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Open Access
Epidemiological studies on physical, chemical, psychological and zoonotic hazards among veterinarians
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Parmar, Tanuja; Upadhyay, A.K.
This study reports physical, chemical, zoonotic and psychological hazards relevant to Indian veterinarians as obtained by a self administered questionnaire. Out of total 392 respondents, 5.1% (20) reported no injury, 47.5% (186) respondents had 1-5 injuries, 32.1% (126) encountered 5-10 injuries and 15.3% (60) veterinarians had more than 10 injuries during last 5 years. Animal related injuries like bite (31.8%), scratch (65.1%), kick (62.8%), horn wound (14%), needle prick (89.2%), injuries due to falling / lifting animals / moving heavy equipments (61.3%) and fracture (3.8%) were commonly reported physical injuries Total no. of veterinarians involved in taking radiographic examinations were 19.1% (75/392). Most of the veterinarians taking x-rays were academic veterinarians. Hundred eighty seven (47.7%) veterinarians were using antineoplastic agents to treat animals and out of those 2.1% (4) veterinarian accidently injected drugs to themselves. Thirty two (8%) veterinarian, experienced adverse effect due to disinfectant and 0.7% (3) veterinarians reported adverse effects due to pesticides. The types of allergy sustained by veterinarians were sneezing, eye/ nose/ throat infection, cough, skin irritation and latex gloves allergy. Ringworm (13.5%) and fungal infection (26.5%) were most common zoonotic infection among veterinarians. Low level stress reported among 45% participants, moderate among 34% and high level of stress among 21% participants. The 43.9% veterinarians used gloves, 39.8% were wearing apron and only 1% respondents were using goggles. 36.7% participants did not use any protective gear. Routine deworming within every 6 months followed by 43.6% of the respondents, 31.1% in the last 1 year and 17.1% had done it once in the last two years. Percentage of the veterinarians did not practice routine deworming was 8.2%. Majority of veterinarians 73% (286) rarely go for medical checkup or only when required. The awareness levels among the veterinary health professionals was near optimal but the need was felt to implement efforts aimed at addressing deterrence of occupational hazards by developing and executing improved safe handling practices and safety measures.
Open Access
Isolation, identification and antimicrobial susceptibility of public health significant organisms present in organic agricultural farms of Uttarakhand
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Rautela, Richa; Maansi
Studies are lacking in India regarding the presence of public health significant organisms in the environment and plant samples at organic farms. The present work was carried out on two highly significant public health organisms i.e. Salmonella and Listeria in five organic agricultural farms located at Kotabagh, Dhamola, Ramnagar and two locations of Pantnagar in Uttarakhand state, India. A total 500 samples, comprising 350 environmental samples viz; soil (n=227), manure (n= 66) and water (n=57) besides, 150 plant samples viz; rhizosphere (n=50), roots (n=50) and leaves/grains (n=50) were processed for isolation of Salmonella and Listeria. Standard protocols were used for isolation of both organisms. The morphological and biochemical characterization was attempted. Genus specific PCR targeting invA (284 bp) gene for Salmonella and prs (370 bp) gene for Listeria was used for molecular confirmation. A total of 11 Salmonella isolates were recovered with an overall prevalence of 2.2% while Listeria could not be isolated representing 0% prevalence. Amidst environmental sources maximum detection (8.7%) was observed from water. Three isolates each were isolated from manure (4.5%) and soil (1.32%).Highest prevalence was noticed at Ramnagar (6.4%) followed by Pantnagar-2 (3.7%) and Pantnagar-1(2.2%). Any isolate could not be detected from Kotabagh and Dhamola. Out of 11 Salmonella isolated, 7 belonged to Group C1; one was confirmed to be Salmonella Gabon and 3 Salmonella Miyazaki. Kirby-Bauer disc diffusion assay was performed for antibiotic sensitivity on all the 11 Salmonella isolates. Most frequent resistance was observed for cefotaxime (72.72%) followed by ciprofloxacin (45.45%), cefazolin (36.36%) and cefoxitin (27.27%). Nalidixic acid, streptomycin, sulphafurazole and tetracycline, each exhibited 18.18% resistance. Only 1 isolate showed resistance against gatifloxacin (9.09%). All the isolates showed susceptibility towards amoxicillin and levofloxacin. Salmonella isolates showing phenotypic resistance were screened for the presence of β lactamase genes (blaTEM, blaCTX-M-9 and blaAmpC) using simplex PCR. Three S. Miyazaki serotypes possessed blaCTX-M-9 gene. None of the isolates exhibited blaTEM and blaAmpC genes. The study focuses on the presence of the public health significant Salmonella and Listeria in environmental and plant samples of the organic agricultural farms which could be significant source of food contamination.
