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ThesisItem Open Access STUDIES ON PESTICIDE RESIDUES IN WATER, SOIL, FODDER AND MILK ALONG MUST RIVER BELT(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2013-10) KOTINAGU, KORRAPATI; KRISHNAIAH, N(MAJOR); MADAHAVA RAO, T; KONDAL REDDY, K; SASHI BHUSHAN, VABSTRACT: A study was conducted to estimate residues of certain pesticides of organochlorines viz., DDT (o,p'- DDE, o,p' - DDD, p,p'- DDT and o,p'- DDT and Dicofol), HCH (Alpha, Beta, Gamma, Delta), cyclodiene compounds (Aldrin, Endosulfan Sulphate and Heptachlor) and organophosphates (Triazophos, Dimetheoate, Chlorpyrifos and Methyl-chlorpyrifos) in soil, water, fodder and milk samples collected from six zones of Musi river belt area. To evaluate the pollution level of Musi river, the river belt was divided in to six zones viz., Zone 1 (Attapur to High court), Zone 2 (Chadhar ghat to Uppal), Zone 3 (Peerzadiguda to Chinna viralla), Zone 4 (Pillai Palli to Alinagar), Zone 5 (Indriyala to Manimadde), Zone 6 (Musi reservoir to Wazirabad). Only soil samples collected hm Zone 1 showed residual levels (in ppm) of 0.06 + 0.005 (0.035 to 0.083), 0.73 * 0.01 (0.675 to 0.791), 1.27 * 0.09 (1.023 to l.893), 0.14 =k 0.015 (0.098 to 0.243) and 0.55 * 0.02 (0.481 to 0.685) for p,p'- DDE, o,p'- DDD, p,p'- DDT, o,p' - DDT and Total DDT respectively. Dicofol was present only in fodder samples of zone 5 at concentration of 0.07 + 0.0007 (0.071 to 0.077). Among HCH compounds only delta HCH was found in soil samples of Zone 1 at a concentration of 0.08 *0.003 (0.065 to 0.098). Water, fodder and milk samples from zone 2 to 6 did not contain any residues of DDT and HCH. None of the samples water, soil, fodder and Milk from all the 6 zones contain the residues of Cyclodiene compounds. Among organophosphorus compounds Triazophos was present in soil samples of zone 1 at a level of 0.03 * 0.001 (0.032 to 0.045) and Dimetheoate was present in milk samples collected from Zone 6 at a level of 0.13 & 0.006 (0.1 11 to 0.167). From this study, it can be concluded that all the pesticide residues in soil were well below the MRL values, whereas Dicofol in fodder and Dimethoate in milk were slightly above the MRL values specified by EU and CODEX.ThesisItem Open Access IN VITRO EVALUATION OF DIFFERENT LACTIC ACID BACTERIAL STRAINS FOR THEIR PROBIOTIC CHARACTERISTICS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2012-11) SRINU, B; MADHAVA RAO, T(MAJOR); SHASHI KUMAR, M; KONDAL REDDY, KABSTRACT : Identifying probiotic characteristics of bacterial strains by in vitro studies forms basis for selection of functional probiotics for commercial use. The objective of study was to collect different cultures and screen to study their functional probiotic characteristics such as acid tolerance, bile tolerance, antibacterial activity, antibiotic sensitivity, temperature tolerance and acidifying activity. Among the eighteen Lactic acid bacterial strains studied, 15 showed good survivability at high bile salt concentration (0.3 to 2 % oxgall) and good growth at a pH of 1.5 to 3.5. These probiotic species showed good survival abilities in acidic pH 2.0 to 3.5 except Lactobacillus delbruckii bulgaricus 281, Bifidobacterium bifidum 232 and Bifidobacterium bifidum 235, which did not grow at pH 2.0. Lactobacillus fermentum 141 and Pediococcus acidolactici 252 was able to grow even at pH 1.5 also. All the 18 lactic acid bacterial strains showed a count (CFU/ml) in the range of 0.23x107 to 2.7x107 at pH 2.0 except Lactobacillus delbruckii bulgaricus 281, Bifidobacterium bifidum 232 and Bifidobacterium bifidum 235, which did not form detectable CFU/ml, whereas Lactobacillus helviticus 194 showed highest count of 2.7x107 CFU/ml at pH 2.0. Among all the Lactobacillus spp. Lactobacillus paraplantarum 321 and Lactobacillus paracasei 22 showed maximum growth at 0.3% oxgall concentrations similarly Bifidobacterium bifidum 233 and Bifidobacterium bifidum 236 showed maximum growth when compared to other