Antimicrobial resistance in Indian isolates of non typhoidal Salmonella of livestock, poultry and environmental origin from 1990 to 2017.

A retrospective antimicrobial resistance study of nontyphoidal Salmonella enterica isolates from India during 1990-2017 was conducted to study the microbial susceptibility to antibiotics. A total of 271 Salmonella enterica isolates from poultry (n = 146), farm animals (n = 55) and environmental sources (n = 70) were tested for susceptibility using 15 antimicrobial drugs. The drug classes include aminoglycosides, phenicols, cephalosporins, penicillins, carbapenems, fluoroquinolones, and sulphonamide-trimethoprim. Study revealed that overall, 133 (49.08%) of 271 isolates were resistant to ≥ 1 antimicrobial drugs and 81 (29.89%) out of 271 isolates were multidrug resistant (resistance to ≥ 3 drugs). Majority (68.96%) of Typhimurium serovars (n = 87) were susceptible to all antibiotics tested, whereas only 5% Kentucky serovars (n = 40) were pan susceptible. All the drugs revealed decreasing trend of susceptibility from 1990 towards 2017 except cephalosporins and carbapenems. Statistical analysis of association between time period and antimicrobial resistance revealed a significance of < 0.05. Molecular detection of genetic determinants associated with antimicrobial resistance revealed the presence of genes like class I integrons, sul1, sul2, catIII, cmlA, dfrA, blaTEM, blaAmpC in the resistant isolates. Furthermore, plasmid mediated quinolone resistant determinants like qnrD and qnrS were also reported in the current study.

On point identification of species origin of food animals by recombinase polymerase amplification-lateral flow (RPA-LF) assay targeting mitochondrial gene sequences.

The present study was aimed to develop and standardize Recombinase polymerase amplification-lateral flow (RPA-LF) assays for on point identification of species origin of food animals viz: cattle, buffalo and pig. Species specific RPA primers sets for cattle, buffalo and pig were designed by homology comparisons of the sequences of mitochondrial cytochrome b gene and d-loop region from common food species viz: cattle, buffalo, sheep, goat, pig and chicken. The RPA assays for designed primers sets were optimized using the reaction components from Twist Amp basic kit and instructions in its manual. Endpoint detection of species specific amplified RPA products were made by gel electrophoresis and designed species specific RPA-LFA strips. The developed assays were evaluated for their specificity, diagnostic sensitivity, and validated on coded samples and binary meat admixtures with relative percentage of 20, 10, 5 & 1% target species. The developed RPA assays resulted in amplification of DNA template exclusively of cattle, buffalo and pig origin to product sizes of 294, 405 and 283 bp respectively. The diagnostic sensitivities of developed assays were up to 10 pg of genomic DNA and highly correlated with species specific PCR assays taken as gold standard. Developed species specific RPA assays also identified the target species in coded samples and binary meat admixture up to 1%.

Paper-based loop-mediated isothermal amplification and lateral flow (LAMP-LF) assay for identification of tissues of cattle origin.

The present study was made with the objectives of development and standardization of cattle specific paper-based loop-mediated isothermal amplification cum lateral flow assay (LAMP-LFA), as a Point-of-care test (POCT) for identification of tissue of cattle origin. The components of standardized LAMP reaction utilizing cattle specific primer sets were lyophilized over paper buttons, identified best as the carrier of LAMP reagents. Based on probable LAMP amplicon, a pair of probes was designed, tagged and its hybridization with the amplified product of paper LAMP reaction was optimized. The components of lateral flow assay for detection of probe hybridized LAMP products were standardized. Analysis of successful amplification was made by using HNB dye, LAMP-LFA strip, and also by the typical ladder-like pattern on gel electrophoresis. The assay was found highly specific for cattle with an analytical sensitivity of 0.1 pg of absolute DNA. Laboratory validation carried out on samples from different individuals of cattle, coded samples, binary meat admixture, and heat-processed cattle tissues substantiated the accuracy of the assay. Comparison with pre-standardized species-specific PCR assay taken as gold standards revealed 100% conformity. The field utility of the developed assay was further established by its compatibility with the commercial kit eliminating the lengthy DNA extraction step and storage stability of LAMP reagent carrier buttons for 4 months under refrigeration. Thus, the developed assay capable of the result within 3 h in resource-limited settings can be used as POCT for identification of tissue of cattle origin.

Prevalence of Campylobacter jejuni and Campylobacter coli in captive wildlife species of India.

Campylobacteriosis is an important zoonotic disease and the prevalence of Campylobacter is largely unknown in the wildlife of India. A total of 370 samples, comprising of 314 fresh faecal samples from apparently healthy captive wild animals and birds, 30 stool swabs from animal care takers and 26 samples of the animals' food and water were collected from G. B. Pant High Altitude Zoo, Nainital, Kanpur Zoo, Wildlife Park, IVRI and the Post Graduate Research Institute in Animal Sciences (PGRIAS), Chennai, Tamilnadu from August 2014 to May 2015. Samples were processed for cultural isolation, direct PCR and multiplex PCR for species confirmation. To decipher the genetic diversity, the 16S rRNA gene was amplified, sequenced and analyzed. Based on isolation, the overall occurrence rate of Campylobacter spp. was 0.8% (3/370), being 2.94% (3/102) for captive wild birds. Three Campylobacter jejuni were isolated from silver pheasants, lady amherest pheasants and saras cranes. Direct PCR assay showed the overall occurrence rate of Campylobacter spp. to be 4.77% (15/315), being 1.58% (2/126) for captive wild ruminants, 5.81% (5/86) for non-ruminants and 7.84% (8/102) for birds. All the isolates were identified as C. jejuni.

