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Theses

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2004 - 20073
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Subject: Veterinary Microbiology × End date: 2009 × Start date: 2004 × Type: Thesis × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen 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.
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    ThesisItemOpen 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 Chandra
    Fowlpox 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.
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    ThesisItemOpen 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.