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Subject: Veterinary Public Health × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Studies on isolation, identification and epidemiology of thermophilic campylobacters from wild mammals and birds
    (G.B. Pant University of Agriculture and Technology, Pantnagar, District Udham Singh Nagar, Uttarakhand. PIN - 263145, 2022-07) Singh, Nawal Kishor; Upadhyay, A. K.
    Campylobacters are one of the foodborne pathogen responsible for mild to severe diarrhoea in young domestic animals and human, indicating its zoonotic nature and public health concern. This study looked for thermophilic Campylobacters in animals' faeces. A total of 521 samples were collected from eight zoos/sanctuaries/national parks of Uttarakhand (n=194), Uttar Pradesh (n=45) and Chhattisgarh (n=282) states of India. Samples included 302 ruminants, 166 non-ruminants, and 53 birds. Among ruminants 71.2% (215/302) belonged to deer family and in non-ruminants, 44.58% (74/166) felidae and 21.68% (36/166) canidae family. Among captive birds, 24.52% (13/53) belonged to Pheasant followed by wild fowl 20.75% (11/53). CBA in microaerophilic condition at 420C temperature yielded highest thermophilic Campylobacter (11.90%), followed by mCCDA (10.56%), BA (8.25%), CA (5.76%) and HCCA (4.22%). Multiplex PCR (mPCR) confirmed 11.71% (61/521) Campylobacter spp., including 58.06% C. jejuni (36/61) and 40.32% C. coli (25/61). Ruminants (59.68%) exhibited highest incidence, followed by non-ruminants (29.03%) and birds (9.68%). Tryptone soy broth with 20% glycerol and -800C temperature could be better preservation media for Campylobacter isolates upto 180 days. Nucleotide sequence analysis (BLAST) and Phylogenetic tree (MEGA 11) confirmed Campylobacters in wild mammals and birds. Current study found that TaqMan assay (qPCR) could detect even a single template copy of pathogen with specificity for Campylobacter genus and reproducible with low SD and CV%. Real time PCR (qPCR) could detect and quantifies Campylobacters in clinical as well as field samples. Due to high sensitivity of Gentamicin (60.00%), Amikacin (64.00%) and Cefotaxim (69.45%) against Campylobacters, we recommend them as drugs of choice for treatment of Campylobacteriosis. The presence of thermophilic Campylobacters in wild mammals and birds as well as in grazing domestic animals indicate its endemicity might be the source of infection in human.
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    ThesisItemOpen Access
    Whole genome sequencing of nontyphoidal Salmonella isolates recovered from humans, swine and environment
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-01) Pathak, Anubha Prashant; Upadhyay, A.K.
    Nontyphoidal Salmonella (NTS) is a serious zoonotic concern worldwide. It is one of the most important causes of diarrhea due to the consumption of food of animal origin such as pork, chicken and eggs. Armed with a battery of virulence factors and equipped with chromosomal and plasmid mediated antimicrobial resistance (AMR), Salmonella has established itself as successful zoonotic pathogen. Whole Genome Sequencing (WGS) is increasingly replacing various molecular typing and subtyping techniques used in bacteria as it offers highest resolution as well as detailed information on the accessory genes. Keeping these assertions in perspective, this work was designed to study the serotypes, AMR genes, virulence genes and plasmids in the Salmonella isolates (n=90) obtained from swine, human and swine rearing environment using WGS. The WGS of all the isolates (n=90) was conducted using Illumnia Miseq platform. The analysis of sequence data using various online and command-line based tools revealed a stereotypically diverse population of bacteria. A total of 15 serotypes were identified. The isolates were further classified using eBGSs (eBurst Groups), MLST (Multi Locus Sequence Typing) and rST (Ribosomal Sequence typing). Isolates grouped into 16 eBGs, 18STs and 25 rSTs offering more information than serotyping alone. The wgMLST was used to construct the phylogenetic tree which revealed serotype-based clustering and rST based sub-clustering. The virulence gene analysis revealed a highly conserved repertoire of genes. A total of 115 genes were detected, all the isolates (100%) carried genes encoding for type three secretion system one (T3SS1) and two (T3SS2) and their effectors, nonfimbrial adherence determinants, macrophage inducible genes and genes aiding in Mg2+transport. However, six genes constituting the T3SS2 effectors gogB, sseK1, sseK2, sseI, sspH1, sspH2 showed a serotype specific distribution pattern. A total of 27.7% of the isolates, all consisting of the serotype Typhimurium, from the three sources, were found to carry IncF plasmid mediated highly virulent genes spv, rck and pef. Typhoidal toxin gene cdtB was reported in 11.1% of NTS isolates. A total of 30 antimicrobial resistance genes encoding for resistance to 9 groups of antimicrobials were detected. The gene sul1 (45.5%) was most commonly detected, followed by tetA (30%) and ant(3'')-Ia (28%). Notably, two recently discovered genes encoding for resistance to the most crucial antibiotics Fosfomycin (fosA7) and Colistin (mcr9) were also reported. Out of the 19 plasmids identified, The pSPCV (IncFII) plasmid was found in 30% of the isolates followed by pSLT-BT (IncFIB) in 27.7% of isolates. A total of 8 groups of coexisting genes were found, ant(3'')-Ia blaCARB-2 floR_2 sul1 tet(G) which is responsible for the penta- resistance (ACSSuT) pattern of Salmonella Typhimurium was the most commonly reported group.