bifidobacterial strains. Among eighteen Lactic acid bacterial strains Lactobacillus casei 17, Pediococcus acidolactici 252 (except Clindamycin), Lactobacillus delbruckii bulgaricus 281 (except for Nitrofurantoin, Clindamycin and Cefpodoxime) and Lactobacillus helviticus 194 (except for Gentamycin, Nitrofurantoin and Streptomycin) were resistant to all the antibiotics tested. Bifidobacterium bifidum 231 (except for Streptomycin and Kanamycin) and Bifidobacterium bifidum 236 (except Norfloxicin) showed resistance to all antibiotics tested. All these Lactic acid bacterial strains were screened for in vitro inhibition of pathogenic microorganisms namely Proteus vulgaris, E.coli ATCC, E.coli 448, Pseudomonas aeruginosa, Klebsialla pneumoneae, Salmonella typhimurium, Bacillus cereus, Salmonella para-B, Staphylococci aureus. All the eighteen strains showed good antibacterial activity against the tested pathogenic bacteria. The strains of Lactobacillus paraplantarum 321, Pedicocci acidolactici 252 and Lactobacillus bulgaricus 281 inhibited the growth of all pathogenic bacteria. Bifidobacterium bifidum 232 and Bifidobacterium bifidum 233 (except Bacillus cereus and Salmonella typhimurium) also inhibited the growth of all pathogens tested. All the eighteen Lactic acid bacterial strains showed growth at all the tested temperatures (15oC, 40oC, and 45oC) except Bifidobacterium bifidum 229, Lactobacillus fermentum 141 and Lactobacillus rhamnosus 18 which have shown growth only at the temperatures of 15oC and 40oC, except at 45oC Among twelve Lactobacillus spp. Lactobacillus helviticus 194 has shown the highest acidifying activity, whereas Lactobacillus casei 17 shown lowest acidifying activity. Similarly Bifidobacterium bifidum 231 shown highest acidifying activity, whereas Bifidobacterium bifidum 236 shown lowest acidification values when compared with the other Bifidobacterial strains. The present study identified functional probiotics for future in vivo studies to commercialize probiotic strains of lactic acid bacteria to promote public health.ThesisItem Open Access STUDY ON CHARACTERIZATION OF SLAUGHTERHOUSE WASTEWATER IN AND AROUND HYDERABAD CITY(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2011-02) ENUMULA KUMAR; MADHAVA RAO, T(MAJOR); KRISHNAIAH, N; GOPALA REDDY, A; PRABU PRASADINI, PABSTRACT : The present study was undertaken to characterize the physical, chemical and microbiological properties of raw wastewater collected from three slaughterer houses located in and around Hyderabad city and to assess the efficiency of the treatment available at the plant and the effect of such wastewater on the environment. In raw wastewater from three slaughterhouses, the physical characteristics such as temperature of 31oC, pH of 6.8, turbidity in the range of 1847-4287 NTU, alkalinity varied between 433-903 mg/L, electrical conductivity in the range of 3.31-3.57 mS/cm, TDS varied between 1705-1896 mg/L, TSS ranged between 3001-5479 mg/L. The biochemical characteristics like DO varied between 0.41-1.45 mg/L, BOD ranged between 3559-4169 mg/L, COD varied between 4812-5308 mg/L, TOC varied between 2046-3345 mg/L, calcium ranged from 369 to 595 mg/L, NH4-N varied between 66-152 mg/L, nitrates in the range of 89-111 mg/L and phosphates of 76 mg/L were recorded. Major heavy metals such as lead, nickel and cadmium detected in wastewater from three slaughterhouses lead content ranged between 5.73 and 10.88 mg/L, nickel content of 1.03 mg/L and cadmium content varied from 0.59 to 1.47 mg/L detected. Except lead, cadmium and nickel values were below the effluent discharges limits recommended by pollution control board. In raw wastewater from three slaughterhouses, the microbiological characteristics such as TVC ranged from 68.21x106 - 105.14x 106 cfu/ml, total coliform count varied between 10.41 x106 - 35.16x106 cfu/ml recorded. The pathogens were isolated and the total count of Salmonella spp. ranged between 11.3-14.1x10 4 cfu/ml, Shigella count varied between 2.67x10 