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THE S E R OLOGICAL AND CULTU RAL PREVALENCE
lN
S H E EPOF
L EPTOSPIRAL I N F ECTION IN THENORTH ISLAND O F NEW ZEALAND
A THES I S P RESENTED I N PART I AL F ULFILMENT
(30°/o)
OF THE REQ U I REMENTS F O R THE D EGREE OF MASTE R OF PH I LOS OPHY I N VETERINA RY PATH OL OGY AND P UBL I C HEALTH AT MASS EY
UN I VERS I T Y , NEW ZEALAND
ABDUL RAN I BAHAMAN
1981
11 .
ABSTRACT
A survey o f sheep from th e N o r t h Is l and of New Z e a l and was c ondu c t e d for l ep t o s p i r a l in fe c t i on . The r e su l t s of the s e rolog i c a l examina t i on showed 20 . 5 percent of the sheep
'::J!il."'
had t i t r e s ( � 1 : 48 ) to Hebdomad i� �erogroup , 3 . 8 percen t t o s e rovar pomona., 2 . 6 percent t o s erovar talc..MJ.:Jovi.., 2 . 3 p e r c e n t t o serovar c.ope.n.ha.ge.n.i.. and 2 . 7 p ercent to s erovar ba.Uum. No t i t res o f 1 : 48 or greater we r e detec ted to s erovar �tk�.
I t wa s shown that a min imum d i lu t i on o f 1:24 ·resu l t ed in many non- s p e c i f i c or cr os s -r e a c t ion . A min imum d i l u t ion o f
1 : 48 wa s mor e accurate for d e t e c t ing the s e ro l o g i c a l
p reva l enc e o f agglu t inins t o l e p to s p i r e s i n ovine s e ra . In the cu l tu r a l s urvey , serovar haJr.djo wa s i s o l a t e d from three anima l s in one group o f s h e e p . It was cons idered tha t the Hebdomad i s t i t r e s were mo r e l ik e ly to repr e s e n t pr ev1ou s infec t i on w i th ha.Jtdjo than w i th ba.l.c.a.n.i..c.a.
B a s ed on th e s e r o l o g i c a l and c u l tural examina t ion s from the general su rvey and a s tudy f arm , a p a t t ern of infe c t ion was recorded . The s er o l ogi c a l p r ev a l ence and the geome t r i c mean t i t r e ( GMT ) o f d i f fe r en t a ge groups o f sheep from
d i fferen t f a rms and the l ack o f suc c e s s on ob t a ining fur th e r i s o l a t e s o f haAdjo ind i c a t e d tha t sheep a r e not t h e ma int enance h o s t for th i s s er ovar in New Z e a l and . Al though inf e c t ion o f
sheep b y s e r ovar haAdjo i s n o t uncommon , i t i s a s porad i c o c currenc e and endemic i n f e c t ion i s un l ike ly t o occur .
P re l iminary inve s t i ga t i on s on the u s e o f rad i o immunoa s s ay in detec t in g l e p t o s p i r e s o r l e p t o s p i r a l an t i gens in ur ine
are pre s e n ted .
111.
ACKNOWLEDGEMEN TS
I wou ld l ike t o expr e s s my s1ncere gra t i tude t o the Depar tment of Ve t e r inary Pa tho l ogy and Pub l i c Hea l th ,
Ma s s ey Un iver s i ty , New Zealand for p rov iding th e o p p o r tun i t y and exc e l l en t fac i l i t i e s for th i s inve s t i ga t ion .
My mo s t s 1n c e r e thank s are due t o my superv i s o r ,
Dr R . B . Mar sha l l , .for h i s enc ouragemen t and gu i danc e throughout the c our s e o f th i s work .
Th e adv i c e and a s s i s t ance o f Pro f e s s o r D . K . B l ackmo r e ,
P r o fe s s o r B .W . 'Mank t e l ow , Dr K . M . Mor i ar ty , Dr A . J. Rob inson , Mr G . V . Pe t e r s en and Dr L . M . Scholium are gra t e fu l l y
acknowledged and apprec iated .
