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ThesisItem Restricted ISOLATION AND MOLECULAR CHARACTERIZATION OF Campylobacter spp. FROM CHICKEN(2021) KAVIN G; TANUVAS; ANANDA CHITRA M; SHOBA K; MEENAMBIGAI TVCampylobacter spp. are the zoonotic bacteria which are the most common cause of food borne gastroenteritis around the world. The link between human campylobacteriosis and poultry has been well established. This study was aimed to isolate Campylobacter spp. from chicken and characterize them with molecular methods like multiplex PCR, Loop Mediated Isothermal Amplification assay (LAMP) and Multilocus Sequence Typing (MLST). A total of 191 chicken caecal mucosal scraping were collected from Chennai, Namakkal, Krishnagiri, Erode and Coimbatore districts of Tamil Nadu. Bacterial isolation was done by plating on blood free Campylobacter broth added with 3% agar agar, CCDA supplement and Campylobacter growth supplement IV. Identification of Campylobacter species was done by multiplex PCR and they were also subjected to LAMP assay. The prevalence of Campylobacter spp., C. coli and C. jejuni was found to be 28% (54/191), 7% (14/191), 0.5% (1/191), respectively. The minimum inhibitory concentration (MIC) values for eight antimicrobials – azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid and tetracycline were obtained by microdilution resazurin assay. Fourteen C. coli isolates showed 100% resistance to nalidixic acid and higher resistance to tetracycline (92.8%), erythromycin (71.4%), clindamycin (71.4%) and azithromycin (64.2%). All C. coli isolates were sensitive to chloramphenicol and higher sensitivity to ciprofloxacin (78.5%) and gentamicin (71.4%) was observed. One C. jejuni isolate was resistant to azithromycin, tetracycline, nalidixic acid and sensitive to all other antimicrobials used. Multilocus sequence typing was carried out for five representative C. coli isolates. Three C. coli isolates had the existing sequence types ST-899, ST- 872 and ST-9108. Two novel sequence types were found in this study and they were ST-10872 and ST-11031. However, all the STs belonged to the same clonal complex of ST-828. This is the first report with characterization of C. coli isolates by MLST in IndiaThesisItem Restricted DEVELOPMENT OF A PEPTIDE BASED IMMUNOASSAY FOR DETECTION OF ANTIBODIES TO PESTE DES PETITS RUMINANTS VIRUS (PPRV)(2021) MAHALAKSHMI N; TANUVAS; THANGAVELU A; THIRUMURUGAAN KG; VIDHYA MThe focus of the present study is to optimize and develop peptide-based immunoassay for the detection of antibodies to PPRV in sheep and goat using synthetic peptides derived from immunoreactive and immunodominant B cell epitopes predicted from Nucleoprotein (N) and Haemagglutinin (H) proteins of PPRV. Four 8 mer B cell epitopes each for N and H proteins of PPRV namely N1, N2, H1 and H2 were predicted using bioinformatics tools such as abcPRED, BepiPRED 2.0, Phyre 2.0 and IEDB using various indices such as linearity, surface probability, surface accessibility, hydrophilicity, flexibility and antigenicity and were custom synthesized by linking two 8 mer antigenic peptides with GGGS linker and tested for their immunoreactivity. Based on immunoreactivity the N1, H2 and a cock tail of N1 and H2 was selected as antigen for peptide ELISA. Serum samples collected at random from sheep and goats vaccinated with live attenuated PPRV vaccine and serum samples collected at specific intervals of 3, 7, 10, 14, 17, 35 days of post vaccination from sheep and goats were used for optimizing the peptide-based immunoassay for PPRV. A total of eighty-eight random serum (Goat = 47, sheep = 41) and Hundred and thirty-five serum samples (Goat = 73, sheep = 62) at specific intervals of vaccination were collected from Post Graduate Research Institute in Animal Sciences (PGRIAS), Kattuppakkam and Instructional Livestock Farm Complex (ILFC), TANUVAS to have a wide range of samples with varying antibody titres. The serum samples were initially screened with competitive ELISA kit (IVRI, Mukteshwar) before proceeding to peptide ELISA. In competitive ELISA, 106 goat serum samples and 96 sheep serum samples were found to be sero-positive. Fourteen goat serum samples and seven sheep serum sample were shown to be sero negative.ThesisItem Restricted MOLECULAR CHARACTERIZATION OF LUMPY SKIN DISEASE VIRUS AND EXPRESSION OF MAJOR IMMUNO-DOMINANT PROTEIN/S FOR THEIR DIAGNOSTIC EVALUATION(2022) PRABHU M; TANUVAS; MALMARUGAN S; BALAKRISHNAN G; LAKSHMI PRASANTH T; RAJAGUNALAN SLumpy skin disease is a transboundary poxviral disease of cattle and Asian water buffalo caused by Lumpy Skin Disease virus (LSDV). India reported its first emergence in 2019 from Odisha state followed by spread to several states including Tamil Nadu. The present study was undertaken to gather necessary data regarding the molecular characterization