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With rapidly increasing urbanization and scarcity of adequate river water, there is a constant rise in the extraction of groundwater and its use for various purposes such as drinking, bathing etc. But due to steep expansion of industrialization there is increase in amount of discharge of untreated sewage water in these water bodies which imposes a high risk of percolation to nearby groundwater sources creating an alarming situation to public health. One such case is of the River Kali in Muzaffarnagar District where untreated water from numerous industries flows in which makes its water unfit for use. The study was carried out to access the effect of river water to groundwater within 500m radius and study its impact on public health.This was an observational study conducted in Muzaffarnagar district where, river water from 4 locations and groundwater from 4 locations (near 500m radius) was taken and tests performed for pH, taste, odor, BOD, Coliform count, heavy metals, hardness, suspended solids using references from Indian standards of drinking water. : The water quality of river Kali is not fit for any purpose and also the Groundwater in its proximity is not fit for drinking.

Shiga toxigenic Escherichia coli (STEC) is one of the most important food-borne zoonotic bacterial pathogens responsible for causing gastrointestinal infections, haemorrhagic colitis and haemolytic uremic syndrome. The present study was aimed to isolate and characterize STEC from neonatal dairy calves, animal handlers and their surrounding environment and to establish the genetic relationship among isolates by multilocus sequence typing (MLST). A total number of 115 samples were collected and processed for the isolation of E. coli. The occurrence rate of E. coli was 92.2% (106/115), of which, 18 were typed as STEC. Antibacterial susceptibility analysis revealed 11 (61.1%) strains as multiple drug-resistant (MDR). MLST analysis has delineated 16 sequence types (STs) including nine novel STs. Among STs, ST58 dominated with three strains and was recovered from the environment and neonatal calves. Strains from neonatal calves and humans showed genetic relatedness with significant bootstrap support values indicative of zoonotic transmission potentiality. Analysis of 211 global isolates belonging to 61 STs indicated predominant STs (ST 21, ST 33 and ST 3416) that can be either host-specific (ST 33 and ST 3416) or can be shared among human and bovine hosts (ST 21). The MLST analysis indicates genetic relatedness among isolates and the results predispose inter-host transmission and zoonotic spread. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00561-9.

Ducks are the "Trojan Horses" for Asian H5N1 avian influenza viruses (AIV) and attain carrier status without displaying overt infection. These birds help in the spread of the virus among the poultry and human population through direct or indirect contact. Preen oil is the secretion of preen gland of water birds such as ducks. In a process called preening, the water birds spread preen oil across their feather and body. Preen oil has been known to play a significant role in the accumulation of various pathogens including Highly Pathogenic Avian Influenza (HPAI) from water onto feathers. However, the studies are scarce on the role of preen oil in the survivability of HPAIV. We conducted a simulative study to analyse the effect of preen oil on the survivability of the HPAI virus (H5N1) on duck feathers. Duck feather samples along with relevant controls were spiked with the H5N1 virus at two different initial concentrations (104 EID50 and 106 EID50 ), stored at 37°C, 25°C and 10°C temperatures and tested at regular intervals for percent infectivity by egg culture method and qRT-PCR. The infectivity and viral load were significantly higher in naturally preened duck feathers in comparison to the three preen oil deficit controls at both low and high initial concentrations of virus (104 EID50 and 106 EID50 ). Maximum persistence was seen at 10°C in naturally preened duck feathers spiked with 106 EID50 concentration of viruses. It was also seen that depletion of preen oil from duck feathers reduced the persistence of the virus. These results demonstrate that preen oil plays a significant role in survivability and protection of HPAIV on duck feathers. This study herein will present new avenues in understanding one of the epidemiological niches of HPAIV.

Shiga toxin-producing Escherichia coli (STEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC) make up an important group of pathogens causing major animal and public health concerns worldwide. The aim of this study was to determine the prevalence of different pathotypes of E. coli in captive wildlife. We analyzed 314 fresh fecal samples from captive wildlife, 30 stool swabs from animal caretakers, and 26 feed and water samples collected from various zoological gardens and enclosures in India for the isolation of E. coli, followed by pathotyping by multiplex PCR. The overall occurrence rate of E. coli was 74.05% (274/370). The 274 E. coli isolates were pathotyped by multiplex PCR targeting 6 genes. Of them, 5.83% were pathotyped as EPEC, 4.74% as STEC, and 1.09% as ETEC. The 16S rRNA genes from the selected isolates were amplified, sequenced, and a phylogenetic tree was constructed. The phylogenetic tree exhibited indiscriminate genetic profiling and some isolates from captive wild animals had 100% genetic identity with isolates from caretakers, suggesting that captive wildlife may serve as a reservoir for infection in humans and vice-versa. The present study demonstrates for the first time the prevalence of these E. coli pathotypes in captive wildlife in India. Our study suggests that atypical EPEC strains are more frequent than typical EPEC strains in captive wildlife. Discovering the implications of the prevalence of these pathotypes in wildlife conservation is a challenging topic to be addressed by further investigations.

