Showing papers by "Chaitanya G. Joshi published in 2011"

Identification and Antibiogram of Microbes Associated with Bovine Mastitis

Abstract: An investigation of Mastitis in cattle was carried out in Anand city and in nearby villages of Gujarat state using California Mastitis Test (CMT) kit. The prevalence of clinical and subclinical mastitis was found to be 5.5% and 15.75%, respectively. Staphylococcus aureus was identified through strain specific polymerase chain reaction; the remaining isolates identified on the basis of molecular analysis by 16S rDNA sequencing and phylogenetic analysis were Staphylococcus species, B. pumilus, Staphylococcus chromogenes, Bacillus species, and Pseudomonas species. In vitro antimicrobial susceptibility pattern of all the isolates was checked against 13 different antibiotics using the agar disc diffusion method. Highest bacterial resistance was observed with penicillin G and oxacillin antibiotics. It was also observed that the patterns of bacterial resistance have not changed in India over the years. The data supports the decrease in the incidence of mastitis but the rate of decrease is minimal. More effective...

Whole-Genome Shotgun Sequencing of an Indian-Origin Lactobacillus helveticus Strain, MTCC 5463, with Probiotic Potential

Abstract: Lactobacillus helveticus MTCC 5463 was isolated from a vaginal swab from a healthy adult female. The strain exhibited potential probiotic properties, with their beneficial role in the gastrointestinal tract and their ability to reduce cholesterol and stimulate immunity. We sequenced the whole genome and compared it with the published genome sequence of Lactobacillus helveticus DPC4571.

Genome Sequence of Pasteurella multocida subsp. gallicida Anand1_poultry

Abstract: We report the finished and annotated genome sequence of Pasteurella multocida gallicida strain Anand1_poultry, which was isolated from the liver of a diseased adult female chicken. The strain causes a disease called “fowl cholera,” which is a contagious disease in birds. We compared it with the published genome sequence of Pasteurella multocida Pm70.

In vitro expression profiling of myostatin, follistatin, decorin and muscle-specific transcription factors in adult caprine contractile myotubes

Abstract: Skeletal muscle is one of the several adult postmitotic tissues that retain the capacity to regenerate, which relies on a population of quiescent precursors, termed satellite cells. Proliferation and differentiation of myoblasts to form mature myotubes in vitro has been a valuable tool in the characterization of the cellular events during myogenesis, which is a multistep process starting with progenitor cell proliferation, followed by their exit from the cell cycle, differentiation, alignment, and fusion to form multinucleated myotubes. A typical feature during muscle differentiation is the variation in expression of various genes along with myogenic factors. In this experiment, mRNA level of myostatin, follistatin, decorin and three muscle-specific transcription factors in adult caprine contractile myotubes have been studied through quantitative real time PCR. We observed that the expression level of myostatin, decorin, Myf5 and myogenin transcripts were significantly higher in contractile myotubes compared to myoblast monolayer (P < 0.05), and follistatin level was similar in both types of cells, whereas MyoD transcript level was significantly high in monolayer culture which might be due heterogeneity of myoblast population. It is concluded that the information generated would provide the base line information as well as monitoring markers to undertake experiments aimed at modulating muscle growth.

Dasytricha dominance in Surti buffalo rumen revealed by 18S rRNA sequences and real-time PCR assay.

Abstract: The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR-RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10(5) vs. 1.3 × 10(4)) in per ml ruminal fluid.

Characterization of 132 bp Repeats BamH1-H Region in Pathogenic Marek's Disease Virus of Poultry in Gujarat, India, Using PCR and Sequencing.

Abstract: A total of 34 clinical samples and four Marek’s disease virus (MDV) vaccines were tested using primer BamH1/BamH2 in layer birds of poultry. Out of 34 samples tested for detection of MDV, 32 samples produced approximately 434 bp product. All the three HVT vaccines as well as SB-1 (MDV-2) vaccine failed to produce the expected amplicons, there by proving negative for the targeted 132 bp repeats of MDV genome by the primers BamH1/BamH2. Resultant PCR products of the field samples were purified and sequenced and resulted in 378 bp long sequences. PCR was found very satisfactory in detecting the presence of MDV either in feather follicle or in tissue samples. Sequencing study has proved beyond doubt that the two representative samples contained two 132 bp repeats indicating the virulent nature of the field virus.

