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Thesis

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2012 - 20133
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Subject: Animal Biochemistry × End date: 2013 × Start date: 2012 × 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 – 517502. (A.P.) INDIA, 2012-08) SAMPATH KUMAR, B; ASHOK, VASILI (Major); 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
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    ThesisItemOpen Access
    EFFICACY OF ALOIN MEDIATED PHYTOGENIC NANOSILVER PARTICLES IN EXPERIMENTAL MURINE MASTITIS
    (SRI VENKATESWARA VETERINARY UNIVERSITY , TIRUPATI – 517502. (A.P.) INDIA, 2013-02) VENKATA CHAITANYA KUMAR, T; ESWARA PRASAD, P (Major); PADMAJA, K; SREENIVASULU, D
    ABSTRACT : An experimental study was conducted to evaluate the efficacy of aloin mediated phytogenic nanosilver particles in experimental murine mastitis. The nanoparticles were synthesized and the synthesized nanoparticles were characterized using UV-visible spectrophotometer, Scanning electron microscope (SEM), DLS (Dynamic light scaterring) Technique and FT-IR analysis and were also evaluated for antimicrobial activity and cytotoxic effects. A total of 40 female mice between 10-15 days of lactation were utilized for the study. The animals were divided into five groups of eight animals each. Group I served as lactating control, groups II to V were inoculated with 20μl of 24h broth culture of S.aureus containing 4.0 x 104 cfu/quarter under ketamine anaesthesia using 33g blunt hamilton needle. After 6h post inoculation, groups III and IV received 20μl of Aloin nano silver (ANS) through intramammary and intra peritoneal routes respectively. Group V received antibiotic Cefepime at a dose rate of 1mg/kg body weight through intra peritoneal route. After 18 h post treatment, serum C reactive protein (CRP) levels, weights of mammary glands, mammary bacterial loads, levels of TBARS, GSH, SOD and CAT, histopathology and ultrastructure studies were conducted. UV Visible absorption spectrum showed a maximum absorption around 200 nm for ANS. SEM images showed that ANS with cubical, rectangular, triangular and spherical morphology of size 287.5 to 293.2 nm. The results of FT-IR analysis indicated the involvement of hydroxyl, carboxyl, amine and nitrile groups in the synthesis of aloin mediated silver nanoparticles. The size of the particles as measured by DLS technique was 67.8 nm. The compound (ANS) showed an MIC of 21.8 ng/ml against Staphylococcus aureus and showed an in vitro spleenocyte viability of more than 80% at the highest concentration of 87.5 ppm per well. The compound was also found to be safe at the limit dose in acute oral toxicity conducted in rats. The bacterial load was significantly reduced in ANS (intramammary), ANS (intra peritoneal) and antibiotic group compared to mastitis control. The levels of C-Reactive Protein were significantly reduced in Aloin treatment groups III and IV compared with antibiotic group V. TBARS was not significantly reduced to the level of normal lactating group, however SOD and CAT levels were lowered by both routes of aloin nanosilver (ANS) treatments. In histopathological sections, significant reduction (98%) in PMN infiltration was observed with ANS than antibiotic (50%). Ultrastructure of mammary gland from aloin treatment groups III and IV indicated moderate progress in the architecture of the secretory epithelial cells showing reconstituting mitochondria and normal inter alveolar septa.
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    ThesisItemOpen Access
    COMPARATIVE EVALUATION OF WOUND HEALING PROPERTIES OF NANOSILVER AND BOSWELLIA OVALIFOLIOLATA MEDIATED NANOSILVER IN WISTAR RATS
    (Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2012-10) APARNA REDDY, N; ESWARA PRASAD, P (Major); PADMAJA, K; ADILAXMAMMA, K
    ABSTRACT : An experimental study was conducted to evaluate and compare the wound healing properties of citrate coated silver nanoparticles and B.ovalifoliolata mediated silver nanoparticles in wistar rats in three wound models. The nanoparticles were synthesized and the synthesized nanoparticles were characterized using UV-visible spectrophotometer, Scanning electron microscope (SEM), DLS (Dynamic light scaterring) Technique and FT-IR analysis and were also evaluated for antimicrobial activity and cytotoxic effects. A total of 90 rats (both sexes) were divided into five groups, each group containing 18 rats and the wound healing activity was evaluated in three different wound models viz., excision, incision and dead space model. Group 1 was treated with normal saline, group 2 with Povidone iodine (1% solution), group 3 with Citrate coated silver nanoparticles (AgNPs) (170 μg per dressing), group 4 received B.ovalifoliolata bark ethanolic extract (50 mg per dressing) and group 5 received B.ovalifoliolata mediated silver nanoparticles (BENS) (85 μg per dressing). The respective concentrations of the drug were prepared as dressings and applied topically. In excision wound model, percentage wound contraction and the periods of epithelialization were recorded. Blood was collected from orbital sinus in EDTA coated tubes and plasma was separated for estimation of Vitamin C, TBARS and protein. Granulation tissue from excision model was processed for histopathological examination. In incision wound model, the wound breaking strength was measured on 10th day. In dead space wound model, the granulation tissue collected on Day 10 was used for the estimation of hydroxyproline, hexosamine, total protein, TBARS, superoxide dismutase, catalase, reduced glutathione and ascorbic acid. UV Visible absorption spectrum showed a maximum absorption between 350-450 nm and around 450 nm for AgNPs and BENS respectively. SEM images showed that AgNPs showed an agglomerated morphology (size 374-718 nm) while, B.ovalifoliolata mediated silver nanoparticles were spherical in shape (size 192.8-346.1 nm). The results of FT-IR analysis indicated the involvement of hydroxyl, carboxyl and amine groups in the synthesis of B.ovalifoliolata mediated silver nanoparticles. The size of the particles as measured by DLS technique was 78.8 and 273.5 nm respectively. The in vitro studies showed that BENS possess highest antibacterial activity against Staphylococcus aureus as evidenced by highest zone of inhibition (17.67 mm) and lowest MIC value (1.77μg/ml). BENS was practically nontoxic at 85 ppm while AgNPs were toxic from 10 ppm onwards. In excision wound model, BENS showed significantly (p<0.05) higher percentage of wound contraction and shortest period of epithelialization compared to control group indicating better wound healing property. The TBARS level was significantly (p<0.05) lower and the levels of vitamin C and protein were elevated in both BE and BENS groups. Histopathological examination of BENS group showed mild cellular infiltration with angiogenesis and more fibroblast proliferation when compared to control. In the incision wound model, BE and BENS groups showed significantly (p<0.05) higher wound breaking strength compared to other groups. In dead space wound model, there was a significant increase in hydroxyproline and hexosamine content with increased catalase and SOD activity and decreased lipid peroxide levels in the granulation tissue of BENS group compared to other treatment groups. Thus in conclusion B.ovalifoliolata mediated silver nanoparticles showed better wound healing properties compared to citrate coated silver nanoparticles and ethanolic extract of B.ovalifoliolata.