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ThesisItem Open Access STUDIES ON MOLECULAR EPIDEMIOLOGY OF BRUCELLOSIS AND TUBERCULOSIS IN CATTLE AND BUFFALOES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2020-09) SANTOSH SAJJAN; SRINIVASA RAO, T (MAJOR); MADHAVAPRASAD, C.B.; RAMANI PUSHPA, R.N.; ASWANI KUMAR, KBrucellosis and tuberculosis are neglected zoonoses having worldwide public health concern. The present study was undertaken to elucidate the prevalence of brucellosis and tuberculosis in cattle and buffaloes, detect and identify the species by PCR, understand the genetic diversity among the detected species, investigate the antimicrobial resistance of pathogens and evaluate the risk factors associated with the occurrence of these diseases. A total of 1070 samples comprising of 330 blood samples, 185 milk samples, 310 nasal swabs and 245 vaginal swabs from 300 cattle and 30 buffaloes which were reared along with cows were collected and examined for detection of brucellosis and mycobacteriosis in different production systems viz., intensive (110), semi-intensive (110) and extensive system (110). The overall prevalence of brucellosis and mycobacteriosis was 6.06% (20/330) and 3.33% (11/330), respectively among the animals studied. The prevalence of both the diseases was higher in farms with intensive production system and larger herd size. Sexually active (2-10 years) and xii aged animals (5-10 years) were at risk of infection for brucellosis and tuberculosis, respectively. Exotic breeds of cattle were more susceptible to mycobacteriosis compared to indigenous breeds. Rose bengal plate test (BPT) and indirect enzyme linked immunosorbent assay (iELISA) tests revealed almost perfect agreement (К=0.92) with each other. While, SIT and IFN-γ ELISA tests revealed substantial agreement (К=0.50) with each other. The species of Brucella recognized by Bruce-ladder multiplex PCR in cattle were found to be B. melitensis (4) and B. abortus (5) out of 09 samples confirmed by PCR. Further, all the Mycobacterium spp. detected by PCR were differentiated as non tuberculous Mycobacterium (NTM) by commercial qPCR kit. Brucella species displayed genetic homogeneity by both PCR-Restriction Fragment Length Polymorphism (RFLP) and Repetitive Element Palindromic (REP)-PCR techniques. Multiple Antimicrobial Resistance (MAR) index for B.abortus and B. melitensis isolates were found to be in the range of 0.66 to 0.83 and 0.5 to 0.91, respectively. Majority of the isolates showed the presence of multiple genes responsible for resistance to rifampicin (rpoB-M4, M5, M6 and +354rB/-720rB gene) fluoroquinalones (gyrA and gyrB). While only one isolate showed the presence of single gene (tetB) responsible for resistance to tetracyclines and one isolate showed presence of single gene {Aac(3)-Ia} responsible for resistance to aminoglycosides. None of the isolates showed presence of catB gene responsible for resistance to chloramphenicol even though all the isolates were resistant to chloramphenicol phenotypically. There was significant association with the individual level (age, sex and breed) and herd level risk factors (production system, herd size, cleanliness and lack of screening of animals) with the occurrence of brucellosis and mycobacteriosis in cattle and buffaloes as evidenced by Odd’s ratio.ThesisItem Open 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, KHuman 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.ThesisItem Open Access STUDIES ON MOLECULAR EPIDEMIOLOGY OF BRUCELLOSIS AND TUBERCULOSIS IN CATTLE AND BUFFALOES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2020-09) SANTOSH SAJJAN; SRINIVASA RAO, T(MAJOR); MADHAVAPRASAD, C.B.; RAMANI PUSHPA, R.N.; ASWANI KUMAR, KBrucellosis and tuberculosis are neglected zoonoses having worldwide public health concern. The present study was undertaken to elucidate the prevalence of brucellosis and tuberculosis in cattle and buffaloes, detect and identify the species by PCR, understand the genetic diversity among the detected species, investigate the antimicrobial resistance of pathogens and evaluate the risk factors associated with the occurrence of these diseases. A total of 1070 samples comprising of 330 blood samples, 185 milk samples, 310 nasal swabs and 245 vaginal swabs from 300 cattle and 30 buffaloes which were reared along with cows were collected and examined for detection of brucellosis and mycobacteriosis in different production systems viz., intensive (110), semi-intensive (110) and extensive system (110). The overall prevalence of brucellosis and mycobacteriosis was 6.06% (20/330) and 3.33% (11/330), respectively among the animals studied. The prevalence of both the diseases was higher in farms with intensive production system and larger herd size. Sexually active (2-10 years) and aged animals (5-10 years) were at risk of infection for brucellosis and tuberculosis, respectively. Exotic breeds of cattle were more susceptible to mycobacteriosis compared to indigenous breeds. Rose bengal plate test (BPT) and indirect enzyme linked immunosorbent assay (iELISA) tests revealed almost perfect agreement (К=0.92) with each other. While, SIT and IFN-γ ELISA tests revealed substantial agreement (К=0.50) with each other. The species of Brucella recognized by Bruce-ladder multiplex PCR in cattle were found to be B. melitensis (4) and B. abortus (5) out of 09 samples confirmed by PCR. Further, all the Mycobacterium spp. detected by PCR were differentiated as non tuberculous Mycobacterium (NTM) by commercial qPCR kit. Brucella species displayed genetic homogeneity by both PCR-Restriction Fragment Length Polymorphism (RFLP) and Repetitive Element Palindromic (REP)-PCR techniques. Multiple Antimicrobial Resistance (MAR) index for B.abortus and B. melitensis isolates were found to be in the range of 0.66 to 0.83 and 0.5 to 0.91, respectively. Majority of the isolates showed the presence of multiple genes responsible for resistance to rifampicin (rpoB-M4, M5, M6 and +354rB/-720rB gene) fluoroquinalones (gyrA and gyrB). While only one isolate showed the presence of single gene (tetB) responsible for resistance to tetracyclines and one isolate showed presence of single gene {Aac(3)-Ia} responsible for resistance to aminoglycosides. None of the isolates showed presence of catB gene responsible for resistance to chloramphenicol even though all the isolates were resistant to chloramphenicol phenotypically. There was significant association with the individual level (age, sex and breed) and herd level risk factors (production system, herd size, cleanliness and lack of screening of animals) with the occurrence of brucellosis and mycobacteriosis in cattle and buffaloes as evidenced by Odd’s ratio.ThesisItem Open 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, KHuman 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.
