j uvant . S u ch v a c c i n e s a c t by s t imul a t i n g b a c t e r i c i d a l
ant ibodi e s which a re di re cted a g a i n s t c e l l s u r f a c e a n t i gens a n d a s

s u c h t end t o b e s e ro-spec i f i c .

I f the number o f se rotypes o f a pat hogen i s l imited t he f o rmulat ion

o f a v a c c ine i s re l a t ive ly s i mple . Howeve r , when s e ve r a l s e r o t ypes

a re i nv o lved as is the c a s e w i t h P . m u l t o c i da i n f e c t i o n s of s ome

ho s t s p ec i e s , mu l t icomponent vacc ines mu s t be u s e d . The s e vacc ines
may not be pract i c a l . For example if a h igh numbe r of c e l l s for each

of s evera l s e r o t yp e s a re a dded t o a vacc ine , t oxic l e ve l s of

endot oxin may b e p re sent o r t he required inocu lum may b e exc e s s ively
l a rge . It w o u l d t he r e f o re be adva n t a ge o u s to p u r i f y immu n i z i ng

a n t i g e n s a nd s u c h a v a c c i ne h a s b e e n p re p a r e d f o r h a emo r rh a g i c

s ept i c a emi a . P u r i f ied capsul a r poly s a c c h a r ide i s not immunogenic in

 48

a l l anima l s ( P enn and Nagy, 1976) but when p u r i f ied f rom s t rains o f
P. mul t o c i da wh ich c a u s e haemo r rh a g i c sept ic aemi a , ie S e rotypes 6B
a nd 6 E ( t h e bovi ne - h a emo rrh a g i c - s ept icaemia s t rains ) , i t h a s been

s h own to b e immunogen i c in c a t t le ( P enn and N a gy , 1 97 6 ) . This has

b e en exp l o i t e d and a mu l t i - s t r a i n v a c c i n e c o n t ain ing t he pur i f ied
a n t igens ( B and E ) is n o w a va i l a b l e c omme r c i a l l y ( Wa l ke r a n d
Foster, 1981) .

The haemo r rhagic sept i c a emia vacc i ne discussed above i s except ional
b e c a u s e W i t h s ome o t h e r a n ima l d i s e a s e s , eg pneumo n i a in s heep

( Came ron a nti Beste r , 1984) , an e f fect ive w h o l e -ce l l mu l t i c omponent

vacc ine h a s been e f fect i ve .

I t has been shown that a combined infection o f

B o rd e t e l l a b r o n c h i s ep t i c a and d e rmo n e c r o t i c - t o x i n - p r o d u c i n g

P. mul t oc i da Type D w i l l induce progre s s i ve a t roph i c rh i n it i s (AR)

in pigs ( see Sect ion 1 . 5) . P e de r s e n a n d B a r f o d ( 1 9 8 2 ) vacc inated

p regnant g i l t s w i t h a whole - ce l l bacterin made from a DNT -produc ing

P. mul t o c i da Type D . T hey demo n s t rated that both t he g i l t and i t s

o f f s p r i n g w e r e p r o t e c t ed aga i n s t AR . H o w e ve r , it has s i nce been

f o u nd t h a t Type A s t r a i n s of P. m u l t o c i da can a l s o p r oduce DNT
( P i j o a n et a l , 1984) w h i c h s ugge s t s t h a t an ef f e c t ive AR v a c c ine

will need t o be composed of more than one s t ra i n o f P . mul t oc i da .

The de s i r a b i l i t y o f p r odu c i ng a n e f fe c t ive n on - t o x i c v a c c ine f o r

P. m u l t o ci da led t o at least one h ig h l y unort hodox a pp r o a c h

( E i s en s t e in [ 1 9 7 8 ] see P h i l l i p s a nd R iml e r , 198 4 ) . T h i s app r o a c h
invo lved t h e ext ra c t i o n o f r i b o s ome s f rom t he b a c t e r i a and u s in g
t hem a s a n immu n i z ing antigen . Contrary t o w h a t might reasonably be

e xpe c t e d , t h i s w a s h i ghly s u c c e s s f u l . Howeve r, in a mo re c r i t i c a l

s tudy o f t h i s app ro a c h , P h i l l ip s a n d Rimle r ( 1 9 8 4 ) reinve s t igated the

use of r i b o s o me s a n d s howed t h a t when h i gh l y p u r i f i e d , t he i r

immun i z ing c apac i t y w a s dimi n i s hed o r l o s t . However t h i s immun i z ing

c apa c i t y w a s r e s t o r e d not o n l y t o P . mul t o cida ribo s ome s but a l s o

Aspergi l l us spp r i b o s ome s a n d a v i a n r ib o s omes provided that t he s e
 49

w e r e exp o s e d t o t r ace amo unt s o f P . mul t o c i da l ipopo l y s a c c ha r i de

( LP S ) .

W i t h h i nds i gh t it is clear that t h e r i b o s ome s act as a potent

a d j uvant for s ome s u r f a ce ant igens of P. m u l t o c i da . Wh i l e t he

o r iginal repo rt that P. m u l t o c i da r i b o s ome s were a n e f f e c t i ve

a n t ige n to p rotect a g a i n s t P . mul t o c i da i s at lea st ( in one sense )
n o n e s enc e , neverthe l e s s i t i s c o r re c t in t h a t t h e y a c t a s potent

v a c c ines because t hey have a s t rong adjuvant e f fect on t race amount s

o f P . mul t o c i da LP S ant igens . This approach therefore seems t o be o f
p o t ent i a l u s e because r ib o s ome s t hemse lve s a re n o t t o x i c a n d t he
t race amount s o f LPS needed to be immunogen i c a re not toxic at these

levels .

Mo re recent l y , i e w i t h i n t he l a s t ye a r , t he po s s ib i l i t y o f a l i ve

P. mul t oci da vacc ine h a s been reexamined . The reade r may rec a l l that
l ive (or a t any rate not intent iona l l y k i l led) P . m u l t o ci da vacc ines
we re used by P a s te ur .

S h imi z u e t a 1 ( 1 9 8 7 ) u s e d u l t r a v i o l et a nd chemi c a l mut agene s i s t o

t re a t a s t r a i n o f DNT - p r oduc i n g P . m u l t o c i da T ype D . They t h u s

obta ined a temperat ure s e n s i t ive ( t s ) mut ant . Wild t ype s t ra ins grow
between 3 0 C and 4 1 C but the mut ant did not grow above 3 4 C . i e it
would not grow at phys i o log i c a l t empera t u re . This imp l i e s that t he
t s s t ra i n could be used a s a l ive vacc ine because it cannot grow in

the h o s t a n ima l and c o n s equent l y c a nnot c a u s e d i s e a s e . They t h e n

u s ed t h i s t s s t r a in t o immu n i z e m i c e a n d f ou nd t h a t the mice we re

p r o t e c t e d a ga in s t t h e w i l d t yp e s t r a i n . T h i s w o r k w a s done o n l y

recen t l y and as y e t t h e a pp r o a c h h a s not been ext e nded t o t he

immun i z a t i on o f a nima l s o f g re a t e r comme r c i a l impo rtance than mice .

I t i s however a promi s in g avenue .

D igiacomo e t a 1 ( 1 9 8 7 ) u s ed select ive media to i s o l a t e a s t reptomyc in

dependent ( Smd ) s t r a i n of P . m u l t oci da Se rotype 1 2 : A . They t hen g rew
t h i s s t r a i n in broth c u l ture a nd admin i s t e re d i t int rana s a lly into
 50

r a b b it s . T h i r t y da y s l a t e r t he y c h a l l e nged h a l f o f t he t reated
rabb i t s with t he equiva lent wild type . The rema inde r of t he t reated
r a b b i t s w e r e c h a l l e n g e d w i t h s t rept omyc i n s e n s i t i ve c e l l s of a

di f f e rent S e r o t ype ( 3 : A) . The rabb i t s t re a t e d w i t h t he w i l d type

1 2 : A a l l devel oped a mild nas a l infect ion which r e s olved a f t e r t wo
wee k s . T h o s e c h a l l e n ge d w i t h t he 3 : A s t r a i n de v e l oped a c h ro n i c
n a s a l i n f e c t i on wh i c h d i d not p r o g r e s s t o t he s e ve re c o n d i t ion .
U n v a c c inated cont ro ls a l l deve l oped s e ve re pa steure l l o s i s and some
died .

The s e re s u l t s s ug ge s t t h at t he S md s t r a i n m a y be e f f e c t i ve at

p r o t ect ing r abbit s aga i n s t seve re disease due t o T ype A s e rotype s o f

P. m u l t oc i da . Howeve r t o g ive complete protect ion t he r e s u l t s would
imp l y that a mixture o f s e rotypes a re necce s s a ry .

1.8 Antibio t i c Re s i stance and the Presence of P l asmids

The amount o f l ite rature c oncerning the antibiotic s e n s i t ivities o f

P. mu l t oc i da i s va s t ( C a r t e r and Ba i n , 1 9 6 0 ) but t he relat ionship o f

t h i s t o p l a smid c a r r i age h a s not been extens ive ly examined . Chang

and Carter ( 1 97 6 ) demon s t r a t e d t h a t mu l t i p l e drug re s i s t ance is
common ly found in s t r a i n s o f P . m u l t o c i da i s o l ated f rom ca t t le and
s w i ne . They showed that over 8 0 % of i s olates po s s e s sed re s i s t ance t o

o n e o r mo re o f the drugs S t reptomyc in , Penic i l l i n , Tet racycl ine and

C h l o r amphen i c o l . Re s i s t a n c e to St rept omycin w a s f ound to be t he mo st

f re quent . Be rman and H i r s h ( 1 9 7 8 ) i s o lated a s t r a in of P . m u l t o c i da

f rom f ow l c h o le r a o f t u r ke y s a nd s h o we d i t t o be r e s i s t ant t o

T e t r a c y c l i ne , S t rept omyc in and s u l p h o n a m i de s . They further

demon s t ra t e d t h a t it c a r r ied t w o re s i s t a n c e - c a r rying p l a s mids [ R

p l a sm i ds ] . One of t he s e was s hown to e n c o de re s i s t ance to

S t rept omyc i n and s u l p h o n amide s . The o t he r coded re s i s t ance t o a l l
t h re e of t he ant ibiot i c s . Both pla smids were s hown t o b e n o n
t r ansmi s s able ( Be rman a n d H i r s h , 1 97 8 ) .
 51

Hirsh, Ma r t i n a nd Rho a d e s ( 1 9 8 5 ) s u rveyed many t u r k e y s t ra i n s o f

P. mult ocida f o r pla smids . Approxima t ely 6 0 % c onta ined p l a smids a nd
1 0 % o f t h e s e enc oded r e s i s t a n c e t o T e t r a c y c l ine , S t rept omycin a nd

s u lphonamide s . T he s e R-pla smids w e r e n o n t r a n s mi s s a b l e . The s e

f i ndings a re s imi l a r t o both t h o s e o f Be rman and H i r s h ( 1 9 7 8 ) [ s ee
above ] who a l s o worked w ith P . mu l t o c i da i s o l a t ed f rom t u rkeys and
S i l ve r et a1 (1979) who demo n s t r a t e d R - p l a s mi d s in s t r a ins of
P. mul t oc i da i s o l ated f rom c a t t l e w i t h pneumonia .

The a b o ve di scuss ion shows that a s i g n i f i cant propo rt ion of

P. m u l t o c i da s t ra ins c a rry o n e o r mo r e p l a smids . F u r t h e rmo re a
s igni f i cant a nd growing propo r t ion o f t he se s t ra ins c a r ry plasmids

which enc ode r e s i s tance ( R- p l a s mids ) to Tet racyc l in e , S t rept omyc i n

and S u lp h o n a mide s . The s e a r e t h e p redomi nant ant ibiot i c s u s e d f o r
t he rapy and pr ophy l a c t i c t r e a t me n t of dis e a s e s c a u s e d by

P. mul t o c i da . T h i s sugge s t s that t he evo lut ion of re s i s t ant s t r a ins

i s cont inuing and in future t hey may become a ma j o r problem a s t he

use o f t h e s e a nt ib i o t i c s i s cont i nued . F o r example S u lph onamide s

have been a dded t o c h i cken feed t o prevent fowl cholera f r om

be coming e s t a b l i s he d in f l o c k s . This h a s p robably a s s i s ted the

selection o f s t rains of P. m u l t o c i da wh i c h c a r r y S u l p h o n amide
res i s t ance ma rkers .

Woodgye r ( 1 9 7 6 ) demo n s t rated t h a t s t r a i n s o f P . mu l t o ci da i s o lated

f r om c a t s i n New Z e a l a nd w e r e not r e s i s t a nt to S t r e p t omyc i n ,

S u lp h o n a m i de , T e t r a c y c l ine , C h l o r amp he n i c o l o r P e n i c i l l i n . This

would appea r t o be t h e only report o f antibiotic sen s i t i v i t ies being

det e rmined f o r P . m u l t o c i da s t r a i n s which have been i s o l ated f rom

New Z e a l and anima l s so it is not s u rpri s i ng that t he p r e s ence of R

pla smids in New Z e a la nd strains a l s o has not been repo rted .

 52

1.9 I s o l at ion a n d Ident i f ic a t i o n o f P . mul t ocida

M a n y s t r a i n s o f P . mu l t o c i da a re h i g h l y p a t hoge n i c f o r m i c e a nd
rabb i t s . Pure cu ltures o f t h e o rga n i sm c a n be rec overed f rom the se
a n ima l s a f t e r i n t rape r i t o n e a l i n o cu l a t i o n ( P ede r s e n a n d N i e l s en ,
1 983) . However t h i s method i s not a s e f f ect ive as u s ing s o l id media
f o r re a s ons wh i c h a re discu s s e d later ( see Sect ion 2 . 1 ) .

The p a s teure l l a e g row we l l on blood a g a r so t h i s i s the medium of

choice for t he i s o l a t ion of P . mu l t o c i d a f r om c l in i c a l spec imens .
P. m u l t o c i da i s di f f i c u l t t o i s o l at e i f the med i um u s e d do e s not
c o nt a i n b l ood ( C a r te r , 1984 ) H o we ve r s e l e c t i ve me d i a h a v e b e e n
de s c r i b e d for the isolat i on of P . mu l t o c i da f rom he a v i ly
cont aminated mat e r i a l s ( S ee Chapt e r 2 ) .

P. m u l t o c i da can , with expe r i e nce , be ident i f ied, i n mixed cult ure

on s o l i d medium . Typical c o l o n ie s , grown on 5 % horse blood aga r , a re

1 - Smm i n diame t e r a ft e r 4 8 h r s growth a t 3 7 C ( s e e F igures 1 . 3 a nd

1 . 4 ) . T hey a re r a i s ed, grey i s h in colou r , non haemo lytic and u s u a l l y

po s s e s s a smoo t h t o mu c o i d appea rance , depending on t he amount of
caps u l e produce d .

The P a s t e u re l l a c e a e a re f a c u l t a t i ve l y a n a e rob i c G r a m n e g a t i ve

coccob a c i l l i . They a re di s t i ngu i s hable f rom t he other two fami l ie s

o f t h i s group [ En t eroba c t e r i a c e a e a n d Vibri ona ce a e ] b e c a u s e t hey a re

oxida s e posit ive , nonmot i le and mo s t species ferment gluc ose to a c id

but w i t hout g a s product ion . T he pasteure l l a s a re d i s t ingu i shed f rom

Haemoph i l us because t hey do not requ i re the g rowt h f a c t o r s NAD a nd

Haemin ( Ca rte r , 1984) . The c o l on i a l morpho logy o f t he a c t inoba c i l l i

i s s im i l a r t o P . m u l t o c i da a nd i t c a n b e dif f i c u l t t o dist ingu ish

bet w e e n t he t w o . Howeve r t he y c a n be di f f e r ent i a t ed b e c a u s e t he
a c t i n o b a c i l l i p o s s e s s a u n i que ' s t i c ky ' coloni l texture ( Smit h ,
1974) .
 53

T h e Gen u s P a s t e u rel l a i s c omp o s e d o f s i x s p e c i e s (Carter , 1 98 4 ) .

P . m u l t o c i da is di s t i n gu i s ha b l e f r om t h e o t he r f i ve s pe c i e s
b i o chemi c a l ly and t he ma i n di st ingu i s hing re a c t i ons a re pre s ented i n
T able 1 . 5 . Colonies o f P . mul t o ci da on blood a g a r have a dist inct ive
o dour use f u l in its iden t i f i cat ion .

T able 1 . 5 D i f ferent i a l Charact e r i s t i c s of Pa s t e urel l a Spe c i e s . a

. ' . . ' ..

Ch aract e t' i st i cs M p H u A G

(3 Haemolys i s +
Growt h o n McConkey A g ar + +
I nd ole Prod uct i on + +
Urease Act i v i t y + + +
Ac i d f r om Lact ose V .. y ll

based or1 Carter ( 1 '384 )
'
' va r i a ble react i on '

LEGEND
M: P. m u l toci da
P: P. pneumo b'opi ea
H : P. haemo l yt i ca
U : P. tweae
A : P. aerogenes
6 : P. ga l l i nar'ttm
 54

CHAP TER 2

I s olat ion of P. mu l t o c i da f r o m t he Na s a l Cavit y : E v a l u a t i on and

Modi f i c a t ion o f a Selective Medium .

2 . 1 Int r oduct ion

P. m u l t o c i da c a u s e s ma ny d i s e a s e s i n di f f e r e n t a n i ma l s pe c i e s
( C a r t e r and Bain , 1960) and i t i s pos s ible t o iso l ate the o rganism
f rom sever a l organs . Depending on t he s ite of infect i o n , it may or

may not be a s soci ated with an exce s s o f other ba c t e r i a . eg it may be

p re s e n t i n pure ( o r nearly pure) c u l t u r e and in h i g h numbe r s in
s ubcut aneous abces s e s s .

I n cont r a s t n a s a l s wabs usua l ly cont ain ma ny species o f bact e r i a in

low numb e r s ( Nga t i a et a l , 1985; Ca rter and Bain , 1960) . The se may
i nc l ude bacteria which a re pre sent in g re a t e r numb e r s o r repl icate

mo re r a p idly than P . m u l t o ci da . Othe rs ( eg some mo t i le spec ies of

En t eroba c t eri a ce a e and Vibri on a ce a e ) can spread on t he s u r f ace o f an
aga r p l a t e . The p r e s e n ce of s u c h o r g a n i sms in t he nasal f l o ra

hamp e r s t he de t e c t ion o f P . m u l t o c i da b e c a u s e t h e y o v e r g r ow t h e

medium ( Morris , 1 9 5 8 ; Smith and Baske rvi l le , 1 9 8 3 ; Knight , P a ine a nd

S pe l l e r , 1983) . This problem can be s o lved by s e lect ing for

P. m u l t o ci da i n vi vo o r i n vi t ro .

Enc ap s u l a t e d s t r a i n s o f P . m u l t o c i d a a re pat hoge n i c f o r mice s o a

pure c u lt u re may be obt a ined by intrape r itoneal inoc u l a t ion o f n a s a l

ma t e r i a l . T h i s me t h od h a s s e v e r a l di s a dvantages . F o r examp l e , if
mo r e t h a n o n e s t r a i n o f P . m u l t o c i da i s present it s e l e c t s t he
s t ra in which i s mo s t virulent f o r mice ( P ede rsen and N i e l s e n , 1983)

and t he l a rge ' . n umbe rs o f m i c e requ i re d f o r rout i n e s c r e e n i ng a nd

i s o l at ion work ma kes this method cumbers ome and expens ive ( Pede rsen
a nd N i e l s e n , 1983)
 ss

Va r i o u s in vi t ro s e lect ive med i a f o r t he i s o lation o f P a s t e urel l a

spec ies h a ve been reported . The s e u s e a ba s a l medium i nc o rporat i ng
t he s e l e c t ive prope r t i e s o f a h i gh pH a n d ant imi c r ob i a l chemica l s

including antibiot ics (Cart e r , 1 9 67 ; Mo r r i s , 19S8 ; Smith a nd

Baskervi l l e , 1983 ; Kn ight , P a i n e a nd S p e l l e r , 1983; De Jong and
Bo r s t , 1 9 8S) . A s e lect ive me d i um ( 8 HPG) de s igned spec i f ica l ly for
the i s o l a t ion of P . mul t oci da f rom the na s a l t ract o f pigs h a s been
de s c r ib e d by Smit h a nd B a s k e rv i l l e ( 1 9 8 3 ) . I t is b a s e d on a l k a l ine

nut r i e n t agar [ pH 8 . 6] which a l s o cont a i ns four a n t ibiot ics vi z

Myc o s t a t i n , S O Uml - 1 ; Bacit ra c i n , SUml - 1 ; P o l ymix i n , 0 . 2ugrnl - 1 a nd

Gent amyc i n , 0 . 0 3 ugml - 1 .

For a s e l e c t ive medium t o be ef fective it mu s t h a ve t w o ma i n

att ribute s : it mu s t e f f i c i ent l y propa g a t e the ta rget o rgan i sm but
must s uppre s s the background f l o ra .

P. mul t o ci da colon i ze s many o f t he anima l species which a re found in

New Z e a l a nd t he re f o re an ide a l s e l e c t ive medium s h o u l d a l low t h e
i s o l a t i o n o f t he o rg a n i s m f r om a l l o f t he s e anima l s . T o eva l uate
s u c h a medium it i s necc e s s a r y to obt a i n i s olates o f P . mul t o c i da
f rom a v a riety o f anima l s , t o t e s t the s e l e c t ive mediums a b i l i t y t o

e f f i c i e n t l y propa g a t e t he i s o l a t e s and t o eva luate t he a b i l i t y of

the s e l e c t i ve me d i u m to inhibit t he growth o f c o n t amin a t ing

o rgani sms , espec i a l l y those p r e s ent i n the upper re s p i r at o ry t ra c t .

This c hapter evaluates t he s e lect i ve medi um of Smith a nd

B a s k e rv i l l e ( 1 9 8 3 ) and de s c r ibe s t he de v e l opment a nd t e s t ing o f a

modi f ie d medium f o r i s o lat ing P . m u l t o c i da f rom Ne w Z e a land an ima l s .

 56

2.2 Mate r i a l s and Methods

2 . 2 . 1 P repa rat ion of Media

BA ( B lood Aga r )

S e e appendix .

BA + I ndiv idual Ant ibiotics

BA p l a t e s con t a i n ing di f f e rent c o n c e n t rat ions o f one o f four

ant ibiotics were prepa red a s fol lows :

Medium Ant ibiot i c Concen t r a t ions

BA plus Mycostat i n : Uml - 1 2.5 5 10 25 so

[ BA+M ]

BA plus Bacit rac i n : Uml - 1 0 .25 0.5 1 2.5 5

[ BA+B ]

BA plus P o lymixin ugml - 1 0 . 01 0 . 02 0 . 04 0.1 0.2

[ BA+P ]

BA p l u s Gent amyc i n : ugml - 1 0 . 0015 0 . 003 0 . 006 0 . 015 0 . 03

[ BA+G ]

BA + 4 Ant ibiot ics ( BA+ 4 )

Ant ib i o t i c S o lut ion ( see 8 H P G Medium)

Liquef ied BA

The 8HPG ant ibiot ic s o l u t i o n wa s u s e d to prepare 1 0 0 % ' BA + 4

Ant ibiot i c ' p lates . D i lut ions o f t h i s antibiot i c s o lut i on were

used t o prepa re BA cont a i n i ng 5 , 10, 15, 20, 25, SO, 75 percent

s t rength media .

BHI B r o t h ( Brain Hea rt Infus ion )

 57

See appendix .

8 HP G Medium ( s elect ive medium as per Smith and Baskervi l l e , 1983)

Mycost a t i n ( Squibb ) : 5 0 0 O O OU
Bacit r a c in ( S igma ) 5 0 O O OU
Po lymix i n B S u lphate ( S igma ) 2 0 0 0 ug

Gentamy c i n Sulpha t e ( S igma ) 3 0 0 ug

D i s t i l le d Wat e r l O Oml
Nut rient Aga r ( pH 8 . 6 )

i. The ant ibio t i c s were di s s o lved in the di s t i l led water a nd

s t e r i l i zed by f i ltrat ion u s ing a 0 . 2 um f i lter .

ii . T h i s a n t ib i o t i c s o l u t i o n w a s s t o r e d at + 4 C i n a da r k

bot t le .

i i i . F o r use lml of the ant ibiotic s o lut ion was mixed with 9 9ml
l i quefied ( 5 0 C ) nut rient aga r . T h i s was dispensed equ a l ly
between f o u r st anda rd pet r i dishes .

