Frik_84_humoral Immune Response of Dogs After Vaccination

8/9/2019 Frik_84_humoral Immune Response of Dogs After Vaccination

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Veterinary Immunology and Immunopathology, 7 (1984) 245--254 245Elsevier Science Publishers B.V., Am sterda m -- Printe d in The Net herlands

HUMORAL IMMUNE RESPONSE OF DOGS AFTE R V ACC INATION A GA INS T LEP TOS PIROS IS MEASUREDBY AN IgM- AND IgG-SPEClFIC ELISAE.G . HARTMAN M. VAN HOUTEN J .F . F R IK1 and J .A . VAN DER DONKi D ep a rt m e nt o f B a c t e r i o l o g y , V e t e r i n a r y F a c u l t y , U n i v e r s i t y o f U t r e c h t ,P .O. Box 80 171, 3508 TD U t rec h t (The N e the r land s)2 Dep ar tme nt o f I mmu no lo gy , V e t e r i n a ry Fa c u l t y , U n i v e rs i t y o f U t r e c h t .P r e s e n t a d d re s s : L a b o r a t o r y o f H i s t o l o g y a n d C e l l B i o l o g y , M e d ic a l F a c u l t y ,U n i v e r s i t y o f U t r e c h t , N i c . B e e t s s t r a a t 2 2 , 35 11 HG U t r e c h t (T he N e t h e r l a n d s )(Accepted 9 March 1984)ABSTRACTHartman , E .G . , van Hou ten , M. , F r i k , J .F . and van de r Donk , J . A . , 1984 .H um ora l im m un e r es p on s e o f d o g s a f t e r v a c c i n a t i o n a g a i n s t l e p t o s p i r o s i smeasured by an IgM- and IgG -s pe c i f i c EL ISA. Ve t . Immuno l. Imm unopa tho l . , 7:245 -254 .

An IgM- and IgG -s pe c i f i c EL ISA was used to measure the a n t ibo dy response s t i -m u la te d i n d o g s b y v a c c i n a t i o n w i t h a l e p t o s p i r a l b a c t e r i n c o n t a i n i n g c h e m i c a l l yi n a c t i v a t e d L e p t o s p i r a i n t e r r o g a n s s e r o t y p e i c t e ro h a e m o r r h a g i a e an d s e r o ty p ec a n i c o l a l e p t o s p i r e s . A l l d o g s p ro du ce d a n t i - l e p t o s p i r a l IgM a nd Ig G. Th e Ig Mp r o d u c t i o n w as o f t h e p r i m a r y r e sp o ns e ty p e a f t e r e ac h v a c c i n a t i o n ( p r i m a r yv a c c i n a t i o n , b o o s te r v a c c i n a t i o n a nd a nn ua l r e v a c c i n a t i o n ) . A s u b s t a n t i a l a n t i -l e p t o s p i r a l Ig G r es p on s e c o u l d be d e m o n st ra t ed o n l y a f t e r t h e f i r s t b o o s t e rv a c c i n a t i o n a nd t h e a n n ua l r e v a c c i n a t i o n . A n n ua l r e v a c c i n a t i o n r e s u l t e d i n ah i g h e r a nd m u c h l o n g e r p e r s i s t i n g Ig G r es p on s e t h a n d i d t h e f i r s t b o o s t e rv a c c i n a t i o n . A r e v i s i o n o f t h e v a c c i n a t i o n s c he m e i s s u g ge s te d .

INTRODUCTIONC a nin e l e p t o s p i r o s i s i s d i s t r i b u t e d w o r l d - w i d e and g e n e r a l l y a s s o ci a te d w i t h

i n f e c t i o n b y L e p t o s p i r a i n t e r r o g a n s , s e r o t y p e i c te r o h a e m o r r h a g i a e an d c a n i c o l a( M i c h n a , 1 9 7 0 ) . I n t h e N e t h e r l a n d s i n f e c t i o n s w i t h s e r o t y p e c a n i c o l a a r e r e -s t r i c t e d t o t h e c a n i n e p o p u l a t i o n , w he re as s e r o t y p e ic t e r o h a e m o r r h a g i a e i ssp read by the brown ra t as i t i s wo r ld - w i de (Har tman , 1977) .

