Immunological factors associated with foot-rot in sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University

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Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

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I M M U N 0 L 0 G I C A L F A C T O R S A S S 0 C I A '1' E D W I '1' H F 0 0 '1' ^- R 0 '1' I N S H E E P

A the s i s pre s ented i � partia l fulfilment of tne requiremen ts f o r Lhe degree o f Do c tor of Phil o s ophy at. Mas s ey Univ ers i ty

Brian Seymour Cooper 1 972

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ACKNOWLEDGEMENTS

I wish to thank my supervisors for their help, interest and

encouragement during this work. In particular I thank Dr. K.M. Moriarty for his suggestions regarding experimental design and for constructive criticism of the work and its presentation. I gratefully acknowledge also the guidance of Prof. B.W. Manktelow and his helpful advice in the preparation of the manuscript.

My appreciation is extended to the many people both at Massey University and in the Department of Scientific and Industrial Research, Palmerston North who have assisted me with discussion, comment and technical advice. In particular, I wish to thank Messrs A. Craig and D. Hopcroft of the Electron Microscope Unit who prepared material for examination and printed the photographs in Chapter II; and Mr. J. Austin, Photographic Unit, Massey University, who photographed and printed most of the figures in the thesis.

Special thanks are also due to my colleagues at Glaxo Laboratories, Palmerston North, in particular Mr. R.E. Lynch for the provision of some bacteriological media including GC broth, the preparation of experimental vaccines and the general supervision of animals under experiment.

Mr. H.V. Brooks assisted with assessment of lesions seen in vaccine trials;

Dr. J.M. Rudge gave valuable advice on vaccine trial design and Mr. D.J.

Jull provided enthusiastic support at all times. Technical assistance with growth inhibition assays was given by Mrs. P. Twentyman.

Finally, I am most grateful to the directors of Glaxo Laboratories (N.Z.) Ltd who provided financial support during my leave of absence, and in particular Messrs R. Stagg and K.D. Huse whose foresight made this project possible.

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CHAPTER XI

CHAPTER XII S UMMARY REFERENCES Append i c e s

De tection o f delayed-type hypers ensi tivi ty as an expre s s i on of c e ll-mediated immunity in sheep wi th foot-rot

Vac c ination as the s o le means o f tre atment in s heep affected with pr ogre s s ive fo ot-ro t : Expe riment The rapy I

General D i s cus s i on

p . 93

p . 99

p . 1 05 p . 1 1 2 p . 1 1 6 Fo ll owi ng p . 1 29

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LIST OF TABLES

Tab le Foll owi ng page

II.I III. I

III.II

The effe c t of di fferent methods o f culture on the haemagg lutinating effect of four F. nodosus s trains

Agglutination ti tres given by an anti-F . nodosus (J3/22 ) ant i s e rum when reac ted wi th F . nodosus ( J 3/22 ) c e l l s grown in biphas i c me uium and sub

s equently treated in various ways

Agglutination ti tres given by ant i sera against e i ther F. nodosus (J3/22 ) o r F. n odosus (M9/4 ) c e l l s grown in biphas i c medium and subs eqnently treated in vari ous ways , o r wi th F . nodosus (J3/22 ) c e l ls grown in biph�s ic medium

III.III Agglutination titres given by an anti-F . nodosus (J3/2 2 ) anti s e rum when reac ted wi th F . nodosus (J3/22 ) c e l l s grown on h oo f agar medium and sub

s equently trea -r,ed i :o.-1 vari ous ways III. IV

III.V

III. VI

Agg luti nation titres given by anti -F . nod osus ( J3/22 ) anti s e rum when reac ted w i th F. ncdo sus (JJ/22 ) c e l l s grown i n hoof broth and sub

s equently treated in vari ous ways

Agglutinati on titres given uy an anti-F. nodo sus (J3/22 ) antiserum when reac ted wi th F . nodo sus (A8/C ) c e l ls grown in bipha s i c medium and sub

Agglutinati on titres given by an ant i -F . nodosus ( J3/2 2 ) anti s e rum when reac t ed with F . nodo sus (J3/2 2 ) c e l l s grown in bulk in GC broth and sub

