A study of Leptospira interrogans infection in deer and goats in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand

the permission of the Author.

A STUDY OF LEPTOSPIRA INTERROGANS

INFECT I ON IN DEER AND GOATS IN

NEW ZEALAND

A THESIS P RES ENTED I N P ART I AL FULF I LMENT OF THE REQUI REMENTS F O R THE D EGREE OF

��STER OF S C I ENCE IN MICROBIOLOGY AT

�;SS EY UNIVERS I T Y , P ALMERSTON NORTH , NEW Z EALAND

STEPHEN HARRY FLINT 1 9 8 8

ABS TRJI.CT

I n o rde r t o de t e rm i n e t he p re v a l e n c e o f lept o s p i ro s i s in dee r and g o a t s b o t h s e r o l o g i c a l te s t s a nd the c u l t u r i ng o f b a c t e r i a f r om u r ine s amp le s we re u s e d . The s e r o logic a l t e s t s enabled a n a s s e sme nt to be made as t o the n a t ure and e xt e nt of a n t i body leve l s . To e n s u re c on f i dence i n t he s e r o l ogical re s u l t s , i t w a s nec e s s a ry to va l ida t e t h e s t a n da r d mic r o s c o p i c a g g l u t i o n a t i o n t e s t (MAT ) . Rep e a t t e s t s d e mo n s t r a t e d r e p r o du c i b l e r e s u l t s t h a t we r e w i t h i n o ne t w o - f o ld s e r i a l d i l u t i o n . E n z ym e l i n k e d immuno s o rb e n t as s a y s (EL I S A ) w e r e i nve s t i g a t e d a s a l t e r n a c i v e s f o r b o t h t he de t ect ion o f ant ibodies a n d L ep t o sp i r a a n t i g e n s . T h e s e a s s a y s d e m o n s t r a t e d g r e a t e r s e n s i t i v i t y t h a n t he MAT f o r t he det ect ion o f ant ibodie s , but were l e s s s e n s i t ive than s t a n da rd me t hods for t he de tec t i o n of a n t i ge n .

U s i n g t h e MAT , a n t i b o d i e s t o c op e n h a g e n i , h a r d j o , p o m o n a ,

a u st ra l i s , ba l l u m , a n d t a r a s s o vi w e r e

b r a t i s l a va , d e t e c t e d i n s e r o l o g i c a l s u rveys o f deer and goats . I n dee r , t he mo s t f requent ly reco rded a n t ibody t i t re s we re t o ba l l um, bra t i s l a va a nd c openh a gen i . A s 8 7% o f the ant ibody t i t re s < 8 0 , there appe a rs t o b e a low leve l o f a c t ive i n fect i o n . I n some a r e a s t he re wa s a h i gh p re v a l e n c e o f a n t ibody t i t re s t o h a rdj o . I n go a t s , 70% were f ound t o h a ve ant ibody t i t re s � 1 0 t o one o r mo re s e r o va r s w i t h a n t i b o d i e s t o ba l l um and b ra t is l a va t h e mo s t f r eque n t l y r e c o r de d . As 90% o f t he a n t ibody t i t re s were < 8 0 , there a ppea r s t o be a l o w leve l o f a c t i ve i n f e c t ion i n goa t s .

A n t i b o d i e s t o bra t is l a va , a s e r o va r that h a s not been i s o la t e d i n N e w Z e a l a n d , we re wide s p read in b o t h dee r a n d goa t s . T h e po s s ib i l it y

iii

t h a t t h e s e r e s u l t e d f r om mixed i n f e c t i o n s w a s c o n s ide red but not r e s o lved . U n s u c e s s f u l a t t emp t s we r e ma de t o p u r i f y mixed c u l t u re s u s i n g s p e c i f i c a n t i s e r a . T h e p o s s i b i l i t y o f s e r o l o g i c a l c r o s s r e a c t i o n s o f a n t i b o d i e s t o o t h e r s e r o v a r s w i t h b ra t i s l a va w a s s upported b y the i nc r e a s ed s e ro l o g i c a l r e s p o n s e o f dee r and go a t s t o v a c c i n a t i o n w i t h h a rdj o a n d pomon a a n t ige n s . We s t e rn b l o t s t udies i dent i f ie d s e ve r a l c ommo n ant ige n s between bra t i sl a va and pomon a .

