Humoral immune response of dogs after vaccination against leptospirosis measured by an IgM- and IgG-specific ELISA

Veterinary Immunology and Immunopathology, 7 (1984) 245--254 Elsevier Science Publishers B.V., A m s t e r d a m -- Printed in The Netherlands

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HUMORAL IMMUNE RESPONSE OF DOGS AFTER VACCINATION AGAINST LEPTOSPIROSIS MEASURED BY AN IgM- AND IgG-SPEClFIC ELISA E.G. HARTMAN1 M. VAN HOUTEN1 J.F. FRIK1 and J.A. VAN DER DONK2 iDepartment of Bacteriology, Veterinary Faculty, U n i v e r s i t y of Utrecht, P.O. Box 80 171, 3508 TD Utrecht (The Netherlands) 2Department of Immunology, Veterinary Faculty, U n i v e r s i t y of Utrecht. Present address: Laboratory of Histology and Cell Biology, Medical Faculty, U n i v e r s i t y of Utrecht, Nic. Beetsstraat 22, 3511HG Utrecht (The Netherlands) (Accepted 9 March 1984) ABSTRACT Hartman, E.G., van Houten, M., F r i k , J.F. and van der Donk, J . A . , 1984. Humoral immune response of dogs a f t e r vaccination against leptospirosis measured by an IgM- and IgG-specific ELISA. Vet. Immunol. Immunopathol., 7: 245-254. An IgM- and IgG-specific ELISA was used to measure the antibody response s t i mulated in dogs by vaccination with a l e p t o s p i r a l bacterin containing chemically i n a c t i v a t e d Leptospira interrogans serotype icterohaemorrhagiae and serotype canicola leptospires. A l l dogs produced a n t i - l e p t o s p i r a l IgM and IgG. The IgM production was of the primary response type a f t e r each vaccination (primary vaccination, booster vaccination and annual revaccination). A substantial a n t i l e p t o s p i r a l IgG response could be demonstrated only a f t e r the f i r s t booster vaccination and the annual revaccination. Annual revaccination resulted in a higher and much longer p e r s i s t i n g IgG response than did the f i r s t booster vaccination. A r e v i s i o n of the vaccination scheme is suggested. INTRODUCTION Canine l e p t o s p i r o s i s is d i s t r i b u t e d world-wide and generally associated with i n f e c t i o n by Leptospira interrogans, serotype icterohaemorrhagiae and canicola (Michna, 1970). In the Netherlands i n f e c t i o n s with serotype canicola are res t r i c t e d to the canine population, whereas serotype icterohaemorrhagiae is spread by the brown rat as i t is world-wide (Hartman, 1977). The f i r s t

successful vaccination experiments in dogs were carried out by

Dalling and Okell (1926) with a k i l l e d serotype icterohaemorrhagiae vaccine and by Ottosen (1946) with a serotype canicola vaccine. Prevention of leptospirosis in dogs through vaccination with a chemically inactivated bacterin of mixed cultures of the above-mentioned serotypes is widely practiced nowadays. Before these bacterins are licenced they must pass a potency test in hamsters as prescribed in the U.S. Code of Federal Regulations (Standard Requirements B-41) and the European Pharmacopoeia. This hamster protection t e s t , however, does not provide information concerning the degree and duration of protection against c l i n i c a l l e p t o s p i r o s i s obtained through vaccination of dogs. The hamsters are challenged only during the 3rd week a f t e r vaccination. 0165-2427/84/$03.00

© 1984 Elsevier Science Publishers B.V.

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The degree of immunity conferred by vaccination is not related to the agglutination titre.

