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ThesisItem Open Access COMBINED EFFECT OF DIETARY AFLATOXIN AND FUMONISIN ON SERUM BIOCHEMICAL PROFILE IN BROILERS AND THEIR AMELIORATION USING ABSORBANTS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2006-02) JAYASRI, K; NARASA REDDY, G.V(MAJOR); SETTY, O.H.; RAJASEKHAR REDDY, A; REDDANNA, PThesisItem Open Access ISOLATION OF BUFFALO URINARY PROTEINS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-09) RAGA SUDHA, MEDICHERLA; Ashok, VASILI(MAJOR); Viroji Rao, S.T.; Narasimha Reddy, YABSTRACT: Presence of proteins in urine is considered pathological, but still certain proteins are excreted in minute quantities into the urine of healthy animals. The normal proteins in the urine are uroplakins (15 to 47kDa) and uromodulin (85 to 90kDa) which are produced by the urinary bladder and the thick ascending limb of Henle’s loop (kidney) respectively. The tissue specific expression of these proteins in the urinary system formed the basis for transforming the urinary system into a bioreactor to produce proteins of economic importance in the urine. In the present day world, the idea of transforming urinary system as a bioreactor and urine as an expression system has growing importance. So, the objective of the present study is isolation and quantification of these normal buffalo urinary proteins. The outline of the work is collection of urine from apparently healthy lactating Murrah buffaloes. Precipitation of proteins from the urine is carried out by protein precipitating agents like acetone, ammonium sulphate and sodium chloride. These precipitated proteins were analyzed by SDS-PAGE. Coomassie blue stained gel revealed a prominent and consistent protein band of molecular weight 85kDa in all the precipitated samples which could be uromodulin, the protein of our interest. Silver nitrate stained gels revealed low molecular weight protein bands (15, 27 and 47kDa) in all precipitated samples in addition to the high molecular weight band, which could be uroplakins. The consistent production of uromodulin by the animal into the urine gives an idea of transforming the kidney into a bioreactor. The reason for inconsistent occurrence of various low molecular weight proteins could be due to the dilution effect of liquid intake by the animal, diet, temperature, climate and various physiological factors which are yet to be ruled out. Quantification of protein precipitates of urine samples and urine as such of 10 different healthy buffaloes was done. The mean protein yields (mg) of urine and precipitates of acetone, ammonium sulphate and sodium chloride are 0.96±10.20, 10.60±0.39, 1.50±0.04 and 1.91±0.13 respectively. Acetone precipitation yielded significantly (p<0.001) higher quantity of protein precipitate. However, the banding pattern is not commensurate with the amount of precipitate. This could be the result of interference of salts and metabolic wastes with the Lowry’s method, yielding higher values. Ammonium sulphate precipitated more number of proteins, evidenced by the gel replicates, but at higher concentration. Sodium chloride selectively precipitates almost a single protein and is required in small quantities when compared to ammonium sulphate for producing good banding pattern. It is concluded that acetone precipitates higher amounts of protein quantitatively. Ammonium sulphate precipitates more number of proteins, where as sodium chloride precipitation yields almost a single protein. However, sodium chloride method has the advantage of selective precipitation over acetone and ammonium sulphateThesisItem Open Access PROTEIN PROFILE OF POULTRY KIDNEY(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-09) KALYANI, P; ASHOK, VASILI(MAJOR); RAJASEKHAR REDDY, A; NAGAMALLESHWARI, YABSTRACT : The extensive prevalence and escalating incidence of Chronic Renal Failure and End Stage Renal Disease worldwide necessitates renal tissue engineering as a practical solution to meet the organ demand for renal transplantation therapy. Renal tissue engineering is a multidisciplinary field, which is supported on three pillars- cells, scaffolds and signaling biomolecules. A critical understanding of the role of signaling biomolecules is necessary for tissue engineering because these biomolecules are differentially expressed with specific spatio-temporal distribution. Signalling biomolecules and signal transduction pathways are highly conserved between species during millions of