Browsing by Author "John Kirubaharan, J"
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ArticleItem Open Access Association of COL1A2 (PvuII) gene polymorphism with risk and severity of dental fluorosis – A case control study(2019) Rahila, C; Aswath Narayanan, MB; John Kirubaharan, J; TANUVASDental fluorosis is a foremost public health problem in many countries, including India. Very few studies investigated gene polymorphism and risk of dental fluorosis. Genetic polymorphisms in Collagen Type I, alpha 2 (COL1A2) gene, found to be linked with bone pathogenesis, may affect the tooth formation resulting in the vulnerability to dental fluorosis. Aim: To assess the association between COL1A2 (PvuII) gene polymorphism and risk as well as severity of dental fluorosis. Methods: The present case control study was conducted among participants with (n = 60) and without (n = 60) dental fluorosis. Dental fluorosis was assessed using Modified Dean’s fluorosis index (1942). The PvuII polymorphisms (in exon 25) inside the COL1A2 gene were genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) procedure. Statistical analysis were carried out with Chi-square test and Odds Ratio (OR) was determined with multivariate logistic regression analysis. Results: The genetic polymorphism in COL1A2 PvuII was found to be associated with the risk of dental fluorosis which was highly significant (p< 0.001). The odds ratio was 31.4 times [OR = 31.9, 95% CI: 3.9–48.7] higher for the homozygous PP genotype group and 4.0 times [OR = 4.0, 95% CI: 1.0–10.7] higher for the heterozygous Pp genotype.ThesisItem Open Access BIOLOGICAL AND PHYLO GENETIC CHARACTERISATION OF PIGON PARMYXOVIRUS (PRMV-1) ISOLATES(TANUVAS, 2016) Akhil, VS; John Kirubaharan, J; TANUVAS; Thangavelu, A; Tirumurugan, KG; Tirumurugan, KGThe focus of the present study is to find out the pathotype, genotype and evolution of two isolates of pigeon paramyxovitus namely D167 and D168, which have been reported to cause nervous lesions and mortality among pigeons. The pathotype identification of D167 and Dl68 have been done by mean death time (MDT) estimation in embryonated chicken eggs, intracerebral pathogenicity index estimation (ICPI) in day old chicks and infection of maternal antibody negative SPF chicks through different routes. In order to demonstrate the pathogenetic difference of chicken and pigeon origin NDV, a comparative study has been done using chicken and pigeon isolates in one month old SPF chicks.ThesisItem Open Access BIOLOGICAL AND PHYLOGENETIC CHARACTERISATION OF PIGEON PARAMYXOVIRES (PPMV-i) ISOLATES(TANUVAS, 2016) Akhila, VS; John Kirubaharan, J; Thangavelu, A; Tirumurugan, KG; TANUVASThe focus of the present study is to find out the pathotype, genotype and evolution of two isolates of pigeon paramyxovitus namely D167 and D168, which have been reported to cause nervous lesions and mortality among pigeons. The pathotype identification of D167 and D168 have been done by mean death time (MDT) estimation in embryonated chicken eggs, intracerebral pathogenicity index estimation (ICPI) in day old chicks and infection of maternal antibody negative SPF chicks through different routes.PresentationItem Open Access Construction of fowlpox virus vector carrying viral 2A-peptide interlinked immunogenic genes of Avian Avulavirus-1 and IL-18 cytokine(2020-02) Rajasekaran, Ranjani; John Kirubaharan, J; TANUVASIn the present study, a Fowlpox virus (FWPV) plasmid vector was developed to carry three heterologous genes, which included two immunogenic genes of Avian Avulavirus-1 (AAv1) namely Fusion (F) and Haemagglutinin-neuraminidase (HN), and IL-18 cytokine. The FWPV plasmid vector, pJFA was constructed in two intermediate plasmid constructs, pJF7F9 and pJFHNIL18G. The construction of pJF7F9 involved cloning of F7 and F9 genes of FWPV with modifications in the F7-F9 intergenic region to carry AscI-SalI