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ThesisItem Open Access EVALUATION OF ANTI RABIES VACCINAL EFFICACY IN FREE RANGING DOG POPULATION IN BENGALURU(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2018-07) EVALUATION OF ANTI RABIES VACCINAL EFFICACY IN FREE RANGING DOG POPULATION IN BENGALURU; Dr. SHRIKRISHNA ISLOOR; (SHRIKRISHNA ISLOOR)The present study was undertaken to evaluate the anti rabies vaccinal efficacy in free ranging dog population in Bengaluru and comparison of an iELISA with RFFIT. Serum samples from 250 free ranging dogs were collected for the study and tested by RFFIT as well as iELISA. In all, 18 wards from the North and South zones of Bengaluru were covered during the sample collection with the help of three NGOs, who claimed that those animals were annually vaccinated. So, the post vaccinal efficacy was studied and it was found that,ThesisItem Open Access EVALUATION OF ANTI RABIES VACCINAL EFFICACY IN FREE RANGING DOG POPULATION IN BENGALURU(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2018-07) LEKSHMI J. DAS; Dr. SHRIKRISHNA ISLOORThe present study was undertaken to evaluate the anti rabies vaccinal efficacy in free ranging dog population in Bengaluru and comparison of an iELISA with RFFIT. Serum samples from 250 free ranging dogs were collected for the study and tested by RFFIT as well as iELISA. In all, 18 wards from the North and South zones of Bengaluru were covered during the sample collection with the help of three NGOs, who claimed that those animals were annually vaccinated. So, the post vaccinal efficacy was studied and it was found that, out of 250 dogs 125 were having a protective anti rabies antibody titre by RFFIT accounting for 50 per cent of seroconversion. Samples from North zone were having a better seroconversion level (65.97%) than south zone of Bengaluru. By iELISA, 126 dogs showed a per cent positivity (PP) more than 57.09 (cut off) accounting for 50.4 per cent of post vaccinal seThesisItem Open Access EVALUATION OF ANTI RABIES VACCINAL EFFICACY IN FREE RANGING DOG POPULATION IN BENGALURU(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2018-07) LEKSHMI J. DAS; Dr. SHRIKRISHNA ISLOORThe present study was undertaken to evaluate the anti rabies vaccinal efficacy in free ranging dog population in Bengaluru and comparison of an iELISA with RFFIT. Serum samples from 250 free ranging dogs were collected for the study and tested by RFFIT as well as iELISA. In all, 18 wards from the North and South zones of Bengaluru were covered during the sample collection with the help of three NGOs, who claimed that those animals were annually vaccinated. So, the post vaccinal efficacy was studied and it was found that, outThesisItem Open Access CONSTRUCTION OF Brucella melitensis GHOST AS A VACCINE CANDIDATE AGAINST BRUCELLOSIS(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDA, 2018-07) SUMATHI, B. R; B. M. VEEREGOWDAhe present study reports the usefulness B. melitensis ghost as a vaccine candidate against sheep and goat brucellosis. An overall seroprevalence of 36.36 per cent (200) by RBPT, 47.27 per cent (260) by c-ELISA and 36.36 per cent (200) by both the tests with highest seroprevalence of 56 per cent in Bengaluru rural for sheep and of 43 per cent in Kolar for goats was observed. On an isolation from aborted foetuses five Brucella spp. were recovered which were confirmed to be B. melitensis biotype 1 by biochemical tests, dye sensitivity tests, agglutination with monospecific serum and by genus specific, species specific and Bruce ladder PCR. The B. melitensis Rev 1 and B. melitensis field isolate ghosts were constructed at a cell concentration of 3.0 x 10 9 CFU/mL of culture with 80 μg/mL GI24 for complete ghost formation was confirmed by absence of growth on BSA, non amplification of specific bands in AMOS multiple PCR and morphological changes observed in scanning and transmission electron microscopy. All three vaccines, were evaluated in mice by IP and oral routes, for humoral immune response by quantifying IgG levels in post vaccinal sera and for cell mediated immune response by lymphocyte proliferation assay and cytokine profiles of IL-10, IL-4, TNF-α and IFN-γ. There was a significant difference in IgG levels between control and vaccinated groups. Further, within the vaccinated groups there was a significant (P<0.05) difference between Rev 1 vaccinated group from Rev 1 ghost and field ghost vaccinated groups in both IP and oral routes. The serum IgG levels were significantly higher in Rev 1 IP route vaccinated group than other groups. There was an interplay of both Th2 (IL-4 and IL-10) and Th1 cytokines (TNF–α and IFN-γ) in eliciting effective immune response, wherein initial response was dominated by Th2 through IL-4 and IL-10 which antagonize the TNF–α and IFN-γ cytokines whose role is very essential to initiate cell mediated immune responses. In later stages decreased levels of IL-4 and IL-10 resulted in surge of TNF–α and IFN-γ cytokines resulting in effective Th1 mediated cellular immune response which is required for effective clearance of Brucella organisms. The ghosts made from B. melitensis Rev 1 and field isolate performed equally well as B. melitensis Rev 1 by eliciting an effective immune response and protecting mice against B. melitensis 16M challenge.ThesisItem Open Access APPLICATION OF POLYMERASE CHAIN REACTION FOR DIAGNOSIS AND MOLECULAR EPIDEMIOLOGY OF ANTHRAX IN LIVESTOCK IN KARNATAKA SHASHIKALA, N.