Loading...
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
6 results
Search Results
ThesisItem Open Access MOLECULAR CHARACTERIZATION OF NEWCASTLE DISEASE VIRUSES PRESENT IN INDIAN CHICKENS AND DEVELOPING VECTOR FOR BIVALENT HVT-ND VACCINE(DEPARTMENT OF ANIMAL BIOTECHNOLOGY COLLEGE OF VETERINARY SCIENCE & ANIMAL HUSBANDRY ANAND AGRICULTURAL UNIVERSITY ANAND, 2017) Subhashchandra J. Jakhesara; Dr. C. G. JoshiNewcastle disease is a highly contagious viral disease continuously haunting poultry industry throughout the globe. Molecular epidemiology and nature of NDVs prevalent in recent outbreaks in India is poorly understood. The present study aimed to determine the nature of NDVs prevalent in vaccinated flocks of India by whole genome sequencing and biological pathotyping.ThesisItem Open Access REGULATION OF ACTIVIN TYPE II RECEPTOR B (ACVR2B) EXPRESSION THROUGH RNA INTERFERENCE IN GOAT MYOBLAST CELLS(AAU, Anand, 2014) PATEL, AMRUTLAL KALUBHAI; Joshi, Chaitanya G.Enhancement of skeletal muscle mass through genetic manipulation has drawn attention to increase the meat production in the farm animals. Among the various techniques of regulating gene expression, RNA interference (RNAi) has been proposed as a promising tool to suppress the target gene expression. Attempts have been made to increase the skeletal muscle mass in transgenic animals through knockdown of Myostatin, a gene with potential negative effect on muscle growth. It has been well established that myostatin mediates its action through binding to its cell surface receptor mainly to activin type II receptor B (ACVR2B). Besides regulating myostatin activity, ACVR2B has also been known to regulate the activity of other Transfonning Growth Factor beta (TGF-(3) superfamily ligands which negatively regulates muscle growth. The inhibition of ACVR2B signaling has shown dramatic increase in the muscle mass to a greater extent than myostatin inhibition. Hence, in the present study we aimed to investigate the possibility of ACVR2B knockdown to enhance the myogenesis in goat through various RNAi methods such as expression of short hairpin RNAs (shRNAs) under U6 and CMV promoters and expression of artificial microRNAs (amiRNAs) under CMV and muscle specific muscle creatine kinase (MCK) promoters. Further we studied effect of ACVR2B knockdown on the expression of myogenic regulators and assessed induction of undesired interferon response against RNAi vectors. Among the seven shRNAs tested, the U6 promoter driven shRNAs showed 63 (p= 0.0004), 76 (p= 0.0001), 75 (p=0.0000), 74 (p= 0.0005), 80 (p= 0.0001), 74 (p= 0.0000) and 57% (p= 0.0013) silencing with sh1 to 7, respectively in HEK293T cells whereas 24 (p= 0.1497), 24 (p= 0.2243), 15 (p= 0.3988), 31 (p= 0.1263), 14 (p= 0.4425), 46 (p= 0.0318), and 26 % (p= 0.1288) silencing of endogenous ACVR2B with shl to sh7, respectively and 53 (p- 0.0005), 32 (p= 0.0171), 38 (p= 0.0025), 66 (p= 0.0002) and 51% (p= 0.0008) with sh3, sh4, sh5, sh6 and sh7, respectively against ectopically expressed goat ACVR2B in goat myoblasts ceils. The knockdown of endogenous ACVR2B resulted in the 116, 105, 84, 64, 119, 102, 121. and 157% expression of MyoD; 131, 128, 128, 123, 104, 103, 69, and 157% of MyoG in sh1 to sh7 transfected cells, respectively. The transfection of U6 driven shRNA resulted in the induction of OAS1, a marker for innate interferon response by 3 to 1861 fold in 293T cells and up to 94 fold in the goat myoblasts cells. In an attempt to overcome the undesired cellular toxicity associated with U6 driven shRNAs as reported in the number of studies, we expressed these shRNAs under CMV promoter. The CMV driven shRNAs showed weak silencing of 37 (p= 0.1622) and 18% (p= 0.4877) by sh1 and sh3, respectively in HEK293T cells whereas 7% (p= 0.5749) by shl and 4% (p= 0.7493) by sh5 in goat myoblasts cells. Unlike