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Subject: Veterinary Microbiology × Author: Tiwari, Aakanksha × End date: 2021 × Start date: 2021 ×
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    ThesisItemOpen Access
    Egg derived antibodies (IgY) against Outer membrane proteins of multi-drug resistant Salmonella Typhimurium: Production and evaluation of therapeutic potential
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-03) Tiwari, Aakanksha; Rajesh Kumar
    Keeping in view, the emerging problem of anti microbial resistance in Salmonella Typhimurium, the present research work was carried out to produce and assess a therapeutic alternative against the bacteria. In the present study, attempts were made to revive glycerol stocks of 150 field isolates and 14 isolates were successfully revived. These isolates were characterized culturally and subjected to ABST. The isolate showing resistance against six groups of antibiotics was selected for further study. This isolate was also characterized molecularly and serotyped as Salmonella Typhimurium with antigen types- 4,5,12:i:1,2. After confirmation of the culture, OMPs were isolated with a concentration of 7.28 mg/ml and SDS-PAGE analysis revealed prominent OMP bands ranging from 11-90 kDa. OMPs were used for hyperimmunizing the RIR layers with a suitable adjuvant subcutaneously. Eggs were collected and IgY was isolated by dextran sulphate method. The purified IgY preparation revealed bands of 63 kDa and 27 kDa in SDS-PAGE, corresponding to the heavy and the light chains. Total protein concentration of IgY preparation by Lowry method was 14.246 mg/ml and specific IgY concentration by RID was 12.023 mg/ml, indicating 84.40% purity. The specificity of IgY against the OMPs was indicated by positive reaction in AGID, CIE, Western blot and Dot Enzyme Immunoassay. Titre of antibody in serum and egg yolk at weekly basis was also determined by ELISA. The maximum antibody titre was observed at 13th week and 11th week in the egg yolk and serum, respectively. It was also observed that IgY was stable at temperatures ranging from -40 ̊ C to 37 ̊ C as indicated by ELISA and SDS-PAGE. In-vitro efficacy testing of different concentrations of IgY against the bacteria showed results in a dose-dependent manner and significant difference was observed between the different combinations. Massive adherence and penetration of bacteria in Vero cells was observed in the negative and culture control unlike in the IgY treated bacterial Vero cells. In vivo experiment in mice, prophylactic group showed no mortality and bacterial count in the faecal swabs and in the organs was significantly less. It indicates that prophylactic activity of IgY is much stronger than the therapeutic activity.