Browsing by Author "Mishra, Adarsh"
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PresentationItem Open Access Assessment of immune response of recombinant glycoprotein D (gD) of infectious laryngotracheitis (ILT) virus expressed in Pichia pastoris(2020-02) Mishra, Adarsh; Thangavelu, A; Roy, Parimal; et al; TANUVASInfectious laryngotracheitis (ILT), caused by Gallid herpesvirus1, is one of the most important diseases of poultry that affects chickens of all age groups. The present study was aimed at elucidation of immune response of recombinant glycoprotein D (gD) of ILT virus through in vitro and in vivo studies. Hence, the truncated ectodomain of gD was cloned in pPICZαA shuttle vector and was integrated into Pichia pastoris strain GS115 yeast chromosomal DNA. The recombinant protein (eu-rgD) was expressed in 72 hours of methanol metabolism and was analyzed by SDS-PAGE followed by western blotting. Further, the protein was confirmed by MALDI-TOF-MS/MS analysis. The immunogenicity of eu-rgD was assessed through immunization trial in specific pathogen free (SPF) chicken model. The antigen induced cellular proliferation of peripheral blood mononuclear cells (PBMCs) was appreciated by lymphocyte proliferation assay. Assessment of the antigen specific immune response by IFN-γ cytokine profiling in PBMCs through competitive ELISA had revealed a significantly higher level of IFN-γ response in PBMCs primed with orally administered antigen to those primed parenterally. On assessment of seroconversion by indirect ELISA developed in the study, it was observed that there was no significant difference across the parenterally inoculated groups (high dose, medium dose and low dose groups), which indicated a lower dose would be sufficient for an efficient seroconversion. However, there was poor seroconversion found in the orally administered group. In the present study, the seroconversion to the yeast expressed recombinant glycoprotein antigen was assessed to forecast its immunogenicity. It was found optimum in producing ideal seroconversion in parenteral route administration strategies with adjuvant.ArticleItem Open Access Detection of Mycoplasma galliseptium infection in chickens from Tamil Nadu State of India(2019-01) Manimaran, K; Mishra, Adarsh; Hemalatha, S; Karthik, K; Ganesan, P.I.; TANUVASMycoplasma gallisepticum (MG) is one of the major respiratory tract pathogens affecting chickens. It causes Chronic Respiratory Disease (CRD) among chickens of various age groups. The present study describes the isolation, molecular detection and histopathological changes associated with CRD in chickens. A total of 790 samples viz., trachea, lungs and air sacs were collected from chickens showing the symptoms of CRD from different parts of Tamil Nadu state. All the samples were processed for isolation and molecular detection of MG. A total of 91 samples were found positive by isolation and 105 samples were found positive through MG specific PCR targeting 16S rRNA gene. The histopathological changes in tissue samples of trachea, sinuses, air sacs and lungs collected from naturally infected M. gallisepticum infection were suggestive of subacute to chronic nature of infection. Though isolation is considered to be a gold standard, still PCR is a rapid, sensitive and cheap method for early diagnosis of MG which can help poultry farmers to avoid severe economic loss.ArticleItem Open Access Infectious Laryngotracheitis in Layer Birds from Tamil Nadu, India(2020-11) Mishra, Adarsh; Thangavelu, Ardhanary; Roy, Parimal; Tirumurugaan, Krishnaswamy Gopalan, et al.,; TANUVASInfectious laryngotracheitis (ILT), caused by Gallid herpesvirus1, is one among the respiratory diseases of poultry gradually spreading worldwide, including Indian subcontinent. Present study was carried out to identify the pathogen from the suspected cases of the disease. The tracheal tissue samples (pooled) were collected from the birds suspected to have died of ILT from 26 commercial poultry farms located in and around Namakkal district of Tamil Nadu state of India. On post-mortem examination, haemorrhagic caseous exudate and