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Sri Venkateswara Veterinary University, Tirupati

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  • ThesisItemOpen Access
    STUDIES ON EMERGING ZOONOTIC BACTERIAL PATHOGENS OF FISH AND SHELLFISH FROM FRESH WATER, MARINE AND ESTUARINE SOURCES OF ANDHRA PRADESH
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2020-08) SUBHASHINI, NELAPATI; SRINIVASA RAO, T (MAJOR); MADHAVA RAO, T; RAMANI PUSHPA, R.N.; ASWANI KUMAR, K
    Human contact with and consumption of fishes presents hazards from a range of bacterial zoonotic infections. The present study was undertaken to characterize Arcobactaer spp., Aeromonas spp. and V. vulnificus of fresh water, estuarine/ brackish and marine origin based on cultural isolation. A total of 420 samples comprising fish and prawn samples from fresh (70 each), marine (70 each) and estuarine environments (70 each) were analyzed to characterize Arcobacter spp., Aeromonas spp. and V. vulnificus and further these samples were screened for antibiotic residues by HPLC. Overall prevalence of Arcobacter spp. was found to be 12.61% (53/420). Out of the 53 Arcobacter spp. isolates, m-PCR revealed 38 (56.71%) to be A. butzleri, 3 (4.48%) to be A. cryaerophilus and 12 (17.91%) to be A. skirrowii. Out of the 53 Arcobacter spp. isolates, virulence genes mviN, irgA, hecA, cj1349, tlyA, pldA, hecB, ciaB and cadF were detected in 75.47%, 9.43%, 3.77%, 30.18%, 98.11%, 92.45%, 11.32%, 94.33% and 81.13% of Arcobacter spp. isolates, respectively. Antibiogram profile of 53 Arcobacter xix spp. isolates revealed natural resistance towards penicillin-G (100%); resistance to vancomycin (75.47%), nalidixic acid (26.42%), erythromycin (18.87%), cefixime and kanamycin (5%) and co-trimoxazole (3.77%). ESBL production was confirmed in 28 Arcobacter spp. isolates by both phenotypic and molecular methods and blaTEM was the only β-lactamase gene detected in all the 28 isolates. A greater degree of molecular heterogeneity was observed among ESBL positive Arcobacter butzleri (16) and A. skirrowii (12) isolates, respectively by ERIC-PCR and REP-PCR. The discriminatory power of the two typing methods for Arcobacter spp. was found to be highly significant (>0.90) i.e. one. Overall prevalence of Aeromonas spp. was found to be 26.42% (111/420). Out of 111 Aeromonas spp. isolates, m-PCR revealed 98 (88.28%) to be A. veroni, 9 (8.10%) to be A. hydrophila, 2 (1.80%) A. media and 2 (1.80%) A. caviae. Out of 111 Aeromonas spp. isolates, virulence genes act, ast, alt, ahyB, fla, lip, aer, ser, gcat and exu were detected in 53.15%, 4.5%, 5.4%, 9.9%, 0.9%, 22.52%, 16.21%, 2.7% 5.4 % and 15.31% of isolates, respectively. Antibiogram profile of 111 Aeromonas spp. isolates revealed natural resistance towards penicillin-G (100%) and resistance to vancomycin (63.06%) and nalidixic acid (50.45%). ESBL production was confirmed in 12 Aeromonas spp. isolates by both phenotypic and molecular methods and blaTEM was the only β-lactamase gene detected in all 12 isolates. A greater degree of molecular heterogeneity was observed among 12 ESBL positive Aeromonas spp. isolates from different sources as 12 different genotypes were observed by ERIC-PCR and REP-PCR. The discriminatory power of the two typing methods for Arcobacter spp. was found to be highly significant (>0.90) i.e. one. xx Overall prevalence of V. vulnificus was found to be 6.19% (26/420). All the V. vulnificus isolates carried vvhA gene and none of the isolates were belonging to Bt2 or serovar E. All the V. vulnificus isolates belonged to Bt1. Antibiogram profile of 26 V. vulnificus isolates revealed natural resistance for penicillin-G (100%) and resistance to vancomycin (61.54%), erythromycin (46.15%), cefixime (46.15%) and nalidixic acid (30.77%). ESBL production was confirmed in 17 V. vulnificus isolates by both phenotypic and molecular methods and blaTEM gene was the predominant gene in 16 isolates and blaSHV gene was detected in only one V. vulnificus isolate. A greater degree of molecular heterogeneity was observed among 17 ESBL positive V. vulnificus isolates from different sources as 16 and 17 different genotypes were observed under ERIC-PCR and REP-PCR, respectively. The discriminatory power of the two typing methods for V. vulnificus isolates was found to be highly significant (>0.90) i.e. 0.9926 and one for ERIC and REP-PCR, respectively. Cluster analysis revealed a greater degree of homogeneity and heterogeneity among different isolates (Arcobacter spp., Aeromonas spp. and V. vulnificus) recovered from various sources and indicating that there is a chance of cross-contamination particularly in the fish markets. All the fish and shellfish samples were negative for antibiotic residues by HPLC.
