Pasteurell a multocida
A Study on the I solation, Ident i fication and Characterization of New Zealand St rains .
A The s i s P re s ented in P a rt i a l F u l f i lme nt o f t h e Requ irement s for t he Degree of Ma s t e r o f S c ience in Microbio logy at Ma s s ey Un ive r s i t y , New Zealand .
RhysJohnJon�
1988
ABSTRACT
P a s t e urella mul t ocida is a sma l l Gram negative bac i l lus which causes dis e a s e in many anima l spe c i e s . I t s pathogenic it y i s a t t r ibuted to two mechanisms : inva s ivenes s and toxin product ion . The proport ion o f s t r a in s w h i c h produce t ox i n i s
a t r o p h i c r h i n i t i s i n s w i n e . m e c h a n i s m o f p a t h o g e n i c i t y po s se s s ion o f a capsule .
low but such s t r a ins c a u s e severe I n v a s i ve n e s s i s t h e p r edomi n a n t a n d i s l a r g e l y d e p e n d a n t o n t h e
P . m u l t o cida i s divided into f ive se rotype s ( A , B , D , E and F ) ba sed on t he antigenic structure of the capsule a nd a c o r relat i on exi s t s between serotype , disease 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 re 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 on o f a n o n a n t i gen i c ( hy a l u ronic a c i d ) caps u l e . The presence o f an ant igenic capsule and a hyaluronic a c id capsule are independant . i e one , both o r n e ither may be present .
T o f a c i l i t a t e t h e i s o l a t i o n o f P . m u l t o c i d a we a s s e s s e d t h e s e l e c t ive medium of Smith and Bas ke rvi lle ( l 9 8 3 ) a n d found that many New Zea land i s olates of P . mul t o c i da grew poorly in the absence o f b l o od ( not p resent i n Smi t h and Bas ke rville ' s medium) . Furt hermore , m a n y i s o l a t e s were inhib it e d by P o l ymixin and t h e high a lkal inity
(pH 8 . 6 ) o f t he medium .
O n t h e b a s i s o f t h e s e o b s e r v a t i o n s w e 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 Polymixin 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 , B a c i t r a c i n a n d Myco s t a t i n ) p r e s e n t i n t h e o ri g i n a l med i um o f Smith and Baske rvil le . T h i s modif ied medium propagated a l l o u r t e s t 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 de r ived f rom s even
iii
animal spec ie s ( f owl , s h e e p , goat , c at t l e , r abbit , dog , c at ) . I t als o suppre 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 the t radit ional is o l at ion t e chnique o f p l at ing specimen s on b l o od agar and the 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 iso l ate s ) , rabbits ( 2 5 ) , cat s ( 1 0) , dogs ( 2 1 ) and deer ( 1) . I s olates we re examined in some det ail .
I s o l ates were serotyped u s ing the indirect haemagglutination as s ay ( I H A ) . T h e y w e r e f o u n d t o be T ype A ( 7 % ) , B ( 1 % ) , D ( 9 % ) o r untypable ( 8 3 % ) . This is similar t o overseas f indings .
The IHA as s ay is a laborious technique s o two pos s ible alternat ives 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 ly ac r y l amide - ge l a n a l y s is ( S D S - P AGE) o f p r o t e in s and r e s t r ic t io n e n do n u c l e as e an alys is ( REA) o f DNA . Both techniques f ailed t o dist inguish between P . mul t o c i da s e rotypes in the s e n s e t h at is o l at e s were e xt reme l y heterogeneous and mos t g ave a unique pattern both with SDS-PAGE and REA .
T h is hete rogeneity allowed the ident ification o f individual s t r ains o f P . m u l t o c i d a a n d c o n s e qu e n t l y S D S - P AG E w a s u s e d in an e pidemiological inves t ig ation which t raced t he origin o f an outbreak of respirat o ry dis e ase in rabbit s and provided evidence t h at the outbre ak was not ( as was earlie r believed) due t o t he int roduction of rabbit 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 rmore , u s ing t his approac h we were able t o show t h at s t r ains which c ause s eve re pneumonia were not c o n f ined t o t he lowe r respirat ory t ract but could al s o be carried in t he nasal c avit y . This is consis tent w it h the accepted hypothesis t h at P . mul t oci da is an opportunis t ic pathogen .