Modi f i ed S e l e ct ive Medium ( SM )

A mixed antibiot ic solut ion w a s prepa red a s fo l lows :

Myc o s t a t in ( Squ ibb ) : 5 0 0 O O OU

Bac i t r a c in ( S igma ) 5 0 O O OU

Gentamycin S u lphate ( S igma ) 3 0 0 ug

D i s t i l led Wat e r l O Oml

lml o f t h i s s o l u t ion w a s mi xed w i t h 9 9ml l ique f ied ( 5 0 C ) BA

and di spensed between four s t a nda rd pet r i di she s .
 58

Nut rient Aga r [ pH 8 . 6 ]

Nut r ient Aga r [pH 7 . 4 ]

T h e me dium [ NA ] (Difco) w a s p r e p a r e d a s p e r manu f a c t u re r ' s

i n s t ruct ions . The pH was adj usted us i ng lM NaOH a nd t he medium

wa s d i s pensed i nt o l O Oml bott les a n d a ut oc l aved . For use t he

medium was me l t e d and 2 5ml a l iquots were dispen s e d between four
st anda rd pet r i d i she s a s requi red .

N . B . P l ates were s t o red at + 4 C f o r not mo re than one wee k .

2 . 2 . 2 P repa ration and St anda rdi zat ion of C u l t ures

Ten c u l t u re s [ s ee b e l o w ] of P. mu l t o c i da f r om a r a nge of h o s t s

l i s t e d b e l o w w e r e g r own , a s s ayed a n d s t o red u s i n g t h e f o l l o w ing

protoco l :

Cultures
1 . Fowl 6 . Sheep

2 . Fowl 7 . Goat
3. Bovine 8 . Cat
4 . S wine 9 . Rabbi t

5 . Dog 1 0 . Swine

i. Cu ltures were removed f rom - 8 0 C s t o rage a nd propagated on

BA f o r 1 2 h r s at 3 7 C .

ii . Colonies w e re t r a n s f e r re d t o Sml BH I b roth u s ing a

b a c t e r i o l og i c a l l o op . This was incub a t e d a t 3 7 C o n a n
orbit a l s haker ( at approximate ly 1 2 0 rpm) unt i l t he culture
had reached mid-logarithmic growth pha s e ( ie O.D = 0 . 2 at
S O O nm) .
 59

i i i . S t e r i le g lyce ro l w a s added t o g i ve a f i n a l concent r a t i o n

of 2 0 % . The mixture was mixed t h o roughly a nd l m l a l iquo t s
were s t o r e d at - 8 0 C .

iv . A l xlml a l iquot f rom each o f the ten cultures was u s e d t o

est abl i s h the viable count o n BA .

2 . 2 . 3 Eva luat ion o f BHPG Medium

i. One c u l t u r e f r om e a c h of ten d i f ferent s t o c k cultures of

P. m u l t o c i da [ s ee 2 . 2 . 2 ] was r emo ved f r om - 8 0 C s t o rage

and t ha wed .

ii . The c u l t u res were di luted in s t e rile BHI broth so that 5 0 -

lOO v i a b l e o r g a n i sms w e r e c o n t a i n e d i n a known v o l ume

within the range o f 2 0 - l O O ul .

iii . A v o l ume [ see ii . ] f rom e a ch of the ten c u l t u re s

suffic ient t o p r oduce 50 to 100 colonies on BA was
ino c u l a ted on to t he surface of ten of each of t he

f o l lowing aga r plat e s : 8 HP G , BA, NA [ pH 8 . 6 ] , NA [ pH 7 . 4 ] .

iv . U s ing a g l a s s s p r e a de r the i n o c u l a we re spread ove r the

surface of the p l a t e s and l e f t to dry .

v. The p l a t e s we re t h e n in ve r t e d and incubated a t 3 7 C f o r

4 8 hrs and the co l ony count s recorded .

2 . 2 . 4 The E f fect o f Ant ibi o t i c s on the G rowth of P . mul t o c i da and

the S uppr e s s ion of Othe r Organi sms

i. The n a s a l cavity o f ten New Z e a land Wh i t e rabbit s ( f rom a

P. m u l t o c i d a - f re e colony) w e r e individu a l ly s wabbed with

 60

cotton buds soaked i n B H I broth . Each s wab was pl aced i n

5ml BHI broth and t ra n s f e rred t o t he labo ra t o ry .

ii . T he b r o t h s c o nt a i n i n g t he s w a b s w e r e a g i t a t e d u s ing a
vortex mixe r .

i i i . One c u l t u r e from each o f t e n d i f f e rent s t o c k c u ltures o f

P. m u l t o ci da [ s ee 2 . 2 . 2 ] w a s remo ved f r om - 8 0 C st o ra ge
a nd t hawed .

iv . T he cultures were diluted in

a . s t e r i l e BHI broth and

b . rabbit nasal ma t e r i a l cont ain ing broth ( se e i i . )

s o that 5 0 - 1 0 0 viable o rgani sms were cont a i ned in a known

volume of di luent w i t h i n the range of 2 0 - l O O u l , for each

dilut ion of the ten c u l tures .

v. A v o l u me of each of the diluted cult u re s was t hen

i n o c u l a t e d on to t he s u r f a ce of s o me or all of t he
f o l lowing media : BA, BA+ 4 , BA+M , BA+B, BA+P , BA+G .

vi . The i n o c u l a we re di s t r i b u t e d o v e r t h e s u r f a ce u s ing a

g l a s s spre ader a nd the plates we re left t o dry .

vii . The plates were then i nverted and incubated 4 8 hr s at 3 7 C

and t he c o l onies o f P . mul t oc i da we re counted .

2 . 2 . 5 I nocu lat ion o f Modi f ied Se lect ive Medium (SM)

i. The n a s a l c avity o f t e n New Z e a l and Wh ite rabb i t s ( f rom a

P. m u l t oc i da - f re e colony) we re i ndividu a l l y s wabbed w i t h
cotton buds which had been s o a ked in B H I brot h . Each s wab
wa s p l a c e d in 5 ml B H I b ro t h and t ra n s f e r red t o the
l abo rat o ry .
 61

ii . The b r o t h s c o n t a i n i n g t h e s wa b s w e r e a g i t a ted u s i n g a
vortex mixe r .

i i i . One c u l t u re f rom e ach o f ten d i f fe rent s t ock cultures o f

P. m u l t o c i da [ s ee 2 . 2 . 2 ] wa s r emo ved f r om - 8 0 C s t o rage
and t h a we d .

iv . The c u l t u re s were di luted in

a . s t e r i le BH I broth and
b . rabb i t n a s a l ma terial cont a in ing broth ( see i i . )

s o that 5 0 - 1 0 0 vi able organi sms were conta ined i n a known

vol ume o f di luent within the r a nge o f 2 0 - 1 0 0 u l , f o r e ach

dilut ion of the ten culture s .

v. A v o l ume of each of the di luted cu ltures wa s t hen

inoculated respect ive ly o n t o t he surface o f each o f a BA
plate a nd an SM plate .

vi . Us ing a gla s s spreader t he i n o c u l a were di s t r ibuted over

the s u r f ace o f the plates and left to dry .

v i i . The p l a t e s were then inve rted, incubated 4 8 hrs at 3 7 C and

colon i e s counted .

2.3 Re sults

2 . 3 . 1 Evalua t i o n of B HPG Medium

An i n i t i a l expe r iment e v a luat ed t he e f f ic iency of B HPG medium f o r

propagat ing a r a nge o f P . m u l t ocida i s o lates de r i ved f rom di f f e rent

species . An i de n t i c a l i n o c u lum of t e n P . m u l t o c i da i s o l a t e s f rom
e i ght dif ferent h o s t spe c i e s was p l a t ed on four d i f ferent media ( vi z

a l k a l i ne n u t r i e n t agar plus f o u r a n t i b i o t ic s ( 8HPG) , a l k a l i ne

 62

nut rient agar ( NA-A) , neut r a l p H nut rient agar with neut ra l pH ( NA ) ,
blood a g a r ( BA ) ) . The number o f result ant co lonies w a s expre s sed as
a pe rcent age o f t h o s e growing o n blood a ga r . The re s u l t s a re shown
on Table 2 . 1 . Note that only 1 o f 1 0 i s o lates grew on B HPG medium .
More i s o l a t e s (7 out o f 1 0 ) g rew on ( NA-A ) which re p r e s e n t s B HPG
medium w i thout ant ibiotics and mo re ( 9 out of 1 0 ) grew if a neut ral
pH w a s u sed in addit i on t o no a n t ibiot ics ( NA) .

*
Table 2 . 1 The Ab i l ity of B H P G Medium to P r opaga te P . m u l t o c i da .

N o - - - - - - - - - - - - - - - - - - - - - - - - -.
... - - - - - - - - - - - - - - - - - - - - - - - l e o l a t e

M e d l 1 2 3 4 5 7 8 g 10

8HPG 0 0 Ill 0 0 0 0 0 0 80

NA-A 0 0 15 55 10 50 0 35 20 75

NA 40 35 20 35 30 10 15 35 0 1 05

BA 1 00 1 00 1 00 1 00 1 00 1 00 1 00 1 00 1 00 1 00

Res u l t s e x pressed as g rowt h [ No co l on i es r o unded t o t h e

nearest 5 ] com pared t o t h e n u m b e r o f co l on i es o n B A .

Legend

Or i g i n o f P. mu l t oc i da C u l t utes

8HPG : S e l ect i ve med i um o f Sm i t h 1. Fow l 6. S h ee p

a n d B a s kerv i l l e ( 1 383 ) 2. Fow l 7. Goat
< see Sect i on 2. 2. 1 ) 3. Bov i ne 8. Cat
NA-A N u t r i ent a g ar E pH 8 . 6 ] 4. Sw i re 9. R a bb i t
NA N u t r i ent a g ar E pH 7 . 4 ] 5. Dog 10. Sw i ne
BA B l ood a g ar
 63

I n a sub s e quent experiment the medium o f Smi t h and Ba s ke rv i l le ( 1 9 8 3 )

was mod i f ied by subst ituting bl ood aga r a t neut ra l pH f o r a l k a l ine

nut rient a ga r . This modi f i ed medium was t e s t e d u s ing a s e r i e s o f

di lut ions o f the mixed antibiot i c s for i t s ab i l ity t o (a) propagate
strains of P. mul t o c i da and (b) suppre s s othe r o rganisms . The
result s a re shown on T able 2 . 2 a nd 2 . 3 . I n Table 2 . 2 it can be seen
that a n a n t ibiot ic c o ncent r a t i o n of 2 5 % o r les s will not suppre s s
t he b a c kground f l o r a whe re a s a c oncent rat i o n o f 5 0 % w i l l s upp re s s

background f l o r a but i s inhibi t o ry f o r 3 o u t o f 1 0 i s o l a t e s ( note

that [ T a b le 2 . 3 , i s o l a te 6] even a c oncent rat ion o f 1 0 % inhibited
t he growth of one sheep s t r a i n ) . We conc lude that no dilut ion o f the
a n t i b i o t i c mi x t u r e ful f i l led the two n e ce s s a ry c riteria (to
propagat e the t a rget bact e r i a but suppre s s o ther o rgani sms ) f o r an
e f f e c t i ve s e l e c t i v e medi um . This led us t o t e s t t h e i n h ib i t o ry
e f fect o f the individual ant ibiotics on P . m u l t ocida i s olat e s .

2 . 3 . 2 The E f fect o f Antibiot ics on the Growth of P . mul t o c i d a on BA

An expe r iment was de s igned where the inhib i t o ry e f fe c t s of the f our

ant ib i o t i c s were t e s ted individu a l ly u s i ng t he f ou r mo s t s e n s i t ive
s t r a i n s of P . mul t oc i da as t e s t o rgan i sms . The re s u l t s ( T able 2 . 4 )

show that three ant ibiotics ( Myc o s t a t i n , Gen t amyc i n a nd Ba c i t r a c i n )

a re not s igni f i cant l y inhibitory even a t concentrat ions u s e d in 8 HP G

medi um . I n cont ra s t , no g r o w t h o c c u r r e d i n t h e p r e s e nce o f t h e

fourth ant ibiot i c ( P o l ym i x i n ) when this was p r e sent at t he

concen t rat ion ( 0 . 2 ugml - 1 ) used in 8HPG medium . T h i s ant ibiot i c was

i n h ib i t o r y t o one o vine s t ra i n o f P . mu l t ocida even at 0 . 0 2 ugml - 1

( one t e nt h o f the concent r a t i o n used in 8 H P G ) .
 64

Table 2 . 2 The E f f e c t s o f D i f fe rent Leve l s o f a Combinat ion o f

Ant ibiot i c s on P . mu l t ocida .

- - - - - - - - - - - - - - - - - - ft t l , l o t l a L e v e l - - - - - - - - - - - - - - - - - - - -

1 00"
I o 1 a t
p N p N p N p N p N

i 1 00 og 75 og 85 70 50 55 0 0

2 1 00 og 1 05 og 1 00 '30 1 05 '30 85 85

3 1 00 og 65 og 0 0 0 0 0 0

4 1 00 og 1 05 og 0 0 0 0 0 0

5 1 00 og 65 og 75 50 70 65 50 45

6 1 00 og 0 og 0 0 0 0 0 0

7 1 00 og '30 og 15 10 0 Ill 0 0

8 1 00 og '30 og 85 75 '30 75 0 5

9 1 00 og 20 og 20 15 0 0 0 0

10 1 00 og 1 10 og 80 75 80 80 '30 85

Res u l t s e x pressed a s I g rowt h [N co l on i es rounded t o t h e

nearest 5J compared t o t h e n u m b e r o f co l on i es on B R .

Legend

Scor i r,g

BR w i t h I 8HPG ant i b i ot i c og : P l at e overg rown b y

com ponent as i nd i ca t ed bac k g round f l or a

I noc u l a Or igi n o f P. m u l t oc: i da C u l t ur e s

P: Pure brot h cu l t ure o f 1. Fow l 6. Sheep

P. mul t oc: i da cont a i n i n g 2. Fow l 7. Goat
5 0 - 1 00c f u per i nocu l um 3. Bov i ne a. Cat
4. S w i ne 9. Rabb i t
N : P. mul t oc: i da f r e e r a b b i t 5. Dog 1 0. Sw i ne
nasa l swa b mat er i a l seeded
w i t h 50- 1 00 cfu P. mul toc:i da
per i nocu l um
 65

Table 2 . 3 The E f f e c t s o f Low Leve ls o f a Combinat ion o f Ant ibiot i c s

o n P . m u l t o c i da .

Note : T h e s e s t r a i n s w e r e t h e f o u r mo s t a n t i b i o t i c
s e n s it ive a s determined f rom Table 2 . 2 .

r - - - - - - - - - - - - - - - - - - A R t t t o t t o L e v e l - - - - - - - - - - - - - - - - - - - -

. . . , . . .
p N p N p N p N p N p N

3 1 00 og 95 og 1 00 og 90 og 85 og 60 og

4 1 00 og 1 00 og 1 00 og 1 00 og 95 og 95 og

6 1 00 og 70 og 0 og 0 og 0 og 0 og

9 1 00 og 95 og 1 00 og 90 og 90 og 20 og

Res u l t s e x pressed a s g rowt h [ N co l on i es round ed t o t h e

nearest 5] com pared t o t h e n u m b e r of co l on i es on B R .

Legend

Scor i ng

BR w i t h 8HPG ant i b i ot i c og : P l at e overg rown b y

component a s i nd i c a t ed bac k g round f l ora

I nocu l a Or igi n o f P. mul t oc i da C u l t u r e s

P: Pure brot h cu l t ure o f 3. Bov i ne 4. Sw i ne

P. m u l t oc i da c o nt a i n i n g 6. Shee p 9. Ra b b i t
50- 1 00cfu per i nocu l um

N : P. m u l toc i da f r e e ra b b i t
nasa l swa b m a t e r i a l seede d
w i t h 50- 1 00 c f u P. m u l t oc i da
per i noc u l um
 66

2 . 3 . 3 Eva luat i on o f Modi f ied Select ive Medium (SM)

From t he above r e s u l t s it was concluded that an e f fect ive s e l e c t ive

me dium might be compo sed of blood a g a r at a neut r a l pH c o nt a i ning
t h re e ant ib i o t i c s ( vi z Myco s t a t i n , B a c i t r a c i n a nd Gent amyc i n ) at
c o n c e n t r a t i o n s e qu a l t o t h a t o f 8 HP G medium . ie with n o P o lymixin
addi t i on . T h i s modi f i e d s e l e c t i ve me d i um ( SM) wa s e va l u a t e d by
p l a t ing s t anda rd inocula of ten P . mu l t o c i da t e s t s t ra in s w i t h and
w i t hout nas a l f l ora . The results a r e s hown in Table 2 . 5 and F igure
2.1. It can be seen ( T able 2 . 5 ) that f o r any test s t ra i n , a t least
6 5 % o f t he o rg a n i sms plated on BA c a n be recovered on SM . That is ,
SM p ropagated P . m u l t ocida with an e f f i c iency which we cons ide red t o
be s imi lar t o t hat of BA . I n a pa r a l l e l exper iment the s ame number
of o rgani sms ( mi xed w i t h nasal swab mat e r i a l ) wa s plated on BA and
SM . It w a s found ( Table 2 . 5 ) that no P . mu l t o c i da c o l o n i e s were

i de n t i f i e d on BA . The s e l e c t i v e me d i um p r o d u c e d i d e n t i f i a b l e
P. m u l t o c i da co lonies which were c ounted ( Table 2 . 5) . On the
s e lect ive medium a number o f the colonies rep re sented t he background
growth of o rg a n i sms wh i c h could not be readily quant ita ted because
t hey were too s ma l l ( s e e F i gure 2 . 1 ) . It should howeve r be noted

t h a t in t he s e lect ive medium w h i c h w a s h e a v i l y cont amin a t e d w i t h

n a s a l s wab mat e rial a t l e a s t 6 0 % o f t he tot a l number o f P . mu l t ocida

c o lonies present on blood agar we re obse rved .
 67

F igure 2 . 1 The S uppr ess ion of N a s a l Bacte r i a l Flora by a

S e lect ive Medium (SM)

A n a s a l s wab w a s t a ken f r om a rabbit a nd broken o f f

into broth . A low concent rat ion ( enough to g i ve
approx i ma t e ly 50 cfu) of P . m u l t o c i da wa s a?ded and

t he prepa rat i on wa s p l a t e d on B l ood Aga r ( le f t ) and

S e l e c t i ve Medium ( r i ght ) a nd in cubated f o r 4 8 h r s at

3 7 c .

Note t hat the s e l e c t ive m e d i um s u pp r e s s e d the

backgr ound flora but pe r m i t t ed the g rowth of
P. mul t o c i da colonies .
 Table 2 . 4 The E f fe c t s o f I n d i v i du a l Ant ib i o t i c s a t D i f f e rent Concent rat ions
on t h e growth of P . mul t o c i da .

Mycost at i n Bac i t rac i n Po l yra i x i n Gentamyc i n

Um l - 1 Um i - 1 ugm i - u g m l - < x h'l - l
I so l at e N BA 2!i 10 25 50 1,25 .50 C!i .0 1 .02 4 .1 0 .20 .1 5 .30 .60 45 4.0 1
3: Cat t l e 1 00 85 95 80 8 0 8 0 9 5 8 5 95 80 7 5 81{) 95 80 0 0 95 81{) 81{) 80 81{)

4: Sw i ne 1 1{)0 80 90 '30 80 80 80 8 5 9 5 9 5 9 5 7 5 95 95 0 0 95 80 80 80 80

6: Sheep 1 00 50 '35 '30 '35 7 5 '35 75 75 95 95 75 0 0 0 0 '35 7 5 95 90 75

'
9: Rabb i t l lll lll '3 5 Bill '35 80 81{) 95 95 91{) 95 95 95 95 91{) 20 I{) 95 95 95 91{) 95

Re s u l t s e x p r e s sed as g rowt h [N co l on i es
r o u n d ed t o t h e nearest 5] compared to the
n u mber o f co l on i e s o n B A .

0\
CD
 69

*
Tab le 2 . 5 . The Ab i l i ty of SM Medium to Re cove r P . mul t oc i da .

r - - - - - - - - - - - - - - - - - - - - - l o l a t e - - - - - - - - - - - - - - - - - - - - - - - - - -

11 . I a 1 ::>
.... 3 4 5 6 7 8 9 10

BA 1 00 1 00 1 00 1 00 1 00 1 00 1 00 1 00 1 00 1 00

SM 95 90 90 1 00 90 65 1 00 90 65 65

BA* er g er g og og og og og og og og

SM* 60 75 85 95 75 60 85 85 60 65

Res u l t s e x pressed a s g rowt h [No co l on i e s rounded t o t h e

nearest 5] compared t o t h e num ber o f co l on i es on B A .

Legend

O r i g i n o f P. nw l t oc i da C u l t u r e s

BA B l ood a g ar i nocu l at ed 1. Fow l 6. Sheep

with p ure c u l t ure ::>
'-'- Fow l 7. Goat
BA* B l ood a g ar i no c u l at ed 3. Cat t l e 8. Cat
w i t h nasal m a t er i a l 4. Sw i ne 9. Rabb i t
SM Mod i f i ed se l ect i ve 5. Dog 1 0. Sw i ne
med i um i noc u l a t e d w i t h
p u t'e c u 1 t ure
SM* Mod i f i ed se l ect i ve
med i um i noc u l a t ed w i t h
nasa l mat e r i a l

Note : The n umb e r s o f c o l o n i e s reco r ded f o r SM * plates did not

inc lude s pe c i e s of b a c kg r ound flora which were not

suppre s sed by the antibiot ic s . However i t c a n b e seen f rom

Figure 2 . 1 that i n spite o f o t he r o rga n i sms P . mul t o c i da

could be di st ingu i s hed .

2 . 4 D i s cu s s ion

The det ect ion of low numb e r s of P . m u l t o c i da i n the nas a l c a v i t y i s

di f f icult . To i n c re a s e t he e f f i c i e n c y o f de t e c t i o n a s e l e c t ive
me d i um c a n be u s ed be c a u s e it has t w o impo r t a n t p r op e r t i e s : it
 70

e f f i c i e n t l y propaga t e s t he requ i red bac t e r i a and supp r e s s e s other

o r g a n i s ms . S mi t h a n d B a s k e r v i l l e ( 1 9 8 3 ) u s e d a s e l e c t i ve me d i um

( 8 HPG ) . This had an nut rient aga r ba s e , w a s modi f i ed by ad j u s tme nt

t o a l k a l ine pH and conta ined f o u r antibiot i c s . It was deve loped f o r
use w i t h pigs .

Our i n i t i a l expe r ime n t s inve s t igated t h e r e l a t ive ab i l it y o f 8 HP G

and non - s e lect ive me dia to p ropagate ( ma i n l y New Zea land) isolates
o f P . m u l t oci da f rom a wide v a r iety o f a n imal spe c i e s . T h e re s u l t s
( Table 2 . 1) indi c a t ed that 8 H P G me d i um f a i l ed t o g r o w 9 of 10
i s o l at e s te sted but it s hould be noted t h a t the only s t r a i n w h i c h
g r e w w a s i s o l ated f rom pigs . T h e othe r porc ine strain d i d n o t grow .
Th i s data demon s t ra t e s that 8 HPG medium i s not sat i s f a ct o ry f o r the

i s o l a t ion of P . mul t o ci da at least f rom spec ies othe r than pigs .

The r e s u l t s ( T able 2 . 1 ) sugge ste d that f o r the e f f i c ient i s o l a t i on

of P. mult ocida it w a s n e c c e s s a r y t o mo d i f y t h e medium by the

add i t i on o f b l o o d a nd b y t he u s e o f a n e ut ral pH . T h i s c omb ined
cha nge gave an inc r e a s ed e f f ic iency of p ropagation of P . mu l t o c i da
f r om a s t a nda rd i n o c u lum ( Table 2 . 1 ) . These changes are c le a r ly
advant ageous f o r p ropagat ing pure c u l t u r e s o f P . mu l t o c i da but t he

me d i um i s not i nh i b i t o r y f o r cont ami n a t ing o r g a n i s ms p r e s e n t in

c l in i c a l mate r i a l such a s nasal swabs . H o weve r , when t he four
ant ibiot ics present i n 8 H P G me dium w e r e a dded t o t h i s mod i f ied

medium it suppre s s ed not only t he background f lora but a l s o mo s t of

t he s t andard test s t ra ins of P . mul t o c i da ( T able 2 . 2 ) . Furthermo r e ,

w h e n t h e mixture o f antibiot i c s were d i l uted t o 5 0 % , 2 5 % o r eve n a

l ow e r concent r a t i o n , t hey rema ined i n h i b i t o ry f o r s ome s t ra in s o f

P. m u l t ocida but below a concent ration o f 5 0 % f a i led t o s uppre s s t he

b a c k g r ound f l o r a . Thus , t he re wa s n o c on cent r a t i on o f t h e mixed

ant ibiotic which was non-inhibitory for P . mul t o cida and s uppre s sed
t he background f l o ra .

