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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access DEVELOPMENT OF A MODIFIED INACTIVATED VACCINE AND ITS COMBINATION WITH A RECOMBINANT PROTEIN AGAINST LEPTOSPIROSIS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-12-30) MANJU SOMAN; M.MiniThe study was taken up with an aim to develop a foolproof technique for prevention of leptospirosis. It involved the development and immunity evaluation of a modified whole cell inactivated vaccine incorporating the predominant leptospiral serovars, Australis, Autumnalis and Pomona in hamsters. The study also ascertained the genus specific immunoreactivity of a truncated recombinant leptospiral LigA protein and its combination with the modified inactivated vaccine, in hamsters. The immunomodulatory effect of incomplete Freund’s adjuvant (IFA) and the aluminium hydroxide gel adjuvant in hamsters was also assessed. Primers were designed for a highly immunodominant region of the ligA gene, spanning nucleotides from 1873 to 3363. The PCR amplified 1491 bp fragment of ligA DNA was cloned into pET -32a vector and expressed in E.coli BL21(DE3). The conditions optimum for expression of this recombinant protein were analysed. Maximum expression was obtained following induction with 2 mM IPTG, at an incubation temperature of 28o C following six hours of incubation at 200 rpm shaking speed. The Ni-NTA purified rLigA protein was used for immunization of hamsters. The optimum concentration of the rLigA protein and modified inactivated vaccine required for immunisation of hamsters, was determined by immunising four sets of hamsters with four different concentrations of the antigens, 14 days apart. It was revealed that the concentration of 80µg/ 40 µg of Lig A protein and 108 leptospires per millilitre gave the maximum IgG ELISA and MAT titres.Six vaccine groups were set up for six different vaccine combinations which included the modified inactivated vaccine, rLigA protein and a combination of the modified inactivated vaccine and rLigA protein. Adjuvants IFA and aluminium hydroxide were used in the study. The serum antibody titres on days 0, 7,14 and 27 were determined by MAT and recombinant IgG ELISA The virulence of laboratory strains of Leptospira interrogans serovars Pomona (homologous) and Icterohaemorrhagiae (heterologous) was enhanced by serial passage in hamsters and these were used as challenge organisms in the study. The LD50, of the serovars Pomona and Icterohaemorrhagiae, in hamsters was determined as 106.893 organisms and 107.38 organisms, respectively and the challenge was carried out with 100 LD50 (≈109 ) organisms, on 28th day post first immunization. Challenge studies revealed maximum protection levels of 80 to 100 per cent in groups immunised by modified inactivated vaccine alone and combination of rLigA and inactivated vaccine. Groups immunised with rLigA protein alone showed 60-70 per cent protection to both serovars. The highest MAT titres to homologous and heterologous serovars were presented by the groups immunized with a combination of rLigA protein and modified inactivated vaccine. These groups elicited higher MAT titres to heterologous serovar Icterohaemorrhagiae compared to whole cell vaccine alone, which indicated the genus specificity contributed by the partial rLigA protein. It also showed that the inactivated vaccine and recombinant protein compliment each other in increasing the respective immunogenicity. The study revealed that IFA adjuvanted rLigA protein could elicit the maximum ELISA titres in hamsters, followed by the group immunised by IFA adjuvanted rLigA protein combined with modified inactivated vaccine. The IFA adjuvanted vaccine groups showed higher ELISA titres compared to those adjuvanted with aluminium hydroxide but the aluminium hydroxide adjuvanted vaccine groups showed consistent increase in antibody titres.ThesisItem Open Access ASSESSMENT OF CELL MEDIATED AND HUMORAL IMMUNE RESPONSE TO SUBUNIT VACCINE AGAINST RIEMERELLOSIS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-12-30) RINSHA BALAN; Priya P. M.Riemerellosis is a bacterial disease among ducks, caused by Riemerella anatipestifer, which has been well documented as a cause of considerable economic loss to the duck production