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Subject: Biochemistry ×

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    ThesisItemOpen Access
    COMPARISON OF INNATE IMMUNITY RELATED GENES IN VECHUR AND CROSSBRED CATTLE AND THEIR EXPRESSION PROFILE IN CLINICAL AND SUBCLINICAL MASTITIS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2016-12-30) LAKSHMI R.; K.K. Jayavardhanan
    Bovine mastitis is considered as the most economically imposing diseases of dairy cattle. Vechur cattle an indigenous breed of Kerala are generally not susceptible to mastitis. Investigation of innate immune mechanism of this breed might provide an insight into the mechanisms involved in the disease resistance. In light of this premise, the present study was carried out to investigate the expression of TLR2, TLR4, and TLR9 in crossbred and Vechur cattle using RT-qPCR, to better understand the immune resistance mechanisms against mastitis and also characterized the promoter and mRNA sequence of TLR genes in Vechur cattle. RT-qPCR analysis showed a significant up fold increase in the TLR2 gene expression in mastitis caused by S. aureus, whereas expression level of TLR4 mRNA was relatively higher in E. coli caused samples. In both S. aureus and E. coli caused mastitis milk samples, relative expression of all three TLRs was found to be significantly high (P ≤ 0.01) in sub-clinical mastitis than the clinical mastitis. So, during early stage of mammary infection these TLRs are expressed at high level to subside the sub-clinical mastitis without precipitating into clinical mastitis. After the challenging the PBMCs with TLR agonist in vitro, relative expression of mRNA of all three TLR genes was higher in Vechur cattle than the crossbred cattle. Furthermore, the expression of TLR2 mRNA was relatively higher in Vechur breed as compared with other TLRs. These findings suggest that one of the reasons for the development of resistance to mastitis in Vechur cattle is associated with the level of expression of TLRs in response to infection. The sequence of promoter region of TLR2 of Vechur cattle with the Bos taurus sequence showed 98 per cent similarity whereas TLR4 and TLR9 revealed 99 per cent similarity. TLR2 and TLR9 revealed variations for three sequence motifs. Significant variants observed for TATA and CAT boxes and multiple putative binding sites in the promoter region of TLR2 and TLR9 genes in Vechur cattle breed, may potentially link the influence the innate immunity response against mastitis diseases. All three TLR mRNA sequences showed 99 per cent homology with Bos taurus sequence and exposed variations for 17 nucleotide in TLR2, 7 nucleotide in TLR4 and 5 nucleotide in TLR9 mRNA. The ectodomain of Vechur cattle displayed 10 LRRs for TLR2, 13 LRRs for TLR4 and 18 LRRs for TLR9. The variation in the extracellular domain of LRRs, which may promote the recognition of pathogen ligand specificity. The primary structure of protein showed highest per cent of leucine amino acid for all three TLRs and alpha helix is the prominent secondary structure seen in all TLRs followed by beta turn and random coil. Phylogenetic tree for TLR genes showed all Bovidae family falling under the same group, indicated conserved nature of TLR genes. The presence of unique structural features and substantial variation for TLR genes in Vechur cattle, may change the confirmation of TLR proteins, which may influence the binding affinity and interaction with pathogen to boost the innate host disease resistance in Vechur cattle.