T h i s led us t o a s se s s the e f fect o f t he antibiot i c s s ingly . I n our

h a n d s t h re e o f t he a n t i b i o t i c s ( vi z Mycos t a t i n , B a c i t r a c i n and
 71

Gent amyc i n ) a t the f u l l concent ration de s c r ibed for 8 HP G medium did

not s ign i f i c a nt l y supp r e s s t he growth of any t e s t s t ra in of

P. mul t o c i da . I n c o nt rast t o t h i s the fourth ant ibiot i c ( P o lymixin )

was h i gh l y i n h i b i t o r y ( see Table 2 . 4 ) . These results led u s t o

f o rmu l a t e a modi f i e d medium ba sed o n 8 H P G . Howeve r t h i s modi f i e d
medium di f f e r s f r om 8HPG medium i n three re spect s : I t i s compo sed o f
a b l o o d aga r base w i t h a neut r a l p H and doe s not cont a i n P o lymixi n .
It r e s emb les 8 HP G medium in t h ree respect s : I t cont a i n s Myco s t a t i n ,
Ba c i t r ac i n a n d Gent amyc in a t t h e concent r a t i ons spe c i f ied b y Smith
a n d B a s ke rv i l le ( 1 9 8 3 ) . T h i s modi f i ed medium had only a ma r g i n a l

i n h i b i t o ry e f f e c t on any o f t he t e s t s t r a i n s but s uppre s s e d t he

b a c k g r ou nd f l o r a p re s ent i n n a s a l s wa b s t a ke n f r om r abbi t s ( s ee
Table 2 . 5 and Figure 2 . 1 ) .
 72

CHAP TER 3

I s o1at ion of P. mul t o cida , S e rotyping o f I s olates and Compa r i s on of

The s e by SDS - PAGE and Re s t riction Endonuc l e a s e Analy s i s ( REA) .

3.1 Int roduct ion

The r a nge o f h o s t s pe c i e s c o l o n i z e d by P . m u l t o c i da i s u n u s u a l l y
wide a nd the o rgan i sm a lmo st certainly c a u s e s s ign i f i c a nt di s e a s e in
a nima l s in New Z e a l a nd . Neve rthe le s s t he impo rt ance of P . mul t o c i da
in t h i s count ry h a s been l i t t le studied a nd is o lates f rom di f fe re nt

ho s t s p e c i e s h a ve n o t b e e n c o mpa r e d . As a cont r ibut i o n t o t he

a s s e s sment o f P . m u l t o c i da s t r a ins present in New Z e a land we have ,

in this sect ion, de s c r i b e d t h e isolat ion of P . m u l t o c i da f r om

s e ve r a l spe c i e s a n d c ompa red t he i s o l a t e s w i t h re f e re n c e t o t he

f o l l owing prope rt i e s :

A . Serotype s .
B . The i r protein a s demonst rated by S D S -PAGE .
C. The c l e a v a ge of t he i r DNA by Re s t r i c t i o n E n d o n u c l e a s e

Ana lys i s .

3 . 2 Materials and Methods .

3 . 2 . 1 I s olation and Ident i fication of P . mul tocida

3 . 2 . 1 1 Mate r i a l s

B l o o d Aga r ( BA )
B r a i n Heart I nf u s i on Broth ( BH I )
Mat e r i a l s f o r I den t i f ication Test s

See appendix
 73

Modi f ied Se lect ive Medium ( SM )

S e e section 2 . 2 . 1

3 . 2 . 1 2 Methods

I. I s o 1ation and Identi ficat i on o f P . mul t ocida f rom P ig and Rabbit

Lungs .

i. P neumo n i c l ungs we re obt a ined f r om pigs at s l aught e r a nd

f rom angora rabbit s which had died f rom pneumon i a .

ii . A sma l l p i e ce o f pneumo n i c l u ng t i s s ue wa s removed and

rubbed ove r one qu adrant of a BA plate . A bact e r io logi c a l

loop wa s t hen u s e d t o s t re a k t he i n o c u l a t e d mat e r i a l t o

obt a i n s ingle c o l onie s . The c u lt u re was incubated f o r
2 4 hours a t 37 C .

i i i . P . m u l t o c i da w a s p r o v i s i o n a l l y i dent i f i e d by c o l o n i a l
mo rphology, Gram and oxida s e re act ions a nd ident i f i c a t ion

was comp leted a s per Section 1 . 9 ) .

iv . P. mul t o c i da i s o l a t e s were c u l t u red in Sml BHI broth f o r

2 4 h r s a t 3 7 C o n a n orb i t a l s h a ke r ( s et a t approxima t e l y

1 2 0 cpm ) . Glyce r o l was added t o a f i n a l c oncent r at ion o f

2 0 % a nd the culture w a s s t o red at - 8 0 C i n 2ml a l iquot s .

II . I s o1ation and Ident ification f rom Rabbit Na s a1 Cavitie s .

i. The n a s a l t ract o f angora rabb i t s was s wabbed with c o t t on

buds s oa ked in BHI brot h .

ii . The b r o t h s cont a in ing the s w a b s we r e a g i t a t e d u s i n g a

vo rtex mixe r .
 74

i i i . 1ml a l iquo t s were inoculated o n t o one SM plate each . T he

inocu l um was spread and left t o dry .

iv . The p l at e s were incubated at 3 7 C for 4 8 h r s a nd examined .

v. P. mu l t o c i da - l i k e c o l o n i e s w e r e ident i f ie d a s de s c r ibed
above .

III . I s o lation and Ident ificat ion from Cat s and Dogs .

i. I s o l a t e s a l ready ident i f ied a s P . mul t o c i da were supp l ied

by t he M i n i s t r y o f Ag r i c u l t u r e and F i s h e r i e s ( MAF ) ,

P a lme r s t o n No r t h a s b l o o d a g a r c u l t u r e s s t r e a ke d f o r
s i ngle c o loni es .

ii . Seve r a l c o l o n ie s were re s t r e a ke d on to BA and propagated

for 2 4 h r s a t 3 7 C . The ide nt i f i cat i on of t he cultures as

P. m u l t o ci da was then con f i rmed as de s c r ibed above .

3.2.2 P rocedures f o r the IHA As say .

3 . 2 . 2 1 Mat e r i a l s

A l s eve rs S o lu t i o n ( pH 6 . 1 )

Glucose : 2 0 . 5gl - 1
T r i s odium C i t rate : S . O g l - 1

NaCl 4 . 2gl-1

C i t r i c Ac id 0 . 5 5gl - 1
D i s t i l led Water

The pH w a s a d j u s t e d u s i n g 0 . 1 M NaOH and t h e s o l ut i o n w a s

autoclave d a nd s t o re d a t + 4 C .
 75

Fo rma l in-PBS

F o rma l i n was mixed with PBS to a f i n a l concentration of 0 . 4%

and s t o red a t + 4 C in a da rk cont a ine r .

1 . 0 % Glutara ldehyde -PES

i. 2ml o f 5 0 % Glut ara ldehyde S o lut ion ( S igma ) was mixed with
9 8ml P B S [ see appendix] .

ii . The s o lu t ion was autocl aved a nd s t o red at + 4 C .

Mic r o t i t re pl a t e s

L inbro Tit retek 1 2 x8x0 . 2 5ml V bott omed p l a t e s . .

0 . 8 5 % NaCl Solut ion

See appendix

PBS ( P hosphate B u f fered S a l ine )

See appendix

P B S -A Solut ion

Bovine Se rum Albumin ( BSA) ( S igma ) : 0 . 2 5g

S odium A z ide ( NaN 3 ) ( S igma ) : l g
PBS t o l O O Oml

The c o mp o n e n t s were di s s o l v e d i n t he P B S , s t e r i l i z e d by

f i l t ra t i o n a nd s t o red a t + 4 C .
 76

P B S -B S o lut ion

T h i s wa s p r e p a red as per P B S -A S o l u t ion ( see above ) with t he

ommi s s ion o f BSA .

3 . 2 . 2 2 Methods

I. P r oduction of P . mul t ocida Antiserum in Hens .

S tep 1 : Prepa rat ion of Ant igen (Ba c t e r in) .

i. A c u l t u r e was removed f r om - 8 0 C s t o r a ge and propagated

1 2 h r s a t 3 7 C on b l ood aga r ( BA ) .

ii . F i ve de x t r o s e s t a r c h a g a r p l a t e s were inoculated with

c o l o n i e s us ing a b a c t e r i o l o g i c a l loop . T h e i n o c u lum w a s

dist ributed over t he surface u s i ng a gla s s spreade r .

i i i . The p l a t e s we re dried and incubated for 1 8 hrs at 3 7 C .

iv . Colonies we re sc raped f rom the p l ates u s i ng a s t e r i le f l at

edged spatula , s u s pended in app roximately t h ree volume s of

F o rma l i n - P B S a nd incuba t e d for 48hrs at 3 7 C with
occa s io n a l mixing .

v. A loop f u l l of the suspen s i o n w a s s t reaked on t o a BA p l a t e

and i n c ub a t e d f o r 2 4hrs at 3 7 C to c o n f i rm t h a t the
culture h ad been s t e r i l i sed .

vi . The s u s pe n s i on w a s diluted w i t h s t e r i le P BS t o a t u rb i dity

equ a l to Mc F a r land ' s St a ndard # 4 .
 77

v i i . The di l u t e d s u spen s i o n [ re f e r r e d t o a s a b a ct e r i n ] was

s t o red i n 3ml a l iqu o t s a t - 2 0 C .

S t ep 2 : Immun i z a t i on P rocedure .

i. 1 - 2ml b l o o d was c o l l e c t e d f r om t h e wing ve in o f each o f

two dome s t i c hens .

ii . Each s e rum was separated, p r e s e r ved ( by a dd i n g N a N 3

( S igma ) t o a f inal concent r a t i o n o f 0 . 1 % ) a nd s t o red at -
2 0 C .

iii . Each hen was inocul ated both i n t r a ve n o u s l y w i t h 1ml

bact e r i n a nd int ramuscularly ( O . Sml into each leg o f e a ch
hen ) with 1ml of ant igen in FCA p repa red a s f o l lows :

Freunds Complete Ad j uvant : 1 . 5ml

Who le Ce l l Bacte rin : O . Sml

* - 1
P u r i f ie d Capsule [ 1 0 0mgml J : O . Sml

* . .
Note : See sect1on I V for prepa rat 1on .

iv . I n t r av e n o u s i n j e c t i o n s o f 1 . 0 m l o f b a c t e r i n were g i ven

twice wee kly for up to three mon t hs or unt i l an a c c eptable

s e rum a n t ibody t i t re was reached . An a c cept able t it re was
de f ined as at lea s t a 1 6 fold i n c rease above the pret i t re

( see S e c t ion VI ) .
 78

S tep 3 : C o l l e c t ion of Ant i s erum .

Hens were e x s anguinated and the s e rum w a s s ep a r a t e d and

p r e s e rved b y a dding NaN 3 ( S i gma ) t o a f i nal c oncen t r a t i o n o f
0 . 1% . I t w a s t hen d i s pe n s ed i n Sml a l iquot s a nd s t o re d a t -
2 0 C .

II . S tandardization of Antisera .

S e r u m a n t i b o dy t i t r e s to T y pe s A, B, a n d D P . m u l t o c i da we re
mea s u red by the f o l lowing modi f i cation of t he techn ique de s c r ibed in
S ect i o n VI :

i. Us ing P B S -A s e r i a l two-fold dilut ions o f a s e rum were made

in t r i p l i c a t e in a microt i t re p l a t e . The d i l u t i o n r ange

used was f rom 2 to 2 0 4 8 fold .

ii . 2 5 u l a l i qu o t s o f T ype A , B a n d D s e n s i t i zed ( ie ant i gen

coated) GRBC s ( s ee S e c t i on V ] w e r e added to one row of

we l l s e a c h .

i i i . The p l a t e w a s roc ked gent l y a n d t h e c e l l s were l e f t to

settle [ approximately two hou r s ] .

iv . The t it re o f ant i s e rum wa s de f ined a s the great e s t s e rum

di lut ion which caused agglut inat ion .

v. Us ing P B S -B s o lut i on , an a l iquot o f each s t ock a n t i s e rum

was d i l u t ed so that it had a t i t re of 8 unit s . ie c a u s ed
agglut i n a t ion up t o a di lut i on o f 1 : 8 .

vi . I f a ny a n t i s e rum c ro s s -reacted w it h a het e ro l ogous ant i gen

it w a s a b s o rbed w i t h t he het e ro l ogous a n t i gen ( b e f o re
di lut ing ) a s f o l lows :
 79

Absorption o f Serum by Heterologous P . mul tocida St rains .

T he ant i s e rum was mixed w ith a 1 / 1 0 t h vo lume s a l ine-ext r a cted

capsule [ see Sect ion Ill] of t h e h e t e r o l o g o u s P . mu l t o c i da
ant igen and w a s retested f o r spec i f ic i ty . I f the c r o s s re a c t i on
w a s not e l imi n ated larger vo l ume s o f capsule we re added unt i l
n o c ro s s rea c t ion wa s obse rved .

III . P reparation o f Saline Extracted Capsule .

i. A c u l t u r e wa s removed f rom - 8 0 C s t o r a ge a nd propagated

1 2 hrs a t 3 7 C on b l ood agar ( BA ) .

ii . Ten dext r o s e s t a rc h aga r p l a t e s were inocu lated with

c o l o n i e s o f prop a g a t ed c u l t u r e u s i ng a b a c t e r i o l og i c a l
loop . The inocu lum w a s di s t ributed ove r the s u r f a c e u s i ng

a g l a s s spreader .

i i i . The p l a t e s were dried and incubated for 1 8 h r s at 3 7 C .

iv . Colonies were sc raped f rom t he p l ates u s ing a s t e r i l e f l at

edged s p a t ula and s uspended in app roximately one volume o f

PBS .

v. Hya l u r o n ida s e [ S i gma ] was a dd e d to g i ve a final

concent rat ion of 2 0 0 Um l - l a n d t he p repa rat ion was

incubated at 3 7 C f o r 2hrs w i t h occas ional mixing .

vi . The dige s t was hea t ed at 1 0 0 C f o r 1 h r , l e f t to c o o l a nd

cent r i f uged at 1 0 O O O xg f o r 2 0mins .

v i i . The s up e r n a t ant w a s c o l l e c t e d , p r e s e rved by adding NaN 3

( S igma ) t o a f i n a l c on c e n t r a t i on o f 0 . 1 % a n d s t o re d a t
+ 4 C .
 80

IV . P reparation o f P urified Capsule .

i. A c u l t u re was removed from - 8 0 C s t o rage a nd propagated on

blood aga r ( BA) for 1 2 hrs at 3 7 C .

ii. Sml BH I broth was inoc u l a t ed with colonies using a

ba c t e r io l o g i c a l l oop and incub a t ed at 3 7 C on an o rb i t a l
shaker ( a t approxima t e ly 1 2 0 rpm) unt i l t h e culture reached
mid- l oga r i thmic growth pha s e . That i s , it had a n opt i c a l

de n s i t y ( OD ) of a p p r o x i ma t e l y 0.2 at 5 0 0 nm u s i n g a
Spe c t r o n i c 2 0 .

i i i . 1 1 de xt r o s e s t a rch b r o t h w a s i n o c u l a t e d with 4ml o f t he

broth c u l t u re and incub a t e d a t 3 7 C for 1 8hrs on an

orbit a l s haker ( at approxima t e l y 1 2 0 rpm) .

iv. NaCl was a dded t o g i ve a final concent rat ion o f 2% .

Fo rma l i n was a l s o added t o give a f i n a l concent rat ion o f

0 . 4% .

v. This prepa rat ion was incub a ted a t 3 7 C f o r a further 2 4 h r s

and cent r i fuged at 1 0 O O O xg f o r 2 0mins .

vi . The supe rnat ant was reta ined and the pe l le t disca rded .

v i i . Hya luronidase [ S igma ] was added t o the s upe rnatant t o give

a f in a l c o ncen t r a t i o n o f 2 0 0 Um l - 1 . It was then incubated

f o r 2 h r s a t 3 7 C in a water b a t h .

v i i i . T he d i g e s t w a s p a s s e d t h r o u g h f i l t e r s o f succe s s ively
sma l l e r po re diame t e r down t o 0 . 4 5um .

ix . The f i l t rate was concent rated t wenty-fold u s i ng an Amicon

filter ( UM 1 0 ) . This was p e r f o r me d at + 4 C using
pre s s u r i z e d nit rogen ( N2 ) .
 81

x. The c o n c e n t r a t e w a s d i a l y s e d a g a inst s e ve r a l ch ange s o f

di s t i l le d water, a t + 4 C f o r Sdays .

xi . Sodium a c e t ate was added t o give a final concent rat ion o f

1% ( t h i s a ids the precipitat ion o f antigens ) .

x i i . 3 vo lume s o f methanol were added . The suspens ion was mixed

and a l l owed t o st and for Smins a t room tempe rature .

x i i i . The p re c i p i t ate w a s removed by f i l t r a t i o n u s ing Whatman

N 1 pape r and t he f i lt rate r e t a i n ed . The p r e c ip i t a t e

( which cont a ined mo s t o f the endo t oxin) was disca rded .

xiv . 1 v o l um e of a ce t o ne wa s a dd e d to the f i lt rate . The

suspe n s i o n wa s a l lowed t o s t and a t 4 C f o r 1 2 hrs . Dur ing
this t ime a precip i t a t e ( c ap s u l a r po lysaccharide ) f o rmed ,
sett led o u t and adhe red to t he s u r f a ce o f t he t ube .

xv . The t ube a nd conte n t s we re c e n t r i f uged a t 10 O O O xg f o r

3 0mins .

xvi . The s upe rnat ant was de canted and d i s c arded . The cent r i f uge

tube , w h i c h cont a i ned the pr e c ip i t ate ( pu r i f i ed caps u l e )

was then l e f t a t 3 7 C f o r 1 2 hrs t o dry .

xv i i . The pre c ip i t ate was removed f rom t he tube s by s c rap ing out
us ing a s p a t u l a , we i ghed and r e c o n s t it uted i n d i s t i l led
wate r t o g ive a 1 0 0mgml -1 s o lut i o n . Thi s was p r e s e rved by

adding NaN 3 ( S igma ) to a f in a l c o ncent ra t i on of 0 . 1 % a nd

stored at + 4 C .
 82

V. P reparation o f Sens it i zed Blood Cells .

S t ep 1 : P repa rat ion of GRBCs .

i. 2 0 ml of s h e e p blood was c o l lected in a h ep a r i n i z e d

' vacut a iner ' .

ii . The blood w a s mixed with l O Oml o f c o ld Al severs s o lut ion

a nd cent r i fuged at 6 5 0 xg at 8 C f o r 2 0mins .

i i i . The c e l l s we re washed s i x t ime s by repe atedly su spending

in 5 vo lume s o f cold 0 . 8 5 % NaCl s o lu t ion and cent r i fuging

a s above .

iv . The ce l l s w e re then s u s pended in ap p r o x i ma t e l y n i ne

vo lume s of s t e r i le c o ld P B S to g i v e an appro x ima t e 10%
suspens ion .

v. An equa l v o l ume o f a 1 . 0 % glut a r a l de hyde - i n- P B S s o lut ion

was a dd e d t o t h e w a s hed blood c e l l suspens ion . This
e f fect ive l y gave a 5 % blood ce l l s u spension .

vi . This was incubated at + 4 C for two hours with s low mixing .

v i i . The c e l l s we re cent r i f uged a t 2 5 C f o r 1 0 mi n s a n d t h e n

washed three t imes with c o ld s t e r i le PBS .

v i i i . After t he f i n a l wash t hey we re s u spended in approxima t e l y

nine volume s o f PBS -B s o lut ion ( t o g ive a n approxima t e 1 0 %

suspen s i o n ) a nd s t o red a t + 4 C unt i l required .

 83

S t ep 2 : Sen s i t i zat ion of GRBCs .

F o r each isolate

i. 0 . 2 ml o f a 1 0 % s u s pe n s i o n o f GARBCs [ f rom S t ep 1 ] we re

mixed with a n equ a l volume o f a 2 5 % s u spens i on of s a l ine

ext racted capsule [ f rom Sect ion I I I ] in PBS .

ii . The mi xtu re wa s in cuba ted at 3 7 C f o r one hour w i t h s lo w

mixing .

i i i . The c e l l s were t he n washed t h re e t ime s i n c o ld s t e r i le

PBS , cent r i fuging at 6 5 0 xg f o r l Omins each t ime .

iv . They were then suspended in 4ml P BS -A solut ion to g ive an

approx ima te 0 . 5 % suspen s i o n o f s e n s i t i zed cells and s t o red

at + 4 C unt i l requi red .

VI . P rocedure f o r the Indirect Haemagglut ination As s ay ( IHA) .

f o r each of Type A, B and D sera :

i. Us ing a microt i t re p l at e , 2 5 u l o f st anda rdi z ed ant ise rum

( cont a in i ng 8 haemagglut inat ing u n it s ) was di luted in t wo

fold s t e p s us ing P B S -A s o lut i o n t o give a 1 i n 8 dilut i o n .

ii . 2 5 ul o f s e n s it i zed GRBCs [ see S e c t ion V ] was added t o each

we l l . T he plate was rocked and left to s et t le

[ appr ox imately t wo hours ] .

i i i . A f t e r t h i s t ime t he caps u l a r po l y s a ccha r ide P . m u l t o ci da

isolate [ see r e s u l t s ] w a s det e rmined f rom t he p a t t e r n o f

agglut i n a t ion .
 84

3 . 2 . 3 P rocedures f o r S D S - PAGE Analys i s .

3 . 2 . 3 1 Ma teria l s

1 0 % Ace t i c Ac id S o lution

A stock s o l ut ion ( 101) was p r epared and s t o red a t r o om

t empe r a t u r e . U s e d s o l u t ion wa s r e c y c l e d by p a s s i ng t h ro ugh
deco louri z ing charcoa l .

Run n i ng Ac ryl amide

Acrylamide : 30 0 . 0g
Met hylene -bi s -Ac rylamide S . Og

D i s t i l led Wa t e r to 1 0 0 0ml

i. The a c r y l a mide wa s added t o a pp r o x i mat e l y 7 0 % o f t h e

D i s t i l le d Wat e r and s t i r red unt i l the s o lut ion retu rned t o

room t emperatu re .

ii . The Me t hylene -b i s -Ac ryl amide wa s added and t he prepa rat ion

was made up to volume with D i s t i l led Wate r , pas sed t h rough

one laye r of Wha tman N 1 pape r a nd s t o red at + 4 C .

S t a c k ing Ac ryl amide

Acrylamide : 3 0 0 . 0gl- 1

Methylene -bi s -Ac rylamide : 1 6 . 0g l - 1

D i s t i l led Wat e r

T h i s mixtu r e w a s prepa red a s per Run n ing Ge l ( see be l o w ) .

 85

Ammo nium P e r s u lphate Solut ion

A 10% s o l u t i on was p repared and kept f o r no l onge r t h a n o ne

hour .

B r a i n He a rt I n f u s ion Broth ( BH I )

See appendix

Cooma s s ie-Blue Reagent

Cooma s s i e -Blue G-2 5 0 O . lg

9 5 % Etha n o l 5 0 . 0ml
8 5 % wv - l P hospho ric Acid : l O O . Qml
D i s t i l le d Water t o l O O Oml

i. The e t h anol a n d phosph o r i c a c id were mixed and t hen the

dye w a s completely di s s o lved in the s o lut i on .

ii . T h e m i x t u r e w a s made up t o v o l ume , p a s s e d t h r o u gh two

l a ye r s of Wha tman N 1 paper and s t o red at r o om

tempe rature .