in Kerala. At least 21 serotypes of the organism have been identified globally. Since vaccination is the mainstay for the control of the disease, a research work was undertaken to prepare subunit vaccine employing recombinant OmpA of R. anatipestifer and assessment of cell mediated and humoral immune responses of the vaccine and also to evaluate the comparative efficacy with that of the developed inactivated vaccine. Broth culture of R. anatipestifer at a concentration of 2.5 OD values at 525 nm with a dose of 1 mL per bird subcutaneously was selected as LD50. L per bird subcutaneously was selected as LD50. A total of 52, day-old ducklings were divided into three treatment groups with ten birds each. They were injected with 0.5 mL of different types of vaccine subcutaneously. Group I (T1) served as control with 22 birds including six birds each for challenge control of inactivated and subunit vaccine. Group II (T2) was injected with an inactivated vaccine (7x109 cfu/mL), which was prepared as per the protocol standardised in the Department of Veterinary Microbiology and group III (T3) and group IV (T4) were administrated with different antigen concentration of subunit vaccine (equal quantity of the rompA protein (250µg and 500µg) and montanide, respectively). A booster dose was given at third week post-primary vaccination to T2, T3 and T4. It was observed that, by using both crude Omp and rOmpA based ELISA, inactivated vaccine birds (T2) produced higher antibody titre during early age while in the subunit vaccine group, the titre was higher during later stage. An early antibody response is required to lower the mortality rate in riemerellosis as the organism targets young ducklings. Thus, it could be inferred from this study that inactivated vaccine was more effective than subunit vaccine A significant CMI response was also shown by inactivated vaccine groups on 14th and 28th day post-vaccination by lymphocyte proliferation assay (LPA). Challenge studies to assess the protective response revealed 100 per cent protection for inactivated vaccine group (T2), 80 per cent protection for T4 group and 70 percent protection for T3 group. All the vaccinated birds were having significantly less gross lesion when compared to the challenge control groups. On analysing the cytokine mRNA expression levels using real-time PCR, the inactivated vaccine group showed significantly higher (p< 0.05) mRNA levels of IL-6, IL-12B and IFN-γ gene on day 28 than the two subunit vaccine groups. It was found that the inactivated vaccine was superior in terms of results obtained from the challenge study, antibody titre, CMI response and gene expression analysis than the subunit one. Hence, it is desirable to advocate the use of inactivated vaccine in field condition owing to its easiness to prepare and low cost.ThesisItem Open Access PRODUCTION AND APPLICATION OF MONOCLONAL ANTIBODIES AGAINST DUCK PLAGUE VIRUS(College of Veterinary and animal Science,Mannuthy, 1999) RAVINDRA DATTATRAYA PADALKAR; V. JayaprakasanMonocloiMl iinlibodies (Mabs) were raised against the vaccine strain of DPV and three strains of DPV Mz, Vaccine (OPV^V). IVRl (DPV-1) and Alleppy strain (DPV-A) were used to raise polyclonal serum in tite present investigation. DPV-V was revived in 11 day old chicken enibiyo and the enibrjo deatli was recorded four to five days PI with congestion all over the body and spleen and nccrotic foci in liver. The cytopathy in CEF cell culture observed was rounding and clumping of the cells, syncytium formation and bridge fonriation with extensive vacuolation m the Cytoplasm. The detachment of the cells was observed at 120 h PI. 1)PV-1 a virulent strain was inocnlaied in the ducklings, death was recorded in all the inoculated birds with extensive hemorrhages on serous membranes, muscles and visceral organs. Nccrotic foci on li\ei, enlargement and congestion of liver and spleen, and white necrotic foci m the gizzard were evident. The virus was further passaged in DDE and cultivated in bulk in DEP cell culture. The Dl'V-V and Dl'V-A were titrated in Clil' cell cultnie and the TCin,o was 4.7 X 10' per nd of the inoeulnm lor DPV-V and 3.2 X lO' for I)PV-A. ni'V-i cultivated in Did' cell culture had a IC 11),,i of the inoculum All the strains were