suggested earlier, we observed significant induction of interferon response to CMV driven shRNAs up to 46 fold in 293T cells and 105.3 fold in goat myoblasts cells. Alternatively, we assessed another RNAi approach using amiRNAs which mimics the endogenous miRNA biogenesis pathway. Among the four amiRNAs tested by placing them in 5'-UTR region of GFP reporter, we observed 64 (p=0.0004), 77 (p=0.0002), 1 (p=0.8712), and 41% (p=0.0115) silencing in 293T cells by ami204, ami318, ami735 and ami878, respectively against exogenously expressed goat ACVR2B; 19 (p=0.3593) and 9% (p=0.4977) by ami204 and ami318, respectively against endogenous ACVR2B and 23% (p=0.0444) by ami318 against exogenously expressed ACVR2B in goat myoblasts cells. Since, amiRNAs placed in 5'-UTR were shown to affect the translation of reporter GFP, we further placed them in 3'-UTR of GFP which resulted in enhanced expression of GFP thereby enabling the monitoring of expression of amiRNAs. The 3'-UTR derived amiRNAs showed 50% (p=0.0002) silencing only by ami3I8 in 293T cells whereas 47 (p=0.0193), 16 (p=0.2959), 19 (p=0.1547), and 28% (p=0.0770) by ami204, ami318, ami735 and ami878, respectively against endogenous and 67%) (p=0.0004) by ami318 against overexpressed ACVR2B in goat myoblasts cells. The expression of myogenic regulators MyoD remained unchanged by amiRNAs cloned in the 5'-UTR whereas expression of MyoG was significantly up regulated by ami878 (p=0.0089). However, amiRNAs cloned in 3'-UTR showed significant down regulation of MyoD by 51 (p=0.0007), 27 (p=0.0232), 29 (p=0.0074), and 31% (p=0.0104) and MyoG by 36 (p=0.0034), 12 (p=0.2532), 22 (p=0.0303), and 37% (p=0.0026) by ami204, ami318, ami735 and ami878, respectively. As observed for U6 and CMV driven shRNAs, CMV driven amiRNAs showed significant induction of interferon response in 293T (up to 121.7 fold) and myoblasts (212.5 fold) cells. As ACVR2B has been shown to be essential for embryonic development, we tested the possibility of its knockdown in skeletal muscle using muscle specific MCK promoter. Among the MCK and MSTN promoters with and without two repeats of MCK enhancer, we observed maximum transcriptional activity by MCK promoter in goat myoblasts cells. We thus tested best amiRNAs (ami204 and ami318) by expressing under MCK promoter which showed 22% silencing efficacy by ami318 in 293T cells and 32% silencing efficacy in goat myoblasts cells by transient transfection assay. Further to test the possibility of ACVR2B knockdown after stable integration of amiRNAs into goat myoblasts genome, we generated lentivirus particles carrying amiRNAs expression cassettes and transduced the goat myoblasts. The myoblasts cells stably integrated with amiRNAs showed ~8% silencing by ami318 which was increased to 34% upon induction of differentiation under muscle specific promoter. Western blot analysis revealed 41% and 57% silencing by 5'-ami204 and 5'-ami318, respectively whereas 14% and 35% silencing by 3'-ami204 and 3'-ami318, respectively in stable myoblasts upon induction of differentiation. Unlike transient transfection assay vv'hich showed positive correlation of expression of ACVR2B with myogenic regulators, its stable knockdown resulted in up regulation of MyoD in 5'-UTR derived ami204 and ami318 and overall down regulation of MRFs in 3'-UTR derived ami204 and ami318 integrated goat myoblasts cells. The 5'- UTR derived ami204 and ami318 showed increased rate of cell proliferation as well as myoblasts fusion in stable goat myoblasts compared to scramble control indicating growth promoting effect of ACVR2B knockdown. The skeletal muscle specific partial knockdown of ACVR2B is unlikely to affect the embryonic survival and it will be interesting to further assess its possible growth promoting effect in adult animal by generating transgenic goat through somatic cell