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2020 - 20222
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Subject: Veterinary Public Health × Start date: 2020 × 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.