of the field isolates with an aim to develop diagnostics. A total of 108 samples were collected from twelve districts of Tamil Nadu and one village from Karnataka for a period of two years from August 2020 to July 2022 from suspected cases of LSD. Initial screening was done by P32 and F gene based PCRs in which, 93.8 per cent of scabs (n=76) and 7.4% of unclotted blood samples (n=2) were found positive. Scab tissues (n=10) positive in PCR selected for virus isolation in Embryonated Chicken Eggs (ECE) revealed the characteristic pock lesions on CAM during third and fourth passages. One isolate initially passaged in ECE was isolated using BHK 21 cells showed CPE after 48-60 hpi during the third passage. The harvested isolates were further confirmed by PCR. Molecular characterization based on P32, F, RPO30 and GPCR genes revealed close proximity of the Indian isolates from the study to other LSDV field isolates reported globally. Whereas the vaccine strains of LSDV clustered separately. Further, though closely related, SPPV and GTPV clustered distantly. The GPCR sequence alignment revealed unique signatures (A11, T12, T34, S99, and P199) to confirm its identity as LSDV. The sequences contained 12 nulceotide insertion (nt94 – 105) and corresponding four amino acid (TILS) (aa 30-33). The obtained isolates were more closely related to Kenyan strains and strains from neighboring countries such as Bangladesh, Myanmar and Nepal. This confirms the common exotic source of LSDV responsible for outbreaks in these countries and the transboundary spread across borders.ThesisItem Restricted DEVELOPMENT OF NANOPARTICLES COUPLED INACTIVATED AND MULTI EPITOPE BASED RECOMBINANT INFECTIOUS LARYNGOTRACHEITIS VACCINE(2022) PONNUSAMY P; TANUVAS; SUKUMAR K; SARAVANAN S; RAJA A; SRINIVASAN PThe focus of the present study was to isolate and characterize the infectious laryngotracheitis virus using ICP4 and TK genes and to develop chitosan and PLGA nanoparticles coupled inactivated ILT vaccine. Another focus was to design and express multi epitope based recombinant protein and to develop chitosan and PLGA nanoparticles coupled multiepitope recombinant vaccine and evaluating its immune responses in chickens. A total of 27 out of 29 ILT suspected flocks were found to be positive by PCR using ICP4 and TK genes and were observed to have amplicon size of 635 and 649 bp respectively. The PCR positive tracheal samples were subjected to isolation of ILTV in 12 days old embryonated chicken eggs through chorioallantoic membrane route. Multiple pock lesions and thickening were noticed in the infected chorioallantoic membrane of embryonated chicken eggs after 120 hr of infection. Histopathological examination of infected CAM showed hemorrhage, congestion, epithelial hyperplasia with lymphocytic and heterophilic infiltration and the presence of syncytial cells with intranuclear inclusion bodies. Out of 27 field isolates, the four field isolates of ILTV were sequenced for both ICP4 and TK genes. The ICP4 gene sequences of field ILTV isolates were observed to have identical nucleotide and amino acid sequences with CEO vaccine and vaccine-like isolates. All the four sequences had six nucleotide mutations at residues 3875 (T→C), 3927(C→T), 3951(T→C), 3982(G→A), 4017(G→A), and 4309(A→T) in comparison to TCO vaccine and vaccine-like strains. The ILTV sequences of this study had three unique amino acid mutations at residues 1292 (P→L), 1328 (G→R), and 1437 (T→S) in comparison to TCO vaccine and vaccine-like strains. The TK gene of four field ILTV sequences was observed to have four nucleotide mutations at positions 441, 540, 594, and 755. The four ILTV sequences of TK gene had one unique amino acid mutation at residues 252. The amino acid threonine was found at position 252 in all the four TK gene sequences of this study as well as low virulent and vaccine reference strains but replaced with methionine in virulent reference strains.ThesisItem Restricted MOLECULAR CHARACTERIZATION OF CIRCULATING PORCINE CIRCOVIRUS 2 (PCV2) AND DEVELOPMENT OF INACTIVATED VACCINE(2022) PARTHIBAN S; TANUVAS; RAMESH A; DHINAKAR RAJ G; HEMALATHA S; ANBU KUMAR KThe present study was aimed at molecular characterization of the circulating porcine circovirus 2 (PCV2) under field condition in southern India and development of a inactivated vaccine. A total of 434 field samples comprising of serum (n=273), post mortem tissues (n=109) and swabs from natural orifices (n=52) collected from swine populations of southern India during the period from 2019 to 2021 were used in this study. Of the 434 field samples screened, 53(12.2%) samples were found positive for PCV2 infections by ORF2 gene based diagnostic polymerase chain reaction (PCR) assay. Statistical analysis of PCV2 positivity within breed, age, sex and vaccination status revealed no significant