III.VII Agg lutination t i tres of ant i -F . nodosus (J3/22 ) ant i s e rum wi th c e l l suspens i ons of di fferent F . nodosus s trains grown by di fferent me thods of culture and subsequently t reated in vari ous ways

30

38

3 8

38

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Tab l e IV . I

IV . I I

IV . I I I

IV . IV

IV . V

IV . VI

IV . VI I

Degree o f acc eptab i l i ty of l o c a l reac ti ons to two doses o f vacc ine f ormulated w i th d i ffe rent adj uvant sys tems

Degree of a c c eptab i l i ty o f local reac ti ons to one or two d o s e s of vacc ine formulated wi th alum or o i l adj uvant

Number of f e e t affe c ted w i th foot-ro t after

attempted infe c ti on by appli cati o n o f swab or horn agar c u l ture ( HAC )

Sus c eptibi l i ty s c ores for individual she e � and fo r gro ups i nj e c ted wi th diffe rent vac c ines and cha l

lenged wi th the homo logous s train in Vaccine Tri al I

Numbers o f sheep and feet showing ac tive and pro

gre s s iv e horn s e parati on on po s t-chal lenge week 4 in Vac c i ne Trial I

Sus ceptib i l i ty s c o re s for i ndiv idual sheep and for groups inj e c ted wi th different vac c ines and chal

lenged wi th the homol ogous s train in Vaccine Trial I I

Numbe rs o f she ep and fe e t s �owing ac ti ve and pro

gre s s iv e horn separation on po s t-cha llenge week 4 in Vac c ine Tria l I I

Fo l l owing page 50

5 0

50

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IV . VIII Sus c eptibi li ty s c ores and numbers o f sheep and f e e t 50

I V . IX

showing ::tc tive and progre s s iv e horn s eparati on on po s t-cha l lenge week 4 in Vac c i ne Tri al Ill -

Homo l ogous c ha l lenge

Susc e p tibi li ty s cores and numbe r s of sheep and f e e t showing a c tive and progre s s ive horn separati on o n pos t-chal l enge week 4 in Vac c ine Trial I l l -

He te r o l ogous challenge

50

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Tab l e V . I

V . I I

V . I I I

V . IV

v.v

V . VI

V . VI I

V . VI I I

VI . I

V I . I I

Fo l l owing page Post-vacc ination agg luti nation ti tres o f " re s i s tant"

o r "sus c eptib l e " sheep and pooled sera from the s ame group in Vac c ine Tri a l I

Post-vac c ination agglutination ti tre s o f " re s i s tant"

or "susc eptible " she ep and po o led sera from the s ame group in Vac c ine Tri a l I I

Pos t-vac c ination agglutination ti tre s o f "re s i s tant"

or "sus ceptib l e " she e }J and po o l ed sera from the same group in Vac c ine Tri al I I I

Homologous cha l lenge - F . nodo sus (JJ/22 )

Post-vac 8inati o n agglutination titre s of " re s i stant"

or "susc eptib l e 11 she ep and poo l ed s e ra from the s ame group in Vac cine Tri a l I II

He tero logous chall enge - F . nodo sus (AS/C )

Eff e c t o f vac c i nation on agglutination titre s of she ep with exist�ng foot-rct

Agglutination reacti ons us ing forma l i n treated c e l l s o f thre e F . nodosus stra ins wi th spe c ific an t i s e ra Agg lutination o f F . nodosus (JJ/22 ) c e l l s by un

abs o rbed arr�ibody present i n homo logous and

hete rologous ant i ::se r a from the previ ous expe riment ( Tabl e V . VI)

Agglutination rsacti ons us ing fo rmalin treated , o r washed and bo i l ed F . nod osus (JJ/22 ) c e l l s wi th the i r re spe c tive and oppo s i te spe c i f i c anti s era

Effe c t of vac c ination on ::serum antiproteo lytic titre s of sheep with existing foot-rot

Antiprote o lyti c ti tre s of poo l ed s e ra from groups of sheep vac c inated ,.,i th vari ous adj uvant/c e l l c ombinati ons i n Vac c ine Tri a l s I , I I , and I I I