years of evolution, which engenders the scope for probing into mammalian kidney biology by using lower organisms as models. Gallus gallus (chicken) is a suitable animal model to study the role of proteins in renal organogenesis, owing to the availability of extensive literature on embryogenesis and the ease of embryo retrieval. Metanephric kidneys were collected from different developmental stages of Vanaraja breed chick embryos viz., on 7th , 9th , 11th , 13th , 15th , 17th , 19th days , day old chicks and adult cock in ice cold PBS. Embryonic kidneys and kidneys of day old chicks were dissected under diascopic stereo zoom microscope. The kidney tissues were homogenized, centrifuged and supernatants were analyzed by SDS PAGE. Prominent, differential protein expression was evident upon comparing the protein bands of tissue extracts. In total, 22 prominent differentially expressed proteins were identified on careful examination of banding pattern from the tissue extracts of different developmental stages. A single high molecular weight protein band was seen only in the adult sample. Some of these prominent protein bands were identified to be MMP9 (97 kDa), nephronectin (79 kDa), osteopontin (76 kDa), MMP2 (72 kDa), pax 8 (57 kDa), pax 2 (50 kDa), BMP 4 (45 kDa), actin (43 kDa), FGF2 (23kDa), pleitrophin (21kDa), activin 1 (20kDa), endostatin (15kDa) based on their molecular weights and temporal distribution. Further confirmation of the identity of these proteins could be done by in vitro cultural tests, protein sequencing and other functional tests. The present study reveals a prominent, differential protein expression at various developmental stages in kidney organogenesis. Based on the banding pattern, it is proposed that most of the architectural design for kidney organogenesis is framed by the end of embryonic day 15 and after that it is refined to perfection to carry out various biological activities. Research on the histology and immuno-histochemistry of the kidney tissue during various developmental stages can confirm the present findings.ThesisItem Open Access EFFICACY OF ACTIVATED CHARCOAL AND YEAST CULTURE ON AMELIORATION OF COMBINED TOXICITY OF AFLATOXIN AND T-2 TOXIN IN BROILERS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-01) RAGHAVENDRA SRIKANTH, N; NARASA REDDY, G.V(MAJOR); SETTY, O.H.; RAJASHEKAR REDDY, AABSTRACT : Aflatoxin and T-2 toxin were produced from Aspergillus parasiticus and Fusarium sporotrichioides (F. tricinctum) respectively and quantified using TLC. The toxins were mixed with broiler feed to attain required concentrations. Four diets for broilers were prepared diet 1 – basal diet (control), diet 2-basal diet + aflatoxin (1 ppm) + T-2 toxin (1 ppm), diet 3 – basal diet + aflatoxin (1 ppm) + T-2 toxin (1 ppm) + activated charcoal (0.4%), diet 4 – basal diet + aflatoxin (1 ppm) + T-2 toxin (1 ppm) + activated charcoal (0.4%) + yeast culture (0.1%). These four diets were fed to 4 groups of day old broiler chicks with four replicates of eight birds in each replicate, in a completely randomized design for six weeks. The body weight gains were significantly (P<0.01) lower on diet 2 containing aflatoxin and T-2 toxin. The body weight gains increased gradually on diet 3 containing activated charcoal and on diet 4 containing activated charcoal and yeast culture. Feed consumption was significantly (P<0.01) higher on basal diet compared to all other diets, which were at par. Feed conversion ratio was the highest on diet 2 compared to other diets which were at par. On diet 2 containing aflatoxin and T-2 toxin, the biochemical profile revealed elevated serum enzyme activities of AST, ALT, ALP, GGT indicating liver toxicity. These studies also recorded decreased levels of serum proteins, glucose, cholesterol, triglycerides, calcium, uric acid and increased levels of serum bilirubin. Increased serum creatinine and BUN recorded indicates toxicity to kidney. The studies on liver and kidney function tests indicate possible damage of these organs in the chicks fed on diet 2. Degenerative changes in liver, kidney and lymphoid depletion in bursa of Fabricius, disruption of cardiac muscle fibres, and disruption of intestinal villi were observed on histopathology in birds fed with aflatoxin and T-2 toxin supporting the biochemical study. The birds on diet 3 showed lower body weight gains and feed consumption compared to that of control diet. Efficiency of feed utilization on diet 3 was comparable