Open Access
Molecular epidemiology of antimicrobial resistant non-typhoidal Salmonella isolated from retail chicken meat shops
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-05) Sharma, Jaishree; Deepak Kumar
The objective of this study was to determine the prevalence, antimicrobial resistance and virulence factors of non-typhoidal Salmonella. A total of742 samples viz. meat swabs (n=188), poultry faeces (n=214), hand swabs (n=78), knife swabs (n=83), meat rinsing water (n=35), cutting surface swabs (n=31), chopping board swabs (n=41), utensil swabs (n=70) and litter (n=2) were collected from 39 retail chicken meat shops located at 6 different cities. The overall prevalence Non Typhoidal Salmonella (NTS) was found to be 9.43% (n= 70). The highest prevalence of Salmonella was observed in chicken meat (28/188, 14.89%), followed by knife (11/83, 13.25%), utensils (8/70, 11.43%), chopping board (3/41, 7.32%), rinsing water (3/35, 8.57%), poultry feces (15/214, 7.01%) and cutting slab surface samples (2/31, 6.45%). Geographically, the highest prevalence was observed in Lalkuan (20.99%, 17/81) followed by Pantnagar (15.60%, 17/109), Nainital(10.83%, 13/120), Rudrapur (9.78%, 9/92), Bareilly (5.83%, 7/120), Haldwani (5.66%, 6/106) and Kiccha (0.88%, 1/114). Three serotypes of Salmonella were identified, Salmonella Kentucky (74.29%; 52/70), S. Virchow (17.14%; 12/70) and S. Typhimurium (7.14%; 5/70). All 70 Salmonella isolates (100%) were multidrug resistant (MDR). Highest resistance was observed against Tetracycline (70/70, 100%) and Erythromycin (70/70, 100%) followed by Nalidixic Acid (69/70;98.57%), Ampicillin (67/70;95.71%), Ciprofloxacin (58/70;82.86%), Gatifloxacin (57/70;81.43%), Cefazolin (53/70;75.71%), Cefotaxime (36/70; 51.43%), Levofloxacin (35/70;50%), Sulfisoxazole (33/70;47.13%), Streptomycin (19/70;27.14%) and Cefoxitin (11/70; 15.71%). Twenty nine (29/70, 41.43%) Salmonella isolates were identified as coresistant to ciprofloxacin and cefotaxime. The most common resistance patterns were NA CIP GAT CZ E AMP TE (7/70, 10%), NA CIP LE GAT CZ CTX E AMP TE (6/70, 8.57%), S NA CIP LE GAT CZ CTX E AMP TE and NA CTX E AMP TE SF (both 5/70, 7.14%).These isolates were also resistant to 4 other antimicrobial agents (NA, E, AMP, TE). Salmonella isolates were screened for 12 antimicrobial resistance genes. Out of 47 resistant and intermediate streptomycin isolates, aadA1 and aadA2 were found to be 80.85% and 2.13% respectively, while strB was present in 6.38% isolates. Sulfisoxazole resistance genes sul1 and sul2 were detected in 82.35% (28/34) and 8.82% (3/34) isolates, respectively. Out of the β-lactam resistance genes, blaTEM was the most predominant (25.37%) followed by blaPSE and blaCMY (1.49%). The tetracycline resistance gene, tetA was detected in all Salmonella isolates (100%, 70/70). All 70 isolates were screened for the presence of virulence genes using PCR. A total of 66 isolates harbored sipA (94.29%) followed by mgtC (52/70; 74.29%), sopE1 (26/70; 37.14%), stn (24/70; 34.29%), sopB (9/70; 12.86%) genes, fliC gene (2/70; 2.86% each), spvC and gipA genes (1/70; 1.43%). Risk factor analysis revealed that none of the potential risk factors included were significantly associated with the Salmonella prevalence in the chicken meat shops. Overall, our study detected very high prevalence of multidrug resistant Salmonella in the chicken meat shop environments. Higher resistance to “critically important” (3rd and 4th generation fluoroquinolones) and “highly important” (1st generation cephalosporins and tetracycline) antibiotics detected in Salmonella isolates of poultry origin is a serious threat to public health which highlights the irrational use of antimicrobials in the poultry production in India.
