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Veterinary Immunology and Immunopathology 159 (2014) 11–15

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

j ourna l h omepa ge: www.elsev ier .com/ locate /vet imm

esearch paper

omparision of efficacy of two experimental bovineeptospira vaccines under laboratory and field

. Balakrishnan, Parimal Roy ∗

epartment of Veterinary Microbiology, Madras Veterinary College, Tamilnadu Veterinary and Animal Sciences University,hennai 600007, Tamil Nadu, India

a r t i c l e i n f o

rticle history:eceived 16 October 2013eceived in revised form 27 January 2014ccepted 3 March 2014

eywords:eptospiraaccinesovinesdjuvants

a b s t r a c t

Two different inactivated vaccines for bovine leptospirosis were prepared with fivedifferent Leptospira serovars namely australis, ballum, hardjo, hebdomadis and pomona andadjuvanted with Montanide ISA 206 (Vaccine-I) or adjuvanted with Aluminium hydroxidegel (Vaccine-II) to evaluate the efficacy of the vaccines. The immunogenicity of the vaccineswas studied in rabbits under experimental condition and in cattle under field conditionfor a period of 180 days. Antibody response against five different leptospira serovarsranged from 6.84 log2 to 9.64 log2 (GM-MAT titres) in rabbits at 180 days after vaccinationwith vaccine I, whereas in vaccine II, the titre value ranged from 5.64 log2 to 7.44 log2.In the case of cattle under field condition, vaccine I showed GM-MAT titres of 6.84 log2

to 7.69 log2 against five different serovars at 150 days following vaccination. Such titres

were maintained following vaccination with vaccine II for 120 days only. It is concludedthat vaccine I is a better vaccine than vaccine II. However both the vaccines showed highimmune response of 5.64 log2 at six months of immunization. Vaccination did not causeany adverse reaction in the vaccinated cattle.

. Introduction

Leptospirosis is a zoonotic disease of a variety of domes-icated and wild animals including cattle. Leptospirosis inattle is characterized by abortion, jaundice, mastitis andubclinical infection (Balakrishnan et al., 2009; Thiermann,984). The disease can be controlled by vaccination andhe purpose of immunization of domesticated food andreeding animals is to protect them from Leptospirosiso maximize the productivity (Bey and Johnson, 1986).onsidering wide host range and complex epizootiology,

erovar specific vaccine is recommended (Bey and Johnson,986; Chen, 1986). Vaccine has been considered to be aost effective agent to control communicable diseases and

∗ Corresponding author. Tel.: +914425551581; fax: +914425551577.E-mail addresses: roy@tanuvas.org.in, parimalroy@hotmail.com

P. Roy).

http://dx.doi.org/10.1016/j.vetimm.2014.03.002165-2427/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

proved to be very effective in the control of Leptospirosisin most developed countries (Faine, 1983). But very oftenit is difficult because of non availability of vaccine withlocally prevalent serovars. Recently a sero-surveillance wasconducted using 3087 cattle and 518 buffaloes in differentparts of India and prevalence of serovars australis (10.77%),ballum (10.93%), hardjo (33.17%), hebdomadis (32.27%)and pomona (12.85%) were observed (Balakrishnan et al.,2011a,b). In the present study, considering the endemicsituation, two pentavalent inactivated vaccines were pre-pared with different adjuvant and their efficacies werecompared under laboratory and field condition.

2. Methods

2.1. Vaccine-1

Culture of Leptospira serovars australis (2 × 109/ml),ballum (2 × 109/ml), hardjo(2 × 109/ml), hebdomadis

nology a

12 G. Balakrishnan, P. Roy / Veterinary Immu

(2 × 109/ml) and pomona (2 × 109/ml) were inactivatedwith 0.4% formalin and adjuvanted with Montanide ISA206 (Seppic, France). Montanide ISA 206 is a commerciallyavailable (Seppic, France) vegetable oil adjuvant whichprovide double emulsion effect (water in oil in water)to provide immunity for longer duration. Leptospireswere grown in low protein (0.2% BSA) EMJH medium. Theinactivated and adjuvanted whole bacterial cells wereused as vaccine.

