Loading...
Theses
Browse
8 results
Search Results
ThesisItem Open Access Isolation, identification and molecular characterization of nontyphoidal Salmonella and listeria SPP. from foods of animal origin(G.B. Pant University of Agriculture and Technology, Pantnagar, District Udham Singh Nagar, Uttarakhand. PIN - 263145, 2022-07) Gunjiyal, Harshita; MaansiNon-Typhoidal Salmonella and Listeria are the two bacterial food-borne organisms that pose a major impact to the food sector worldwide. In addition, the emergence of multi-drug resistant organisms makes food-borne illnesses more severe. In view of this, the present study aims to ascertain the occurrence of non-typhoidal Salmonella and Listeria organisms in foods and their antimicrobial resistance profiles isolated from animal origin foods of four districts belonging to Kumaon region of Uttarakhand. A total of 250 samples comprising raw milk (n=141), milk products (n=59) and poultry meat (n=50) were collected randomly from multiple vendors, dairy farms, locality, butcher shops and screened for the presence of non-typhoidal Salmonella and Listeria organisms. The bacteria were isolated using culture method and biochemical identification was performed as per conventional method. Further, molecular characterization was done for confirmation. Antibiotic susceptibility testing was performed for the obtained isolates against a set of 12 antibiotics belonging to 9 different classes for Salmonella spp. and 8 different classes for Listeria spp. using the standard Kirby Bauer disc diffusion method. Salmonella spp. was detected in 7.2%; 18/250 and Listeria spp. in 2.4%; 6/250 of the 250 food samples studied. None of the Listeria isolates was found to be belonging to L. monocytogenes. Serotyping of Salmonella isolates revealed that S. Typhimurium and S. Weltevreden correspond to the dominant serotypes recording (4/18; 22.22%) higher serovar occurrence than S. Kentucky (2/18; 11.11%), S. Infantis (2/18; 11.11%). Rest were untypable (6/18; 33.33%). U.S Nagar harbored more Salmonella spp. (12.5%) followed by Nainital district (3.90%). On the other hand, Nainital district (3.12%) was found to harbor more Listeria spp. than U.S Nagar (1.9%). On subjection to antimicrobial susceptibility testing, Salmonella isolates showed varying degree of sensitivity to Co-trimoxazole (55.55%), co-resistance to Gentamicin and Chloramphenicol (44.44%). Complete phenotypic resistance (100%) was found for Cefotaxime and Erythromycin followed by Nalidixic acid (72.22%). Out of 18 obtained Salmonella isolates, 14 isolates (77.77%) were multi-drug resistant. A total of 12 different antimicrobial resistance patterns were observed. On the other hand, Listeria spp. were completely susceptible (100%) to Vancomycin, Chloramphenicol and Ampicillin. Complete resistance (100%) was found for Kanamycin and Tetracycline followed by Amikacin (83.33%). Out of six obtained isolates for Listeria spp. five isolates (5/6; 83.3%) were multi-drug resistant. A total of 5 different antimicrobial resistance patterns were observed which can be related to the non-judicious administration of antibiotics during both prophylaxis and treatment. Therefore, this study warrants careful consideration towards adopting hygienic measures and consumption of properly cooked food along with judicious use of antibiotics.ThesisItem Open Access Epidemiology and molecular characterization of Cystic echinococcosis in cattle and Cysticercosis in pigs(G.B. Pant University of Agriculture and Technology, Pantnagar, District Udham Singh Nagar, Uttarakhand. PIN - 263145, 2022-06) Pathak, Ayushi; Upadhyay, A.K.The present study was undertaken to study the epidemiology of Echinococcosis in cattle and Cysticercosis in pigs through slaughter house study of Uttarakhand. During this study, 264 buffaloes and 100 pigs were studied. The overall prevalence rate of Hydatidosis was 13.26% {Nainital (n=172)- 14.53%, Bageshwar (n=25), 12%, Almora (n=18)-11.11%, US Nagar (n=38), 7.89% and Rampur (n=11)- 18.18%}. For Cysticercosis, the overall prevalence rate was 3% {US Nagar (n=60)-1.66%, Nainital (n=30)-3.33% and Bageshwar (10)-10%}. Both the diseases were found to be independent of the geographical location of the animals. The average prevalence recorded in lungs, liver and both lungs & liver was 14.28%, 71.42% and 14.28%, respectively without significant statistical difference. Genderwise prevalence revealed no significant difference statistically and was higher in females