Genome wide host gene expression analysis in mice experimentally infected with Pasteurella multocida.

Pasteurella multocida causes acute septicemic and respiratory diseases, including haemorrhagic septicaemia, in cattle and buffalo with case fatality of 100%. In the present study, mice were infected with P. multocida (1.6 × 103 cfu, intraperitoneal) to evaluate host gene expression profile at early and late stages of infection using high throughput microarray transcriptome analyses. Several differentially expressed genes (DEGs) at both the time points were identified in P.multocida infected spleen, liver and lungs. Functional annotation of these DEGs showed enrichment of key pathways such as TLR, NF-κB, MAPK, TNF, JAK-STAT and NOD like receptor signaling pathways. Several DEGs overlapped across different KEGG pathways indicating a crosstalk between them. The predicted protein-protein interaction among these DEGs suggested, that the recognition of P. multocida LPS or outer membrane components by TLR4 and CD14, results in intracellular signaling via MyD88, IRAKs and/or TRAF6 leading to activation of NFκB and MAPK pathways and associated cytokines.

Changes of haemogram and serum biochemistry in neonatal piglet diarrhoea associated with porcine rotavirus type A.

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 < 0.001) decreased and concentrations of ALT, AST, BUN and creatinine were significantly increased (P < 0.001) in the RVA-infected piglets. Haemogram showed marked haemoconcentration (P < 0.001), leukopenia (P < 0.01) and neutropenia (P < 0.01) in the presence of RVA infection than healthy piglets. The results indicated a possible extra-intestinal spread of RVA in piglets during neonatal diarrhoea. The finding might be helpful to clinicians and while treating such type of clinical cases, incorporation of organ protective drugs will be helpful for better response in the treatment schedule.

A double-stranded probe coupled with isothermal amplification for qualitative and quantitative detection of avian reovirus.

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.

Spontaneous Ocular Autoimmunity in Mice Expressing a Transgenic T Cell Receptor Specific to Retina: A Tool to Dissect Mechanisms of Uveitis.

The "classical" EAU model induced by immunization of mice with the retinal protein IRBP or its peptides has been very useful to study basic mechanisms of ocular inflammation, but is inadequate for some types of studies due to the need for active immunization in the context of strong bacterial adjuvants. We generated transgenic (Tg) mice on the B10.RIII background that express a T cell receptor (TCR) specific for IRBP161-180. Three strains of TCR Tg mice were established. Spontaneous uveitis developed in two of the three strains by 2-3 months of age. Susceptibility correlated with a higher copy number of the transgenic TCR and a higher proportion of TCR Tg T cells in the peripheral repertoire. Even in mice with uveitis, peripheral IRBP-specific CD4(+) T cells displayed mostly a naïve phenotype. In contrast, T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells. These mice thus provide a new and distinct model of uveitis from the "classical" EAU, and may represent some types of uveitis more faithfully. Importantly, this new transgenic model of uveitis can serve as a template for therapeutic manipulations, and as a source of naïve retina-specific T cells for a variety of basic and pre-clinical studies. Several examples of such studies will be discussed.

Essential oils as natural food antimicrobial agents: a review.

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.

Aetiologies of Acute Undifferentiated Febrile illness in Adult Patients - an Experience from a Tertiary Care Hospital in Northern India.

INTRODUCTION: Acute undifferentiated febrile illness (AUFI) is a common clinical entity in most of the hospitals. The fever can be potentially fatal if the aetiology is not recognized and appropriately treated early. AIM: To describe the aetiology of fever among patients in a tertiary care hospital in Northern India. MATERIALS AND METHODS: A one-year retro-prospective, observational study was conducted in adults (age>18years) presenting with undifferentiated febrile illness (of duration 5-14 days). Diagnosis was confirmed by suitable laboratory tests after exhaustive clinical examination. RESULTS: A total of 2547 patients with AUFI were evaluated. Of these, 1663 (65.3%) were males and 884 (34.7%) were females. Dengue (37.54%); enteric fever (16.5%); scrub typhus (14.42%); bacterial sepsis (10.3%); malaria (6.8%); hepatitis A (1.9%); hepatitis E (1.4%); leptospirosis (0.14%); were the main infections while no specific diagnosis could be delineated in 11%. Mixed infections were noted in 48 (1.9%) patients. CONCLUSION: A good clinical acumen supported by the basic investigations can help diagnose the cause of fever with reasonable certainty.

Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle.

BACKGROUND: 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. OBJECTIVE: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Human milk banking guidelines.