Severe a t roph ic rhi 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 ins of P . mul t ocida . The di sease is respons ible f o r muc h e conomi c l o s s ove r s e a s but h a s not been reported in New Zealand . We obta ined a toxigenic s t rain of P . mul t oc i da and used it t o va l i d a t e an in v i t r o ( ma mma l i a n c e l l cu l t u r e ) me t h o d f o r de t e ct i n g t oxin -produ c i ng s t r a i n s . T h i s me t hod w a s t he n u s ed t o examine New Zea land i s o lates o f P . mul t ocida . None o f these were s hown t o be t ox igen i c . T h i s sugge s t s t h a t t ox i g e n i c s t ra in s , i f present in New Zea land, are not c ommon .
D iseases due to P . mu l t o ci da are 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 , Aminoglyc o s ides and Sulphonamides . St rains which are res i s t ant to these agents are p resent in overseas c o u n t r i e s a nd r e s i s t a n c e is a s s o c i a t e d w it h t h e p r e s e n c e o f pla smids .
We dete rmined the min imum inhib i t o ry concent rat ions ( M I C ) , o f four a n t imi c r o b i a l s ( P e n i c i l l i n G , S t r e p t o my c i n , T e t r a c y c l i n e a n d S u lphadi a z i ne ) f o r New Z e a land i s olates . Con s iderable variat ion wa s f o und b e t w e e n i s o l a t e s but no i s o l a t e w a s o f s u f f i c i e n t l y h igh re s i stance to be cons ide red " res i s t a nt " . This was de spite the fact t h at 1 2 of 73 ( 1 6 % ) of t he f i e l d i so l a t e s p o s s e s s ed pla smids of a s imil a r s i ze ( lMda l to SMda l ) to plasmids known to c a rry ant ibio t i c re sistance markers .
De spit e it s import ance a s a pathogen of swine , cat t le , s heep , fowl and rabbit s , P . mul t o c i da has been l it t le s t u died in New Zealand . T h is the s i s represents a pre l iminary stage t o a fuller unde rstanding of the importance of P . mul t ocida in this coun t r y and the best means o f c o n t r o l . The p o s s i b i l it y o f o u t b r e a k s of dis e a s e due t o P . mul t o ci da , eg at rophic rhinit i s , may wel l s t imulate further work on this organism .
V .
ACKNOWLEDGMENTS
I am indebted t o the D epa rtment o f Mic rob 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 ject.
I wish to thank my supe rvi s o r , John Cla rke , f o r his cont r ibut ion to this work . His zeal and gramma t i c a l dext e r i t y were mos t apprec iated .
T ha n k s go t o Ron T u c ke r and P a u l Hocqu a r d , f o r t he i r e f f o r t s in s upplying mate r ia l s , and to the f o l lowing people and o rgan i z at ions f o r donat ing mat e r i a l s a nd providing financ i a l a s s i s t ance :
The Leona rd Conde l l Trust
Min i s t ry of Agriculture and F isheries Rex P at t e rson (Gibco NZ Ltd)
Drs J.M . Rutt e r and P . D . Luther (ARC I n s t i t ute, United Kingdom)
I w o u l d l i k e t o t h a n k G e o r g e I o n a s f o r g e ne r o u s l y p r o v i d i n g t echnic a l a dvice . I a m a l s o indebted t o h im f o r h i s phi l o s ophi c a l t eachings , especially h i s balanced concept o f a utopian s o c iety .
F inally I thank my c l a s smates Mike , Nicky, S a l l y and Steve for the i r f riendship and to the t e chnica l s t a f f f o r ente rtaining u s a l l .
TABLE OF CONTENTS
T IT LE P AGE . . ABS TRACT
ACKNOWLEDGEMENTS . . TABLE OF CONTENTS L I S T OF F IGURES L I ST OF TABLES INTRODUC T I ON . .
Page
i i i
V
vi xi . . x i i i 1
CHAPTER 1 : Historical Review
1 . 1 Clas s i f i cation and Nomenc lature 4
1 . 2 Serological Cha racterisat ion o f P . mu l t ocida
1 . 3 1 . 4
1 . 2 . 1 E a rly Attempts at Grouping . . 9 1 . 2 . 2 D i scove ry o f Capsu l a r T ypes 12 1 . 2 . 3 D iscovery o f Soma t ic Ant igen ic Groups 1 6 1 . 2 . 4 Agreement between Typing Methods . . 1 9
1 . 2 . 5 Bacteriophage Typing . . 2 0
Ant igeni c St ruct ure o f P . mul t ocida Toxigenic St rains o f P . mul t oc i da . .