I s opropa nol S t a i n

I s opropy l Alcoho l ( P ropan-2 -o l ) 2 5 0ml

Ace t i c Ac i d ( Gl a c ia l ) l O Oml
Cooma s s ie B lue R-2 5 0 ( B r i l l iant B lue ) 0 . 4g

D i s t i l le d Water to l O O Oml

i. The I s opropyl Alcohol a nd Ace t i c Ac id were mixed t ogethe r .

ii . T h e Cooma s s ie B l u e dye w a s di s s o lved comp l e t e l y in t he

mixt u re be f o re ma k in g up t o v o l ume w i t h D i s t i l le d Water
a nd s t or ing at room tempe r a t u re .
 86

SDS Sample Bu f fe r

-Me rcapt oethanol : l O . Oml

Upper T r i s Bu f f er : 2 5 . 0ml
SDS : 6 . 0g
D i s t i l le d Wat e r t o l O Oml

The c omponent s were mixed and s t o red at room tempe rature .

1 0 % SDS S o lut ion

-l
A 10% wv s o l u t i on of S o d i um D o de c y l Sulphate ( Lauryl

Su lphat e ) ( S igma ) w a s prepared i n D i s t i l led Wat e r and s t ored at

room t empe rature .

TEMED

N , N . N 1 , N 1 -Tet ramethy lethylenedi a mine ( S igma )

T r a c k ing Dye

Bromophenol Blue : O . l Og

Glyce r o l : 8 . 0ml

D i s t i l le d Water t o 1 0ml

The c omponents were mixed and s t ored at room tempe r a t u re .

 87

T r i s -Glyc i n e Re servo i r Bu f f e r ( pH 8 . 3 )

T r i zma Base : 6 . 07g

Glyc i n e : 2 8 . 8g
SDS 2 . 0g
D i s t i l led Wat e r t o 2 0 0 0ml

The c omponent s were mixed a nd s t o red at room tempe r a t u re .

Lower T r i s B u f fer ( pH 8 . 8 )

T r i zma Base 18 . 17g

1 0 % SDS S o lution 4 . Oml

D i s t i l led Wat e r t o 1 0 0ml

This mixture wa s prepa red a s per Uppe r T r i s Bu f fe r ( s ee be l o w ) .

Uppe r T r i s Buf fer (pH 6 . 8 )

T r i zma Base 6 . 0 6g
1 0 % SDS S o lution 4 . 0ml

D i s t i l led Water t o 1 0 0ml

i. The T r i zma Base was added t o approximately 9 0ml D i s t i l led

W a t e r a n d s t i r re d u n t i l t he m i xt u re r e t u r n e d t o r o om

t empe rature .

ii . T he pH was adj usted with 1 2 N H C l a nd t h e m i x t u re wa s

b rought up t o vo lume and s t ored a t + 4 C .

 88

3 . 2 . 3 2 Methods

I. P reparation o f Samples f o r P rotein As s a y .

f o r e ach sample

i. A l O Oml B H I broth c u l t u re o f P . m u l t o c i da ( wh ich was in

t h e mid- l og a r ithmic p h a s e of g r o w t h ) wa s c e n t r i fuged at
10 O O O xg for 2 0mi ns , wa shed twice with cold PBS , and the
pel let s u spended in app roximately one vo lume o f c o ld PBS .

ii . The s u s p e n s ion w a s p l a ced in a n i c e bath a n d t h e ce l l s

were dis rupted by s o n i c vib ration at 7 0 cps f o r 3min s .

iii . The lysed cell suspens i on was s t o red at - 2 0 C .

II . P rotein As say .

i. The lysed cell mat e r i a l wa s removed f rom - 2 0 C s t o rage and

a O . lml a l iquot wa s di l uted ten- f o ld, thirty- f o ld and one
hundred- f o l d in 0 . 2 M NaOH .

ii . O . lml a l iquots o f t h e above d i l ut i o n s were heated f o r 3

mins in a boil ing w a t e r bath and left to c o o l .

iii . 5ml o f C o o ma s s i e - B lue Re agent w a s added t o e a c h o f t he

O . lml s amples and mixed .

iv . The a b s o rbance o f e a ch s amp le a t 5 9 5 nm w a s mea s u red and

t h e i r p r o t e in c o n t e n t c a l c u l a t e d f rom a s t a ndard c u rve

wh i ch wa s c on s t ructed f rom t he a b s o rbance s of a range o f

Bovine S e rum Albumen ( BSA) ( 0 - l O O ug/ O . lml ) w h i c h h a d been

a s s ayed u s ing t he met ho d de s c r ibed above .

 89

III . P reparat ion o f Gels . ( 1 7 0 mmx l 3 0mmxl 5mm)

A) Mould P reparat ion

Ge l moulds we re prepa red a s per I onas ( l 9 8 3 ) . The s e con s i sted o f

two suitable s heet s of g l a s s h e l d t ogethe r by 1 5 mm- t h i c k
pe rspex space r s coated w i t h pet roleum j e l ly .

B) P repa r a t ion and Pouring o f G e l Componen t s

Run n i n g Gel : A 10% a c r y l a mi de r u n n i n g ge l w a s p r e p a red a s

f o l l ows .

Lower Tris Bu f fe r (pH 8 . 8 ) 5 . Oml

Run n ing Acryl amide 6 . 7ml
D i s t i l led Wat e r 8 . 3ml
Ammon ium P e r s u lphate Soln O . lml
TEMED O . O lml

i. T h e bu f f e r , a c r y l a m i d e and d i s t i l l e d w a t e r w e r e mixed
toge ther by swir ling so a s not t o create bubbl e s .

ii . The ammo n i um p e r s Jp h a t e and T EMED were m i x e d in t o t he

mixt u re a nd an l l c m l o ng ge l w a s immed i a t e l y pou red by

decant ing suf f i c ient into the prepared mou ld .

iii . The mou l d was rocke d t o create an even s u r f a ce on t he t op

o f the ge l . A sma l l vo lume ( l . Oml ) o f di s t i l led water was

then di s t r ibuted ove r the s u r f a ce o f the mixture and t he

ge l was left to polyme r i z e .
 90

S t a c king Ge l : A 3 0 % a c ry l amide / 1 . 6 % Met hylene -b i s - a c rylamide

s t a c k ing ge l w a s prepared a s f o l lows .

Uppe r T r i s Bu f fe r ( pH 6 . 8 ) 2 . 5ml
S t a c k ing Ac rylamide l . Sml

D i s t i l led Wat e r 6 . 0ml

Ammonium P e r s u lphate Soln 0 . 0 5ml

TEMED O . O lml

i. T he b u f f e r , a c r y l a m i de and di s t i l l e d w a t e r we re mixed

togethe r by s w i r l ing s o a s not t o c reate bubb l e s .

ii . The ammo n ium p e r s u ph a t e and T EMED were mixed in t o t he

mixt ure and the s u r f a ce o f the ( po lyme r i z ed ) running gel

was was hed with app ro xima t e l y 0 . 5ml of the mixt ure after
t he wat e r ove r lay h a d been de c a nted . The mould was then
f i l led w i t h st acking-gel mixt u re to 5 . 0mm f r om i t s t op , a
1 0 t ooth c omb was i n s e rted to a depth o f 1 0 - 1 5mm and left

unt i l the ge l had polymeri zed .

i i i . The comb was care f u l ly removed, t he we l l s we re then was hed

out with 1 0 ml of T r i s -Glycine Buffer a n d t h e b o t t om

perspex spacer was removed

iv . T h e a s s emb l y w a s a t t a tched t o a di s c on t i nu o u s -bu f f e r

vert i c a l e l ect roph o re s i s t a nk ( s ee I o na s , 1983) and the
tanks were f i l led w i t h T r i s -Glycine bu f fe r .

IV . P reparation of Samples f o r E lectrophore s i s .

i. To a 1 0 0 u l al iquot o f lysed ce l l mat e r i a l wa s added 1 2 5 u l

o f S D S S amp le Bu f f e r a n d 2 5 u l T ra c k ing Dye . T h e mixture
was agit ated us ing a vortex mixe r and hea t e d at 1 0 0 C for

3mins ( t o solub i l i z e t he prot e i n ) .

 91

ii . The p repa ra t i o n was then coo led for Smi n s at room

temp e r at u re and cent r i f uged a t 2 0 0 0 xg for 1 0min s at room

tempe rature .

iii . U s i n g a m i c r o p i p e t t e , v o l u me s o f s u p e r n a t a n t w h i c h
*
cont a i ned 8 0 ug protein we re added t o the a c rylamide we l l s
( Se c t ion I I I ) .

*
8 0 ug had pre v i o u s l y been de t e rmined ( re s u l t s not
s hown ) as t he opt imum l oading o f P . m u l t o c i da protein
for a 1 0 % polyacrylamide ge l .

V. Gel Elect rophores i s and Staining .

i. The l oaded bu f f er tank wa s c o nne cted to a s u i t ab l e power

supply ( anode to t op tank ) .

ii . The g e l was e lect rophoresed at a constant cur rent o f 1 5mA

un t i l t he t r a c k i ng-dye f ront r e a c h e d t h e s t a c k i n g - ge l

runn i n g - ge l i n t e r f a c e . T he cu rrent w a s t h e n reduced t o

l O mA unt i l t he dye f r ont w a s 1 cm f rom t h e bottom o f the

ge l . That is, t h e dye f r o n t had e l e c t ropho r e s e d a c r o s s

1 0 cm o f running ge l .

i i i . The powe r source was swit ched o f f and the ge l wa s removed

f r o m t h e mo u l d , imme r s e d i n I s op r opano l S t a i n a n d l e f t

ove r n ight o n a shake r .

iv . T h e g e l w a s de s t a i n e d i n 1 0 % Ac e t i c A c i d s o l u t i o n and

phot ographed u s ing Koda k Techpan f i lm .

 92

3 . 2 . 4 P rocedures for Restriction Endonuc1ease Ana1ys i s (REA) .

3 . 2 . 4 1 Mat e r i a l s

0 . 2M EDTA

EDTA ( di - s odium s a lt ) : 2 2 . 3 3g
Gla c i a l Ace t i c Ac id
D i s t i l le d Wat e r to 3 0 0ml

i. T h e EDTA w a s mixed w i t h a p p r o x i ma t e l y 2 8 0 m l D i s t i l l ed

Wat e r , ad j u s t ed t o pH 7 . 2 w i t h G l a c i a l Acet i c Ac id and

b r o ught t o vo lume with D i s t i l led Wat e r .

ii . T h e s o l ut i o n was ste r i l i z ed by aut o c l aving and s t o red at

+ 4C .

E lect ropho re s i s (E) Bu f fe r l O x Concentrated

T r i zma B a s e ( S igma ) : 9 6 . 8 8g
EDTA 7 . 4 4g

S odium Acetate 8 . 2 0g
G l a c i a l Acetic Ac id

D i s t i l le d Water to 2 0 0 0ml

i. The Tri zma Ba s e , EDTA and Sodium Acet ate were d i s s o lved in
approximat e l y 1 8 0 0ml D i s t i l led Wat e r and a d j u s ted t o pH
7 . 8 u s ing Glacial Ace t i c Acid .

ii . The mixture was made up t o volume w i t h D i s t i l le d Wat e r and

s t o ed at + 4 C . For use i t w a s di l uted l O X in di s t i l led

water .
 93

-
P ronase 2 5mgml l S o lut ion ( S igma )

A s o lut ion w a s prepa red i n di s t i l led wat e r , incubated f o r 2 h r s

( t o remove DNAse act ivity a n d st o red at -2 0 C .

Re s t r i c t i on Endonuc lease (RE) B u f fer

As p r e s c r i be d by t he manu facturers of the rest r i ct ion

endonuclease used .

Runn ing B u f f e r

lM T r i s -HCl : O . l Oml
0 . 2M EDTA : O . O Sml
Glycerol : 2 . 0 0ml
1 0 % SDS Solut ion : O . O Sml

D i s t i l led Wat e r to l O . Oml

The componen t s were mixed and st o red at room t empe rature .

1 0 % S D S S o lution

A 1 0 % wv - l S odium Dode c y l S u lpha t e ( Lauryl S u lphat e ) ( S igma )

s o l u t ion was p repa red w i t h di s t i l led wat e r and s t ored at room

t empe rature .
 94

Sa l in e - T r i s -EDTA (S TE) Bu f fe r l O x Concentrated

SM NaCl Solut i o n : 2 0 . 0ml

lM T r i s -HCl B u f fer : S O . Oml

0 . 2 M EDTA : S . Oml

D i s t i l led Wat e r t o l O Oml

T he component s were mixed , ste r i l i zed by autocl aving and s t o red

*
at room tempe r a ture . F o r use it was di luted l O x .

*
Note : T h i s d i l u t i o n i s t a ke n i n t o a c c ount w i t h mo s t o f t h e
bu f fe r formu lae de s c r ibed in t h i s section .

T r i s - EDTA (TE) Bu f f e r

l M Tris -HCl B u f f er : 4 0 . 0ml

0 . 2M EDTA : 2 0 . 0ml
D i s t i l led Wate r to 4 0 0 0ml

The component s were mixed f resh l y f o r use .

lM T r i s -HCl Bu f f e r

T r i zma HCl : 4 7 . 2 8g

SM NaOH

D i s t i l led Wat e r to 3 0 0ml

i. T he T r i zma HCl was mixed with app r o x i ma t e l y 2 8 0 ml

D i s t i l le d Wate r , a d j u s t ed t o p H 7 . 5 w i t h G l a c i a l Ace t i c
Ac id a n d b rought t o volume w i t h D i s t i l led Water .

ii . The s o l u t ion was s t e r i l i z ed by autoclaving and s t o re d at

+ 4 C .
 95

3 . 2 . 4 2 Met hods

I. Extraction o f DNA .

i. A 5 0ml BH I broth culture o f P . mu l t o ci da which wa s i n the

late - l ogarithmic phase o f growth ( that is , had a n opt i c a l
de n s i t y ( OD ) of a pp r o x i ma t e l y 0 . 6 at 5 0 0 nm ) was
cent r i f uged at 1 3 S O O xg f o r 2 0mins , wa s hed twice w i t h 2 0ml
cold P B S , and t he pe l l e t s u s p ende d in S a l i ne - T r i s -EDTA
( STE ) buf fer t o approxima t e l y Sml .

ii . The s u s pe n s i o n was placed in a 1 5 ml c a pped c e n t r i fuge

t ub e , 1 0 0 u l of a 2 5mgml - 1 s o l u t i o n o f P r ona s e ( S i gma ) ,
2 00ul of a 10% SDS s o l u t i o n a n d 5 u n i t s o f DNAs e - f r e e

Ribo n u c l e a s e w a s a dded a n d t he m i x t u r e w a s incubated

ove rnight at 5 0 C i n a wat e r -bath .

i i i . 0 . 3ml o f a 3 . 4M s o lut ion o f s odium pe rch l orate w a s added

to the t ub e s and re incubated for 1hr . During this
i n c u b a t i o n pe r i o d a 2 5 : 2 4 : 1 phe n o l - c h l o r o f o rm- i s o amy l
a l c o h o l s o lut i o n ( E xt r a c t i on M i xt u r e ) w a s p re p a red a nd

equ i l i b r a ted by a dding 1 / 1 0 t h vo l ume o f S T E b u f f e r a nd

st a nding for 3 0mins .

iv . Sml o f Ext r a c t i o n Mixt u r e wa s adde d to t he t reated c e l l

suspe n s ion , t h e t ubes were capped a n d then rocke d unt i l a

milky emu l s ion was fo rmed .

v. The t ubes were left t o s t a nd ( ve r t i c a l l y ) f o r 1 0mins and

cen t r i fuged a t 10 O O O xg a t + 4 C for 2 0mins .

vi . Us ing s e r o l og i c a l pipe t t e s t he upp e r laye r ( aqueou s ) was

removed, re-ext racted unt i l c le a r a nd di a lysed a g a i n s t T E

bu f f e r a t + 4 C f o r t w o days .
 96

vii . T h e d i a l y s e d f ract ion was de c a n t e d int o s t e r i le 4ml

b o t t le s , incubated at 6 5 C f o r 1 0 mi n s ( t o n e u t ra l i z e
DNAs e ) and s t o r e d at + 4 C .

II . As s ay o f DNA .

i. 1 ml o f a 1 / 2 0s o l u t i o n o f dia lysed DNA i n TE bu f fe r was

*
p l a ced in a cuvette and it s ab s o rba n c e at the f o l l owing

wave lengths was me asured us ing TE bu f f e r as a blank :

2 5 8 nm . Abs o rbance o f DNA .
2 7 0 nm . Abs o rbance o f Pheno l .

3 0 0 nm . Abs o rbance o f P rote in .

* ' '
Us lng a Cec l l CE2 7 2 Spect rophot ometer .

ii . S u i t ably ext ra cted DNA prepa r a t i o n s we re deeme d to have

2 5 8 / 2 7 0 and 2 5 8 / 3 0 0 values of > 1 . 0 .

i i i . The DNA c o n t e n t o f t he p repa r a t i o n w a s c a l c u lated us ing

' *
t he fol lowlng f o rmu la :

[ DNAmgml - 1 ]

x dilut ion fact o r .

20

whe re OD opt i c a l dens ity at de f ined wave lengths .

20 DNA absorbance const ant ( 1mgml - 1 DNA has a n OD
of 2 0 absorbance units at 2 5 8 nm ) .

*
Brenner and Fa lkow ( 1 9 7 1 )

I n the above c a s e the d i l ut ion f a c t o r i s 2 0 s o t he f o rmul a

c ondenses t o :

[ DNAmgml - 1 ]
 97

III . Digest ion o f DNA with Restriction Endonuclease .

i. A volume of DNA s o lution cont a ining Sug DNA was p laced in

a 2ml Eppendo rf Mic rof uge t ube with RE Bu f fe r and mixed .

ii . 5 U Re s t r i ct i o n E n zyme ( i e 1 U per l ug DNA) w a s a dded ,

m i x e d by t a pp i n g t h e t u b e , pu l s e d i n a M i c r o f u g e and

incubated at 3 7 C f o r 4 5mins .

iii . The t ube was then incubated at 6 5C for l Omins (to

ina c t i vate t h e en zyme ) a n d c o o l e d r apidly a t - 2 0 C (to
prevent reanne a l ing o f the cut DNA ) .

iv . A 1 / 2 0 volume o f SM NaCl w a s added and mixed by t apping

t he t ube . 2 v o l ume s of c o l d ( - 2 0 C ) ethanol was a dded,

mixed by inve r s ion and le ft a t -2 0 C for at lea s t 2 h r s f o r

t h e DNA t o prec ipitate .

v. The t ube was cent r i fuged f o r l Omins ( in a Micro fuge ) , the

supe rnatant was de canted and discarded , the pellet washed

once w ith cold ethanol and left for 3 0mins at 3 7 C to dry .

vi . The DNA was s o l ub i l i zed by a dding 4 5 u l o f Run n i ng Bu f f e r

and m i xing by t apping t h e t ube . I t was t hen cent r i fuged

for l Omins in a Microfuge and a 4 0ul a l i qu o t of
s upe rnatant was removed, p l a ced into a we l l i n a n agarose

ge l and elect rophoresed .

IV . Preparat i o n of 0 . 7 % Agarose Gel s .

i. 0 . 7 g Ult rapure Aga r o s e (B i o -Rad) w a s mixed w i t h l O Oml E

Bu f f e r in a r o u n d - b o t t ome d 2 5 0 ml flask, b o i l e d unde r
re f lu x f o r Smins on a heat ing mant le and le f t t o c o o l t o
approximate l y 5 0 C .
 98

ii . U s ing Ce l l o t a pe a g e l mo uld w a s p r epared b y s t i c k i n g a

length o f t ape a round the pe r ime t e r o f a 2 0 cmx1 6 cm g l a s s

plate and s e a l ing one f a c e ( i e t he bottom o f t he p l a t e ) .

iii . The mould w a s leve l led a nd the coo led gel mixt ure decanted
in to it . Bubbles were removed u s ing a hot wire and a 1 0
t ooth ( 2mmx 1 0mm) comb was pre s sed i n t o on end o f the ge l
and left t o set .

iv . The comb and Cellot ape was removed . The plate and ge l were
p l aced i n a continuo u s -b u f f e r h o r i z ontal e lect rophore s i s
tank a nd E B u f f er w a s added unt i l t he ge l wa s s ubme rged .
DNA s amp l e s we re added to t he w e l l s and t he s y s t em w a s
conne cted t o a suitable powe r s o u r c e ( we l l s a t anode end)

for elect rophore s i s a s f o l lows .

V. Elect rophore s i s and Photographing Gel s .

i. A const ant volt age o f B O v was app l i e d a c r o s s the ge l unt i l

*
the bu f f e r f ront had t rave l led 1 5 cm . The power s ource was

di s c onne c t e d and t he ge l v i s u a l i z e d by s l iding o f f t he

g l a s s plate on to an u l t ra violet l ight box .

ii . A permanent record w a s made b y expos ing with T r i -X f i lm

f o r 1 5 s e c s a t an ape r t u re o f 4 . 5 a n d deve l op i ng a s p e r

manufacture r s instructions .

*
T h i s i s taken a s being RNA which h a s been p l aced in
a spa re we l l and visua l i zed us ing l ong-wavelength UV
l ight .
 99

3 . 3 Re sult s

3 . 3 . 1 I s o l at ion of P . mu l t ocida and Some Epidemi ologi c a l Aspects

S ome s t r a i n s of P . m u l t o c i da w e r e obta ined f rom ove r s e a s c u l t u r e

c o l lection s . The s e prototype s t r a i n s had been i s o lated f rom a range

o f anima l s ( fowl , buf f a l o , pig , goat , dog and s heep ) . Three of these
s t ra ins ( fowl , bu f f a l o and p i g ) were s e r o t ype s A, B and D
r e spect ive ly and were used to produce st a nda rd a ntise rum f o r the IHA
a s s ay ( s ee Sect ion 3 . 2 . 2 ) .

T o obt a i n f i eld i s o l a t e s f r om New Zea land a n ima ls we a t t empted t o

i s olate P . m u l t o c i da f rom a ra nge o f dome s t i c a n ima l s a n d obta ined a
t ot a l o f 7 2 i s o l ates f rom f ive spe c i e s . The s e were ( w ith numbe r of

i s olates i n b racket s ) : Dog ( 2 1 ) , Cat ( 1 0 ) , Deer ( 1 ) , Rabb it (25) and

S w ine (15) .

C a n i ne s t r a i n s w e r e i s o l a t e d f r om p rema t i n g v a g i n a l s wa b s . N i ne

f e l i ne s t r a i n s we r e i s o l a t e d f r om w o u n d s . The rema i n i n g f e l i ne
i s olate wa s f r om a pne umonic lung . The c e rv i ne s t r a i n wa s i s o l a ted
f rom a lung . These dog , cat and dee r s t r a ins were s uppl ied t o us by

The Mini s t ry of Ag r i c u l t u re and F i sheries ( MAF ) at P a lme rston North .

*
H oweve r mo s t rabbit and swine s t ra ins were i s o lated in t he course

of t he pres ent st udy .

*
One rabbit and one swine i s o l a t e were

suppl ied by MAF .

f
... -
. .
' (
 100

Isolates o f P . mul t oc ida from Rabbit s .

Isolates were obt a i ned f rom t h ree r a bb i t colonies l oc a t ed i n

d i f f e r e n t a re a s ( Patea , Wa i t o t a r a Va l l e y a nd Uppe r Hutt ) . Hea l t hy

a nima l s and anima l s which had c l inical s igns of re s p i r a t o ry d i s e a s e
we re c h o s e n a t ra ndom and t he i r na s a l c a v i t ie s s wabbe d . The s wab
mat e r i a l w a s p l a t ed on s e l ect ive medium ( s ee Chapter Two ) for t he
i s o l a t i o n of P . mu l t o ci da . I f the anima l h a d died f rom pneumo n i a a
piece o f pneumonic (ie conso l idated) l ung t i s s ue was p l ated on b l ood
aga r .

P. mul t o ci da w a s i s o l a t ed f r om t he n a s a l t ra c t o f r a bb i t s in a l l
three c o lonies . The results ( T able 3 . 1 ) show that the propo rt ion o f
d i s e a s e d rabb i t s w h i c h s h owed c a r r i age o f P . mul t oc i da was highe r
than t h a t found in healthy anima l s . This di f ference w a s s ign i f i c a nt

(p< 0 . 0 1 ) when the c omb i n e d r e s u l t s of t he t hree colonies were

analyse d . The di f f e rence was a l s o s ign i f icant f o r t he P a t e a ( p< 0 . 0 1 )

and Uppe r Hutt (p< 0 . 0 2 5 ) dat a when t reated s eparate l y . The rema i n ing

dat a ( Wa it otara Va l l e y ) was not a large enough s amp le s i ze to apply

s t a t i s t i c a l a n a l ys i s . F u r t h e rmo re ( r e s u l t s not shown ) , seve r a l of
the P a t e a rabb i t s w e re s u f f e r i ng f r om t o r t i c o l l i s , a s ympt om o f

inne r e a r infec t i on which is a ssociated with P . m u l t o c i da ( see

S e c t i o n 1 . 5 . 5 ) . T h e o rganism wa s i s o l ated f rom t h e n a s a l cavity o f

a l l o f t he s e anima l s .
 1 0 1"

T a b le 3 . 1 P . mul t o c i da S t rains I s o l a ted f rom Rabbit s .

