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ThesisItem Open 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, NABSTRACT : 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 urineThesisItem Open 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, KABSTRACT : 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.ThesisItem Open Access EFFECT OF Caesalpinia sappan EXTRACTS ON PRESERVATION OF MEAT(Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2011-12) VENKATASAIKUMAR, T; ESWARA PRASAD, P (Major); PADMAJA, K; JAGADEESH BABU, AABSTRACT : An experiment was conducted to study the effect of C.sappan extracts on preservation of meat. This study was carried out to evaluate the antimicrobial and antioxidant activities of C.sappan extracts ( aqueous, methanolic), methanolic extract of C.sappan along with antibiotic (chlortetracycline) as natural food preservative in chicken, pork and mutton The results were compared with conventional methods like acetic acid and chlorinated water treatment on meat preservation. The experiment was carried out in two phases. In phase I, aqueous and methanolic extracts of C.sappan, methanolic extract of C.sappan along with antibiotic (chlortetracycline), acetic acid and chlorinated water at different dilutions were prepared and were evaluated to find MIC using agar diffusion assay. The results obtained in the phase I revealed that the aqueous extract of C. sappan did not exhibit any inhibitory effect on pathogenic organisms tested and hence it was not used for further study. However methanolic extract C.sappan, methanolic extract of C.sappan along with antibiotic (chlortetracycline), acetic acid and chlorinated water have exhibited high inhibitory action on pathogenic bacteria at dilutions of 1:5, 1:5, 4% and 300ppm respectively. In second phase, a control and four treatment groups namely - methanolic extract of C. sappan plus antibiotic (T1), methanolic extract of C. sappan (T2), acetic acid (T3) and chlorinated water (T4) were formed to study their effect on preservation of different species of meat. Raw chicken, pork and mutton samples were obtained from local retail meat market, each sample was treated with methanolic extract of C. sappan plus antibiotic (T1), methanolic extract of C. sappan (T2), acetic acid (T3) and chlorinated water (T4) and effect of the treatments were evaluated using different parameters. There was no significant difference found between T1 and T2 for the parameters such as pH, TBARS, free fatty acid, peroxide, yeast and mould count, colour and flavour. For the parameters such as total platecount psychrophiles, coliforms, MRSA, Salmonella counts all treatments differed significantly(P<0.05). The effect in reduction of microbial load in meat samples was highest in T1 followed by T2, T3 and T4. The mean values of parameters (pH, TBARS, free fatty acid, peroxide, yeast and mould count, total platecount, psychrophiles, coliforms, MRSA, Salmonella counts) increased significantly from 0 to 8th day irrespective of type of treatment. The values for color and flavour decreased significantly as the period of storage increased. The results of the experiment concludes that methanolic extract of C. sappan and methanolic extract of C. sappan with antibiotic significantly exhibited beneficial effects on meat preservation as compared to other conventional treatments and these effects were almost similar on different species of meat samples. The results also indicated that pH, TBARS, free fatty acid, peroxide value and microbial count increased along with period of storage irrespective of treatment.ThesisItem Open Access MOLECULAR DETECTION AND CONTROL OF SALMONELLA IN ANIMAL FEEDS(Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2010-10) SRAVANTHI, MANNEM; ESWARA PRASAD, P (Major); SUDHAKARA REDDY, K; SREENIVASULU, D; ADILAXMAMMA, KABSTRACT : Feed and feed ingredients serve as a carrier for a range of bacteria, which can adversely affect the performance of animal. One such important contaminant of feeds is Salmonella species. Traditional cultural methods used for identification of Salmonella involves use of pre-enrichment, selective enrichment broths (Selenite cystine broth or Rappaport Vassiliadis broth), selective enrichment media (BGA, MCA, XLD etc.) and confirmation of isolates by biochemical tests. Among 109 feed samples tested, seven (6.42%) positive Salmonella isolates were obtained. The incidence of Salmonella contamination in animal feed samples was screened as 10.81% in Kadapa where as 4.4% in Chittoor district. The traditional cultural methods used for Salmonella isolation were time