A s t udy o f a dee r f a rm showed a h i gh p re v a l e n ce o f a n t ibody t i t re s t o h a rdj o c o r r e s p o nd i n g to a s im i l a r p reva l e n c e o f a n t ib ody t i t re s t o bal can i ca be l ie ve d t o b e due t o t h e a n t i ge n i c s im i l a r i t y o f t h e s e t wo s e ro v a r s . Bal can i c a was i s o l at e d f rom u r i n e s amp l e s f rom t h e s e deer and i s bel ieved t o b e the f i r s t i s o la t i o n of t h i s s e rova r f rom dee r .

S t u d i e s o f s i x g o a t f a rm s s h o w e d l o w a n t i b o dy l e v e l s a n d n o L ept o spi ra we re i s o l a t e d f rom u r i ne samp le s .

ACKNOWLEDGEMENTS

I am i ndebted to my supe r vi s o r s , D r s . Roge r M a r s ha l l and Mary Nu l s�n f o r t h e i r e n c o u r a g e me n t a n d gu i de n c e a n d t o t h e D e p a r t me n t s o f Ve t e r i n a ry Patho l ogy and P ubl ic H e a l t h and M i c rob i o l ogy and Gen e t i c s f o r p ro viding the f a c i l i t i e s w h i c h enabled t h i s study .

I a p p r e c i a t e d t h e a dv i c e , a s s i s t a n c e a n d f r ie nds h i p o f my e o­

w o r ke r s , I nge B o l t a n d Ray C o r ne r .

T h e h e l p o f D r . P . R a ma d a s i n t h e c l a s s i f i c a t i o n o f i s o l a t e s p ro v ide d a n e s s e n t i a l contribu t i o n t o t h i s t he s e s .

I w i s h t o t h a n k D r . C o l i n Ma c k i n t o s h f o r h i s adv i c e w h i c h he l ped launch t h i s p roje c t a nd f o r h i s e f f o r t s in o b t a ining s e rum s amp l e s . I am a l s o great f u l t o the Mi n i s t ry o f Ag r i c u l t u re and F i she r i e s a t M o s g i e l , Ru a k u r a a n d Wa l l a c e v i l l e f o r p r o vid i n g me w i t h s e r u m s amp l e s .

John Achten and T re v o r S h akes p r ovided t he i r s k i l l and expe rience in a n ima l handl ing wh i c h proved inva luble in the colle c t i o n o f s amp l e s .

The t e c h n i c a l adv i c e o f Geo rge I o n a s w a s muc h appre c i a t ed .

I w i s h t o thank J a n S c h rama f o r h e r e f f i c i e n c y in the p repara t i o n o f e qu ipme n t and P e t e r Wi ldbo re f o r helping me overspend my budge t .

The generous s uppo r t o f Coope r s An ima l H e a l t h Ne w Z e a land Lt d . w a s o f c o n s ide rable a s s i s t ance . I n p a r t icu l a r , I would l i ke a c kno w l edge t he e n c o u rageme n t p r o vided by t he l a t e D a v i d L i a rde t .

V

K a r y n Mc K i n t y re a nd P a u l S c o t t we r e o f g r e a t a s s i s t a nc e i n p r o o f readi ng .

I o we t h a n k s t o my f e l l o w s t ude n t s , M i k e , Ni c k y , Je a n , Rh y s and S a l l y who pro vided c ompa n i o n s h i p o ve r t h e p a s t t wo ye a r s .

Spe c i a l thanks t o H e len fo r he r mo r a l s up p o r t .

FRONTP I ECE Lep t ospira interrogans

LIST OF CONTENTS

PAG::::

TITLE PAGE... .., ABSTRACT. . . . . . . . . . . . . ii AKNOWLEDGEMENTS ...^..^.... i-.

LIST OF CONTENTS ...........^............ ^.. v:...:.

LIST OF FIGURES .........^... X:.

LIST OF TABLES .......^..... xiii INTRODUCTION ... .

