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Subject: Veterinary Microbiology ×

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    ThesisItemOpen Access
    EPIDEMIOLOGY AND DEVELOPMENT OF RECOMBINANT ANTIGEN BASED DIAGNOSTICS FOR BOVINE LEPTOSPIROSIS
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2022) SONALI MENAMVAR; B. M. VEEREGOWDA)
    The present study was aimed to study seroepidemiology of leptospirosis and to express the leptospiral surface adhesion (rLsa 44) protein of pathogenic Leptospira interrogans serovar Pomona in E. coli and its evaluation as a potential diagnostic antigen. The observed seroprevalence of bovine leptospirosis at animal level and farm level in Telangana state was 41.36 per cent and 77.61 per cent respectively. The highest seropositivity (80.95%) was observed in Wanaparthy and Rangareddy and the lowest seropositivity (20%) was observed in Mancheriala district. The prevalent leptospiral antibodies were predominantly against the serogroups Icterohemorrahgiae (32.4%), Pomona (22.16%), Javanica (19.07%), Australis (17.01%), Bataviae (15.46%), Bankinang (12.89%), Hebdomadis (12.89%). Further, the breed of the animal (p=0.03) and the health status of the animal (p=0.03) are the significant risk factors associated with the prevalence of leptospirosis. Furthermore, multivariate statistical analysis of farm factors revealed that the size of the farm (p=0.05), presence of the dog in and around the farm (p=0.039), presence of rodents in the farm (p=0.011), farms using fodder from wet soils (p=0.043) and farms closer to the water bodies (p=0.041) were significantly associated with the prevalence of bovine leptospirosis. Leptospiral surface adhesion (Lsa 44) gene was amplified employing designed primers by polymerase chain reaction and the purified amplicon was initially cloned in pGEM-T Easy vector in a pET vector expression in E. coli. The expressed recombinant protein after induction with IPTG was purified through Ni-NTA column and characterized by SDS-PAGE and western blot, which confirmed the expressed reactive protein is Leptospira specific with a molecular weight of ~44 kDa. The antigen was coated on latex beads and assessed for its suitability as diagnostic antigen in latex agglutination Test (LAT) for the development of new diagnostics. Further, the evaluation of rLsa 44 LAT beads revealed a relative diagnostic sensitivity and diagnostic specificity of 93.55 per cent and 90.62 per cent respectively with an accuracy of 92.06 per cent against MAT while testing known MAT positive and negative bovine sera samples (n=126) collected from cattle and buffaloes associated with the reproductive problems. This rLsa 44 LAT is an extremely simple and rapid test and can be used as a diagnostic tool at the field level. This study appears to be the first to assess the prevalence of bovine leptospirosis at the farm level in endemic conditions and to use rLsa 44 protein for the detection of anti-leptospiral antibodies in cattle and buffaloes as a preliminary screening test.
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    ThesisItemOpen Access
    ISOLATION AND MOLECULAR CHARACTERIZATION OF ANTIMICROBIAL RESISTANT Escherichia coli IN PORK PRODUCTION CHAIN IN AND AROUND BENGALURU
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2022) SHIVAKUMARA, R.; SHRIKRISHNA ISLOOR
    The present investigation was carried out to evaluate the occurrence of AMR E. coli in pork production chain in and around Bengaluru. Initial survey indicated that majority of the pig farmers were small and marginal farmers (66%) and kitchen / hotel waste was the major source of feed. The major antibiotics used were tetracycline followed by enrofloxacin and sulphonamides (Co-trimazole) and antibiotics were administered by themselves (80%). A total of 230 samples (40 each from piglets, weaners and adults, 30 each of feed, water, hand swabs and 20 boot socks) from 10 pig farms were screened and 366 E. coli isolates were used for further characterization. The average E. coli count ranged from 5.811 to 6.282 log10 cfu / g. The highest per cent of AMR E. coli counts as proportion of total E. coli was observed in kitchen waste (66.16%) followed by water trough (55.55%), human hand swabs (52.94%), outside boot socks (50.87%) and piglet (45.37%) samples as compared to other samples in this study. The overall occurrence of E. coli, tetracycline, fluoroquinolone and ESBL resistant E. coli in the entire pork production chain was 75.65, 63.48, 46.52 and 20.87 per cent, respectively. Of the 366 isolates, 355 isolates (96.99%) carried either one or the other gene, whereas, 3.01 per cent of the isolates did not harbor any of the genes screened. Majority of the isolates carried tetA (97.54%), followed by qnrB (89.34%), sul1 (82.791%), blaCTX-M (42.08%), cmlA1 (13.11%) gene and none of the isolates carried colistin resistance gene (mcr1 to mcr5). Antimicrobial susceptibility testing revealed that majority of the isolates showed resistance to tetracycline (93.14-95.19%) followed by fluoroquinolones and complete sensitivity to carbapenem followed by aminoglycosides. It was observed that 84.91 per cent of E. coli isolates were MDR and PFGE analysis revealed a high genetic diversity at 80 per cent similarity indicating that dissemination of AMR genes within the farm premises.
