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ThesisItem Open Access Prevalence, seroprevalence and postvaccinal antibody response of Peste des Petits ruminants virus in goats(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-11) Netam, Amisha; Tewari, AnujPeste des petits ruminants (PPR) is an economically important transboundary disease of sheep and goats caused by Peste des petits ruminants virus (PPRV). In India, the disease is endemic and therefore, Government of India has initiated PPR control program (PPR-CP) under which kids more than 4 months of age are vaccinated. At the same time it is also important to know the seroprevalence of PPR in unvaccinated goats to know the virus circulation. Therefore, this study was conducted to know the seroprevalence of PPR in unvaccinated goats around the Pantnagar region of Uttarakhand and also to understand the virus distribution in the region. In addition, study also included antibody response and kinetics in Pantja goats vaccinated against PPRV. Total 212 serum samples from goats were collected randomly from various villages from three district (Udham singh Nagar, Nainital, and Almora) of Uttarakhand. Serum samples were tested for anti-PPRV antibody by a commercially available kit from IDvet. 41 animals from various villages were found positive with a prevalence rate of 19.33%. At the same time, PPR outbreaks were also reported from the Pantnagar area. Blood, nasal, oral and rectal swabs were collected from the 19 goats suspected/showing clear sign of PPR. RNA was extracted from the swabs and was subject to one step RT-PCR. The amplified PCR product confirmed PPR in 8 goats with a PPRV prevalence rate 42.10%. Two representative swab samples (one pooled swab and one nasal swab) were subjected to virus isolation in Vero cells. Swabs from both goats showed typical cytopathic effect of PPRV in the first passage and led to complete detachment of the cell monolayer in 48-72 hours in comparison to the vaccine strain Sungri 96 which showed complete cytopathic effect in 4-5 days without complete monolayer detachment. Post vaccination antibody response in Pantja goats vaccinated against PPRV varied from 7-10 days and the antibody response was maintained upto 91 days. Hence the present study signifies that PPRV is circulating in the Tarai region of Uttarakhand and there is an urgent need of mass vaccination to increase the herd immunity to substantial level.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.ThesisItem Open Access Isolation and characterization of fowlpox virus of poultry(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2007-07) Madanpal; Rao, V.D.P.Fowl pox is a contagious and slow spreading viral disease. It affects the birds of all age, sex and breeds. The disease is manifested in three different clinical forms–cutaneous, diphtheritic and oculo nasal forms. The mucosal lesions involving the mouth, esophagus and trachea can be confused with other respiratory diseases like infectious laryngotrachaetis, coryza etc. Keeping in view of the impact of disease on economics of poultry industry, the present study was undertaken to isolate the virus from scab lesions of birds of a poultry farm near Barielly following isolation of virus on chorioallantoic membrane (CAM), of developing chicken embryos, further characterization was carried by studying cytopathogenicity in cell culture and sero diagnostic tests. It was observed that virus was successfully adapted to CAM, CEF cells as well as in BHK 21 cell line. Characteristic pock lesion in CAM and CPE in unstained and stained preparations confirmed the presence of virus. In MGG staining, the cytopathic changes characterized by rounding of cells 24 hrs PI and the cytoplasmic vacuolation and syncytia formation by 48 hrs PI. In few cells, the nucleus occupied eccentric position and degenerative changes in the nucleus characterized by fragmentation of nuclear membrane in the infected CEF cells while the cytopathic changes in infected BHK 21cells were characterized by rounding of cells 36 hrs PI and the cytoplasmic vacuolation and syncytia formation by 48 hrs PI. The infectivity titre was calculated to be log104.25/ml (EID50/ml) on CAM and log10 9.79/ml (TCID50/ml) in CEF cell culture. The agar gel precipitation test (AGPT) revealed the precipitation band, which confirms the presence of antigen and antibody. Counter immunoelectrophorasis (CIE) showed a precipitation line within