consuming. Among the several alternate analysis strategies, Polymerase Chain Reaction has been found to be a highly specific molecular diagnostic tool. PCR technique was standardized targeting fimA gene of Salmonella. A 29 cycle PCR reaction with an annealing temperature of 58°C and MgCl2 concentration of 3 mM were found optimum for amplification of 85 bp product of fimA gene. To overcome the production losses due to the presence of bacteria in feed samples, suitable control methods have to be followed. Three different control methods viz., formaldehyde treatment, UV treatment and sun drying were adopted. Formaldehyde at two concentrations (0.5% and 1%) and UV light at a wavelength range of 250-260 nm for 10 minutes were used for treatments. Feed samples were kept for sun drying for a period of 6 days. After treatments, feed samples were again processed for Salmonella isolation and total plate counts. All the three treatment methods were effective in completely decreasing the Salmonella levels in feed samples. For reducing total bacterial counts formaldehyde treatment found to be the most effective method, followed by UV treatment and sun drying. ABST was carried out to determine the drugs, effective for the therapy of Salmonella. The drugs, ofloxacin, ciprofloxacin, and ceftriaxone were found to be effective for all isolates of Salmonella. The results of the experiment conclude that PCR technique with fimA gene is found to be effective in detecting Salmonella from feed samples at a shorter time and 1% formaldehyde treatment is highly effective in reducing the total counts of bacteria. Results of ABST conducted indicated ofloxacin, ciprofloxacin and ceftriaxone are highly effective against Salmonella as compared to the other antibiotics.ThesisItem Open Access A STUDY ON AMELIORATING EFFECT OF CAESALPINIA SAPPAN L IN LEAD INDUCED OXIDATIVE STRESS IN RATS(Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2010-09) HEMALATHA, K; ESWARA PRASAD, P (Major); PADMAJA, K; ADILAXMAMMA, K; SREENIVASULU, DABSTRACT : An experiment was conducted to study the ameliorating effect of Caesalpinia sappan in lead toxicity using a total of 24 male wistar rats which were randomly divided into four groups consisting of six animals in each group. Group I was maintained on basal diet as control and group II on lead acetate @ 90 mg/kg orally along with basal diet as toxic control group. Group III was maintained on methanolic extract of Caesalpinia sappan stem bark @ 100 mg/kg along with the basal diet and group IV on lead acetate @ 90 mg/ kg and methanolic extract of Caesalpinia sappan @ 100mg/ kg along with the basal diet for 28 days. The serum biomarkers were estimated for assessing the damage caused to liver, kidney and heart due to lead toxicity. Parameters like total protein, ALT, troponins, AST, creatinine and BUN values were measured on Day 14 and Day 28 from freshly collected blood samples. Tissue samples were collected and preserved at -20ᵒC for analysis of tissue antioxidant profile (TBARS, glutathione, GST, SOD, catalase, membrane ATPases, Vitamin E and C) and lead content. Blood samples were collected and haematological parameters (RBC, WBC, Hb and PCV) were estimated at the end of 28th day of experiment. The results indicated that lead toxic group (group II) showed a significant (p<0.05) decrease in serum total proteins and significant (p<0.05) increase in ALT, troponins, AST, creatinine and BUN values revealing that lead has induced damage to the liver, kidney and heart tissues. Significant decrease in total RBC count, haemoglobin and PCV were observed in group II which showed the evidence of damaging effect of haemopoietic system due to lead toxicity. The mean values of TBARS were significantly increased in lead control group where as the levels of glutathione, GST, SOD, total membrane ATPases and Vitamin E were significantly decreased in group II indicating lead induced oxidative damage to the tissues. Significant increase in the levels of lead was observed in liver and kidney in group II. Treatment with methanolic extract of Caesalpinia sappan stem bark (group IV) resulted in improvement in above parameters as compared to lead toxic group (group II). Lead quantity of the liver was reduced slightly in group IV compared to group II but could not bring the levels to normal. Thus, it is concluded that lead induces biological damage to liver, kidney and heart tissues and further caused oxidative stress and Caesalpinia sappan offered amelioration which is attributed to its antioxidant activity but it could not able to reduce the lead content of the tissues completely.