Molecular cloning and characterization of rabbit myostatin gene

Abstract: Revised on: Accepted on: Published on: make comparative studyshowed only one variation (a>G) in exon I of a soviet chinchilla (S11) and one variation (g>A) in exon III of a resequencing; codon Corresponding author: Email: drajais@gmail.com; Tel: +91–2692 261201, 261486; Fax: +91–2692 261201, 261486 [I] INTRODUCTION Rabbits are small mammals in the family Leporidea of the order Lagomorpha found in several parts of the world and used for many purposes mainly meat, fur production etc. Several DNA markers associated with production traits in livestock have been already identified through candidate gene approach [1], which is based on the fact that variability within genes coding for protein products involved in key physiological mechanisms and metabolic pathways directly or indirectly involved in determining an economic trait (e.g. feed efficiency, muscle mass accretion, reproduction efficiency, disease resistance, etc.) and might probably explain a fraction of the genetic variability for the production trait itself [2]. differentiation factor 8), is specifically expressed during embryonic development, expressed at high level in adult skeletal muscle and controls skeletal muscle growth [3]. Molecular analysis of the myostatin gene in different species has showed that it consists of three exons and two introns

Duplex-PCR for identification and differentiation of cattle and buffalo processed meat.

Abstract: A simple and reliable duplex polymerase chain reaction (duplex-PCR) technique is proposed to identify and differentiate cattle and water buffalo DNA using primers that were tested on mitochondrial DNA (mtDNA) extracted from meat muscle samples. Different levels of autolysis were experimentally produced by putrefaction and heating the samples at various temperatures and conditions to simulate the various meat processing technology. The optimized PCR amplified 113 bp and 152 bp fragment of cyt b gene from mtDNA. This test was successful in detecting up to 1 pg adulteration in cattle-buffalo meat mixture. The test is a valuable tool for meat authentication and screening of cooked, putrefied and mixed samples of cattle and buffalo.

Evaluation of buffalo bull spermatozoal dna damage using single cell gel electrophoresis

Abstract: Deoxyribonucleic acid was influenced by sperm ageing and sperm storage temperature. In this study, the effect on DNA integrity at prefreeze and postfree ze stages was analyzed by single cell gel electrophoresis during winter and summer seasons. Traditionally, the diagnosis of male infertility depends on microscopic assessment and biochemical assays to evaluate quality of semen. These tests are needed to provide the basic information for initial diagnosis. However, none of these parameters gives information about sperm function and their fertilization capability. The fertility of these sperms is questionable. The evaluation of sperm DNA damage is a promising technique to diagnose the male infertility. It has been shown to be closely associated with negative relationships with fertilization, embryo quality, implantation and positive relationships with abortion. Here we report the effect of preservation temperature and season on sperm DNA damage, using the single cell gel electrophoresis to measure DNA fragmentation. The significant difference (P<0.05) in DNA damage among Surti buffalo bulls spermatozoa were more pronounced at postfreeze stage as compared to the prefreeze stage. In addition, the significant difference (P<0.05) in DNA damage was observed among Surti buffalo bulls spermatozoa during summer season as compared to the winter season. The changes on DNA integrity were dependant on temperature of preservation, period of storage and seasonal variation. The single cell gel electrophoresis can be used to detect low levels of DNA damage in Surti buffalo bulls spermatozoa. Therefore, screening of the sperm DNA damage may be used as an additional test of sperm quality that can have diagnostic importance in the male infertility.

DNA polymorphism of 3’ UTR of Nramp1 gene in Malvi breed of cattle

Abstract: The natural resistance-associated macrophage protein 1 gene (Nramp1), which is a member of large family of metal ion–transport protein. Nramp1 gene plays a critical role in innate immunity favoring bacterial killing by macrophages in addition to its influence on adaptive immunity. The aim of the present investigation was to identify the genetic variations in the 3’UTR (Untranslated region) of Nramp1 gene in the Malvi breed (Bos indicus) cattle, using the technique PCR-SSCP and by sequencing. PCR-SSCP (Single Strand conformational polymorphism) of 440 bp amplicon of Nramp1 gene revealed three common SSCP patterns in Malvi breed. A total of 3 SSCP patterns viz Pattern I, Pattern II and Pattern III were observed with frequency of 0.361, 0.426 and 0.213 respectively. The patterns variations were confirmed by cloning and sequencing, which showed total 6 mutations in 3 patterns.

Polymorphism in 3′UTR region of Slc11a1 gene in Indian breeds of cattle.