2.2. Vaccine-II

Culture of Leptospira serovars australis (2 × 109/ml),ballum (2 × 109/ml), hardjo (2 × 109/ml), hebdomadis(2 × 109/ml) and pomona (2 × 109/ml) were inactivatedwith 0.4% formalin and adjuvanted with aluminiumhydroxide gel. Leptospires were grown in low protein(0.2% BSA) EMJH medium. The inactivated and adjuvantedwhole bacterial cells were used as vaccine.

2.3. Potency test

Potency testing of both vaccines I and II were done inguinea pigs following OIE (2008). After 14 days of vacci-nation of 50 guinea pigs with vaccine I, 10 guinea pigseach were challenged intraperitonially with five differ-ent serovars namely australis, ballum, hardjo, hebdomadisand pomona with predetermined challenged dose of 104

ID50. Challenged organisms were prepared by repeatedpassage in guinea pigs (Balakrishnan, 2009). Control unvac-cinated 50 guinea pigs were challenged in same way.Similarly, potency testing was done for vaccine II. Mor-tality, and sero-conversion was recorded and live guineapigs were sacrificed humanly at 20 days post inocula-tion and examined for presence of leptospira organismsin liver and kidney samples and sero-conversion by MAT(Balakrishnan, 2009).

2.4. Immunogenicity in rabbits

Fifteen sero-negative rabbits (4 weeks old) wereselected for the study; they were maintained in experi-mental house with feed and water ad libitum. Rabbits weredivided into three groups. Group I, consisted of 5 rabbits,vaccinated subcutaneously with 0.5 ml of the vaccine I onday 0 and day 30. Group II consisted of 5 rabbits, vacci-nated subcutaneously with 0.5 ml of the vaccine II on day0 and day 30. Group III consisted of 5 rabbits administeredsubcutaneously with 0.5 ml of low protein medium (EMJHcontaining 0.5% BSA) only on day 0 and day 30. Blood sam-ples were collected on day 0 (before the first vaccination),day 3, day 7, day 30, day 60, day 90, day 120, day 150 dayand 180 to assess antibody levels against different serovarsby microscopic agglutination test (MAT).

2.5. Immunogenicity in cattle

A field trial in HF-Jersey crossbred cows in a privatedairy farm was conducted. Sero-negative sixty crossbredcows were selected for the study. They were divided into

nd Immunopathology 159 (2014) 11–15

three groups. Group I, consisted of 20 cows, vaccinated sub-cutaneously with 2 ml of the vaccine I on day 0 and day 30.Group II consisted of 20 cows, vaccinated subcutaneouslywith 2 ml of the vaccine II on day 0 and day 30. Group III,consisted of 20 cows as unvaccinated controls. Blood sam-ples were collected on day 0 (before the first vaccination),day 3, day 7, day 30, day 60, day 90, day 120, day 150 day180 to assess antibody levels against different serovars byMAT.

2.6. Microscopic agglutination test (MAT)

Antigen for MAT was prepared in liquid culture asdescribed earlier (Balakrishnan et al., 2011a,b; OIE, 2008).A 5–8 day old liquid culture containing density of 2 × 108

leptospires per ml was used as antigen for MAT. The anti-gens were prepared individually using Leptospira serovarsaustralis, ballum, hardjo, hebdomadis and pomona.

Standard MAT was performed for the assessment ofagglutination in serial two-fold serum dilutions in ‘U’bottom 96 well microtitration plates as described ear-lier (Balakrishnan et al., 2011a,b). In short, 20 �l of PBSwas added in all the wells. In the first well of each row,20 �l of 1:25 diluted (initially diluted in PBS in a sepa-rate deep well dilution plate) serum samples were added,mixed well and equal volume (20 �l) was serially trans-ferred upto 12 wells. From 12th well 20 �l was discarded.For antigen control, only PBS 25 �l was added to whichrespective antigen was added. A constant volume of 20 �lof the respective Leptospira antigen (2 × 108 per ml) wasadded in each row incubated at 37 ◦C for 2 h. Each serasample was tested against 5 different antigens. A drop(5 �l) of mixture from final dilution (50, 100, 200, 400,800, 1600, 3200, 6400, 12,800, 25,600, 51,200, 102,400)was placed on grease-free slide and the wet preparationwithout cover slip was screened using 20× objective ofthe dark field microscope (M/s. Nikon, 200E Japan) and theresults recorded for the presence of agglutination and/orreduction in number of organism in comparison with therespective antigen control. A 50% reduction in the num-ber of free leptospires in the test sample comparable withthe respective antigen control was considered positive withor without agglutination was recorded as the respectivetitre.