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65

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Tab l e V I I . 1 2

VI I . 1 3

VI I . 14

VI I . 1 5

VI I . 1 6

V I I . 1 7

V I I . 1 8

VI I . 1 9

VI I . 20

VII . 2 1

VII . 22

Fo l l owing page Reacti ons betwe e n untreated c e l l extract , and

e i ther spe c ifi c antis era , I gG frac t i o n , or poo l s o f s e ra from di fferent treatment groups in

Vac c i ne Trial Ill

Compari s on of reac ti ons between untreated c e l l extract and poo l s of s e ra from she ep inj e cted with one o r two do s e s of alum pre c i pi tated vacc ine s Compari so n of reacti ons ?e tween b o i led c e l l

extract and poo l s o f s era from sheep inj ected wi th one o r two do s e s of alum pre c i p i tated vacc ine s Compa r i s on of reactions b e tween untreated c e l l extra c t and s �ra of e i the r lamb o r dam about the time o f partur i ti on

Compari s on o f reac ti ons b e tween b o i led c e l l extract and s e ra o f either lamb o r dam about the time of partur i ti on

Re a c t i ons b etween cultur e supernatant and s e ra o f s e l e cted " re s i s tant " o r " sus c epti b l e " sheep i n Vac c i ne T r i a l I

Rea c t i ons bet1veen culture supe rna tant and spe c i a l anti s era as deta i l ed

Reacti ons betwe en culture supe rnatants of different pro t e o lyti c ti tres wi th anti-F . nodosus (J3/22 ) ant i s e rum

Reac t i ons between culture supernatants of s tandard proteo lyti c t i tre wi th anti-F . nodo sus (J3/2 2 ) ant i s erum

Rea c t i ons between untreated c e l l washings and e i ther pre co l o s tral or po s tc o l o s tral l amb s era Rea c t i ons betwe en bo i led c e l l washings and homo l ogous ant i s erum ei ther b e f o r e o r after absorpti o n

8 0

80

.80

80

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Tab l e

VI I I . I Eff e c t o f e i the r antibody absorp t i o n or various heat tre atments on the growth-inhibi tion titres of s era from e i ther no rmal or vac c i nated she ep VI I I . I I Effec t o f naturally acqui red or experimental ly

induced foo t-rot on the growth-inhibi tion titres o f s e ra o f affec ted and unvac c i nated she ep

VI I I . I I I Growth-inhib i t i on titres of heat treated s era o f groups of sheep inj ected with diffe rent adj uv ant/

c e l l c omb inations in Vacc ine Trial I I I IX . I

X . I

X . I I

X . I I I

X I . I

X I . I I

Induced change in F . nodosus c o l ony type on hoof agar when e xpo s ed t o s e rum of a " re s i s tant "

sheep

Degree of l o cal reaction in o ne norma l she ep and one s he ep affec ted wi t h beni gn fo ot-rot , i n re spons e to a n intrade rmal i nj e c ti on of F. nodosus culture extract

Extent of c orneal opac i ty in two foot-rot in

fec ted she ep in response t o intra c o rneal inj e c t i o n of e i ther F. nod o s us untreated c e l l extract o r E . c o l i untreated c e l l extract Degree and extent of c orneal opac i ty in either no rmal o r foot-rot infected she ep , in r e sponse to intra c orneal inj e c t i on o f e i th e r untreated or boi led c e l l extrac t

Re spons e o f foot-rot infec ted sneep to e i the r one or two do s e s of Alum ( one per c en t . ) pre c ipi tated who l e cul ture vaccine

Re sponse o f foot-rot infected she e p to e ithe r one or two d o s e s of Alum ( one per c e nt . ) prec ipi tated who l e cul ture vac cine

Fo l l owing page 88

88

92

98

1 04

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Figure II .1

I I . 2

I I . 3

I I . 4

II .6

II . 7

I I . 8

I I . 1 0

LIST OF FIGURES

F . nodo sus ( J3/22 ) organi sms taken from a rough c o l ony cultured for 4 days on hoof agar