4-5.3x10 4 cfu/ml, S. faecalis count ranged between 8.58 x104 - 10.91x104 cfu/ml, S. aureus varied between 18.96x104 - 21.19x104 cfu/ml, B. cereus varied between 9.17 x104 - 10.19 x104 cfu/ml, and L. monocytogenes varied between 1900-6300 cfu/ml. The two slaughterhouses (i.e. II and III) have no treatment facilities and slaughterhouse I only having the UASB and DAF treatment facilities. The treated wastewater from slaughterhouse I has shown insufficient reduction in wastewater strength which was above the recommended limits for effluent discharges. The UASB reactor significantly reduced the wastewater strength. It has shown the removal rates of turbidity, TSS, BOD, COD, TOC, NH4-N, nitrates and phosphates as 71%, 80%, 79%, 71%, 63%, 70%, 53% and 42%, respectively. Whereas the DAF unit has shown the removal rates as 53%, 47%,42%, 55%, 48%, 36%, 49% and 77% respectively. The treatment of wastewater by UASB and DAF units has not been shown any effect on the levels of heavy metals. During UASB and DAF treatments the heavy metal followed an irregular trend with slight variation in levels. Further, the efficiency of UASB reactor has shown efficient removal rates for microbiological properties like TVC, TCC, total count of Salmonella spp., Shigella spp. S. faecalis, S. aureus, B .cereus and L. monocytogenes as 78%, 87.4%, 92%, 87%, 85%, 76%, 78% and 72% respectively, whereas the DAF unit has shown these removal rates as 82.6%, 85%, 64%, 70%, 72%, 58% and 72% respectively. The effect of the treated and raw wastewater from slaughterhouses on some physical and chemical properties of the soils was investigated. The results obtained revealed that both raw and treated slaughterhouse wastewater decreased the soil pH to 6.95 and to 6.88 from pH of 7.34 (control soil) and increased the electrical conductivity of soils to 1.59 mS/cm and to 1.48 mS/cm from 0.62 mS/cm (control soil). The raw and treated wastewaters also increased the organic carbon of control soils (1.75%) to 5.29% and to 6.24% and the available nitrogen of control soils (339.04 kg/ha) to 752.64 kg/ha and to 815.36 kg/ha. The heavy metal (Pb, Ni, and Cd) levels in soils polluted with raw and treated wastewaters are significantly increased to 4.11 mg/L, 0.38 mg/L and 0.54 mg/L when compared to that of control soils such as 11.4 mg/L, 0.05 mg/L and 0.13 mg/L, respectively.ThesisItem Open Access STUDIES ON THE INCIDENCE OF SHIGELLA IN LIVESTOCK PRODUCTS AND ENVIRONMENTAL SAMPLES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-07) DURGA RAMA DEVI, N; KRISHNAIAH, N(MAJOR); MADHAVA RAO, T; ANJANEYULU, YABSTRACT: Out of 60 samples each of stools, drinking water, farm water, beef, chicken, egg and fish, Shigella spp. were found in 12 ,4, 15, 4, 3, 4, 3 and 5 respectively. Out of 12 positive stool samples for Shigella, 8 were S. flexneri, 3 were S. sonnei and 1 was S. dysenteriae. Out of 7 and 15 positive samples of drinking and farm water, S. flexneri was found in 4 and 10, S. sonnei 2 and 3 respectively and S. dysenteriae was one in each. Out of 4 positive samples each of beef and milk. S. flexneri was found in 3 and 2 S. sonnei one each and S. dysenteriae was 1 in milk and not found in beef. Out of 3, 3 and 5 positive samples of chicken, eggs and fish, S. flexneri was found in 2, 2 and 4 respectively. S. sonnei was found in one each and S. dysenteriae was not found. Out of 480 total different samples 53 were positive for Shigella spp., out of which 35 were S. flexeneri, 13 were S. sonnei and 4 were S. dysenteriae. For isolation and identification of Shigella spp., BHI broth enrichment was good for S. flexneri and SB broth was good for S. sonnei and S. dysenteriae. The isolation with or without enrichment and plating has shown that XLD and HEA media are superior over DCA, MAC and SS agar for S. flexneri and S. sonnei, where as for S. dysenteriae XLD, DCA, HEA and MAC has given moderate growth, with no growth on SS medium. In general, all the three enrichment broths have given better growth compared to direct