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20176
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Subject: Veterinary Microbiology × End date: 2017 × Start date: 2017 × Type: Thesis ×
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    ThesisItemOpen 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.
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    ThesisItemOpen 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.
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    ThesisItemOpen 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 ISLOOR
    The 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.
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    ThesisItemOpen Access
    BACTERIOLOGICAL STUDY OF WATER FROM MAJOR WATER RESOURCES OF BANDIPUR AND NAGARAHOLE NATIONAL PARKS OF KARNATAKA
    (Karnataka Veterinary Animal And Fisheries Sciences University, Bidar, 2017) VARDHAMAN PATIL
    Bandipur national park established in 1974 as a tiger reserve under â Project Tigerâ ? is located in Gundlupet taluka of Chamarajanagara district in Karnataka
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    ThesisItemOpen Access
    COMPARATIVE EVALUATION OF LATERAL FLOW ASSAY AND LOOP MEDIATED ISOTHERMAL AMPLIFICATION WITH ACETONE AND FORMALIN FIXATION BASED DIRECT FLUORESCENT ANTIBODY ASSAY FOR DIAGNOSIS OF RABIES IN ANIMALS
    (Karnataka Veterinary Animal And Fisheries Sciences University, Bidar, 2017) TAJUNNISA, M
    Rabies is considered to be a re-emerging zoonosis in many parts of the world, particularly in Asia, Africa and Latin America
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    ThesisItemOpen Access
    ISOLATION AND MOLECULAR CHARACTERIZATION OF SALMONELLA SPECIES FROM DIARRHOEIC CASES OF PIGS
    (Karnataka Veterinary Animal And Fisheries Sciences University, Bidar, 2017) RAMESH KANKANAWADI
    Pig rearing is one of the traditional activities in India carried out by rural folk. Among various livestock activities