Ori g i n Date o f Cor.d i t i or A n i ma l s PM

o f St t a i n I so l at i on o f An i ma l Tested Pos i t i ve

Pat ea June Hea l t h y 20 1

1 98 7 D i seased 33 17
Tot a l 53 18

Wa i t ot ara JLme Hea l t h y 3 0

V a l l ey 1 987 D i seased 1 1
Tot a l 4 1

Upper August Hea l t h y 43 1

Hut t 1 98 7 D i sea sed 13 4
Tot a l 56 5

PM = P. mu l t oc i da

P orcine I s olates

S w ine s t r a i n s were i s o l a t ed f rom pneumonic lung t i s s ue , obta ined at

s l a ughte r ( Kiwi Bacon Company ) , f r om pigs wh i c h came f rom at lea st

f i ve di f f e rent f a rms in t he Manaw a t u . Pneumo n i c lungs we re s amp led

once in w i n t e r and once i n s umme r . The re s u l t s a re s h o wn on T able

3.2.
 102

Table 3 . 2 The P r opo r t i o n s o f P n e u m o n i c L e s i o n s f r o m wh i c h

P. mul t oci da was I s o l a te d in Wint e r and S umme r .

Dat e Tot a l N L Lm g s w i t h Recovery o f

E x am i ned of P i gs L e s i ons P. m u l t oc i da

_t!O " __No "

J une 300 20 7 3 15
1 '386

December 1 00 30 30 14 47
1 '386

The propo r t ion o f l ungs with les i o n s in December ( 3 0 o u t o f 1 0 0 ) was

s igni f i c a n t l y di f fe rent (p< 0 . 0 1 ) than the pr oport ion f ound in June

( 2 0 out o f 3 0 0 ) . T he propo r t i o n of pneumo n i c lungs w h i c h conta i ned

P. mul t o c i da in December ( 1 4 out o f 3 0 ) w a s h ighe r than t hat f ound
in June ( 3 out of 2 0 ) but t h i s w a s not s ignif icant at t h e 9 5 % leve l
(p< O . 1 ) .
 103

3 .3.2 Se rotyping : Va l idat ion o f ! HA As s ay and its Appl icat ion t o t he

Typing of F ie l d S t r a ins

Us ing t he !HA a s s a y ( see Section 3 . 2 . 2 2 ) P. mul t o c i da i s o l ates were

s e rotyped . ie They we re a s s igned t o Type A, B, D o r were rega rded a s
Non-typable ( NT ) . T he re s u l t s of s e r o t y p i n g a re s hown i n t h e
f o l lowing two section s :

3 . 3 .21 Va l idating t he !HA Assay

We i n i t i a l l y a t t e mp t e d t o p r o du c e type- spec i f i c a n t i s e rum i n

rabbit s . This wa s unsat is factory ( see d i s cuss ion ) but we were
succes s f u l i n produc ing typing s e rum i n hens b y the me thod de s c r ibed

in Sect ion 3 . 2 .

P rototype s t ra ins ( se e Section 3 . 3 . 1 ) we re examined by t he s t andard

!HA a s s a y used to e s t a b l i s h the s e ro type o f isolat e s . The re s u l t s

a re sho wn in Table 3 . 3 .

Table 3 . 3 Serotyping P rototype S t ra ins o f P . m u l t o c i da by !HA As s ay .

A n i ma l of No of Res u l t of I HA
I so l at i on S t ra i ns Serot y p e Serot y p i n g

Bov i ne 1 s B

Fowl 2 A , r.k A, A

S w i ne 2 o ' D D, D

Sheep 1 nk A

Goat 1 nk D

Dog 1 nk NT

Legend NT : Non-t y pa b l e ( see Sect i on 3. 3. 2 )

nk : Serot y p e not known b y s u p p l i e r
: St ra i ns u sed t o prod uce st and ard ant i serum f o r I HA a s s a y
 104

T he a nt i s e r um prep a red aga i n s t t he p r o t o t ype s t r a i n s w e re Typ e

spec i f ic (ie Ant i - A s e rum r e a c t e d w i t h Type A b u t n o t Type B o r D
a nt i g e n etc) We c o n c l u de t h a t i n o u r h a nds t he I HA a s s a y c a n
dist ingu i s h A , B and D s t ra i n s . The rema ining four prototype s t ra ins
( vi z Fow l , S w ine , S h eep a n d G o a t ) were f ound to be of t he mo s t
p robable s e r o t ype c o n s ide r i n g t h e s pe c i e s f r om wh i c h t h e y were
i s o l ated . T hu s , sheep and go a t s a re known to be a s s oc i a t e d w i t h A

a nd D s t r a i n s which i s con s i s t an t w i t h our result s and f u rthe rmo re ,

dog s t r a i n s tend t o b e rough a n d there f o re untypable . We conc lude

that a l t hough the numbe r o f s t r a i n s o f known serotype i s sma l l t he

resu l t s v a l idate t he I HA a s s a y .

The I HA a s say was then used to s e r o t ype f ie l d isolates of

P. m u l t o c i da ( s ee S e c t ion 3 . 3 . 1 ) . The re s u l t s a re s h own i n T a b l e

3.4.

3 . 3 . 2 2 Se rotyping o f F ie ld I s o l a t e s

Table 3 . 4 S e rotype s o f Field I s o l a t e s as Determined by t he

I HA As say.

Host N of I so l at es of Each Serot y pe Tot a l

A B D NT* -

Dog 2 0 0 19 21

Cat 3 1 1 5 10

Pi g 0 0 4 11 15

Deer 0 0 1 0 1

Ra b b i t 0 0 0 25 25

NT = Non-t y p a b l e
 105

The s e re s u l t s a re dis c u s sed late r ( see Sect i o n 3 . 4 ) but t he f inding

t hat a l l (25) rabb i t i s o la t e s a re untypab l e was unexpect ed because
t he l i t e r a t re indi c a t e s that mo s t rabbit P . mul t o c i da i s o lates a re
t ypable . H o weve r , o u r re s u l t s might be e xp l a ined i f t h e i s o l a t e s
repre s en t ed one o r a f e w s t r a ins which had become dis s eminated . To

inve s t igate t h i s po s s ib i l ity w e examined a n d compared t he proteins

o f rabb i t i s olates by SOS -PAGE .

3 . 3 . 3 S O S -PAGE Ana lys i s o f S t r a i n s o f P . mu l t ocida

The p r o t e i n p a t t e r n s of a v a r i e t y o f m i c r o o r g a n i s ms h a ve b e e n
compa r e d u s i ng S O S - P AGE but no work h a s appa rent ly been done with
P. mu l t o c i da apa rt a l im i t e d s t udy of memb r a n e proteins of
exo t o x i g e n i c strains f r om s w i ne ( Lu g t e nburg , 1984) and the
c h a r a c t e r i zat ion o f l i popo l y s a c c ha r i de s in s ome r a b b i t s t r a i n s

( Ma n n i n g e t a l , 1986) . I f P . m u l t o c i d a w a s a homoge neous s pe c i e s

with respect t o prot e i n patterns then the t e chn ique could b e used t o
i dent i f y t he o rgan i s m . I f i s o l a t e s f rom a va riety o f host spe c i e s
w e r e he t e r oge nous between spe c i e s but homogeneous w i t h i n a spe c i e s
it c o u l d be used to ide n t i f y t h e s pe c i e s o f o r i g i n . If (as we
init i a l ly and corre c t ly a s sumed i n the present wo r k ) i s o lates f rom a

s ingle species of h o s t anima l a re heterogeneous it could be u s ed t o

ident i f y s t ra ins .

Th i s s ect ion examined the 25 rabbit i s o lates by S O S - P AG E to

e s t ab l i sh i f t he y represented few o r many s t rains o f t he o rga n i s m .

H o w e ve r , t o ob t a i n an o ve r a l l p i c t u r e o f the h e t e r ogene i t y o f
P. m u l t o c i da i n i t i a l e xpe r ime n t s examined s t r a i n s f r om di f f e re n t
h o s t s pecies .

One repre sentat ive s t ra in f rom each of f o u r hos t s was compa red . The
re s u l t s a re s hown i n F igure 3 . 1 . It can be seen that s ome bands a re
common t o a l l f o u r s t r a i n s . H oweve r the o ve r a l l p a t t e rns show t h a t
P. m ul t o c i da i s hete rogeneous when i s o l a te d f rom di f fe rent spe c i e s .
106

F igure 3 . 1 S O S -PAGE P rotein Ana lys i s o f P. mu l t ocida I s olated

f rom Several An imal Spec i e s .

The isolates ( left to r ight ) we re obt a i n e d f r om a

rabbit , pig, cat and dog .

N o t e t h a t t h e i s o l a t e s c a n be d i s t ingu i s he d de s p ite
t he presence o f some common bands .
 107

I n f u r t h e r expe r ime n t s ( re s u l t s not s h o wn ) some common bands were

f ound bet ween P . m u l t ocida and P . h a emolyt i ca so SD S -P AGE is not an
appr op r i a t e met hod t o use t o ide nt i fy the bacte rial s pe c ie s .

Eight r e p re sent a t ive st r a i n s f r om e a c h o f three h o s t s ( s wine , dog

and c a t ) were t h e n examined . T he re s u l t s ( F igure s 3 . 2, 3 .3, 3 . 4)

show t h a t s t ra ins i s olated f r om within e a c h of t he h o s t spec ies a re

het e r o geneous in that although many ba nds were common , e a ch o f t he

e ight i s o l ates had a unique ove r a l l pattern of bands .

Three s t rains o f P . mu l t o c i da wh ich had been i s o l a t ed f r om rabb i t s

of t h re e di f fe re n t colonies i n N e w Z e a l a nd we r e e x a m i n e d . The
re s u l t s a re s hown in F igure 3 . 5 whe r e it can be s een that t he
s t ra i n s a re d i f f e rent with respect to banding pat t e rn s . We t he re f o re
c o n c l ude that s t r a i ns o f P . m u l t o c i da i s o l a ted f rom rabb i t s ( l i ke

c a n i ne , f e l ine and porc ine s t ra i n s ) a re hete rogene o u s . T h i s led us

t o examine s t r a i n s f rom within indi vidu a l rabbit c o l o n ie s .

F o u r representat i ve s t ra ins o f P . mul t o c i da f rom e a c h o f two rabbit

colonies ( P a t e a a nd Uppe r H u t t ) w e r e c ompa red by S D S - P AGE . The
re s u lt s a re shown in F igu res 3 . 6 where it can be seen that s t r a ins

i s o l a ted f rom t he same rabb i t co lony were homogeneo u s . This f inding

has i mp l i c a t i o n s wh i c h a r e c o n s ide red in t he d i s c u s s i o n ( S e c t ion

3 . 4)

3 . 3 . 4 Epidemio l ogical Trac ing of P . mul t ocida by S D S - PAGE

S o f a r SDS-PAGE examin ation of P . mul t o c i da stra i n s f rom rabb i t s was

l im i t e d to n a s a l i s o l a t e s . T h e y we r e homoge n e o u s w i t h i n r a bbit

c o l o n i e s but hete rogene ous between colonie s . It is gene r a l ly a s s umed

t h a t du r i ng t h e c o u r s e o f di s e a s e P . m u l t o c i d a s p r e a d s f r om t he

n a s a l cavity t o the lungs ( p a r t i c u l a r l y with r abb i t s in a s t re s sed

condit ion ) . T h i s imp l i e s that the s t r a i n s c a r r i e d i n t he n a s a l
c a v i t y should b e iden t i c a l t o those which colon i z e t he l ungs . This
108

F igure 3 . 2 SDS -PAGE Analys i s o f P. mu l t o ci da Is olates from Swine .

Note t h a t t he i s o l a t e s c a n be di s t ingu i s hed de spite

t he pres ence o f s ome common bands .

109

Figure 3 . 3 SD S -PAGE Analys i s o f P. mu l t ocida I s o lates f rom Dogs .

Note t h a t t he i s o l a t e s c a n be d i s t ingu i s he d de spite

the pre se nce o f s ome common bands .

 -::.

0
110

F igure 3 . 4 SDS -PAGE Analysis o f P. mult ocida Isolates f rom Cats .

The i s o lates can be distinguished de spite the pre sence

o f s ome common bands .

111

F igure 3 . 5 SOS -PAGE Analys i s o f P. mult ocida Is olates from

Rabbits .

The i s o lates we r e de r i v e d f r om t h re e c olonies of

rabb i t s f rom ( le f t to right ) Patea , Wa i t o t a r a Va l ley
a nd Upper Hutt .

Note that the i s o l a t e s c a n be di s t ingu i s hed de s p ite

the pre sence of s ome common bands .

1 12

F ig u r e 3 . 6 SDS -PAGE Analys i s of P. mul t o c i da from Rabbit s .

Seve ra l I so l a t e s Derived f rom Rabbit s in Each o f Two

Colonies are Compared .

Four is olates ( left ) we re f r om a P a t e a r abbit co lony

and four ( r ight ) f rom an Upper Hutt colony .

Note t hat the i s o lates f r om a n y one r a bb i t c o l ony

c a n n o t be d i s t i n gu i s h e d b u t isolates f r om t h e t w o

colonies a re d i f fe rent .
 11s

w a s t e s t ed b y comp a r ing n a s a l a nd l ung i s o l a t e s de r ived f r om the

s ame r abbit c o l onies .

We c ompa red t w o n a s a l s t r a i n s o f P . mu l t ocida with t wo s t rains which

h a d been i s o l a t e d f rom t he l u n g s o f rabb i t s w i t h pneumo n i a . The

re s u l t s ( F igure 3 . 7 ) i nd i c a t e t h a t t he n a s a l a nd lung s t r a i n s a re
h omogene o u s w i t h respect t o t he i r p r o t e in patterns s o we conc lude
t h a t t hey rep res ent the s ame s t ra in .

3 . 3 . 5 The Sou rce of a P . mu l t oc i da I s o late Assoc iated with an

. . *
Epldeml c o f Snu f f l e s

The isolation of P. m u l t o c i da f r om P a t e a rabbit nasal s wabs

c o i n c ided w i t h a n outbre a k o f snu f f le s . This outbreak was exp l a ined
by t he impo r t a t ion of a new batch of rabbit s f rom Germany in Ma rch
1987 . S ince P. mu l t o c i da is a h e t e r o gene o u s spe c i e s and v a r i e s
b e t ween c o l o n i e s in New Z e a l a nd i t c a n be rea sonably a s s umed that

t he pat hogen i c s t rain o f P . mul t o ci da imported f rom Germany would be

d i f f e r e n t to local s t ra in s . The MAF l a bo r a t o r i e s had p re v i o u s l y

i s o l ated ( September 1 9 8 6 ) a s t r a i n o f P . mul t o c i da f r om t h i s co lony
( P a t ea ) . I f t he outbreak of snu f f l e s was cau sed by the int roduct ion

o f a new s t r a i n of P . m u l t o c i da de r i ved f rom the infected rabb i t s

t he n it w o u l d be reasonable t o a s s ume t h a t the MAF i s o l ate obta ined

be f o re impo r t a t ion would di f fe r f rom t he new st r a i n de r ived f rom t he

imp o rted rabb it s .

U s i ng SOS-PAGE we compared the protein pattern o f t he rabbit i s o late

w i t h t he pa t t e rns o f t h ree recent ( Ma rch 1 9 8 7 ) nasal isolates of
P. m u l t o c i da f rom t h e s ame rabbit c o lony ( P a t ea ) . T h e re s u l t s a re

s hown in F igure 3 . 8 . It c a n be seen that , cont r a r y t o expe c t a t i on s ,

t he s t r a i n s were homogene o u s . T h i s i s discussed l a t e r ( Sect ion 3 . 4 ) .

* See S e c t ion 1 . 5 . 5
114

F i gu r e 3 . 7 SDS -PAGE Analys i s of P. mu l t o cida from Rabbit s .

A comparison o f lung and nasal t ract isolates .

Four i s o l a t e s , f rom one rabb i t c o l o n y , a re compa red .

Two i s o l a t e s ( le ft ) were obt a i ned f r om t h e l ungs o f

= bb i t i h h rl rl i e d o f pneumon i a . T h e rema i n ing
i s o lates ( right ) o r i g i n a t e d f r om n a s a l s wa b s t a ken

f rom rabb i t s with resp i ra t ory disease .

The l ung and n a s a l t ract i s o l a t e s a re

indi s t inguishable .
115

F ig u r e 3 . 8 SDS -PAGE Analys is of P. mu l t o cida I s olates f rom

Rabbit s .

A comparison o f i solates obta ined be fore and a fter the

int roduction of anima l s , f r om ove r s ea s , to a rabbit

co lony .

F o u r i s o l at e s , f rom one rabbit co lony , a re compa red .

One i s o l a t e ( le ft ) wa s o b t a i ned f rom t h e l u n g o f a

r a bb i t , which h a d d i e d o f p n e umo n i a , before the

int roduct ion o f impo rted rabb i t s into t he co lony . The

rema i n ing i s o l a t e s ( r ight ) o r iginated f r om n a s a l s wabs
t a ke n f r om i mp o r t e d r a bb i t s wh i c h had cont racted

r e s p i ratory d i s e a s e a f t e r int roduct ion t o t h e c o lony .

Note that a l l f o u r i s o l a t e s a re indist i ngu i s h able .

 . ' .
... ..,. .
- - -

-
 116

3 . 3 . 6 Compa r i s o n of P . mu l t ocida Se rotype s w i t h SDS-PAGE a n d REA

T h e de t e c t i o n o f t he s e r o t ype o f a n i s o l a t e o f P . m u l t o c i da is a
l ab o u r - i nt e n s ive proce s s . I s o l ates of P . m u l t o c i da can be
d i s t inguished By SDS-PAGE a nd REA c an be u s ed t o dist ingu i s h othe r

o rg a ni sms . W i t h t h i s in mind we examined s e rotypes o f P . mul t o c i da

to see i f they could be c o r r e l a t e d w i t h e i t h e r S D S - P AGE o r REA

p a t t e rns .

U s i n g S D S - P AGE we ex ami ned repre s e n t a t ive s t r a ins o f P . m u l t o c i da

Type s A , B and D . The r e s u l t s a re s h own in F i gure 3 . 9 . It can be
s e e n that s ome bands a re common to a l l s e rotype s , howeve r no bands
w e r e un ique to any one s e rotype so we c o n c l ude t ha t " s e r o t yp i ng"

P. m u l t o c i da by SDS-PAGE of prote ins is not p o s s ible .

I t i s po s s i b l e ( e ven i f not p a r t i c u l a r l y l i ke ly ! ) that REA dige s t

p at t e rn s c o u l d be c o r r e l a t e d w i t h P . m u l t o c i da s e r o t yp e s s o we
e x amined represent at ive s t ra ins of P . mu l t o ci da Types A a nd D by REA
( see S e c t i o n 3 . 2 . 4 ) u s ing EcoRl . The re s u lt ( F igu res 3 . 1 0 and 3 . 1 1 )

s how that a l though cert a in bands a re common t o a l l t e s t s t ra ins o f

P. mul t o c i da , n o band i s un ique t o one s e rotype . T h i s s ituat ion w a s

a l s o f ound u s ing a di f f e rent s i x - b a s e c u t t ing enzyme ( Xho - 1 ) ( s ee

F igures 3 . 1 2 and 3 . 1 3 ) .

S ince ne i t he r t he S D S - P AGE or the REA p a t t e rn produced c o r re l at ions

in any obvious way w i t h the s e r o t ype o f P . m u l t o c i da we c o n c l ude

that P . m u l t o c i da c annot be " s e r o t yp e d " b y e i t he r S D S - P AG E o f

proteins o r by REA o f DNA .

1 17

F i gu re 3 . 9 S D S -PAGE Ana lys is of P. mult ocida S e ro type s A, B and D .

Note that t he protein banding p a t t e r n s cannot be

rel ated to their s e r o t ype s . It follows t ha t the
s e r o t ype o f a new i s o l a t e c a n n o t b e e s t a b l i s hed by
e xamining the p rote ins by SDS-PAGE .
118

F igure 3 . 1 0 Restrict ion Endonuclease Analys i s of P. mul t ocida .

a. A comparison o f s e rotype A i s olates us ing Eco Rl .

b . A comparison o f s e rotype D i s olates u s ing Eco Rl .

c. A comparison of serotype A i s olates us ing Xho 1 .

d . A comparison o f se rotype D i s olates us ing Xho 1 .

The le f t hand t ra c k in each phot ograph ( dige s t ed DNA

f r om P h a g e L a mb d a ) repre s e n t s mo l e c u l a r w e i gh t
ma rke r s .

Heterogeneous patterns we re obt a i ned for all

is olate s . i e n o t wo i s o l a t e s were ide n t i c a l . This

ext reme hete rogene ity indicates that t he REA pat terns

ca nnot be correlated with the s e rotype . i e REA o f the

DNA gives no informa t i on about t he serotype .

 a
b

Mr ( M dil l )
M r (Mdal )

c d
 119

3 . 4 D i s cuss ion

P. m u l t ocida is an o rganism which causes disease in many spec ies o f

a n ima l s . We i s o l a te d P . mul t o c i da f rom t h e spec ies i nve st igated s o
c l e a r ly P . m u l e o c i da i s u b i qu i t o u s i n N e w Zea l and anima l s . In our
l im i t e d s t udy t h e di s t r i b u t i o n o f s e r o t yp e s i n N e w Z e a l a nd w a s
s imi l a r t o that f ound ove r s e a s and in rabb i t s at lea s t , t h e s t ra ins
which co loni zed t he lungs were ident i c a l to n a s a l i s o lates .

The ma j o r problem in our hands wa s the production o f type - spec i f ic

ant i s e ra . Repeated attempt s ( data not shown ) were made at produ c ing

ant i s e rum in rabb i t s but w i t h l it t l e s u c ce s s . This prob lem had a l so

been encount e red by P r ince ( 1 9 8 5 ) when she a t tempted t o produce t ype

s pe c i f i c ant i s e rum t o P . h a emolyt i ca . She f ound that s a t i s factory
a n t i s e r u m c o u l d be produced in dome s t i c hens a l though c ross

react ions occurred . Fol lowing t he work o f P r ince ( s ee above ) we u s ed

dome s t i c h e n s t o p r oduce s a t i s f a c t o r y t ype - spec i f i c a n t i s e r a t o
P. m u l t ocida . Howeve r w e a l s o obt a ined c r o s s re act ing se rum but t h i s

wa s r emoved b y abs o rpt ion with heterologous ant igen s .

The h ope that the S D S -PAGE and REA pa t t e r n s could be co rrel ated with

the s e rotype s wa s not rea l i s e d so at present there i s no a lternat ive

to s e rotyping by the c l a s s i c a l met hod .

T h e S D S pa t t e rn o f individu a l i s o l a t e s w e re s o heterogeneous that

i n d i v i du a l s t ra i n s c o u l d be differen t i ated . This a l l o w e d s ome

ep idemi o log i c a l a spects of disease in rabb i t s to be s t udied . T h i s i s

c o n s i de red in t h e gene r a l di s c u s s ion .
 120

CHAP TER 4

The Detection o f T oxigenic Strains of P . mul t ocida Derived from New

Zealand Anima l s .

4 . 1 I n t roduct ion

At rophic Rh i n i t i s ( s ee Sect ion 1 . 5 . 6 ) i s a ma j o r c a u s e o f economic

l o s s in Europe an s wine he rds a nd a mi ld f o rm has been recogn ised in

New Z e a l a nd . T he cause of t h i s di s e a s e h a s been att ributed to

s t r a i n s o f P . mu l t o c i d a wh ich produce a heat labile exot oxin . This
exotoxin c a n be demon s t r a t e d in vi t r o because it p r o du c e s a
cytopathic e ffect ( C PE ) in Emb ryo n i c B o v ine Lung ( EBL) cells ( s ee
S e c t i on 1 . 4 ) .