The inva luab l e and wi l l ing as s i s t ance from the s t a f f o f the Departmen t o f Ve t e r inary Pathology and Pub l i c H e a l th 1n prepar a t i on o f me d i a , g l a s sware , pho t ograph i c work , admini s t ra t ive a s s i s t an c e and techn i c a l as s i s tance are gr e a t l y appr e c i a t e d .
The provi s i on s o f b l ood and k i dney s amp l es from the Longburn F r e e z ing Wo rks and F e i l d ing Fre e z ing Works are acknowl edged w i th gra t i tude . I wou l d a l s o l ike t o thank Mr and Mr s B . M . C l are o f Wa i tuna We s t in prov i d ing s amp l es from sheep on the ir farm .
S p e c ial thank s t o my eo-worke r s , Mr C . G . Mackin t o sh ,
Mr P . Ramada s s and M i s s T . M . B a l a s s u who s e fr i end s h i p and d i s cu s s ions have added so much t o th i s s tudy and t o i t s c our s e .
F inal ly , I w i sh t o ext end my thanks t o Mr s V . Lobb for the exce l l en t t y p ing o f th i s thes i s .
TABLE OF CONTENTS
Ab s t rac t
Acknowledgemen t s
Tab l e o f Con t en t s L i s t o f Tab l e s L i s t o f F i gu r e s
CHAPTER ONE
CHAPTER TWO
- Ovine l e p t o s p i r o s i s : A rev 1 ew o f the l i tera ture .
- The s e r o l og i c a l preva l ence o f sheep t o natural l y-oc c urr ing l e p t o s p i r a l infe c t ion .
CHAPTER THREE - The c u l tural preva l ence 1n sheep
on na t u ra l ly-occurring l e p t o s p i r a l in fe c t i on .
CHAPTER FOUR - An e p i demi o l og i c al s tudy o f l e p t o s p i ro s i s due t o Le.pto.op.VC.a.
-i.n.tVtJtoga.M s erovar h.a!tdjo in a par t i c u l ar f l ock o f sheep .
v .
P a ge
11
111
V
V11
1X
1
1 2
4 1
6 5 CHAPTER F IVE - D iagno s i s o f l ep to s p i ral in fec t ion . 8 5 CHAPTER S IX - Gene r a l d i s cu s s i on. 1 1 6
APPENDIX I - L i s t o f equi pmen t and apparatus . 1 1 9 APPENDIX I I - Li s t o f ma t e r i a l s. 1 2 1 APPENDIX Ill - Prepara t i on o f bu f fe r s (Formu l a) . 1 2 3
TABLE OF CONTENTS CONT I NUED
APPEND IX IV
APPENDIX V
APPENDIX VI
- Prepar a t i on of J S med ium according to the me thod o f J ohns on and
S i e t er ( 1 9 7 7 ) .
- P roduc t i on o f rabb i t hyper irnmune s erum.
- Produ c t i on o f the immunogl obu l in frac t i ons .
APPENDIX V I I - Prepar a t i on o f ce l lulose-haJtdjo ant ibody c omp l exe s .
APPENDIX V I I I - Rad i o- l abe l l ing o f the garnmaglobu l in ( an t i - haJtdjo I g G ) w i th sod ium
V1.
Page
1 2 5
1 28
1 2 9
1 3 1
iod ide ( 1 2 5I) . 1 3 3
TABLE 2 - 1
2 - 2
LIST O F TABLE S
Wo r l d surveys of the preva l enc e o f l ep to s p i ra l infe c t ion i n sheep .
The var iou s lep t o s p iral s er ovar s r e po r t ed t o have i n fec ted s h e e p .
2 -3 The sero l o g i c a l preva l enc e o f l e p t o s p i r a l i n fec t ion in dome s t i c anima l s and w i l d l i fe in New Zea l and .
2 -4 D i s t r i bu t i on o f t i t r e s to l e p to s p i r a l an t i gens i n a s e r o l ogi�l s u rvey o f sheep from the N o r th I s l and of New Z e a land . 2 - 5 Preva lence o f agglu t in a t ing an t ibod i e s
t o lep t o s p i ral i n fe c t ion 1 n individu a l f l ocks o f sheep .