Open Access
Studies on prevalence, virulence genes and antimicrobial resistance of thermophilic campylobacters isolated from poultry farms of Kumaon region
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-07) Garhia, Geetika; Maansi
Thermophilic campylobacters are leading cause of food-borne gastroenteritis worldwide and considered a major food safety concern. With highly complex epidemiological cycle and multiple sources of contamination, it is critically important to create a framework of effective measures to control these organisms. The present work was carried out to study the prevalence of thermophilic campylobacters in eight poultry farms located in the Kumaon region (Haldwani, Pantnagar, Bazpur, Ramnagar, Kiccha, Jawaharnagar and Bindukhatta) of Uttarakhand state, India. A total 545 samples comprising 345 poultry faecal and 199 environmental samples (51 litter, 52 feed, 50 water and 46 manure) were processed for Campylobacter isolation. Respective protocols were used for Campylobacter isolation from poultry faeces and environmental samples. The organisms with typical morphological and staining characteristics were confirmed biochemically. Genus specific PCR targeting 16S rRNA gene (816 bp) and a multiplex PCR targeting lpxA gene for simultaneous identification of C. jejuni (331 bp) and C. coli (391 bp) was used. A total of 67 Campylobacter isolates were recovered with an overall prevalence of 12.29%. Highest prevalence was detected in Bazpur farm (31.4%) followed by Pantnagar farm 2 (25.0%), Pantnagar farm 1 (24.4%), Haldwani farm (16.3%), Bindukhatta farm (7.5%) and Jawaharnagar farm (5.6%). No isolate was detected from Kiccha and Ramnagar farms. Out of total 67 Campylobacter isolates, 51 (76.11%) were identified as C. coli and 16 (23.88%) as C. jejuni. C. coli was found to be more prevalent than C. jejuni. Only 48 isolates were screened for detection of virulence genes. The cadF and flaA gene was present in all 48 isolates, while 6 (12.5%), 44 (91.66%) and 11 (22.9%) isolates harboured ciaB, cdtB and cgtB genes, respectively. None of the isolates was positive for wlaN gene. Kirby Bauer disc diffusion assay was used for antibiotic sensitivity testing of 42 Campylobacter isolates. Most frequent resistance was observed for cefoxitin (97.9%), followed by ciprofloxacin (64.28 %), nalidixic Acid (33.33 %), ampicillin (28.5%), ceftriaxone (14.28), tetracycline (4.76%), clindamycin (2.38%), sulfafurazole (2.38%) and erythromycin (2.38%). All isolates were susceptible to levofloxacin and gentamicin. Campylobacter isolates showing phenotypic resistance were screened for the presence of corresponding antimicrobial resistance genes (ARGs). β-lactam resistance gene blaOXA-61 was detected in 18 (58.06%) isolates. Resistance genes cmeB and tet(O) were detected in 19 (79.16%) and 2 isolates (100%) respectively. The ermB gene was absent in a single erythromycin resistant isolate. Risk factor analysis revealed significant association between the use of feed purchase from market and Campylobacter positivity at farm. The study focuses on the presence of the Campylobacter in poultry faeces and its associated environment which could be significant sources to humans. Higher resistance to cefoxitin and ciprofloxacin along with detection of multiple antimicrobial resistance genes in poultry faecal and environmental isolates warrants strict measures for judicious use of antimicrobials in poultry farms.
Open Access
Isolation, identification and molecular characterization of Salmonella Sarovars from poultry and domesticated animals
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2016-08) Chauhan, Rahul; Singh, S.P.
Open Access
Molecular characterization and antimicrobial susceptibility of thermophilic campylobacters isolated from humans, farm and laboratory animals
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2016-07) Rawat, Neelam; Maansi
Open 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.
Open 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.

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