restriction enzyme (RE) sites. The construction of pJFHNIL18G involved sequential cloning of F, HN and IL- 18 genes under a synthetic early late promoter (P E/L). These three heterologous genes were interlinked with two viral 2A-peptides P2A and T2A. Immediately downstream of these three heterologous genes, a marker gene AcGFP was cloned under a late promoter (P11). This entire construct PE/L-F-P2A-HN-T2A-IL18-P11- AcGFP in pJFHNIL18G was released and was cloned into AscI-SalI RE site of pJF7F9 to obtain pJFA plasmid. ThepJFA plasmid upon transfection in chicken embryo fibroblast (CEF) cells previously infected with wild FWPVshowed AcGFP fluorescence from 48 hours post transfection. The expression of immunogenic genes F and HN was observed at 72 hours post transfection by western blotting at 66 kDa and 74 kDa respectively. Similarly, the expression of IL-18 cytokine was confirmed by ELISA. Thus, the expression of F, HN and IL-18 in the constructed viral 2A-peptide based FWPV vector confirms the usage of this strategy for future poultry vector vaccine constructions.ThesisItem Open Access EFFECT OF SUPPLEMENTATION OF YEAST ON PRODUCTION PERFORMANCE OF COMMERCIAL BROILER CHICKEN(TANUVAS, 2016) Atul Shankar, Patane; Premavalli, K; TANUVAS; Omprakash, AV; John Kirubaharan, JThe study was undertaken to evaluate the effect of yeast supplementation on the production performance of commercial broiler chicken. A total of 288 day old (Vencobb 400) chicks were randomly assigned in to four treatments with three replicates with 24 chicks in each replicate for 42 days experimental period.ThesisItem Open Access GENERATION OF RECOMBINANT NEWCASTLE DISEASE VIRUS (D58 Strain) BY REVERSE GENETICS(TANUVAS, 2016) Vidhya, M; John Kirubaharan, J; TANUVAS; Thangavelu, A; Kumanan, K; Ganesan, PIArticleItem Open Access Impact Study of Frontline Demonstration (FLD) on Efficacy of Oral Pellet Vaccine (D58) in Namakkal Chicken-1 in Backyard System of Poultry Rearing in Tiruvannamalai District(2020-03) Durairajan, R; Rajkumar, R; Rajalakshmi, S; John Kirubaharan, J; TANUVASOral pellet vaccine at the dose rate of one grain orally was dispensed to one month old Namakkal chickens-1 and 0 day, 21 days post vaccination blood sample in lter paper was collected for titre estimation using Haemagglutination inhibition test (HI). TANUVAS D58 oral pellet ND vaccine in the present study elicited protective antibody titre without interfering production and weight gain.ArticleItem Open Access Impact Study of Frontline Demonstration (FLD) on Efficacy of Oral Pellet Vaccine (D58) in Namakkal Chicken-1 in Backyard System of Poultry Rearing in Tiruvannamalai District(2020-03) Durairajan, R; Rajkumar, R; Rajalaksmi, S; John Kirubaharan, J; TANUVASOral pellet vaccine at the dose rate of one grain orally was dispensed to one month old Namakkal chickens-1 and 0 day, 21 days post vaccination blood sample in lter paper was collected for titre estimation using Haemagglutination inhibition test (HI). TANUVAS D58 oral pellet ND vaccine in the present study elicited protective antibody titre without interfering production and weight gain.PresentationItem Open Access In-vitro and in-vivo TLR-7 mRNA levels in response to lentogenic and velogenic pathotypes of Avian Avulavirus-1(2020-02) Chinju, C; Rajasekaran, Ranjani; Balakrishnan, G; Meenambigai, TV; John Kirubaharan, J; TANUVASTranscriptional studies in response to Avian Avulavirus-1 (AAv1) have gained importance in the recent past and have provided insight towards disease pathogenesis in chickens. The present study was focused towards elucidation of TLR-7 mRNA levels in response to D58 (lentogenic vaccine strain) and D162 (velogenic isolate) isolates of AAv1 in chicken embryo fibroblast cells (CEF) and chicken spleen at 1, 2, 3, 4 and 5dpi. The mRNA levels were determined by real-time PCR using SYBR-green chemistry. The TLR-7 mRNA levels were significantly up