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2008 - 20095
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Subject: Veterinary Public Health × End date: 2009 × Start date: 2008 × Type: Thesis ×
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    ThesisItemOpen 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, A
    ABSTRACT : 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.
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    ThesisItemOpen 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, Y
    ABSTRACT : 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.
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    ThesisItemOpen 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, Y
    ABSTRACT : 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.
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    ThesisItemOpen 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, K
    ABSTRACT: 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.
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    ThesisItemOpen Access
    MICROBES OF PUBLIC HEALTH SIGNIFICANCE IN SHRIMPS AT KAKINADA PORT AREA
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-03) ANNIE SUPRIYA, R; VENKATESWARA RAO, L(MAJOR); KRISHNAIAH, N; Dhanalakshmi, K
    ABSTRACT: A study was under taken to assess microbiological status and incidence of pathogens of Public Health significance in three varieties of shrimps (flower, White and Tiger) at Kakinada port area in market as well as processing centres samples. Five stages of processing i.e. head removal, sizing and grading, final rinse, arrangement and water filling and packing were selected for analysis. The SPC count in market and processing plants was high in flower shrimp (7.6x107 and 5.7x105), low in Tiger shrimp (4.2x107 and 2.8x106) and moderate in White shrimp (5.4x107 and 4.1x106) respectively. During processing SPC counts decreased after first step, increased after second step and decreased upto final step with counts of 4.9, 3.6 and 2.1x106 in Flower, White and Tiger varieties. The total coliform count in market and processing centres are high in flower shrimp (51.2 and 27.2) low in Tiger shrimp (38.2 and 22.8) and moderate in White shrimp (45.2 and 25.1). During processing coliform count reduced after first step, increased after second step and continuously decreased after further steps reaching counts of 12.8, 15.9 and 16.8 for Tiger, White and Flower shrimp. The Yeast and Mould count in market and processing centres are high in Flower shrimp (8.6x104 and 5.6x103), low in Tiger shrimp (3.0x104 and 2.6x103) and moderate in White shrimp (4.8x104 and 3.6x103). The yeast and mould counts reduced after first step, increased after second step and continuously reduced upto fifth step of processing reaching 2.6x103, 1.2x103 and 1.4x103 in Flower, Tiger and White shrimps. The incidence of Salmonella in samples from market and processing centres is high in Flower (36% and 22%), low in Tiger shrimp (24% and 14%) and moderate in White (28% and 18%) respectively. The incidence during processing reduced after first step, remained same after second step and reduced afterwards reaching 8% in all the three varieties. The incidence of Vibrio cholerae and Vibrio parahaemolyticus in market samples are high in flower (6% and 32%), low in Tiger (4% and 26%) and moderate in White (4% and 28%). The incidence is zero for Vibrio cholerae in processing centres and Vibrio parahaemolyticus is 24%, 16% and 20% for flower. The incidence of Vibrio parahaemolyticus remained the same after first and second steps, reduced after third step, reduced after final step reaching 12%, 8% and 6% in Flower, White and Tiger shrimps respectively. The Staphylococcus aureus counts of samples from market and processing centres were low in Tiger (3.9x105 and 4.1x103), high in Flower (5.8x105 and 5.4x103) and moderate in White (4.6x105 and 4.8x103) respectively. These counts reduced after first step, increased after second step and reduced after further steps reaching 2.0, 2.1 and 2.2x103 in Tiger, White and Flower shrimps. In general the microbiological parameters are high in market samples compared to processing centres. Tiger shrimp had least, high Flower and moderate in White varieties.