in both the diseases (hydatidosis- 15.43% and cysticercosis-3.03%) and lower in males (hydatidosis-9.8% and cysticercosis-2.94%). Statistical significant difference could not be observed on age-wise prevalence considerations {Hydatidosis- 6-7y (16.66%), 4-5y (14.63%) and 2-3y (8%); cysticercosis-1-2y (4.16%) and 3-4 years (2.77%)}. Various types of cysts were recovered from different organs without statistical significant difference in rate of infection {single (lungs-80%, liver-80%) as well as multiple (lungs-20%, liver-20%)}. The overall fertility rates found in the cysts were 71.42% (lungs-40%, liver-80%, lungs & liver-60%). The overall viability rate of the cysts recovered from different organs was 80% (lungs-50%, liver- 80%, lungs & liver-100%). Significant difference was not observed in sizes of cyst and their viability {3-6cm (85.71%); >6cm (77.78%)}. DNA was extracted from 25 Echinococcus samples and 3 T. solium samples. Cox1 gene amplification fragment length observed was 440bp in Echinococcus and 420bp in Cysticercus. Purification of PCR products lead to clear products of 340 bp for US Nagar isolate; 338bp for Nainital isolate; 319 bp for Bageshwar isolate; 330 bp for Almora isolate; 339 bp for Rampur isolate for Echinococcus. For Cysticercus, clear products of 322 bp for US Nagar isolate; 418 bp for Bageshwar isolate and 354 bp for Nainital isolate were obtained. Phylogenetic analysis of the isolates show that Echinococcus isolates from the present study are 100% similar to the isolates from South Korea, Iran, Italy, Israel, Turkey and Iraq (G1 isolate). The T. solium isolates share 97.92% resemblance with Mexico isolate (TsChb4), 98.02% with Bangalore isolate, 98.59% with Meghalaya isolate (Ts-MI) and, 97.99% with Uttar Pradesh isolate (TsInd D). The Bageshwar isolate of T. solium is shown to be the root of the phylogenetic tree with other isolates being the branches. This is the first report of prevalence of G1 isolate of Echinococcus in buffaloes. Asian isolate of T. solium is found in the pigs of Uttarakhand, thus explaining the significance of this study in understanding the zoonotic potential of both the parasites.ThesisItem Open 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.ThesisItem Open Access Epidemiological studies on rabies through meta-analysis(G.B. Pant University of Agriculture and Technology, Pantnagar, District Udham Singh Nagar, Uttarakhand. PIN - 263145, 2022-07) Chauhan, Ram Swaroop; Upadhyay, A.K.In the present study, meta-analysis on rabies in India by the use of a random-effect model was done to estimate the prevalence of the disease in India. The data is obtained from the peer-reviewed articles and publications on this disease during 2010-2020. The data which is used in the present study includes the studies in which the samples are completely random. The Meta-analysis for the epidemiology and sero-prevalence of rabies was done on a total of 32 studies. Further subgroup analysis was done like analysis for species, geographical regions, and diagnostic tests.The total sample size for prevalence estimation in humans are 49828 and sero-prevalence estimation in dogs by ELISA and RFFIT are 1856 and 689 respectively. Pooled prevalence of published papers using random-effect model for rabies in humans was estimated 65% (95%CI: 40%-86%) and in dogs by ELISA and RFFIT was estimated 53%(95%CI: 33%-73%) and72%(54%CI: 95%- 86%) respectively.Publication bias for rabies in humans through regression test revealed significant publication bias (z = 0.6947, p> 0.05). Sero-prevalence of rabies in dogs by ELISA by rank correlation test showed non-significant (Kendall’s tau = 0.1111, p> 0.05) and regression test revealed significant publication bias (z= 0.2142, p>0.05). For seroprevalence of rabies in dogs by RFFIT the rank correlation test showed non-significant (Kendall’s tau = 0.4667, p > 0.05)and regression test revealed significant publication bias (z= 0.3222, p>0.05). The majority of bite victims were between the ages of 0-20 (21.49%) followed by(20.30%) the ages of 21-40. In the studies that were mentioned, males were disproportionately more likely (71.87%) to have been bitten by a dog than females (28.13%). The majority of victims suffer animal bites on their extremities. Maximum dog bites were recorded in the evening (62.9%). According to a survey, 15% of dogs in the nation were vaccinated. With the help of definite and precise clinical history with epidemiological rates, relation