Mukta M. Shukla

Hydrophilic-interaction chromatography of complex carbohydrates

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

Effect of dietary supplementation of phytobiotic on growth and immunocompetence of commercial broilers

I with 328.73 million hectares geographical area is endowed with 485 million diverse livestock wealth. Currently, India is the largest milk producer in the world with105 million metric tons per annum. Despite this, there is deficit of milk and meat production to meet the recommended daily allowances (RDA) of milk and meat consumption for human. Hence, there is a need to enhance livestock production, which justifies the increasing the livestock population. But the main impediments are low genetic makeup of indigenous livestock and qualitative and quantitative insufficiency of feed and fodder resources for livestock feeding.I keratoconjunctivitis (IKC) is a disease of worldwide economic importance causing blepharospasm, corneal opacity and conjunctivitis in ruminants. Recovered animal may develop corneal opacity and blindness. Although, Moraxella bovis is the major cause of IKC in bovines, Moraxella ovis has been implicated in epizootics of IKC in sheep and goats worldwide. Isolation of M. ovis has been reported from both healthy sheep and those with IKC; however we confirm for the first time, isolation and molecular confirmation of M. ovis and Moraxella spp. isolates from IKC cases in sheep in India. Out of a freeranging nomadic herd, six cases of IKC in sheep were randomly sampled. Conjunctival swabs for bacterial culture were plated on 5% Columbia blood agar and MacConkey agar. Out of twenty isolates 10 oxidase-positive isolates which showed microscopic morphology of Gram-negative cocci in pairs were biochemically processed. Three non-motile, nitrate positive, indole negative and non-saccharolytic isolates were identified as Moraxella ovis. Two isolates subjected to 16S rRNA PCR sequencing. Clones of 16S rRNA gene were sequenced to complete >1.5 bp, which was subjected to phylogenetic analysis. By RDB database analysis sequence showed a sequence similarity of 99.24% with Moraxella ovis ATCC33078, and formed a cluster with M. ovis strains, however the other isolate Moraxella spp. clusterd close to Moraxella boevrei DSM14165. The M. ovis isolate was resistant to penicillin and cloxacillin. Phenotypic, biochemical, molecular and phylogenetic evidence identified isolates as M. ovis and Moraxella spp. This isolate with first molecular confirmation of M. ovis isolation from sheep in India, and has been accessioned in VTCC repository.A five year old pleuriparous cow was presented to the Veterinary Ambulatory clinic Mylardevpally with the history of reduced feed intake, reduced milk yield, salivation, cough, swelling of neck and bilateral mucopurulent nasal discharges. Upon clinical examination, the cow was dull and depressed with higher temperature, congested mucosal membranes, labored breathing and had ruminal atony(0/3minutes).Blood smear examination revealed bipolar organisms. The cow was treated with Inj. Enrocin@5 mg/kg b.wt IV, Inj. Melonex power @ 3ml IM, Inj. Tribivet @ 12 ml i/m,Inj.DNS@1 litre slow IV. The above treatment was continued for 5 days and clinical improvement was noticed after 3 days.T Mithun (Bos frontalis) according to Phanchung and Roden (1996) is believed to have its origin from Gaur (Bos gaurus). The Mithun looks more like a transition between cattle and buffalo, but genetic analysis of the Mithuns chromosomes by Winter et al.(1984), Gupta etal. (1995) and Mukherjee et al. (2012) shows a diploid number 58 (2n) compared to 60 in cattle and 48+ in buffalo. Mithun is specifically reared by the tribal community and is reared on free range, grazing at altitudes of between 1000-3000m AMSL.B is considered as one of the most widespread zoonosis in the world. For complete eradication of infection from herds, strain-specific identification is essential to know the source of infection. In the present study, MLVA-15genotyping scheme was applied to differentiate the field strains of B. abortus.A total of 13 B. abortus strains along with 4 representative reference strains (B. abortus 544 (ATCC 23448), B. abortus strains S19, S99 and 1119-R) were taken from the repository of the Brucella Laboratory, Div. of VPH, IVRI, Izatnagar. For MLVA-15 genotyping, 15 sets of primers (8 minisatellites and 7 microsatellites markers) were used for the amplification of 15 VNTR loci in all the strains that resulted in13 different genotypes. Of these, 10 profiles were unique while remaining 3 profiles were shared by at least 2 strains. In B. abortus, Bruce 6, 8, 11, 21, 42, 43, 45 and 55 were found to be most conserved with no length polymorphism. Bruce 4, 9, 12, 16 and 18 showed moderate length polymorphism. Bruce 7 showed significant length polymorphism whereas highest length polymorphism was seen at locus Bruce 30.The dendrogram based on MLVA data was generated using the UPGMA clustering method implemented by START vs. 1.0.5 software. Through clustering analysis using UPGMA, the B. abortus isolates were grouped in 6 clusters showing a 90% similarity. Allelic diversity for each of the locus was calculated using HGD index. In the present study, the MLVA-15 genotyping clearly discriminated Indian field isolates of Brucella abortus.S contamination of seafood with pathogens of human health significance is a major problem that compromised the safety of seafood. The problem is confounded when the pathogens found in seafood are also antibiotic resistant. In the present study, the prevalence of extended spectrum β-lactamase producing enterobacteria in seafood in Mumbai was studied. All samples of seafood, comprising of shellfish and finfish, harboured enterobacteria. A total of 215 isolates of enterobacteria were obtained, the predominant species being E.coli (30.69%). Using chromogenic medium and antibiotic disc diffusion methods, the ESBL-positive phenotype was detected in 169 (78.60%) of the isolates. Of these, 165 (97.63 %) isolates were resistant to cefotaxime, followed by 156 (92.30%) to cefpodoxime, 154 (91.12%) to ceftazidime, 109 (65.08%) to aztreonam and 69 (40.82%) to piperacillin/tazobactam. Resistance to imipenem was observed in 18 (10.65%) isolates, while 27 (15.97%) isolates were resistant to ciprofloxacin.PCR analysis of ESBL-encoding genes detected blaCTX, blaSHV, blaTEM and blaNDM genes in resistant isolates. blaCTX gene was the most common ESBL gene detected in 76.92% of the isolates. Multiple ESBL genes were found in majority of the isolates. The recently discovered New Delhi metllo-β-lactamase gene (blaNDM) was found in 4 seafood isolates of enterobacteria. The NDM gene from E. coli (EC-114)which was resistant to cephalosporins, fluorquinolones, carbapenems but sensitive to aminoglycosides, colistin, polymyxin B and nitrofurantoinwas sequenced and was found to be NDM-5 variant gene.The widespread occurrence of multiple antibiotic resistant enterobacteria is a health concern and compromises the safety of seafood and may implicate seafood as potential carriers of antibiotic resistant bacteria.