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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.
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ThesisItem Open Access TRANSCRIPTOME PROFILING TO EVALUATE EFFECT OF HERBAL PLANT EXTRACT ON BULL SPERMATOZOA(Department of Animal Biotechnology College of Veterinary Science and Animal Husbandry Anand Agricultural University, Anand, 2019) Jignesh M. Italiya; Prakash G. KoringaThe mammalian ejaculated spermatozoa are highly differentiated terminal cells with an extremely compacted nucleus of haploid genome. The mammalian spermatozoon transfers coding as well as non-coding transcripts to the oocyte during fertilization. Spermatozoal transcript composition and expression levels are associated with spermatogenesis, functional parameters of spermatozoa, early embryonic development and pregnancy outcomes. Putranjiva roxburghii wall which is known as child’s amulet tree or child-life tree. Bryonia laciniosa is an herb, which has been included in Vrishya rasayana category in Ayurveda texts. It is also used traditionally as an aphrodisiac and pro-fertility compound. Hence this ethno-herb has immense potential of research in the field of infertility of either sex. On the basis of hypothesis that, any environmental disturbance in form of given treatment of herbal seed extract to spermatozoa that makes change in transcriptome profile, the current study is planned to establish the fact about effect of herbal preparations on sperm mortality, viability and RNA profiling.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 TOLL LIKE RECEPTORS (TLR) EXPRESSION IN MILK SOMATIC CELLS DURING MASTITIS IN CATTLE USING REAL-TIME PCR(AAU, Anand, 2011) GHADIGAONKAR, DINESH DILIP; Rank, D. N.The dairy sector in India has shown remarkable progress in the recent years and consequently the country has now become the largest milk producer and valueadded milk products in the world. Though the country is the largest producer of milk, milk production per animal is much less compared to agriculturally developed countries. One of the reasons for this less milk production per animal is loss of milk production capacity because of inflammation of the mammary gland or mastitis in dairy animals. Mastitis is defined as an inflammatory reaction of the parenchyma of the mammary glands to bacterial, chemical, thermal or mechanical injury which is characterized by a range of physical, chemical and usually bacteriological changes in the milk and pathological changes in the glandular tissue which is the most common and the most expensive disease of dairy cattle throughout the world. Mastitis is classified as subclinical and clinical mastitis. The cows that have subclinical mastitis are reservoirs of organisms that lead to infection to other cows. Most clinical cases start as subclinical; thus, controlling subclinical mastitis is the best way to reduce the clinical cases. Somatic cells consist of leukocytes and epithelial cells exfoliated from the mammary epithelium. Mastitis is associated with an influx of inflammatory cells; hence somatic cell count of milk increases. Enumeration of somatic cell counts and bacterial culture of milk has been suggested as a standard method for detecting subclinical udder infections in dairy cows. Genes have major impact on health status of animals. Genetic variability of mastitis resistance is well established in dairy cattle. Resistance to mastitis is a complex function involving various biological pathways, molecules and cells. Study of the expression of genes involved in mastitis resistance is major tool for early diagnosis of disease and genetic improvement to superior stock. Toll Like Receptors (TLR) are cell-surface receptors that recognizes a broad class of pathogen-associated molecular patterns, activates immune system, and induces the over expression of inflammatory factors, which participate in irmate immune responses to confer disease resistance. The bovine TLR genes have been studied in recent years. Hence the study was planned with the objective to investigate expression of TLR-2, TLR-4, TLR-9 in somatic cells in healthy and mastitic udder by Real Time PCR in cattle. The study was undertaken to assess different TLRs (TLR-2, TLR-4 and TLR- 9) in three cattle breeds namely Gir, Kankrej and Triple crossbred (Kankrej x Jersey x Holstein Friesian) with sub-clinical mastitis. A total of 65 