Thus, an animal may be protected for a considerable period a f t e r

serum a g g l u t i n i n s have declined below detectable levels. Leptospiral bacterins often induce protection without at 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 levels of agglut i n a t i n g antibodies. Using the a g g l u t i n a t i o n reaction as the sole measure of immuno-protection, one could f a l s e l y conclude that the bacterins had f a i l e d to be s u f f i c i e n t l y immunogenic (Heath and Box, 1965; Hanson et a l . , 1972; Hanson, 1973) y e t , the discrepancy between a g g l u t i n a t i o n and protection is a r e s u l t of the d i f f e r e n t properties of the various classes of immunoglobulins produced a f t e r antigenic s t i m u l a t i o n through vaccination. IgM antibodies are the f i r s t to be detected a f t e r antigenic s t i m u l a t i o n and can be seen as a primary response d i r e c t l y proportionate to the antigen concentration f o r r e l a t i v e l y short periods f o l l o w i n g each exposure to the same ( i n a c t i v a t e d ) antigen. IgG antibodies can be detected somewhat l a t e r a f t e r primary antigenic s t i m u l a t i o n and respond anamn e s t i c a l l y f o l l o w i n g secondary exposures (Pike, 1967; Hood et a l . , 1978). Due to t h e i r high valency IgM antibodies are 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 , whereas IgG antibodies are more closely associated with n e u t r a l i z i n g a c t i v i t i e s and phagocytosis and only p a r t i a l l y contribute to the a g g l u t i n a t i o n reaction (Hood et a l . ,

1978). From the above-mentioned one can conclude that the degree and

duration of the production of s p e c i f i c a n t i - l e p t o s p i r a l

IgG is the most impor-

tant c r i t e r i o n to determine the q u a l i t y of (humoral) immunity a f t e r vaccination. The aim of the present i n v e s t i g a t i o n was to determine the production of specific anti-leptospiral

IgM and IgG a f t e r vaccination of dogs against l e p t o s p i -

rosis using the ELISA technique, which has been shown to be a very useful tool for t h i s purpose (Hartman et a l . ,

1984).

MATERIALS AND METHODS Dogs Primary vaccination.

Eight purebred beagles ( s i x females 7 to 9 months of

age and two males 18 months of age) were vaccinated subcutaneously on the thoracic wall with one recommended dose of a commercial bacterin. The bacterin consisted of a chemically i n a c t i v a t e d whole c u l t u r e mixture of Leptospira interrogans serotypes icterohaemorrhagiae and canicola. The vaccination was repeated three weeks l a t e r . Annual revaccination. Seven purebred beagles ( a l l females circa 2½ years of age), which had been vaccinated one and two years before, were subcutaneously vaccinated f o r the t h i r d time with a single recommended dose of the same commerc i a l bacterin. A l l dogs were vaccinated with bacterin of the same l o t . Serum sampling.

Serial serum samples were collected from each dog before and

a f t e r vaccination and stored at -70°C u n t i l use.

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Microscopic a g l u t i n a t i o n test (MAT) The MAT (Turner, 1968) was carried out in Cooke M i c r o t i t e r plates. Leptospires were c u l t i v a t e d in a polysorbate-80 bovine albumin medium (Difco). Cultures of Leptospira interrogans serotypes icterohaemorrhagiae s t r a i n Kantorowics and canicola s t r a i n Hond Utrecht IV were used as antigen (density approximately 108 leptospires per ml). Serial two-fold d i l u t i o n s of serum s t a r t i n g with I in 20 were made in Chang buffer (Chang, 1947). The t i t r e

is ex-

pressed as the reciprocal of the highest d i l u t i o n of serum at which at least 50% of the leptospires were agglutinated (WHO, 1967). ELISA The ELISA was carried out in m i c r o t i t r e plates (Cooke Dynatech, Microelisa) coated with the outer envelope antigen of Leptospi ra interrogans serotypes icterohaemorrhagiae s t r a i n Kantorowics and canicol a s t r a i n Hond Utrecht IV. In consecutive steps (1) two-fold d i l u t e d dog serum,(2a) rabbit anti-canine IgM (~ chain s p e c i f i c ) or (2b) r a b b i t anti-canine IgG (~ chain s p e c i f i c ) , then (3) goat a n t i - r a b b i t IgG (H+L) horse-radish peroxidase conjugate (Miles Laboratories) and f i n a l l y

(4) substrate (ortho-phenylenediamin) were added. After each

incubation step the plates were thoroughly washed with tapwater-Tween-20. The test was read on a Dynatech Microelisa Minireader MR 590 (Pope et a l . , Hartman et a l . ,

1982;

1984).