regulated to 18.90+0.03 in CEF cells and 12.33+0.02 in chicken spleen in response to lentogenic D58 at 1dpi. Similarly, in response to velogenic D162, TLR-7 mRNA levels were significantlyunregulated to 52.40+0.03 in CEF cells and 34.62+0.02 in chicken spleen. Later, in both CEF cells and chicken spleen, the mRNA levels of TLR-7 gradually declined from 2dpi in response to both lentogenic D58 and velogenic D162. TLR-7 has been reported to be a protein that plays specific role in detecting single stranded viral components and stimulating pro-inflammatory and anti-viral immune response. In the present study, the significantly up regulated levels of TLR-7 mRNA at 1dpi confirms the fact that TLR-7 levels were induced so as to stimulate necessary pro-inflammatory and anti-viral immune response against AAv-1. Further, it was observed that the virulence of AAv-1 pathotype also affected TLR-7 mRNA levels. This shows that TLR-7 mRNA levels vary with varying virulence of AAv1.ThesisItem Open Access INDUCTION OF INVATE IMMUNE RESPONSE BY INFECTIOUS BRONCHITIS VIRUS IN CHICKENS(TANUVAS, 2016) Baskar, VR; John Kirubaharan, J; TANUVAS; Thangavelu, A; Senthilkumar, TMAThe intention of the present work was to study the expression of certain proinflammatory, antiviral and immune regulatory cytokines by avian infectious bronchitis virus. The important objective is to determine the relative changes in the expression of certain genes involved in inflammation, virus elimination, immune regulation responsible for inducing innate immune response and comparing them with the Cell mediated immune responses and the humoral immune responses in trachea, lungs and spleen of chickens.PresentationItem Open Access Mutational Analysis of Nucleocapsid (N) Protein of Newcastle Disease Virus Using a Minigenome System(2020-02) Vidhya, M; John Kirubaharan, J; Rajalakshmi, S; Rajasekaran, Ranjini; TANUVASThe Nuclepcapsid protein (N) protein of Newcastle disease virus (NDV), synonymous with Avian Avula virus-1(AAv-1) functions primarily to encapsidate the virus genome for transcription, replication and packaging. In addition to its role in replication, N gene is the major immunogenic and conserved gene of NDV. Identifying mutation permissive regions in the N protein provides a potential approach for NDV marker vaccine. In the present study mutations introduced in to the NP gene in the form of epitope replacement between amino acid positions 446-453 was analysed using an intracellular NDVD58-GFP minigenome assay for its effect on minigenome RNA synthesis and encapsidation.Site directed mutagenesis was carried out in pCIneo N plasmid to replace the NDV epitope 446(QFLDLMRAV)453 with a marker epitope TAVSPTTLR.The mutated pCIneo N plasmid was co-transfected with other NDV expression plasmids pCIneo L, pCIneo P and pNDVD58-GFP minigenome plasmid in BSR/T7 cells. GFP expression was observed 72 hours post transfection indicating a functional minigenome. Further the presence of encapsidated minigenome RNA in transfected cell supernatants were also confirmed by Real time PCR. These results indicate that the mutations did not affect the functionality of the Nucleoprotein and can serve as marker epitopes for NP based ND marker vaccine.ThesisItem Open Access PATHOLOGY, PATHOGENESIS, MOLECULAR DIAGNOSIS AND ASSESSMENT OF PROTECTIVE IMMUNITY OF VIBRO ALGINOLYTICUS VACCINE IN COBIA (RADIYCEUTRON CANASUM)(TANUVAS, 2016) Ramesh Kumar, P; Balachandran, C; TANUVAS; Vairamuthu, S; Dhinakar Raj, G; John Kirubaharan, JThe objectives of the study are (i) to rccord the natural occurrence of vibriosis in cobia, (ii) to investigate the pathological and histomorphological features in experimental Vibrio alginolyricus infection in cobia (Rachycentron canadum) and (iii) to evaluate the protective effect of vaccine against Valginolyticus infection.PresentationItem Open Access Pathotyping of Newcastle disease virus by