T he f i r s t s u c c e s s f u l v a c c i n a t i o n e x p e r im e n t s i n d o g s w ere c a r r i e d o u t byD a l l i n g a n d O k e l l ( 19 2 6) w i t h a k i l l e d s e r o t y p e i c te r o h a e m o r r h a g i a e v a c c i n e an db y O t to s e n ( 1 94 6 ) w i t h a s e r o t y p e c a n i c o l a v a c c i n e . P r e v e n t i o n o f l e p t o s p i r o s i si n do gs th r o u g h v a c c i n a t i o n w i t h a c h e m i c a l l y i n a c t i v a t e d b a c t e r i n o f m ix edc u l t u re s o f t h e a b o v e -me n t i o n e d s e ro t y p e s i s w id e l y p ra c t i c e d n o wa d a y s . B e f o ret h e s e b a c t e r i n s a re l i c e n c e d t h e y mus t p as s a p o t e n c y t e s t i n h ams te rs a sp re s c r i b e d i n t h e U .S . Co d e o f Fe d e ra l R e g u la t i o n s (S t a n d a rd Re q u i re me n t s B-4 1)and the European Pharmacopoeia. Th is hamste r p r o t ec t io n te s t , however , does no tp r o v i d e i n f o r m a t i o n c o n c e r n i n g t h e d e gr ee a nd d u r a t i o n o f p r o t e c t i o n a g a i n s tc l i n i c a l l e p t o s p i r o s i s o b t a i n e d t h r o u g h v a c c i n a t i o n o f d o gs . T he h am ste rs a r ec h a l le n g e d o n l y d u r i n g t h e 3 r d w e e k a f t e r v a c c i n a t i o n .0165 -242 7/84 /$03 .00 1984 Elsevier Science Publishers B.V.


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The d e g re e o f immu n i t y c o n f e r re d b y v a c c in a t i o n i s n o t r e l a t e d t o t h e a g g lu -t i n a t i o n t i t r e . T h u s , a n a n im a l may b e p r o t e c t e d f o r a c o n s i d e r a b l e p e r io d a f t e rs eru m a g g l u t i n i n s h av e d e c l i n e d b e lo w d e t e c t a b l e l e v e l s . L e p t o s p i r a l b a c t e r i n so f te n i n du c e p r o t e c t i o n w i t h o u t a t a l l s t i m u l a t i n g s i g n i f i c a n t l e v e l s o f ag g lu -t i n a t i n g a n t i b o d i e s . U s in g t h e a g g l u t i n a t i o n r e a c t i o n a s t h e s o l e m ea su re o fi m m u n o - p r o t e c t i o n , on e c o u l d f a l s e l y c o n c lu d e t h a t t h e b a c t e r i n s h ad f a i l e d t obe s u f f i c ie n t l y immunogen ic (Hea th and Box , 1965 ; Hanson e t a l . , 1972 ; Hanson ,1 97 3) y e t , t h e d i s c r e p a n c y be tw ee n a g g l u t i n a t i o n a nd p r o t e c t i o n i s a r e s u l t o ft h e d i f f e r e n t p r o p e r t i e s o f t h e v a r i o u s c l a s s e s o f i m m u n o g l o b u li n s p ro du ce da f t e r a n t i g e n i c s t i m u l a t i o n t h ro u g h v a c c i n a t i o n . IgM a n t i b o d i e s a r e t he f i r s tt o b e d e t e c t e d a f t e r a n t i g e n i c s t imu la t i o n a n d c a n b e s e e n a s a p r ima ry re s p o n s ed i r e c t l y p r o p o r t io n a t e t o t he a nt ig e n c o n c e n tr a t io n f o r r e l a t i v e l y s h o r t p er io d sf o l l o w i n g e ac h e x p os u re t o t h e s am e ( i n a c t i v a t e d ) a n t i g e n . IgG a n t i b o d i e s c an bed e t e c t e d s om e wh at l a t e r a f t e r p r i m a r y a n t i g e n i c s t i m u l a t i o n a nd r es p on d an am -n e s t i c a l l y f o l l o w i n g s e c on d a ry e x p os u re s ( P i k e , 1 96 7; H o o d e t a l . , 1 9 7 8) . D uet o t h e i r h i gh v a l en c y IgM a n t i b o d i e s a r e h i g h l y e f f e c t i v e a g g l u t i n a t o r s , w h e re -as Ig G a n t i b o d i e s a r e m o re c l o s e l y a s s o c i a te d w i t h n e u t r a l i z i n g a c t i v i t i e s andp h a g o c yt o si s an d o n l y p a r t i a l l y c o n t r i b u t e t o th e a g g l u t i n a t i o n r e a c t i o n (H oo de t a l . , 1978 ) . F rom the above -m en t ioned one can conc lude th a t the degree andd u r a t i o n o f t h e p r o d u c t io n o f s p e c i f i c a n t i - l e p t o s p i r a l IgG i s t h e m ost im p o r -t a n t c r i t e r i o n t o de te r m in e th e q u a l i t y o f ( h um o r a l) i m m u n it y a f t e r v a c c i n a t i o n .

T he a im o f t h e p r e s e n t i n v e s t i g a t i o n w as t o d e t e r m i n e th e p r o d u c t i o n o f s p e-c i f i c a n t i - l e p t o s p i r a l IgM and IgG a f t e r v a c c i n a t i o n o f do g s a g a i n s t l e p t o s p i -r o s i s u s i n g t h e E L I S A t e c h n iq u e , wh i c h h as b e e n s h o wn t o b e a v e ry u s e f u l t o o lf o r t h i s p u rp o s e (Ha rtma n e t a l . , 1 9 8 4 ) .

