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PresentationItem Open Access Quantification and initial profiling of salivary and cervico vaginal fluid proteins in murrah buffalos during estrus and proestrus using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(2020-02) Kavitha, K; Suresh Kumar, R; Krishnakumar, K; Sarath, T; Vijayarani, K; Arunmozhi, N; Balasubramaniyan, S; Leena, KB; Abhinaya, K; TANUVASSaliva and cervico vaginal fluid (CVF) are considered good non invasive biomarkers for detection of estrus in buffalo as external signs are not significant in these animals. Based on the linkage between salivary proteins and estrus, the present study was aimed to profile the salivary and CVF proteins in Murrahbuffaloes (atproestrus and estrus) using single dimension (1-D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).It was observed that the saliva collected from sevenestrus buffalo had proteins with the molecular weight of 26kDa, 31kDa, 50kDa, 70kDa and 76kDa whereas saliva from eight proestrus buffalo had proteins with molecular weight of 48kDa, 62kDa, 70kDa and 75kDa. Based on published data by Shashikumaret al., (2018) the proteins in the estrus and proestrus correspond to short palate lung and nasalepithelium carcinoma-associated protein 2B (26.5kDa), tRNA-methyltransferase catalytic subunit TRM61A, CCDC85A protein (50kDa), Heat shock 70kDa protein (70kDa), Testin (48kDa), Max-binding protein MNT and Rho guanine nucleotide exchange factor 37 (76.1kDa) or Serine/threonine-protein phosphatese 2A 65kDa regulatory subunit A beta isoform (76.7kDa). Molecular weight of proteins observed in the CVF collected during estrus (n=4) where predominantly of 70kDa and 78kDa which correspond to Heat shock 70kDa protein and Receptor-type tyrosine-protein phosphatase epsilon (78.49kDa) respectively based on the published data by Muthukumaret al.(2014). Thus these findings provide a lead for further analysis and evaluation of the proteins for estrus detection in buffalo.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.PresentationItem Open Access Infectious bursal disease and ndv infection in a brown layer farm(2020-02) Jayanthi, N; Jaisree, S; Bharathi, R; Kalai Selvi, G; Karthik, K; Anand Chitra, M; Senthilkumar, TMA; Harikrishnan, TJ; Dhinakar Raj, G; TANUVASInfectious bursal disease (IBD), an acute, highly contagious viral disease affects the immune system of young chickens, caused by infectious bursal disease virus (IBDV) belonging to the genus Avibirnavirus within the family Birnaviridae. Though heavy outbreaks are less it continues to lurk in Tamil Nadu. A brown layer chicken farm situated in Thiruvallur with a history of sudden death, clinically ill birds showing dullness and watery diarrhoea was presented for necropsy. The birds were vaccinated as per schedule for NDV. The farm had a stock of about five hundred birds of which there was a mortality percentage of 14.4 when the birds were presented for necropsy. Necropsy examination revealed severe hemorrhages in the breast and thigh muscles, pale liver mildly oedematous enlarged pale bursa with stray petechiae in the proventriculus. Molecular screening for viral and bacterial diseases revealed the presence of IBD by PCR and NDV by RT- PCR while it was negative for IB virus. E.coli could be detected in heart blood swab. Antibiogram revealed nil sensitivity among the antibiotics commonly used. It was intermediate sensitive to cefotaxime, amoxyclav, norfloxacin and ciprofloxacin and was resistant to gentamicin,sulphadimidine and tetracycline. Histopathological examination revealed lymphoid cell depletion in the bursa, lymphocytolysis and few cystic spaces. This case documents the presence of IBD infection in areas around Chennai. Infection of NDV as known was probably due to immunosuppression caused by IBDV as the flocks were vaccinated for NDV as per schedule.PresentationItem Open Access Cardiomyopathy in a chestnut- fronted macaw (Ara severus)(2020-02) Jayanthi, N; Saahithya, R; Pazhanivel, N; Venkata Sudhakar Rao, Ganne; TANUVASThe life expectancy of macaw ranges from 35- 50 years. Pet birds are many times deprived of conveniences as compared to their free-living counterparts. Captivity leads to boredom due to restricted area, lack of exercise and psychological changes in spite of good maintenance and precipitates into diseases in the long run of which one such condition is the risk of cardiovascular disease. A female severe macaw (chestnut fronted macaw) aged 7.5 years was presented for necropsy with a history of severe loss of feathers, wasting, chronic illness and death with a suspicion of avian tuberculosis. External examination revealed aberrations on the skin, erosions on the beak and injury on the right wing suggestive of self-mutilated wound. Grossly, carcass appeared dehydrated, blanched mucous membrane, thin watery blood, faecal material aroundthe cloaca and matting of feathers. The right leg was twisted and slightly deformed. Liver was swollen, dark brown in colour with enhanced lobular pattern and diffuse pale areas. Heart had a roughly elongated patchy pale area (approx. 