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2012 - 20152
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Subject: Biochemistry × End date: 2019 × Start date: 2011 × Type: Thesis ×
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    ThesisItemOpen Access
    CONJUGATION OF ANTIBIOTIC RESIDUES PREVALENT IN MILK WITH CARRIER PROTEIN AND RAISING OF POLYCLONAL ANTIBODIES AGAINST THEM
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2012-08) SAMPATH KUMAR, B; ASHOK, VASILI; ESWAR PRASAD, P; Krishnaiah, N
    ABSTRACT : Antibiotics are the drugs used to kill or inhibit the growth of bacteria. Presence of antibiotic residues in milk has significant public health importance as they have adverse side effects on the health. The present study was undertaken to produce polyclonal antibodies against the most commonly used antibiotics in large animal practice so that a diagnostic test can be developed to screen milk samples for the presence of antibiotic residues. Ampicillin, ceftiofur, enrofloxacin and gentamycin were conjugated with carrier protein BSA by carbodiimide method in which EDC was used as a crosslinking agent. Successful conjugation was confirmed by SDS PAGE in which antibiotic conjugates revealed high molecular weight protein bands of 90kDa, 85kDa, 78kDa, 74kDa for gentamycin, ampicillin, ceftiofur and enrofloxacin respectively in contrast to 68kDa band of BSA. Sprague Dawley rats aged 7-8 weeks were immunized with the antibiotic-BSA conjugates by subcutaneous route according to the standard immunization schedule. A total of four blood samplings were done from each group (3 animals) as follows: first three samples serially at 15 days time interval after 1st immunization plus 1st booster, 2nd booster, 3rd booster and the 4th sampling one and half month after the third booster. The mean total protein content in the antisera of ampicillin, ceftiofur, enrofloxacin and gentamycin groups were 12±1.15 g/dL, 30±1.20 g/dL, 24±1.73 g/dL and 8.25±0.20 g/dL respectively and in the control group serum it was 6.66±0.01 g/dL. The mean albumin concentrations were 3.44±0.020 g/dL, 3.36±0.173 g/dL, 3.21±0.003 g/dL & 2.93±0.014 g/dL for ceftiofur, ampicillin, enrofloxacin and gentamycin groups respectively compared to 3.36±0.173 g/dL in the control group. The mean globulin content in the antisera of ampicillin, ceftiofur, enrofloxacin and gentamycin groups were 8.64±0.10 g/dL, 26.56±1.18 g/dL, 20.79±0.08 g/dL and 5.32±0.05 g/dL respectively and it was 3.30±0.04 g/dL in the control group. A/G ratios were less than the normal value (>1) in antisera of ampicillin, ceftiofur, enrofloxacin and gentamycin groups. Indirect ELISA was standardized with antibiotic-casein conjugate as coating antigen to detect antibody titres. The optimum antigen concentration was chosen as 20ng/ml (which was in the range of Maximum residue limits) and optimum antiserum dilution was chosen as 1/100 by checker board titration. The antibody titres in the test samples of 4 immunization cycles for each antibiotic were determined by indirect ELISA at various serum dilutions ranging from 1/100 to 1/6400. Cut off value of 0.3 was calculated from PNT base line constructed with M+3SD values of control serum samples. All the four antibiotic-BSA conjugates elicited positive immune response in rats. The immune response in BSA-ampicillin conjugate immunized rats was maximum with the highest antibody titre of 2.577 (OD450) followed by BSA-ceftiofur conjugate immunized rats, BSA-enrofloxacin conjugate immunized rats and BSA-gentamycin immunized rats with OD450 values of 2.072, 1.723, and 0.928 respectively at the antiserum dilution of 1/00. These polyclonal antibodies are the efficient tools for developing immunoassays to detect specific antibiotic residues in biological fluids like milk and urine samples
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    ThesisItemOpen Access
    EFFECT OF BISPHENOL-A ON OXIDATIVE STRESS DURING EMBRYONIC DEVELOPMENT IN CHICKS
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2015-01) SRAVANI, J; PADMAJA, K(MAJOR); ESWARA PRASAD, P; PUNYA KUMARI, B
    ABSTRACT: An experiment was conducted to study the effect of Bisphenol-A (BPA) on oxidative stress during embryonic development in chicks. Fertile eggs were procured and eighty (80) eggs in two sets were assigned randomly into 4 groups of20 each. Group I served as control and received 10 μl of distilled water, groups II to IV were treated with BPA dissolved in distilled waterin three concentrations of 100μM, 250μM and 500μM respectively. A single dose of 10 μl volume injected into the aircell using microsyringe separately to 11thday old and 14th day old chick embryos. The developed embryos were sacrificed after 24 and 48 hours of BPA exposure and liver and brain tissues were collected and preserved at -20ºC for estimation of Thiobarbituric acid reacting substances (TBARS), antioxidant enzyme activities (Superoxide dismutase (SOD),Catalase (CAT),Glutathione peroxidise (GPx) and Glutathione-S-transferase (GST)), Reduced glutathione (GSH), Deoxy ribonucleic acid (DNA), Ribonucleic acid (RNA) and protein levels. The liver and brain tissues were preserved in 10% formalin for histopathological studies. The results showed a significant dose dependent increase in TBARS after 24 and 48 hours in both 11th and 14th day treated embryos in liver and brain tissues. Glutathione levels were elevated after 24 and 48 hours in both liver and brain tissues of 11th and 14th day treated embryos. Protein levels were not much altered in both liver and brain tissues, whereas a significant induction in GPx activity was observed after 24 and 48 hours in group IV followed by group III, group II compared to group I in both 11th and 14th day BPA exposed chick embryos. A significant increase in GST activity was observed after 48 hours compared to 24 hours with BPA treatment in both 11th and 14th day old chick embryos. A significant increase in SOD activity was noticed after 24 hours in all the treatment groups compared to control in liver of both 11thand 14th day BPA administered chick embryos. Whereas CAT activity was not altered much after 24 hours, but a significant increase was noticed after 48 hours in liver and brain of 11th and 14th day BPA treated chick embryos. A dose dependent decrease in DNA levels was observed in all treatment groups compared to control after 24 hours in both 11th and 14th day BPA treated chick embryos compared to control. A significant dose dependent decrease in RNA levels was observed after BPA exposure compared to control after 24 hours in both 11th and 14thday BPA treated chick embryos. In histopathological sections, degenerative, necrotic changes with mononuclear cell infiltration, congestion and proliferation of fibroblasts are prominent in liver of both 11th and 14th day BPA treated chick embryos. Neuronophagia, satellitosis, extensive demyelinating changes, proliferation of the capillaries, perineuronal vacuolation was observed in brain of BPA treated chick embryos. The study concludes that the toxic effects of BPA are dose dependent and are inversely effective with age during the embryonic development in chicks.