Exotoxigenic s t r a i n s of P . mul t ocida have not been i s o l ated f rom New

Z e a l a n d swine he rds nor have t hey been i s olat ed f r om othe r spec i e s
in t h e world . It i s n o t c l e a r howeve r i f exotoxigenic s t r a i n s a re
c o n f ined t o pigs o r wh e t h e r t he a b s e n c e o f repo r t s o f t o xige n i c
strains in othe r s pe c i e s re flects t he a pp a re n t absence of a

s y s t emat i c sea rch f o r t oxin produc ing i s o l at e s f rom other spe c i e s .

This s e c t i o n e s t a b l i s he s a n in vi t ro a s s a y for t he exotoxin of

P. m u l t ocida u s i n g a s t anda rd o f exotoxigenic P . mul t o ci da a n d b a sed

o n t h e me t h od de s c r ibed by Rut t e r and Lut he r ( 1 9 8 3 ) . It h a s been

f u r t h e r modi f i e d b y u s i n g t h e me t h o d de s c r i b e d b y S a w a t a a n d
Kume ( 1 9 8 5 ) f o r p re pa ring c e l l - free f i l t r a t e s of the t e s t o rgan i s m .

Us i n g t h i s a s s a y s t ra in s w h i c h h a d b e e n i s o l a t ed f rom p i g s i n New

Z e a l and, ove r s e a s s t ra ins and Ne w Zea l a nd st ra ins o t he r t han s wine

were examined .
 121

4 . 2 Materials and Methods

4 . 2 . 1 P repa r a t i o n o f Reagent s and Media

Ac idic Gent ian Violet Solution

A 0 . 2 % wv - 1 s o l u t i o n o f gent ian v i o let in 2% acet i c a c id was

prepared and s t o red at room t empe r a t u re .

B i c a rbonate S o lu t ion (4 . 4 %)

S odium Hydrogen Carbonate 4 . 4g

D i s t i l led Wa t e r to 1 0 0ml

The compo nent s were mixed and dispensed in 2 0ml a l iquot s . The

l ids were f i rmly t i ghtened, t he p repa r a t i o n s were aut o c la ved

and then s t o red at + 4 C .

B l o od Aga r ( BA )

See appendix .

BH I Broth

See appendix .

Bovine Se rum

T h i s wa s c l a r i f ie d , s t e r i l i z e d b y p a s s i ng t h r o u g h a 0 . 2 um

f i lter and s to red i n 5 0ml volume s a t - 2 0 C .

Ce l l Cult u re Mic r o t i t re P l a t e s

F a lcon 9 6 -we l l f lat bot t om dispo s ab le .

 122

E agle s Base

This was prepared a s pe r manu f a c t u rers ( Gibco ) dire c t i o n s f rom

dr i - f o rm m a t e r i a l ( Gibco ) , s t e r i l i z e d by pa s s i ng t h r o u gh a
0 . 2 um f i l t e r and s t o red i n 3 8 0ml a l iquots at + 4 C unt i l

requ i red .

Min ima l Eagles Medium (MEM )

4 . 4 % B i c a rbonate Solut i on 2 0 . 0ml

TPB 4 0 . 0 ml
PSK 1 . 0ml
Eagles B a s e 3 8 0 . 0ml
Amphot e r i c i n B ( Smgml - 1 ) 0 . 2ml

Bicarbona t e S o l u t i o n , TPB, PSK, Eagle s B a s e a nd were prepa red

sepa rately ( see individual headings ) .

All components were added t o the Eagles Base .

N . B . The c o l o u r o f t h e me dium s ho u ld be t ange l o o r a n g e when

used . I f not then it i s requ i red t o restore the c o r rect pH
by bubbl ing s t e r i l e co2 t h rough the medium . Wa rm medium t o

3 7 C be f o re us ing .

Complete MEM Medium

This was p repa red a s per MEM w i t h t he addition o f 4 0ml B ovine

Se rum ( t o g ive a f in a l concent r a t i o n o f 1 0 % ) .

F i xing S o lut i o n

A 1 2 % s o l u t ion o f f o rma l i n i n P B S was prepa red a nd s t o red a t

room tempe rature .
 123

F re e z ing Mixt u r e

Complete MEM Medium 3 3 . 5ml

Bovine Se rum 6 . 5ml
D imethyl s u lphoxide ( DMS O ) l O . Oml

T h e c omp o n e n t s we r e di s s o l v e d i n t h e d i s t i l l e d w a t e r . The

p r epa r a t i o n w a s t h e n s t e r i l i z e d b y p a s s ing t h rough a 0 . 2 um

f i lt e r and stored i n 5ml volume s at -2 0 C .

P h o sphate Bu f f e red S a l ine ( P BS )

See appendix

P S K ( Pen/ Str / Ka n )

Pen i c i l l i n lxl 0 7 IU
St reptomycin l Og
Kanamyc i n l Og
PBS t o l O O Oml

The c ompo nent s were dis so lved in the PBS . The prep a r a t ion was
then s t e r i l i zed by p a s s ing th rough a 0 . 2 um f i l t e r and s t o red in

2ml volume s at - 2 0 C .
 124

Tryps in Ve r sene S o l ut ion ( TV)

NaC l 0 . 8g
KCl 0 . 0 4g
Glucose O . lg
NaHC0 3 0 . 058g
Phenol Red 0 . 0 2ml
T ryp s in 0 . 05g
EDTA 0 . 0 2g
D i s t i l led Wa t e r t o l O Oml

T h e c omp o n e n t s w e r e di s s o l v ed i n t h e di s t i l l e d w a t e r . The
p r e p a r a t i o n w a s t h e n s t e r i l i z e d b y p a s s ing t h r o u gh a 0 . 2 um
f i l t e r and s t o red in 2ml v o l ume s at - 2 0 C .

Trypt o s e P hosph ate Broth ( TP B )

T rypt o s e (Difco) : 2 0 . 0 gl

Glucose 2 . 0 gl
NaCl S . Ogl
N a 2 HP0 4 2 . 5gl
D i s t i l led Wat e r t o l O O Oml

The c o mp o n e n t s were mixed, dispen s ed in 4 0ml a l i qu o t s ,

a u t o c l aved and t hen s t o red a t + 4 C .

4 . 2 . 2 Met hods

I. Cultivation of Embryonic Bovine Lung ( EBL) Cells .

Not e : An Emb ryonic Bovine Lung w a s k indly donated by Gibco

(NZ) Ltd . This l ung was s t o red for t w e l ve hours ( on
i c e ) be fore u s e .
 125

i. A s ma l l port ion of t i s sue ( approx 5 cmx5 cmx1 cm) wa s removed

f r om a lobe o f a f o e t a l c a l f l u ng ( t he f o e t u s w a s aged
b e t ween one and two mont h s f rom t e rm ) .

ii . The t i s sue w a s wa s hed w i t h MEM .

i i i . Sma l l piece s ( app rox 5mm3 ) were removed, washed in f r e s h

T ryps in Ve r s ene S o l u t i o n ( TV ) a n d t r ans f e red t o a 2 5 0ml
c o n i c a l f l a s k which conta i ned a sma l l magnet ( " f le a " ) .

iv . The pieces were washed t w i ce in TV . F resh TV w a s a dded and

s t i rred at 3 7 C u s i ng t he ' flea ' wh i c h was s pun
e c cent rica l l y ( t o a i d d i s rupt ing t he t i s s ue ) u nt i l t he
medium bec ame turbid due t o individual cel l s .

v. T h e medium w a s then t r a n s f e r red, u s ing a p i p e t t e , to a

s t e ri le c e n t r i f u ge t ub e in an ice bat h . The removed

( t u rbid) medium wa s rep l a c ed with f resh TV and t h e proce s s
w a s repeated f i ve t ime s .

vi . The pooled s u spended c e l l s were cent r i fuged f o r 1 0 minut e s

a n d the s upe rnatant di s c a rded .

vii . The pe l let was re s uspended in approximately 3 0 v o lume s o f

Complete MEM Medium .

viii . The re s u s p e n ded c e l l s were p o o l ed . Sml a l i qu o t s were

di spensed i n t o 5 0ml t i s s ue culture f l a s ks a nd inc ubated i n

5 % co 2 a tmo sphere a t 3 7 C .

ix . F l a s ks were examined da i l y f o r t he adherence and growth o f

t h e c e l l s i n t o monolaye r s .

x. Monolayers not requi red f o r immediate use were p a s s aged t o

o b t a i n a s t o c k o f c e l l s which were t hen s t o red ( s ee next
s e c t ion) in l i quid N 2 .
 126

II . S t o r age o f Cu1tured Ce11s .

i. The mon o layer was t ryps i n i zed and the c e l l s were suspended
in 5ml Complete MEM Medium .

ii . The ce l l s we re di l u t e d t o g i ve a f i n a l c o n cent rat ion of

1x1 0 7 ml - 1 .

i i i . 0 . 5ml a l i quot s o f F re e z ing M i x t u re w e r e d i spensed i n t o

s t e r i l e g l a s s ampou l e s . These were placed on i c e a n d 0 . 5ml
of c e l l s u spen s ion w a s added t o each .

iv . The ampoules we re s e a led, placed in an ins u l ated conta ine r

which was placed f o r 1 2 h r s at - 8 0 C .

v. The ampoules we re t hen t r a n s fe r red t o LN 2 .

III . S ource of P . multocida I s o lates .

S t r a ins o f P . m u l t o c i d a were obt a i ned f rom a ra nge o f anima l s

as de s c r i b e d i n S e c t i o n 3.3.1. A t ot a l of 7 2 N e w Z e a l a nd

i s o lates we r e obt a i n e d f r om f i ve s pe c i e s . T h e s e were

I
( w ith

number o f i s o lates in b r a c ket s ) : Dog ( 2 1 ) , Cat ( 1 0 ) , Deer (1) ,

Rabbit (25) a nd Swine ( 1 5 ) . In addit ion e ight prototype st rains

we re imported . The s e s t r a i n s had been i s o l ated f rom a range o f

a n ima l s ( fo w l , bu f f a lo , p i g , goa t , dog a nd sheep ) . One porcine

s t ra i n , k n o w n to p r o d u c e e x o t o x i n , was used a s a p o s i t i ve
c o nt rol .

IV . P reparation o f Cell-Free F i ltrates o f P . mul t ocida .

i. A culture was removed f rom - 8 0 C s t o rage a n d propagated on

blood agar ( BA ) f o r 1 2 h r s a t 3 7 C .
 127

ii . Co l onies were i nocul ated into 1 0ml o f B H I b roth and t h i s

wa s t he n i n c u b a t e d at 3 7 C on an o rb i t a l shaker ( at
app roximately 1 2 0 rpm) f o r 4 8 h rs .

i i i . The cells were dis rupted by sonic vibration f o r 1 0mins at

7 0 cp s and the p repa rat ion was c e nt r i fuged for 6 0mins at
10 O O Oxg at 4 C .

iv . The s upe r n a t a n t w a s c o l l e c t e d , p a s s e d t h r o u g h a 0 . 2 um
f i l t e r and s t o r e d at + 4 C f o r up to 4 8 h rs .

V. P rocedure for the in vi tro As s ay of P . mu l t o c i da in Embryonic

Bovine Lung Cells ( EBL as say) .

i. A s uspens ion o f EBL c e l l s con t a ining between 1 . 5 x 1 0 5 and

2 . 0 x 1 0 5 c e l l s . ml - 1 in Complete MEM w a s prepa red .

ii . P. mul t ocida c e l l - f ree f i lt rates ( s ee above ) were diluted

10, 30, 100, 300 and 1 0 0 0 f o l d u s i ng t h e d i l uted c e l l

s u spens ion a s d i luent . Toxin -free cont r o l s were included .

i i i . A 0 . 1 5ml a l iqu o t o f e a c h d i lu t i o n wa s dispen sed into t he

we l l s of a c e l l culture mi c rot i t re p l ate a nd incubated at

3 7 C in a humidi f ied- 5 % co 2 a tmo s ph e re unt i l mon o layers

were f o rme d i n t he we l l s which r e p re s e n t e d n e g a t ive

cont rols .

iv . The cells were then f ixed, stained ( see below) and

e xamined mic r o s cop i c a l l y f o r t he p r e sence o r absence o f a

cytopathic e f fe ct .

Fixing artd Staining EBL Cel l Cultures

i. Medium was removed f rom the plat e s .

 128

ii . 2 - 3 drops of a s o lut ion conta ining equ a l volume s o f F ixing

S o l u t i on and Acidic Gent ian V i o let Solution were di s pensed

i nt o e a c h we l l and the p l a t e w a s i n c ub a t e d f o r 2 h r s at
3 7 C in a humidif ied cont a i ne r .

i i i . The we l l s were washed with w a t e r and dried at 3 7 C .

4 . 3 Results

Va l i dation o f the EBL As s ay and i t s Appl i cat ion to the S c reen ing o f
F ield Stra in s o f P . mul t o ci da for Exo t oxin P roduction .

I n i t i a lly we a t t empt ed t o produce a CPE in " s ensit i zed" Vero c e l l s

u s ing a k n o w n t o xige n i c s t r a i n o f P . m u l t o c i d a ( da t a not s hown ) .
This wa s uns u c ces sful but we we r e able t o p r o d u c e a p o s i t i ve
r e a c t ion in c u l t u red EBL c e l l s u s i n g t he proce du re de s c r i obed i n
Sect ion 4 . 2 .

U s ing the EBL a s say ( see Section 4 . 2 . 2 ) P. mul t ocida i s o la t e s were

s c r e e n e d f o r e xo t o x i n p r oduct i o n . The r e s u l t s a re s hown in t he

f o l l owing two s ect ions .

4 . 3 . 1 Va l ida t i o n of the EBL Assay

P rot otype s t r a i n s ( s ee Sect ion 4 . 2 , P a rt I I I ) were examined by t he

EBL a s s a y . T he re s u l t s a re shown in T a b le 4 . 1 .

B o t h po r c i n e p r o t o t ype s t ra in s c a u sed a CP E in EBL ce l l s ( see

F igures 4.1 and 4 . 2 ) a l t hough ( re s u l t s n o t s h o wn ) t h e p o s i t ive

c on t rol p r o du c e d a h igher t i t re . The i s o lates obt a ined f rom othe r

species did n o t produce a CPE . It w a s c o n c l uded t h a t t h e a s s a y

c ondit ions u s e d we re adequate to det e c t P . mul t oci da exot oxin .

129

F igure 4 . 1 Ce l l Culture Mono l aye r of Emb ryonic Bovine Lunq Cells .

A c e l l - f ree ext ract of a n o n - t o x i ge n i c strain of

P. m u l t ocida was a dded t o the culture .

Note t hat t h e mo n o l a y e r i s int a c t a f t e r f o u r days

i n c u b a t ion . T h e c e l l s a re l a rge w i t h p a l e s t a i n ing
nuc l e i (compa re w i t h F igure 4 . 2 ) .

4 0 x Magn i f i cat ion .

S t a i ned with Gen t i an Violet .

F ig u r e 4 . 2 Ce l l Culture Mono l aye r of Emb ryonic Bovine Lung Cells .

The cytopathic e f f ect of P . mult ocida exotoxin .

A c e l l - f ree ext r a c t o f a t o x i n - p r oduc i n g s t r a i n o f

P. m u l t ocida was a dded t o the culture .

Note t hat the mon o laye r i s incomp lete a f t e r four days

i n c u b a t ion . The c e l l s a re s ma l l a nd s p i n d l e s haped
w i t h darkly s t a in i ng nuc l e i c ompared with norma l c e l l s
( s ee F igure 4 . 1 ) .

4 0 x Magn i f i c a t ion .

S t a ined with Gent ian Violet .

 1 30

Table 4 . 1 A s s ay o f P rot otype S t rains o f P . mul t ocida f o r Exot ox i n

P rodu c t i o n

An i ma l of No o f CPE
I so l at i on St ra i ns Prod uct i o n

-
Bov i ne 1

- -
Fow l 2 '

Sw i ne 2 +" '
+

-
Sheep 1

-
Goat 1

-
Dog 1

Legend +: CPE prod uct i on

. No CPE prod uct i on
: Known t o x i gen i c s t r a i n < Pos i t i ve cont rol >

4 . 3 . 2 A s s ay o f F ie ld I s o l a t e s

The EBL a s s ay wa s t h e n u s e d t o s c reen f ie l d i s o l ates o f P . mul t o c i da

( s e e S e c t i on 4 . 2 , Part III) . The re s u l t s a r e shown in T a b l e 4.2

whe re it can be s een that no New Zealand i s o l a t e s produced exotoxin .

Th i s i s di s c u s s e d in Section 4 . 4 .
 131

Table 4 . 2 As s ay o f F i e ld I s o l ates of P . mul t o c i da f o r Exot oxin

P rodu c t ion

An i rn a l Exotox i n
Host Prod uct i on Tot a l

+ -

Do g 0 21 21

Cat 0 10 10

Pig 0 15 15

Deer 0 1 1

Ra b b i t 0 25 25

Legend +: E x ot o x i n prod uct i on

-: No e x ot o x i n prod uct i on

4 . 4 D iscu s s ion

N o New Zea land f ie ld i s o l ate s we re s hown t o produce exot o x i n . This

s u g ge s t s that e it h e r t oxigenic strains o f P . m u l t o c i da a re n o t

p r e s ent i n New Z e a l and o r a re present a t a f reque ncy which i s too

l o w t o be det e c t e d with the s amp le t e s t e d .

I t must be app rec iated however that du r ing the course of t h i s s t udy
we w e re not a b l e t o ident i f y a n y h e r d of pigs w h i c h h a d At rophic

Rh i n i t i s . We c o n c l u de that a r e a s s e s sme n t of t he p r e s e n c e o f

t oxigenic s t r a i n s i n pigs should be unde rtaken in t he event that an

outbreak o f At rophic Rhin i t i s occurs .

 132

CHAP TER 5

Comparison of New Zealand I s olates of P . mul t ocida With P a rt icular

Ref e rence to P l asmids .

5 .1 Int roduc t i o n

P re v e n t i o n a n d t re a t me n t o f di s e a s e s d u e t o P . m u l t o c i da may be
e f fected us ing t he ant ibiot ics Penicillin, Te t r a c yc l ine ,

S t r e p t omyc i n a nd S u l ph o n amide s . The r o u t ine u s e o f t h e s e a ge nt s

overseas has led to an i n c re a s e i n t h e p r e v a l e n c e o f re s i s t ant

st r a ins o f P . m u l t oc i da a nd re s i s t a nce h a s been as soci ated w i t h the

po s s e s s ion o f p l a smids ( H i r s c h , Ma r t i n a nd Rhoades , 1985) wh i ch a re
between 1 Megada lton ( Mda l ) and 5 Mda l s in s i ze .

P l a smids can c a rry informat ion f o r t h e s ynthe s i s o f prot e i n s . This

s e c t ion examin e s s t ra i ns of P . mul t oc i da i s o l ated in New Z e a l a nd for
the pre sence of pla smids ( between 1 Mda l and 5 Mda l s i n s i z e ) and
at tempt s to e s t ab l i s h i f a re lat ion s h ip exi s t s between t he c a rriage
of p l a smids , a nt ib i o t i c r e s i s t a nc e a nd the pre sence o f u n i que

proteins a s e v i denced by SOS-PAGE a n a l y s i s .

5 . 2 Materials and Methods

Source o f P . mul t o c i da Isolates .

S t r a ins o f P . mul t o c i da were obt a ined f rom a ra nge o f anima l s

a s de s c r i be d i n S e c t i o n 3 . 3 . 1 . A total of 70 Ne w Z e a l a nd

isolates were o b t a ined f r om f i ve s pe c ie s . The s e w e r e ( w ith

numbe r of isolates i n b r a c ke t s ) : C a n ine (19) , Fel ine (10 ) ,

Cervine (1) , Lapr i ne (25) and P o r c ine ( 15 ) . In a ddi t i o n t h ree

prot otype ( 1 canine , 2 s wine ) s t r a i n s were tested .
 133

5 . 2 . 1 Examina t ion o f P. mult ocida f o r P lasmid Carriage .

5 . 2 . 1 1 Mat e r i a l s

See Sect i o n 3 . 2 . 4

5 . 2 . 1 2 Met hods

i. D N A w a s ext r a c t e d a n d a s s a ye d a s de s c r i b e d i n S e c t ion
3.2 . 4, P a rt s I. and I I . DNA was a l s o ext r a c t e d f r om a

c u l t ure o f E . coli V5 1 7 which was used a s a ma rker s t r a i n

be c a u s e i t cont a in s e ight pla smids of k n o w n mo l e c u l a r
weight s .

ii . A vo lume which con t a i ned 5 u g DNA was pla ced i n t o s t e r i l e

Eppe n do r f t ube s , a 1/20 v o l ume o f 5M NaCl w a s added and

the p repa r a t i o n s w e r e mi xed by t apping t he t ubes . 2
v o l ume s o f cold ( - 2 0 C ) e t h a n o l we r e a dde d , mi xed by
inve rs ion and left at - 2 0 C for a t least 2hrs for t he DNA
to p recipitat e .

i i i . T h e t ubes were cent r i f uged f o r 1 0 m i n s ( i n a M i c r o fuge ) ,

the supernat a n t s were dec an t ed and disca rded, t he pellet s

w a s hed once with cold ethanol and l e f t f o r 3 0mins at 3 7 C

t o dry .

iv . The DNA was s o l ubi l i zed by adding 4 5 u l o f Running Bu f fe r

a nd mixing by t ap p i n g the tube s . The y were then

c e n t r i fuged f o r 1 0min s i n a M i c r o fuge a nd a 4 0 u l a l iquot

o f s upern a t a n t were remove d , p l a ced in we l l s in a 0 .7%

a g a r o s e ge l which had been prepa red a s de s c r i be d i n
S e c t ion 3 . 2 . 4 , P a rt I V .

v. The s a mp l e s we re t hen e lect rophoresed, s t a i n e d a nd

phot ographed a s de s c r ibed i n S e c t i on 3 . 2 . 4 , P a rt V .
 134

5.2.2 P. mu l t oci da : Det e rminat ion o f the Minum Inhibitory

Concent rations * o f Antibiot ics .

* B a s ed on the Aga r D i lut ion Te st o f Wa sh ington

and Barry a s de s c r ibed b y Lennet t e , Spau lding

and Truant ( 1 9 7 4 ) .

5 . 2 . 2 1 Ma t e r i a l s

Ant ib i o t i c S e n s i t ivity P l ates

MHA plates were prepa red which c o n t a i ne d one of three

a nt i b i o t i c s ( Te t r a c yc l ine , S t reptomycin and S u lphadi a z ine ) at

concen t rat ions ra nging f rom 0 . 2 5 ugml - 1 to 2 0 4 8 u gml - 1 . Further

plates were prepa red wh i c h c o n t a i n ed Penici llin at
concent rat ions ranging f r om 0 . 2 5 IUml - 1 t o 2 0 4 8 IUml - 1 .

Bl ood Aga r ( BA)

See appendix

B r a in He a rt I n fus ion Broth ( BH I )

See appendix

Mue l le r-Hinton Aga r ( MHA)

Mue l l e r -Hinton Broth ( D i f c o ) was prepared as per man u f a c t u re r ' s

ins t r u c t ions w i t h the a ddi t ion o f 1 7 g l - 1 aga r . T he medium was
di spe n s ed into 1 0 0ml bot t le s , autocl aved and 2 5ml a l i quots we re
di spens ed between four s t anda rd pet r i dishes as requ i red .
 135

NaC l S o lut ion ( 0 . 8 5 % )

See appendix

5 . 2 . 2 2 Me t hods

i. A c u l t u re o f P . m u l t o c i da wa s removed f rom - 8 0 C s t o rage

a nd propagated on b l ood agar ( BA ) f o r 1 2 hrs a t 3 7 C .

ii . l Oml BH I b roth was inocu la ted w i t h colonies and incubated

at 3 7 C o n an o r b i t a l s haker ( a t approxima t e l y 1 2 0 rpm )
u n t i l the c u l t u re r e a c hed mid - l o g a r i thmic g ro w t h pha s e .
That is , it had an opt i c a l den s it y (OD ) o f app roxima t e l y
0 . 2 at 5 0 0 nm us ing a S p e c t r o n i c 2 0 . A culture o f E . col i

( ATCC 2 5 9 2 2 ) wa s a l s o g r own and u s e d as a cont r o l because

t he MIC of seve r a l ant ibiotics t o this s t r a i n were known .

i i i . T he turbidity of the c u l t ure was adj us ted t o a Mc F a r l a nds

*
S t a nda rd # 1 u s i n g 0 . 8 5 % N a C l S o l u t i o n a n d d i l u t e d by
adding approximat e l y one volume of diluent .