Abstract: The solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1 (Slc11a1) also called natural resistance-associated macrophage protein 1 gene (Nramp1) is a member of the large family of metal ion-transport proteins. It encodes a divalent cation (Fe+ & Mn+) transporter that localizes in the phagolysosome membrane in macrophages. Slc11a1 gene plays a critical role in innate immunity favoring bacterial killing by macrophages in addition to its infl uence on adaptative immunity. Polymorphism at the 3’ untranslated region (3’UTR) of Slc11a1 gene is associated with natural resistance against brucellosis in cattle. Such polymorphisms are associated with variations in the number of GT repeats. This study aimed to discover polymorphism in the 3’UTR region of the Slc11a1 gene in the Indian cattle breeds Nimari and Kenkatha. Polymerase Chain Reaction Single Strand Conformation Polymorphism (PCR-SSCP) of 440 bp amplicon of Slc11a1 gene revealed three common SSCP patterns in these breeds, which was also confi rmed by detecting point mutation in sequences of these patterns. The study will augment the information available and be useful in further studies to determine the role of the Slc11a1 gene in disease resistance and for the selection of brucellosis resistant animals.

SNP EXPLORATION IN THE OXIDISED LOW DENSITY LIPOPROTEIN RECEPTOR 1 (OLR1) GENE IN Bubalus bubalis

Abstract: Single nucleotide polymorphisms (SNP) are the most common type of DNA sequence polymorphisms and of mutation in vertebrates. Keeping in view the involvement of the oxidised low density lipioprotein receptor 1 ( OLR1) gene in lipid metabolism and the higher milk fat percentages in Bubalus bubalis, the present study was carried out to explore the SNPs in OLR 1 intron I in two breeds of buffalo viz. Jaffarabadi and Surti by gene cloning and sequencing. 3 SNPs were detected and all the SNPs identifi ed followed the breed lineage.

Haplotype and phylogenetic analysis of OLR1(Intron I) gene in Jaffarabadi and Surti buffalo.

Abstract: The present study was carried out to reveal haplotype and phylogenetic analysis of OLR1 gene in Jaffarabadi and Surti breeds of buffalo. Twenty nucleotide sequences generated from our previous study (Shabir, 2009) were used to reveal the haplotypes in OLR1 (Intron I) gene in the population of Jaffarabadi and Surti buffalo. Four haplotypes viz. H1 (CTA), H2 (TCT), H3 (CTT) and H4 (TTT) were observed with frequencies of 0.05, 0.1, 0.15 and 0.7 respectively in the population studied. Phylogenetic analysis of these twenty sequences with the same region of Bos taurus distributed the sequences into four clusters based on the homology between them. Cluster I contained two sequences (jb3 and jb8) bearing a common SNP at nucleotide position 843. Cluster III contained s3, s4 and s7 since they possessed a common SNP at nucleotide position 423, however s3 remained as an out group in this cluster since it contained an additional SNP at position 866. The remaining sequences had highest homology and fell in cluster II while as the sequence of Bos taurus remained as an out group on account of the difference in bases at many loci.

Somatotropin-mediated gene expression profiling of differentially displayed ESTs during lactation in Indian buffalo (Bubalus bubalis).

Abstract: The study of bovine mammary gland functional genomics requires appropriate cDNA library collections to access gene expression patterns from different developmental and physiological stages. The present study was undertaken with the objective to identify candidate genes involved in the process of increased milk synthesis following 0, 48 and 96 h of recombinant bovine somatotropin (rbST) treatment to Surti buffalo (Bubalus bubalis) through differential display reverse transcriptase PCR (DDRT-PCR). Of a total 50 sequenced DD bands, 64% of ESTs were differentially expressed (appeared only in post-treatment samples, i.e. 48 h and 96 h) and 36% were up-regulated after rbST treatment. Of the ESTs 32%were found to be located on Bos taurus chromosome 24 (equivalent to buffalo chromosome 22), whereas 16% of ESTs could not be mapped, indicating that they are specific to buffalo. Quantitative real time PCR assay of 15 ESTs revealed transcript level surge in 13 ESTs, and decline in one EST, while one showed up-regulation in expression level at 48 h while down-regulation at 96 h. This study indicates more than 30 novel transcripts, with unknown function, involved in increased milk synthesis and also the involvement of many more genes in the physiology of milk production than once thought.

Molecular characterization of exon 2 of leptin gene in Bos-indicus.

Abstract: Milk production traits are quantitative traits controlled by numerous genes and environmental factors. The 167 amino acid protein product of the ob gene was named leptin and consists of three exons of which the first exon is not transcribed into the leptin protein of 16-Kilo Dalton. The bovine leptin gene is located at BTA4q32 and is involved in the growth and metabolism of animals, and plays an important role in the regulation of feed intake, energy metabolism, growth, production and reproduction of cattle. This study aimed to find out the polymorphism in the leptin gene of Nimari (n=70) and Gaolao (n=75) bovine breeds. PCR-SSCP (Single Strand conformational polymorphism) of 317 bp amplicon of leptin gene revealed SSCP patterns I, II and III in both the breeds. The chi-square test revealed that difference between observed frequencies of different patterns in Nimari and Gaolao were non-significant at 5% level of significance.