(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR – 585 401, 2018-01) SHASHIKALA, N.; Dr. SHRIKRISHNA ISLOORThe present study employed the microscopy and the PCR for the detection of B .anthracis by targeting virulence genes for protective antigen (PA) and capsule (CAP) located on two plasmids, pXO1 and pXO2 respectively. Further, Ba813 gene of B. anthracis and nhe gene of B. cereus were targeted for species specific identification. In all, 14 blood smears from 5 different locations (Bellary, Koppal, Davangere, Doddaballapura and Chamarajnagar) were subjected to Gram’s and Polychrome methylene blue (PMB) staining. Of these, 2 smears from Doddaballapura were negative for anthrax bacilli by both the staining techniques. These blood smears and the blood samples were also found negative by PCR. Further, out of 10 blood samples from Bellary, Koppal, Doddaballapura and Chamrajnagar, 5 were PCR positive. Out of 14 blood smears from Davanagere, Doddaballapura and Chamarajanagar, only 4 smears revealed PCR positivity. All the 5 ear peice samples collected from Bellary, Koppal and Tumkur were found negative by PCR. None of the 5 soil samples from Bellary, Koppal and Chamarajnagara were PCR positive. The B. cereus isolate showed amplification for both PA as well as nhe genes. Sequencing and phylogenetic analysis of the PA gene revealed that the PA gene sequences of the B. cereus were homologous to that of B. anthracis. This indicated close genetic relatedness between B. anthracis and B. cereus. The high genetic homology (98%) among B. anthracis isolates was revealed. The Ba813 and CAP gene based phylogenetic analysis indicated probable prevalence of two strains of B. anthracis in the outbreak of anthrax in BellaryThesisItem Open Access APPLICATION OF POLYMERASE CHAIN REACTION FOR DIAGNOSIS AND MOLECULAR EPIDEMIOLOGY OF ANTHRAX IN LIVESTOCK IN KARNATAKA SHASHIKALA, N.(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR – 585 401, 2018-01) SHASHIKALA, N.; Dr. SHRIKRISHNA ISLOORThe present study employed the microscopy and the PCR for the detection of B .anthracis by targeting virulence genes for protective antigen (PA) and capsule (CAP) located on two plasmids, pXO1 and pXO2 respectively. Further, Ba813 gene of B. anthracis and nhe gene of B. cereus were targeted for species specific identification. In all, 14 blood smears from 5 different locations (Bellary, Koppal, Davangere, Doddaballapura and Chamarajnagar) were subjected to Gram’s and Polychrome methylene blue (PMB) staining. Of these, 2 smears from Doddaballapura were negative for anthrax bacilli by both the staining techniques. These blood smears and the blood samples were also found negative by PCR. Further, out of 10 blood samples from Bellary, Koppal, Doddaballapura and Chamrajnagar, 5 were PCR positive. Out of 14 blood smears from Davanagere, Doddaballapura and Chamarajanagar, only 4 smears revealed PCR positivity. All the 5 ear peice samples collected from Bellary, Koppal and Tumkur were found negative by PCR. None of the 5 soil samples from Bellary, Koppal and Chamarajnagara were PCR positive. The B. cereus isolate showed amplification for both PA as well as nhe genes. Sequencing and phylogenetic analysis of the PA gene revealed that the PA gene sequences of the B. cereus were homologous to that of B. anthracis. This indicated close genetic relatedness between B. anthracis and B. cereus. The high genetic homology (98%) among B. anthracis isolates was revealed. The Ba813 and CAP gene based phylogenetic analysis indicated probable prevalence of two strains of B. anthracis in the outbreak of anthrax in Bellary.ThesisItem Open Access DEVELOPMENT AND EVALUATION OF INACTIVATED Mycobacterium avium subspecies paratuberculosis VACCINE IN SHEEP(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2018-07) RAMESHA, C. B.; Dr. D. RATHNAMMAThe present study was undertaken to evaluate autogenous inactivated MAP vaccine of sheep origin with inactivated MAP S5 vaccine of goat origin in Mandya breed of sheep. The clinical symptoms attributable to JD were not observed in any of the vaccinated animals, whereas unvaccinated animals showed progressive loss of body weight, emaciation, weakness, pasty diarrhoea and rough hair coat after 90 days post vaccination (DPV). In Lymphocyte transformation test, PBMCs from the vaccinated animals had higher stimulative index. In serum nitric oxide assay, more of serum nitric oxide was observed in vaccinated animals. In serum IFN γ assay, vaccinated animals had significantly higher level of IFN γ. All three groups of animals showed