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    ThesisItemOpen Access
    DETERMINATION OF ANTIMICROBIAL RESISTANCE AND CHARACTERIZATION OF ASSOCIATED GENES IN SALMONELLA AND Escherichia coli IN POULTRY AND ITS ENVIRONMENT
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2022) M. NASIM; RATHNAMMA)
    A study was conducted to monitor the presence, antimicrobial resistance (AMR) phenotype and genotype of Salmonella and E. coli in the entire commercial broiler supply by collecting 232 samples from Broiler breeder farms (BBF), hatcheries, commercial broiler farms (CBF) and retail meat shops (RMS). It was evident that enrichment in TTB followed by colony PCR was rapid and specific; however, BGA and XLT4 were more specific media for isolation of Salmonella. The overall presence of Salmonella and E. coli in complete poultry supply chain was 20 and 72 per cent, respectively. A significant higher (P<0.05) presence of Salmonella was observed in RMS (46%) followed by CBF (19%), and hatcheries (10%). The presence of E. coli in BBF, hatcheries, CBF and RMS were 48, 83, 70 and 91 per cent, respectively. Antimicrobial resistance based on disc diffusion assay and MIC revealed that all the E. coli and Salmonella isolates were resistant to at least one of the antibiotics and 75 & 76 per cent of the isolates were multidrug resistant (MDR) respectively. In complete poultry supply chain E. coli and Salmonella showed highest resistance to doxycycline (97 & 94%), followed by fluoroquinolone, gentamicin, β lactam, sulphonamides and colistin and lesser resistance to phenicols and neomycin. All E. coli and Salmonella isolates were screened for the presence of 35 antimicrobial resistant (AMR) genes belonging to seven groups of antibiotics by PCR and it was observed that the overall presence of AMR genes among the E. coli and Salmonella isolates was 69 and 60 per cent respectively. Among the AMR genes, highest presence was recorded for tetA (69%), followed by blaTEM, mcr2-5, aac(6’)-Ib-cr, qnrS, qnrD, cmlA1, sul1, catA2, qnrB, blaCTX-M, blaSHV, mcr-1, catA1, blaOXA and OqxAB in Salmonella and qnrB, qnrS, blaTEM, aac(6’)-Ib-cr, blaCTX-M, mcr1-5 (19.59%), qepA, mcr-4, cmlA1, sul1, catA1, blaSHV, catA2, qnrD, blaOXA and sul2, blaCTXM group2, gropu9, &gropu8/25 in E. coli. There was fair to significant correlation between AMR phenotype and genotype. AMR E. coli and Salmonella and genes encoding AMR were present throughout the poultry supply chain, however CBF and RMS were the major focal points of AMR. No significant difference in presence of AMR E. coli could be observed during the crop cycles in CBF, as presence was observed at all the intervals indicating the need for efficient monitoring and control strategies for effective prevention of AMR in complete poultry supply chain.