F . nodosus (A8/C) organi sms taken f rom a rough c o lony cultured for 4 days on hoof agar

Smear o f nec rotic material from a fo ot-rot infec ted foot showing two F . nodo s us o rgani s ms near a mas s o f pal e s tained filaments pos s i b ly o f F . nec rophorus Smear o f necrot i � material from a f oot-rot infec t ed foot showing two F . nodo s�s o rgani s ms (arrowed) surrounded by ot her sma l l er rods in c harac teri s ti c

" row-boat" arrangement

Smear of F . nodo sus o rgani sms rultured for 48 hr in biphas i c med ium showing an amo rphous m�trix containing short rod shaped s truc tures and typi c al F . nodosuf:

Smear o f necroti c material from a foot-rot infec ted foot showing a large F. nod o s us organi sm wi th

darkly s tained polar regions . Cel l div i s i on may be taking pJ'lc e

Smear c f necrotic ma terial from a foot-ro t infec t ed foot showi ng a s i ngle F . �odosus o rgani sm con

taining two dis tinct darkly s tained granules in the body o f the c el l

S ec t i on o f interdigi tal nec rotic materi al showing c el l bo rder area with a s e c ti oned spi ral organi sm pos s i b ly Sp. peno rtha

Sec t i on o f interdigital necrot i c material wi th a pi l i ated F . nodo sus-like o rgani sm pos sibly wi thin a c el l and showing one po lar granule

Sec t i on o f interdigi tal necroti c material showing a p i l i a t ed F . nodosus-like o rgani sm wi th s everal dens e granules wi thin the nuc l eoplasm

Fo l l owing page

30

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Figure I I . 1 1

I I . 1 2

II . 1 J

I I . 1 4

I I . 1 5

I I . 1 6

I I . 1 7

I I . 1 8

11. 19

I I . 20

F . nodo sus c e l l s cultured for 4 days on hoof agar, showing 4 laye rs of c e l l env e lope and 2 large granules in the nuc l e oplasm

F . nodo sus c e l l cultured for 4 days on hoof agar , s howing p i l i with terminal enlarg2ments and

s ev e ra l medi um s i zed granules in the nuc leoplasm

F. nodosus c e l l cul tured for 4 days on hoof agar , s howi ng l onge r p i l i with terminal enlargeme nts F . nodo sus c e l l cultured for 4 d8,ys on hoof agar , showing tuft of fine filamentous appendage s a r i s i ng from one po l e

F . nodosus c e l l cultured fo r 4 days on ho o f agar , s howing 2 long filaments ari s ing from the pole and nume rous sma l l pili

F . nodosus ce l l cultured for 4 days on hoof agar , s howing an area o f conv o luted membr anes inter

posed betwe en the cytoplasmi c membrane and th e outer 'c e l l wal l '

F . nodosus c e l l s cultured fo r 48 hr in biphas ic medium , showi ng clearly s eparated laye rs o f the

c e l l env e lope and wrinkled appe arance

F. nodosus c e l l cultured for 48 hr in biphas i c medium , showing wrinkling o f the s eparated layer s of c e l l env e lope and two d i s crete areQs of nuc l e oplasm

F . nodosus c e l l s cu ltured for 48 h r in biphas i c medium , s howing lack o f c e l l env e lope detai l and

one large dens e granul e as s o c iated wi th the nuc leoplasm

F. nodosus c e l l s cultured fo r 24 hr in hoof broth , showing c e ll div i s i on and almo s t c omplete lys is

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Figure I I . 2 1

VI I .1

VII . 2

VI I . 3

VII .4

VII . 5

VI I . 6

VI I . 7

VI I . 8

VI I . 1 0

VI I . 1 1

Fo l lowi ng page F . nodosus c e lls cultured for 24 hr i n hoof bro th ,

showing one c e l l containing vari ous s i zed vacuo les and ano the r undergo ing divi s i on

Reac t i ons b e tween untreated c e l l extrac t and s era from normal and vac c i nated sheep

Rea c ti ons b e tween untreated c e l l e �trac t and s e ra from v a c c i nated she ep - Vac c ine Trial I and one spe c i al anti s e rum