lactating cows comprising 16 Gir, 29 Kankrej and 20 Triple crossbred animals were screened for presence of mastitis using Electronic Somatic Cell Counter and bacteriological culture examination. Total RNA was extracted from milk somatic cells from 15 positive and 6 healthy quarters from each breed using TRIZOL method. The RNA was treated with DNase enzyme to remove any traces of genomic DNA. cDNA was synthesized from RNA using Qiagen's Omniscript reverse transcriptase kit and random hexamer primers. The amplification of cDNA template of TLR 2, TLR 4 and TLR 9 genes was carried out using gene specific primers. Expression of TLR 2, TLR 4 and TLR 9 genes mRNA was quantified by Real Time PCR and analysed using Applied Biosystems 7500 SDS software. Relative expression study of these genes was carried out using GAPDH as internal control. Results indicated that there was upregulation of TLR 2, TLR4 and TLR9 gene expression in animals affected with subclinical mastitis compared to healthy animals. Targeted amplification of 421 bp TLR 2, 108 bp TLR 4 and 108 bp TLR 9 was confirmed by agarose gel electrophoresis. Prevalence of subclinical IMI was higher in Triple crosbred cows (65%) compared to Gir (50%) and Kankrej cows (27.59 %). The mean SCC of infected quarters was significantly higher than that of noninfected quarters (P < 0.05) in all three breeds. The average relative expression of all three genes i.e. TLR2, TLR4 and TLR9 was higher (ranged from 7 to 35 folds) in mammary gland with subclinical intramammary infection than those measured in the uninfected glands. The concomitant increase in somatic cell count and upregulation of TLR2, TLR4 and TLR9 gene expression was observed during subclinical mastitis in all three breeds. Comparison of SCC upregulation between breeds indicated that, there was no significant difference between breeds in the SCC in the diseased quarter during subclnical mastitis in Gir, Kankrej and triple crossbred cows. In Gir cows, TLR2 gene expressions level in diseased quarters was found to be upregulated with an average 10.54 (10.54 ± 7.12) fold compared to pooled healthy quarters. In Kankrej cows, TLR 2 gene expressions level in diseased quarters was found to be upregulated with an average 12.22 (12.22 ± 11.61) folds compared to pooled healthy quarters. In Triple crossbred cows, TLR 2 gene expression level in diseased quarters was found to be upregulated with an average 7.13 (7.13 ± 10.57) folds compared to pooled healthy quarters. In Gir cows, TLR 4 gene expressions level in all diseased quarters was found to be upregulated with an average 18.43 (18.43 ± 24.230) fold upregulation compared to pooled healthy quarters. In Kankrej animals, TLR 4 gene expressions level in diseased quarters was found to be upregulated with an average 31.59 (31.59 ± 18.74) folds compared to pooled healthy quarters. In Triple crossbred cows, TLR 4 gene expression level in diseased quarters was found to be upregulated with an average 23.817 (23.817 ± 27.6963) fold compared to pooled healthy quarters. In Gir cows, TLR 9 gene expression level in diseased quarters was found to be upregulated with an average 6.193 (6.193 ± 8.19) fold compared to healthy quarters. In Kankrej cows, TLR 9 gene expression level in diseased quarters was found to be upregulated with an average 5.44 (5.44 ± 8.14) folds compared to Pooled healthy quarters. In Triple crossbred cows, in all diseased quarters, TLR 9 gene expression level was found to be upregulated with an average 19.29 (19.29 ± 16.31) fold compared to pooled healthy quarters. During subclinical mastitis SCC was found to be positively correlated with the transcriptional activities of TLR2, TLR4 and TLR9 gene in Gir and Triple crossbred cow. In Kankrej cows TLR 2 and TLR 9 gene expressions were positively correlated with s e c but TLR 4 gene expression was not correlated with SCC. The level of infection as reflected by number of somatic cells had significant effect on level of upregulation in gene expression. However, there was no significant effect of a breed on level of upregulation of TLR 2, TLR 4 and TLR 9 gene expression.ThesisItem Open Access Evaluation of Gender Pre-selection through Sperm Enrichment Techniques Using Real-Time PCR in Bovines(AAU, Anand, 2013) ROY, LATISH CHANDRA; Panchal, M. T.The present work was undertaken to assess the efficiency of the classical X and Y sperm enrichment methods, viz., sperm