Devlopment and Evaluation of Formalized killed Adjuvant Brucella melitensis biovar 3 Vaccine
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Date
2016-04-23
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U.P. Pandit Deen Dayal Upadhyaya Pashu-Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, (DUVASU), Mathura – 281001
Abstract
The present study was conducted to develop a stable, safe and effective vaccine against caprine brucellosis. For
that three well approved and recognized adjuvants viz., MontanideTM IMS, MontanideTM GEL 1 and
MontanideTM ISA61VG were incorporated with virulent Brucella melitensis biovars 3 IND1 (Accession no.
VTCCBAA228) bacterial strain, isolated from stomach content of aborted fetus of infected goat to develop three
different formalized killed adjuvant B. melitensis biovar 3 IND1 vaccines viz. NPV (Nano particle based
vaccine), PGV (Polymer gel based vaccine) and OAV (oil adjuvant based vaccine), respectively to make it
1.47X108 and 1.47X1010 CFU per shot in mice and goats vaccine. These vaccines were tested for sterility and
then safety in adult female inbred BALB/c mice and were kept at different temperature to assess its stability.
Sterile, safe and stable all three vaccines were inoculated 10μl vaccine with 1.47X108 CFU of Brucella in per
dose intra nasally (NPV & PGV), Subcutaneously (PGV) and intra muscularly (OAV) in adult female BALB/C
mice in the group of 10 for efficacy. 50% of mice were vaccinated with single vaccination where as remaining
50% mice were given booster on 21st day of initial vaccination. Vaccinated mice were challenged on 28th day of
vaccination and booster vaccination, respectively with live virulent B. melitensis biovar 3 cultures (109 CFU)
through I/P route and sacrificed on 7th day of challenge. During mice experiment, blood was collected at 7th, 14th
,
28th and 35th days of booster vaccination (28th, 35th, 49th and 56th day of first vaccination) for serum as well as
whole blood. The blood erythrocytes were used for the estimation of oxidative stress biomarker parameters,
plasma for plasma cytokine level whereas serum was used for the status of serum antibodies against Brucellosis
by RBPT and indirect ELISA. After sacrifice, mice organs were collected for live weight whereas spleen and
liver were also used for live Brucella count and molecular confirmation of B. melitensis by amplification of 16S
rRNA and Omp31 genes. Splenocytes proliferation and expression of cytokines in spleen by Real time PCR
were done. Analysis of all the parameters revealed all the vaccines produced efficacy as desired in OIE
guidelines and European pharmacopeia. The sterile and stable vaccines which were found to be safe and
effective in inbred BALB/c mice (NPV, PGV, OAV) were further used in homologous host (three pure bred
apparently healthy non pregnant Brucella free Jamunapari adult female goats in each group) with 1.47X1010
CFU per shot with intra nasal, subcutaneous and intra muscular routes, respectively and compared with standard
Rev.1 (IIL, Hyderabad). Vaccinated animals were subjected to blood collection on 0, 14th and 28th day of
vaccination for serum as well as whole blood. On 28th day, animals were challenged with live virulent B.
melitensis biovar 3 cultures (109 CFU) through subcutaneous route and monitored for physical, physiological
and other adverse reactions and blood samples were collected 14th, 28th, 60th and 90th days post challenge till
animals were sacrificed on 90th day of challenge. Serum separated from blood samples were used for detection
of serum antibodies by RBPT, STAT and indirect ELISA along with serum enzyme chemistry. The whole blood
was used for blood hematology, plasma for cytokine levels and estimation of oxidative biomarker parameters
was done in erythrocytes. The vital organs collected immediately after sacrifices were subjected to
histopathology to observe the changes produced by challenge, estimation of oxidative biomarker parameters,
live Brucella load in spleen and liver and molecular confirmation of B. melitensis by amplification of 16S rRNA
and Omp31 genes. The splenocyte proliferation and expression of TLR by Real time PCR in spleen, liver, supra
mammary lymph node and uterus tissues were also examined. On the basis of the findings of present study we
can conclude the following:
1. Brucella melitensis biovar 3IND1 can be used as a vaccine candidate for the control of caprine
Brucellosis in India.
2. Three vaccines developed with formalized killed Brucella melitensis biovar 3IND1and nano particle
(NPV), polymer gel (PGV) and oil adjuvant (OAV) were found stable for the duration of 12 months
under refrigeration temperature (4-8 °C).
3. Brucella melitensis biovar 3IND1based formalized killed vaccines (NPV, PGV and OAV) confer good
serological as well as cell mediated immune response in mice and goats.
4. Nano particle, polymer gel and oil adjuvant can be used as adjuvants to improve immunogenicity in
caprine.
5. The persistence of antibodies due to killed vaccination is for shorter duration in comparison to Rev.1 in
caprine.
6. The protection against virulent Brucella melitensis biovar 3IND1in goats vaccinated with killed
vaccines was comparable to Rev.1 for the duration of 90 days.
7. Among three killed vaccines attempted in present study, OAV revealed better efficacy and safety in
comparison to other two vaccines (NPV & PGV).
8. The OAV was followed by PGV in efficacy and safety parameters.
Based on laboratory findings as well as trial on homologous host OAV can be further recommended for field
trial.
Description
Development and Evaluation of Formalized killed Adjuvant Brucella melitensis biovar 3 Vaccine