Reac t i ons o f ident i ty be twe en F . nodosus (J3/2 2 ) and F . nodosus (Mc M1 98) untreated c e l l extrac ts , and the i r r e spe c tive and oppo s ite an ti s e ra

Rea c t i ons o f identity between F . nodosus (JJ/22 ) and F . nod o sus (McM1 98) boi l e d c e l l extrac ts , and the i r respec tiv e and oppo s i t e anti s era

Reac t i ons b e tween untreated c e l l extrac t and a s e r i e s o f " re s i s tant" s era from Vac c ine Trial I

Reac t i ons between untreated c e l l e xtrac t and a s e r i e s o f " sus c ept i b l e " s e ra from Vac c ine T:..i a l I Reac t i ons between untreated c e l l extrac t and s e ra from sheep i n Vac c i ne Trial I I

Reacti ons b e tween untreated c e l l ext�act and s e ra from shee p i n Vac c ine Tri a l I I

Reacti on s between untreated c e l·l e xtract and s e ra from she ep i n Vac c ine Tri al I I

Reac t i ons between b o i l ed c e ll extrac t and s e ra from she ep in Vac c int Trial I I

Compari s o n o f pe rs i s tenc e o f pre c i pi tating activi ty agains t untreated c e l l extract i n poo l s of s era from d i fferent treatment groups in Vac cine Trial I ll

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80

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80

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Figure VII . 23

VI I . 24

VI I . 2 5

VII .26

VII .27

VI I . 2 8

VI I . 2 9

VI I . 30

VI I . 31

VI I . 32

Reacti ons b e tween untreated c e l l extract and anti s e rum rai s ed by e ither forma l in treated c e l l s o r boi led c e l ls

Reacti ons b e tween boi led c e l l extra c t and se rial s e rum s amp l e s taken from a s he ep wi th foot-rot

Effe cts o f absorpti on using F . nodosus ( J3/2 2 ) c e l l s o n reacti ons betwe e n F . nodo sus (J3/22 ) boi led c e l l extrac t and homo logous ant i s e rum

Effe c ts o f absorption us ing F . nodo sus (J3/2 2 ) c e l l s on reactions betwe en F . nodos us (J3/22 ) boi led c e ll extract and hP.terc logous anti s e rum

Effe cts o f abs o rption using F . nod o s us (J3/22 ) c e l l s o n reactions between F . nod o s us ( McM198 ) boi led c e ll ex�ract and heterologous an tiserum

Effe cts o f absorpti on us ing F . nodosus (J}/22 ) c e l l s o n reactions betwe e n F. nodo sus ( McM198 ) boi l ed c e l l extract and homo Jo gous anti serum CompRr i s on o f reacti ons betwe�n F . nodo sus boi led c e l l extra c t and s e ra from e i ther " re s i stant" o r

" sus c e ptib l e " sheep i n o ne gr oup o f Vae:c ine Trial I

Compari s on o f reacti ons b e tween F . nodosus bo i led c e l l extract and s e ra from e ithe r " r e s i stant " o r

" susceptib l e " sheep in Vac c ine T r i a l I I

Compa r i s o n of reactions b e twe e ri F . riodosus b o i l ed c e l l extract and s era from e i ther " re s i stant" o r

" suscepti b l e " sheep in Vac c ine Tri a l II

Reacti ons betwe en F . nodo sus boi led cell extract and poo l s o f s e ra from sheep wi th existing foot

rot and that had been vac c i nated with e i ther one or two d o s e s of the Alum ( one pe r cent . ) wh ole culture formulati on

Fol lowing page 80

80

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Figure VII. 33

VII. 34

VII . 35

VII . 36

VII. 37

VII. 38

X.1

Reactions between F . nodo sus untreated c e l l extract and frac t i ons of anti-F. nodo sus anti - s erum

Re actions between F . nodo sus boi led c e l l extract and fracti ons of anti-F . nodosus anti s e rum

Reacti ons b e twe en rabbi t ant i -sheep se rum and e i ther normal s e rum o r a11ti-F . nodosus ( J 3/2 2 ) ant i s e rum and i ts frac ti ons