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    ThesisItemOpen Access
    DEVELOPMENT OF FIELD AND LABORATORY BASED IMMUNODIAGNOSTIC ASSAYS FOR RABIES IN ANIMALS
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR., 2022) KAVITHA, G.; SHRIKRISHNA ISLOOR
    The present study emphasized on the development of field and laboratory based immunodiagnostic assays for rabies in animals. The N protein was purified from CVS 11 strain of RABV in both cell supernatant and infected BHK 21 cell pellet by ultracentrifugation and sonication respectively to know its reactivity with the MAb specific to N protein of RABV developed at IIL, Hyderabad. Western blot analysis of purified N protein revealed immunogenic band of 50 kDa with the aforementioned MAb. Further, the MAb was conjugated with FITC and in-house DFA test was standardized with 1:10 as the optimal dilution which is same as that of the standard DFA conjugate. The in-house DFA was evaluated with 131 brain samples against the standard DFA and the results found to be having high correlation with diagnostic sensitivity of 90.8 and diagnostic specificity of 93.18 per cent. Simultaneously, using the biotinylated MAb resourced again from IIL, Hyderabad, in-house dRIT was standardized with 1:20 to be the optimal dilution. The in-house dRIT was evaluated for its performance with the same brain samples tested using the CDC dRIT and the results were in concordance with 100 per cent diagnostic sensitivity and specificity. Finally, both the in-house assays were validated by intra and inter laboratory comparisons. The comparisons with respect to inhouse DFA had some discrepancies but for in-house dRIT, the results were in complete agreement. The in-house dRIT might have potential future applications in robust surveillance of rabies in animals as part of National Action Plan for dog mediated Rabies Elimination by 2030 (NAPRE).
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    ThesisItemOpen Access
    ISOLATION, IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF Pasteurella multocida FROM DOMESTIC ANIMALS
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2022) KAVITHA KANDIMALLA; BASAVARAJ AWATI)
    In the present study, a total of 583 samples were collected from various infected, dead and apparently healthy animals of different species. Pasteurella multocida was recovered in pure cultures from 28 samples thus showing an isolation percentage of 4.8%. All the isolates were tentatively identified as P. multocida based on cultural, morphological and biochemical characteristics of P. multocida. For confirmation, Pasteurella multocida species specific PCR (PM-PCR) was done and all the 28 isolates were found positive. Out of these, 25% of the isolates were identified as capsular type A and 75% of the isolates were identified as capsular type B. The overall prevalence of virulence genes was found to be 100% for fur, nanB, sodA, pilB, tadB, rcpA and flpD, 92.85% for ptfA, 89.28% for ompH, oma87 and fimA, 78.57% for tbpA and 17.85% for toxA. The occurrence of ptfA, ompH, oma87, fimA and toxA varied among isolates from buffaloes and small ruminants. The prevalence of ompH and oma87 was detected in 81.25% of buffaloe isolates and 100% in sheep and goat isolates. The occurrence of toxA differed among capsular type A and B isolates of small ruminants. The toxA gene was detected in majority of capsular type A (71.42%) isolates, whereas none of the capsular type B isolates harboured toxA. Nucleotide sequencing of virulence genes of NLR1 isolate indicated a great homology and the query sequence showed 100% identity with the sequences of P. multocida available in GenBank from different geographical regions and from different host species. In antibiogram study, the isolates were 100% sensitive to cefoperazone/sulbactum, ceftiofur, ceftriaxone, cloxacillin, levofloxacin and tetracycline (30 μg/disc). Resistance to 126 gentamicin, tetracycline (10 μg) erythromycin, co-trimoxazole, nalidixic acid, methicillin, lincomycin, enrofloxacin, ampicillin, kanamycin, penicillin, cefotaxime, ciprofloxacin and amoxiclav was found to be in the order 46.42%, 42.85%, 32.14%, 28.57%, 28.57%, 25%, 21.42%, 17.85%, 17.85%, 14.28%, 14.28%, 10.71%, 7.14% and 7.14% respectively. In the present study, real-time PCR and multiplex PCR were used for detection of genes (pilB, tadB, flpD and rcpA) associated with biofilm formation by P. multocida and found 100% sensitivity of the developed PCRs for detection of these genes. Only nine isolates produced weak biofilm by Congo red method but the genes associated with biofilm formation were detected in all the 28 isolates by multiplex PCR. It is further observed that, all the nine biofilm producing isolates were conferring resistance to at least four of the antibiotics. From the present study, it can be concluded that P. multocida was found to be a major etiological agent in respiratory infections of domestic animals. Detection of virulence genes, biofilm forming abilities along with alarming percentage of antibiotic resistance suggests need for development of suitable vaccine and prophylactic measures against respiratory infections of all domestic animals.