JUSTIFICATION: WHO and UNICEF state that the use of human milk from other sources should be the first alternative when it is not possible for the mother to breastfeed. Human milk banks should be made available in appropriate situations. The IYCF Chapter is actively concerned about the compelling use of formula feeds in the infants because of the non availability of human breast milk banks. PROCESS: A National Consultative Meet for framing guidelines was summoned by the IYCF Chapter and the Ministry of Health and Family Welfare, Government of India on 30th June, 2013, with representations from various stakeholders. The guidelines were drafted after an extensive literature review and discussions. Though these guidelines are based on the experiences and guidelines from other countries, changes have been made to suit the Indian setup, culture and needs, without compromising scientific evidence. OBJECTIVES: To ensure quality of donated breast milk as a safe end product. RECOMMENDATIONS: Human Milk Banking Association should be constituted, and human milk banks should be established across the country. National coordination mechanism should be developed with a secretariat and technical support to follow-up on action in States. Budgetary provisions should be made available for the activities.

New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination.

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.

Enhancing hepatoprotective bioactives of phyllanthus amarus through immobilization by growth promoters and media changes.

The present paper describes enhancement of lignans, phyllanthin and hypophyllanthin present in Phyllanthus amarus plant. Supplementation was done to enhance secondary metabolites by modifying the media and treatment with different growth promoters and abiotic elicitors to increase the content of hepatoprotective bioactives in immobilized cell cultures, after incubation for 21 days. MS medium was supplemented with gibberellic acid and to make whole process commercially viable, when coconut water, sugarcane juice and water-melon extract were treated, it was revealed that watermelon extract, enhances maximum phyllanthin and hypophyllanthin yield followed by sugarcane juice, coconut water and gibberellic acid after estimation by HPTLC. The present method was found to be accurate, economical and viable to enhance the content of phyllanthin and hypophyllanthin in P. amarus for large-scale commercial production.

Cloning and sequencing of biofilm-associated protein (bapA) gene and its occurrence in different serotypes of Salmonella.

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.

Infant and young child feeding guidelines: 2010.

JUSTIFICATION: The first National Guidelines on Infant and Young Child Feeding (IYCF) were formulated by Ministry of Women and Child Development (Food and Nutrition Board) in 2004, and the same guidelines were revised in 2006. India is committed to halving the prevalence of under weight children by 2015 as one of the key indicators of progress towards the Millennium Development Goals (MDG). By the end of 2009 nutritional achievement goals did not make for happy reading. So there was need to revise the existing guidelines and to have more viable and scientifically accepted national guidelines on Infant and Young child feeding. PROCESS: A National Consultative Meet was organized by Indian Academy of Pediatrics at Gurgaon in 2009 where members of IYCF and Nutrition Chapters of IAP, BPNI, WHO, UNICEF, USAID, WFP were present. Each group made detailed presentations after reviewing recent literature on the subject. After extensive discussions a consensus was reached and the guidelines were formulated. OBJECTIVES: To formulate, endorse, adopt and disseminate guidelines related to Infant and Young Child feeding from an Indian perspective (including infant feeding in the context of HIV infection). RECOMMENDATIONS: Optimal infant and young child feeding: Early initiation of breastfeeding, exclusive breastfeeding for the first six month of life followed by continued breastfeeding for up to two years and beyond with adequate complementary foods is the most appropriate feeding strategy for infants and young children. Adequate nutrition and anemia control for adolescent girls, pregnant and lactating mother is also advocated.

Limb splinting for intravenous cannulae in neonates: a randomised controlled trial.

OBJECTIVE: To evaluate the efficacy of peripheral intravenous (IV) cannula site joint immobilisation by splint application on functional duration of peripheral IV cannula in neonates. DESIGN: Randomised controlled trial. SETTING: Neonatal intensive care unit of a tertiary care hospital. PARTICIPANTS: Neonates requiring continuous IV infusion for an expected duration of more than or equal to 72 hours. INTERVENTION: Eligible cannulations were randomised to either "splint" or "no-splint" group. In the splint group, a cardboard splint was used to immobilise the joint at peripheral IV cannula site. No attempt was made to immobilise the limb in the no-splint group. OUTCOME MEASURE: Functional duration of a peripheral IV cannula measured as interval from time of insertion to the development of predefined sign of removal (extravasation, blockage, inflammation). RESULTS: A total of 69 peripheral IV cannulations in 54 neonates were randomised to either the splint (n = 33) or no-splint group (n = 36). Both groups were comparable in birth weight, gestation, site of cannulation and nature of fluids administered. Mean functional duration of cannula was lesser in the splint group compared to the no-splint group (h; 23.5 (SD15.9) vs 26.9 (SD15.5), mean difference: -3.3 h, 95% CI -11.02 to 4.3 h) although the difference was not statistically significant (p = 0.38). Extravasation at cannula site was found be the commonest indication for cannula removal in both the groups (84% vs 76.5%). CONCLUSION: Joint immobilisation with splint at cannula site did not improve the functional duration of peripheral IV cannula.