Reacti ons betwe en rabbit anti-she ep se rum and sera o f e i ther new born lamb s , o r from an adult sheep with longstanding foot-rot

Reactions between rabbi t anti -she e p IgG and e i ther s e ra o f new born lamb s o r anti-F. nodo sus

(J3/22 ) anti s e rum and its fracti ons

Reacti ons betwe en rabbi t anti -sheep IgG and

e i ther pre co l o s tral lamb s e rum o r ant i -F. nodosus ( J3/2 2 ) ant i s e rum and its frac ti ons

Corneal opac i ty in response to i atrac orneal in

j e ction o f F . nodo sus b o i led c e l l extract admini s tered 48 hr previ ous ly . The she ep had s evere foot-rot

Fo l lowi ng page 80

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98

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P R E F A C E

Foot-rot in sheep has troubled farmers for many years not because of any spectacular epidemics associated with high mortality but because the chronic lameness associated with foot infection, and its consequences, frequently interfere with farm husbandry . Methods of control and eradica

tion have been lmown since 1 940 but as they generally involve many hours of strenuous work, the development of more efficient methods of foot-rot control has been the aim of different research groups .

Two findings which suggested that foot-rot organisms might be accessible to blood-borne inhibitors helped change the attitude of re

searchers involved with this problem . The first was the successful use of parenterally administered antibiotics against foot-rot infection and the second was the demonstration that serum globulins were able +.o make contact with Fusiformis nodosus1 in situ . The results of these experiments carried out at the McMaster Institute, Sydney led to a reappraisal of

existing research projects and the formation of a new rclicy leading to

wards the development of experimental vaccines .

By 1 970 when the writer's course of study began, it had been shown that bench scale vaccines conferred some protection again�t foot-rot

infection and that vaccinution was effective in stimulating resistance even after infection had become established . There were no methods a�.railable other than direct sheep challenge exp8riment� for evaluating different types of vaccine and although the existence of anti-F . nodosus bacteri-

cidal antibody was known, there was still conside:cable douht about the immune mechanisms involved in protection . There had been no investigation of the antigenic potential of F. nodosus products of growth or indeed of the bacterial cells themselves and as a consequence the influence of media components on the immunogenicity of the resulting culture had received little attention.

Before the foot-rot vaccines could be applied under New Zealand field conditions, two major problems had to be overcome. Firstly ^a large scale method of culture had to be developed that would produce economic yields of F. nodosus while retaining those antigens important for pro-

Fusiformis nodosus was shown to be the primarily important and trans

mitting organism of sheep foot-rot .

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te c t i on o f sheep . S e c ondly , an adj uvant system had to be cho s en that would be e ffective i n s timulating an adequate antigen i c re spons e but would cause minimal damage to the carcas s .

To obtain the e s sential information upo n which large s ca l e modi

fi cati ons might be b a s ed , a s e r i e s of exp e r iments were de s igned to

improve understanding o f s ome bas i c aspe c ts of foot-rot vac c i ne e ffe c tive

nes s . As a starting p o int fo r the inve s tigation di fferent me thods of bact e ri a l culture were used and the r esulting F . nodosus cells were c om

par e d for gros s morpho logy and ultrastructure as we l l as for the i r degree o f i mmunogeni c i ty after inc orporati on into various vac c i ne f ormulations . The s e r o logi cal respons e by the sheep to vac cinati on admini ste re d 0e fore and during F . nodo sus i nf e c tion was i nves tigated to find a laboratory t e �t system that would r e f lect the immune s tatus o f the animal s. Exper iments were a l s o carr i e � out to test t he pos s i bi l i ty of a humo ral prote ctive sys tem , and thP. other c o ntributor to spe c i f i c re

s i stanc e , ce ll mediated i ��unity , was i nve�tigated in normal and natural ly i nf e c ted she ep .

Thi s programme o f res earch produc ed r e sults ,that with the c on

c lusi ons that were drawn from them, form the maj or rart of the f o l l o wi ng the s i s .

Immunological factors associated with foot-rot in sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University

TABLE OF CONTENTS

CHAPTER XI

LIST OF TABLES

LIST OF FIGURES