mean death time and TaqMan MGB probe real time PCR assay; a comparative study(2020-02) Parthiban, S; John Kirubaharan, J; Ramesh, A; Vidhya, M; et al.; TANUVASNewcastle disease (ND) is a standout amongst the most significant diseases of poultry throughout the world. The disease is associated with serious respiratory, gastrointestinal, and neurological lesions in chicken. The etiological agent of the disease is an Avian avulavirus 1, synonymously referred as Newcastle disease virus (NDV). In vivo pathogenicity assaying is sensitive and specific pathotyping tools for detection and identification of NDV used until the recent past. In this study twelve NDV isolates (Isolate numbers 463, 464, 475, 476, 122-17C, 122-17D, 122-17E, 128-17A, 128-17D, 137, 139, 141) available in the department of Veterinary Microbiology, Madras Veterinary College (MVC), Chennai are subjected for pathotyping using mean death time (MDT) in specific pathogen free (SPF) embryonated eggs and TaqMan minor groove binding (MGB) probe real time PCR assay. Pathotyping based on the MDT revealed two NDV isolates (isolate no. 476 and 128-17D) as velogenic strains and remaining ten NDV isolates as lentogenic strains. Pathotyping based on TaqMan MGB probe real time PCR assay revealed six NDV isolates (476, 128-17D, 463, 464, 475, 137) as velogenic/mesosogenic strains and remaing six NDV isolates (122-17C, 122-17D, 122-17E 128-17A, 139, 141) as lentogenic strains. Four NDV isolates (463, 464, 475, 137) which are pathotyped as lentogenic strains by MDT were pathotyped as velogenic/mesosogenic strains by real time PCR assay this shows the higher sensitivity of TaqMan MGB probe real time PCR assay in pathotyping of NDV over conventional MDT.ArticleItem Open Access Phylogenetic Analysis of 4b Core Protein Gene of Fowlpox Virus Field Isolates and Commercial Vaccines of Indian Origin(2019-03) Rajasekaran, Ranjani; John Kirubaharan, J; Rajalakshmi, S; Vidya, M; TANUVASThe highly conserved P4b gene Was ampli- fied from two field isolates (FWPV-W1 and FWPV-W2) and two commercial vaccines (FWPV-G and FWPV-V) of fowlpox virus and was subjected nucleotide sequencing. The nucle- otide sequence (512 nucleotides) showed 100% sequence similarity in BLAST analysis with other FWPV sequences available in GenBank. Phylogenetic analysis of partial P4b gene (495 nucleotides) sequences of both the field isolates and both the commercial vaccines was done using Neighbour-joining method with 1000 bootstrap replications. In the P410 partial gene based phylogenetic tree, both the field isolates and both the commercial vaccines were grouped under sub-clade A1 along with other fowlpox viruses.ArticleItem Open Access Pro-inflammatory cytokine and apoptotic gene mRNA levels against lentogenic and velogenic Newcastle disease virus pathotypes in in-vivo and in-vitro biological systems(2019-04) Ranjani, Rajasekaran; John Kirubaharan, J; Vidhya, M, et al.; TANUVASKnowledge on the influence of pro-inflammatory cytokine and apoptotic gene mRNA levels in the pathogenesis of Indian field isolates of Newcastle disease virus (NDV) is little. In this study, cytokine mRNA levels were elucidated in spleen of chickens (in-vivo) and chicken embryo fibroblast cells (in-vitro) infected with lentogenic D58 strain and viscerotropic velogenic D165 isolate until five days post infection (dpi). In spleen of chickens infected with D165, maximum upregulation of pro-inflammatory cytokines (IL-1, IL-6, TNF-), chemokine (IL-8) and apoptotic gene (Caspase-8) at 3dpi correlated with the onset of severe clinical signs and necrotic histopathological lesions in spleen, proventriculus, intestine and caecal tonsil of chickens. Similarly, in CEF cells infected with D165, upregulation of pro-inflammatory cytokine and apoptotic gene mRNA levels correlated with the appearance of CPE. In spleen of chickens and CEF cells infected with D58, there was comparatively