CHAPTER 1 LITERATURE REVIEW

1.1 Introduction and Classification .^..^..........^... ^� 1.2 Leptospirosis - The disease in Animals ..^.....^... 4 1.3 Leptospirosis The disease in Humans ... . 1.4 Leptospirosis in New Zealand ... . 1. 4 .1 Cattle ... ^.

1. 4.2 1. 4.3 1.4.4 1. 4.5 1. 4. 6 1. 4. 7 1. 4. 8

Sheep... E Pigs ......^....^................. 1:

Goats ...^........^....... 1:..

Deer ... 1�

Other Domestic Animals ....^....^{. ·-} Wildlife ....^....... 1.:

Humans ....... 1:;

CHAPTER 2 VALIDATION OF THE MICROSCOPIC AGGLUTINATION TEST FOR THE DETECTION OF ANTIBODIES TO LEPTOSPIRA

2 .1 Introduction. . . . . 2:;:

2 .2 Materials and Methods ........^.....^{. .}..^..... ^{. .}. 2:!

2.2.1 Culture Medium ...^....^.... 2:::

vii i

2 . 2 . 2 Ant igens ^{. . . . . . . .. . . . . . . . . .} . ^{. . .} . ^{.. . . . .} . ^{.. . . . . . . . .} 24

2 . 2 . 3 S e ra . . . 2 4 2 . 2 . 4 E

qwp

ment Val idation . . . 25

2 . 2 .5 The Microscopic Agglut inat ion Test . . . 25

2 . 3 Re sults ^{. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . .} 28

2 . 4 D i scus s ion . . . 38

2 . 5 Conclus ion . ^. . . . 42

CHAPT E R 3 LEPTO S P I RO S I S IN GOAT S 3 . 1 Int roduction . . . 43

3 . 2 Materials and Methods . . . 44

S ample Select ion . ^. . . . . . 4 4 Sero logy . . . 4 5 Culture Prepa rat ion ^{. . . .} . ^{. . . . . . . .. . . . . . . . . .} . .^{. .} . ^. 47

3 . 2 . 1 3 . 2 . 2 3 . 2 . 3 3 . 3 Results . . . 47

3 . 3 . 1 S e rological S u rvey ^{. . . . . . . . . . . . .} . ^{. . . . . . . . . . . . . . . . .} 47

3 . 3.2 F a rm studies . . . 4 9 3 . 4 D is cuss ion . . . 5 8 3 . 5 Conclusion . . . 6 5 C HAPT E R 4 LEPTO S P I ROS I S I N DE2R 4 . 1 I nt roduct ion . ^. . . . 6 6 4 . 2 4 . 2 . 1 4 . 2 . 2 4 . 2 . 3 4 . 2 . 4 4 . 3 4 . 3 . 1 4 . 3 . 2 4 . 4 4 . 5 Materials and Methods ^{. . . .} . ^.. . ^. . ^{. . . . . . . . . . . . . . . . . .} 67

Sample Select ion . . . . . . . . . 67

Sero logical Techniques ^{. . . .} . ^{. .}. . . ^{. . . . . ..} . ^{. .} . ^{. .} . ^. 68

Culture Preparation ^{. . . . . . . . . . .}. ^{. . . . . . . . . . . . . . . .} 6 9 Culture T yp ing ^{. . . . . . . . . .. .} . ^{. .} .^{. . . . . . . . . . . .. . . . .} 6 9 Result s . . . 72

Se rological Survey . . . 72

Farm Study . . . 8 0 D iscuss ion . . . 8 9 Conclusion ^{. .} . ^{. . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .}9 3

C HAPT E R 5 THE U S E OF ENZYME L I NKED IMMUNOS ORBEMT AS S AY FOR THE DETECT ION OF ANTI BOD I E S TO LEP TOSP IR A AND LEP TOSP IRA ANT I GEN

5 . 1 Int roduct ion . ^. . . . 9 5 5 . 2 Materials and Methods ^{. . . . . . .} . . ^{. . . . . . . . . . . .} . ^. . ^{. . . . .}1 0 2