MATERIALS AND METHODSDogs

P r ima ry v a c c in a t i o n . E ig h t p u re b re d b e a g le s ( s i x f e ma le s 7 t o 9 mo nth s o fage and two ma les 18 mon ths o f age) w ere vacc ina te d s ubc u tane ous ly on the tho -ra c i c w a l l w i t h o ne re co mmen de d d os e o f a c omme rc ia l b a c t e r i n . The b a c t e r i nc o n s i s te d o f a c h e m i c a l l y i n a c t i v a t e d w h o le c u l t u r e m i x t u r e o f L e p t o s p ir ai n t e r r o g a n s s e r o ty p e s i c t e r o h a e m o r r h a g i a e a nd c a n i c o l a . T he v a c c i n a t i o n w asr e pe a te d t h r e e w e e k s l a t e r .

A n n u al r e v a c c in a t i o n . S e v e n p u re b re d b e a g les (a l l f e ma le s c i r c a 2 y e a rs o fa g e ) , wh i c h h ad b e e n v a c c in a t e d o ne an d t wo y e a rs b e f o re , we re s u b c u t a n e o u s l yv a c c in a t e d f o r t h e t h i r d t ime w i t h a s i n g le re co mmen de d d os e o f t h e s ame c omme r-c i a l b a c t e r i n . A l l d o g s w ere v a c c i n a te d w i t h b a c t e r i n o f t he s am e l o t .

Se rum sam p l ing . S e r i a l se rum samples were co l le c t ed f rom each dog be fo re anda f t e r v a c c i n a t i o n a nd s t o r e d a t - 70 C u n t i l u se .


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M i c r o s c o p ic a g l u t i n a t i o n t e s t (M AT )The MAT (Tu rn e r , 1 96 8) was c a r r i e d o u t i n Co o k e M ic ro t i t e r p l a t e s . L e p t o -

s p i r e s w ere c u l t i v a t e d i n a p o l y s o r b a t e - 8 0 b o v i n e a lb u m i n m e diu m ( D i f c o ) .C u l t u r e s o f L e p t o s p i r a i n t e r r o g a n s s e r o t y p e s i c t e r o h a e m o r r h a g i a e s t r a i nK a n t o ro w i c s a nd c a n i c o la s t r a i n Ho n d U t re c h t IV we re u se d a s a n t i g e n (d e n s i t ya p p r o x i m a t e ly 108 l e p t o s p i r e s p e r m l ) . S e r i a l t w o - f o l d d i l u t i o n s o f seru ms t a r t i n g w i t h I i n 2 0 we re ma de i n Ch a n g b u f f e r (Ch an g, 1 9 4 7 ) . Th e t i t r e i s e x -p re s se d as t h e r e c i p r o c a l o f t h e h i g h e s t d i l u t i o n o f s e r um a t w h ic h a t l e a s t50% o f t h e l e p t o s p i r e s we re a g g lu t i n a t e d (W HO, 1 9 6 7 ).

ELISAT he E L IS A w as c a r r i e d o u t i n m i c r o t i t r e p l a t e s

c o a t e d w i t h t h e o u t e r e n v e l o p e a n t i g e n o f L e p t o s p ii c t e r o h a e m o r r h a g i a e s t r a i n K a n t or o w i cs an d c a n i c o l

(Co o k e Dy n a t e c h , M i c ro e l i s a )r a i n t e r r o g a n s s e r o t y p e sa s t r a i n H o nd U t r e c h t I V. I n

c o n s e c u t i v e s t ep s ( 1 ) t w o - f o l d d i l u t e d d og s e r um , ( 2 a) r a b b i t a n t i - c a n i n e I gM( ~ c h a in s p e c i f i c ) o r ( 2b ) r a b b i t a n t i - c a n i n e IgG ( ~ c h a in s p e c i f i c ) , t he n (3 )g o a t a n t i - r a b b i t Ig G (H + L) h o r s e - r a d i s h p e r o x i d a s e c o n j u g a te ( M i l e s L a b o r a to -r i e s ) a nd f i n a l l y ( 4 ) s u b s t r a t e ( o r t h o - p h e n y l e n e d i a m i n ) w ere a dd ed . A f t e r e ac hin c u b a t i o n s t e p t h e p l a t e s we re t h o r o u g h l y wa s he d w i t h t a p wa t e r -Twe e n -2 0 . Thete s t was read on a Dynatech M ic ro e l i s a M in i r ea de r MR 590 (Pope e t a l . , 1982 ;Ha rtma n e t a l . , 1 9 8 4 ) .