  • ThesisItemOpen Access
    STUDIES ON EMERGING ZOONOTIC BACTERIAL PATHOGENS OF FISH AND SHELLFISH FROM FRESH WATER, MARINE AND ESTUARINE SOURCES OF ANDHRA PRADESH
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2020-08) SUBHASHINI, NELAPATI; SRINIVASA RAO, T(MAJOR); MADHAVA RAO, T; RAMANI PUSHPA, R.N.; ASWANI KUMAR, K
    Human contact with and consumption of fishes presents hazards from a range of bacterial zoonotic infections. The present study was undertaken to characterize Arcobactaer spp., Aeromonas spp. and V. vulnificus of fresh water, estuarine/ brackish and marine origin based on cultural isolation. A total of 420 samples comprising fish and prawn samples from fresh (70 each), marine (70 each) and estuarine environments (70 each) were analyzed to characterize Arcobacter spp., Aeromonas spp. and V. vulnificus and further these samples were screened for antibiotic residues by HPLC. Overall prevalence of Arcobacter spp. was found to be 12.61% (53/420). Out of the 53 Arcobacter spp. isolates, m-PCR revealed 38 (56.71%) to be A. butzleri, 3 (4.48%) to be A. cryaerophilus and 12 (17.91%) to be A. skirrowii. Out of the 53 Arcobacter spp. isolates, virulence genes mviN, irgA, hecA, cj1349, tlyA, pldA, hecB, ciaB and cadF were detected in 75.47%, 9.43%, 3.77%, 30.18%, 98.11%, 92.45%, 11.32%, 94.33% and 81.13% of Arcobacter spp. isolates, respectively. Antibiogram profile of 53 Arcobacter spp. isolates revealed natural resistance towards penicillin-G (100%); resistance to vancomycin (75.47%), nalidixic acid (26.42%), erythromycin (18.87%), cefixime and kanamycin (5%) and co-trimoxazole (3.77%). ESBL production was confirmed in 28 Arcobacter spp. isolates by both phenotypic and molecular methods and blaTEM was the only β-lactamase gene detected in all the 28 isolates. A greater degree of molecular heterogeneity was observed among ESBL positive Arcobacter butzleri (16) and A. skirrowii (12) isolates, respectively by ERIC-PCR and REP-PCR. The discriminatory power of the two typing methods for Arcobacter spp. was found to be highly significant (>0.90) i.e. one. Overall prevalence of Aeromonas spp. was found to be 26.42% (111/420). Out of 111 Aeromonas spp. isolates, m-PCR revealed 98 (88.28%) to be A. veroni, 9 (8.10%) to be A. hydrophila, 2 (1.80%) A. media and 2 (1.80%) A. caviae. Out of 111 Aeromonas spp. isolates, virulence genes act, ast, alt, ahyB, fla, lip, aer, ser, gcat and exu were detected in 53.15%, 4.5%, 5.4%, 9.9%, 0.9%, 22.52%, 16.21%, 2.7% 5.4 % and 15.31% of isolates, respectively. Antibiogram profile of 111 Aeromonas spp. isolates revealed natural resistance towards penicillin-G (100%) and resistance to vancomycin (63.06%) and nalidixic acid (50.45%). ESBL production was confirmed in 12 Aeromonas spp. isolates by both phenotypic and molecular methods and blaTEM was the only β-lactamase gene detected in all 12 isolates. A greater degree of molecular heterogeneity was observed among 12 ESBL positive Aeromonas spp. isolates from different sources as 12 different genotypes were observed by ERIC-PCR and REP-PCR. The discriminatory power of the two typing methods for Arcobacter spp. was found to be highly significant (>0.90) i.e. one. Overall prevalence of V. vulnificus was found to be 6.19% (26/420). All the V. vulnificus isolates carried vvhA gene and none of the isolates were belonging to Bt2 or serovar E. All the V. vulnificus isolates belonged to Bt1. Antibiogram profile of 26 V. vulnificus isolates revealed natural resistance for penicillin-G (100%) and resistance to vancomycin (61.54%), erythromycin (46.15%), cefixime (46.15%) and nalidixic acid (30.77%). ESBL production was confirmed in 17 V. vulnificus isolates by both phenotypic and molecular methods and blaTEM gene was the predominant gene in 16 isolates and blaSHV gene was detected in only one V. vulnificus isolate. A greater degree of molecular heterogeneity was observed among 17 ESBL positive V. vulnificus isolates from different sources as 16 and 17 different genotypes were observed under ERIC-PCR and REP-PCR, respectively. The discriminatory power of the two typing methods for V. vulnificus isolates was found to be highly significant (>0.90) i.e. 0.9926 and one for ERIC and REP-PCR, respectively. Cluster analysis revealed a greater degree of homogeneity and heterogeneity among different isolates (Arcobacter spp., Aeromonas spp. and V. vulnificus) recovered from various sources and indicating that there is a chance of cross-contamination particularly in the fish markets. All the fish and shellfish samples were negative for antibiotic residues by HPLC.
  • 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.