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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

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2019 - 201912020 - 20231
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Subject: Biochemistry ×
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    ThesisItemOpen Access
    Studies on thermostable Newcastle Disease Virus induced cytokine expression in chicken embryo fibroblas
    (LUVAS Hisar, 2023-04) Neetu Bala; Sangwan,Nirmal
    Newcastle Disease (ND) is one of the endemic viral diseases of poultry worldwide and is caused by Newcastle Disease Virus (NDV). It is member of the genus Avulavirus in the family Paramyxoviridae. It can not only cause the death of birds but also has a great negative impact on the productivity of surviving birds, such as impaired growth, poor feed conversion, reduced egg production, and decreased fertility and hatchability of eggs. NDV has the ability to reproduce in a variety of cell types, including chicken embryo fibroblasts. Infecting host cells is essential for viral pathogenicity. Cytopathic effects (CPE) can be detected following NDV infection, and these effects are linked to the strain's virulence. ND is being managed and prevented via vaccines. About all commercially marketed ND vaccines need to be refrigerated and these start to lose their effectiveness after 1-2 hours at room temperature. Therefore, the present research was planned to study the role of cytokines in immune response to thermostable NDV, and to find out an effective potential candidate for a thermostable vaccine. The thermostable strain of NDV was developed in the laboratory which was isolated from duck and characterized as lentogenic strain. For this, analysis to elicitate cytokine response was necessary to characterize the thermostable strain of NDV, therefore a comparison was done with conventional lentogenic strain. Both the strains were cultivated in 9 days old embryonated SPF eggs and strains confirmation was done with hemagglutination titer (HA) test, outcomes of HA titer were 1:4 for thermostable strain, 1:8 for lentogenic strain and 1:16 for mesogenic strain. Chicken embryo fibroblast (CEF) culture was used to study cytokine expression of the thermostable and lentogenic strains of NDV. The CEF cells were infected with thermostable strain with 103 TCID50 and lentogenic strain with 104 TCID50. On RNA extraction from CEF cells, after being harvested at 0, 24 and 48 hours post infection, a real-time quantitative PCR was performed, to analyze expression of anti-viral (IFN-α), pro- inflammatory (IL-6) and anti- inflammatory (IL-10) cytokines. Relative expression analysis revealed non-significant change in expression of IL6 in infected CEF cells. However, relative expression of IFNα and IL10 were higher. On the basis of relative expression analysis of immune related cytokines, it can be concluded that the thermostable strain of NDV capable to elicit antiviral and anti-inflammatory cytokine response in CEF cells. Overall, our data signifies the potential of thermostable strain of NDV as vaccine candidate.
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    ThesisItemOpen Access
    Exploration of detoxification mechanisms of acaricide resistance in Ixodid ticks
    (LUVAS, 2019) Surbhi; Nirmal Sangwan
    To study the acaricide resistance status and possible mechanisms of action in conferring resistance to commonly used acaricides (Deltamethrin and Coumaphos), Hyalomma anatolicum and Rhipicephalus (Boophilus) microplus ticks were collected from gaushalas and commercial dairy farms of Hisar, Bhiwani and Charkhi Dadri districts of Haryana. Using standard AIT-DD against deltamethrin and coumaphos, higher degree of resistance was observed in H. anatolicum ticks against coumaphos as compared to deltamethrin whereas it varied in case of R. (B.) microplus against both the acaricides. H. anatolicum and R. (B.) microplus ticks larvae collected from Charkhi Dadri were found to be susceptible to both the acaricides used. Level I resistance was reported in 4 isolates collected from Shahpur, Dobhi, Mangali, Kaimri and level II in one isolate of Mingnikhera against coumaphos in H. anatolicum, whereas in R. (B.) microplus level I resistance was observed in 3 isolates collected from Dobhi, Kaimri and Mingnikhera. Kaimri isolates of both H. anatolicum and R. (B.) microplus ticks showed level I resistance against deltamethrin. H. anatolicum ticks having higher values of resistance factor (RF) against coumaphos were found to have higher α-esterase, β-esterase, gultathione-s-transferase (GST) and mono-oxygenase activities whereas the monoamine oxidase activities did not show any constant trend of either increase or decrease. With the increase in RF value against coumaphos in resistant isolates of R. (B.) microplus, the levels of enzyme activities increased. β-esterase activities were more prominent as compared to α-esterase in resistant H. anatolicum and R. (B.) microplus ticks. Development of resistance in R. (B.) microplus against coumaphos showed significant correlation with α-esterase, β-esterase, gultathione-s-transferase and mono-oxygenase activities whereas in H. anatolicum significant correlation only with GST was reported. Native PAGE analysis of esterases showed a total of 9 types numbered as EST-1h to EST-9h in H. anatolicum ticks whereas 6 types in R. (B.) microplus numbered as EST-1b to EST-6b irrespective of susceptible and resistant ones. When inhibition studies were undertaken, pCMB and CuSo4 (arylesterase inhibitor) didn’t inhibit the activities of enzymes indicating that arylesterases were not involved in the development of resistance against coumaphos in both the ticks. Further analysis of electrophoretogram showed that EST-6h and EST-8h in highly susceptible H. anatolicum ticks did not appear. However, these esterases were very prominent in resistant ticks and were found to be inhibited by PMSF (serine esterase inhibitor) indicating presence of serine residue at the enzyme’s active sites. EST-5b activity in R. (B.) microplus was observed only in resistant isolates collected from Dobhi, Kaimri and Mingnikhera and was found to be inhibited by eserine sulphate indicating development of resistance due to expression of acetylcholine esterase. The mechanism of resistance development in both the ticks against coumphos was different where in H. anatolicum it appeared due to more expression of EST-6h and EST-8h having serine residue at their active sites whereas in R. (B.) microplus due to more expression acetylcholineseterase. Organophosphate (carboxylesterase) and Pyrethroid (Domain II of sodium channel) genes were investigated partially for mutations in resistant isolates of H. anatolicum and R. (B.) microplus ticks and did not find any mutation in both the genes indicating possible mechanism for development of resistance was due to increased expression of detoxification enzymes as observed in the present study.