partially purified at 100000 g for 4.5 h at 4" C in Heekman ultra centrifuge and the protein concentration of the virus was estimated by biuret method and was found to he 1 1 mg. 8 mg and 7 mg for DPV-V, A and I respectively. All the three strains of DPV were inoculated in mice to raise polyclonal serum. Four mice out of five inoculated with DPV-V showed FJdSA titres more than 1:12800, one mouse showed a litre of 1:6400. The mice inoculated witi* DPV-A showed a litre of more than 1:12800 and those inoculated with DPV-I, 1:6400 FddSA was used to test the sera samples of the miee inoculated with DPV strains. The test was found to be highly sensitive, easy to perlorm and less time consuming. The test therefore can be recommended for routine diagnosis of DPV I'olyclonal seiuin was used to study ll'.c cross reactivitv of the three strains oC I)PV with FIJSA. DI'V-V polyclonal scrum reacted with the liomologous virus with a titre of 1:12800. It also showed similar titer with other two heterologous strains. I'olyclonal serum raised against DI'V-A had a titre of 1:12800 with homologous and hcterologuus strains of DI'V. DPV-I reacted with Immnhigous strain at a titre uf 1:6400 Similar titrcs were observed with hertologous strains. Immiino pero.xidase test was used for the detection of the tissue antigens in DPV infected CEF monolayers and in liver and spleen sections. Polyclonal serum raised against DPV-V detected homologous virus in the CF.F monolayers and hetrologous virus in the liver and spleen sections. The staining reaction was observed as dark brown deposits at the virus localization. However background tissue was also stained brown to faint yellow in the stained preparation. The virus neutralization test was used to study the cross neutralization by employing polyclonal .serum raised against the three strains of DPV. Polyclonal serurTi raised against DPV-V showed a VN F of 64 with a VNl of 1.8 with homologous virus and a VN F 45 with VNI 1.65 with other two strains of DPV. Polyclonal serum raised against DPV-A neutralized the homologous virus at a titre of 32 with a VNl of 1.5. it also neutnrlized the vacciiic strain nf DI'V with same VNT and VNI, However it neutralized DPV-1 with VN I' 24 and VNI 1.35. Dl'V-i poiyclonal serum neutralized the homologous virus at a titre of 45 with a VNI of 1.65 . I he s.iiue neutralized DPV-V and DPV-A with a VN T 32 and VNI 1.5.ThesisItem Open Access ASSESSMENT OF IMMUNITY TO DUCK PLAGUE VIRUS (DUCK VIRUS ENTERlTlS) ON VACCINATION(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 1993) DIWAKAR DATTATRAYRAO KULKARNI; P.c. JamesDuring 1991, six outbreaks clinically duck plague (DP) with 33 per cent morbidity mortality were investigated. Duck plague from each outbreak. The isolates were able lesions and death of the duck embryos but fai chicken embryos during initial passages. suspected to be and 26 per cent was isolated to produce the led to kill the One of the strain, named DP-S was par by 10 passages in chicken embryos following duck embryos. Though the attenuated strain did pathogenicity index was reduced from 1.9 to 1. DP-S under transmission electron microscope of herpes virus morphology. Two DP vaccines - commercial vaccine vaccine having virus titres 0.74 and 3.5 respectively, were separately inoculated into ducklings respectively, two groups receiving two receiving double dose of corresponding interval of four weeks. Another group of duckl as control without vaccination. tially attenuated 20 passages in kill ducks, its 23. The isolate revealed virions and lab-adapted log 10 ELD 50/ml four groups of single dose and vaccines at an ings was kept (ii) challenged with Thre e ducks in each group were virulent DPV at four,eight and 20 weeks post-vaccination. The birds in all the five groups were screened at regular intervals for studying the immune response by virus neutralization (VN), leucocyte migration-inhibition (LMI) and passive haemagglutination (PHA) test. The challenged and survived birds were the carrier status of DPV by examination of their for virus isolation. In an organized farm, 180 ducks were gi vaccine at one year of age and were scre antibodies, LMI response and PHA titres before post-vaccination. Randomly selected two birds six weeks post-vaccination. The findings of