Loop-mediated isothermal amplification (LAMP) is a promising, simple, rapid and sensitive molecular detection method. In the present study, LAMP assay was developed for detecting Clostridium perfringens in chevon. Primers were designed to detect the cpa gene of C. perfringens. A panel of 19 bacterial strains, including 3 C. perfringens and 16 other strains, were included in this study to standardize and evaluate the LAMP assay. No false positive amplification was observed indicating 100% specificity of the assay. The detection limit of LAMP and conventional PCR in the DNA extracted from pure C. perfringens was 0.34 pg and 3.4 pg, respectively. This revealed that LAMP assay is 10 times more sensitive than conventional PCR. The sensitivity of the LAMP assay for the detection of C. perfringens in raw chevon was found to be 1.2 × 102 CFU/g after 6-h enrichment and 1.2 × 105 CFU/g without enrichment in artificial spiking studies. Improved C. perfringens detection of 12 CFU/g within 12 h was obtained proving that LAMP assay is significantly faster than traditional methods that take >2 d. The developed LAMP assay also detected the targeted organism in clinical and environmental samples with the sensitivity and specificity of 97% and 84%, respectively with Kappa agreement of 0.824 respects to PCR assay. This method shows immense potential for routine diagnosis and monitoring of C. perfringens in food, environment and clinical samples. This is the first report in which the LAMP assay was optimized for the detection of C. perfringens in chevon.

The present study was aimed to develop and evaluate dot–blot assays for rapid detection of staphylococcal enterotoxin-A (SEA) in food. Dot blots were developed in two formats, indirect and sandwich utilizing mouse monoclonal anti-SEA and rabbit polyclonal anti-SEA antibodies. In indirect dot–blot format, recombinant SEA was directly coated on NCM dot–blot strip and detection was carried out by anti-SEA antibodies. In sandwich dot–blot format, SEA was trapped between anti-SEA capture and detection antibodies. Both the dot–blot assays exhibited a sensitivity of ~48 ng ml−1 when tested in different food matrices. The developed assays were highly specific as no cross-reactivity was detected with other classical staphylococcal enterotoxins, toxigenic bacteria and foodborne pathogens. Sensitivity and specificity of developed indirect and sandwich dot–blot assays with respect to PCR was found to be 100 and 99%, respectively. The results shows that the developed dot–blot assays can be used as rapid preliminary screening tests for detection of SEA in food or determining the toxigenic potential of staphylococci, especially in resource-limited settings.

A non-blinded randomized clinical trial was conducted to assess the immunomodulatory potential of β-glucan (BG) in piglet diarrhoea associated with type A rotavirus infection. A total of 12 rotavirus-infected diarrheic piglets were randomly divided into two groups: wherein six rotavirus-infected piglets were treated with supportive treatment (ST) and other six rotavirus-infected piglets were treated with BG along with ST (ST-BG). Simultaneously, six healthy piglets were also included in the study which served as control. In rotavirus-infected piglets, marked increase of Intestinal Fatty Acid Binding Protein-2 (I-FABP2), nitric oxide (NOx), Interferon-γ (IFN-γ) concentrations and decrease of immunoglobulin G (IgG) were noticed compared to healthy piglets. The faecal consistency and dehydration scores were significantly higher in rotavirus-infected piglets than healthy piglets. The ST-BG treatment progressively reduced the I-FABP2 and increased the IgG concentrations over the time in rotavirus-infected piglets compared to piglets received only ST. A pronounced enhancement of NOx and IFN-γ concentrations was observed initially on day 3 and thereafter the values reduced on day 5 in ST-BG treated piglets in comparison to piglets which received only ST. Additionally, ST-BG treatment significantly reduced faecal consistency and dehydration scores on day 3 compared to ST in rotavirus-infected piglets. These findings point that BG represents a potential additional therapeutic option to improve the health condition and reduce the piglet mortality from rotavirus associated diarrhoea where porcine rotavirus vaccine is not available.