minimal upregulation of pro-inflammatory cytokine and apoptotic gene mRNA levels which did not cause histopathological changes in tissues and CPE formation in CEF cells. In both in-vivo and in-vitro systems, upregulation of anti-inflammatory cytokine IL-10 showed inhibitory effects on the mRNA levels of pro-inflammatory cytokines. Thus, this study reports variation in the cytokine mRNA levels elucidated in response to two different pathotypes isolated from India and associates the same with the clinical signs and pathological lesions produced during the course of ND.ThesisItem Open Access SEROLOGICAL, VIROLOGICAL AND MOLECULAR DETECTION OF JAPANESE ENCEPHALITIS VIRUS INFECTION IN DOMESTIC ANIMALS(TANUVAS, 2016) Kamei Kakhulan; Ravikumar, G; TANUVAS; John Kirubaharan, J; Gomathinayagam, SJapanese encephalitis (IE) is an emerging zoonotic and arboviral disease of public health significance and endemic in South East Asia and India. Among domestic animals, pigs are the main amplifying host in which it can cause reproductive failure in pregnant sows. Horses and human exhibit encephalitis and arc dead-end hosts. There is no effective diagnostic assay developed in lndia and it is important to develop rapid, sensitive assay to detect JE virus specific antibody and viral RNA to prevent future threat to the state and the country.ArticleItem Open Access STUDIES ON TAIL AND FIN - ROT (BACTERIOSES PINNARUM) DISEASE IN LOMMON CARPS (CYPRINUS CARPlS)(1995) John Kirubaharan, J; Raveneswaran, K; Justin William, B; Balachandran, S; TANUVASAn outbreak of tail and fin-rot was reported in common carp (Cyprinuss carpis) fishes reared in irrigation tanks of Agricultural College and Research Institute, Kullikulam. The mortality rate was upto 50%. Affected fishes developed erosions and ulcers in the fin and tail. with impaired movement.PresentationItem Open Access SYBR Green-based duplex real time PCR method for detection of Infectious bronchitis virus, Mass serotype(2020-02) Rajalakshmi, S; Shilpa, P; John Kirubaharan, J; Vidhya, M; et al.; TANUVASInfectious bronchitis (IB) is a highly contagious economically important viral disease of poultry, caused by Infectious bronchitis virus (IBV) belonging to the family Coronaviridae. The Massachusetts (Mass) serotype of IBV is being used worldwide for regular vaccination against IB since several years. The continuous application of the Mass strain of IBV for vaccination and mutating nature of virus has yielded a selection pressure, resulting in the emergence of field strains of non-Mass serotypes. Currently, rapid detection of IBV infection in the poultry flocks amidst other upper-respiratory avian pathogens is a major challenge, which demands sensitive and rapid diagnostic methods. The SYBR Green – based RT PCR assay has been proven to be one of the most effective and sensitive tools for such a diagnosis. A duplex SYBR Green – basedreal time RT PCR assay has been designed for the simultaneous detection of IBV and identification of the Mass serotype in a single reaction based on melt curve analysis. IBV-specific UTR primers and IBV-Mass- S1-specific primers used in the duplex real time RT-PCR yielded curves of amplification with two specific melting curves (Tm1 = 81oC ± 0.5oC and Tm2 = 84oC ± 0.7oC) for IBV – Mass serotype. Owing to the fact that IBV serotypes other than Mass do not exist in India, the assay was standardised using a known Mass serotype (H120 vaccine strain) and a well-characterised field isolate IBV –B17. The assay did not yield amplification curves when viruses other than IBV were assessed. This duplex realtime PCR can thus be used as a rapid molecular diagnostic tool for identification of IB outbreaks. Further evaluation of the assay for the simultaneous detection and differentiation of IBV Mass and non-Mass serotypes would throw more light for the effective control of IB, especially in vaccinated flocks.