*
Note . Ant ib i o t i c plates were inoc u l a t e d with t e s t
s t r a i n s w i t hin 3 0min s f rom t h i s t ime .

iv . 0 . 5ml vo lume s of each t e s t s t r a i n were de l ivered into t he

we l l s of a " Steers Rep l ic a t o r " and Antib i o t i c Sens it ivity

P l ates were t hen st ampe d .

vi . T he p l a t e s were d r i e d , incub a t e d f o r 7 2 h r s a t 3 7 C and

e xamined f o r growt h .

v. T he Min imum I nh ib i t o ry Concent r a t i o n o f t he a n t i b i o t i c s

w a s de s ignated a s t he l o we s t concent rat i o n o f ant ib i o t i c
w h i c h prevented g rowt h o f t he t e s t s t rain .
 136

5 . 2 . 3 P ro c edure s f o r SOS-PAGE Ana1ysis

See Sec t i o n 3 . 2 . 3

5 . 3 Res u l t s

S e v e n t y t h re e (73) P. mul t o c i d a i s o l a t e s f rom s e ve r a l di f fe rent

spe c i e s of a n i ma l s ( see Sect i on 5 . 2 .2) were s c reened f o r t he
pre sence of sma l l ( i e between 1 Mda l a n d 5 Mda l ) p l a smids . The
re s u lt s ( T able 5 . 1 ) s how that p l a smids were present i n i s o l ates of

P. mu l t o c i da f rom c a t s a nd do g s but were not detected i n i s o l a t e s

f r om r a bb i t s , p i g s o r de e r ( s e e Table 5 . 1 ) . I n t h e c a se o f t hose
isolates wh i c h c o n t a ined p l a s mi d s ( s ee Table 5 .2) t w o p l a smid
spe c i e s we re inva r i ably present .

Table 5 . 1 As say of S t rai ns of P . mu l t o c i da for P l asmids .

An i ma l of N o f N with
I so l at i on St ra i ns P l asm i ds

Dog 20 5

Cat 10 7

Rabb i t 25 0

Pig 17 0

Deer 1 0

The p l a smids within an i s o l a t e were di s t inguished by t h e i r mob i l it y

but t he mob i l i t y o f plasmids f rom d i f f e rent i s olates w e r e s imi l a r i f
not i de n t i c a l ( see F i gu r e s 5 . 1, 5 .2 and T a b l e 5 . 2) with t wo
except i o n s ( C S a nd D S ) .
137

F i gure 5 . 1 Agaro s e - Gel Elect rophoresis o f DNA from P la smid

Cont aining Feline I s o lates of P . mu l t ocida .

The mob i l i t y o f p l a smids c o n t a ined in s e ve n f e l ine

i s o l a t e s were compa red .

The le f t hand t ra c k ( p l a smids recovered f r om E . c o l i

V5 1 7 ) represent s mo lecular we ight ma rkers .

N o t e t h a t any o n e i s o l a t e p o s s e s s e d t w o s pe c i e s o f

p l a smid which a re s imi l a r , i f not ide n t i c a l , t o the

p l a sm i d s found in the othe r s i x fe l i n e isolates .

S imi l a r plasmids we re present in some c a n i ne i s o l a t e s
( see F igure 5 . 2 ) .
138

F igure 5 . 2 Agarose-Gel Electrophores i s o f DNA from P l a smid

Cont a ining Canine I s o lates o f P . mul t ocida .

The mo b i l i t y o f p l a smids conta ined i n f ive c a n i n e

i s o la t e s were c ompa red .

The l e f t hand t ra c k (pla smids recovered f r om E . c o l i

V5 1 7 ) repre sents mo le cu l a r we ight ma rke r s .

Note that any one i s o l a t e p o s s e s s ed t w o s pe c i e s o f

p l a smid w h i c h a re s imi l a r , i f not ide n t i c a l , t o the

p l a s mids f o und in t h e o t h e r f o u r c a n i n e isolates .
S imi l a r pla smids were present in s ome f e l ine i s o l ates
( s ee F igure 5 . 1 ) .
Mr (Mdal)
 139

T able 5 . 2 An Eva luat ion of P l a smids Ca rr ied by Strains o f

P. mu l t oc i da .

Ani mal eo f I so l at e P l asm i d

I seo l at i eon I d ent i t y D escr i pt i on

____M Mr l fY1t'2

Cat Cl 2 2. 4 1. 4
C2 2 2. 4 1. 4
C3 2 2. 4 1. 4
C4 2 2. 4 1. 4
C5 2 1. 8 (1. 4
C6 2 2. 4 1. 4
C7 2 2. 4 1. 4

Dog 01 2 2. 4 1. 4
D2 2 2. 4 1. 4
03 2 2. 4 1. 4
04 2 2. 4 1. 4
D5 2 2. 5 1. 5

Legend Mr 1 : A p prox i ma t e mo l ecu l ar w e i g h t < Md a l ) o f l ar g e s t p l asm i d

Mr2 : A p preox i ma t e mo l ecu l ar we i g h t ( fYld a l > o sma l l es t p l asm i d

5 . 3 . 1 The Antibiot ic Sensitiv ity o f S t r a i ns o f P . mu l t o c i da

T he antib i o t ic sensit ivity ( M I C ) o f iso l a t e s were det e rmined and the

r e s u l t s obt a ined for s t ra ins with a nd without plasmids were compared

( see Table 5 . 3 ) . The s t ra ins va r i e d in the i r sens i t i v i t i e s but none

*
w e re " re s i s t a n t " t o any o f t he a n t ibiot i c s in the s e n s e norma l ly

u s ed in a d i agnost i c l abora t o ry to de f i ne r e s i stance . F u rthermo re ,

t he re w e r e n o c o n s i s t a nt d i f f e r e n c e s i n a n t ib i o t i c sensitivity

between s t r a ins which c a r r ied p l a smi ds a nd t hose wh ich did not c a rry
p l a smids .

*
A s t rain o f P . mul t oc i da is cons ide red t o be re s i s t ant i f
it grows in the presence of : 3 2 I Um l - 1 P e n ic i l l in ,
3 2 u gm l - 1 Tetracyc l ine, 3 2 u gm l - 1 S t r e p t o my c i n or
1 0 2 4ugml - 1 S ulphonamide .
 140

Table 5 . 3 Min imum I n h ib itory Concent rations o f Ant ib iot i c s F o r

P. mul t oc i da .

An i ma l of I so l at e M i n i m um I nh i b i t ory Concent rat i on

I so l at i on I d ent i t y Pe Te Sm Su
I Um i - 1 ug m i - 1 ug m i - 1 u gm i - 1

Cat Cl 1 16 2 64
C2 i0. 2 5 8 1 64
C3 i0. 25 2 2 16
C4 .5.0. 25 8 2 8
C5 0. 5 16 2 256
C6 8 16 0. 5 256
C7 .i0. 2 5 8 1 256
ea 1 16 2 64
C9 0. 5 16 2 256
C10 .i0. 25 16 1 128

Dog D1 .i0. 2 5 2 2 256

D2 1 16 2 1 28
D3 8 16 0. 5 256
D4 0. 5 16 2 1 28
D5 1 16 2 1 28
D6 1 .i0. 25 4 16
D7 1 16 2 64
DB 0. 5 32 32 1 28
D9 0. 5 8 2 256
::>
D10 4 16 L. 32
D1 1 .i0. 25 2 2 512
D12 1 1 28 4 256
D13 8 16 0. 5 64
D14 .5.0. 2 5 8 0. 5 1 28
D15 .i0. 25 8 2 256
D16 1 16 2 1 28
D17 1 16 2 256
D18 .5.0. 25 2 2 32
D19 1 16 2 256
D20 .i0. 2 5 8 1 1 28

Pi g P1 1 .5.0. 25 2 32
P2 1 .5.0. 25 2 64
P3 1 .5.0. 25 2 64
P4 0. 5 32 4 64
P5 0. 5 8 2 1 28
P6 1 16 2 1 28
P7 0. 5 16 2 1 28
PB 1 16 2 64
P9 1 16 2 64
P10 1 2 2 1 28
P1 1 1 16 2 256
 141

Table 5 . 3 Cont d

P12 i.0. 2 5 16 2 256

P13 .i 0 . 25 8 1 256
P14 .i 0 . 2 5 8 1 1 28
P15 0. 5 8 1 1 28
P16 i.0. 25 8 1 1 28
P17 0. 5 8 2 1 28

Rabbi t R1 0. 5 8 1 256
R2 i. 0 . 2 5 4 1 256
R3 i.0 . 25 4 1 256
R4 .i0. 2 5 2 1 256
R5 0. 5 2 1 256
R6 .5. 0 . 25 2 1 1 28
R7 0. 5 2 1 256
R8 0. 5 2 1 256
R9 .i 0 . 25 2 1 64
R10 .5.0. 25 4 1 256
R1 1 0. 5 4 1 256
R12 .5. 0 . 25 2 1 256
:::>
R13 .5. 0 . 25 L. 1 256
R14 0. 5 4 1 256
R15 .5. 0 . 25 2 1 256
R16 0. 5 4 1 256
R17 .5. 0 . 25 4 1 256
R18 0. 5 2 1 256
R 1 '3 .5. 0 . 2 5 2 , .
1 256
R20 .5. 0 . 25 2 1 256
R2 1 0. 5 4 2 1 28
R22 0. 5 2 2 1 28
R23 .5. 0 . 2 5 4 2 1 28
R24 0. 5 2 2 1 28
R25 0. 5 2 2 1 28
.

Deer X1 0. 5 8 1 256

Legend : A n t i b i ot i cs Not es

Pen i c i 1 1 i n G < Pe > R 1 - R 1 9 from Pat ea co l ony

T e t racyc 1 i r.e H C l < Te > R20 from Wa i t ot ar a Va l l ey co l ony
S t rept omyc i n so. < Sm > R 2 1 - R25 from Upper H u t t co l ony
S u l ph ad i a z i ne <Su>

Canine , f e l ine and porc ine s t ra i ns ( s ee Table 5 . 3 ) o f P . mul t o c i da

s h o wed mo re v a r i a t ion in t he i r s e n s i t i vi t i e s t o a n t i b i o t i c s t ha n
rabbit i s o l a t e s . T h i s can b e s een in Figure 5 . 3 and i s di s cus sed i n
S e c t ion 5 . 4 .
 142

*
Figure 5 . 3 Va r i a t i o n of MICs Between S t r a ins of P . m u l t o c i da .
*
See Legend

I. Fe l i n e S t r a i ns

1 00 Pen i c i 1 1 i n 1 00 Tetracyc l i ne
" "
90 90
5 5
t 80 t 80
r t'
a 70 a 70
i i
r1 60 n 60
5 5
50 50
5 5
e 40 Hi!!i e 40
r H!!H n
5 30 !!!!!! 5 30
i !!!!!! i
t 20 H!i!i t 20
iiiiii::::::::::::
i . . . . . . . . .. . .......
i
V 1 0 !!!H!iii!!i!!!!H V 10
e !!H!!!iiii!H!!!! e
0
llllllllllllllllll 0

-2- 1 0 1 2 3 4 5 6 7 8 9 1 0 -2 - 1 0 1 2 3 4 5 6 7 8 9 1 0
Ant i b i ot i c C o ncent rat i on Ant i b i ot i c Concent rat i on
< Lo g . I Um l - 1 l < Lo g 1 ugml - 1 l

1 00 Strept omyc i n 1 00 Sul phad i a z i ne

" "
90 90
5 5
t 80 t 80
r r
a 70 a 70
i i
n 60 n 60
5 5
50 50
5 5
e 40 e 40
r n
5 30 5 30
i i
t 20 t 20
i i
V 10 V 10
e e
0 0

-2- 1 0 1 2 3 4 5 6 7 8 9 1 0 - 2 1 0 1 2 3 4 5 6 7 8 9 10
-

A n t i b i ot i c Concent rat i on Ant i b i ot i c Concent rat i on

( Lo g . ugml- 1 ) < Loga ugml- 1 l
 143

II. Can i ne St ra i ns

1 00 Pen i c i l l i n 1 00 Tetracyc l i ne

90 90
s s
t 80 t 80
r r
a 70 a 70
i i
I'"I 60 n 60
s s
50 50
s s
e 40 e 40
n n
s 30 !!!!!! s 30
i !!!Hi i
t 20 mm iiiiii t 20
i !!!!!! iiiiii i
V 1 0 !iiiii::::::iiiiii V 10
e i!iii!iiiiii!iiiii e
0
llllllllllllllll!! 0
$
/./"

-2- 1 0 1 2 3 4 5 6 7 8 9 1 0 -2- 1 0 1 2 3 4 5 6 7 8 9 1 0
A n t i b i ot i c C o ncent rat i on Ant i b i ot i c Concent rat i on
< Log a I Um i - 1 > ( Lo g e u g rn i - 1 >

1 00 Strept omyc i n 1 00 S u l phad i a z i ne

90 90
s s
t 80 t 80
r r
a 70 a 70
i i
n 60 n 60
s s
50 50
s s
e 40 e 40
n n
s 30 s 30
i i
t 20 t 20
i i
V 10 V 10
e e
0 0

-2- 1 0 1 2 3 4 5 6 7 8 9 1 0 -2- 1 0 1 2 3 4 5 6 7 8 9 1 0
A n t i b i ot i c Concent rat i on Ant i b i ot i c Concent rat i on
( Lo g a ugm l - 1 ) < Lo g a u gm l - 1 )
 144

I11. Porc i ne St ra i r1s

1 00 Pen i c i 1 1 i n 1 00 Tet racyc l i ne

" "
90 90
s s
t 80 t 80
r r
a 70 a 70
i i
Yl 60 n 60
s s
50 50
s s
e 40 e 40
Yl Yl
s 30 s 30
i i
t 20 t 20
i i
V 10 V 10
e e
0 0
m
-2- 1 0 1 2 3 4 5 6 7 8 9 1 0 -2- 1 0 1 2 3 4 5 6 7 8 9 1 0
A n t i b i ot i c Concent rat i on Ant i b i ot i c Concent rat i on
< Lo g . I Urn l - 1 > < Lo g . u g rn l - 1 >

1 00 Streptomyc i n 1 00 Su l ph ad i az i ne
" "
90 90
s s
t 80 t 80
r r
a 70 a 70
i i
Yl 60 Yl 60
s s
50 50
s s
e 40 e 40
Y"l n
s 30 s 30
i i
t 20 t 20
i i
V 10 V 10
e e
0 0

-2-1 0 1 2 3 4 5 6 7 8 9 10 -2- 1 0 1 2 3 4 5 6 7 8 9 1 0
A nt i b i ot i c Concent rat i on Ant i b i ot i c Concent rat i on
< Lo g . u g rn l - 1 > < Lo g . u grn l - 1 )
 145

I V. Rabb i t St r a i ns

1 00 Peni c i l l i n 1 00 Tet racyc l i ne

" "
90 '30
s s
t 80 t 80
r r
a 70 a 70
i i
Yl 6 0 n 60
s 5
50 50
5 5
e 40 e 40
r n
s 30 s 30
i i
t 20 t 20
i i
V 10 V 10
e e
0

-2- 1 0 1 2 3 4 5 6 7 8 '3 1 0 - 2 - 1 0 1 2 3 4 5 6 7 8 '3 1 0

A nt i b i ot i c Concent r a t i on Ant i b i ot i c Concent rat i on
< Lo g 1 I Urn l - 1 ) < Lo g a ugml- 1 )

1 00 St reptomyc i n 1 00 S u l phad i a z i ne

'30 '30
s s
t 80 t 80
r r
a 70 a 70
i i
n 60 n 60
s s
50 50
s s
e 40 e 40
Yl n
s 30 s 30
i i
t 20 t 20
i i
V 10 V 10
e e
0 0

-2- 1 0 1 2 3 4 5 6 7 8 9 1 0 -2- 1 0 1 2 3 4 5 6 7 8 9 1 0
Ant i b i ot i c Concent rat i on Ant i b i ot i c Concent rat i on
<Log, ugm l - l ) < Lo g , ugm l - 1 )

Legend : Ant i b i ot i cs :
Pen i c i l l i n G <Pe>
Tet racyc l i ne H C l < Te )
St rept omyc i n so.. ( S rn >
S u l ph ad i a z i ne <Su>
 147

5 . 3 . 2 Compa r i s on of P rote i n s o f P . mul t ocida . P l a smid C a r rying

S t ra in s Ve rsus Non - P l a smid C a r rying S t ra in s

T h e proteins o f 1 6 s t r a i n s o f P . mul t o c i da (8 c a n ine , 8 f e l ine ) we re

c omp a red by S D S - P AGE . H a l f o f the 16 s t r a ins t e s ted (4 c a n ine , 4

f e l i ne ) c a r r i e d pla smids . The re s u l t s a re shown in F igu res 5 . 4 a nd

5.5. The S D S - P AGE pat t e rn of s t r a i n s with and without p l a smids did
not a l low us to di st ingu i s h between the two groups . We conclude that

if t he p l a s m i ds c o de f o r prote ins t h e n t h e s e a r e not readi ly

det e c t able by SDS -PAGE .

5 . 4 Discus sion

I s o l ates o f P . mul t oc i da f rom New Zeala nd anima l s were s c reened for

p l a smids . We found that s ome canine and fel ine s t rains were the only

i s o l ates t o c a rry plasmids ( Table 5 1 ) .

. It was interest ing t hat all

of t he s e p l a smid- c a r r y i ng isolates had two p l a smids and t hat

p l a s mids f r om both t he dog and c a t s t r a i n s w e r e , w i t h t w o s l ight
except ions , t he same s i z e ( ie 2 . 4 Mda l and 1 . 4 Mda l , see Table 5 . 2 ) .
This s ugge s t s t h a t t h e p l a smids o f di f f e r e n t i s o l a t e s might be

re l a ted or a re ident i c a l .

Ant i b i o t i c r e s i s t a nce c a n be a s s o c i a t ed w i t h p l a smid c a r r i age i n

P . m u l t o c i da ( S i l ve r et al , 197 8 ) . We e xamined s t r a ins of

P . m u 1 t o c i da f o r the i r sens it iv it ies (MIC s ) to the f o u r ma i n

t he r apeut ic a n t ibiot ics ( P enic i l l in , Tetracyc l in e , S t rept omycin a nd

S u l p honamide s ) and found t hat no i s o late was re s i st ant . Furthermore ,

t he p l a smid- c a r rying i s o l a t e s s howed s imi l a r s e n s i t i v i t i e s t o those

i s o l ates which did not c a r ry pla smids . We c o n c lude that t he pla smids
d i d not c a r ry antibiotic re s i s t a n c e m a r k e r s at lea s t to t he
ant ibiotics t e s ted ( P en ic i l l in , T e t r a c yc l i n e , S t r e p t o my c i n a n d

S u lp honamide s ) .
148

F igure 5 . 4 SOS-PAGE Ana lys is o f P. mul t o cida I s olates f rom Dogs .

A comp a r i s o n of p l a smid- c o nt a i n i ng i s o lates with

i solates which do not contain plasmids .

F o u r p l a s m i d- c o n t a i n i ng i s o l a t e s ( left ) and four

i s olates ( r ight ) which did not p o s s e s s p l a smids were

compa red .

T h e pa t t e r n s w e r e e x amined f o r t he p r e sence o f an

ext ra band ( o r bands ) common t o t he pla smid-cont a ining

i s olates . N o such ba nds were det e c t e d .

 . ..
ci .
 :.

149

F i gure 5 . 5 S D S -PAGE Ana lys is of P. mu l t ocida I so lates from Cat s .

A c omp a r i s o n of p l a smi d - c o n t a i n i n g is olates with

i s o lates which d o not contain p la smids .

F o u r p l a sm i d - c o n t a i n i ng isolates ( left ) a nd f o u r

i solates ( r ight ) which did not po s s e s s p l a smids were

c ompa red .

The patterns we re e x amined f o r t h e p r e s e n c e o f a n

ext ra band ( o r bands ) common t o t h e p l a smid-cont a i n ing

i s o l ates . No s uch bands were de tected .

 150

I t w a s obse r v e d ( T able 5 . 3 ) t h a t s t r a i n s o f P . mul t oc i da i s o l ated

f rom rabb i t s exhibited very s imi l a r leve l s of s ens i t ivity to a l l of
t h e t e s t a n t i b i ot i c s . T h i s was in c o n t r a s t to fe l ine , c a n i n e and
p o r c ine s t r a i n s which s howed c o n s i de r a b l y mo re v a r i a t ion . E a r l ier
work ( see S e c t ion 3 . 4) had shown that i solates o b t a i n e d f r om
i n d i v idu a l r a b b i t c o l o n i e s we re homo gene o u s w it h r e s pe c t t o the
p r oteins (as demon s t r a t e d b y S D S - P AG E ) a n d t h e re f o r e p r o b a b l y
r e p r e s e n t mu l t i p l e i s o l a t e s o f o n e s t ra in . The s imi l a r i t y i n t h e
ant ibiot ic sensitivit ies of rabbit isolates s uppo rt s this
c o n c lusion .
 151

CHAP TER 6

General D i s c u s s ion .

Pa s t e u rel l a m u l t oc i da , a s its name impli e s , c a u ses di s e a s e in many

di f f e rent a n ima l s . It i s the r e f o r e not surpris ing t hat f o r de cade s

the o rganism h a s been de s ignated by diffe rent names bec a u s e of i t s

wide h o s t range and the many disea s e s wh ich it causes . T h e di s cove ry

( w i t h i n t he l a s t t h i r t y ye a r s ) that P . m u l t o c ida i s c o mpo sed o f

di f f e r e n t s e r o t yp e s led to the re c o gn i t i o n t h a t isolates f r om
va r ious host species a nd di s e a s e s b e l onge d to a single spec ies a nd
under l ines t h e patholog i c a l s igni f i c a nce of t h i s bact e r i a l species .

Be c a u s e of i t s impo rta nce as a pathogen P . mul t ocida h a s been mu ch

st udied ove r s e a s but , perhaps s u r p r i s ingly, h a s been l i t t le studied

i n New Z e a l a nd de s p i t e t he e c onomic importance of anima l s in t h i s

c o u nt ry . T h i s t he s i s s h o u ld be r e g a rded a s a c ont r ibut i o n t o t h e

u n de r s t a nd i n g o f t he d i s t r i bu t i o n o f and t h e va r i a t i o n s between
P. mul t o c i da s t ra ins recovered f rom a nima l s in New Z e a l and . Thus we

de s c ribed t h e i s o l a t i o n of P . m u l t o c i da f r om diffe rent species of

a n ima l i n N e w Z e a land and c ompa red t he s e i s o l a t e s u s i n g s e ve r a l

t e c hnique s .

Typ ica l ly P . m u l t ocida is found in a n ima l s in a s s oc i a t i o n w i t h other

b a c t e r i a w h i c h a re u s u a l l y p r e s e n t in l a rge exce s s , so e f f ic i ent

i s o lat ion of P . mul t o c i da requ i r e s a select ive medium . Cons equent ly

we a s s e s s e d t h e v a l ue of s e l e c t i ve me d i um 8 H PG w h i c h h a s b e e n

de s c r ibed b y S m i t h a n d B a s ke rv i l l e ( 1 9 8 3 ) wh o u s e d i t t o i so late

P. m u l t o c i da f r om t h e n a s a l t r a c t of pigs . I n o u r h a nd s it was
un s a t i s f a ct o ry be cause it inhibited the growt h o f many New Z e a l and

i s o lates of P . mul t o c i da de r ived f rom a ra nge of h o s t s pe c ie s ( s ee

Table 2 . 1 ) We t h e r e f o r e a t t empt e d to f o rmu l a t e a mo r e s u i t ab l e
medium .
 152

6 . 1 The Deve 1opment of a Se1ective Medium f o r the Iso1ation

of P . mu l t ocida in New Zea1and

The c ompo n e n t s of 8 H P G me d i u m w e r e e x a m i n e d i n d i v i du a l l y ( see

Sect ion 2 . 3 ) and i t w a s found t h a t the b a s a l medium ( Nut r ient Aga r )
f a i led t o p r op a g a t e s e v e r a l o f o u r t e s t s t r a i ns a n d o t h e r s were
i n h i b i t e d b y t he h i g h a l ka l i n i t y o f 8 H PG me dium . In a dd i t ion t o
thi s , 8 HP G medium conta ined polymixin wh ich inh ibited a s ignif icant
proport ion of t he New Zea land i s o l a t e s o f P . mul t ocida .