2- 6 D i s t r ibu t i on o f t i tr e s t o one or more s e rovar s in a random s amp l e o f Nor th I s l and ovine s e r a .
2 - 7 D i s t r ibu t i on o f s ingle and dua l t i tre s t o the s even s erovars a t s er um d i lu tion o f 1 : 24 .
2 - 8 D i s t r ibu t i on o f s ingle and d u a l t i tr e s t o t h e s even s e rovar s a t s e r um
d i lu t ion o f 1 : 48 . 2 - 9
2 - 1 0
The preva l ence o f l e p to s p i r a l infe c t i on 1n the three age -grou p s of she e p .
The geome t r i c mean t i t re ( GMT ) o f
l ep to s p i r a l infe c t ion 1 n t h e three age groups o f sheep .
Vll.
PAGE
1 3
1 5
1 7
2 2
2 5
2 8
34
34
36
36
L I S T OF TABLE S Tab le
3 - 1 L e p to s p i r a l serovars tha t have b een i s o l a ted f r om sheep over s e a s .
3 - 2 Lep t o s p i r a l se rovars tha t have b een i s o l a t ed 1n New Zea l and .
3-3 Ma i n tenan c e and s e c ondary h o s t s for l e p t o s p i r e s 1n New Z e a l and .
3-4 S e ro t yp ing o f the i s o l a te s u s ing rabb i t hyper immune sera .
3-5
3- 6
3 - 7
Percentage o f t h e kidneys examined cu l tu ra l ly pos i t ive to l e p t o s p i ral infec t ion .
The s e ro l o g i ca l and c u l tura l p r eva l enc e o f sheep in Flock No . 5.
The s ero l o g i c a l and cul tura l s tud i e s o f hams t e r s b e ing infec t e d wi th the ov ine
ha.Jc.djo i s o l a tes .
4- 1 D i s t r ibu t i o n of t i t r e s to l e p t o s p i r a l an t i gens i n a sero l o g i c a l s urvey o f sheep from a s tudy farm .
4-2 The p r eva l ence o f lepto s p i r a l infect ion and the g e ome t r i c mean ti t re in the three
V1 1 1 .
Page
4 2
4 4
4 6
5 1
5 2
5 5
5 7
6 9
age grou p s o f sheep from a s tudy farm . 7 1 4-3 Propor t i on o f farms wi th sheep with
t i tre s
(
� 1 :48 ) t o d i f feren t s erova r s . 7 61X .
LIST O F FIG U R E S
F IGURE P a ge
2 - 1 Or i g in o f the f l ocks o f sheep examined . ( Numbers ind i c a t e the numb er o f f l ocks examine d ) .
2 - 2 The propor t i on s o f sheep s era demon s t r a t ing t i t r e s ( � 1 : 4 8 ) to the s even l e p t o s p i r a l an t i gens .
2 - 3 D i s t r ibu t i on o f t i t r e s to t h e an t igens t e s t e d a c c o r d ing to the d i f fe r e n t age group s of she e p .
3 - 1 The proc e du r e invo lved 1 n the c u l tur ing o f k i dney s amp l e s .
3 - 2 Re s t r i c t i on e ndonuc l e a s e ana l y s i s o f one (S5-7) of the i s o l a t e s and the s even s e rova r s tha t have b e e n i s o l a t ed i n New Z e a l and .
From l e f t t o r i gh t : .tambd.a., ba.ll.um, sheep ha.Jtdjo i s o l a t e (S5-7), haJtdjopJc.a.jdno,
bai.c.a.n-<..c.a., c.ope.nha.ge.n-i., a.u..6Vta.Li-6, ta.Jr..M.Mv-i. and pomona..
3-3 Re s t r i c t i on endonuc l e a s e ana l y s i s o f the three sheep haAdjo i s o l a te s and f ive
d i f feren t s e r ovar s o f the Hebdoma d i s sera
group . F r om l e f t to r i gh t : .f.ambda., ha.e.mo.fy:U.c.a., me.da.ne.M..W, .�.>zwa.j-i.za.k, he.bdoma.d..W, lvte.Yr�MtM, shee p ha.Jtdjo i s o la t e s S5-7, S5-9 and S5-10.