Data analysis All antibody t i t r e s are expressed as the arithmetic mean of the loglO of the reciprocal t i t r e s and presented as a l i n e a r function of time in weeks (Figures 1-4). RESULTS Primary vaccination Serological examination of the 8 beagles before the primary vaccination experiment demonstrated no a g g l u t i n a t i o n t i t r e s equal to or exceeding 20; ELISA IgG t i t r e s w e r e < 2 0 and ELISA IgM t i t r e s were ~ 4 0 in a l l dogs and should be considered to be negative (Hartman et a l . ,

1984). The antibody response of the

primary vaccinated beagles is shown in the Figures I and 2. IgG response.

Primary vaccination did not stimulate a substantial production

of IgG to e i t h e r serotype icterohaemorrhagiae or serotype canicola during the three weeks preceding the booster vaccination. In only two dogs a t i t r e

of 40

was demonstrated to serotype canicola and in one dog to serotype i c t e r o haemorrhagiae. A f t e r the f i r s t

booster vaccination a sharp r i s e of the IgG t i t r e

was demonstrated: an eleven-fold rise of the mean a n t i - c a n i c o l a IgG t i t r e an e i g h t - f o l d r i s e of the mean anti-icterohaemorrhagiae IgG t i t r e .

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248

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Fig. 2. Antibody response to the icterohaemorrhagiae component of a bivalent bacterin as measured by the MAT and the IgM-, resp. IgG-specific ELISA in eight dogs after primary vaccination: f i r s t vaccination (1) and booster vaccination (II). . . . . . IgM; IgG; ...........MAT.

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Fig. 3. Antibody response to the canicola component of a bivalent bacterin as measured by the MAT and the IgM-, resp. IgG-specific ELISA in seven dogs a f t e r the 2nd annual revaccination (arrow). . . . . IgM; IgG;-" MAT.

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Fig. 4. Antibody response to the icterohaemorrhagiae component of a bivalent bacterin as measured by the MAT and the IgM-, resp. IgG-specific ELISA in seven dogs after the 2nd annual revaccination (arrow). . . . . . IgM, IgG; ~ M A T .

250

of the mean IgG t i t r e to both serotypes was attained one week after the booster vaccination. Thereafter the mean IgG t i t r e to both serotypes gradually declined to reach the lowest detection level 1 in 20 (1.3) at 4½ months and 6 months post-vaccination, respectively for serotypes canicola and icterohaemorrhagiae; however, the i in 40 (1.6) level (one d i l u t i o n step above the lowest detection level) was already reached within 10 weeks after the booster vaccination (both serotypes). The immune response of the individual dogs varied to some extent. The maximal IgG t i t r e varied from 40 to 320 (icterohaemorrhagiae)

and from 80 to 320

(canicola). In two dogs ("low responders") the IgG t i t r e declined to 20 within three weeks (icterohaemorrhagiae) and seven weeks (canicola) following the booster vaccination. After half a year in only two dogs an IgG t i t r e of 80 could s t i l l

be demonstrated to both serotypes. One year after the primary

vaccination a l l dogs had IgG t i t r e s ~ 4 0 . IgM response. The IgM response to both serotypes was of the primary response type after the f i r s t and booster vaccination. The mean IgM t i t r e to both serotypes attained i t s maximum one week after the f i r s t vaccination (varying from 160 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 within 10 weeks after the booster vaccinatiQn. MAT response. The mean MAT t i t r e pattern with respect to serotype icterohaemorrohagiae closely followed the mean IgM pattern and declined to the lowest detection level (1 in 20) within 10 weeks after the booster vaccination. The mean MAT t i t r e to serotype canicola did not exceed the lowest detection level for one year following primary vaccination. In only two dogs an anti-canicola MAT t i t r e ~ - 2 0 (80 to 160) was detected during the f i r s t two weeks after the f i r s t and booster vaccination. Annual revaccination The 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 r e to both serotype icterohaemorrhagiae and canicola to attain a maximum 2 to 3 weeks after the revaccination. In the individual dogs the maximum varied from 320 to 2560 to both serotypes, with the exception of one dog (probably a "low responder") which demonstrated a maximal IgG t i t r e of only 80 to serotype icterohaemorrhagiae and 40 to serotype canicola. Although the mean IgG t i t r e to both serotypes declined gradually after attaining the maximum, i t remained at a higher level than before the revaccination for at least one year. However, in

251

two dogs including the "low responder", the a n t i - c a n i c o l a IgG t i t r e decreased to~40

by the end of the year f o l l o w i n g the revaccination.