Porcine rotavirus type A (RVA) is a major cause of neonatal piglet mortality in India. The effect of the disease on haemogram and serum biochemical profile is not well established in piglets. Accordingly, we assessed the haemogram and serum biochemical profile in the neonatal piglet diarrhoea with RVA infection (n = 17). The diagnosis of RVA was confirmed using RNA-polyacrylamide gel electrophoresis (RNA-PAGE), commercially available enzyme-linked immunosorbent assay (ELISA) kit and reverse transcription-polymerase chain reaction (RT-PCR). Non-infected healthy piglets (n = 6) served as control. The concentrations of total protein, albumin, alanine amino transaminase (ALT), aspartate amino transaminase (AST), blood urea nitrogen (BUN) and creatinine in serum were measured by spectrophotometric method. Haemogram was done in the blood using sodium ethylenediaminetetraacetic acid (Na2 EDTA) as anticoagulant. The mean values of total protein, albumin and globulin concentrations were significantly (P

The present study was carried out to find out the occurrence and types of Salmonella present in street vended foods and associated environment, and their resistance pattern against various antibiotics. About 1075 street vended food and associated environment samples were processed for isolation and confirmation of different Salmonella spp. by targeting gene specific invA gene and serotype specific Sdf I, Via B and Spy genes by PCR. Selected Salmonella isolates were screened for antibiotic resistance by using Baeur-Kirby disk diffusion test. Out of 1075 samples, only 31 (2.88%) isolates could be amplified the invA gene of which 19 could be recovered from meat vendors; 8 from egg vendors while remaining 4 from milk vendors. Though, majority of Salmonella recovered from raw foods the ready-to-eat food like chicken gravy and rasmalai also showed its presence which pose a serious public health threat. Overall, 19, 6 and 1 isolates of S. Typhimurium, S. Enteritidis and S. Typhi could be detected by PCR while remaining 5 isolates could not be amplified suggesting other type of Salmonella. Selected Salmonella isolates were completely resistance to Oxacillin (100%) followed by Cefoxitin (30.43%) and Ampicillin (26.10%). Thus, it is observed that the street vended foods of animal origin and associated environment play an important role in transmission of food borne pathogens including Salmonella.

Salmonella is found to be a major causes of food borne diseases globally. Poultry products contaminated with this pathogen is one of the major sources of infections in humans. Outer membrane protein C (OmpC) of Salmonella Typhimurium is a promising DNA vaccine candidate to mitigate Salmonella infection in poultry. However, the large-scale production of bioactive recombinant OmpC (rOmpC) protein is hindered due to the formation of inclusion bodies in Escherichia coli. The objective of this work was to attain high level expression of rOmpC protein, purify and evaluate its functional properties. The ompC gene was optimized and fused with small ubiquitin-related modifier (SUMO) gene for high level expression as soluble protein. The fusion protein with ~58 kDa molecular weight was observed on SDS-PAGE gel. The expression levels of rOmpC fusion protein reached maximum of 38% of total soluble protein (TSP) after 8 h of 0.2% rhamnose induction. Protein purification was carried out using nickel nitrilotriacetic acid (Ni-NTA) purification column. Western blot were performed to analyse expression and immunoreactivity of rOmpC fusion protein. The results indicate that SUMO fusion system is ideal for large scale production of functional rOmpC fusion protein expression in E. coli.

We applied a probe-based real-time loop-mediated isothermal amplification (Cy5-RTqLAMP) technique targeting the avian reovirus (ARV) S3 gene to develop a rapid, sensitive, and specific method for virus detection and quantification. This test specifically detected the presence of ARV, but not other viruses or bacteria present in clinical or artificially spiked samples, including Newcastle disease virus, infectious bursal disease virus, fowl adenovirus, Marek's disease virus, Escherichia coli, and Salmonella spp. This test can detect ARV in less than one hour with an analytical sensitivity of 10 viral gene copies and 1 fg of total cDNA. The Cy5-RTqLAMP does not yield false positive results and is 100 times more sensitive than conventional PCR. This test was shown to be able to detect the presence of ARV in clinical samples. A similar strategy may be used for detection of other important human and animal viral pathogens.

The enteric pathogen Salmonella Typhimurium (ST) survives inside the oxidative environment of phagocytic cells. Phagocyte generated oxidants primarily target proteins and modify amino acids in them. These modifications render the targeted proteins functionally inactive. Conversion of Asp to iso-Asp is one of the several known oxidant mediated amino acids modifications. By repairing iso-Asp to Asp, protein-isoaspartyl methyltransferase (PIMT) maintains the activities of proteins and thus helps in cellular survival under oxidative stress. To elucidate the role of PIMT in ST survival under oxidative stress, we have constructed a pimt gene deletion strain (Δpimt strain) of ST. The Δpimt strain grows normally in various culture media in vitro. However, in comparison to wild type ST, the Δpimt strain is found significantly (p