Da t a a n a l y s i sA l l a n t i b o d y t i t r e s a r e e x p re s s ed as t h e a r i t h m e t i c m ea n o f t he l o g l O o f t h e

r e c i p r o c a l t i t r e s an d p r e se n t ed as a l i n e a r f u n c t i o n o f t i m e i n w e ek s ( F i g u r e s1 - 4 ) .

RESULTSP r i m a r y v a c c i n a t i o n

S e r o l o g i c a l e x a m i n a t i o n o f t h e 8 b e a gl es b e f o r e t he p r i m a r y v a c c i n a t i o ne x p e r i m e n t d e m o n st ra te d n o a g g l u t i n a t i o n t i t r e s e q ua l t o o r e x c e e d in g 20 ; E LIS AIg G t i t r e s w e r e < 2 0 a nd E L I S A IgM t i t r e s w ere ~ 4 0 i n a l l d o g s a nd s h o u ld bec o n s id e re d t o b e n e g a t i v e (Ha rtma n e t a l . , 1 9 8 4 ) . The a n t i b o d y re s po n s e o f t h ep r ima ry v a c c in a t e d b e a g le s i s s h o wn i n t h e F ig u re s I a n d 2 .

Ig G r e sp o n se . P r im a r y v a c c i n a t i o n d i d n o t s t i m u l a t e a s u b s t a n t i a l p r o d u c t i o no f Ig G t o e i t h e r s e r o t y p e i c te r o h a e m o r r h a g i a e o r s e r o t y p e c a n i c o l a d u r i n g t h et h r e e w e e k s p r e c e d in g t h e b o o s t e r v a c c i n a t i o n . I n o n l y tw o d og s a t i t r e o f 4 0was d e mo n s t ra te d t o s e ro t y p e c a n i c o la a n d i n o n e d og to s e ro t y p e i c t e ro -h a e m o rr h ag ia e . A f t e r t h e f i r s t b o o s t e r v a c c i n a t i o n a s h a rp r i s e o f th e I gG t i t r ew as d e m o n s t ra t ed : an e l e v e n - f o l d r i s e o f t h e m ea n a n t i - c a n i c o l a Ig G t i t r e a nda n e i g h t - f o l d r i s e o f t h e m ea n a n t i - i c t e r o h a e m o r r h a g i a e Ig G t i t r e . T he m axim um


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Fig. 2. Antibody response to the icterohaemorrhagiae component of a bivalentbacterin as measured by the MAT and the IgM-, resp. IgG-specific ELISA in eightdogs af te r primary vaccination: f i r s t vaccination (1) and booster vaccination( I I ) .. . . . . IgM; IgG; ...........MAT.


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of the mean IgG t i t re to both serotypes was attained one week af ter the boostervaccination. Thereafter the mean IgG t i t re to both serotypes gradual ly declinedto reach the lowest detection level 1 in 20 (1.3) at 4months and 6 monthspost-vaccination, respectively for serotypes canicola and icterohaemorrhagiae;however, the i in 40 (1.6) level (one di lut ion step above the lowest detectionlevel) was already reached wi th in 10 weeks af te r the booster vaccination (bothserotypes).

The immune response of the individual dogs varied to some extent. The maximalIgG t i t re varied from 40 to 320 (icterohaemorrhagiae) and from 80 to 320(canicola). In two dogs ("low responders") the IgG t i t re declined to 20 wi th inthree weeks (icterohaemorrhagiae) and seven weeks (canicola) fo llowing thebooster vaccination. After ha lf a year in only two dogs an IgG t i t re of 80could st i l l be demonstrated to both serotypes. One year af ter the primaryvaccination al l dogs had IgG t i t re s ~40.

IgM response. The IgM response to both serotypes was of the primary responsetype af ter the f i r s t and booster vaccination. The mean IgM t i t re to both sero-types attained i ts maximum one week af ter the f i r s t vaccination (varying from160 to 640 to both serotypes) and also one week after the booster vaccination(varying from 80 to 320 and 640 respectively to canicola and icterohaemorrhagiae)and declined below the 1 in 40 level wi thin 10 weeks af ter the booster vaccina-tiQn.

MAT response. The mean MAT t i t re pattern with respect to serotype ictero-haemorrohagiae closely fol lowed the mean IgM pattern and declined to the lowestdetection level (1 in 20) wi th in 10 weeks af ter the booster vaccination. Themean MAT t i t re to serotype canicola did not exceed the lowest detection levelfor one year fo llowing primary vaccinat ion. In only two dogs an anti -canicolaMAT t i t re~-20 (80 to 160) was detected during the f i rs t two weeks af ter thef i r s t and booster vaccination.

