Thumbnail Image

Anand Agricultural University, Anand

Anand Agricultural University (AAU) was established in 2004 at Anand with the support of the Government of Gujarat, Act No.(Guj 5 of 2004) dated April 29, 2004. Caved out of the erstwhile Gujarat Agricultural University (GAU), the dream institution of Sardar Vallabhbhai Patel and Dr. K. M. Munshi, the AAU was set up to provide support to the farming community in three facets namely education, research and extension activities in Agriculture, Horticulture Engineering, product Processing and Home Science. At present there seven Colleges, seventeen Research Centers and six Extension Education Institute working in nine districts of Gujarat namely Ahmedabad, Anand, Dahod, Kheda, Panchmahal, Vadodara, Mahisagar, Botad and Chhotaudepur AAU's activities have expanded to span newer commodity sectors such as soil health card, bio-diesel, medicinal plants apart from the mandatory ones like rice, maize, tobacco, vegetable crops, fruit crops, forage crops, animal breeding, nutrition and dairy products etc. the core of AAU's operating philosophy however, continues to create the partnership between the rural people and committed academic as the basic for sustainable rural development. In pursuing its various programmes AAU's overall mission is to promote sustainable growth and economic independence in rural society. AAU aims to do this through education, research and extension education. Thus, AAU works towards the empowerment of the farmers.

Browse

2008 - 200912010 - 20151
Reset filters
Subject: Animal Biotechnology × Author: AHIR, VIRAL B. ×
Reset filters