Clostridium perfringens is a globally recognized zoonotic pathogen. We report isolation and genotyping of C. perfringens from neonatal calves, dairy workers and their associated environment in India. A total of 103 fecal samples from neonatal calves, 25 stool swabs from the dairy workers and 50 samples from their associated environment were collected from two dairy farms. C. perfringens was detected in 26 out of 103 (25.2%) neonatal calf samples, 7 out of 25 (28%) human stool samples and 17 out of 50 (34%) environmental samples. C. perfringens type A strains were predominant in neonatal calves (24/26; 92.3%) and associated environment (15/17; 88.2%). In contrast, strains from dairy workers mostly belonged to type F (5/7; 71.4%), which also carried the beta2 toxin gene. Seventeen strains were analyzed by multilocus sequence typing (MLST) for studying genotypic relationship along with 188 C. perfringens strains available from public databases. A total of 112 sequence types (STs) were identified from 205 C. perfringens strains analyzed. A Clonal complex (CC) represented by three STs (ST 98, ST 41 and ST 110) representing predominantly type F (18/20 strains) were mostly associated with human illnesses. Among predominant STs, ST 54 was associated with enteritis cases in foals and dogs and ST 58 associated with necrotic enteritis in poultry. Seventeen Indian strains were assigned to 13 STs. Genetic relatedness among strains of calves, dairy worker and associated environments indicate inter-host transfers and zoonotic spreads.

Occurrence of Salmonella spp. in captive wild animal species in India is largely unknown. The purpose of this study was to determine the occurrence of different Salmonella serotypes, antimicrobial resistance patterns and genotypic relatedness of recovered isolates. A total of 370 samples including faecal (n = 314), feed and water (n = 26) and caretakers stool swabs (n = 30) were collected from 40 different wild animal species in captivity, their caretakers, feed and water in four zoological gardens and wildlife enclosures in India. Salmonellae were isolated using conventional culture methods and tested for antimicrobial susceptibility with the Kirby–Bauer disc diffusion method. Salmonella isolates were serotyped and genotyping was performed using enterobacterial repetitive intergenic consensus (ERIC) PCR and 16S rRNA sequencing. Animal faecal samples were also subjected to direct PCR assay. Salmonella was detected in 10 of 314 (3.1%) faecal samples by isolation and 18 of 314 (5.7%) samples by direct PCR assay; one of 26 (3.8%) feed and water samples and five of 30 (16.7%) caretakers stool swabs by isolation. Salmonella was more commonly isolated in faecal samples from golden pheasants (25%; 2/8) and leopard (10%; 2/20). Salmonella enterica serotypes of known public health significance including S. Typhimurium (37.5%; 6/14), S. Kentucky (28.5%; 4/14) and S. Enteritidis (14.3%; 2/14) were identified. While the majority of the Salmonella isolates were pan-susceptible to the commonly used antibiotics. Seven (43.7%; 7/16) of the isolates were resistant to at least one antibiotic and one isolate each among them exhibited penta and tetra multidrug-resistant types. Three S. Kentucky serotype were identified in a same golden pheasants cage, two from the birds and one from the feed. This serotype was also isolated from its caretaker. Similarly, one isolate each of S. Typhimurium were recovered from ostrich and its caretaker. These isolates were found to be clonally related suggesting that wildlife may serve as reservoir for infections to humans and vice versa. These results emphasise the transmission of Salmonella among hosts via environmental contamination of feces to workers, visitors and other wildlife.

Blackleg, an economically important and highly fatal disease of ruminants, is caused by anaerobic bacillus, Clostridium chauvoei. Identification and differentiation of the causative agent is crucial for implementation of therapeutic and control measures in real time. Most of the diagnostic tests available for blackleg are PCR based, and only a couple of serological tests have been reported. In this study, we targeted flagellin, an important immunogenic protein of C. chauvoei, to develop a sandwich ELISA for detection of C. chauvoei. Sequence analysis of flagellin gene of related Clostridium species showed that central region of flagellin gene is unique to C. chauvoei. Hence, we cloned and expressed central region of flagellin in a prokaryotic expression system. Antiserum against recombinant flagellin was generated in rabbits and chickens. A sandwich ELISA was developed, in which rabbit anti-flagellin antibodies were used as capture antibodies and chicken anti-flagellin antibodies as detecting antibodies. The test was specific and sensitive in detection of up to 10(4) CFU/ml of C. chauvoei. This study shows that assay developed can be used for detection of C. chauvoei in suspected samples.

Escherichia coli causes diarrhea and extraintestinal infections in humans and animals. Here, we report the draft genome sequences of Escherichia coli strains 360/16 and 646, isolated from neonatal calves.