Annual revaccinationThe antibody response of the annually revaccinated beagles is shown in the

Figures 3 and 4.IgG response. Revaccination of the beagles, which had been vaccinated

annually two times before, resulted in an anamnestic rise of the IgG t i t re toboth serotype icterohaemorrhagiae and canicola to attain a maximum 2 to 3 weeksaf te r the revaccination. In the ind iv idual dogs the maximum varied from 320 to2560 to both serotypes, with the exception of one dog (probably a "low respond-er") which demonstrated a maximal IgG t i t re of only 80 to serotype i ctero-haemorrhagiae and 40 to serotype canicola. Although the mean IgG t i t re to bothserotypes declined gradually after at ta in ing the maximum, i t remained at ahigher level than before the revaccination for at least one year. However, in


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two dogs inc ludi ng the "low responder", the ant i- ca ni co la IgG ti tr e decreasedto ~4 0 by the end of the year foll owin g the revaccinati on.

IgM response. The IgM response to both serotypes was of the primary responsetype as it was af te r the fi r st and booster vacci nati on of the primary vaccinateddogs. The mean IgM t i t re at ta ined i ts maximum als o one week aft er the vacc ina-ti on . The maximal IgM t i t re varied from 80 to 320 wi th respect to both sero-types. The mean IgM t i t re to both serotypes dec lined to a negative IgM leve l(~ 40 ) wi th in seven weeks af te r the reva ccinat ion. The low IgG responder showeda "normal" IgM response.

MAT response. The MAT response to serotype can icola cl os el y resembled theIgM response to the same serotype at ta in ing a maximum two weeks aft er the re-vac cinat ion. The mean MAT t i t r e decl ined to below the lowest de tec tion l eve lwi th in si x weeks. The MAT response to serotype icterohaemorrhagiae was more pro-nounced, reached a maximum one week af te r the revacc inat ion and only gr adua ll ydeclined ther ea ft er . It pa ra ll el le d the IgG response to serotype icterohaemorrha-giae to a ce rtai n ext ent . The maximal MAT t i t r e to serotype icterohaemor rhagiaevar ied from 160 to 640, whereas the maximal MAT t i t re to serotype canicolavar ied from 20 (the same "low responder") to 160.

DISCUSSIONThe immune responses to le pt os pi ra l bacterins have been d i f fi c ul t to evaluate

using the MAT because the majority of the vaccinated animals develop either alow agglut i nat ion t i t re or fai l to develop any agglut i nat ion t i t r e at al lfollowing vaccination. However, results of hamster protection test and challengestudies show tha t pro tec tiv e an tibod ies do develop af te r vaccina tion and per sis taf te r the ag gl ut in at io n t i t r e has disappeared (Heath and Box, 1965; Negi et a l . ,1971; Huhn et a l . , 1975).

Immunoglobulin G is the pr incip al class of antibody for pro tec tio n, whereasthe IgM class of anti body is pr im ar il y associated with agglut inati on (Hanson,1973). Negi et a l. (1971), using the hamster passi ve- pro tec tio n test , demon-strated that protective antibodies were present in both the IgM and IgG frac-ti on of c a l f serum, being more prevalen t, however, in the IgG fr ac ti on . Hamsterpas siv e-p rot ecti on tests are laboriou s and expensive and not suited fo r rout inetest in g of the humoral immune response a ft er vac cinati on. Although Huhn et al .(1975) have demonstrated the re la t ion between the res istance induced in vac ci -nated dogs and tha t induced in vaccinated hamsters by a challenge two weeksaf te r vacc inat ion and Desmecht (1982) found that hamsters were pro tec ted f or atlea st 18 months af te r two vaccinati ons three weeks apart wit h 1/10 of the doseof a vaccine for dogs, hamster protection tests as prescribed in the U.S. Codeof Federal Regulations and the European Pharmacopoeia in principle provide noinformation concerning the duration of immunity in dogs.