2 0 2 6 1 . 5 Disease As sociat ions and Epidemiology o f
P . mul t ocida
1 . 5 . 1 1 . 5 . 2 1 . 5 . 3 1 . 5 . 4 1 . 5 . 5 1 . 5 . 6 1 . 5 . 7 1 . 5 . 8 1 . 5 . 9
Gene r a l Catt le Sheep Goa t s . . Rabbi t s S wine . . Dee r
Cat s and Dogs Man . . . . . .
2 9 3 1 3 3 3 5 3 7 3 8 3 9 3 9 4 0
CHAPTER 2 :
1 . 6 The Re l a t ionship of Type o f P . mul t oc i da t o Hosts a n d Diseases . . . .
1 . 7 Immuni t y and Vaccines . .
1 . 8 Ant ibiotic Resist ance and the P resence o f P lasmids . . . . . . . . . . . . . .
1 . 9 Iso l a t i on and Ident ification o f P . mu l t o ci da
Is olation of P. mul t ocida from the Nas a l Cavity:
Evaluat ion and Modi fication of a S elective Medium
2 . 1 2 . 2
2 . 3
2 . 4
Int roduct ion . . . . . . Mate r i a ls and Methods 2 . 2 . 1 P repa ration of
. .
Media
. .
. . . .
. . . .
. . . .
. . 2 . 2 . 2 P repa rat ion and S t anda rdizat ion of
Cultures . . 2 . 2 . 3 Eva luat ion 2 . 2 . 4 The Effect
. . . . . of 8HPG
. . . . Medium
of Ant ibiot i cs on the G rowth of P . mul t o c i da a nd the Suppression of
2 . 2 . 5 I noculation of Medium ( SM) Results
2 . 3 . 1 Evaluat ion of 2 . 3 . 2 The Ef fect of
Othe r Organisms . . Modi f ied Select ive
. . . . . . . .
8 HP G Medium Ant ibiotics
. .
on . .
the Growth o f P . mu l t o c i da on BA 2 . 3 . 3 Evaluat ion of Modif ied S e lect ive
Medium ( SM) D iscussion
. .
. .
. .
. .
. .
. . . .
. .
. . v i i
4 0 4 5
5 0 5 2
5 4
5 6
5 8 5 9
5 9
6 0
6 1
6 3
6 6 6 9
CHAP TER 3 :
3 . 1 3 . 2
Is olat ion of P . mul t ocida, Serotypinq o f I solates and Comparison of Thes e by SDS-PAGE and Res t r iction Endonuclease Ana lys is (REA)
Introduct ion . . . Mat e r i a ls and Methods . .
3 . 2 . 1 Isolation and Ident i f ic a t ion o f P . mul t ocida . . . .
3 . 2 . 1 1 Materials 3 . 2 . 1 2 Methods
3 . 2 . 2 P rocedures for the IHA A s s ay 3 . 2 . 2 1 Materials
3 . 2 . 2 2 Methods
3 . 2 . 3 P rocedures for SDS-PAGE Analys i s 3 . 2 . 3 1 Materials
3 . 2 . 32 Methods
3 . 2 . 4 P rocedures for Re s t rict i on Endonuclease Ana lys i s ( REA)
3 . 2 . 4 1 Mate rials 3 . 2 . 4 2 Methods . .
7 2 7 2
7 3 7 4 7 3
7 4 7 6
8 4 8 8
9 2 9 5 3 . 3 Result s
3 . 3 . 1 I solat ion o f P . mul t oc i da and Some Epidemio logical Aspect s . . . .