IgM response.

The IgM response to both serotypes was of the primary response

type as i t was a f t e r the f i r s t

and booster vaccination of the primary vaccinated

dogs. The mean IgM t i t r e attained i t s maximum also one week a f t e r the vaccinat i o n . The maximal IgM t i t r e varied from 80 to 320 with respect to both serotypes. The mean IgM t i t r e to both serotypes declined to a negative IgM level ( ~ 4 0 ) w i t h i n seven weeks a f t e r the revaccination. The low IgG responder showed a "normal" IgM response. MAT response.

The MAT response to serotype canicola closely resembled the

IgM response to the same serotype a t t a i n i n g a maximum two weeks a f t e r the revaccination. The mean MAT t i t r e declined to below the lowest detection level w i t h i n six weeks. The MAT response to serotype icterohaemorrhagiae was more pronounced, reached a maximum one week a f t e r the revaccination and only gradually declined t h e r e a f t e r . I t p a r a l l e l l e d the IgG response to serotype icterohaemorrhagiae to a certain extent. The maximal MAT t i t r e to serotype icterohaemorrhagiae varied from 160 to 640, whereas the maximal MAT t i t r e to serotype canicola varied from 20 (the same "low responder") to 160. DISCUSSION The immune responses to l e p t o s p i r a l bacterins have been d i f f i c u l t

to evaluate

using the MAT because the majority of the vaccinated animals develop e i t h e r a low a g g l u t i n a t i o n t i t r e or f a i l to develop any a g g l u t i n a t i o n t i t r e at a l l fo l l o wi n g vaccination. However, results of hamster protection test and challenge studies show that protective antibodies do develop a f t e r vaccination and persist a f t e r the a g g l u t i n a t i o 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 p r i n c i p a l class of antibody for p r o t e c t ion, whereas the IgM class of antibody is p r i m a r i l y associated with a g g l u t i n a t i o n (Hanson, 1973). Negi et al. (1971), using the hamster passive-protection t e s t , demonstrated that p r o t e c t i v e antibodies were present in both the IgM and IgG fraction of c a l f serum, being more prevalent, however, in the IgG f r a c t i o n . Hamster passive-protection tests are laborious and expensive and not suited for routine t e s t i n g of the humoral immune response a f t e r vaccination. Although Huhn et a l. (1975) have demonstrated the r e l a t i o n between the resistance induced in vaccinated dogs and that induced in vaccinated hamsters by a challenge two weeks a f t e r vaccination and Desmecht (1982) found that hamsters were protected f or at least 18 months a f t e r two vaccinations three weeks apart with 1/10 of the dose of a vaccine f o r dogs, hamster protection tests as prescribed in the U.S. Code of Federal Regulations and the European Pharmacopoeia in p r i n c i p l e provide no information concerning the duration of immunity in dogs.

252

A simple reproducible serological t e s t to measure the IgG response would facilitate

the evaluation of the humoral immune response a f t e r vaccination.

Tripathy et al.

(1973) described the use of the g r o w t h - i n h i b i t i o n test to detect

antibody to leptospires in c a t t l e vaccinated with l e p t o s p i r a l bacterins and found that the g r o w t h - i n h i b i t i n g a c t i v i t y was l a r g e l y associated with the IgG antibodies. The major disadvantage of t h i s test is that an incubation period of approximately two weeks is required before results can be read. Using our ELISA technique the r e s u l t s can be read w i t h i n f i v e hours. The r e s u l t s presented by Bey and Johnson (1978, 1983b), using the microscopic a g g l u t i n a t i o n t e s t and the l e p t o s p i r i c i d a l assay to monitor the humoral immune response of dogs a f t e r vaccination, were p a r t i a l l y i n c o n s i s t e n t with the results described in the present paper. They found that both IgM and IgG were produced in s i m i l a r amounts in vaccinated dogs over an observation period of two years. In our experiment, using the ELISA technique, a n t i - l e p t o s p i r a l