fibrinous pseudo-membrane adhered to tracheal mucosa were noticed. A total of 22 farms were found positive through PCRs targeted against ICP4 gene and the thymidine kinase (TK) gene followed by confirmation through sequencing. Histopathology examination revealed decilliation, hyperplasia, degeneration and/or loss of tracheal epithelium with severe submucosal edema. There was infiltration with numerous lymphocytes, macrophages and plasma cells in milder infections, whereas presence of fibrinous exudates admixed with numerous erythrocytes and inflammatory cells like heterophils and lymphocytes were seen indicating the severe acute form of the disease. A fibrinous pseudomembrane was seen firmly attached to the inflamed and necrotic mucosa in subacute cases. Further, virus was isolated from randomly selected 5 PCR positive tracheal tissue samples in embryonated chicken eggs through chorioallantoic membrane (CAM) route. The typical pock lesions were observed on CAM along with engorged blood vessels and thickening of the membrane. Present study has reported the disease ILT among poultry flocks in the Namakkal district of Tamil Nadu and raises the concern for thorough investigation of the nature of prevailing pathogen in the region.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF INFECTIOUS LARYNGOTRACHEITIS VIRUS FIELD ISOLATES AND IMMUNOMICS OF ITS MAJOR GLYCOPROTEIN(TANUVAS, Chennai, 2017) Mishra, Adarsh; Thangavelu, A.; Shoba, K.; Tirumurugaan, K.G.; TANUVASPresent study was carried out to characterize the infectious laryngotracheitis virus (ILTV) from the suspected cases of ILT by employing molecular tools. Further, an immunodominant glycoprotein of ILTV was utilized for cloning and expression studies in prokalyotic as well as eukaryotic (yeast) expression systems. In addition, the immune response of the recombinant glycoprotein was assessed by in vitro and in vivo experiments.ThesisItem Open Access Studies on the respiratory tract infections in poultry in relation to mycotoxins in feed(Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 2013) Mishra, Adarsh; Dr. Tejinder Singh RaiA total of fifty two (52) isolates of bacteria associated with various respiratory tract infections in chicken were isolated from twenty two (22) different outbreaks in and around Ludhiana comprising of Staphylococcus aureus (46%), avian pathogenic E. coli (APEC, 29%), Proteus sp. (9%), Klebsiella pneumoniae (6%), Streptococcus equi subsp. zooepidemicus and Pseudomonas aeruginosa (each 4%) and Citrobacter koseri (2%). Antibiogram showed an increasing trend of resistance to various antimicrobial drugs and emergence of multi-drug resistant bacteria. Phenotyping of APEC showed all the isolates as Congo red binding positive (Crb+) suggesting their invasive nature. Virulence typing of APEC showed 7/15 isolates to contain all the five plasmid associated virulent genes. Present study also reported molecular detection of the organism Avibacterium paragallinarum (1.29%), although the organism could not be isolated. Occurrence of Mycoplasma gallisepticum was 59.15% while that of Mycoplasma synoviae was 33.8% confirmed through PCR. Multiple sequence alignment of partial sequence data revealed 99%-100% sequence homology with respect to M. gallisepticum and/or M. synoviae. A total of fifty (50) poultry feed samples from the affected farms were analyzed initially for presence of any mycotoxins by screening through pressure mini columns (PMCs) in which the level of aflatoxin was in the range of 25 ± 25 ppb to 600 ± 25 ppb. Results of thin layer chromatography (TLC) revealed occurrence of aflatoxin B2 to be the highest (88%) followed by aflatoxin B1 (84%), ochratoxins (74%) and citrinin (10%). The levels of aflatoxin B1 and B2 were found in the range of 25-600 ppb with an average of 178.57 ± 23.8 ppb and 202.27 ± 25.43 ppb, respectively. Ochratoxins were in the range of 50-600 ppb with an average of 206.08 ± 20.6 ppb while citrinin was in the range of 50-200 ppb with an average of 150 ± 31.56 ppb. Histopathology findings revealed a marked immunosuppression in the birds with respiratory tract affections.