3 . 3 . 2 S e rotyping : Va lidat ion o f IHA As s a y a nd its Application t o t he Typing o f F ield St rains
9 9
3 . 3 . 2 1 Va l idat ing the !HA Ass a y 1 0 3 3 . 3 . 2 2 Serotyping o f F i e ld I s o l a t e s 1 0 4 3 . 3 . 3 SDS -PAGE Ana lys i s o f S t r a ins o f
P . mul t oci da . . . . . . . . 1 0 5 3 . 3 . 4 Epidemio logical Tracing o f
P. mul t ocida by SDS-PAGE . . . . . 1 0 7
CHAPTER 4 :
CHAP TE R 5 : 3 . 4
4 . 1 4 . 2
4 . 3
4 . 4
5 . 1 5 . 2
ix
3 . 3 . 5 The Source of a P . mul t ocida Isolate Associated with an Epidemic of
Snuff les . . . . . . . . . 1 1 3 3 . 3 . 6 Compa rison o f P . mul t o cida Serotypes
with SOS-PAGE and REA . . D iscussion
The Detect ion of Toxigenic S t rains of P . mul t ocida Derived f rom New Zealand Animals
1 1 6 1 1 9
Int roduct ion . . . . . . 1 2 0 Mat e r ials and Methods
4 . 2 . 1 P repa rat ion of Reagents and Media . . 4 . 2 . 2 Methods . . . . Resul ts
4 . 3 . 1 Va lidat ion of the EBL Assay . . 4 . 3 . 2 Assay o f Field Iso l ates
D iscussion
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 Plasmids
I nt roduction . . . . Mat e r ials and Methods . .
5 . 2 . 1 Examination of P . mul t ocida for P lasmid Carriage
5 . 2.1 1 Mate rials 5 . 2 . 1 2 Methods . .
5 . 2 . 2 P . mul t ocida : Dete rminat ion o f the Minimum I nhibi t o ry Concent rat ions of Ant ibiot ics
1 2 1 1 2 4
1 2 8 1 3 0 1 3 1
1 3 2 1 32
1 3 3 1 3 3
5 . 2 . 2 1 Materials 1 3 4
5 . 2 . 2 2 Methods 1 3 5
5 . 2 . 3 P rocedures for SOS -P AGE Analysis 1 3 6
5 . 3 Results . . 136 5 . 3 . 1 The Ant ibiotic Sensit 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 lasmid Carrying S t r a ins Versus Non-
P lasmid C a r rying S t r a i ns . . 147
5 . 4 D iscussion 147
CHAPTER 6: Gene ral Discus s io n . . . . . . . . . . . . . . . . . . . . . . 151 6 . 1 The Development o f a Select ive Medium f o r
the Isolat ion o f P . mult o c i da i n New Zealand . . 152 6 . 2 Iso l a t ion and Se rotyping o f P. mul t ocida
f rom New Zealand Animals . .
6 . 3 S D S - P AGE Ana lysis of Field Isolates o f
6 . 4
P . multocida.. . . . . . . . . Compa rison o f P. mu lt ocida S e rotypes by
. . . . 152
. . . . 154
Rest r ict ion Endonuclease Ana lysis ( REA) . . .. . . 155 6 . 5 Assay f o r Exotoxigenic S t r a i ns o f
P . multocida .. .. .. .. .. . .
6 . 6 Ant ib iotic Resist ance and its Relat ionship to P lasmids in New Zea land S t rains o f P . multocida ..
6 . 7 Out look
. . 156
157 158
APP END IX . . . . 159
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
LIST OF FIGURES
F igure
1 . 1 Gram S t a i n o f P . mul t o c i da
1 . 2 Ant igenic Cha racte r i s t i c s of P . mu l t ocida
x i
Page 5
1 8
1 . 3 Type A Colon ies of P . mult ocida . . . . . . . . . . . . . . . . . . 22
1 . 4
2 . 1
Type D Colonies o f P . mu l t ocida . . . .
The Suppre s s ion of Na s a l Bacte r i a l F lora by a Select ive Medium ( SM) . . . . . . . . . . . . . .