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A s im p le re p ro d u c ib l e s e ro l o g i ca l t e s t t o m ea su re t h e I g G re sp o n se wo u ldf a c i l i t a t e t h e e v a l u a t i o n o f t h e h um ora l i mm un e r es p on s e a f t e r v a c c i n a t i o n .T r i p a t h y e t a l . ( 19 73 ) d e sc r ib e d th e use o f t he g r o w t h - i n h i b i t i o n t e s t t o d e t e c ta n t ib o d y t o le p t o s p i r e s i n c a t t l e v a c c i n a t ed w i t h l e p t o s p i r a l b a c t e r i n s a ndf ou n d t h a t t h e g r o w t h - i n h i b i t i n g a c t i v i t y w as l a r g e l y a s s o c i a te d w i t h t h e IgGa n t i b o d i e s . T he m a j or d is a d v a n t a g e o f t h i s t e s t i s t h a t a n i n c u b a t i o n p e r i o do f a p p ro x im a t e l y t wo we e ks i s r e q u i re d b e f o re re su l t s can b e re a d . U s in g o u rE LIS A t e c h n i q u e th e r e s u l t s c an be r ea d w i t h i n f i v e h o u r s . T h e r e s u l t s p r e -se n t ed b y B ey a nd Joh nson (1 9 7 8 , 1 9 8 3 b ) , u s i n g t h e m ic ro sc o p i c a g g l u t i n a t i o nt e s t a nd t h e l e p t o s p i r i c i d a l a ssay t o m o n i t o r t h e h um o ra l im m un e re sp o n se o fd og s a f t e r v a c c i n a t i o n , w ere p a r t i a l l y i n c o n s i s t e n t w i t h t h e r e s u l t s d e sc r ib e din th e p re sen t paper . They found tha t b o th IgM and IgG were p roduced in s im i l a ra m ou nts i n va c c in a t e d d o g s o ve r a n o b s e rva t i o n p e r i o d o f t wo ye a rs . I n o u r e x -p e r i m e n t , u s i n g t h e E L IS A t e c h n i q u e , a n t i - l e p t o s p i r a l IgM c o u l d o n l y be d e t e c te df o r s h o r t p e r i o d s o f t im e f o l l o w i n g e a ch v a c c i n a t i o n , wh ere as a s u b s t a n t i a lp r o d u c t i o n o f a n t i - l e p t o s p i r a l Ig G w as n o t d e te c t e d b e f o r e on e w e e k a f t e r t h ef i r s t b o o s t e r v a c c i n a t i o n ; t h e I gG t i t r e s b e i ng f a r m o r e p ro n ou n ce d a nd o f am uch l o n g e r d u r a t i o n a f t e r t h e an n u al r e v a c c i n a t i o n . T he f a c t t h a t t h e Ig Gr es p on se m o r e o r l e s s p a r a l l e l l e d t h e Ig M r es p on s e a f t e r t h e f i r s t b o o s t e rva cc in a t i o n i s i n a g re em e nt w i t h t h e re su l t s o f B e y a nd Joh nson (1 9 7 8 , 1 9 8 3 b ) .H ow ev er, I gM t i t r e s c a n n o t q u a n t i t a t i v e l y b e c om p a re d w i t h Ig G t i t r e s d ue t od i f f e r e n t a v i d i t i e s o f b o t h i m m u n o g l o b u l i n s .

As p r o t e c t i o n i s p r i m a r i l y a s s o c ia t e d w i t h Ig G a n t i b o d i e s a nd as a n t i - l e p t o -sp i ra l Ig G t i t r e s o f m ax im a l 80 co u ld o n l y b e d e m o n s t ra t e d f o r a l im i t e d n um b ero f w e e k s a f t e r a s i n g l e v a c c i n a t i o n , a s w as d e m o n s tr a te d i n f o u r o t h e r d o g s,p r im a r y v a c c i n a t i o n o f d og s a g a i n s t l e p t o s p i r o s i s s h o u ld i n c l u d e a b o o s te rv a c c i n a t i o n . T he r a p i d d e c l i n e o f t he IgG t i t r e a f t e r t h e f i r s t b o o s te r ( F ig u r e sI and 2 ) m igh t r e s u l t in a too low lev e l o f humoral imm un i t y and an inc rea sedr i s k o f i n f e c t i o n . R e v a c c i n a t i o n b e f o r e t h e e nd o f t h e 3 rd m on th a f t e r t h ef i r s t b o o s te r c o u ld p r o b a b ly p r e v e n t t h i s r i s k o f i n f e c t i o n . A nn ua l r e v a c c i n a -t i o n , r e s u l t i n g i n th e p r o d u c ti o n o f r e l a t i v e l y h ig h a n t i - l e p t o s p i r a l IgG t i t r e sp e r s i s t i n g f o r a t l e a s t o ne y e a r i n t h e m a j o r i t y o f t h e d o gs , as s h o w n i nF i g u r e s 3 an d 4 , s ee ms s a t i s f a c t o r y .

A s d e sc r i b e d f o r o t h e r b a c t e r i n s (Ha n so n , 1 9 7 3 ) , t h e ca n i co la co mp on en t o ft h e b a c t e r i n , u se d i n t h i s e x p e r i m e n t , d i d n o t s t i m u l a t e a d e t e c t a b l e MAT t i t r e( ~ 2 0 ) i n t he m a j o r i t y o f t he do gs a f t e r t h e p ri m a r y v a c c i n a t i o n (+ b o o s t e r ) ,wh erea s t h e i c t e ro h a e m o r rh a g ia e com p on en t s t im u la t e d a MAT re sp o n se s im i l a r t ot h e Ig M re sp o n se t o t h a t se ro t yp e a s d i d t h e ca n i co la com p on en t i n t h e g ro u pa n n u a l l y r e v a c c i n a te d b ea gl es ( F i g u r e 3 ) . A g g l u t i n a t i o n t i t r e s t o s e ro t yp ec a n i c o l a w ere c o n s i d e r a b l y l o w e r t ha n a g g l u t i n a t i o n t i t r e s t o s e r o ty p e i c t e r o -h a e m or rh a gi ae . L i v e s e r o t y p e c a n i c o l a a n t i g e n a p p a r e n t l y c a n n o t e a s i l y b e


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t ra p p ed by a g g l u t i n a t i n g a n t i b o d i e s ( I gM ) i n s p i t e o f r e l a t i v e l y h i g h a n t i -l e p t o s p i r a l Ig M t i t r e s ( F i g u r e i ) a s d e t e c t e d by t h e E L IS A u s i n g t h e o u t e r e n -v e l o p e a n t i g e n .

