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Subject: Animal Biotechnology × Author: DEURI, NIRAB KUMAR × End date: 2019 × Start date: 2014 × Type: Thesis ×
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    ThesisItemOpen Access
    CHARACTERIZATION OF Clostridium perfringens MEMBRANE VESICLES AND THEIR IMMUNOGENIC POTENTIAL IN MICE
    (Assam Agricultural University, Khanapara,Guwahati, 2016-07) DEURI, NIRAB KUMAR; SHARMA, R. K.
    The present study was undertaken with a view to characterize as well as evaluate the immuno-protective potential of the Membrane Vesicles (MVs) of Clostridium perfringens Type ‘A’. A total of five isolates of C. perfringens from the repository of Department of Microbiology, College of Veterinary Science, Khanapara, Guwahati were revived and reconfirmed based on gross morphology, staining characteristics and amplification of 16S rRNA and cpa genes by polymerase chain reaction. One isolate of C. perfringens belonging to Type A was selected and grown in TPGB and RCMB media. MVs were extracted at 4, 8, 12 and 24 hrs of growth. Study of protein profile based on SDS-PAGE revealed the appearance of one band at 4 and 8 hrs of growth and 7 bands in MVs extracted at 12 hrs of growth on TPGB. No bands were observed in MVs extracted at 24 hrs of growth in TPGB. Molecular weight of the protein bands were ranging from 43.3 kDa to 75.2 kDa. The MVs extracted from RCMB media revealed the appearance of one protein band in 4 and 8 hrs and 6 bands from 12 hrs of culture growth. The 24 hrs growth culture in RCMB also revealed no bands in SDS-PAGE. The RCMB protein bands were also ranging from 43.3 kDa to 75.2 kDa. The extracellular proteins and cell lysate proteins were extracted from the cultures grown on BHI broth and compared the protein profile of MVs as revealed by SDS-PAGE. The protein profile revealed 15 distinct bands in cell lysate proteins 11 bands in MVs and 4 bands in extracellular proteins. Cell lysate protein bands were ranging from 17.2 kDa to 75.0k Da, whereas MVs and extracellular protein bands were ranging from 43.3 kDa to 75.0k Da and 44.1 kDa to 75.0 kDa, respectively. The DNA content of MVs was released by GES reagent and PCR was done targeting to 16S rRNA and cpa (alpha toxin) genes yielding two distinct bands of 795 bp and 324 bp. Immunization of mice to group A1 and B1 was done with the vaccine prepared from MVs at the dose rate of 20 µg/0.2 ml while group A2 and B2 was done with that of 40 µg/0.2 ml through intraperitoneal (i/p) route at 0, 14, and 28 days. The protective efficacy of the MVs vaccine was studied by challenging the immunized mice on 42nd day post-primary vaccination with 3x108 CFU and 6x108 CFU of C. perfringens Type A. The challenge trial revealed that the 20.0 µg and 40.0 µg /0.2ml dose of vaccines could confer 100.0 percent protection in the immunized mice following homologous challenge with 3 x 108 CFU. However, the challenge trial with 6 x 108 CFU of homologous strain of C. perfringens Type A revealed only 66.66 and 33.33 percent protection in the mice immunized with 40.0 µg and 20.0 µg /0.2 ml vaccine doses, respectively.