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    ThesisItemOpen Access
    MOLECULAR STUDIES ON HEAT SHOCK PROTEIN 70 GENE IN DOMESTIC FOWL AND DUCK
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) LIJO JOHN; Sisilamma George
    A study has been carried out to analyse the promoter and 3’UTR of hsp70gene in different breeds of chicken (Naked neck (NaNa), Kadaknath (KAD), White Leghorn (WL), Rhode Island Red (RIR) and White Plymouth Rock (WPR)) and duck (Kuttanadu (KUT) and White Pekin (WP)). Plasma corticosterone level was also estimated in two different (summer and rainy) seasons. Promoter and 3’ UTR of Hsp70 gene were amplified by PCR and the products were sequenced. Sequence analysis of the promoter region showed 100% homology between three breeds of chicken, WL, RIR and KAD and the duck breeds. These breeds exhibited only 99.7% identity with WPR and NaNa Sequence alignment (with Gene bank accession no. J02579) revealed a point mutation in the CAAT box, where an ‘A’ is deleted at position -312 in all the breeds of chicken and duck except NaNa. Another ‘A’ deletion could also be detected at position -329 from a heat shock element in WPR. In all the breeds under study, TATA box (TATAA) was found at position -134. Three inverted CAAT boxes, ‘ATTG’ and two variants of GC boxes, one having GGCG motif (6 numbers) and another having GGGCGG motif (2 numbers) were identified in both chicken and duck sequences. Two variants of consensus heat shock elements (HSE), NGAAN (single unit) and ‘NGAAGAAN’ (double unit) were detected in the promoter region of all breeds of chicken and duck under study except WPR, in which a point mutation (‘A’ deletion) was noted in one of the double units. Restriction analysis showed that only Bgl І has a site in the amplified region of the promoter of both the species. Due to the presences of point mutations, three alleles for the promoter region of the gene could be detected. Sequence analysis of 3’ UTR showed varying levels of sequence polymorphism between chicken breeds, where only 80 to 95.8 % identity could be observed. Between duck breeds, KUT and WP, 97.7 % identity was observed. Analysis between chicken and duck revealed 79 to 99.5% identity where 97 to 99.5% identity was observed between duck breeds and WPR. Two consensus, CAAC sequence and a variant of poly adenylation signal sequence could be identified in 3’ UTR. A variant of poly adenylation signal sequence, TATAAA could be identified in all breeds except RIR, in which two point mutations (transversion) were observed (TAAAAA) when compared to the consensus sequence (AATAAA). A second poly adenylation signal sequence, which was again a variant (AATAAT) was detected only in NaNa. The number and position of CAAC motifs varied (2 to 4 numbers) between species and breeds under study. In both the species, rather than a consensus sequence, a variant of K box (GGTGAT) and Brd box (TGCTTA) could be identified. Several AT (AU in mRNA) rich regions could be identified in the 3’ UTR of both chicken ad duck breeds. However, an additional ATATA motif is also detected in RIR and NaNa. Restriction enzyme analysis of 3’ UTR revealed that NaNa, RIR and WL have no cutting site for any of the common enzymes while a single cutting site for Bgl ІІ was observed in KAD, showing two alleles in chicken breeds. Duck breeds also showed two alleles where the enzyme, Bam HI has a restriction site at different positions. Plasma corticosterone level in chicken and duck showed considerable variation in different breeds of chicken both in summer and rainy seasons. Among the chicken breeds WPR showed the lowest level of corticosterone in summer season, which did not differ significantly from that of rainy season. Comparison between seasons showed a highly significant (P<0.01) increase in summer in all breeds except WPR. Duck breeds also showed a similar trend. Significant (P<0.01) increase in the plasma corticosterone level was noticed in summer. However, between breeds, no significant variation was observed in each season. Hormone responsive elements (HRE) for corticosterone could not be detected in the promoter region of any of the breeds under study. Any correlation between the sequences and the plasma corticosterone level could not be detected in both chicken and duck breeds under study. Although, two point mutations were detected in the promoter region of WPR, it could not be correlated with the plasma corticosterone level.
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    ThesisItemOpen Access
    METABOLIC PROFILE OF CROSSBRED DAIRY COWS DURING TRANSITION PERIOD
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2017) ANISHA, J. PERUMBILLY; Shynu, M.