plating. S. flexneri was highly sensitive to Kanamycin (94.28%) and Nalidixic acid (84.7%) and highly resistant to Gentamycin (97%) and Ampicillin (91.4%), where as S.sonnei was highly sensitive to Kanamycin (92.3%), Ampicillin (84.6%), Chloramphenical (69.23%) and highly resistant to Gentamycin (92.3%), Nalidixic acid (76.9%) and Cotrimaxozole (69.23%). S.dysenteriae was sensitive only to Kanamycin (75%) and Ciprofloxacin (25%) and resistant to Ampicillin, Gentamycin, Tetracycline , Nalidixic acid , Erythromycin, Chloramphenical and Cotrimoxazole (75%).ThesisItem Open Access DETECTION OF VIBRIO CHOLERAE IN FOODS BY PCR TECHNIQUE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-06) MAHESHWARI, MANI; KRISHNAIAH, N(MAJOR); MADHAVA RAO, T; ANJANEYULU, YABSTRACT: The present study was undertaken to standardize PCR assay for detection of Vibrio cholerae, its serogroups (O1 and O139) and cholera toxin from foods and compare its efficacy with conventional cultural methods. Primers derived from ompW gene, rfbO1 and rfbO139 genes and ctxA gene were used which gave specific amplification products of 304, 192, 449, 302 bp for V.cholerae, its serogroups (O1 and O139) and cholera toxin respectively. To determine their suitability to PCR, four different template preparation methods viz. genomic DNA extraction, heat lysis, lysis buffers-1 and 2 were compared, of which heat lysis was found to be efficient and convenient. The specificity for V.cholerae, its serogroups (O1 and O139) and cholera toxin was tested using primers from ompW, rfbO1, rfbO139 and ctxA genes with V.cholerae and nine other non-V.cholerae strains, which gave specific products at 304, 192, 449 and 302 bp for V.cholerae, its serogroups and cholera toxin respectively. The minimum detection level was 2.5 cfu for V.cholerae, its serogroups and cholera toxin. Evaluation of two non-selective broths, i.e Luria Broth (LB) and Tryptic Soy Broth (TSB) and two selective broths, i.e Alkaline Peptone Water Broth (APW) and Salt Polymyxin Broth (SPB) for PCR compatibility, it was revealed that APW was superior followed by SPB, LB and TSB. Spiking studies were carried out by inoculating with pure culture of V.cholerae in selective enrichments broths (8h and 18h), which revealed that earliest detection time for V.cholerae by PCR was 18h and APW was the most suited selective broth for PCR assay. Screening of 245 naturally contaminated samples (35 each of water, fish, crab, shrimp, meat, milk and clinical stool samples) revealed 80 positive for V.cholerae (water-19, fish-16, crab-20, shrimp-6, meat-4, milk-3, and clinical stool samplres-12) out of which 45 belonged to O1 serogroup (water 11, fish-9, crab-12, shrimp-3, meat-2, milk-1 and clinical stool samples-7), 25 belonged to O139 (water-6, fish-5, crab-6, shrimp-2, meat-1, milk-1 and clinical stool samples-4). Out of 45 and 25 positive samples for O1 and O139, 35 and 20 were positive for cholera toxin respectively.ThesisItem Open Access DETECTION OF YERSINIA ENTEROCOLITICA IN LIVESTOCK PRODUCTS BY PCR TECHNIQUE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-09) SHASHIKALA, SANKARAMADDI; KRISHNAIAH, N(MAJOR); MADHAVA RAO, T; GOPALA REDDY, AABSTRACT : The present study was undertaken to standardize PCR assay for detection of Yersinia enterocolitica and Yersinia heat stable enterotoxin from livestock products and compare its efficacy with conventional cultural methods. Primers derived from ail gene and yst B genes used which gave specific amplification products of 425 and 146bp for Y. enterocolitica and yersinia heat stable enterotoxin (yst B) respectively. To determine their suitability to PCR, three different template preparation methods viz. heat lysis, lysis buffer-1and 2 were compared, of which heat lysis was found to be efficient and convenient. The specificity for Y. enterocolitica and yersinia heat stable enterotoxin were tested using primers from ail and yst B genes with Y. enterocolitica and seven other non-Y. enterocolitica strains, which gave specific