nuclear transfer of goat myoblast cells stably integrated with amiRNAs.ThesisItem Open 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.ThesisItem Open Access IN VITRO MYOSTATIN GENE SILENCING BY shRNA IN CHICKEN EMBRYO MYOBLAST CELLS(AAU, Anand, 2012) TRIPATHI, AJAI KUMAR; Joshi, Chaitanya G.Myostatin (MSTN), a member of transforming growth factor-P (TGF-P) superfamily, is a negative regulator of the skeletal muscle growth as it suppresses the proliferation and differentiation of myoblast cells. Scientists have reported that mice genetically engineered to lack myostatin activity have about twice the amount of muscle mass throughout the body. Dysfunction of MSTN either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the muscle mass in manmialian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN knockdown by developing short hairpin RNA (shRNA) construct against it. It is considered that in vitro knockdown of MSTN gene in chicken embryo myoblast by shRNA expressing constructs would help in devising suitable in vivo strategies for MSTN gene knockdown. This, in turn, might help to produce transgenic chicken with increased muscle mass. Apart from meat quantity, the quality of meat will also be improved as the knock down of myostatin gene will result in more lean type of meat which is advisable for the safe and healthy meat consumption. In the present study, shRNA induced myostatin gene silencing in in vitro chicken myoblast culture was evaluated using seven different shRNA expressing constructs by quantitative Real Time PCR. Myostatin silencing efficiency of shRNA constructs was first evaluated in Human embryonic kidney cell-line 293T (HEK 293T) cells, which are frequently used for its extreme transfectability by the various techniques and exogenously express target proteins. Seven antimyostatin constructs were used in this study, of which six were designed from online tool using GU075928 sequence as input, and one siRNA was selected form literature. The antisense strands were checked for the presence of any secondary structure. Out of seven; one construct formed secondary structures in its antisense strand, whereas no secondary structures were found in rest six constructs. These constructs were cloned into pSIREN vector between EcoRI and BamHl restriction sites. Sequencing results of shRNA constructs revealed no mutation in any of the shRNA constructs. Full length chicken myostatin gene was amplified from total RNA extracted from 11-12 days WLH chicken embryo, and cloned in to pcDNA3 expression vector and synthesized pcDNA3_cMSTN constructs. This expression vector was cotransfected with each shRNA into HEK293T cells. HEK 293T cells were seeded 2.5 x 10 power of 5 cells per well in a 12-well plate, and transfections were performed in triplicates. Transfection efficiency of the combinations did not vary much (80.05±0.73%). Total RNA was extracted from transfected cells after 36 h of transfection. First strand cDNA was synthesied and qPCR was performed using SYBR Green master mix, gene specific primers for GAPDH (endogenous control) and MSTN, OASl, IFNp and PKR (targets); and cDNA as template. Amplified PCR products were electrophoresed on 2% agarose, which revealed single compact band of 257 bp in GAPDH, 245 bp in MSTN, 209 bp in OASl, 159 bp in IFN-p and 165 bp in PKR. These shRNA constructs were having efficient myostatin silencing effect, ranging from 37.5% to 95.6%. The induction of interferon genes {OASl, IFN J3 and PKR) expression was significant (1.1 to 23.5 folds, pThesisItem Open Access STUDY OF SHORT HAIRPIN RNA INDUCED MYOSTATIN GENE SILENCING IN IN-VITRO CAPRINE MYOBLAST CULTURE(AAU, 2012) RAMANI, UMED V; DR. C. G. JOSHIThesisItem Open Access “STUDY OF SHORT HAIRPIN RNA INDUCED MYOSTATIN GENE SILENCING IN IN-VITRO CAPRINE MYOBLAST CULTURE”(AAU, 2012) RAMANI, UMED V; DR. C. G. JOSHI