F o l lowing a s s e s s me n t o f the i n h ibit o ry e f fect s of i n d i v i du a l

a n t i b i o t i c s o n a range o f New Z e a l a nd s t r a i n s o f P . m u l t o c i da we
f o rmul ated a modi f ied s e lect ive medium ( SM ) which had a b l ood agar
base at neut ral pH ( S ect i o n 2 .2 .1) and c o n t a i n e d B a c i t r a c i n ,

Ge n t amy c i n a nd My c o s t a t i n a s de s c r ib e d f o r t he o r i g i n a l medium

( 8HPG) . SM ( s ee T a b l e 2 . 5 ) was f o und to p r opagate a l l of the New

Z e a l a nd t e s t s t r a i n s of P . m u l t o c i da a nd s uppressed mo s t bacte ria

( s e e F i gu re 2 . 1) wh i c h were p r e s e n t i n n a s a l swabs de r i ve d f rom

rabbit s . It wa s t he r e f o re u s e d to i s o l a t e P . m u l t o c i da f rom t h e
na s a l c a v i t y o f rabb i t s inc l uding he a l t hy a n imals a nd a n ima l s with
r e s p i r a t o ry d i s e a s e . We be l i eve t h at t h i s me dium has c o n s iderable

pot ent i a l for use in rout ine vete r i nary inve s t igat i on s . I t could f o r

example be u sed t o s c reen t h e na s a l f l o r a o f impo rted p i g s f o r t he

pre s ence o f P . mu l t o ci da . I s o l a t e s obt a i ned could t hen be examined

f o r t h e p r o du c t i o n o f ex o t o x i n ( u s i ng t he cell c u l t u r e me t h o d

de s c ribed i n Chapt e r 4 ) and thus p revent t h e introdu c t i o n o f s evere

at r oph ic r h i n i t i s into the count ry .

6.2 I s o 1 a t i on and S e r o typinq o f P. mu l t o c i da f rom N e w Zea 1and

Anima1 s

Us ing t radit ional i s o l a t i o n met h ods ( p l a t ing on b lood aga r )

augmented w i t h our s e lective medium, P. mul t oci da wa s i s o l ated f rom
seve r a l s p e c i e s of a n imal in New Z e a land ( i e Cat , Dog , Rabb i t , P ig
and Dee r ) .
 153

I t w a s f ou n d ( s ee T a b le 3 . 1 ) t h a t a h i ghe r p ropo r t i o n o f rabb i t s

w i t h c l i n i c a l r e s p i r a t o ry t ra c t i n fe c t i on c a r r ied P . mul t ocida i n

t he i r n a s a l t ract than did hea lt hy cont ro l s . This is c o n s i s t ant w i t h
the g e n e r a l l y ac c e p t e d c o n c e p t t ha t P . m u l t o c i da c a r r i e d i n t he

uppe r re s p i ra t o ry t ra c t can ( e s p e c i a l l y i n the presence of s t res s )

spre a d t o t he lungs and c ause pneumonia . F u rthe r expe r iment s which
examined i s o l a t e s by SDS -PAGE a n a lys i s ( s e e S ect ion 6 . 4 ) confi rmed
that the s t ra i n s i s o l a ted f rom t he l ungs of di seased rabb i t s we re
indi s t ingu i s hable f rom the st r a i n s i s o lated f rom the n a s a l cavit y .

The i ndi r e ct haemagglut inat i o n a s s a y ( I HA ) i s the a c cepted method

f o r t yp i n g s t r a i n s o f P . mul t o c i da . T h i s te chn ique , which has not
p r e v i o u s l y been u s e d in New Z e a l a nd , had to be v a l i d a t e d i n o u r

hands be f o r e f ield i s o lates c o u l d b e typed . A ma j o r prob lem w a s t o

produce t ype - s pe c i f i c a n t i s e r u m . As t h e l i t e r a t u r e w i l l ve r i f y

( B r o gde n a n d P a c ke r , 197 9) , t h i s p r oce s s i s di ff i c u l t beca use , on

its own , t h e t ype - s p e c i f i c a n t i g e n ( c a p s u l a r po l y s a c c h a r i de ) is
poo r ly a n t igenic o r non antige n i c .

Using w h o l e c e l l s and pu r i f ied capsula r ant igen of P . mul t oc i da with

F re u n d ' s Complete Ad j uvant as a n immun i z i ng ant igen we f a i l ed t o

produce t ype- spec i f i c ant ise rum i n rabb i t s but later p roduced t yping

ant i se r a of adequate spe c i f i c i t y by immun i z ing dome s t i c hens . Wh i le

muc h o f t he l i t e r a t u re s ugge s t s t h a t rabbit s a re s a t i s factory f o r

producing P . mul t o c i da typing s e r um the f a c t t h a t s ome other worke r s

used hen s , which a re not common expe riment a l anima l s , s ugge s t s t h a t

o t h e r s h a ve expe r i enced di f f ic u l t y in immu n i z ing r a bb i t s w i t h t h i s

ant i gen .

Us i ng t h e IHA a s s a y and avian a n t i s e ra we were able t o dist ingu i s h

between s e rotypes A , B and D ( s e e Table 3 . 3 ) .

I s o l a t e s o f P . mul t o c i da f rom dogs , c a t s a nd p i g s i n New Ze a l and

we r e p re dominant l y T ypes A a n d D so we c oncluded, u n s u rp r i s ingly ,

 154

t h a t t he s e r o t yp e s c a r r ie d b y t h e s e h o s t s i n Ne w Z e a l a nd a re t he
s ame a s t h o s e c a r r i ed by t he s ame s pe c i e s o f an ima l ove r s e a s . A

s ingle i s o l ate f rom a deer p r oved to be Type D . We a re a w a re of only

one dee r i s o l at e be ing typed oversea s . I t a l so was Type D .

T w o f i n d ings we r e unexpe c t e d ( s ee Table 3 . 4 ) : A f e l ine i s o l a t e o f

P. m ul t o ci da wa s Type B . The s e a re norma l l y a s s o c i a t e d w i t h c a t t l e
and b u f f a loe s . A l l t h e rabbi t i s o l a t e s ( f rom t hree r a b b i t colonie s )
we re u n t yp a b l e w h i c h i s i n c o n t r a s t t o ove r s e a s f i n d i n g s where
approximately 5 0 % of rabbit s t rains a re untypable and the rema inder
a r e Type s A and D .

No t w i t h s t a n d i n g t he l a t t e r r e s u lt , w h i c h i s di s c u s s e d be l o w , we
conclude that the New Zeal and r e s u l t s mi rror ove rsea s f indings .

6 . 3 SOS-PAGE Analys i s o f Field Isolates o f P . mul t o c i da

The e f f o rt invo lved in s e rotyping P . mu l t o c i da by I HA is

cons i de rable and a n alterna t i ve me thod i s not a va i l ab le . I t s eemed

p o s s i b l e t h a t t h e r e might b e a c o r r e l a t ion b e t w e e n p r o t e i n s of
P. m u l t o c i da ( a s examined by S O S -PAGE analys i s ) and t he s e rotype of
a n i s o l a t e . We examined this p o s s ib i l ity but found that P . m u l t o c i da

i s o l a t e s were h e t e rogene o u s n o t o n l y when i s o lated f rom diffe rent

host species ( F igure 3 . 1 ) but a l s o w h e n mu l t i p l e i s o l a t e s were

o b t a i n e d f r om a s i ngle h o s t s pe c i e s ( F igures 3 . 2, 3.3 a nd 3 . 4 ) .

There f o re , our a t t empt to c o r relate proteins with s e r o t ypes was not

s ucce s s f u l wh ich pe rhaps accounts f o r the absence of d a t a conce rning

S D S - P AGE pro f i le s o f P . mul t o c i da in the l iterature .

T h e e x t reme h e t e r ogene i t y o f S D S - P AGE p a t t e r n s of P. m u l t o c i da

a l lowed us t o u s e S D S - P AG E analysis t o c o mp a re n a s a l a n d l u ng

i so la t e s f rom rabb i t s ( see F ig u re 3 . 7 ) a nd we were a b l e t o s how that

t he s ame s t ra in s were involved ( s ee S e c t i o n 3 . 3 ) wh i c h s uppo rt s t he
c o n c e p t t h a t p n e umon i a is c a u s e d b y t h e di s semi n a t i o n o f n a s a l

s t ra i n s .
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Exami n a t ion o f P . mul t o c i da p roteins b y SDS-PAGE a l s o enabled u s t o

c a s t l i ght o n t h e source o f P . mul t oc i da which caused a n outbre a k o f

re s p i r a t o ry disease i n r a bb i t s . D i s e a s e h a d o c c u r r e d a f t e r t he
int rodu ct i on of imported rabb i t s into a co lony s o i t was c oncluded

that a newly int roduced s t r a in o f P . mu l t dc i da had caused the

o u t b re a k . Howeve r S D S - PAGE ana lys is of isolates o b t a i n e d f r om
di s e a s ed rabb i t s be f o re a nd a f t e r t he import a t ion o f new rabb i t s

( F ig u r e 3 . 8 ) s howed t hat the s t r a ins we re ind i s t ingu i s hable . S ince

i s o l a t e s f r om d i f f e rent f l o c k s a re hete rogeneous we conclude that
the p a t hogen i c s t r a i n o f P . m u l t o c i da was pre s e nt i n New Z e a l a n d
be f o r e t he impo r t a t i o n o f t h e new r a bb i t s and t h a t the disease
o u t b re a k was p r obably u n c o n n e c t e d w i t h t he int rodu c t i o n o f a new

i s o l a t e o f P . mul t ocida .

As a l l u ded t o a b o ve it wa s f o und t h a t all rabbit i s o l a t e s were

u n t y p a b l e when e xamined by t he I HA a s s a y . T h i s w a s unu s u a l ( s ee

Chapt e r 3 ) so we specu lated that these i s o l a t e s repre sented mu lt iple
i s o l a t ions of o ne , or at at any r a t e , a sma l l numb e r o f s t r a i n s .
T h i s w a s s uppo r t e d be c a u s e when exami ned by SDS -PAGE ( F igure 3 . 6 )
i s o l a t e s f r om i n dividu a l r a b b i t c o l o n i e s were homogene o u s . Thus ,
s in c e only three rabbit c o l o n ies were examined our rabbit i s o lates

probably rep resent only three s t ra ins .

I n c o n c l us ion , S D S - P AGE w a s f o und t o b e a u s e f u l t o o l t o s t udy

e p i demi o logi c a l a spec t s of di s e a s e ( at least in rabb i t s ) cau sed by

P. m u l t o c i da but could not be used to d i s t ingu i s h between se rotype s .

6 . 4 Compa rison o f P. m u l t o c i da Serotype s by Res trict ion Endonuclease

Analys i s (REA)

A s d i s c u s s e d a bove t h e I HA a s s a y i s a l a b o r i o u s t e c h n i qu.e s o a n

a l t e rnat ive met hod o f serotyp ing P . mul t o ci da would be advantageous .

S D S - P AGE ana lys i s did not c o rrelate w i t h s e rotype s o we cons ide red

REA a s a p o s s ible a lt e rna t i ve because it had been s u c c e s s f u l l y used

 156

b y Ma r s ha l ! , W i l t on and Robe r t s o n (1981) t o d i f fe rent iate between

s e r o v a r s of Lep t osp i ra . H o w e ve r , the e l ect roph o re s i s p a t t e rn s o f
dige s t ed DNA f rom P . mul t oc i da ( s ee f igures 3 . 1 0 , 3 . 11, 3 . 12 , 3 . 13 )
di d n o t c o r r e l a t e w i t h s e r o t yp e s . The hete rogene o u s p a t t e rn s o f
di f fe rent i s o l ates would, l i ke S D S - P AGE , a l l o w e p i de mi o l o g i c a l

s t u di e s o f i nd i vidu a l s t r a i n s but w e did not pursue t h i s approach

w i t h re spect to REA .

6 . 5 As say for Exotoxigenic St rains of P. mul t oci da

P ro g r e s s ive a t r ophic rhin i t i s ( AR) i s an impo r t a nt d i s e a s e o f pigs

i n o ve r s e a s c o unt r i e s and a mild f o rm has b e e n d e s c r i bed in New

Ze a l a n d . The d i s e a s e i s c a u s ed by e x o t oxin produ c ing s t r a i n s of
P. m u l t o c i da . These can be det e c t e d in vi t r o by p r o du c i n g a

cytopathic e f f e c t ( CP E ) in cultured t i s s ue ce l l s by expo s ing t hem to

ce l l - f ree ext ra c t s of toxin-p roduc ing st ra ins o f P . mul t oc i da .

We s o ught t o e s t a b l i s h i f t ox i g e n i c s t r a i n s o f P . m u l t o c i da a re

present in thi s count ry u s in g cell cult ure s a s a s c reening met hod .

Th i s me t h o d h a d t o be v a l i d a t e d in o u r h a nds and has not been

previ o u s ly used in New Zea land .

Emb ry o n i c Bov ine Lung ( EB L ) cel l s and " sen s i t i zed" Vero c e l l s have
b e e n u s ed t o de t e c t ex o t o x i g e n i c s t ra in s of P. m u l t o c i da ( see

Sect ion 1 . 4 ) . I n our h a n d s a CPE wa s not v i s i b l e i n " s e n s i t i z ed"

Ve r o cells ( da t a not shown ) but w a s p r o du c e d i n EBL c e l l s ( s ee

F i g u r e s 4 . 1 and 4 . 2 ) . C o n s e quen t l y the EBL ce l l c u lt u re a s s ay was

used to s c reen f ield i s o l a t e s for exotoxin product ion .

N o n e o f t he N e w Z e a l a nd s t r a i n s o f P . mul t ocida which were t e sted

( se e Table 4 .2) pr odu c e d a CPE in E B L c e l l s . ie n o ne p r o du c e d
exotoxin . Th i s w a s n o t unexpected because none o f our New Zea l and
i s o l a t e s had been recove red f rom pig s with AR a nd we were unable t o

ide n t i fy a n y h e rd w i t h the d i s e a s e du ring t he c o u r s e o f t h i s s t udy .

We c o n c lude t h a t the pre s ence of t oxigenic s t r a ins o f P . mul t o c i da

 157

mus t rema in a n open que s t ion unt i l e ither a n outbreak o f AR occurs

and i s o l ates f rom the na s a l t ract o f these pigs can be examined or

in t he absence of the disease . This r e qu i r e s a pro ject wh i c h

s y s t emat i c a l l y s c reens anima l s , espe c i a l ly pigs , i n New Z e a land f o r
the ca rriage of P. mu l t o c i da a n d examine s i s o l ates f o r t he

product ion o f endotoxin .

6 . 6 Ant ibi o t i c Re s i s t a n c e and i t s Re lat ionship to P l asmids in New

Zealand S t rains o f P . m u l t ocida

The occurrence of ant ibiot ic re s i s tance in P . mu l t o c i da i s a problem

in s o me count r ie s and has been att ributed t o the ( l a r ge l y

i n d i s c r im i n a n t ) use of a n t ib i o t i c s . Penic i l l i n , Tet r a c y c l ine ,

S t reptomyc in and Su lphonamides , a re often u s ed t o t reat disease due

t o P . mul t o c i da in dome s t i c anima l s in New Ze a l and so it t he re f o re

s eemed p o s s i b l e that " re s i s t ant " st rains of P . mul t ocida , ie s t ra ins

w i t h a s en s it i vity below a de fined leve l ( s ee Sect ion 5 . 3 . 1 ) could

be i s o lated f rom anima l s i n this count ry .

We d e t e rmi n e d t h e se n s i t i v i t y ( MIC ) of New Zea land s t ra i ns of

P. mul t o c i da but found t h a t none had a re s i s t ance h igh enough t o be

de s i gnated " re s i s tant " . C a t , dog and pig i s o la t e s p o s s e s s e d var iable

s e n s i t i v i t i e s but rabb i t i s o l a t e s w e re h omogene o u s . This latter
re s u lt wa s n o t surp r i s ing because it h a d previ ous ly been concluded

( s e e Sect ion 3 .4) t ha t o u r rabb i t is olates r e p r e s e n t e d mu l t i p l e

i s o l at ions o f a total o f t h ree s t ra i n s only .

The presence o f ant ibio t ic re s i s t a n c e i n P . m u l t o c i da i s o ften

a s s oc iated w i t h pla smids between 1 Mda l a n d 5 Mda l s in s i ze ( Hirsch,

M a r t in a n d R h o ade s , 1 985) . Met hods f o r e xt r a c t i n g p l a smids f rom

b a c t e r i a n o rma l l y involve a techn ique ( eg Ekha rt method) which i s
gen t le enough t o ext ract , in a n intact form , r e l a t ive ly l a rge ( ove r

l O O Mda l s ) p l a smids . Howeve r , we u s ed a harsher method ( s ee Sect ion

5 . 2 . 1 2 ) , wh i c h ext racted t o t a l DNA for use in both the p l a smid a s s ay
and REA . This me t h o d did not r e c o ve r l a r ge p l a smids but we
 158

d e mo n s t r a t e d ( see F igu res 5.1 and 5.2) that p l a smids up t o

a p p r oximat e ly 5 Mda l s i n s i z e w e r e p r e s ent . There f o re t h i s met hod
w a s sui table f o r detect ing re s i s t ance pla smids in P . mul t o c i da .

We found t h a t seve r a l c a t and dog s t rains o f P . mul t oc i da po s se s sed

p l a smids wh i c h had mo l e c u l a r we ight s of approximate ly 1 . 4 Mda l s and
2 . 4 Mda l s . H oweve r , s ince the s e s t r a ins did not p os s e s s " re s i stance"
t o the t e s t a nt i b i o t i c s we c o n c l u de that t h e s e p l a smids , u n l i ke
s im i l a r p l a smids in P . m u l t o c i da i s o l a ted ove r s ea s , do not c a r r y
r e s ist ance ma r ke rs .

6 . 7 Out look

Despite i t s impo rtance a s a pat hogen of s w ine , cat t le , sheep , fowl

a nd rabbit s , P. m u l t o c i da h a s been l i t t le s t udied in New Zeal and .

T h i s the s i s repre sent s a prelimina ry step to a f u l le r unde r s t anding

o f the imp o r t a nce o f P . mul t oc i da in t h i s c ount ry and t o t he be s t

me ans o f cont rol . The po s s ib i l i t y o f outbre a k s o f a t rophic rhinitis

due t o t he i n t roduc t i on o f t o x i g e n i c s t r a i n s of P . m u l t o c i d a i n

" c a r r i e r pigs " may we l l s t imulate furthe r w o r k o f t h i s nature .

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APPENDIX

B l ood Aga r 5 %

Defibrina ted Horse B l ood 5 0mll - 1

Blood Aga r Base

i. The Blood Aga r Base was me l t ed and c o o l ed t o 5 0 C .

ii . Defibrinated Horse B l o od wa s a dd e d t o g i ve a f inal

concent rat ion o f 5 % a nd 2 5ml vo lume s of t h i s preparat ion
were dispensed i n st anda rd pet ri dishe s .

B l o o d Aga r B a s e ( D i f co )

T h e me d i um w a s p r e p a r e d a s pe r manu f a c t u re r s i n s t ruct i o n s ,
autocl a ved in 1 0 0ml bot t l e s and s t o red at room tempe rature .

B r a in-He a rt Inf usion Brot h ( D i f c o )

The medium was prepa red a s per manu f a c t u r e rs i ns t ruct ions and

aut o c l a ved in appropriate cont a i ners .

D e xt rose St a rch Aga r ( D i f c o )

T h e me d i um w a s p re p a re d a s pe r ma n u f a c t u re r s i n s t r u c t i o n s ,

aut o c l a ved in 1 0 0ml bot t l e s and s t o red at r oom tempe rature .

 160

Dext r o s e S t a rch Broth ( pH 7 . 3 )

P roteose Peptone N 3 (Difco ) : 1 5 . Ogl - 1

B a c t o Dext rose ( Difco) : 2 . 0 gl - 1
S o l uble S t a rch : 1 0 . 0 gl - l
NaCl 5 . Ogl- 1
D i s odium Phospha t e 3 . 0 gl - 1
D i s t i l led Wat e r

The compone n t s we re mixed and the pH a d j u s ted u s ing NaOH . The

prepa rat ion w a s then auto c l aved in app r opriate cont a iners a nd
s t o red at + 4 C .

*
Ko vacs Re agent

Amy l , Isoamyl , or I s obutyl Alcoho l 1 5 . 0ml

. . **
pa r a - D lme thylamlnobenza ldehyde 1 . Og

Concentrated Hydroch loric Acid 5 . 0ml

The a ldehyde wa s disso lved complet e l y in t he a l cohol . The a c id

wa s t hen added ( s lowly) and the prep a r a t ion was s t o red at + 4 C

in a da rk cont a i ne r .

*
Based on B l a i r et a 1 ( 1 9 7 0 ) .

**
The raw a l d e h y de s h o u l d be

l ight o f c o lou r .
 161

c Conkey Aga r ( D i f c o )

T h e me d i u m w a s p r e p a r e d a s pe r ma n u f a c t u re r s i n s t r uc t i o n s ,
a ut oc laved i n 1 0 0ml bot t le s and s t o re d at room tempe r a t u re . As ,
requi red 1 0 0ml of medium was me lted a nd dispensed between f ou r
s t andard pet r i dishe s .

*
Mot i l i ty Medium

P eptone 1 0gl-1
NaCl Sgl-l
D a v i s Aga r : 3gl- l
D i s t i l led Wa t e r

T he component s were di s s o lved i n t h e D i s t i l led Wat e r , a d j u s ted

to pH 7 . 5 u s ing NaOH , di spensed i n t ubes , autocl aved and s t o red

a t + 4 C .

* .
B a s e d on B l a l r e t a 1 ( 1 9 7 0 )

NaCl S o lut ion (0 . 85%)

NaCl was d i s s o l ved in Dist i l le d Wat e r to give a f inal

c oncent r a t i o n o f 0 . 8 5 % . The s o l ut i o n w a s then autoc laved a nd
s t o red at + 4 C .
 1 62

*
Oxida s e Reagent

i. A fre s h 1% s o lut ion of Di- or T e t r a -met h y l - p

pheny l e n e d i am i n e d i c h 1 o r ide w a s p re p a red i n d i s t i l l e d
wate r .
ii . As c o rb i c a c id ( a st abil i z e r ) w a s added t o g i ve a f in a l
concent r a t ion o f 0 . 1 1 % .
i i i . 5 mmx 5 0mm st rips of Whatman N 1 f i lte r paper were s o a ke d i n
the prepa rat ion , a i r dried a t 3 7 C a n d s t o r e d at 4 C i n a
sea led c ontaine r .
* .
Based on B l a 1 r e t a 1 ( 1 9 7 0 ) .

PBS ( P hospha te Bu f f ered S a l ine )

NaCl : B . Sgl - 1

NaH 2 Po 4 . 2 H 2 0 0 . 4 gl - 1

D i s t i l led Wat e r

The component s were mixed, aut o c l a ve d and s t o red at + 4 C .

Pept one Wa ter ( pH 7 . 2 )

P eptone 1 0 g l-1

NaCl Sgl-1

D i s t i l led Wat e r

The component s were mixed and the p H w a s a d j u s t e d us ing NaOH .

The prepa ra t i on was t hen aut o c laved a nd s t o red a t + 4 C .
 163

Pept one Water S uga rs

Bromocre s o l Purple : 2 5ml l - 1

Peptone Wat e r (pH 7 . 2 - 7 . 3 )

i. The ingredient s were mixed a nd Sml vo lume s were dispensed

in l o z bot t le s .
ii . A du r h am tube was a dded to each bottle and the

prep a r a t ions were autoc l aved .

i i i . The p r eparations were s t o red a t + 4 C . At the t ime o f use ,
0 . 2 5 ml of ster i le 1 0 % s u g a r s o lut i on was added t o e a c h
bott le .

T r iple Suga r I ro n Aga r ( T S I )

The medium was prepared a s per ma nufacturers in s t ruc t i on s .

T rypt o - S oy Aga r (Difco)

The me d i um w a s p r e p a red a s p e r ma n u f a c t u r e r s i n s t r uc t i o n s ,
autoc laved in l O Om! bottles and s t o red at room tempe r a t u re . As

requ i red l O Oml of medium was me lted and di spensed between four

s tanda rd pet r i dishe s .

T rypt o - S oy Broth (Difco)

The medium w a s prepa red a s pe r manufacture r s inst ruct i on s .

U r e a Aga r ( D i fc o )

T h e me d i u m w a s p r e p a red a s p e r m a n u f a c t u re r s i n s t r u c t i o n s .
 164

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