the study are briefly l Six duck plague outbreaks were investigat isolated, and characterized. It was in duck and chicken embryos. partially The commercial vaccine could elicit very response as compared to laboratory adapted immunity could not last long even upto ei single vaccination and 20 weeks in double screened for rectal swabs ven commercial ened for VN and eight weeks were challenged isted as under: ed, the virus attenuated poor immune vaccine. The ght weeks in vaccination. (iii) * Single vaccination is not effective as c vaccination given four weeks apart. The assessment of antibody-mediated (AMI) and cell mediate d (CMI) immune responses indicated that both and CMI are involved in protection of ducks against duck plague. The vaccinated or vaccinated and infected show carrier status as attempts to isolate rectal swabs collected after vaccination were unsuccessful. The PHA has been standardized for diagnosis of duck plague.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF Pasteurella multocida ISOLATE^ FROM DUCKS IN KERALA(College of Veterinary and Animal Sciences, Mannuthy., 2004) P. X. ANTONY; Dr.J G. Krishnan NairTwenty-five isolates from ducks and one from fow P. multocida using standard bacteriological procedures. strain (LKO) obtained from IVR1 was used for comparisor were found to be pathogenic for mice. Variation in fe dulcitol, mannitol, sorbitol and trehalose allowed the 27 into ten biovars. Two biotypes P. multocida subsp. septlca subsp. multocida were observed among the avian examined uniformly sensitive to enrofloxacin, chloramphenicol isolates from ducks and one from fowl have been sero specific PCR assay was used to confirm the identity of PCR on template DNA prepared from blood smears was fo| rapid method of diagnosis for pasteurella. Dtyped the could N o polymorphism within the KMT1 gene restriction analysis of PM-PCR product with Hae III analysis of genomic DNA of all the avian isolates w revealed three profiles each. The fowl isolates had a majority of the duck isolates. prof le were characterized as A reference chicken . All the avian isolates •mentation patterns of isolates to be grouped , and P. multocida All the isolates were ind pefloxacin. Eight as A:l. A species isolates. Performing und to be an extremely be demonstrated by Restriction endonucleases itjh Hpa II and Hha I that was common to Eighty-eight per cent of the isolates carried p profiles were observed among the isolates examined. showed a single REP-PCR profile indicating a high level them. asmids. Two plasmid All the avian isolates of homogeneity among avi£.n Th e outer membrane protein profiles of all the Two protein fractions with molecular weights of 37.1f identified as the major OMPs by SDS-PAGE. isolates were similar. and 26.36 kDa were A 37.15-kDa protein was identified the major of avian isolates as of P. multocida by Western blotting a molecular mass of 31.33 and 26.36 kDa were also this technique. antige Tw found gene The OmpH gene of all the avian isolates as well were amplified using primers designed based on sequence of a chicken isolate of P. multocida. The am with restriction endonucleases Dra I and Hinf I fragments respectively. The similarity of the profiles for; P. multocida, suggested a high degree of homogeneity region. Restriction analysis of the OmpH-PCR products and B:2 with Dra I generated profiles which were distinct three serotypes. This technique can be helpful for serotypes of P. multocida. Studies on the OmpH gene( cholera vaccine revealed similarity with the amplified re isolate of P. multocida. Since this gene codes for the maj P. multocida the vaccine can be expected to confer immu against pasteurellosis. serotypes A:3 and B:2 blished OmpH gene plified product digested •ated four and three the avian isolates of amongst the amplified The sequence the OmpH-PCR product has GenBank and has been assigned the accession No showed 98 per cent identity with P. multocida strain X membrane protein (OmpH) gene. lie fraction of OMPs o other proteins with to be antigenic using )f serotypes A:l, A:3 rom each other for the erentiation of various s) of inactivated fowl non of the local duck sr antigenic fraction of -lily to ducks in Kerala bjeen submitted to the AY 506823. The sequence 73 (serotype A:l) outer