swim-up, gravity sedimentation, Sephadex filtration, and discontinuous Percoll density gradient centrifiigation, using the modem molecular technique of Real-Time PCR or qPCR in buffalo and cow bulls, in the Department of Animal Biotechnology and Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science & AH, Anand Agricultural University, Anand. Total of 12 semen ejaculates six each from buffalo and cow bull, diluted at the rate of 1:10 with Tris-fructose-yolk- glycerol (TFYG), were collected from the breeding bulls stationed at Semen Station, Amul Research and Development Association (ARDA), Ode, Anand and subjected to the different enrichment techniques. In sperm swim-up four time bound fractions were collected using a special swim-up tube. Similarly with gravity sedimentation, three fractions, with Sephadex filtration four fractions and with discontinuous Percoll density gradient centrifugation the bottom sperm pellet was collected. DNA was extracted from all the collected fractions and the control semen samples and was further used for Real-Time PCR. Four different neat semen dilutions were taken as standards for the Real-Time PCR, viz., 100.00, 75.00, 50.00 and 25.00 per cent, consisting of 50.00, 37.50, 25.00 and 12.50 per cent X-chromosome bearing sperms,' respectively, considering the theoretical ratio of 1:1 for X and Y sperms in an ejaculate. With gravity sedimentation, the X-chromosome bearing sperm percentage in the three collected fractions of semen samples, ranged from 19.96 to 43.99 (31.90±4.44), 41.10 to 46.93 (45.01±1.32) and 38.90 to 48.07 (43.67±1.40) per cent, respectively and 21.09 to 51.32 (35.22±4.00), 35.46 to 51.32 (43.26±2.19) and 34.86 to 42.31 (39.19±1.14), respectively, in buffalo and cow bulls with an overall mean Xchromosome bearing sperm percentage of 40.19±2.08 and 39.22±1.67 in buffalo and cow bull semen, respectively. In the four semen fractions obtained post-filtration through Sephadex (G-lOO) gel, X-chromosome bearing sperm percentage values ranged from 42.51 to 52.08 (46.08±1.42), 42.75 to 52.51 (47.69±1.68), 41.43 to 50.20 (45.96±1.22) and 42.36 to 49.17 per cent (44.80±0.98) as compared to control semen samples having the range of 46.76 to 50.20 per cent (48.48±0.4) in buffalo bull and 27.82 to 48.00 (41.99±3.00), 33.02 to 52.59 (43.75±2.67), 35.09 to 52.79 (43.48±2.35) and 33.32 to 50.15 (42.50±2.20) per cent, compared to the control with the range of 45.94 to 50.11 (47.67±0.76) in the cow bull semen samples. The overall mean was obtained to be 46.13±0.67 and 42.93±1.21 per cent, in buffalo and cow bulls, respectively. The X-chromosome bearing sperm percentage in the three swim-up fractions retrieved ranged from 42.00 to 59.57 (48.62±2.60), 42.16 to 50.61 (45.91±1.29) and 30.13 to 51.93 (44.09±3.15) per cent, respectively, and 33.60 to 45.84 (42.30±1.80), 34.61 to 47.28 (42.86±1.84) and 31.23 to 62.58 (44.07±4.17) per cent, respectively, with an overall values of 46.21± 1.41 and 43.08±1.54, in buffalo and cow bulls, respectively. For control buffalo and cow bull semen samples the X-chromosome bearing sperm values ranged from 46.76 to 50.20 (48.48±0.48) and 45.94 to 50.11 (47.67±0.76) per cent, respectively. With discontinuous Percoll gradient centrifugation, the X-chromosome bearing sperm percentage obtained in the bottom pellet ranged from 43.79 to 51.83 (48.93±1.93) percent against the control value of 45.07 per cent in buffalo bulls and 48.20 to 56.89 (52.42±1.23) per cent, compared to the 50.56 per cent of the control, in cow bull semen samples. No detrimental effect was observed on individual motility of the sperms following any of the sperm enrichment procedures. None of the four methods evaluated proved efficient enough in altering the sex ratio of the sperms. No significant differences in X-chromosome bearing sperms were observed in any of the methods as compared to control, except in gravity sedimentation, where, a highly significant difference was found between the different fractions, both in buffalo and cow bulls.ThesisItem Open Access METHYLATION DETECTION IN H19 GENE IN BUFFALO [Bubalus bubalis) BY BISULFITE SEQUENCING(AAU, Anand, 2011) PATEL, HIREN MAVJIBHAI; Joshi, Chaitanya G.It is increasingly clear that translation of the genetic code into proteins is not the only way that our genes influence our growth, development and health. Another system of influence on the expression of genes is