5 . 2 . 1 S o l id Phase ^{. . . . . . . .} . ^{. .} . ^{. .. . . . . . .} . ^. . ^{. .} . ^{. . . .} . ^{. . . . .} . 1 0 2 5 . 2 . 2 Ant igen and Antisera Preparation f o r Plate

Coat ing ^{. . . . . . .} . . . ^{. . . . . . . .}. ^{. . . . . . . . . . . . . . . . .} . . ^{. . . .}1 0 2 5 . 2 . 3 Test Ant isera Collection . ^{. . . . . . . . . . . . . . . . . . . . .} . ^{. .}1 03 5 . 2 . 4 Antisera Production for Conjugate Preparat ion ^{. .} . . 1 03 5 . 2 . 5 Conjugate Preparation ^{. . . .} . ^{. . . . . . . . . . . . . . .} . . ^. . ^{. . . .}1 0 9 5 . 2 . 6 Tests for Con j ugate Act ivity ^. . . ^{. . . . . . .} . ^{. . . . . . . . . .}1 1 1

5 . 2 . 7 Chequerboard T itrations ^. . ^. . ^{. . .} . ^{. . . . . . .} . ^{. .} . . . ^{. . . . .}1 1 2

5 . 2 . 8 An Indirect ELISA Procedure for the Detect ion

of Goat Ant ibodies t o Lept ospira . . . 115

5 . 2 . 9 A Double Antib ody S andwich Procedure to Detect

Lept ospira ^. . . ^. . . ^.. . ^. . ^{. . . .} . . . ^. .^. . ^{. . . . . . . . . . . . . . .}1 1 6

5 . 2 . 1 0 A Single Ant ibody ELISA Procedure t o Detect

Lept ospi ra . . . ^. . ^{. . . . . . . . . .} . ^. . ^{. . . . . . . . . . . . . .} . . ^{. . . .}1 1 7 5 . 2 . 11 Tests Pe rfo rmed ^{. . . . . . . . . . . .} . . . ^. . . ^. . . . ^{. . . . .} . . 12 1 5 . 2 . 12 S t atistic a l Methods ^{. . . . . . . . . . .. . .} . ^{. . .} . . ^{. . . . . . . . .}1 2 2 5 . 3 Re sults ^{. . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .}1 2 3 5 . 3 . 1 Conjugate Preparation ^. . . ^. . ^{. . . . . . . . . . . . . . . . . . . . . . .}1 23 5 . 3 . 2 Cheque rboard T itrat ions ^{. . . .} . ^. . ^{. .} . ^. . . . ^. . . ^{. .} . ^{. . . . . .}1 2 7 5 . 3 . 3 Tests f o r Goa t Ant ibodies t o Lept osp i r a . . . 1 3 0 5 . 3 . 4 Detection o f Ha rdjo . ^{. . . . . . . . . .} . ^{.. . .} . ^{. . . . . . . . . . .} . ^. 133

5 . 4 D i s cuss ion . ^. . ^. . . . ^. . . ^{. . . . . . .} . ^.. . ^. . . ^. . ^{. .} . ^{. . . . . . . . .} .^. 1 3 6 5 . 5 Conclus ion . ^. . ^{. . . . . . . . . . . . . . .} . ^{. . . . . . . . . . . . . . . .} . ^{. . . .}1 43 CHAPTE R 6 S TUD I E S OF THE ANT I GENIC S IM I LAR I T I E S BETWEEN

BR ATISL AVA AND OTHER S E ROVARS

6 . 1 Introduction ^{. .} . ^{. .} . ^. . ^{. . . .} . ^{. . . . . . . . . .} . ^{. . . . . . . . . . . . . .}1 4 4 6 . 2 Materials and Methods ^{. . . . . . . . . . . . . .} . ^{. . . . . . . . . . . . . .}1 4 6 6 . 2 . 1

6 . 2 . 2

Growth Inhibit ion us ing Specific Ant iserum ^{. . . . .}1 4 6 The Sero logical Re sponse o f Dee r and Goats

Following Vaccination ^{. . . . . . . . . .} . . . ^. . ^{. . . . .}. ^{. . . . .}1 4 7

6 . 2 . 3 The S eparat ion o f Lept ospiral P ro t e ins using Sodium Dodecy1Sulphate P o lyacrylamide Gel

