B Senthilkumar1, K Anbarasu2, D Senbagam3, M Rajasekarapandian4. 1. Centre for Biotechnology, Muthayammal College of Arts & Science, Rasipuram-637 408, Tamil Nadu, India. Electronic address: senthilmicro@yahoo.co.in. 2. Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India. 3. Department of Biotechnology, Vivekanandha College of Engineering for Women, Tiruchengode-637 205. Tamil Nadu, India. 4. Department of Zoology, Arignar Anna Government Arts College, Namakkal-637 001, Tamil Nadu, India.
Abstract
OBJECTIVE: To develop attenuated strains of Salmonella enterica serovar Typhi (S. typhi) for the candidate vaccine by osmolar stress. METHODS: S. typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Namakkal, Tamil Nadu, India. Both strains were grown in LB (Luria Bertani) medium supplemented with various concentration of NaCl (0.1-0.7M) respectively. The effect of osmolar stress was determined at molecular level by PCR using MGR 06 and MGR 07 primers corresponding to ompR with chromosomal DNA of S. typhi SS3 and SS5 strains. Attenuation by osmolar stress results in deletion mutation of the S. typhi strains was determined by agglutination assays, precipitation method, SDS PAGE analysis and by animal models. RESULTS: The 799 bp amplified ompR gene product from wild type S. typhi SS3 and SS5 illustrate the presence of virulent gene. Interestingly, there was only a 282 bp amplified product from S. typhi SS3 and SS5 grown in the presence of 0.5, 0.6 and 0.7 M NaCl. This illustrates the occurrence of deletion mutation in ompR gene at high concentration of NaCl. Furthermore, both the wild-type and mutant S. typhi outer membrane SDS-PAGE profile reveals the differences in the expression of ompF, ompC and ompA proteins. In mice, wild type and mutant strains lethal dose (LD50) were determined. The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU.mL(-1). When the mice were injected with the mutants in same dosage, no clinical symptoms were observed; whereas the serum antibody titre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response. These results suggest that S. typhi SS3 and SS5 may be used as good candidate strains for the development of live attenuated vaccine against salmonellosis. CONCLUSIONS: This study demonstrates that the S. typhi strains were attenuated and could be good vaccine candidates in future.
OBJECTIVE: To develop attenuated strains of Salmonella enterica serovar Typhi (S. typhi) for the candidate vaccine by osmolar stress. METHODS:S. typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Namakkal, Tamil Nadu, India. Both strains were grown in LB (Luria Bertani) medium supplemented with various concentration of NaCl (0.1-0.7M) respectively. The effect of osmolar stress was determined at molecular level by PCR using MGR 06 and MGR 07 primers corresponding to ompR with chromosomal DNA of S. typhi SS3 and SS5 strains. Attenuation by osmolar stress results in deletion mutation of the S. typhi strains was determined by agglutination assays, precipitation method, SDS PAGE analysis and by animal models. RESULTS: The 799 bp amplified ompR gene product from wild type S. typhi SS3 and SS5 illustrate the presence of virulent gene. Interestingly, there was only a 282 bp amplified product from S. typhi SS3 and SS5 grown in the presence of 0.5, 0.6 and 0.7 M NaCl. This illustrates the occurrence of deletion mutation in ompR gene at high concentration of NaCl. Furthermore, both the wild-type and mutant S. typhi outer membrane SDS-PAGE profile reveals the differences in the expression of ompF, ompC and ompA proteins. In mice, wild type and mutant strains lethal dose (LD50) were determined. The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU.mL(-1). When the mice were injected with the mutants in same dosage, no clinical symptoms were observed; whereas the serum antibody titre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response. These results suggest that S. typhi SS3 and SS5 may be used as good candidate strains for the development of live attenuated vaccine against salmonellosis. CONCLUSIONS: This study demonstrates that the S. typhi strains were attenuated and could be good vaccine candidates in future.