    Dairy cows are considered to be most prone to diseases during the period from late pregnancy to onset of lactation, i.e. during the transition period. The present investigation was carried out in blood/serum collected at fortnightly intervals from 15 crossbred dairy cows, from eight weeks before the predicted date of calving till eight weeks after calving, with the objectives of generating a metabolic profile and for evaluating the antioxidant status. Concentrations of glucose, NEFA, BHB, cholesterol, urea, albumin, ceruloplasmin and haptoglobin were determined; differential leukocyte count was done; and assessment of oxidative stress was also performed. The mean concentration of glucose (47.35±1.32 mg/dL) and cholesterol (95.83± 3.62 mg/dL) during transition period was significantly lower than pre and post-transition period. NEFA (0.576 ± 0.08 mmol/L) and BHB (0.638 ± 0.05 mmol/L) concentrations reported significant increase during transition when compared to pre-transition period. Concentration of indicators of protein status viz. albumin and urea were 3.39 ± 0.09 g/dL and 12.26 ± 0.66 mg/dL respectively during transition period and did not differ significantly from pre or post-transition period. Out of the two acute phase proteins measured, ceruloplasmin did not show significant variation during the study period, but a significant increase was shown by haptoglobin during transition period. The level of MDA, an indicator of oxidative stress was higher during transition period, indicating significant oxidative stress during the period. TAS did not show significant change but the antioxidant status of the animals could not be considered optimum, as eleven out of fifteen animals exhibited diseases albeit transient during the period. A significant increase in the number of neutrophils and monocytes and a highly significant decrease in the number of lymphocytes observed during transition period could be due to the influence of corticosteroids. A comprehensive study involving more number of transition animals, maintained under different managemental conditions shall help in establishing reference intervals for various analytes during period.
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    ThesisItemOpen Access
    ISOLATION AND CHARACTERIZATION OF LACTOFERRIN FROM COLOSTRUM OF GOATS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2017) Sinchu Vijayan; UMA, R.
    Lactoferrin, an 80 k Da iron-binding protein, primarily present in milk, is well known for its multifunctional properties such as antibacterial, antifungal, antiviral, antioxidant, immunomodulatory and anticancer activities. The present study focussed on the isolation and characterization of lactoferrin from the colostrum of Malabari, Attappady Black and crossbred goats of Kerala as well as assessment of the antimicrobial potential of the lactoferrin isolated. Colostrum samples collected from the three goat breeds maintained at University Goat and Sheep Farm, College of Veterinary and Animal Sciences, Mannuthy were processed to remove fat globules and casein. The whey obtained after processing was fractionated with ammonium sulphate to remove globulins from the sample. Fraction containing albumin and remaining proteins including lactoferrin was separated out, dialysed against equilibration buffer, loaded on to CM- Sephadex C-50 cation exchanger column and eluted with a step gradient of 0.4, 0.6 and 0.8 M NaCl. The fractions with high OD280 values were analysed using 12 per cent SDS-PAGE to identify their protein components in comparison with standard protein. The protein fractions with high absorbance at 280nm, eluted with 0.6M NaCl, could be visualised as a single 80 kDa Coomassie Brilliant Blue-stained band. The total iron content in the isolated lactoferrin samples was estimated by atomic absorption spectrophotometry and it was found to be 820 ppm for Malabari and Attappady Black lactoferrin whereas 1100 ppm for crossbred goat lactoferrin. The concentration of Malabari lactoferrin (mgLf), Attappady Black lactoferrin (agLf) and crossbred goat (cgLf) as estimated by Lowry’s method was found to be 10.94, 12.93 and 11.22mg /L of colostrum respectively. These isolated samples of caprine lactoferrin were found effective to inhibit the growth of both Gram-positive and negative organisms. The assay depicted that the minimum inhibitory concentration (MIC) of mgLf and agLf against E. coli and S. aureus was 275µg/mL and 550µg/mL respectively. The MIC of cgLf against both E. coli and S. aureus was found to be 550µg/mL. The indigenous as well as crossbred goat lactoferrin exhibited the same intensity of antibacterial activity against Gram-positive bacteria. Against Gram-negative organism, lactoferrin of indigenous goats were found to be more potent when compared to the crossbred goat lactoferrin.