3. Results

3.1. Potency test

These was no adverse reaction or clinical signs in all thevaccinated guinea pigs and geometric mean MAT antibodytitre in vaccine I inoculated group at 20 days after challengeranged between 7.44 log2 and 9.14 log2 but vaccine II inoc-ulated group the corresponding titre values were between7.04 log2 and 8.24 log2. Unvaccinated control guinea pigschallenged with serovars australis, ballum and pomona died

within 7–10 days after inoculation and leptospira orga-nisms could be detected in tissues. Unvaccinated guineapigs that were challenged with hardjo and hebdomadis didnot cause any death but geometric mean MAT antibody

nology and Immunopathology 159 (2014) 11–15 13

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itre at 20 days post challenge was 7.94 log2 and 8.04 log2gainst hardjo and hebdomadis, respectively.

.2. Immunogenicity of the vaccines in rabbits underaboratory condition

The antibody response observed in this study isxpressed in geometric mean MAT titre (log2 base). Theabbits in the group I showed seroconversion to australis7.24 log2), ballum (5.84 log2 log2), hardjo (6.44 log2), heb-omadis (7.24 log2) and pomona (7.84 log2) in response toaccine I by day 7 (Table 1). The peak antibody titres inroup I was observed against australis (11.44 log2), ballum9.84 log2), hardjo (10.4 log2) and hebdomadis (10.4 log2)nd pomona (11.4 log2) on day 60. The titres valuesgainst australis, ballum, hardjo, hebdomadis and pomonaave fallen to 8.04 log2, 6.84 log2, 7.64 log2, 8.24 log2 and.64 log2 respectively by day 180 of the observation period.

The rabbits in the group II showed seroconversion toustralis (6.64 log2), ballum (5.84 log2), hardjo (6.04 log2),ebdomadis (6.04 log2) and pomona (6.24 log2) in responseo vaccine II by day 7 (Table 1). In group II, the peak titresgainst ballum (7.44 log2), hardjo (7.64 log2), hebdomadis7.84 log2) were noticed on day 60. In the case of australis9.04 log2) and pomona (8.64 log2), the peak titres wereound on day 90. The titres values have declined gradu-lly to 7.44 log2, 5.64 log2, 5.64 log2, 6.64 log2 and 7.24 log2gainst australis, ballum, hardjo, hebdomadis and pomona onay 180 of observation period. The rabbits in the group IIIemain seronegative throughout the observation period of80 days.

.3. Immunogenicity of the vaccines in cattle under fieldondition

The antibody response observed in this study isxpressed in geometric mean MAT titre (log2 base). Theows of group I showed seroconversion to all the 5 serovarsamely australis (5.84 log2), ballum (5.64 log2), hardjo5.79 log2), hebdomadis (6.24 log2) and pomona (6.29 log2)n day 7 in response to vaccine I (Table 2). The peakitres were observed against hardjo (7.24 log2), hebdomadis7.54 log2) and pomona (7.59 log2) component of the vac-ine I in group I on day 90. Whereas the peak titres againstustralis (7.99 log2) and ballum (7.64 log2) were observedn day 120. The titres values gradually declined to 7.44 log2,.84 log2, 5.84 log2, 7.04 log2 and 7.04 log2 for against aus-ralis, ballum, hardjo, hebdomadis and pomona on day 180f observation period.

The group II which received vaccine II, showed serocon-ersion to the serovars namely australis (5.74 log2), ballum5.49 log2), hardjo (5.79 log2), hebdomadis (5.84 log2) andomona (5.84 log2) on day 7 (Table 2). The maximum anti-ody response in group II against ballum (6.69 log2) hardjo6.84 log2), hebdomadis (7.19 log2) and pomona (7.19 log2)ere noticed on day 90 and for australis (7.64 log2) on day

20. The titres values were declined to 7.44 log2, 5.64 log2,.64 log2, 6.64 log2 and 6.89 log2 against australis, ballum,ardjo, hebdomadis and pomona respectively on day 180 ofhe observation period. The cows in the group III remain Ta

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4

6.89

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0

6.29

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7.34

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6

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6

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490

7.74

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3

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0

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6

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nd Immunopathology 159 (2014) 11–15

sero-negative throughout the observation period of 180days.