X

Elect ropho re sis ( SD S -PAGE ) ^{. . . . . . . . . .. . . . . . . . . . .}1 4 8 6 . 2 . 4 We stern B l o t s ^{. . .} . ^{. . . . . . . .} .^{. .. . . . . . . . . . . . . . . . . . .}1 5 1

6 . 3 Re s ults ^{. . . . . . . . . . . . . . . . . . . . .} . ^{. . . .. . . . . . . . . . . . . . . . .}1 5 4 6 . 3 . 1 Growt h I nhibition us ing Specific Antis erum ^{. . . . .}1 5 4 6 . 3 . 2 The S erological Re sponse o f Deer and Goats t o

6 . 3 . 3

Vaccinat ion ^{. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .}1 5 8

The S eparat ion o f Leptospiral P roteins by Sodium D odecyl S ulphat e P o lyacrylamide Ge l

Elect ropho resis ( SD S -PAGE ) ^{. . . . . . . . . . . . . . . . . . . . .}1 5 9 6 . 3 . 4 We stern B l o t Analys is o f the P rotein Antigens ^{. .}1 5 9 6 . 4 D i scuss ion ^{. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .}1 6 3 6 . 5 Conclus ions ^{. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .}1 6 6 C HAP TER 7 GENE RA L DISCUS S I ON . . . 1 6 7 AP P E ND I C I E S ^{. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .}1 79 B I B L I OGRAP H Y . . . 2 1 4

LIST OF FIGURES

FIGURE PP..GE

2 . 1 The Micros copic Agglut ination Tes t Procedure . ^. . . . ^. . . 2 7

2 . 2 A Compa rison of Re s ult s f rom Two Labo rat o ries

us ing the MAT ^{. .} . ^. . . . ^. . . . ^. . . . ^. . . . 37

4 . 1 BRENDA of I s o lat e s f rom Deer o f the

Glads tone Property . . ^{. .} . . ^{. .} . . . ^. . . ^{. . . .} . . . ^. . . ^{. .} 8 8

5 . 1 Immun o electropho re s is Plate . . . ^. . . . ^. . . . ^. . 1 0 7

5 . 2 Ge l D i f fus ion Plate . . . ^. . . . ^. . ^. . ^{. . . .} . ^. . ^. . . . _llO

5 . 3 Chequerboard Tit rat ion . . . ^.. ^{. .} . . . ^. . . . ^. . ^. . . . 1 1 4

5 . 4 The E L I SA for Detect ing Ant ibodies t o Lep t osp i ra . . ^{. .} . . ^. 1 1 8

5 . 5 The D o uble Antibody S andwich Procedure . . . . ^. . . . ^. . ^{. .} . . . 11 9

5 . 6 The S ingle Antibody ELISA Procedure . . . ^{. .} . ^{. .} . . . 1 2 0

5 . 7 E lut ion Peak from Deer Sera Ads o rbed onto

DEAE TRYSACRYL M . . . . ^. . . . . ^. . ^. . ^{. .} . . . ^. . . ^. . . ^. . . . ^. 12 4

5 . 8 Immunoelect ropho re s is Plate o f Fractionated Serum . ^. . . ^{. .}1 2 5

5 . 9 S t andard Curve f o r Protein E s t imat ion . . . ^{. .} . . . ^. . . . 1 2 6

5 . 10

6 . 1

6 . 2

6 . 3

6 . 4

A Chequerboard T i t ration to Det ermine the Opt imum Concentration o f Con j ugate and Test Sera f o r use

xii

in the ELISA to D etect Ant ibodies to Hardjo . ^{. . .} . . . . ^{. . . .}1 2 9

BRENDA Patterns o f the Iso lates from the G rowth

Inhibit ion Trial . . . ^{. . .} . ^. . . . ^. . . . ^. . . . ^. . . . ^. . ^{. .}1 5 7

The Titration o f P omona P rotein in SDS -PAGE ^. . . ^{. . . . .} . ^. . . 1 6 0

SDS -PAGE o f E ight Diffe rent S t rains o f Lep t o spira ^. . ^. . . . 1 6 1

Wes t e rn Blots o f Bra t i slava and Pomona ^. . . ^. . . ^. . ^{. . . .} . . ^{. . .}1 6 2