The diversity of toxin-genotypes of C. perfringens in neonatal calves was determined in this study. A total of 682 fresh faecal samples comprising 559 healthy and 123 diarrheic neonatal calves (cattle and buffalo) were collected from various farms in Northern India. The samples were processed for isolation of C. perfringens and toxin-genotyping by multiplex PCR. The overall prevalence of C. perfringens was 37.2%. The most predominant toxin-genotype was type A (59.7%) and the least prevalent was type C. There was no association between toxin genotypes and diarrhea of cattle and buffalo neonatal calves (P > .05). Also, 38 (14.6%) and 16 (6.1%) isolates out of the 259 carried enterotoxin (cpe) and beta 2 toxin (cpb2) genes, respectively. Ten different toxin-genotypes were identified, and iota toxin gene was not detected in any of the sample.

Aim: The present study was aimed at polymerase chain reaction (PCR) amplification and cloning of NAD-dependent betahydroxybutyryl coenzyme A dehydrogenase (BHBD) gene of Clostridium chauvoei. Materials and Methods: C. chauvoei was cultured and confirmed by 16-23S rDNA spacer region primers. The primers for nad-bhbd gene of C. chauvoei were designed to aid in cloning into pRham-N-His SUMO-Kan vector, and nad-bhbd gene was amplified by PCR. The amplified nad-bhbd gene was purified and cloned into pRham-N-His SUMO-Kan expression vector. The recombinant plasmid was transformed into E. cloni 10 G cells and the clone was confirmed by colony PCR using the pRham-SUMO-NAD-For and pRham-SUMO-NAD-Rev primers and also by sequencing. Results: PCR amplification of nad-bhbd gene yielded a product length of 844 base pairs which was cloned into pRham-NHis SUMO-Kan vector followed by transformation into E. cloni 10G chemically competent cells. The recombinant clones were characterized by colony PCR, sequencing, followed by basic local alignment search tool (BLAST) analysis to confirm the insert. Conclusions: Immunogenic protein NAD- dependent BHBD of C. chauvoei was cloned and the recombinant clones were confirmed by colony PCR and sequencing analysis.

Graphical abstract, Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.•Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.•To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.•LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV.

Food-borne illnesses pose a real scourge in the present scenario as the consumerism of packaged food has increased to a great extend. Pathogens entering the packaged foods may survive longer, which needs a check. Antimicrobial agents either alone or in combination are added to the food or packaging materials for this purpose. Exploiting the antimicrobial property, essential oils are considered as a "natural" remedy to this problem other than its flavoring property instead of using synthetic agents. The essential oils are well known for its antibacterial, antiviral, antimycotic, antiparasitic, and antioxidant properties due to the presence of phenolic functional group. Gram-positive organisms are found more susceptible to the action of the essential oils. Essential oils improve the shelf-life of packaged products, control the microbial growth, and unriddle the consumer concerns regarding the use of chemical preservatives. This review is intended to provide an overview of the essential oils and their role as natural antimicrobial agents in the food industry.

The present study was carried out to find out the occurrence and types of Salmonella present in street vended foods and associated environment, and their resistance pattern against various antibiotics. About 1075 street vended food and associated environment samples were processed for isolation and confirmation of different Salmonella spp. by targeting gene specific invA gene and serotype specific Sdf I, Via B and Spy genes by PCR. Selected Salmonella isolates were screened for antibiotic resistance by using Baeur–Kirby disk diffusion test. Out of 1075 samples, only 31 (2.88%) isolates could be amplified the invA gene of which 19 could be recovered from meat vendors; 8 from egg vendors while remaining 4 from milk vendors. Though, majority of Salmonella recovered from raw foods the ready-to-eat food like chicken gravy and rasmalai also showed its presence which pose a serious public health threat. Overall, 19, 6 and 1 isolates of S. Typhimurium, S. Enteritidis and S. Typhi could be detected by PCR while remaining 5 isolates could not be amplified suggesting other type of Salmonella. Selected Salmonella isolates were completely resistance to Oxacillin (100%) followed by Cefoxitin (30.43%) and Ampicillin (26.10%). Thus, it is observed that the street vended foods of animal origin and associated environment play an important role in transmission of food borne pathogens including Salmonella.

The prevalence of Clostridium perfringens in captive wildlife in India has not been reported. The objective of the study was to determine the fecal prevalence of C. perfringens in captive wildlife in India. The prevalence in captive wild ruminants, non-ruminants, birds and caretakers were 34.1%, 36%, 22.5% and 6.7%, respectively. Toxinotyping of C. perfringens indicated that the predominant type was type A with a prevalence rate of 69.7%, followed by type A with cpb2 gene (28.3%) and type B (2.%).

