Mudit Chandra

A single step multiplex PCR for identification of six diarrheagenic E. coli pathotypes and Salmonella

E. coli is generally a commensal but includes some highly pathogenic strains carrying additional genes in plasmids and/or the chromosome. Based on these genes the pathogenic strains are divided into pathotypes including enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC) and diffusely adherent (DAEC) E. coli. Here, previously developed multiplex PCR strategies for these strains were integrated into one single step multiplex that differentiates all these E. coli pathotypes, usually based on multiple characteristic PCR products. This multiplex PCR works reliably for colony PCR. Two additional markers were added: one to detect most Enterobacteriacea, which acts as a positive control for successful PCR, and one to distinguish Salmonella. The multiplex correctly classified a set of 45 reference strains by colony PCR and 71 (45+26) strains by in silico PCR. It was then used to interrogate 44 clinical strains from bovine hosts resulting in detection of EAEC and DAEC determinants.

Interaction of Salmonella enterica Subspecies enterica Serovar Typhimurium and Mung Bean (Phaseolus aureus) Plants

The effect of Salmonella enterica subspecies enterica serovar Typhimurium, a zoonotic serovar, on mung bean (Phaseolus aureus) cultivar Pant Mung-3 plants was studied. Inoculation of mung bean seeds with Salmonella Typhimurium (7.2 x 10(5) CFU/ml) reduced sprouting rate (P < 0.07). This effect was more pronounced at higher levels of contamination. In the soil inoculated with Salmonella Typhimurium (7.2 x 10(6) CFU/g), germination was retarded and the number of defective sprouts was also significantly higher (P < 0.002). Salmonella Typhimurium grew inside germinating seeds and plant tissues and persisted in seedlings, adult plants, and harvested seedlings dried and stored at room temperature (30 degrees C) up to 45 days. Phaseolus aureus plants grown in sterile soil was resistant to Salmonella Typhimurium infection at 15 days of age and cleared Salmonella from all the aerial parts within 3 h of infection. However, Salmonella Typhimurium could be reisolated from the basal area of the stem and from soil even after 45 days of exposure to the pathogen.

Isolation of a bacteriophage against Salmonella Dublin and determination of its physical resistance under varied in vitro conditions

°C and direct sunlight beyond 5 days was found to be deleterious for survival of the phage.

Multiplex real-time PCR for identification of canine parvovirus antigenic types.

Canine parvovirus (CPV) is an important disease causing gastroenteritis and/or haemorrhagic gastroenteritis in dogs. There are four antigenic types of CPV reported worldwide viz. CPV 2, CPV 2a, CPV 2b and CPV 2c. The diagnosis of CPV with the identification of the antigen type responsible remains problematic. In the present study, identification as well as antigenic typing of CPV was done using a de novo multiplex real time PCR to combat the problem of antigenic type identification. From the study it could be concluded that the here developed multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types.

Prevalence of Multidrug-Resistant Salmonella on Ready-to-Eat Betel Leaves (Paan) and in Water Used for Soaking Betel Leaves in North Indian Cities

The purpose of this study was to determine the prevalence of Salmonella in the water used by Paan vendors in 11 North Indian cities. The analysis of 776 water samples and 120 samples each of preprocessed Paan (from vendor stock) and ready-to-eat Paan collected from Bareilly revealed that four of the ready-to-eat Paan and 34 of the water samples contained multidrug-resistant Salmonella strains. The isolates belonged to five different serovars, i.e., Salmonella Newport (1), Salmonella Paratyphi B (1), Salmonella Teko (1), Salmonella Virchow (3), and Salmonella Saintpaul (32), and could also be classified into 18 different resistotypes. All of the isolates were sensitive to cotrimoxazole, and 97.27% of the isolates were sensitive to chloramphenicol, imipenem, ciprofloxacin, ceftriaxone, and neomycin. Multidrug resistance (against 5 to 18 antibiotics) was common, particularly for nalidixic acid (65.79%), cephalothin (68.42%), cefoperazone (57.89%), sulfamethizole (52.63%), furazolidone (65.79%), kanamycin (68.42%), doxycycline (50.00%), and cefotaxime (44.74%). Bacteriological analysis of 16 Salmonella-positive and 23 Salmonella-negative samples revealed that the presence of Salmonella in water samples had a negative correlation (r = -0.35) with coliform counts and a positive correlation (r = 0.55) with nonlactose fermenter counts. We determined that centrifugation was a rapid and cheap method for concentrating large samples of Salmonella. The presence of multidrug-resistant strains of zoonotic Salmonella on ready-to-eat Paan is a public health concern and may be one of the factors responsible for the hyperendemic status of salmonellosis in India.