1 9
2 3
38
49
54
5 6
x.
L I ST OF F I GURE S CONT INUED
F i gure PAGE
4 - 1 The propor t i on o f s h e e p in the s tudy farm demon s tra t ing t i t r e s (� 1:48 ) t o the s even l ep t o s p i r a l an t i gens .
4- 2
4-3
The dynami c s o f the ha.Jr..djo infe c t ion �n the s tudy farm .
The percen tage o f sheep po s i t ive t o
lep t o s p i r a l in fec t ion ( t i t res 1 : 48 or more ) .
4-4 The preva l en c e of l e p t o s p i r a l agglu t ina t ing an t ibod i e s to �djo among the three age group s of s h e e p in the s t udy farm and genera 1 s urvey .
5 - 1 Pr inc i p l e o f the s o l i d phase radio immunoas say . 7 0
7 2
7 5
7 7
(From Thorn l ey �t ai, 1 9 7 9 ) . 9 0
5- 2 As s ay No . 1 - Rad i o immunoas say o f l e p t o s p i r a l c e l l s ( su s p ended � n ur �ne , d i s t i l l ed wa t e r and J S med ium) .
5 - 3
5-4
s-s
5 - 6
Ass ay No . 2 - Rad i o immunoas s ay o f l e p t o s p i r a l c e l l s �n ur �ne .
As s ay No . 3 - Rad i o immunoas s ay o f
l ep t o s p i r a l c e l l s s u s p ended � n a s s ay bu f fer .
As s ay No . 4 - Rad i o immunoa s say o f
l ep to s p i r a l c e l l s su spe·.1ded � n JS med i um .
As s ay No . 5 - Rad i o immunoas say o f s on i c a ted l e p t o s p i r a l c e l l s s u s p ended �n u r �ne .
•
9 5
9 6
9 7
9 8
100
L I ST OF F IGURE S CONT INUED F i gu r e
5 - 7 As say No . 6 - Rad i o immunoa s s ay o f son i c a ted and lyophi l i s ed l e p t o s p i r a l ce l l s su spended i n buf fered s a l ine .
5 - 8 As s ay No . 7 - Rad i o immunoa s s ay o f s on i c a ted and lyoph i l i s ed l e p t o s p i r a l c e l l s sus pended i n buf fered s a l ine ( 1 00 ul of norma l rabb i t s e rum ins t ead of 20 u l ) .
5 -9 As say No . 8 - Rad io immunoa s s ay o f s on i c a ted and l yoph i l i s ed l ep t o s p i r a l ce l l s suspended i n ur i ne . ( 1 00 u l o f norma l rabb i t s e rum ins t e ad o f 2 0 u l ) . 5-1 0 As s ay No . 9 - Rad i o immuno a s s ay o f
son i c a ted and l yophi l i s ed l e p t o s p i r a l c e l l s sus pended in ur ine .
5 - 1 1 As s ay No . 1 0 - Rad io immunoas s ay o f s on i c a ted and lyoph i l i s ed l e p t o s p i r a l ce l l s sus pend ed i n ur �ne . ( T ime o f incuba t ion r edu c e d from 2 0 hours t o one hour ) .
5 - 1 2 As s ay No . 1 1 - Rad io immunoa s s ay o f s on i c a ted and l yoph i l i s e d lep t o s p i ra l c e l l s sus pended i n bu f fe red s a l ine
( 1::. fu l l a s s ay c oun t s , 0 coun t s of 300 u l a l iquot s ) .
5 - 1 3 As s ay No . 1 2 - Rad i o immunoa s s ay o f s on ic a ted and l yoph i l i s ed lep t o s p i r a l c e l l s sus pended i n buf fered s a l ine.
( Labell ed an t i b ody c omp l ex d i l u t e d four t ime s ) .
X � .
PAGE
10 1
1 0 2
1 03
1 0 5
1 0 6
1 0 7
1 0 8