A total of 19 Salmonella strains isolated from diverse sources were subjected to DNA fingerprinting by repetitive sequence PCR using ERIC and (GTG)5 primers for the discrimination of Salmonella enterica isolates at the serotype level. Fingerprints were binary scored and dendrogram was constructed for ERIC and (GTG)5 fingerprint bands. ERIC PCR produced unique patterns consisting of 4 to 16 bands ranging between 0.15 kb to 1.5 kb. Two distinct polymorphic bands at 210 bp and 300 bp were found in all the serotypes. (GTG)5 PCR fingerprint consist of 3 to 15 bands ranging from 0.29 kb to 1.25 kb. All the serotypes tested revealed single polymorphic bands at 900 bp. ERIC and (GTG)5 dendrogram presented fingerprint for all the 19 isolates and serotypes were grouped into three major branches A, B and C which was further subdivided into small groups. The results obtained in the present study indicated ERIC-PCR and (GTG)5-PCR fingerprinting were not so efficient in discriminating Salmonella isolates.

The present study was conducted to study the antibiotic resistance pattern among nontyphoidal Salmonella isolated from human, animal and meat. A total of 37 Salmonella strains isolated from clinical cases (human and animal) and meat during 2008-2009 belonging to 12 serovars were screened for their antimicrobial resistance pattern using 25 antimicrobial agents falling under 12 different antibiotic classes. All the Salmonella isolates tested showed multiple drug resistance varying from 5.40% to 100% with 16 of the 25 antibiotics tested. None of the isolates were sensitive to erythromycin and metronidazole. Resistance was also observed against clindamycin (94.59%), ampicillin (86.49%), co-trimoxazole (48.65%), colistin (45.94%), nalidixic acid (35.10%), amoxyclave (18.90%), cephalexin, meropenem, tobramycin, nitrofurantoin, tetracycline, amoxicillin (8.10% each), sparfloxacin and streptomycin (5.40% each). Isolates from clinical cases of animals were resistant to as many as 16 antibiotics, whereas isolates from human clinical cases and meat were resistant to 9 and 14 antibiotics, respectively. Overall, 19 resistotypes were recorded. Analysis of multiple antibiotic resistance index (MARI) indicated that clinical isolates from animals had higher MARI (0.25) as compared to isolates from food (0.22) and human (0.21). Among the different serotypes studied for antibiogram, Paratyhi B isolates, showed resistance to three to 13 antibiotics, whereas Typhimurium strains were resistant to four to seven antibiotics. Widespread multidrug resistance among the isolates from human, animal and meat was observed. Some of the uncommon serotypes exhibited higher resistance rate. Considerable changes in the resistance pattern were also noted. An interesting finding was the reemergence of sensitivity to some of the old antibiotics (chloromphenicol, tetracycline).

The present study was undertaken to detect antigens of bovine rotavirus (BoRV), bovine coronavirus (BoCV), E. coli F5 attachment factor and Cryptosporidium from faecal samples of healthy and diarrheic neonatal calves (0-3 months). A total of 132 faecal samples comprising 38 diarrheic and 94 non-diarrheic calf samples collected from various places of North India (Bareilly, Hisar, Meerut and Mathura from August 2015 to May 2018) were screened by using multiplex ELISA kit (Bio-X Diagnostics, Belgium) for antigenic detection of above mentioned pathogens. Study revealed the antigenic detection of Cryptosporidium spp. in 19 (14.39%), rotavirus in 11 (8.33%), coronavirus in 3 (2.27%), E. coli K5 in 2 (1.51%) and samples. Overall, 11 (28.94%) of 38 diarrheic samples and 24 (25.53%) of 94 non-diarrheic samples were positive for different pathogens. A representative number of positive and negative samples were tested by RT-PCR for BoRV and BoCV virus as well as staining for Cryptosporidium spp. The results were consistent with that of multiplex ELISA kit. Therefore, the ELISA technique used in the study was found reliable, simple and quick to use and is particularly suited for analyzing large number of samples.

Aims: Salmonella spp. has the capability to form biofilm on various surfaces. Biofilm-associated protein (bapA), a large surface protein has been shown to play a leading role in the development of biofilm in Salmonella. Objective of this study was to investigate the presence of bapA gene in different serotypes of Salmonella spp. and to characterize DNA fragment encoding bapA protein of Salmonella Enteritidis. Methods and Results: Sixty-seven Salmonella strains belonging to 34 serovars isolated from diverse sources in India were screened for the presence of bapA gene employing a primer designed for the purpose. All the strains yielded a positive amplification indicating that the bapA gene is well conserved in Salmonella spp. The amplified gene fragment of bapA was cloned in Escherichia coli (DH5 α) cells by using pGEM-T easy cloning vector. On partial sequence analysis, the product exhibited 667 base pairs, corresponding to 218 amino acids. Conclusions BapA gene was found to be highly conserved in Salmonella. Partial sequence analysis of this gene from a strain of Salm. Enteritidis revealed close association with serotypes of poultry origin and also with some other animal/zoonotic serotypes. Significance and Impact of the Study: BapA gene can be targeted for the genus-specific detection of this organism from different sources. Antigenic index of bapA protein indicates its protective and diagnostic potentials.