products at 425 and 146bp for Y. enterocolitica and Yersinia heat stable enterotoxin respecitvely. The minimum detection level with pure Y. enterocolitica culture was 2.0 cfu. Evaluation of two non-selective broths (Tryptic Soya broth and Brain heart infusion broth) and two selective broths (Yersinia selective enrichment broth and Irgasan-ticarcillin-potassium chloride broth) for PCR compatibility, it revealed that ITC was superior followed by YSE, BHI and TSB. Spiking studies were carried out by inoculating with pure culture for Y. enterocolitica in selective enrichment broths (24hr), which revealed that earliest detection time by PCR of Y. enterocolitica was 24 hr and ITC was the most suited selective broth for PCR assay. Screening 300 naturally contaminated samples (25 each of pork, pork tonsils, pork tongues, pork surface swabs, swine faeces, swine farm water, milk, beef, cattle faeces, dairy farm water, chicken and poultry farm water samples) revealed 132 (14, 23, 23, 10, 9, 21, 4, 5, 4, 8, 6 and 5 respectively for the above samples) positive for Y. enterocolitica, out of which 56 samples (5,10,9,4,2,9,2,2,1,5,4 and 3 respectively) were positive for yst B gene.ThesisItem Open Access DETECTION OF VIBRIO PARAHAEMOLYTICUS IN FOODS BY PCR TECHNIQUE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-09) SUBHASHINI, NELAPATI; KRISHNAIAH, N(MAJOR); MADHAVA RAO, T; ANJANEYULU, YABSTRACT : The present study was undertaken to standardize PCR assay for detection of Vibrio parahaemolyticus and their toxins from foods and compare its efficacy with conventional cultural methods. Primers derived from toxR gene and tdh and trh genes were used which gave specific amplification products of 368, 623 and 460 bp for V. parahaemolyticus and toxins (tdh and trh) respectively. To determine their suitability to PCR, four different template preparation methods viz. genomic DNA extraction, heat lysis, lysis buffers-1 and 2 were compared, of which heat lysis was found to be efficient and convenient. The specificity for V. parahaemolyticus and toxins were tested using primers from toxR, tdh and trh genes with V. parahaemolyticus and nine other non-V. parahaemolyticus strains, which gave specific products at 368, 623 and 460 bp for V. parahaemolyticus and toxins respectively. The minimum detection level was 2.5 cfu for both V. parahaemolyticus and toxins. Evaluation of two non-selective broths, i.e Brain Heart Infusion Broth (BHI) and Tryptic Soy Broth (TSB) and two selective broths, i.e Alkaline Peptone Water Broth (APW) and Salt Polymixin Broth (SPB) for PCR compatibility, it was revealed that SPB was superior followed by APW, BHI and TSB. Spiking studies were carried out by inoculating with pure culture of V. parahaemolyticus in selective enrichments broths (8h and 18h), which revealed that earliest detection time for V. parahaemolyticus by PCR was 18h and SPB was the most suited selective broth for PCR assay. Screening of 280 naturally contaminated samples (35 each of fresh water fish, sea fish, fresh water prawn, marine shrimp, crab, fresh water, marine water and mutton samples) revealed 165 positive for V. parahaemolyticus (fresh water fish-17, sea fish-20, fresh water prawn-24, marine shrimp-33, crab-27, fresh water-14, marine water-28 and mutton-2) out of which 24 carried only tdh (fresh water fish-2, sea fish-4, fresh water prawn-3, marine shrimp-4, crab-4, fresh water-2, marine water-5 and mutton-nil), 95 carried only trh (fresh water fish-12, sea fish-16, fresh water prawn-9, marine shrimp-13, crab-15, fresh water-12, marine water-18 and mutton-nil) and 15 carried both tdh and trh (fresh water fish-1, sea fish-2, fresh water prawn-1, marine shrimp-3, crab-2, fresh water-2, marine water-4 and mutton-nil) simultaneously.ThesisItem Open Access DETECTION OF LISTERIA MONOCYTOGENES IN LIVESTOCK PRODUCTS BY PCR(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-09) SIVA SWETHA, CHINTA; MADHAVA RAO, T(MAJOR); KRISHNAIAH, N; NARASIMHA REDDY, YABSTRACT : The present