Loading...
Theses
Browse
4 results
Search Results
ThesisItem Open Access Studies on virulence gene profiling, serotyping and epidemiology of shigatoxigenic and enteropathogenic e. coli isolated from captive and free ranging wild mammals and birds(G. B. Pant University of Agriculture and Technology, Pantnagar, 2022-08) Shukla, Namita; Rajesh KumarIndia has great wildlife biodiversity and is home to myriad wildlife fauna. Wildlife plays important role in of the planet ecosystem however, it often represents an important risk in emerging zoonosis, scant information is available about the occurrence of zoonotic pathogens in wildlife worldwide. Shigatoxigenic and Enteropathogenic Escherichia coli are two important classes of zoonotic enteric pathogens. Epidemiology of STEC and EPEC is largely unknown in most of the developing countries including India. Recent, studies in have underlined the importance of wildlife surveillance, as large number of emerging zoonotic pathogens are found to be of wildlife origin. Thus, wild animal should thoroughly be monitored, as they can potentially cause a spillover or spillback to human and other domestic animals. In present study, 770 fecal sample were obtained from 598 wild and 172 domestic animal during January 2021 to March 2022 from zoological gardens situated in three different geographical locations of the India and adjoining village near Accanakmar sanctuary Bilaspur Chhattisgarh respectively. Total 515 (86.12%) isolates were identified as E. coli from wild animals subjected to molecular screening for stx1, stx2, eaeA, and ehlyA genes by multiplex PCR. Total 75 (14.56%) isolates were successfully pathotyped and among these isolates STEC/AE-STEC and EPEC virulence genes were detected in 42 (8.73%) and 33(6.40%)E. coli isolates respectively. All 33EPEC isolates were found to be atypical EPEC carrying only the eaeA gene. All 75 STEC / EPEC isolates were serogrouped in29 different serotype, 3 isolates were untypeable (UT), and 1 isolate was found to be rough strain. Serogroup O157 AE-STEC was detected in two isolates from wild animals, one was isolated from red deer and other from peacock. Present study attempts to investigate transmission of STEC and EPEC among domestic animal, poultry and free- ranging wild animal in shared agroecosystem. One AE-STEC O157 isolates each was isolated from peacock, poultry and goat. Which showed similar virulence profile All 75 isolates(STEC and EPEC) were subjected to invitro antibiotic sensitivity assay against 14 commonly used antibiotics (CLSI 2008), which are showing varying resistance to tetracycline Norfloxacin cefuroxime, Gentamicin, Chloramphenicol, Co-Trimoxazole, Ampicillin and Azithromycin, however, no resistance was observed for cephalothin, ciprofloxacin, ceftriaxone, Ticarcillin+ clavulanic acid and Piperacillin + tazobactam, Amikacin. SETC/EPEC isolates (12) from free-ranging wild animals were susceptible to all 14 of antibiotics tested.AE-STEC, STEC and EPEC isolates differed in their ability to form biofilm according to the temperature, time of incubation and surface used and are classified as no biofilm, weak, moderate, or strong biofilm producers. The biofilm formation was more at 37°C than 20°C during initial 24 hrs. incubation and decreased with increasing incubation time at 37°C, as majority of isolates developed highest biofilm mass at 24 hrs., post inoculation. All the AE-STEC, STEC and EPEC isolates were capable to form biofilm. Our findings emphasize the role of wild animals as reservoir of potentially pathogenic STEC and EPEC.ThesisItem Open Access Studies on immunogenicity of recombinant fiber protein of Inclusion Body Hepatitis-Hydropericardium Syndrome (IBH-HPS) virus with special reference to formulation of a candidate subunit vaccine(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-02) Pandey, Garima; Rajesh KumarIn the present study, recombinant fiber protein of strain FAdV- 2/11 of Fowl adenovirus D isolate PANTNAGAR/HA-14/R-21 was studied for its immunogenicity in terms of effect of dose and adjuvant on the immune response of chicken against IBH-HPS virus. SPF chickens were immunized with different doses of recombinant fiber protein with FCA and 25μg/bird dose provided best protection. In second experiment, broilers were immunized with 25μg/bird along with different adjuvants viz ; montanide, resiquimod, saponin and FCA and different immune parameters were studied. Macrophage function test revealed that resiquimod group has released maximum concentration of nitric oxide. Cell mediated and humoral immune responses were analyzed by cutaneous basophil hypersentivity test, lymphocytes proliferation test and serum neutralization test, respectively. Maximum thickness of foot web was observed in montanide group at 72hrs post inoculation of DNCB. Montanide group showed maximum Tcell response at 21st DPI. Neutralization index of montanide group was highest at 28th DPI. Viral DNA in faeces was detected in all groups at 7th DPC, in montanide group 3 out of 6 faecal samples were positive while on 10th DPC only challenged control group was positive for viral DNA in faeces. 