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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 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 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.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. RATHNAMMAThe 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.ThesisItem Open Access DEVELOPMENT OF IN-HOUSE ELISA AND ITS COMPARATIVE EVALUATION WITH RFFIT FOR ESTIMATION OF ANTI-RABIES VACCINAL ANTIBODIES IN DOGS(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCIES UNIVERSITY, BIDAR, 2017-10) A. K. SANTOSH; Dr. SHRIKRISHNA ISLOORThe study was undertaken to develop in-house ELISA and it’s comparision with RFFIT. The recombinant pETRVL-G plasmid having G gene of Dr. Largi’s strain of rabies virus was cloned into pFastBacTM1 vector in DH5α E.coli. The recombinant pFastBacTM1 was confirmed by colony PCR and RE digestion (EcoR I & Sal I) and transposed into DH10 Bac. Recombinant bacmid was selected based on blue-white colonies and confirmed by PCR. Further, the DNA extracted and transformed into Sf-21 cells. The resultant recombinant baculovirus were used to obtain P2 and P3 stock virus and P3 stock used for expression. The expressed recombinant rabies virus G protein was analysed by SDS-PAGE, western blot and doublet bands of 62 and 59 kDa were observed. The crude lysate of baculovirus infected Sf-21 cells was used in ELISA and optimised to arrive at 500ng/100 µl of antigen, 1:100 dilution of serum and 1:15,000 dilution of anti-dog HRPO conjugate. The serum samples from vaccinated dogs (n=247) were tested by RFFIT and in-house ELISA. By RFFIT, 212 serum samples were found to have protective neutralising antibody titre accounting for 85.82 per cent protection. By in-house ELISA , 199 serum samples were found reactive, showing per cent, positivity (PP) more than 36.29 as cut off accounting for 80.56 per cent protection. The diagnostic sensitivity and specificity of in-house ELISA was 90.56 and 80 per cent, respectively with the kappa value of 0.614 revealing good agreement. Usage of purified recombinant rabies virus G protein may be suitable for in-house ELISA in sero-monitoring of antirabies virus neutralising antibodies.