0.5mm by 0.85mm) in the left ventricular myocardium, solitary nodule in the distal femur with cheesy exudate, meningeal congestion and strikingly all the bones were highly brittle and porous with grey white bone marrow. The cheesy exudate was negativefor acid fast organisms by Ziehl–Neelsen staining. Histopathologically the chief lesion was multifocal necrosis of cardiac myocytes and perivascular degeneration of fat in the cardiac vessels suggestive of cardio myopathy. In addition, multifocal coagulative necrosis of the liver and diffuse congestion of the lungs was noticed. This documentation highlights the need for more studies on avian cardiology as clinical signs on cardiac compromise seems to be subtle and owners fail to recognise these changes.PresentationItem Open Access Age related changes in microanatomical features of the stomach in guinea pig (Cavia porcellus)(2020-02) Raja, K; Ushakumary, S; Rajathi, S; Kannan, TA; Satyamoorthy, OR; Ramesh, Geetha; TANUVASThe present work was conducted on the stomach of guinea pigs from neonate to adults (0-2 wks, 2-8 wks, 8-16 wks and 16-32 wks ) to study the histological changes in three regions of stomach. In the present study Tunica mucosa of cardiac region was lined by Non keratinized stratified squamous epithelium with visible stratum granulosum. The mucous membrane of fundic and pyloric parts of the stomach was lined by simple columnar epithelium. Simple tubular cardiac glands were lined mainly by mucous cells along with few parietal cells, enterochromaffin and undifferentiated cells. In fundic glands mucous cells and parietal cells were predominantly in both upper and lower half of the glands where as chief cells and enteroendocrine cells were predominantly occupied in the lower half of the fundic glands. Pyloric glands were simple branched tubular glands were lined by mucous acinar cells. Tunica muscularis of cardiac region comprised of skeletal and smooth muscle fibres whereas other two regions were comprised of smooth muscle fibres. The mean thickness of tunica mucosa was the maximum in fundic region and the minimum in pyloric region The mean thickness of tunica muscularis externa was the maximum in cardiac region and the minimum in pyloric region.PresentationItem Open Access A novel doorstep canine haemoprotozoan diagnostic service – A preliminary survey in Chennai(2020-02) Venkatesan, M; Nivetha, S; Azhiahanambi, P; Raman, M; Dhinakar Raj, G; TANUVASCanine vector-borne diseases pose a major health problem in dogs globally. A door-step diagnostic service for diagnosis of canine haemoprotozoan diseases by polymerase chain reaction (PCR) was launched in collaboration with the Translational Research Platform for Veterinary Biologicals (TRPVB). The aim of this service was to provide door-step diagnosis to practicing veterinarians by collecting the samples from their clinics and providing results with 24 hours. Initially 2 PCRs were done to identify the presence of Babesia and E. canis genomes through amplification of 619 bp and 377 bp products respectively. Then a nested PCR was done independently on all samples to identify the Babesia species, Babesia gibsoni and / or Babesia canis genomes through amplification of 195 bp and 197 bp nested PCR products respectively. Of the 55 samplestested, 17 samples were positive for Babesia genus specific primers and 4 for E. canis. When nested PCR was used for detection of Babesia sp., 31 was positive for Babesia gibsoni and 10 for Babesia canis. Six samples were positive for both Babesia gibsoni and Babesia canis. Three samples were positive for both Babesia gibsoni and E. canis. None of the samples were positive for Babesia canis and E. canis. While 35 samples were positive for either Babesia in nested PCR only 17 were positive in single PCR. This suggests that use of nested PCR is more sensitive and hence more applicable in clinical settings in both Babesia identification and speciation.PresentationItem Open Access Expression of hepcidin and ferroportin in full term swine placenta(2020-02) Rajamanickam, K; Leela, V; Suganya, G; Hayath Basha, Sabiha; Parthiban, M; Pazhanivel, N; Mangala Gowri, A; TANUVASIn swine, even though the