A one step, single tube, accelerated probe based real time loop mediated isothermal amplification (RT LAMP) assay was developed for detecting the invasion gene (InvA) of Salmonella. The probe based RT LAMP is a novel method of gene amplification that amplifies nucleic acid with high specificity and rapidity under isothermal conditions with a set of six primers. The whole procedure is very simple and rapid, and amplification can be obtained in 20min. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of DNA binding dye at the end of the reaction results in colour difference and can be visualized under normal day light and in UV. The sensitivity of developed assay was found 10 fold higher than taqman based qPCR. The specificity of the RT LAMP assay was validated by the absence of any cross reaction with other members of enterobacteriaceae family and other gram negative bacteria. These results indicate that the probe based RT LAMP assay is extremely rapid, cost effective, highly specific and sensitivity and has potential usefulness for rapid Salmonella surveillance.

Chicken (Gallus gallus), duck (Anas platyrhynchos), turkey (Meleagris gallopavo), guinea fowl (Numida meleagris) and quail (Coturnix japonica) are the common poultry species consumed as meat throughout the world. In this work, a molecular technique has been developed for identification and differentiation of meat originating from these species. This tool helps in detection of misrepresentation of different poultry meats. The technique involves the extraction of DNA from the given sample, polymerase chain reaction (PCR) amplification of mitochondrial 12S rRNA gene using universal primers, restriction analysis with selected restriction enzymes, followed by identification of meat species based on restriction fragment length polymorphism (RFLP) pattern. In this study, we used HinfI, Mph1 103I, MvaI, and Eco47I to identify and differentiate to poultry species referred to above. This species identification technique has also been applied successfully to processed meat products including those cooked at 120 degrees C for 30 min. Simplicity of interpretation of results combined with versatility makes this a convenient and appropriate technique in the hands of meat analysts for identifying poultry meat species.

Four enrichment broths were evaluated for their compatibility with the polymerase chain reaction (PCR) for detection of Listeria monocytogenes from raw meat after single-step enrichment. Standardized PCR protocols for listeriolysin O (hlyA) gene were used for the species-specific identification of L. monocytogenes. Four broths, namely, modified University of Vermont broth (MUVM), Listeria enrichment broth (LEB), Fraser broth (FB) and polymyxin, acriflavin, lithium chloride, ceftazidime, aesculin, mannitol, egg yolk broth (PALCAM) , were inoculated with L. monocytogenes. The enriched cultures were subjected for PCR. Similarly, meat samples were artificially spiked with various concentrations of L. monocytogenes, these spiked samples were enriched in the above-mentioned four broths and subjected to PCR to determine the medium that was most compatible for PCR-based detection of L. monocytogenes. The aliquots taken during different incubation periods were subjected to three different procedures for the concentration of the target organism for use in PCR. Results revealed that MUVM was better than other broths for the detection of L. monocytogenes by both PCR and cultural method; moreover, it was able to support the growth of as low as 10 cfu/g of meat. Concentration of the target organisms by centrifugation and washing with PCR buffer was the most suitable method for improving PCR performance for detection of L. monocytogenes. Goat (n = 67) and buffalo (n = 45) meat samples from local markets were also screened by both PCR and cultural method to validate the results obtained from the spiking studies. Both results were in agreement in spiking studies as well as screening of market meat samples.

A study was undertaken to assess the prevalence of Clostridium perfringens in meat and to characterize the isolates obtained in the study for virulence factors. A total of 211 meat samples of different animals (70 each of buffalo and goat and 71 of poultry) were screened and the highest occurrence of C. perfringens was observed in goat (91.4%) followed by poultry (70.4%) and buffalo (65.7%). Among the 116 isolates (buffalo-32, goat-37 and poultry-45) of C. perfringens screened for the presence of enterotoxin gene by PCR, 9.3, 32.4 and 15.5% isolates of buffalo, goat and poultry, respectively, were found to possess enterotoxin gene. Screening of 15 enterotoxin gene possessing isolates for verocytotoxicity revealed that 12 isolates exhibited cytopathic effect while 3 isolates did not show any cytopathic effect in spite of the presence of enterotoxin gene. A total of 115 C. perfringens isolates were screened for other virulence markers, i.e., lecithinase and hemolysin. The results revealed that the majority of the isolates expressed these activities. Antibiogram studies of C. perfringens isolates using 16 antibiotics displayed multidrug resistance. The isolates showed resistance to streptomycin, ceftazidime, colistin sulfate, cephalothin, ampicillin and gentamicin. Whereas 100% sensitivity to ciprofloxacin, ofloxacin and nitrofurantoin was seen, moderate sensitivity was observed with tetracycline and sulfatriad.