to that of diet 1. The biochemical parameters like AST, ALP, GGT, serum calcium, serum uric acid were moderately improved but there was no improvement in creatinine and bilirubin compared to diet 2. There was mild improvement in serum proteins cholesterol, glucose and triglyceride levels compared to diet 2. Complete alleviation of toxic symptoms was noticed in terms of serum ALT and BUN. Broilers on diet 4 showed an increase in body weight gains compared to diet 2. Feed conversion ratio on diet 4 was comparable to that on diet 1. The biochemical profile of cholesterol total protein, glucose, uric acid, calcium and triglyceride showed moderate improvement while the other parameters like ALP, bilirubin, ALT, GGT and AST were comparable to those of control diet. Histopathological studies were comparable to those of control group fed on toxin free diet. These results indicate that aflatoxin and T-2 toxin in the diet caused deteriorated effects on growth, biochemical profile and caused damage to vital organs. The combination of activated charcoal and yeast culture was more effective in counteracting the combined toxicity of aflatoxin and T-2 toxin compared to the activated charcoal alone.ThesisItem Open Access DETOXIFICATION OF CITRININ AND FUMONISIN IN FEED AND ITS EFFECT ON SERUM BIOCHEMICAL PROFILE IN BROILERS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-01) ASHOK KUMAR, DEVARASETTI; ASHOK, VASILI(MAJOR); NARASA REDDY, G.V; ANJANEYULU, YABSTRACT : Citrinin and fumonisin toxins were produced from Pencillium citrinum and Fusarium moniliforme respectively and both were quantified using Thin Layer Chromatography (TLC) method. The toxins citrinin @ 50 ppm and fumonisin @ 10 ppm were mixed in broiler feed to attain required concentrations. Four diets for broilers were prepared diet 1 – basal diet (control), diet 2 – basal diet + citrinin (50 ppm) + fumonisin (10 ppm), diet 3 – basal diet + citrinin (50 ppm) + fumonisin (10 ppm) + activated charcoal (0.4%) and diet 4 – basal diet + citrinin (50 ppm) + fumonisin (10 ppm) + activated charcoal (0.4%) + lyophilized yeast culture (0.1%). Each diet was fed to a group of day old broiler chicks with four replicates having eight birds in each replicate in a completely randomized design for six weeks. The weekly body weights and weekly body weight gains were significantly (P < 0.01) lower on diet 2 containing citrinin (50 ppm) + fumonisin (10 ppm). The weekly body weights and weekly body weight gains increased gradually on diet 3 containing activated charcoal. Further significant (P < 0.01) improvement was recorded on diet 4 containing activated charcoal and yeast culture. Feed consumption was significantly higher (P < 0.01) on basal diet compared to other test diets. Feed conversion ratio was the highest on diet 2 when compared to other test diets. On diet 2 containing citrinin and fumonisin, the biochemical profile revealed elevated serum enzyme activities of AST, ALT, ALP, GGT indicating liver toxicity. These studies also recorded decreased levels of serum proteins, albumin, glucose, cholesterol, triglycerides, calcium, uric acid and increased levels of serum bilirubin. Increased serum creatinine and BUN recorded indicates toxicity to kidney. These studies on liver and kidney function tests indicate possible damage of these organs in the chicks fed on diet 2. Further, supporting the biochemical study, degenerative changes and central venous congestion in liver and marked degenerative changes in kidney and lymphoid depletion in bursa of Fabricius, disruption of cardiac muscle fibres, disruption of intestinal villi were observed on histopathology in birds fed with citrinin and fumonisin. The birds on diet 3 containing citrinin, fumonisin and activated charcoal showed significant (P < 0.01) improvement in body weight gains and feed consumption. Efficiency of feed utilization on diet 3 was comparable to that of diet 1. The serum enzymes like AST, ALT, ALP and GGT were markedly improved and serum creatinine, BUN, calcium, serum uric acid were moderately improved when compared to diet 2. There was mild improvement in serum proteins, cholesterol, glucose and triglyceride levels compared to diet 2. All other biochemical parameters and histopathological findings showed improvement compared to toxin fed group (diet 2). This indicates that activated charcoal (0.4 %) had partial amelioration on toxic effects of citrinin and fumonisin. The birds fed on diet 4 recorded the serum