25μg/bird dose with montanide showed best immune response in broilers against challenged with virulent FAdV-2/11. Immunoinformatics analysis of Fiber protein of FAdV-2/11 revealed 21 continuous B cell epitopes with 13 epitopes having surface accessibility and 19 epitopes were antigenic as predicted by BepiPred, Emini surface accessible and Kolaskar and Taogankar antigenicity method, respectively. Out of four models predicted by SWISS MODEL, model 1 was best and verified by different servers. Three discontinuous epitopes were predicted by Ellipro tool. Sixteen epitopes, strongly linked with the MHC alleles were determined by MHC Class-I binding tool and six core peptides have been predicted by MHC Class-II binding tool. Secondary and tertiary structures were predicted by PesiPred V4.0 and Phyre2 tool, respectively. Physico-chemical properties of fiber protein were predicted by ProtParam tool. 0.5002 antigenicity score was predicted by VaxiJen v2 server and thus, fiber protein was proved antigenic and immunogenic. Therefore, fiber protein can be used for development of promising peptide vaccines. Present study concludes that a recombinant subunit vaccine candidate containing recombinant fiber protein (25μg/bird) with montanide as adjuvant may be formulated for further field trials.ThesisItem Open 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 KumarKeeping 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.ThesisItem Open Access Studies on immunogenicity of recombinant fiber protein of Inclusion Body Hepatitis- Hydropericardium Syndrome (IBH-HPS) virus with special reference to formulation of a candidate subunit vaccine(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-02) Pandey, Garima; Rajesh KumarIn the present study, recombinant fiber protein of strain FAdV- 2/11 of Fowl adenovirus D isolate PANTNAGAR/HA-14/R-21 was studied for its immunogenicity in terms of effect of dose and adjuvant on the immune response of chicken against IBH-HPS virus. SPF chickens were immunized with different doses of recombinant fiber protein with FCA and 25μg/bird dose provided best protection. In second experiment, broilers were immunized with 25μg/bird along with different adjuvants viz ; montanide, resiquimod, saponin and FCA and different immune parameters were studied. Macrophage function test revealed that resiquimod group has released maximum concentration of nitric oxide. Cell mediated and humoral immune responses were analyzed by cutaneous basophil hypersentivity test, lymphocytes proliferation test and serum neutralization test, respectively. Maximum thickness of foot web was observed in montanide group at 72hrs post inoculation of DNCB. Montanide group showed maximum Tcell response at 21st DPI. Neutralization index of montanide group was highest at 28th DPI. Viral DNA in faeces was detected in all groups at 7th DPC, in montanide group 3 out of 6 faecal samples were positive while on 10th DPC only challenged control group was positive for viral DNA in faeces. 25μg/bird dose with montanide showed best immune response in broilers against challenged with virulent FAdV-2/11. Immunoinformatics analysis of Fiber protein of FAdV-2/11 revealed 21 continuous B cell epitopes with 13 epitopes having surface accessibility and 19 epitopes were antigenic as predicted by BepiPred, Emini surface accessible and Kolaskar and Taogankar antigenicity method, respectively. Out of four models predicted by SWISS MODEL, model 1 was best and verified by different servers. Three discontinuous epitopes were predicted by Ellipro tool. Sixteen epitopes, strongly linked with the MHC alleles were determined by MHC Class-I binding tool and six core peptides have been predicted by MHC Class-II binding tool. Secondary and tertiary structures were predicted by PesiPred V4.0 and Phyre2 tool, respectively. Physico-chemical properties of fiber protein were predicted by ProtParam tool. 0.5002 antigenicity score was predicted by VaxiJen v2 server and thus, fiber protein was proved antigenic and immunogenic. Therefore, fiber protein can be used for development of promising peptide vaccines. Present study concludes that a recombinant subunit vaccine candidate containing recombinant fiber protein (25μg/bird) with montanide as adjuvant may be formulated for further field trials.