L I S T OF TABLES

T AB L E P AGE

2 . 1 Ant igens used in the Micros copic Agglut i nat ion Test ^{. . . .} 2 4

2 . 2 Agglutinat ion Tests Compar ing Ant ibody T it re s

t o Hardjo us ing Two D ispensers ^{. . . . . . .} . ^{. . . . . . . . . .. . . . . . .} 2 9

2 . 3 Reproducib ility o f the MAT . Antibody T i t re s

t o Hardjo. ^. . . . 2 9

2 . 4 MAT Re sults f rom Batch 'A' o f 55 Deer Sera ^{. . . . . . . . . .} . ^{. .} 2 9

2 . 5 Re sults from T r iplicate Test s on Ten Dee r Sera

f rom Batch 'A' . . . . 3 0

2 . 6 MAT Re sult s f rom a Second Batch 'B' o f 5 5 Deer Sera ^{.. . .} 3 0

2 . 7 Re sults from Triplicate Test s on Ten Deer Sera

f rom Batch 'B' ^{. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .} 3 0

2 . 8 Reproducib i l ity o f the MAT u s ing Goat S e ra ^{. . .} . ^{. . . . .. . .}. 3 1

2 . 9 Ant ibody Titre s t o Hardjo with Ten Fold Va riat ions

in the Ant igen Concent rat ion ^{. . . . . .} . ^{. .} . ^{. . . . . . . . . .} . ^{. . . . . .} 3 2

2 . 10 Ant ibody Titres to Hardjo Obta ined Us ing D i f ferent

Ant igen Concentrations ^{. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .} 3 3

xiv

2 . 1 1 Comparison of L ive and Inact ivated Antigen . . . . ^{. .} . . . 33

2 . 12 A Comparison o f Antibody T it re s Obta ined Us ing

D i f ferent Ant igen Strains . . . ^. . . ^. . . . 3 4

2 . 1 3 Comparison o f Ant ibody Titre s Obt ained U s ing Cultures

of D i f ferent Age s . . .^. . . . ^{. .} . ^. . . . ^. . . . 3 4

2 . 1 4 Ant ibody Titres Obt ained at D i fferent Incubation

T ime s at 2 9 °C . . . ... . . ... . . . 35

2 . 1 5 A Compa rison o f Ant ibody Titre s Obta ined Us ing PBS

or Culture Medium as D i luents . . . . ^. . . . ^{. . .} . . . 3 6

3 . 1 Ant igens used in the S e rogroup S c reening Test . . . 4 6

3 . 2 S e ro logica l S urvey o f the Nat ional Herd . ^.. . ^. . . . 4 8

3 . 3 S erological S urvey o f the Nat ional He rd

Ant ibody Titre s t o Multiple S erovars . . . ^. . . . ^. . . . 4 9

3 . 4 F a rm Study 1 Sero logy Re sult s . . . ^. . . ^. . . . ^. . . 50

3 . 5 F a rm Study 2 S e rology Re sults . . . ^. . . . ^. . . . 5 1

3 . 6 F a rm Study 3 Serology Re sult s ^. . . . .^. . . . ^. . . . 53

3 . 7 F a rm S tudy 4 S e ro logy Re sults ^. . . . ^. . . . ^. . ^. . . 5 4

3 . 8 F a rm Study 5 S e ro logy Re sults . . . .. . . 5 5

3 . 9 F a rm Study 5 Age Dist ribution o f Ant ibody Titre s . . . 5 6

3 . 1 0 F a rm Study 6 S e rology Results ^. . . . ^.. . . 5 7

I nver.may Deer Herd . ^. . . . 7 5

4 . 3 S e ro logy Re s u l t s for the Mosburn Deer . . . 7 6

4 . 4 Age Dist ribut i on of Antibody Titre s �10 for the

Mosburn Deer ^{. . . . . . . . . . . . . . . . . . . . . .} . . ^. . . ^{. . . . . . . .. . . . . .. .} 7 7