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    ThesisItemOpen Access
    Studies on virulence, antimicrobial resistance and genetic diversity of Salmonella typhimurium isolates from North india
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2016-08) Prasanna Kumar, V.; Singh, S.P.
    Salmonellosis is one of the most frequently reported food-borne diseases world-wide commonly caused by Salmonella Typhimurium and Salmonella Enteritidis serovars. The present study was undertaken to understand the pathogenic nature, antimicrobial resistance pattern and heterogeneity of S. Typhimurium isolates (n=94) of diverse origin from northern states of India (Uttar Pradesh, Uttarakhand, Bihar, Assam and Jammu & Kashmir). All the 94 isolates were confirmed to be S. Typhimurium through various morphological, biochemical, serological and molecular methods. Salmonella Typhimurium isolates revealed varying distribution pattern of virulence viz., invA (100 %), sipA (100 %), sopE1 (61.7%), fliC (80.85%), mgtC (100 %), spvC (12.76%). stn (89.36%), sopB (94.68%) and gipA (45.74%). The presence of virulence determinants located on prophages (sopE1 and gipA) and plasmids (spvC) of S. Typhimurium isolates from North India was found to be less compared to the presence of virulence genes located on SPIs (invA, sipA, fliC, mgtC, stn and sopB). Antimicrobial susceptibility testing revealed higher resistance against nalidixic acid and sulfafurazole (28.72% each) followed by tetracycline (14.89%), ampicillin and ciprofloxacin (12.76% each), levofloxacin and norfloxacin (11.7% each). This appears to be the first study to report the emergence of S. Typhimurium isolates (animal origin) resistant to third generation fluroquinolones in Patna (Bihar) and Pantnagar (Uttarakhand). The findings also suggest the presence of MDR strains of S. Typhimurium in northern states of India except Assam. The present study also detected various antimicrobial resistance genes corresponding to the respective antibiotic classes viz., blaTEM, strA, strB, tet(A), sul1 and sul2 along with class 1 integron. The class 1 integron were identified with two type of gene cassette viz., dfrA5 and aac(3-Id)-aadA7 0.4 kb and 1.3 kb size, respectively. Genetic diversity analysis performed by ERIC-PCR revealed a discriminative index of 0.7817 while, one of the isolates (S145) from Guwahati (Assam) was found untypable. The PFGE analysis of 16 isolates revealed a discriminatory power of 0.9 and it was proved to be the more efficient tool in comparison to ERIC-PCR. The genetic similarity among the isolate from different sources and locations indicate their common origin.
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    ThesisItemOpen Access
    Immunogenicity Of Major Soluble Antigens Of Burcella Abortus S-99
    (Govind Ballabh Pant University of Agriculture and Technology;Pantnagar, 2004) Vaid, Rajesh Kumar; Thapliyal, D.C.
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    ThesisItemOpen Access
    Molecular identification of animal species using Polymerase Chain Reaction based techniques
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2008-06) Karabasanavar, Nagappa; Singh, S.P.