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    ThesisItemOpen Access
    DETECTION OF POLYMORPHISM IN BOVINE PREIMPLANTATION ACTIVE GENES AND THEIR ASSOCIATION WITH SEMEN QUALITY
    (AAU, Anand, 2008) AHIR, VIRAL B.; Panchal, M. T.
    Embryo development in mammals is marked by distinctive biological processes that occur during the preimplantation and early post implantation periods. Preimplantation development encompasses the period from fertilization to implantation, which occurs in different times in various species and it is marked by a number of critical events. This development is a mammalian-specific event, and is vital for successful implantation and pregnancy. Bovine preimplantation embryo development is under constant control of genes activated from either maternal or embryonic genome. Large-scale association studies by genotyping many single nucleotide polymorphisms (SNPs), in individuals with well characterized phenotypes, are considered as promising methods to identify the cause of many complex traits. The present study was undertaken to study the polymorphism of bovine preimplantation active genes loci by PCR-RFLP and PCR-SSCP techniques and their association with various semen quality traits in Murrah buffalo bulls belonging to ARDA, Ode during January to June 2008. The mean and standard error of mean (SEM) for various semen quality parameters, viz., volume (ml), concentration (106/ml), motility (%), motility after thawing(%) and live and dead count (%) was found to be 3.35+0.27, 1511.07+112.25, 73.54+1.05, 54.39+0.66 and 85.44+0.47, respectively. Semen DNA was extracted from 41 Murrah buffalo bulls by Proteinase K method as per standard protocol. Bovine COX-2, CD9, DSC2, AKRIBI and CDHl genes specific primers (COX-2 F: 5'-TGA TCT ACC CGC CTC ATG TT-3' and COX-2 R: 5'-CCC TTT GCC TGG TGA ATG-3'); (CD9 F: 5'-GAG GCA AAA CTC CAA AAC CA-3' and CD9 R: 5'-CTC CAC TGT CGT TTG TCG TG -3'); (DSC2 F: 5'-AAA GTG CAA GAC ATG GAT GG-3' and DSC2 R: 5'-CCT TCA TTG GTT TGG GAA TC-3'); (AKRIBI F: 5'-ACC AGG GCT TAC CTG GAA GT-3' and AKRIBI R: 5'-GGT CAA TGG GCC TTA GGA TT-3') and (CDHl F: 5'-CGC ACA ACA AAA TGT TCA CC-3' and CDHl R: 5'-GGC CTC AAA TCT CCA GAC AA-3') were used to amplify bovine preimplantation active genes loci. PCR was carried out in 25 µl volume for 35 cycles of denaturation at 94°C, annealing at appropriate temperature (COX-2 locus at 52°C, DSC2 and CDHl loci at 51°C, AKRIBI locus at 58°C) and extension at 72°C. Initial denaturation was carried out at 94°C for 5 minutes, while the final extension was performed at 72°C for 5 minutes. For the CD9 locus "Touch down PCR" was performed to avoid spurious priming during PCR amplification. Amplified products were electrophoresed on 2% Agarose gel at 80 V for for 60 minute. For RFLP analysis Amplified PCR product of COX-2, CD9, DSC2 and AKRIBI loci were digested with Alu I, Dra I, Rsa I and Nde /restriction enzymes respectively, by incubating them at 3 7°C for 14-16 hours except for Rsa I which was incubated at 37°C for 12 minute and electrophoresed on 2% Agarose gel at 80 V for 60-90 minutes to reveal the restriction pattern. Monomorphic pattern was observed for the COX-2, CD9 and DSC2 loci and only TT, TT and AA genotypes, respectively, were found in all Murrah buffalo bulls at these loci. The allelic frequencies of T, T and A alleles were 1.00, with absence of A, A, and G alleles, respectively. For AKRIBI locus, 18 samples were found to be homozygous AA and 23 samples were heterozygous AG, with allelic frequency of 0.725 and 0.275 for A and G alleles, respectively. For SSCP analysis, PCR products of CDHl locus were denatured and electrophoresed on 6% non-denaturing PAGE for 4-5 hours at constant 5W. Analysis revealed four different banding patterns with frequencies for pattern 1, pattern 2, pattern 3 and pattern 4 to be 0.463, 0.146, 0.292 and 0.097, respectively. Since, loci COX-2, CD9 and DSC2 were found to be monomorphic, h was not possible to correlate them with the semen quality traits. Loci AKRIBI and CDHl were found to be polymorphic but, statistical analysis revealed no significant association (P>0.05) of this loci with any semen quality parameters.
  • Thumbnail Image
    ThesisItemOpen Access
    WHOLE GENOME SEQUENCE CHARACTERIZATION OF PASTEURELLA MULTOCIDA ISOLATED FROM DIFFERENT ANIMAL SPECIES
    (AAU, Anand, 2015) AHIR, VIRAL B.; JHALA, M. K.
    Pasteurella multocida is a commensal microorganism of the upper respiratory track of many animal and avian species and is responsible for wide range of diseases in domestic animals and poultry. Despite vaccination of the dairy animals particularly against Haemorrhagic Septicaemia (HS), several outbreaks occur regularly in Gujarat as well as in other parts of India. Whole genome sequencing is a recent advanced approach for understanding of genetic makeup of an organism as well for identification of virulence genes/factors responsible for the disease process in host. In order to sequence whole genome of P. multocida and to elucidate virulence associated genes, five isolates of P. multocida were sequenced using pyrosequencing based approach of 454 GS FLX Titanium. All the five isolates viz. P52 vaccine strain (P52VAC), poultry (Anand l_poultry), goat (Anand l_goat), buffalo (Anand l_buffalo) and cattle (Anand 1 cattle) were identified and characterized based on biochemical and cultural characters and subsequently confirmed by PM-PCR. For sequencing of the whole genome of organisms, dsDNA libraries were prepared for all the five isolates and quantity as well as quality checks were done using Agilent Bioanalyzer as well as TBS flurometer. dsDNA library of each of the five isolates was amplified using emPCR and positive clonal amplified DNA beads were used for sequencing after annealing of sequencing primers. After completion of the sequencing run, data generated in form of images were converted into reads using GS Run Browser. After signal processing, total 118843, 113997, 105729, 134886 and 31346 reads were generated which yielded 42,598,100 (42.59Mb), 29,000,497 (29.00Mb), 21,890,353 (21.89Mb), 39,756,349 (39.75Mb) and 7,429,658 (7.42Mb) of sequence bases for P52VAC, Anandl_poultry, Anandlgoat, Anandl buffalo and Anandlcattle, respectively. Coverage obtained for P52VAC, Anandl_poultry, Anandlgoat, Anandl buffalo and Anandlcattle was 18.87, 12.85, 9.70, 17.61 and 3.29 respectively. All the reads after signal processing were mapped with the reference genome available for a poultry isolate Pm70 at NCBI using GS Reference mapper. Mapping of the isolates P52VAC, Anandl poultry, Anandlgoat, Anandl buffalo and Anandl_cattle resulted in 38,079,806 (89.52%), 20,085,356 (87.38%), 19,867,143 (90.81%), 25,095,466 (63.22%) and 6,145,156 (82.87%) mapped bases with 105327, 97674, 95092, 86765 and 24967 mapped reads. Remaining reads which were not mapped by GS Reference Mapper, were used for de novo