Antimicrobial Resistance: Its Surveillance, Impact, and Alternative Management Strategies in Dairy Animals

Antimicrobial resistance (AMR), one among the most common priority areas identified by both national and international agencies, is mushrooming as a silent pandemic. The advancement in public health care through introduction of antibiotics against infectious agents is now being threatened by global development of multidrug-resistant strains. These strains are product of both continuous evolution and un-checked antimicrobial usage (AMU). Though antibiotic application in livestock has largely contributed toward health and productivity, it has also played significant role in evolution of resistant strains. Although, a significant emphasis has been given to AMR in humans, trends in animals, on other hand, are not much emphasized. Dairy farming involves surplus use of antibiotics as prophylactic and growth promoting agents. This non-therapeutic application of antibiotics, their dosage, and withdrawal period needs to be re-evaluated and rationally defined. A dairy animal also poses a serious risk of transmission of resistant strains to humans and environment. Outlining the scope of the problem is necessary for formulating and monitoring an active response to AMR. Effective and commendably connected surveillance programs at multidisciplinary level can contribute to better understand and minimize the emergence of resistance. Besides, it requires a renewed emphasis on investments into research for finding alternate, safe, cost effective, and innovative strategies, parallel to discovery of new antibiotics. Nevertheless, numerous direct or indirect novel approaches based on host–microbial interaction and molecular mechanisms of pathogens are also being developed and corroborated by researchers to combat the threat of resistance. This review places a concerted effort to club the current outline of AMU and AMR in dairy animals; ongoing global surveillance and monitoring programs; its impact at animal human interface; and strategies for combating resistance with an extensive overview on possible alternates to current day antibiotics that could be implemented in livestock sector.

Evaluation of Yucca schidigera extract as feed additive on performance of broiler chicks in winter season.

Aim: Yucca schidigera extract has been successfully used as feed additives in the poultry industry. It enhances the growth and productivity in broiler production. Hence, the present study was designed to analyze the effect of Y. schidigera extract in growth, carcass quality and behavior along with its economical utility in broiler rearing. Materials and Methods: Total, 120 numbers of day-old broiler chicks of equal sex ratio were randomly divided into Yucca supplemented treatment and control group, each having 60 birds in three replications of 20 numbers. The feeding management and rearing conditions were similar for all the groups as per the standard except the Yucca supplementation in the treatment group @ 125 mg/kg of feed. The parameters with respect to growth, carcass, behavior, and litter content were recorded as per standard procedures. Results: The Yucca supplementation can effectively enhance growth of 173 g in 6th week by utilizing lesser feed intake than control group, which ultimately proves better feed conversion rate, protein efficiency ratio, and energy efficiency ratio in broiler production. Eviscerated weight of 58.50% for the treatment group was significantly higher (p<0.05) than 54.10% in the control group. The breast meat yield of Yucca group (32.23%) was significantly higher (p<0.05) than control (30.33%). More frequency of agonistic behavioral expressions was noticed in the control group than the treatment group. A profit of 43.68% was received by usage of Yucca supplementation in the diet on live weight basis. Numerically, lower percentage of moisture was present in Yucca treated group than the control. Conclusion: From this study, it can be concluded that Yucca supplementation has an important role in augmenting broiler‘s growth performance, efficiency to utilize feed, protein and energy, and survivability. Hence, use of Yucca powder in broiler ration could be beneficial to maintain the litter quality, which directly enhances the productivity in broiler production without any adverse effect.

Comparative mutant prevention concentration and antibacterial activity of fluoroquinolones against Escherichia coli in diarrheic buffalo calves.

Abstract Owing to emerging threat of antimicrobial resistance, mutant prevention concentration (MPC) is considered as an important parameter to evaluate the antimicrobials for their capacity to restrict/allow the emergence of resistant mutants. Therefore, MPCs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin, and norfloxacin were determined against Escherichia coli isolates of diarrheic buffalo calves. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were also established. The MICs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin and norfloxacin were 0·009, 0·022, 0·024, 0·028, and 0·036 μg/ml, respectively. The MBCs obtained were very close to the MICs of respective drugs that suggested a bactericidal mode of action of antimicrobials. The MPCs (μg/ml) of ciprofloxacin (4·2×MIC), moxifloxacin (4·8×MIC), and norfloxacin (5·1×MIC) were approximately equal but slightly lower than enrofloxacin (7·6×MIC) and levofloxacin (8·5×MIC) against clinical isolates of E. coli. The MPC data suggested that enrofloxacin has the potential for restricting the selection of E. coli mutants during treatment at appropriate dosing.

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis

Aim: The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type. Materials and Methods: A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV. Results: Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type. Conclusion: It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2.

ISMap02 element targeted nested polymerase chain in the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples of cattle and buffaloes

Background and Aim: Johne’s disease is chronic granulomatous enteritis which affects ruminants. There are many diagnostic approaches for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) of which molecular detection methods using various elements are less time consuming and more accurate. The present study was conducted using ISMap02 element for nested polymerase chain reaction (nPCR) based detection of MAP in fecal samples. The aim was to test the sensitivity and specificity of the ISMap02 element and also to use this element for the detection of MAP in fecal samples of cattle and buffaloes. Materials and Methods: A total of 211 fecal samples of cattle and buffaloes from different herds around Ludhiana aged between 2 and 13 years were collected, and DNA extraction was done from these samples. The nPCR was carried out for the detection of MAP in fecal samples. Results: The ISMap02 element was specific for the detection of MAP only and showed a sensitivity of detection of 7.6 fg/µL of the standard genomic DNA. Among the 211 fecal samples of cattle and buffaloes tested for the ISMap02 element, 18 samples (8.5%) were positive for MAP. Conclusion: The ISMap02 element is specific and sensitive for the detection of MAP in various samples, and when used in nPCR format, it can increase the sensitivity of detection.