    Species-specific PCR assays were developed for the authentic identification of cattle, buffalo, sheep, goat, pig and chicken. The mitochondrial D-loop was targeted in cattle, buffalo, sheep, goat, and pig; while nuclear 5-aminolevulinate synthase gene was used as target for amplification in chicken. The developed species-specific PCR assays yielded products specific to the species studied. In order to exclude the possibility of cross amplification, 25 animal species including different breeds were considered that consisted of mammals, birds, rodent and fish species covering most of the domestic, pet and wild animals. The suitability of the species-specific PCR assays was confirmed in raw (n=20), cooked (60,80 and 100oC for 30 min), autoclaved (121oC for 30 min) and the micro-oven processed meat and meat products such as kabab, mutton curry, chevon curry, pork sausage, chicken patties, chicken products (samosa, nuggets and loaves). The sensitivity of the PCR was established to be at 0.1% in all species studied for the detection of adulteration and the limit of detection was 0.1 pg in cattle and goat, 1 pg in sheep and 10 pg in buffalo, pig and chicken. Based on the present investigation it was concluded that, the species specific PCR assays developed in this study could be used for the authentication of raw, cooked (up to 121oC) and adulterated (up to 0.1%) animal tissues and their products for the specific identification of cattle, buffalo, sheep, goat, pig and chicken. For the simultaneous detection of cattle and buffalo a multiplex PCR was developed using species-specific forward and common reverse primers. Further, for the identification of an unknown species an alternative approach was used, that involvedisolation of total DNA from the cells, PCR amplification of a region of mitochondrial 12S rRNA gene, sequencing of the PCR amplicon and analysis of the sequence. Using this approach, nine species of animals (leopard, Bengal and Siberian tigers, goral, muntjak, sika deer, camel, parakeet and a black kite) were unambiguously identified. This research work presents novel diagnostic primers for 6 animal species and validates the sequence analysis as a tool for identification animal species.
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    ThesisItemOpen Access
    Molecular cloning, expression and In-silico characterization, immunogenicity, Salmonella typhimurium, polymerase chain reaction, isolates, immunity
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2012-01) Rajeev Kumar; Singh, S.P.
    The present investigation was undertaken to isolate and characterize the immunogenic Omp28 protein from S. Typhimurium. The Genus-specific PCR assay was standardized with aview to amplify specific region of 16S rDNA gene and subsequently subjected for molecular characterization for 28 different field isolates of Salmonella from different sources. Theisolates were S. Abortuseqaui, S. Anatum, S. Agona, S. Bareilly, S. Bonariensis, S. Bredeney,S. Berta, S. Bergen, S. Choleraesuis, S. Derby, S. Enteritidis, S. Gallinarum, S. Greenside, S. Heidelberg, S. Illinois, S. Infantis, S. Java, S. Kentucky, S. Newport, S. Paratyphi-B, S. Pollurum, S. Rubislaw, S. Saintpaul, S. Stanley, S. Seftenberg, S. Typhi, S. Typhimurium and S. Weltevreden along with standard Salmonella Typhimurium culture (MTCC-3214). All the samples revealed a specific banding pattern of amplified PCR products with a presence of specific 574bp band, for genus Salmonella. In order to determine Salmonella genus specific Omp28 gene, PCR condition was standardized, gene was amplified and sequenced. All these serovars were also subjected to PCR for determination of presence of immunogenic Omp28 gene in the form of specific amplified products size specifically of 341bp. The outer membrane protein was analyzed using SDS-PAGE which revealed the presence of low molecular weight of 12.32kDa main protein as outer membrane protein of Salmonella Typhimurium. The Immunogenic Omp28 gene of S. Typhimurium was successfully cloned in cloning Vector(pUC19) and confirmed by nucleotide sequencing. The construct pUC19-Omp28 was further Sub-cloned in expression vector pET32a(+). The expression construct pET32a(+)-Omp28 was then used for expression of protein through induction with IPTG and expressed fusion recombinant protein was further characterized through SDS-PAGE analysis, which shows about 29.59 kDa fusion expressed recombinant proteins, consisting of about 17.27 kDa fusion tag of pET32a(+) expression vector and about 12.32 kDa of expressed protein of Omp28 gene of S. Typhimurium which was inserted in expression vector. The sequenced Omp28 gene of S. Typhimurium, S. Enteritidis and S. Paratyphi-B was subjected to in-silico characterization and structure determination (primary, secondary and 3D structure of amino acid sequence derived from sequenced gene). Further phylogenetic tree analysis, B-cell epitope, MHC I, II binding prediction with Allele HLA (Human) and homology modeling was also carried out with predicted amino acid of proteins. Analysis of Pfam showed that the protein belong to Pfam A family, hdeB super family [PRK11566]. The immunogenicity of protein was computed through antigenic plot and found antigenicity index of Omp28 protein were about 2.2 (0.1-2.2) for S. Typhimurium and S. Enteritidis and for S. Paratyphi-B about 2.1(0.1-2.1). More than 1.2 antigencity index suggests that all isolates possessing Omp28 gene protein can trigger the immune system and produce antibody in vivo. i.e. this expressed protein from Omp28 gene could be immunogenic in nature.