Leptospira Cultures Maintenance

Leptospira Cultures Maintenance RESULTS The Leptospira serovars maintained in the Department of Veterinary Microbiology were used in the present study. For maintenance, EMJH medium (Difco) with albumin supplement was employed and subcultured at seven day intervals and incubation was carried out at 28-30Â °C. In addition, the stock cultures were maintained in semi-solid medium with subculturing at one month interval. IDENTIFICATION OF LEPTOSPIRES Under dark field microscope, the live leptospiral organisms were found tightly coiled and actively motile. The motility observed was of both spinning and bending. In highly concentrated cultures, the organisms formed entangled masses. No contaminants were observed in most of the time when streaked on blood agar plates for purity checking of the cultures. In case of contamination they were purified by filtration. RECOMBINANT PROTEIN PRODUCTION Preparation of template DNA from Leptospira The genomic DNA isolated from Leptospira interrogans serovar Australis had a DNA concentration ranging from 40- 60Â µg/ml. The purity of the extracted DNA was checked by measuring the ratio of absorbance (O.D of DNA preparation at 260 and 280 nm). The value of the ratio obtained was found to be in the range of 1.8 to 1.93, indicating that the preparations were almost free of proteins.(in mat and methods). Amplification of lipl21 gene and lipl32 genes The amplification of lipl21and lipl32 genes were carried out and observed amplicons of 507 bp and 767 bp, respectively. (Fig 1) Cloning of the lipl21 and lipl32 gene The colonies of E.coli Dh5∞ cells transformed with lipl21 and lipl32 genes was picked up separately and tested for the presence of the two genes. It was observed that lipl21 clones yielded an amplicon size of 507 bp and lipl32 with 767 bp. These confirmed clones were preserved for further studies (Fig 2) Induction of recombinant protein The above clones were subcultured in LB broth containing Ampicillin (100Â µg/ml) and expression was optimized with 2 mM IPTG for LipL21 and 1mM IPTG for LipL32. The induced recombinant cells were harvested after six hours and five hours for LipL21 and LipL32, respectively. Uninduced controls were set for each. The cells were then pelleted and lysed. The expression of recombinant 21 kDa (rLipL21) and 32 kDa outer membrane proteins (rLipL32) were confirmed in comparison with that of the uninduced cells where there was no significant protein expression (Fig 3) Purification of recombinant lipl21 and lipl32 proteins The rLipL21 and rLipL32 proteins were purified by Nickel chelating affinity chromatography without any contamination. The protein concentrations were estimated to be 0.69 mg/ml and 2.07mg/ml for rLipL21 and rLipL32, respectively. Immunoreactivity of the proteins The immunogenicity of both rLipL21 and rLipL32 proteins were tested using MAT positive canine sera and observed that both the proteins were reacting. Further the protein did not react when blotted with hyper immune serum raised against the different bacteria namely E.coli, Staphylococcus aureus and Pasteurella multocida. DIAGNOSIS Microscopic Agglutination Test A total of 124 canine serum samples from Leptospira suspected dogs were tested using MAT and among this 22 (17.74 per cent) were found to be positive for leptospirosis. Serum samples having a titre of 1:400 and above were considered as positive. The infecting serovars identified with canine leptospirosis are depicted in Table 3 Enzyme Linked Immunosorbent Assay Indirect ELISA was done in separate microtitre plates employing rLipL21 and rLipL32 as antigens and the results were compared with that of MAT. Checker board analysis Using checker board analysis the optimum concentration of antigen, antibody and conjugate were estimated. The optimum concentration of antigen was found to be 50 ng/well and 150 ng/well for rLipL21 and rLipL32, respectively. The rabbit anti-canine IgG HRP conjugate concentration estimated was 1:2500 and 1:2000 for rLipL21 and rLipL32, respectively. A 1:50 dilution of test serum was used as optimum working dilution in both ELISAs. Determination of cut off values In IgG ELISA, the mean OD and standard deviation for the negative sera samples (n=44) was 0.49 and 0.11 for rLipL21 and 0.59 and 0.09 for rLipL32, respectively. The cut off value obtained was 0.82 for rLipL21 and 0.86 for rLipL32. Test proper The results of rLipL21 and rLipL32 based IgG ELISA are given in Table 4. Among 47 positive samples obtained by rLipL21 ELISA, only 20 found positive with MAT. In case of rLipL32 ELISA, 40 samples were recorded positive out of which 18 found positive with MAT. Comparison of MAT and rLipL21IgG ELISA The results of rLipL21 IgG ELISA were compared with that of MAT. Among 124 canine sera examined, 47 (37.90 per cent) showed OD more than the cut off value i.e. 0.82 and were considered positive for leptospirosis by rLipL21 IgG ELISA. The sensitivity, specificity and accuracy of rLipL21 IgG ELISA as relative to MAT was calculated to be 90.90 per cent, 73.52 per cent and 76.61 per cent, respectively (Table 5). On statistical analysis, it was found that there exists a significant difference between the two tests, ie, rLipL21 ELISA and MAT even at 1 per cent level of significance. Comparison of MAT and rLipL32 IgG ELISA The results of the IgG ELISA using rLipL32 as the antigen were compared with that of MAT. Among 124 canine sera examined, 40 (32.25 per cent) showed OD more than the cut off value i.e. 0.86 and were considered positive for leptospirosis by IgG ELISA. The sensitivity, specificity and accuracy of rLipL32 IgG ELISA as relative to MAT was calculated to be 81.81per cent, 78.43 per cent and 79.03 per cent, respectively (Table 6). Statistical analysis revealed that there exists a significant difference between the two tests, ie, rLipL32 ELISA and MAT at 1per cent level of significance. Comparison of rLipL21 and rLipL32 IgG ELISA On statistical analysis using Cochran’s Q test, at 1 per cent level of significance it was observed that there exists no significant difference between rLipL21 and rLipL32 ELISA, as the P value was found to be greater than 0.01. Table 3 Infecting serovars identified with MAT Table 4 Results of MAT, rLipL21 ELISA and rLipL32 ELISA Table 5 Comparison between rLipL21 ELISA and MAT Sensitivity = a/a+c = 90.90% Specificity = d/b+d = 73.52% Accuracy =a+d/a+b+c+d = 76.61%