referred to as epigenetics. Any biochemical modification of the DNA or chromatin that can account for epigenetics gene expression system, only one candidate mechanism that is site-specific DNA methylation, has received experimental support to date. Methylation of DNA in mammalian cells occurs at cytosine residues in CpG dinucleotides. Improper or absence of DNA methylation of imprinted gene may lead to abnormal growth or cancerous condition. The best candidate for the epigenetic mark at the H19/Igf-2/Ins-2 locus is paternal-specific DNA methylation of the H19 and its 5' flank. Methylation status of H19 influences the expression of IGF-2. Keeping in consideration that methylation status ofH]9 influences the IGF2 expression, present study was carried out to detect methylation status in H19 promoter in Jaffarabadi, Surti and Mehsani buffalo by bisulfite sequencing. DNA extraction from blood was carried out by phenolxhloroform method from seven sample of each breed which were subjected to bisulfite treatment by sodium bisulfite. Quality and quantity of DNA was checked by ND-1000 spectrophotometer and 0.8% agarose gel electrophoresis. Locus specific primers for normal DNA and for bisulfite treated DNA were used to amplify a gene fragment of H19 in three breeds of Bubalus bubalis. Purification of PCR product was carried out by eluting desired PCR product from agarose gel by using by QIAquick® Gel Extraction Kit and later the PCR products were ligated in pTZ57R/T vector of InsT/Aclone TM kit (Fermentas). Ligated recombinant vector was transformed in competent E. coli (DH5-α) cells. Recombinant colonies were identified by blue-white screening and white conies were subjected to Ml3 colony PCR for confirmation of positive recombinant clones. White colony containing recombinant clone was grown in LB broth and recombinant plasmids were isolated using QIAprep® Spin Miniprep Kit and used for cycle sequencing, that were later processed for sequencing. Multiple sequence alignment was done for all 21 samples using BioEdit v 7.0.7.1, which revealed no difference between all samples. Four clones from each bisulfite treated samples were sequenced and subjected to methylation study by BiQ Analyzer and BISMA softwares. In H19, 20 CpGs were revealed in all three breeds and were subjected to methylation study. In all three breeds, highest methylation per cent was found to be 94.12, while in some sample it showed no methylation at any CpGs site. The overall methylation per cent of HI9 gene in Jaffarabadi, Surti and Mehsani buffaloes were observed to be 50.19, 70.85 and 52.24, respectively. The incidence of mean per cent methylation in H19 gene in all three breeds was found to be 57.36. Unexpected nucleotide conversion (T>C, A>G and G>A) and deletion (A and G) after bisulfite sequencing were observed. Methylation of non CpG cytosine was observed which were found at CpA and, only in one animal at CpT. In some case, three CpG out of twenty were not analyzed due to non specific conversion of CG>TA. For estimation of association of milk production traits with methylation pattern, animals were grouped into high and low group with respect to milk yield and milk fat percentage. Statistical analysis indicated that there was no significant difference in high and low group with respect to milk yield and milk fat per cent among methylation pattern in all three breeds. Screenings of more number of animals are needed to get reliable data for estimation of association of milk production traits with methylation pattern.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 METHYLATION DETECTION IN H19 GENE IN BUFFALO (BUBALUS BUBALIS) BY BISULFITE SEQUENCING(Anand Agricultural University, Anand, 2011) HIREN MAVJIBHAI PATEL; Dr. C. G. JoshiIt is increasingly clear that translation of the genetic code into proteins is not the only way that our genes influence our growth, development and health. Another system of influence on the expression of genes is referred to as epigenetics. Any biochemical modification of the DNA or chromatin that can account for epigenetics gene expression system, only one candidate mechanism that is site-specific DNA methylation, has received experimental support to date. Methylation of DNA in mammalian cells occurs at cytosine residues in CpG dinucleotides. Improper or absence of DNA methylation of imprinted gene may lead to abnormal growth or cancerous condition.