between associated factors may help in the identification of the highest disease burden that helps us to improve our knowledge to develop a plan of action for effective control and prevention measureThesisItem Open Access Prevalence and characterization of non-typhoidal Salmonella isolates obtained from retail fish meat shops(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-02) Sana Parveen; Sana Parveen; Sana Parveen; Maansi; Maansi; MaansiNon-Typhoidal salmonellosis stands among the major food illnesses. Outbreaks from contaminated fishes have been witnessed worldwide. This study was performed to assess the presence of Non -Typhoidal Salmonella in fishes and associated environmental samples of 22 retail fish meat shops of 4 locations of Uttarakhand. For this, a total of 368 samples (fish meat swabs(n=46), gill swabs(n=23), skin swabs(n=25), intestine swabs(n=23), hand swabs(n=44), knife swabs(n=27), rinsing water(n=22), fish water(n=41), floor swabs(n=22), chopping board swabs(n=24), utensil swabs(n=24) and container swabs(47) were collected. The overall occurrence of Non-Typhoidal Salmonella was found to be 4.35% (16/368). The highest prevalence of Salmonella was observed in rinsing water (18.18%,4/22) sample followed by knife swabs ( 11.11%, 3/27) , chopping board swabs (8.33%, 2/24) , meat swabs (6.52%, 3/46) , floor swab (4.54%,1/22) , gill and intestine swab (4.34%, 1/23), fishwater (2.43%,1/41). Geographically, the highest prevalence was observed in Lalkuan (6.52%, 3/46) followed by Haldwani (5.55%, 10/180), Kiccha (2.12%, 1/47) and Pantnagar (2.08%, 2/96). Of 16 Salmonella isolates, confirmed using ompC (204 bp) gene, 14 were revealed as S.Typhimurium (87.5%, 14/16) while 2 did not exhibit the typh gene (401bp) amplicon size. All the 16 isolates screened for the presence of virulence genes using PCR commonly harbored sipA gene (87.5%, 14/16) followed by stn (75%, 12/16), sopB ( 68.75%, 11/16), sopE1 ( 56.25%, 9/16) mgtC ( 43.75%, 7/16) and spvC and gipA ( 12.5%, 2/16) genes each. Highest resistance was observed against Tetracycline and Ampicillin (93.75%, 15/16), Nalidixic acid (50%, 8/16), Ciprofloxacin (37.5%, 6/16) Ofloxacin, Cefotaxime and Sulfisoxazole ( 25%, 4/16) each, Chloramphenicol (12.5%, 2/16) and Streptomycin ( 6.25%, 1/16). Ten Salmonella isolates were multi drug resistant (MDR). Ten different antimicrobial resistance patterns were observed. Of these, only one pattern (TE, AMP, NA, CTX , OF, SF) was found common in 2 Salmonella isolates belonging to knife and meat swab sample. All phenotypically resistant and intermediate resistant isolates were screened for 6 corresponding antimicrobial resistance genes. The most commonly occurring resistance gene was gyrA (92.30%, 12 /13), blaTEM (53.33%, 8/15), aadA1 and strA (50%, 2/4), sul1 (30.76 %, 4/13) while tetA was not found in any of the isolates. Overall, our study detected occurrence of Non-Typhoidal Salmonella in the fish retail meat shops. Resistance to critically important flouroquinolones and highly important Cephalosporin and Tetracycline antibiotic detected in Salmonella isolates is a serious threat to public health which highlights the indiscriminate use of antimicrobials.ThesisItem Open Access Whole genome sequencing & bioinformatics of Campylobacter isolated from animals(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-11) Roma; Upadhyay, A.K.Campylobacter species are one of the leading cause of bacterial foodborne zoonoses. Worldwide, Pathogenic Campylobacter species are responsible for causing over 400–500 million infections cases each year. These pathogenic Campylobacter species are grouped into major human enteric pathogens (C. jejuni, C. jejuni subsp. jejuni (Cjj), C. jejuni subsp. doyley (Cjd), C. coli and C. fetus) minor pathogens (C. concisus, C. upsaliensis, C. lari and C. hyointestinalis) and major veterinary pathogens (C. fetus subsp. venerealis (Cfv) and C. Fetus subsp. fetus (Cff)). A total of 498 samples consisting of poultry caeca (120), litter (40), poultry rectal swabs (24), poultry skin (24), water samples (38),faeces of pigs (30), meat swabs (32), clinical dog faecal ssample (20), goat (32) and lab animals faeces (92) were screened for the presence of C. jejuni and C. coli using conventional isolation and identification procedures. The overall occurence of Campylobacter was 8.83 %. The samples collected were screened for campylobacters. Highest isolation rate was observed from lab animals (19.56 %, 18/92) followed