peak levels at 90 DPV and maintained up to 180 DPV. Indirect ELISA results revealed that vaccinated animals had significantly higher antibody levels. Both the vaccines successfully inhibited localization of MAP in the intestine and in turn faecal shedding of MAP was significantly low in vaccinated animals up to 300 days. The shedding of MAP increased steadily after 90 days post challenge in control animals. Vaccinated animals remained healthy and gained significantly higher body weights compared to control. Gross and microscopic lesions were not observed in vaccinated animals, control animals showed thickening and corrugation of small intestine and enlarged lymph nodes, infiltration of mononuclear and epitheloid cells in intestine and lymph nodes. None of the vaccinated animals were positive for AFB. Further 33.33 per cent unvaccinated challenged animals were positive for AFB. Whereas, 16.66 per cent vaccinated and 50 per cent unvaccinated animals were positive for MAP antigen by immunohistochemistry. The CD4+ and CD8+ T cells in lymph nodes by immunohistochemistry revealed high concentration of CD8+ cells in vaccinated animals. There was no significant difference in expression of CD4+ T cells. Both inactivated MAP vaccines had elicited cellular and humoral immune responses and effectively inhibited the localization of MAP.ThesisItem Open Access EVALUATION OF ANTI RABIES VACCINAL EFFICACY IN FREE RANGING DOG POPULATION IN BENGALURU(KARNATAKA VETERINARY, ANIMAL AND FISCHERIES SCIENCE UNIVERSITY, BIDAR, 2018-07) LEKSHMI J. DAS; Dr. SHRIKRISHNA ISLOORThe present study was undertaken to evaluate the anti rabies vaccinal efficacy in free ranging dog population in Bengaluru and comparison of an iELISA with RFFIT. Serum samples from 250 free ranging dogs were collected for the study and tested by RFFIT as well as iELISA. In all, 18 wards from the North and South zones of Bengaluru were covered during the sample collection with the help of three NGOs, who claimed that those animals were annually vaccinated. So, the post vaccinal efficacy was studied and it was found that, out of 250 dogs 125 were having a protective anti rabies antibody titre by RFFIT accounting for 50 per cent of seroconversion. Samples from North zone were having a better seroconversion level (65.97%) than south zone of Bengaluru. By iELISA, 126 dogs showed a per cent positivity (PP) more than 57.09 (cut off) accounting for 50.4 per cent of post vaccinal seroconversion. A kappa value of >0.80 suggested a perfect agreement between the results of RFFIT and iELISA. The sensitivity and specificity of iELISA was found to be 94.4 per cent and 95.2 per cent respectively. This ELISA can be used in large population surveys instead of RFFIT to overcome the disadvantages associated with it. The present study emphases regular rabies vaccination followed by seromonitoring of free ranging dogs in Bengaluru.ThesisItem Open Access DEVELOPMENT OF IN-HOUSE ELISA FOR THE DETECTION OF Mycobacterium avium subspecies paratuberculosis INFECTION IN ANIMALS(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCIES UNIVERSITY, BIDAR, 2017-10) PANNAGA, P; Dr. D. RATHNAMMA)The present study was under taken to develop an in-house ELISA using a local Mycobacterium avium subspecies paratuberculosis (MAP) isolate and to investigate the seroprevalance of Paratuberculosis in animals. The MAP protoplasmic antigen was extracted by sonication and quantified by ‘Nanodrop’ spectrophotometer and 4.193 mg/ml antigen was obtained. Extracted MAP antigen was characterized by SDS - PAGE and immune-blot analysis. The SDS-PAGE profile of MAP revealed 25, 35, 57, 72, 100 and 193 kDa proteins and immunoblotting revealed that 20, 29, 35, 45, 70 and 193 kDa as immunogenic proteins. ELISA was standardized with MAP antigen of 0.125 μg / well, serum dilution of 1:50 and Protein A-HRP conjugate dilution of 1:2500. 1032 serum samples from sheep, goat and cattle were screened for MAP infection with in-house ELISA and IDEXX ELISA kit. 113 (10.94 %) serum samples were positive by in-house ELISA and 111 (10.75 %) serum samples were positive by IDEXX ELISA kit. Cut off values were determined as SP ratio ≥ 80 per cent as Positive, ≤ 60 per cent as Negative and 60 - 80 per cent as suspected. Seroprevalence of MAP in sheep, goat and cattle was 8.52, 5.95 and 6.6 per cent respectively. The relative sensitivity and specificity of in house ELISA was 76.58 per cent and 96.96 per cent respectively with Kappa value of 0.7296 showing good agreement. Comparative evaluation of in-house ELISA with IDEXX ELISA kit was statistically analysed by Pearson’s correlation with significant correlation of 0.7725 (P=0.0001) between in-house ELISA and IDEXX ELISA kit. Receiver operating characteristics (ROC) curve was drawn with excellent area under curve of 0.992 was obtained. The study indicated the superiority of in-house indirect ELISA using native MAP antigen as against purified protoplasmic MAP antigens used in commercial ELISA kit in terms of cost effectiveness.