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    ThesisItemOpen Access
    ISOLATION AND MOLECULAR CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS IN POULTRY FROM NORTH-EAST KARNATAKA
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2022) GOPALA LUNAVAT; BASAVARAJ AWATI
    This study details the characterization of 34 NDV isolates recovered from 126 samples collected from 4 avian species of 2 different orders. Samples were collected from both infected and apparently healthy poultry and dead pigeons of India. Isolation of NDV was attempted using embryonated chicken eggs and the isolates were confirmed by HI using hyperimmune serum raised against LaSota and RT-PCR targeting partial fusion gene. Biological (ICPI and MDT) and molecular characterization assays classified seven isolates as velogenic pathotypes. Phylogenetic analysis based on complete fusion gene sequences confirmed that velogenic isolates belongs to class II sub genotype XIII. Deduced amino acid sequences of F proteins of the study isolates diverged from vaccine strains by 10-12.9 %. Analysis of F protein uncovered substitutions at several amino acid residues in domains essential for virulence. NDV isolates confirmed by HI and RT-PCR were further characterized by propagating in Chicken Embryo Fibroblast (CEF) cultures. Velogenic isolates obtained from different avian species induced typical ND infection in the infected birds and efficiently transmitted to in-contact birds. This study further highlights the role of other avian species in maintenance and transmission of NDV and the need for enhanced biosecurity in commercial poultry operations.
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    ThesisItemOpen Access
    GENOMIC SEQUENCING AND PHYLODYNAMICS OF RABIES VIRUS FROM ANIMALS
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2022) ASHOK V. BHOSALE; (BASAVARAJ AWATI)
    Total of 27 rabies cases in large ruminants were analysed for onset of clinical signs after history of dog bite, incubation period recorded was 15-20 days. Noticeable observation was the fatality rate i.e., 100% in the dog bite cases above the neck region despite of vaccination post bite. The reporting of Rabies in cattle is under reported and mostly neglected. Need efforts to increase the reporting and following strict post exposure prophylaxis along with use of antisera at local site, may reduce or prevent the fatality. RTPCR was considered to be the best test for detection of rabies virus in 100 % cases, coupled with dFAT. Amino acid variation was recorded in G protein amino acid, whereas great diversity observed in G-L intergenic region. The diversity in G protein in a long term is matter of concern. Though the variations are very scanty, the efficacy of vaccine on development of neutralizing antibodies needs to be ascertained in future studies. The occurrence of rabies was more in cattle after the onset of rainy season, may be due to breeding season in dogs and increase in dog bite cases, whereas the peak achieved on the onset of winter season, subsiding the cases in summer season. The evolutionary analysis revealed the origin of present day rabies virus in India tracing back to China as origin of virus.