by poultry caeca (13.33%,16/120), pig faeces (10%,3/30), meat swabs (9.37 %, 3/32) ,poultry faeces (7.14%, 3/42) and litter (2.5%, 1/40). Since loads of virulence genes are responsible for bacterial pathogenecity, resilence, stress response and environmental persistence. The individual detection of every gene by techniques like PCR is a tedious process. The next generation sequencing concept play an extremely important role in this context. Among next generation sequencing also, whole genome sequencing (WGS) and assembly is best method for the characterization of isolates as it can detect every gene of the organism coding for it’s identification, virulence, pathogenicity and antibiotic resistance.The study characterised the entire genome of the infectious isolates by analysis of WGS sequence data via use of both web based and command based tools. Virulence genes pattern was observed using web based analysis tools namely VFDB Virulence Factor Database (http://www.mgc.ac.cn/VFs) and wgMLST (Whole-genome MLST) which is pangenome based tool was used using core genome genes/loci and all acessory genes/loci to detect lineage – specific genes/loci. A total of 23 genes were detected by VFDB and 33 virulence genes were detected via SRST2. Only resistance to beta – lactam were found. Overall, our study detected only one antimicrobial resistance gene that is blaₒₓₐ -₄₈₉ commonly found in Campylobacter spp. indicating good antibiotic management in areas of Pantnagar, Rudrapur and BariellyThesisItem Open Access Molecular genotyping of multidrug-resistant Salmonella typhimurium isolates using repetitive sequence-based PCR fingerprinting technique(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-08) Sivakumar, V.; MaansiNon-typhoidal salmonellae, responsible for salmonellosis in humans, are a threat to food safety impacting human health in the form of hospitalizations and in severe cases, fatalities. Such an impact also results in huge economic losses. Several food borne outbreaks resulting from zoonotic non- typhoidal Salmonella serovars have been reported, of which, S. Typhimurium has shown predominance. Epidemiological investigations of an outbreak requires accurate identification of the infection source and the transmission route for effective implementation of preventive measures against microbial food-borne pathogens. For this, techniques which can detect differences among the isolates at genomic level are applied. Therefore, in the present investigation, multidrug-resistant isolates of Salmonella Typhimurium were genotyped using repetitive sequence-based PCR (rep-PCR) fingerprinting. A total of 45 MDR S. Typhimurium isolates belonging to various sources and locations were procured from the Department of Veterinary Public Health and Epidemiology, CVASc, GBPUAT, Pantnagar. All the isolates were tested for purity and were confirmed on the basis of cultural, biochemical, serotyping and molecular (duplex PCR targeting genus specific ompC gene and serovar specific typh gene) assay. The isolates were subjected to ERIC, REP, BOX and GTG5-PCR analysis after DNA extraction and PCR confirmation. ERIC, REP, BOX and GTG5-PCR fingerprinting generated reproducible band patterns ranging from 3-10, 1-5, 6-14 and 2-10 separate bands, respectively. Cluster analysis revealed 9 types from ERIC-PCR, 7 types from REP-PCR, 16 types from BOXPCR and 9 types from GTG5-PCR. Hundred percent typeability was obtained with all genotyping techniques except REP-PCR, which differentiated 39 Typhimurium isolates only (86.6%). The isolates’ sharing similar band patterns is suggestive of a genetic similarity between them directing towards the multifactor involvement in the transmission of Salmonella. Faecal isolates and meat swab isolates sharing identical band patterns and grouped into a single cluster is indicative of a likely cross contamination. No unique pattern was observed in penta resistant (ACSSuT) profile isolates by our typing techniques. DI value of BOX-PCR was highest (0.940) followed by GTG5-PCR (0.862), REP-PCR (0.846) and ERIC-PCR (0.843). On comparing the techniques, the BOX-PCR exhibited good DI value, typeability and complex band pattern on gel in differentiating the isolates. Hence, to conclude, the BOX-PCR fingerprinting technique was found useful in the genotyping of isolates suitable enough to find its application in an epidemiological investigation during an outbreak.ThesisItem Open 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.