enzymes like AST, ALT, ALP and GGT in the liver nearer to the control group (diet 1). All the biochemical parameters and histopathological findings were also nearer to that of the control group and within the normal range indicating that activated charcoal and lyophilized yeast culture had a complementary effect in ameliorating the toxic effect of citrinin and fumonisin. These results indicate that activated charcoal (0.4 %) could adsorb and ameliorate the toxins (citrinin and fumonisin) to some extent and activated charcoal (0.4 %) and lyophilized yeast culture (0.1 %) showed complementary effect in amelioration of the toxic effect caused by citrinin and fumonisinThesisItem Open Access COMBINED EFFECT OF DIETARY AFLATOXIN AND CITRININ ON SERUM BIOCHEMICAL PROFILE IN BROILERS AND THEIR AMELIORATION USING ADSORBENTS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2005-12) PRIYADARSHINI, C.H.; NARASA REDDY, G.V(MAJOR); SETTY, O.H.; ANJANEYULU, YABSTRACT: Aflatoxin was produced from Asprgillm parasiticus and cihinin was produced from Penicillium cirri- ,dtd h were quantifd using thin layer chromatography. The toxins, aflatoxin at the rate of lppm and citrinin at the rate of 25 ppm, were mixed in broiler feed. Activated charcoal (0.4%) and/or lyophilized yeast culture (0.2%) were added as adso&ents. Four diets for broilers were prepared - Diet 1- basal diet (control). Diet 2- basal diet added with aflatoxln (I ppm) and citrinin (25ppm). Diet 3- basal diet added with aflatoxin (Ippm), citrinin (25 ppm) and activated charcoal (0.4%) and Diet 4- basal diet added with aflatoxin (Ippm), citrinin (25ppm). activated charcoal (0.4%) and lyophilized yeast culture (0.2%). These four diets were fed for 6 weeks to four group of broiler chicks with four replications of eight birds in each gnwrp using completely randomized design. The body weight gains. feed consumption and feed conversion ratio were significantly (Pa.01) lower on diet 2 containing aflato.. in and citrinin. These values were significantly improved on diet 3 containing aflatoxin. citrinin and activated charcoal. Further significant improvement was nxoded on diet 4 containing aflatoxin. citrinin. activated charcoal and lyophiliztd yeast culrure but these values were significantly lower than control gmup- On diet 2 containing aflatoxin and citrinin. the biochemical profile of liver function and kidney function of birds showed hepatotoxicity and nephrotoxicity respectively as evidenced by increased serum enzyme activities of AST. ALT. alkaline phosphatase and GGT concentration in the liver and increased levels of serum creatinine, blood urea nitrogen and serum uric acid. Other biochemical parameters revealed decreased serum protein, albumin, glmse, cholesterol, triglycerides, calcium and phosphorous but increase in serum bilirubin. By these studies on liver and kidney function tests, chicks fad on diet 2 indicate the possible damage of tissues. Further, supporting the biochemical study, degenerative changes and central vein congestion in liver and marked degenerative changes in kidney, lymphoid depletion in bursa of Fabricius and depleted germinal centres in spleen were observed on histopathologv. On diet 3 containing aflatoxin, citrinin and activated charcoal, the serum enzyme activities of AST, ALT. AP and GGT in the liver were slightly decreased and serum --.. creatinine, blood urea nitrogen and serum uric acid levels were slightly decreased compared to toxin group (diet 2). All other biochemical parameters and histopathological findings showed improvement compared to toxin fed group. This indicates that activated charcoal (0.4%) had partial amelioration on the toxic effect of aflatoxin and citrinin. The birds fed on diet 4 recorded the serum enzyme activities of AST, ALT, AP and GGT in the liver nearer to the control group (basal diet). All the biochemical parameters and histopathological f i n d i i were also nearer to that of the control group and within the normal range indicating that activated charcoal and lyophilized yeast had a complementary effect in ameliorating the toxic effect of atlatoxin and citrinin. These results indicate that activated charcoal (0.4%) could adsorb and ameliorate the toxins (aflatoxin and citrinin) to some extent and activated charcoal (0.4%) and lyophilized yeast cultw <0.2O/') showed complementary effect in amelioration of the toxic effect caused by aflatoxin and citrinin.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.