A c e r t a i n a m oun t o f b i o l o g i c a l v a r i a t i o n , r e s u l t i n g i n v a r y i n g a n t i b o d y r e s -p on se s t o v a c c in a t i o n , was d e mo n s t rat e d i n t h e g ro up o f b e a g le s u s e d i n t h ep r e s e n t v a c c i n a t i o n e x p e r i m e n t . T h i s v a r i a t i o n c o u l d p r o b a b l y b e m a sk ed w he nla r g e r a mou nts o f a n t i g e n were u s e d i n t h e v a c c in e (Hu h n e t a l . , 1 9 7 5 ).

CONCLUSIONST he E L IS A t e c h n i q u e d e t e c t i n g s p e c i f i c a n t i - l e p t o s p i r a l IgM a nd Ig G p ro v e d

t o b e a v e ry s u i t a b l e t e s t t o me as ure th e h umo ra l immu ne re sp o n se i n d o g s a f t e rv a c c i n a t i o n w i t h a c h e m i c a l ly i n a c t i v a t e d l e p t o s p i r a l b a c t e r i n .

V a c c i n a t i o n t i t r e s do n o t d i s t u r b t he s e r o di a g n o s is o f a c ute l e p t o s p i r o s i sb e ca u se , as a r u l e , d o g s s u f f e r i n g f ro m a c u te l e p t o s p i r o s i s d e m o n st ra te a r e l a -t i v e l y h ig h a n t i - l e p t o s p i r a l IgM t i t r e t o g et h e r w i t h a lo w o r n eg a t i ve a n t i -l e p t o s p i r a l Ig G t i t r e (H artm a n e t a l . , 1 9 84 ). S u c h t i t r e s w ere f o un d o n l y d u r in gt h e t im e b etw ee n t h e f i r s t a nd t h e b o o s t e r v a c c i n a t i o n ( i n p r i m a r i l y v a c c i n a t e ddogs) .

T he r e s u l t s o f t h e p r e s e n t i n v e s t i g a t i o n s u g ge s t t h e n e c e s s i t y o f a r e v i s i o no f t h e v a c c i n a t i o n s c h em e . A d d i t i o n o f a n a d j u v a n t t o th e l e p t o s p i r a l b a c t e r i n scou ld pe rhaps improve the deg ree and du ra t io n o f the immune response as wasdemons t ra ted by Bey and Johnson (1982a) . However, as the p r im ary v ac c i na t io ne x p e r im e n t w as c a r r i e d o u t i n o l d e r d o gs , t h e r e s u l t s o f t h e p r e s e n t e x p e ri m e n ta r e n o t n e c e s s a r i l y a p p l i c a b l e t o p u p p i e s o f c i r c a t h r e e m o n t h s o f a g e .ACKNOWLEDGEMENTS

The a u t h o rs w i s h t o e x p re s s t h e i r g r a t i t u d e t o Mrs . G .M.S . v a n Dam-K nu bb enf o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . P r o f . D r . W . A . de V o og d v an d e r S t ra a t e n f o rs t a t i s t i c a l a s s i s ta n c e and D r s. S .H . L o e f f l e r f o r l i n g u i s t i c a d v ic e . T he s t a f fo f t h e De pa rtme nt o f S ma ll A n ima l O b s t e t r i c s a nd G y n e c o lo g y , V e t e r i n a ry Fa c u l t y ,U n i v e r s i t y o f U t r e c h t ( he a d P r o f . D r . C . H .W . de B o i s ) i s g r a t e f u l l y a ck no wle dg edf o r t h e p e r m i s s i o n t o u s e t h e b e a g l e c o l o n y f o r t h e v a c c i n a t i o n e x p e r i m e n t .

REFERENCESBey, R.F . and Johnson , R.C . , 1978 . Hum ora l immune response o f dogs vac c ina tedw i t h l e p t o s p i r a l p e n t a v a l e n t o u t e r e n v el o pe and w h o le c u l t u r e v a c c i n e s .Am .J .Ve t . Res . , 39 : 831 -836 .Bey, R.F . and Johnson , R.C. , 1982a. Lep tos p i r a l vacc ine s in dogs : Im mu nogen ic i t yo f wh o le c e l l a nd o u t e r e n v e lo p e v a c c in e s p re p a re d i n p ro t e i n - f r e e m e diu m.Am .J .Ve t . Res . , 43 : 831 -834 .