one hr of electrophoretic run. The indirect fluorescent antibody technique (IFAT) was used to demonstrate the virus in infected cell culture and cell line. The infected chicken embryo fibroblast cells revealed small particulate fluorescence in the cytoplasm of the cells. These tests confirmed that the virus isolate as fowlpoxvirus SDS-PAGE analysis of cell culture supernatant infected with FPV isolate revealed 11 polypeptides with molecular weights ranging from 120k Da to 15 kDa.ThesisItem Open Access Studies on immune status of indigenous and exotic breeds of domestic fowl(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-08) Yadav, Renuka; Rajesh KumarPresent study was carried out for comparative evaluation of some indigenous and exotic breeds of domestic chicken with regard to the innate immune response, adaptive immune response and resistence to infection following challenge with infectious agent. One exotic (RIR) and two indigenous breeds (Kadaknath, Uttara fowl) were used for study. All the birds were reared in deep litter system with ad-libtum feed and water throughout the experiment. The experiment lasted for 60 days. Significant difference was observed among parameters studied viz; mean total leukocyte count, differential leukocyte count, leukocrit, serum globulin, A/G ratio, phagocytic activity and opsonocytophagic index, among various breeds of chicken at all stages and all the innate and adaptive immune parameter was significantly higher in RIR compared to Kadaknath and Uttara fowl breed of chicken. The RIR showed highest hemagglutination inhibition titre following NDV immunization till the end of the experimental period followed by Kadaknath and Uttara fowl. Cell mediated immune (CMI) response was also observed to be higher for RIR breed compared to Kadaknath and Uttara fowl. The lymphocyte stimulation index was highest in RIR and the lowest in Uttara fowl. Maximum mortality was observed in Uttara fowl up to 15 days post challenge with a virulent virus and antibody titre was maximum in RIR breed at 3 and 7 days post challenge but at 14 days post challenge kadaknath showed highest antibody titre.ThesisItem Open Access Studies on vaccine strain of Fowlpoxvirus with special reference to its adaptation in primary cell cultures and its characterization(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2006-08) Tripathi, Ashok Kumar; Rajesh ChandraFowlpox is an economically important and widespread disease of poultry that causes heavy morbidity and mortality in the affected birds resulting in heavy losses in the form of low egg and meat production. It affects the birds of all age, sex and breeds. The disease is caused by Avipoxvirus of the family Poxviridae. The disease is manifested in three different clinical forms–cutaneous, diphtheritic and oculo nasal forms. The mucosal lesions involving the mouth, esophagus and trachea can be confused with other respiratory diseases like infectious laryngotrachaetis, coryza etc. Keeping in view the economic impact of disease on poultry industry, the present study was undertaken to adapt the vaccine strain of FPV in chorioallantoic membrane, chicken embryo fibroblast cell culture as well as in chicken kidney cell culture and evaluate the various serological tests for rapid diagnosis of FPV infection. Fowlpoxvirus was successfully adapted to CAM, CEF and CK cells. Characteristic pock lesions on CAM and CPE in unstained and stained preparations confirmed the presence of virus. In MGG staining, the cytopathic changes were characterized by rounding of cells 12 hrs PI and the cytoplasmic vacuolation and syncytia formation by 18 hrs PI. In few cells, the nucleus occupied eccentric position and degenerative changes in the nucleus were characterized by fragmentation of nuclear membrane in the infected CEF cells, while the cytopathic changes in infected CK cells were characterized by rounding of cells 36 hrs PI and the cytoplasmic vacuolation and syncytia formation by 48 hrs PI. The infectivity titre was calculated to be log107.14 EID50/ml on CAM and log10 9.25TCID50/ml in CEF cell culture. The agar gel immunodiffusion test revealed the precipitation band, which confirms the presence of antigen and antibody. The serum neutralization test using the beta (constant virus serum dilution) procedure, revealed an antibody titre of 1:160. CIE showed a precipitation line within one hr of electrophoretic run. The indirect fluorescent antibody technique (IFAT) demonstrated the presence of virus in infected cell culture. The infected embryonic chicken kidney cells as well as the infected chicken embryo fibroblast cells revealed small particulate fluorescence in the cytoplasm of the infected cells.ThesisItem Open Access Studies on epidemiology, isolation and molecular analysis of fowl adenoviruses from the domestic chicken with respiratory disease conditions(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-08) Bisht, Ritika; Rajesh KumarFowl adenoviruses (FAdV) are the ubiquitous and cause extensive damage to the poultry industry. They are known to be associated with various disease conditions such as Inclusion body hepatitis, Hydropericardium Syndrome, respiratory disease, tenosynovitis, impaired growth, reduced egg production, aplastic anemia, atrophy of bursa and thymus, enteritis and conjunctivitis in chickens and other birds. Present study was undertaken to study epidemiology, to isolate and perform molecular analysis of fowl adenoviruses from the chickens with the respiratory disease conditions. Natural outbreaks investigated during the study revealed 2 –15% mortality among the affected birds aged 3-6 weeks. General postmortem findings were enlarged and friable liver with necrotic foci, enlargement of kidney, congestion and haemorrhagic trachea, congested lungs. Four isolates of fowl adenoviruses were propagated in primary CEL cell culture. The cytopathic effects were characterized by rounding of the cells, degeneration, increase in the refractive index of the cells and microplaques formation. AGPT, CIEP, IFAT, Dot-ELISA and Sandwich ELISA determined the presence of the virus in infected tissues and cell culture. Intensely basophilic intra nuclear inclusion bodies were also visualized following MGG staining of CEL cells. The genomic DNAs were extracted from the infected CEL cells and PCR was carried out for amplification of L1 region of the hexon gene showing amplicons of the ~900 bp. On digestion with MluI, three isolates were digested and the two bands of 400 and 500 bp were visualized and with the enzyme StyI, only one isolate was digested and two bands of 480 and 420 bp were obtained. None of the isolates were digested by the enzymes BglI, BsiWiI and ScaI. RE analysis revealed that isolates belong to two serotypes 5 (R-66) and 8 (R-53, R-43, R-63). Thus, it can be concluded that these serotypes are involved in respiratory disease of domestic chickens in India.ThesisItem Open Access Comparative protein profiles of Salmonella & E. coli(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2004-05) Gupta, Meenal; Sharma, V.D.Salmonella is one of the most common foodborne pathogens around the world; its detection is a difficult proposition. Conventional method adopted for its detection in foods is cumbersome and time taking. In the present study, attempts were made to compare the protein profiles of selected Salmonella serovars and E. coli to identify the genus specific protein for Salmonella. The commonly prevalent four Salmonella serovars, namely, S. Bareilly, S. Gallinarum, S. Typhimurium and S. Weltevreden were chosen for the study. E. coli O78 that was isolated most frequently from dead/ailing birds was also included in the study. Bacterial growth of these bacteria were treated with cefotaxime and the supernatant obtained was designated as cefotaxime extract (CE).The CE was precipitated with ammonium sulphate at 100% level and the precipitate was designated as PDP (100%). SDS-PAGE analysis of PDPs (100%) yielded 11, 15, 15, 11 and 14 bands in S. Bareilly, S. Gallinarum, S. Typhimurium and S. Weltevreden and E. coli O78 respectively. S. Weltevreden shared 7 bands with E. coli O78. A protein of molecular weight 20.89 kDa was found in all Salmonella PDPs but not in E .coli O78. In order to further purify this protein, proteins precipitated between 50 and 80% salt concentration (PDP 50-80%) were isolated and subjected to gel filtration using Sephacryl S-200 HR gel matrix. Gel filtration chromatographic analysis revealed 4, 2 and 5 peaks in PDPs (50-80%) of S. Gallinarum, S. Weltevreden and E. coli O78 respectively. All three PDPs (50-80%) and the major peaks obtained by gel filtration, i.e. 2nd peak of Salmonella serovars and 1st peak of E. coli O78 were run on SDS-PAGE. Again the protein with molecular weight 20.89 kDa was seen in all Salmonella serovars along with some other proteins suggesting its further purification.