IgM could only be detected

f o r short periods of time f o l l o w i n g each vaccination, whereas a substantial production of a n t i - l e p t o s p i r a l first

IgG was not detected before one week a f t e r the

booster vaccination; the IgG t i t r e s being f a r more pronounced and of a

much longer duration a f t e r the annual revaccination. The fact that the IgG response more or less p a r a l l e l l e d the IgM response a f t e r the f i r s t

booster

vaccination is in agreement with the r e s u l t s of Bey and Johnson (1978, 1983b). However, IgM t i t r e s cannot q u a n t i t a t i v e l y be compared with IgG t i t r e s due to d i f f e r e n t a v i d i t i e s of both immunoglobulins. As protection is p r i m a r i l y associated with IgG antibodies and as a n t i - l e p t o s p i r a l IgG t i t r e s of maximal 80 could only be demonstrated for a l i m i t e d number of weeks a f t e r a single vaccination, as was demonstrated in four other dogs, primary vaccination of dogs against l e p t o s p i r o s i s should include a booster vaccination. The rapid decline of the IgG t i t r e

a f t e r the f i r s t

booster (Figures

I and 2) might r e s u l t in a too low level of humoral immunity and an increased risk of i n f e c t i o n . Revaccination before the end of the 3rd month a f t e r the first

booster could probably prevent t h i s r i s k of i n f e c t i o n . Annual revaccina-

t i o n , r e s u l t i n g in the production of r e l a t i v e l y high a n t i - l e p t o s p i r a l

IgG t i t r e s

p e r s i s t i n g f o r at least one year in the majority of the dogs, as shown in Figures 3 and 4, seems s a t i s f a c t o r y . As described f o r other bacterins (Hanson, 1973), the canicola component of the bacterin, used in t h i s experiment, did not stimulate a detectable MAT t i t r e ( ~ 2 0 ) in the majority of the dogs a f t e r the primary vaccination (+ booster), whereas the icterohaemorrhagiae component stimulated a MAT response s i m i l a r to the IgM response to that serotype as did the canicola component in the group annually revaccinated beagles (Figure 3). A g g l u t i n a t i o n t i t r e s to serotype canicola were considerably lower than a g g l u t i n a t i o n t i t r e s to serotype i c t e r o haemorrhagiae. Live serotype canicola antigen apparently cannot e a s i l y be

253

trapped by a g g l u t i n a t i n g antibodies (IgM) in spite of r e l a t i v e l y high a n t i leptospiral

IgM t i t r e s

(Figure i) as detected by the ELISA using the outer en-

velope antigen. A certain amount of 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 in varying antibody responses to vaccination, was demonstrated in the group of beagles used in the present vaccination experiment. This v a r i a t i o n could probably be masked when larger amounts of antigen were used in the vaccine (Huhn et a l . , 1975). CONCLUSIONS The ELISA technique detecting 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 proved

to be a very s u i t a b l e test to measure the humoral immune response in dogs a f t e r vaccination with a chemically inactivated leptospiral bacterin. Vaccination t i t r e s do not d i s t u r b the serodiagnosis of acute l e p t o s p i r o s i s because, as a r u l e , dogs s u f f e r i n g from acute leptospirosis demonstrate a relat i v e l y high a n t i - l e p t o s p i r a l l e p t o s p i r a l IgG t i t r e