assembly using GS De Novo Assembler for finding sequences which code for plasmid of P. multocida. None of the de novo assembled sequences matched to plasmid. For sequence analysis and finding of virulence associated genes in P. multocida, two different annotation pipelines were used viz. Rapid Annotation using Subsystem Technology (RAST) and Prokaryotic Genome Automatic Annotation Pipeline (PGAAP). For RAST analysis, all the contigs generated after reference mapping with Pm70 uploaded at RAST server. RAST is a subsystem based annotation pipeline which generated 2,273,366bp (2.27Mb), 2,227,943bp (2.22Mb), 2,285,382bp (2.28Mb), 2,045,610bp (2.04Mb) and 1,438,517bp (1.43Mb)of genome with 209, 489, 349, 2188, and 3152 contigs for P52VAC, Anandl_poultry, Anandlgoat, Anandl buffalo and Anandl cattle, respectively and 68, 54, 54, 40 and 0 RNA. Based on RAST analysis, highest abundance of subsystem were assigned to 'amino acids and derivatives', 'carbohydrates', 'protein metabolism' and 'cofactor and vitamins, prosthetic groups and pigments'. As expected, no subsystem was assigned to 'photosynthesis' and 'motility and chemotaxis' group as Pasteurella is a nonmotile organism and is not photosynthetic. Due to less coverage (3.29X) obtained for the Anandl_cattle isolate, it was omitted from the RAST based comparative analysis. Subsystem based genes/proteins assigned to the other four isolates under 'virulence, disease and defence' category ranged from 47 to 54 in number. There were presence of DedA, DedD and toxin under 'colicin and bacteriocin production' in P52 vaccine strain, poultry and goat isolates. Genes gyrA, gyrB, Pare and ParD under 'resistance to fluroquinolones' were present in all the four isolates. There was also presence of negative regulator of betalactamase expression, BLR gene leading to resistance expressed by this organism as well as multidrug resistance efflux pump cluster genes, MATE (Multidrug and toxin extrusion), MacA and MacB (Macrolide specific efflux protein) in P52 vaccine strain, poultry and goat isolates. For PGAAP analysis, all the reads generated after sequencing run were submitted to the PGAAP pipeline of NCBl after removing sequences less than 200bp. PGAAP analysis revealed genome size of 2,273,366bp (2.27Mb), 2,227,943bp (2.22Mb), 2,285,382bp (2.28Mb), 2,045,610bp (2.04Mb) and l,438,517bp (1.43Mb) with 40.40%, 40.20%, 40.50%, 40.90% and 41.00% of G+C contents for P52VAC, Anandl _poultry, Anandlgoat, Anandl _buffalo and Anandl _cattle, respectively. Total number of coding sequences (CDS) were 2066, 2337, 2319, 3258 and 3623; total number of protein encoding genes (PEG) were 2194, 2284, 2266, 3218 and 3590, and total number of RNA assigned were 64, 53, 53, 41 and 33 for P52VAC, Anandl_poultry, Anandlgoat, Anandl buffalo and Anandl cattle, respectively. Deciphering virulence mechanism is one of the most useflil application of bacterial genomics to understand the molecular intricacies involved in disease mechanism as well as'for understanding host-pathogen interactions. For this purpose, genes associated with virulence were downloaded from annotation files available at (http://www.ncbi.nlm.nih.gov/genome/genomes/912?) in 'Protien' column/section to find out gene locus/id. After manually searching for the virulence associated genes, 55 important genes were selected based on the available literature. These 55 genes grouped under seven broad categories viz. capsule, fimbriae and adhesion, iron metabolism, outer membrane protein, superoxide dismutase, sialic acid metabolism and transcription regulation. Out of these seven categories, all the five genes falling under three categories i.e. SodA and SodC under superoxide dismutase, NanH and NanB under sialic acid metabolism and Fis under transcription regulation category were present in all the five isolates. Nine genes involved in capsule production were found, out of which, PglA and Kmtl were present in all the five isolates, while HyaE was present only in the goat isolate. HexA and HexC genes were absent in buffalo and cattle isolates, while HexB and HexD were absent in goat and cattle isolates. KpsF gene was absent in poultry and cattle isolates. Gene LctP was present only in goat and cattle isolates. Sixteen genes were found under the category of fimbriae and adhesion, of which, Hsf, PfliBl, PfliR, PflB, PlpB, and Plp4 genes were found in all the five isolates of P. multocida studied. HofC gene was absent may in vaccine strain, wliereas PlpE gene was absent in cattle as well as buffalo isolates. ComE gene was absent in P52 vaccine strain, while TadE was absent in buffalo isolate. PfhBl and PlpP genes were absent in cattle isolate, while RcpA and RcpB were absent in buffalo isolate. ClpB gene was absent in P52 vaccine strain and cattle isolates, whereas TadF was absent in buffalo and cattle isolates. For iron metabolism, 16 genes were found, of which, ExbB, FbpB, HbpA, HgbA, HemU, OmpW, Rjh and RffG genes were present in all the five isolates studied. Genes FbpA and TonB were absent in cattle isolate, TbpA and TonB dependent lactoferrin and transferrin receptor were absent in goat isolate. Gene FbpC was absent in buffalo isolate. TonB-dependent receptor was present in poultry and goat isolates only. P52 vaccine strain was having a presence of translocation protein TolB, whereas HemR gene was absent. For outer membrane proteins, out of nine genes found, HasR, LppB, LspB, OmpH and PtfA genes were present in all the five isolates of P. multocida. Outer membrane protein LolB was found in P52 vaccine strain as well as in cattle isolate. VacJ gene was absent in goat and cattle isolates. Oma87 gene was absent only in poultry isolate. Gene PfhA was found present only in P52 vaccine strain. This study is apparently the first attempt in India involving local P. multocida isolates from four different species and a vaccine strain for the purpose of identifying virulence genes/virulence associated genes using modem biotechnological tools like pyrosequencing based whole genome sequencing. The study aids in data of whole genome sequencing of bacterial pathogens particularly for P. multocida and also provides new insight into their genomic characters and possible molecular mechanisms involved in disease process. The present findings would provide a much needed base for fijrther screening of virulence associated genes and identification of certain markers for early diagnosis as well as characterization of P. multocida, which continues to pose challenges as a menace against the health management of animals. Genes which have been found in all the P. multocida isolates under the study can be explored as specific probes for the early diagnosis of the disease. Further, future scientific endeavors targeting the vaccine design for P. multocida may get a scientific support from this data, so as to formulate modern and more effective vaccines, for better animal health.