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Bey, R.F . and Johnson, R .C. , 198 2b. Immunogenic ity and hum ora l and ce l l -m ed iate dimmune responses to lep tos p i r a l who le ce l l , ou te r en ve lope , and p rotop lasmacy l in d er vacc ines in hamsters and dogs. Am.J.Vet . Res . , 43: 835-840.Ch an g, S .L . , 1 9 4 7 . S tu d ie s on L e p to sp i ra i c te ro h a e m o r rh a g ia e . J . I n f . D i s . ,81: 28.Da l l i n g , T . a nd O ke l l , C .C . , 1 9 2 6 . Va cc in e t re a tme n t o f ca n in e l e p to s p i ra lj a u n d i ce . J .Pa th . Ba c t . , 2 9 : 1 3 1 .Desmech t, M. , 198 2 . Dur~e de l ' imm un i t6 con fe re e par le vacc in de la lep to sp i -rose can ine c he z le hamste r . Ann .M~ d.V6 t . , 12 6 : 55 -58 .Ha nso n, L .E . , T r i p a th y , D .N . a nd K i l l i n g e r , A .H . , 1 9 7 2 . Cu r re n t s t a tu s o flep to sp i ro s is immun iza tion in sw ine and ca t t l e . J .A .V .M.A . , 1 61 : 1235-1243 .Hanson , L .E . , 197 3 . Immuno log ic p rob lems in bov ine lep to sp i ro s is . J .A .V .M.A . ,163: 919-921.Ha rtma n , E .G ., 1 9 7 7 . L e p to sp i ro se . T i j d s ch r . D ie rg e n e e sk . , 1 0 2 : 4 5 -5 2 .Hartman, E.G. , van Houten, M. , van de r Do nk, J .A. and F r i k , J . F . , 1984.De te rm in a t io n o f sp e c i f i c a n t i - l e p to sp i ra ] immu n o glo b ulin s M a nd G i n se rao f e xp e r im e n ta l l y i n f e c te d d o g s b y so l i d -p h a se e n zyme - li n ke d immu n osorbe n tassay. Vet. lmmun ol.lmmun opathol. , 7: 43- 51.He ath , K .R . a nd Bo x , P .G ., 1 9 6 5 . Immu n i t y t o l e p to s p i r o s i s : An t i b o d ie s i n va c -

c ina ted and in fec ted dogs. J .Comp. Pa tho l . , 75 : 127-136 .Hood, L . E. , Weissman, I .L . and Wood, W.B. , 197 8. Immunology. The Benjamin/Cummings Pu b l ish ing Company, In c . , Me n lo Park , C a l i fo rn ia .Hu h n, R .C . , Ba ld w in , C .D . a nd Ca rd e l l a , M .A . , 1 9 7 5 . Imm u n i ty t o l e p to s p i r o s i s :Ba cte r ins in dog s and hamsters. Am.J.Vet . Res . , 36: 71-74.Michna , S .W. , 1970 . Le p to sp i ros is . Ve t . Rec . , 86 : 484-496 .Neg i , S .K. , Myers, W.L . and Segre , D . , 197 1 . An t ibody response o f c a t t l e toL_eptospira pomona: Response as measured by h em ag glu t ina t ion , micro scop icag g lu t in a t io n , and hamste r p r o te c t io n te s ts . Am.J.Ve t. Res . , 32: 1915-1920 .Ot tosen , H .E. , 1946 . Om vac c ina t ion mod lep tos p i ro se hos hund . Med lemsb l.danske dy la eg e fo ren . , 29 : 187-194.P i ke , R .M., 1 9 6 7 . An t ib o d y h e te ro g e n e i t y an d se ro lo g i ca l re a c t iO n s .Ba c te r i o l .Re v . , 3 1 : 1 5 7 -1 7 4 .Pope , V. , Hun te r , E .F. and Fee ley , J .C . , 198 2 . Eva lua t ion o f the microenzyme-l i n ke d immu n o so rb e nt a ssa y w i t h T re p on e ma p a ] l id u m a n t i g e n . J .C l i n . M ic r o b io l .15: 630-634.Tr ipa thy , D .N. , Smi th , A .R. and Hanson , L .E . , 197 5 . Immunoglobu lins in c a t t l eva cc in a te d w i t h l e p to s p i ra l b a c te r i n s . Am .J .Ve t .Re s . , 3 6 : 17 3 5-17 3 6.Tu rne r , L .H . , 196 8 . Le p to sp i ro s is I I . Trans . R .Soc .TropoMed.Hyg ., 62 : 880-899 .Wo rld He a l th O rg a n i za t i o n , 1 9 6 7 , Cu r re n t p ro b le ms i n l e p to sp i ro s i s re se a rch .Wld. H l th .O rg . tec hn . Rep .Ser . , 380 .

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