IgM t i t r e

(Hartman et a l . ,

the time between the f i r s t

together with a low or negative a n t i 1984). Such t i t r e s were found only during

and the booster vaccination (in p r i m a r i l y vaccinated

dogs). The results of the present i n v e s t i g a t i o n suggest the necessity of a revision of the vaccination scheme. Addition of an adjuvant to the l e p t o s p i r a l bacterins could perhaps improve the degree and duration of the immune response as was demonstrated by Bey and Johnson (1982a). However, as the primary vaccination experiment was carried out in older dogs, the results of the present experiment are not necessarily applicable to puppies of circa three months of age. ACKNOWLEDGEMENTS The authors wish to express t h e i r gratitude to Mrs. G.M.S. van Dam-Knubben for e x c e l l e n t technical assistance. Prof. Dr. W.A. de Voogd van der Straaten for statistical

assistance and Drs. S.H. L o e f f l e r for l i n g u i s t i c advice. The s t a f f

of the Department of Small Animal Obstetrics and Gynecology, Veterinary Faculty, U n i v e r s i t y of Utrecht (head Prof. Dr. C.H.W. de Bois) is g r a t e f u l l y acknowledged for the permission to use the beagle colony for the vaccination experiment.

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Bey, R.F. and Johnson, R.C., 1982b. Immunogenicity and humoral and cell-mediated immune responses to leptospiral whole c e l l , outer envelope, and protoplasma cylinder vaccines in hamsters and dogs. Am.J.Vet. Res., 43: 835-840. Chang, S.L., 1947. Studies on Leptospira icterohaemorrhagiae. J . I n f . D i s . , 81: 28. Dalling, T. and Okell, C.C., 1926. Vaccine treatment of canine leptospiral jaundice. J.Path. Bact., 29: 131. Desmecht, M., 1982. Dur~e de l'immunit6 conferee par le vaccin de la leptospirose canine chez le hamster. Ann.M~d.V6t., 126: 55-58. Hanson, L.E., Tripathy, D.N. and K i l l i n g e r , A.H., 1972. Current status of leptospirosis immunization in swine and cattle. J.A.V.M.A., 161: 1235-1243. Hanson, L.E., 1973. Immunologic problems in bovine leptospirosis. J.A.V.M.A., 163: 919-921. Hartman, E.G., 1977. Leptospirose. Tijdschr. Diergeneesk., 102: 45-52. Hartman, E.G., van Houten, M., van der Donk, J.A. and Frik, J.F., 1984. Determination of specific anti-leptospira] immunoglobulins M and G in sera of experimentally infected dogs by solid-phase enzyme-linked immunosorbent assay. Vet. lmmunol.lmmunopathol., 7: 43-51. Heath, K.R. and Box, P.G., 1965. Immunity to leptospirosis: Antibodies in vaccinated and infected dogs. J.Comp.Pathol., 75: 127-136. Hood, L.E., Weissman, I.L. and Wood, W.B., 1978. Immunology. The Benjamin/ Cummings Publishing Company, Inc., Menlo Park, California. Huhn, R.C., Baldwin, C.D. and Cardella, M.A., 1975. Immunity to leptospirosis: Bacterins in dogs and hamsters. Am.J.Vet. Res., 36: 71-74. Michna, S.W., 1970. Leptospirosis. Vet. Rec., 86: 484-496. Negi, S.K., Myers, W.L. and Segre, D., 1971. Antibody response of cattle to L_eptospira pomona: Response as measured by hemagglutination, microscopic agglutination, and hamster protection tests. Am.J.Vet. Res., 32: 1915-1920. Ottosen, H.E., 1946. Om vaccination mod leptospirose hos hund. Medlemsbl. danske dylaegeforen., 29: 187-194. Pike, R.M., 1967. Antibody heterogeneity and serological reactiOns. Bacteriol.Rev., 31: 157-174. Pope, V., Hunter, E.F. and Feeley, J.C., 1982. Evaluation of the microenzymelinked immunosorbent assay with Treponema pa]lidum antigen. J.Clin.Microbiol. 15: 630-634. Tripathy, D.N., Smith, A.R. and Hanson, L.E., 1975. Immunoglobulins in cattle vaccinated with leptospiral bacterins. Am.J.Vet.Res., 36: 1735-1736. Turner, L.H., 1968. Leptospirosis II. Trans. R.Soc.TropoMed.Hyg., 62: 880-899. World Health Organization, 1967, Current problems in leptospirosis research. Wld. Hlth.Org.techn. Rep.Ser., 380.