difference (P>0.05) but there was a significant difference (P<0.05) in the positivity of PCV2 among healthy and suspected swine populations. ELISA based PCV2 specific antibody to capsid protein screening in 176 serum samples collected from non-vaccinated animals (based on history) revealed seropositivity of 44.8% (n=79). Screening for PCV1 and PCV3 circulation in the field and their co-infection studies revealed, 2.99% (n=13) positivity for PCV1 genome and 0.69% (n=3) positivity for PCV3 genome. The PCV3 incidence was documented for the first time in southern part of India. The PCV2 co-infection analysis revealed, 11.32% (n=6) co-infection of PCV2 with PCV1 and 1.88% (n=1) co-infection of PCV2 with PCV3.ThesisItem Restricted DEVELOPMENT OF DNA VACCEVE FOR AVIAN INFECTIOUS LARYNGOTRACHEITIS(2021) Jaisree S; TANUVAS; Shoba K; Ramesh A; Vijayarani K; Vairamuthu SThe present study was carried out to analyse the genetic relationship of infectious laryngotracheitis virus (ILTV) isolates and to develop DNA vaccine for avian infectious laryngotracheitis (ILT). A total of thirty three ILTV suspected samples were included in this study. Affected birds showed clinical signs like depression, gasping, nasal discharge with characteristic pump handle respiration. Haemorrhagic tracheitis and the presence caseous plug in tracheal lumen were the characteristic post mortem lesions. On histopathological examination, trachea from affected birds showed desquamation and necrosis of tracheal epithelium, syncytia, haemorrhage and fibrinous exudate, infiltration of mononuclear cells and eosinophilic intranuclear inclusion bodies. The ILTV suspected samples were screened by polymerase chain reaction (PCR) targeting partial ICP4 gene. Out of thirty three samples, sixteen yielded PCR product size of 635 bp and were found positive. Samples that were positive by PCR from a single farm were pooled and inoculated into 10-12 days old specific pathogen fi'ee embryonated chicken eggs via CAM route. Out of ten samples, isolation was successful with nine. The lesions observed on CAM include congestion and haemorrhage in first passage, thickened CAM in second passage and pock lesions.ThesisItem Restricted MOLECULAR CHARACTERIZATION OF FOWL ADENOVIRUS IN CHICKEN(2019) Chitradevi, S; TANUVAS; Sukumar, K; Suresh, P; Balasubramaniam, GA; Kannan, DThe present study was undertaken to characterize indigenous fowl adenovirus in chicken and assess the pathology and viral load of fowl adenovirus infected tissues. The incidences of FAdV infection in commercial broiler birds were observed in the age group of day old to 42 days. The maximum incidence was noticed in the age group of 30 to 40 days (55%) followed by 20 to 30 days (20%), 40 to 50 days (12.5%), 0 to 10 days (10 %) and 10 to 20 days (2.5 %). The maximum mortality of 20 per cent was recorded in 30 to 40 days of age group. In case of broiler breeder mortality of 2.5 per cent was observed between 49 and 56 days and in commercial layer grower chicken, the age group affected was 42 to 91 days with mortality ranging from 0.3 to 7.7 per cent.ThesisItem Restricted ISOLATION AND DETECTION OF PATHOGENIC LEPTOSPIRA SPECIFIC NUCLEIC ACID SEQUENCES BY POLYMERASE CHAIN REACTION(2018) Eazhisai, R; Ravikumar, G; Senthilkumar, TMA; Manimaran, K; TANUVASLeptospirosis is a globally emerging zoonotic disease that has significant impact on animal and human health. A study was carried out to isolate and detect pathogenic Leptospira specific nucleic acid sequence by polymerase chain reaction. Out of 133 samples collected from various animals, two isolates from rat urine sample was obtained. The overall percentage of isolation of this study was low as 1.5, whereas the percentage of isolation in rats were 12.5. The genotypic characterisation of the two rat isolates and isolates of dog, cow and human from the Zoonoses Research Laboratory revealed amplification in G1/G2 (285 bp), LipL32 (756 bp), LipL21 (561 bp) and Loa22 (257 bp), which indicated that all the isolates belonged to pathogenic type. Also, the positioning of isolates in the pathogenic clade was further analysed by gene sequencing with partial rpoB gene and confirmed that all the isolates were pathogenic type.ThesisItem Open Access QUANTITATIVE PROFILING OF MAREK’S DISEASE VIRUS IN VACCINATED LAYER CHICKEN(TANUVAS, Chennai, 2017) Senthilkumar, K.; Suresh, P.; Sukumar, K.; Saravanan, S.; TANUVASThe present study was undertaken to quantify MDVl, MDV2 and MDV3 load in vaccinated commercial layer flocks at 7, 14, 21, 28, 35 and 60-90 days post vaccination (dpv) and correlate the MDVl load with vaccine viral load (MDV2, MDV3). A total of 25 commercial layer flocks were selected in and around Namakkal district of Tamil Nadu. The feather and blood samples were collected from 25 commercial layer flocks at 7, 14, 21, 28, 35 and 60-90 dpv.