study was undertaken to standardize PCR assay for detection of Listeria monocytogenes and Listeriolysin O from livestock products and compare its efficacy with conventional cultural methods. Primers derived from iap and hlyA genes were used which gave specific amplification products of 131 and 456 bp for L. monocytogenes and Listeriolysin O respectively. To determine their suitability to PCR, three different template preparation methods viz. genomic DNA extraction, heat lysis, lysis buffer method were compared, of which heat lysis was found to be efficient and convenient. The specificity of L.monocytogenes and Listeriolysin O were tested using primers from iap and hlyA genes with L.monocytogenes and seven other non-Listeria monocytogenes strains, which gave specific products 131 and 456 bp for L.monocytogenes and Listeriolysin O respectively. The minimum detection level with pure L. monocytogenes culture was found 4 cfu. Evaluation of two non-selective broths (Brain Heart Infusion broth and Tryptone Soya Broth) and two selective broths (LEB and PALCAM) for PCR compatibility, it revealed that LEB was superior followed by PALCAM, BHI and TSB. Spiking studies were carried out by inoculating with pure culture of L.monocytogenes in selective enrichment broths (18 h and 24 h), which revealed that the earliest detection time by PCR for L.monocytogenes was 24 hr and LEB was the most suited selective broth for PCR assay. Screening 200 naturally contaminated samples (25 of milk, 15 of ice cream, 10 of dairy farm water, 25 each of pork, pork swabs, chicken, chicken swabs, fish, fish swabs samples) revealed 23 positive results for L.monocytogenes and Listeriolysin O by PCR.ThesisItem Open Access DETECTION OF ESCHERICHIA COLI O157:H7 FROM LIVESTOCK PRODUCTS BY PCR TECHNIQUE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-10) BINDU KIRANMAYI, Ch; KRISHNAIAH, N(MAJOR); VENKATESWARA RAO, L; DHANA LAKSHMI, KABSTRACT: The present study was undertaken to standardize PCR assay for detection of Escherichia coli O157:H7 and Shiga-Toxigenic Escherichia coli (STEC) from livestock products and compare its efficacy with conventional cultural methods. Primers derived from HlyA gene and Stx1 and Stx2 genes were used which gave specific amplification products of 361, 614 and 779 bp for Escherichia coli O157:H7 and STEC (Stx1 and Stx2) respectively. To determine their suitability to PCR, four different template preparation methods viz. genomic DNA extraction, heat lysis, lysis buffers-1 and 2 were compared, of which heat lysis was found to be efficient and convenient. The specificity for Escherichia coli O157:H7 and STEC were tested using primers from HlyA, Stx1 and Stx2 genes with Escherichia coli O157:H7 and nine other non-Escherichia coli O157:H7 strains, which gave specific products at 361, 614 and 779 bp for Escherichia coli O157:H7 and STEC respectively. The minimum detection level with pure Escherichia coli O157:H7 culture was found 1.7 cfu. Evaluation of two non-selective broths (Brain Heart Infusion broth and Buffered Peptone Water) and two selective broths (modified E.coli broth and modified Tryptic Soy Broth) for PCR compatibility, it revealed that mTSB was superior followed by mEC, BHI and BPW. Spiking studies were carried out by inoculating with pure culture of Escherichia coli O157:H7 in selective enrichments broths (10h and 18h), which revealed that earliest detection time by PCR of Escherichia coli O157:H7 was 18h and mTSB was the most suited selective broth for PCR assay. Screening 600 naturally contaminated samples (50 each of mutton, mutton swabs, sheep faeces, sheep farm water, beef, beef swabs, beef and dairy cattle faeces, milk, dairy farm water, chicken and poultry farm water samples) revealed 110 positive for Escherichia coli O157:H7 out of which 40 carried only Stx1, 18 carried only Stx2 and 9 carried both Stx1 and Stx2 simultaneously. Out of 600 samples 248 samples were positive for STEC of which 178 were Stx1, 70 Stx2 and 40 Stx1 and Stx2 simultaneously.