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    ThesisItemOpen Access
    STUDIES ON ANTIMICROBIAL RESISTANCE (AMR) IN PREDOMINANT STAPHYLOCOCCI AND Escherichia coli ASSOCIATED WITH BOVINE MASTITIS AND COMBINATORIAL APPROACH FOR MITIGATION OF AMR
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERISTY, BIDAR, 2021-03-01) SARITHA, N. S.; SHRIKRISHNA ISLOOR
    The present study was carried out with an objective to study the antimicrobial susceptibility of S. aureus, S. epidermidis and E. coli from bovine mastitis, identification of AMR genes, effect of AMPs, bacteriophages and nanoparticles. Out of 211 isolates, a total of 96 S. aureus, 50 S. epidermidis and 65 E. coli isolates were subjected for species specific PCR and 85 S. aureus, 23 S. epidermidis and 47 E. coli isolates were confirmed. On screening for AMR genes of 85 S. aureus isolates, blaZ was detected in 61.17%, mecA in 3.53%, aacA-aphD in 4.7%, tetM in 16.4%, tetK in 3.52%, strA in 4.7%, strB in 7.05% and ermC in 4.7% of the isolates. Among 23 S. epidermidis, blaZ was detected in 43.47%, mecA in 30.43% and aacA- aphD in 13.04%, tetM in 30.43%, tetK in 8.69%, ermC in 13.04%, strA in 4.34% and strB in 21.73% of the isolates. Among 47 E. coli, blaTEM, tetA genes were detected in all isolates, whereas blaSHV was detected in 31.9%, blaOXA30 in 12.76%, tetM in 2.12%, tetB in 2.12%, strA in 31.9% and strB in 51.06% of the isolates. On MIC testing for seven antimicrobials, out of 31 S. aureus isolates, 9.7% and 29% were sensitive, 90.3% and 70.9% were resistant to penicillin and cefotaxime respectively and all were resistant to gentamicin, streptomycin, tetracycline and oxacillin. On MIC testing of S. epidermidis isolates for penicillin, cefotaxime, tetracycline, gentamicin, streptomycin and oxacillin, all the isolates exhibited resistance. Out of 20 E. coli isolates, 85% were sensitive and 15% were resistant to cefotaxime and all were resistant to penicillin, enrofloxacin, tetracycline, streptomycin. Among 37 S. aureus isolates, 54% were sensitive and 45.9% isolates were resistant to FNDR-01, while out of 10 S. epidermidis isolates, 2 and 8 isolates exhibited sensitivity and resistance respectively. All E. coli isolates exhibited resistance to FNDR-01. S. aureus ATCC culture and four S. aureus isolates showed lytic activity for Phage P3. E. coli ATCC culture showed lysis to five phages whereas, Phage 2, 3 and P4 lysed 8 E. coli isolates. All isolates exhibited resistance to all NPs and one S. epidermidis isolate was resistant to the combination of oxacillin and SNPs.
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    ThesisItemOpen Access
    MOLECULAR EPIDEMIOLOGY OF NEWCASTLE DISEASE VIRUS FROM DIFFERENT AVIAN SPECIES
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2020-12-01) BALAM DEEPTHI; D.RATHNAMMA
    This study details the characterization of 36 AAvV-1 isolates recovered from 223 samples collected from 11 avian species of 8 different orders. Samples were collected from both infected poultry flocks and apparently healthy non- poultry avian species (spot- billed pelicans, pigeons, emus, turkeys, geese and ducks) of India. Isolation of AAvV-1 was attempted using embryonated chicken eggs and the isolates were confirmed by HI using hyperimmune serum raised against LaSota and RT-PCR targeting partial fusion and matrix genes. Biological (ICPI and MDT) and molecular characterization assays classified thirty two and four isolates as velogenic and lentogenic pathotypes respectively. Phylogenetic analysis based on complete fusion gene sequences confirmed that velogenic isolates belong to class II sub genotype XIII 2.2 and lentogenic isolates to genotype II. Deduced amino acid sequences of F and HN proteins of the study isolates diverged from vaccine strains by 10-12.9 per cent and 11.3-14.5 per cent respectively. Analysis of F and HN proteins uncovered substitutions at several amino acid residues in domains essential for virulence. AAvV-1 isolates confirmed by HI and RT-PCR were further characterized by propagating in CEF cultures. The AAvV-1 isolates of this study were evaluated for their ability to infect and transmit to naïve chicken by experimental inoculation of 3-week old chicks. Velogenic isolates obtained from different avian species induced typical ND infection in the infected birds and efficiently transmitted to in- contact birds, while chicks infected with lentogenic isolates were apparently normal. Pathogenicity of the field isolates was compared in vivo by studying relative expression of various cytokines in spleen of infected chicks by qPCR and in vitro using MTT assay. This study further highlights the role of non- poultry avian species in maintenance and transmission of AAvV-1 and the need for enhanced biosecurity in commercial poultry operations.