4. Discussion

4.1. Potency test

In the potency test, there was 100% protection andactive as observed for 20 days in guinea pigs in both thevaccinated groups. There was 100% mortality in the con-trols challenged with australis, ballum, and pomona andthis observation concur with the description of OIE (2008).But challenge with serovars hardjo and hebdomadis didnot cause death of the unvaccinated controls animals butinduce specific antibody titre. It is known that the serovarsthat are not lethal to hamster or guinea pig, such as hardjo,potency is measured by induction of a suitable antibodytitre (OIE, 2008). Both the vaccine fulfilled the criteria asdescribed in OIE (2008) but efficacy was compared sero-logically.

4.2. Immunogenicity of the vaccines in rabbits underlaboratory condition

The vaccines prepared in this study (vaccine I and vac-cine II) were inoculated into rabbits to assess the antibodyresponse for a period of 180 days. Both the vaccines induceddetectable level of antibody response against all the fiveserovars on day 7 which indicated the satisfactory levelof early immune response (Table 1). The GM-MAT titrevalues following vaccine I was always higher comparedto vaccine II. The antibody response on day 30 to day 90for australis and day 30 to day 180 against hardjo, heb-domadis and pomona were more in vaccine I compared tovaccine II and the results were statistically highly signifi-cant (P ≥ 0.001). The duration of the immunity in rabbits forboth vaccines was found to be more than 180 days of obser-vation period. However based on seroconversion it wasobserved that vaccine I is better than vaccine II since vac-cine I always showed higher immune response (Table 1).The variation in efficacy is certainly due to the effect ofadjuvant (Montanide ISA 206) which evoked immunity forlonger duration.

4.3. Immunogenicity of the vaccines in cattle under fieldcondition

The antibody response of the vaccines prepared in thisstudy was also assessed in cattle under field condition fora period of 180 days. For, both the vaccines, first vacci-nation was done at about four month old animals andbooster immunization was done four weeks later as sug-gested by several earlier workers (Bey and Johnson, 1986;Palit et al., 1996; Samina et al., 1997). Vaccination did notcause any adverse reaction in the vaccinated cattle. Therewas no drop in milk production or swelling at the siteof vaccination or rise in body temperature in post vacci-

nated animals. Vaccine I induced peak titres of 7.24 log2to 7.99 log2 against different five serovars by 90 to 120days, while in comparison vaccine II induced peak titresof 6.69 log2 to 7.64 log2 by 90 to 120 days. As per OIE

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G. Balakrishnan, P. Roy / Veterinary Immu

2008) a good vaccine should provide immunity for sixonths. Both the vaccines in the present study showed

ood immune response as detected by MAT titre values.M-MAT titre values of as high as 5.64 log2 to 7.44 log2ere obtained after six months of vaccination. As described

arlier Tripathy et al. (1976), a good vaccine should evokeAT titre of 10 to 100 and that is considered as good indica-

or of potency. In an earlier study Stalheim (1971), observedrotection in cattle when the animal had MAT titre of 10.onsidering the above description both the vaccines were

ound good and provided satisfactory immunity for a longereriod. Earlier antibody titres of 1:100 or above providedatisfactory immunization (Faine, 1983). When vaccine Ind vaccine II were compared, vaccine I (Montanide adju-anted) showed geometric mean antibody (MAT) titre ofore than 6.64 log2 (≥1:100) upto 150 days. But such val-

es were observed in vaccine II for only upto 120 days ofaccination. Hence it is concluded that vaccine I is betterhan vaccine II. The superiority of vaccine I is due to itsdjuvant which provided double emulsion effects to evokemmunity for longer duration.

eferences

alakrishnan, G., 2009. Development of experimental inactivated vaccinefor bovine leptospirosis. In: Ph.D. Thesis. Tamilnadu Veterinary andAnimal Sciences University, Chennai, India.

nd Immunopathology 159 (2014) 11–15 15

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Balakrishnan, G., Roy, Parimal Roy, Govindarajan, R., Ramaswamy, V.,Murali Manohar, B., 2011b. Seroepidemiological studies on leptospi-rosis among bovines in an organized farm. Int. J. Agro Vet. Med. Sci.,511–519.

Bey, R.F., Johnson, R.C., 1986. Current status of leptospiral vaccines. Prog.Vet. Microbiol. 2, 175–197.

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