The emergence of Verotoxin-producing Escherichia coli (VTEC) as zoonotic foodborne pathogens in recent years has become a public health concern because of its life threatening human diseases. In the present investigation, out of 87 strains of E. coli, 22 (25%) belonging to 13 different serotypes isolated from raw buffalo meat and its products were found to be verotoxic as tested by Vero cell cytotoxic assay. Serotype 026 followed by O153 and 0157 were the predominant VTEC. All the VTEC strains were found positive for vt genes by polymerase chain reaction (PCR). Among the vt genotypes, vt 2 (77%) was most predominant followed by vt 1 (14%) and both vt 1 and vt 2 (9%). Production of enterohaemolysin on washed sheep blood agar supplemented with CaCl 2 showed 19 (86%) VTEC strains to be positive. Presence of VTEC in cooked buffalo meat products, namely shami kabab and kabab, appears to be a matter of concern and a potential threat to public health. VTEC detection by different methods suggests that PCR can be useful to evaluate the distribution of virulence genes (vt 1 or vt 2 or both) in E. coli isolates from buffalo meat and its products.

Aim To evaluate the efficacy of recombinant outer membrane proteinC (rOmpC) based enzyme-linked immunoassay (ELISA) for the diagnosis of salmonellosis in poultry. Materials and methods Three antigens were prepared, and the indirect ELISA was standardized using the antigens and the antiserum raised in chicken against Omp and rOmpC. Sera were collected from a total of 255 apparently healthy field chickens and screened for the presence of Salmonella antibodies by this ELISA. Results The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Omp revealed major polypeptides at 36, 42 and 52 kDa, and the rOmpC was evident by a single protein band of 43 kDa. The Omp and rOmpC antigen revealed an optimum concentration of 78 and 156 ng, respectively, in the assay, while the whole cell antigen gave an optimum reaction at a concentration of 10(6) organisms/ml. The test was found to be specific as it did not react with any of the antisera of seven other organisms. The developed ELISA detected Salmonella antibodies from 22 (8.62%) samples with rOmpC antigen, while 24 (9.41%) samples gave a positive reaction with both Omp and whole cell antigens. Conclusion We suggest rOmpC based indirect ELISA as a suitable screening tool for serological monitoring of poultry flocks.

Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level.To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle.LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR).The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye.The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.

Contagious agalactia is a highly infectious disease affecting sheep and goats, mainly caused by Mycoplasma agalactiae. Although various tests are available for diagnosis of contagious agalactia, none of them is credited with the capacity to provide rapid and cost-effective diagnosis. This article reports the development of loop-mediated isothermal amplification (LAMP) test targeting the p40 gene of M. agalactiae, for the diagnosis of classical contagious agalactia. Optimum amplification was obtained at 58 °C in 70 min. The developed test was found to be 100-fold more sensitive than PCR and detected up to 20-fg level of DNA. The test was also superior to conventional PCR in detecting from artificially contaminated milk, i.e. 10(4)-fold more sensitive. The developed LAMP test could detect up to 10 cfu/ml of artificially contaminated milk, indicating its potential for being developed as a field test for rapid and sensitive diagnosis.

Good husbandry practices at farm level form an essential component of the production of quality and safe food. It encompasses all the measures adopted at the farm, from procuring and rearing healthy animals, their welfare, to final slaughter or milking. Farm management is done in such a way as to keep animals in a healthy condition, provide adequate and contamination-free feed and water and optimum living conditions. Animals are raised on the basis of risk analysis and control of these risks is exercised for safe food production. Proper records are maintained for easy traceability.

Food-borne diseases caused by Salmonella enterica from poultry sources represent an important public health problem and no reliable control by vaccination has proved effective despite research. The aim of the present study was to evaluate the use of recombinant OmpC protein for immunization of birds to elucidate its protection against virulent Salmonella Typhimurium. The recombinant OmpC protein was prepared after cloning and expressing ompC gene and was characterized by SDS-PAGE and Western blot analyses. The protein preparations were tested as vaccine candidate in layer birds by comparing the immune response, protection and organ clearance against crude lysate and control. The biologically functional recombinant 43 kDa truncated OmpC protein proved to be a good immunogen which induced a significantly high humoral immune response than control. At the same time, it primed a stable cell-mediated immune response. A protective index (based on faecal shedding of organism) of rOmpC based preparations ranged between 50 and 75% as observed for 3 weeks after challenge. Therefore, the protein preparations conferred satisfactory protection against challenge infections with virulent strains of S. Typhimurium as evidenced by limited faecal shedding and minimal detection of Salmonella from edible tissues and eggs. These findings suggest the possibility to explore the use of S. enterica OMP protein for the production of novel vaccine.