4 . 5 The P roperty D i s tribut ion o f Ant ibody Titres f rom

the Mosburn Deer . ^. . . . 7 8

4 . 6 S e ro logy Re sults for the Nelson Dee r . . . 7 9

4 . 7 S e ro logy Res u l t s for the Ruakura Deer . . . 8 1

4 . 8 S erology Re sults from the Taranaki Deer . . . 8 2

4 . 9 S e rology Re s u l t s from the First S ampl ing o f Deer

f rom the Glads tone P roperty . . . 8 3

4 . 10 Age Dist ribut ion of Ant ibody Titres �10 f rom the

First S ampling of Deer f rom the Gladstone P roperty ^{. . . . .} 8 4

4 . 11 Sero logy Re sults from the Second S ampl ing o f Deer

f rom the Glads tone P roperty . . . 8 5

4 . 12 Age Dist ribut ion of Antibody Titres �10 f rom the

S econd S ampling of Dee r f rom the Gladstone P roperty . . . 8 6

5 . 1 Contingency T able for the Analys i s o f S e ro logical

Data ^{. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .}122

xvi

5 . 2 A Chequerboard T itration t o Determine the Optimum Concentration o f Con j ugate and Test S e ra f o r use

in the ELISA to Detect Ant ibodies t o Hardjo . ^. . . . ^{. .} . . ^. 1 2 8

5 . 3 Rep roducibility o f the E L I SA for the Detection

of Antibodies to Rardjo . ^. . . . ^{. . .} . . . ^. . ^{. .} . . . . ^. . ^. . . . 1 3 0

5 . 4 Comparison o f t he ELISA and MAT ^. . . . ^. . . . ^. . . . ^{. .} . 1 3 0

5 . 5 Cont ingency T able o f Results for the ELISA

and the MAT . ^{. . .} . . . ^{. . . .} . . . ^. . . . ^{. .} . . . ^. . 1 3 1

5 . 6 A Compa rison o f the ELISA ( us ing the titration method) and the MAT f o r Detect ing Antibodies t o Hardjo ^. . . . 1 3 2

5 . 7 A Comparison o f the ELISA and the MAT for the

5 . 8

5 . 9

5 . 1 0

5 . 1 1

5 . 1 2

Detection o f Antibodies t o Pomona ^{. . .} . ^. . . . ^{. . .} . . . . ^. . . . 1 3 2

Cont ingency T able f o r the Comparison o f the ELISA and MAT for the Detection of Antibodies to Pomona .^{. . . . . .} . . . 1 3 3

E L I SA Results for the D etect ion o f Hardjo . . . ^{. . .}1 3 4

E L I SA Results for the Detection o f Hardjo us ing

D if ferent Coat ing Sera . ^. . . ^. . .^{. . .} . . ^. . ^. . . . ^{. .} . ^. . 1 3 4

A Comparison o f Dif ferent D ilut ions o f P l ate Coat ing S e ra and Con j ugate for use in the E L I SA

t o Detect Hardjo ^. . . ^{. .} . . . ^. . . . . ^. . . . . ^{. . .} . . . . ^{. . . .} . . ^. . . . ^. 1 3 5

Comparing the ELISA t e s t t o Detect Hardjo a s Whole Ce lls and Cells D i s rupted through Sonic

T reatment . ^{. .} . ^. . . ^. . ^. . . . . ^{. . . .} . . . ^. . ^{. .} . . . ^{.. .} . . ^{. .} . . . ^{. .} . . . ^. . . 1 3 5

5 . 1 3 The S ingle Ant ibody ELISA for the Detect ion

o f Hardjo ^{. . . . . .. . . . . . . .. . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . .}1 3 6

6^.1 We stern Blots ^{. . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .}1 5 3

6 . 2 Microscopic Examination o f the Growth Inhib ition

T rial ^{. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .}1 5 5

6 . 3 Se rotyping o f Iso lates f rom the Growt h Inhib ition

T rial ^{. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. .. . . . . .}1 5 6

6. 4 The Serologi c a l Re sponse o f Deer t o Vaccination ^{. . . . .. . .}1 5 8

6 . 5 The Sero logical Re sponse o f Go ats t o Vacc ination ^{. . . . . . .}1 5 8