3 . 1 SDS-PAGE P rotein Ana lys i s of P . mu l t o ci da I s o lated
22
6 7
f rom Seve r a l Anima l Species . . . . . . . . . . . . . . . . . . . . 1 0 6
3 . 2 SDS -PAGE Ana lysis o f P . mult ocida I so lates f rom Swine . . 1 0 8
3 .3 SDS-PAGE Ana l y s i s o f P . mult o c i da I s o lates f rom Dogs . . . . 1 0 9
3 . 4 SDS -PAGE Ana lys is o f P . multocida I s olates f rom Cat s . . . . 1 1 0
3 . 5 SDS -PAGE Ana l y s i s o f P . multocida I s o lates f rom Rabbi t s . . 1 1 1
3 . 6 SDS-PAGE Ana lys is of P . multocida f rom Rabbits . . . . . . . . 1 1 2
3 . 7 SDS-PAGE Ana ly s i s o f P . mult 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 nas a l t ra c t i so lates . . . . . . . . 1 1 4
3 . 8 SDS -PAGE Ana lysis o f P . mul t ocida I s o lates f rom Rabbits : A compa r i s o n o f i s o l a t e s obta ined before and a f t e r
the int roduct ion o f anima l s , f rom ove rsea s , t o a
rabbit c o l ony . . . 1 1 5
3 . 9 SDS -PAGE Ana lysis o f P . mul t ocida se rotype s A, B and D . . 1 1 7
3 . 1 0 Rest 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 . . . . . . 1 1 8
4 . 1 Cell Culture Monolaye r o f Embryonic Bovine Lung Cells . . 1 2 9
4 . 2 Cell Culture Monolaye r o f Embryonic Bovine Lung Cells : The cyt opathic e f fect o f P . mu l t o c i da exot oxin
5 . 1 Agarose -Ge l E lect rophore s i s o f DNA f rom P lasmid-
. . 12 9
Conta ining F e l ine I s o lates o f P . mu l t ocida . . . . . . . . . . 1 3 7
5 . 2 Aga rose-Ge l E lect ropho res i s o f DNA f rom P lasmid-
Conta ining Canine I s olates of P . m u l t ocida . . . . . . . . . . 1 3 8
5 . 3 Variat ion o f MICs Between S t rains o f P . mul t oc i da . . . . . . 1 4 2
5 . 4 SDS-PAGE Ana lysis o f P . mul t ocida I s o lates f rom Dogs :A compa r i s on of plasmid-cont aining i so lates with
i solates whi ch do not contain pla smids . . . . . . . 1 4 8
5.5 SDS-PAGE Ana lys i s o f P. mul t ocida I s o lates f rom Cats : A comparison o f plasmid-conta in ing i so lates with
i solates which do not contain pla smids . . . 1 4 9
T able
1 . 1
1 . 2
1 . 3
LIST OF TABLE S
P. mul t o c i da Soma t i c Groups and the i r Re lation t o Capsu l a r Type . . . . . . . . . . . . . . . . . . . . .
Host Range o f P. mul t oci da
Relationsh ip o f P. mul t ocida Capsu l a r Type to D isease
1 . 4 Re lationship of P . mu l t ocida Serot ype to Host and D isease . .
x i i i
P age
1 8
2 9
4 1
4 4
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 Species . . . . 53
2 . 1 The Abi l ity of 8HPG Medium to P ropagate P. mul t ocida . . . . 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 . . . .
2 . 3 The Ef fects o f Low Leve ls o f a Comb inat ion o f Ant ibiot ics o n P . mu l t ocida . .
2 . 4 The E f fects o f Individual Ant ibiot i cs at D i f f e rent Concent rat i o ns on the Growth o f P . mul t ocida
6 4
6 5
6 8
2 . 5 The Abi l it y o f SM Medium to Recover P. mul t o c i da . . .. . . 6 9
3 . 1 P. mul t oc i da Stra ins Isolated f rom Rabbits . . . 1 0 1
3 . 2 The P roport i ons o f Pneumonic Lesions f rom which
P . mul t oc i da was Isolated in Winte r and Summe r . . . 1 0 2
3 . 3 Serotyping P rototype S t r a ins o f P . mul t ocida by IHA Assay
3 . 4 Serotypes o f Field I s o lates a s Dete rmined by the IHA Assay
4 . 1 As s ay o f P ro t otype St rains of P . mu l t ocida for
. . . . 1 0 3
. . 1 0 4
Exotoxin P roduction . . . . . . . 1 3 0
4 . 2 Assay o f F i e ld I s olates o f P . m u l t o c i da for Exotoxin
P roduction . . 1 3 1
5 . 1 Assay o f S t r a ins o f P . mul t oc i da f o r P la smids . . . . . 1 3 6
5 . 2 An Evaluat ion o f P l asmids Carried by St rains o f
5 . 3
P . mult ocida . . . 1 3 9
Minimum Inhibitory Concent rations o f Ant ibiot i c s For
P. mul t o c i da . . . . . . . . . . . . . . . . . . . . . . . . 1 4 0