pregnant sows were with iron abundance, the inborn iron reserve of piglets were compromised indicating the insufficiency of the molecular machinery involved in placental iron transfer from sow to fetus. Hepcidin and ferroportin are the two important peptides regulating systemic iron homeostasis. Hepcidin is synthesized from heptocytes in response to changes in iron level, inflammation and oxidative stress conditions. It controls the iron exportation from cells by acting on membrane iron exporter, ferroportin. Here we investigated, for the first time, the molecular and morphological expression of iron regulatory proteins like hepcidin and ferroportin in placenta of full term pregnant sows and their association with placental iron abundance. Presence of hepcidin and ferroportin sequence in placental DNA was confirmed through PCR and sequencing (Accession number: Hepcidin - MN579557, Ferroportin - MN565887). Hepcidin and ferroportin proteins have also been revealed by western blot analysis and immunohistochemistry. The immunoreactivity for both proteins was observed in the cytoplasm and membrane of the trophoblastic cells of the placenta. Placental mRNA abundance of hepcidin was associated with local stimuli of hepcidin production like oxidative stress and pro-inflammatory cytokines expression. Hepcidin expression was also positively associated with the placental non-heme iron reserve. This indicates that hepcidin has a role in materno-fetal iron transfer by controlling local iron flux. Further studies are warranted to elucidate the use of hepcidin and ferroportin for improving iron transfer to the developing fetus and thereby inborn iron reserve in piglets can be increased, which may prevent the incidence of piglet anaemia.PresentationItem Open Access Growth curve analysis in Mecheri sheep(2020-02) Balan, C; Kathiravan, G; Thirunavakkarasu, M; Jeichitra, V; TANUVASLivestock keeping is an important avocation for the poor in the developing world, especially in India, contributing to rural livelihood and offering pathways to come out of poverty. Among the livestock, the sheep is an economically important species contributing greatly to the Indian agrarian economy, playing a vital role for the livelihood of a large number of small and marginal farmers and landless labourers. In addition to meat and wool, manure from sheep is an important source of organic fertilizer, especially in southern states where they are penned on fallow lands for increasing soil fertility. Mecheri sheep are one of the recognized breeds of sheep in Tamil Nadu and reared mainly for meat with the primary by-product as skin. They constitute 24.28 per cent of total sheep population of Tamil Nadu.Data on growth traits of Mecheri lambs born between the years 1991 and 2014 in Mecheri Sheep Research Station, Pottaneri were utilized for this study. The overall least-squares means (± S.E.) of body weights at birth, 3, 6, 9 and 12 months of age were 2.37 (± 0.008), 9.61 (± 0.062), 12.56 (± 0.071), 15.32 (± 0.085) and 17.87 (± 0.102) kg, respectively. Lambs born during the months of July-September had the highest (2.44±0.018 kg) birth weight and the least birth weight (2.14 ± 0.014 kg) was found among lambs born during the months January-March. Similarly, lambs born during the periods July-September and January-March had the highest (10.06 ± 0.127 kg) and least (7.74 ± 0.099 kg) weaning weights, respectively. Also, the highest yearling weight (18.70 ± 0.216 kg) was found in lambs born during the months July-September and the least weight (16.48 ± 0.177) was found during the months January- March. The overall least-squares means of Absolute Growth Rate (g/day) measures over the age intervals of birth to 3 months, 3 to 6 months, 6 to 9 months and 9 to 12 months were 79.97 ± 0.56, 40.01 ± 0.44, 33.73 ± 0.46 and 31.34 ± 0.86, respectively. Among non-linear growth models fitted to evaluate growth pattern, the Brody function was found to be the best fitted model in describing the growth curve patterns of both ram and ewe lambs of Mecheri sheep, which was followed by Von Bertlanffy, Gompertz, Logistic and Richards models in the order of the goodness of fit. The predicted body weights through Brody model at birth, 3, 6, 9 and12 months of age were 2.57, 8.92, 13.03, 15.70 and 17.50 kg, respectively in Mecheri sheep. The study showed that the Brody function adequately fitted the growth rate of Mecheri sheep, which is a medium sized breed with moderate growth. Further, Influences of environmental factor such as months of lambing and sex of the lambs born were well captured by the Brody model.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.