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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 ISOLATION, IDENTIFICATION AND S1 GENE SEQUENCING OF NEPHROPATHOGENIC INFECTIOUS BRONCHITIS VIRUS(AAU, Anand, 2014) PATEL, BHARATKUMAR HIRALAL; JHALA, M. K.Infectious bronchitis (IB) is a highly contagious acute viral disease of chicken characterized by respiratory signs in growing chickens. IB is a worldwide respiratory disease of major economic importance associated with losses from production inefficiencies and mortality. Some strains of the virus are nephropathogenic and produce interstitial nephritis and mortality. IB is caused by the virus belonging to the Coronaviridae family, Coronavirus genus with more than 26 serotypes. Present work was aimed to carry out isolation, identification, molecular detection and characterization of SI geneof ncpliropathogenicIBV. Fifty tissues samples of kidneys, lungs, trachea and cloaca were collected from dead birds at the Poultry Complex, AAU, Anand suspected for IBV infection during postmortem examination. Tissues from 10 birds were pooled and processed as single sample, and 5 such pooled samples were processed for isolation of IBV in SPF egg embryo. Five tissue homogenates, vaccine sample and PBS (negative control) were passaged thrice in Specific Pathogenic Free (SPF) embo^onated eggs of 9-11 days incubation. Isolates and vaccine sample produced typical lesions of dwarfing and curling in embryos suggestive of presence of IBV in the samples. RNA isolation was done from the allantoic fluids collected after third passage by using QIAamp viral RNA mini kit. RT-PCR was carried out from the infected allantoic fluids of 5 pooled samples, vaccine sample and negative control by using primers XCE2(F) and XCE2(R), targeting SI gene of IBV. All the five samples and the vaccine sample produced the expected amplicons of approximately 466 bp by RT-PCR, where as no amplification was observed in negative control. Direct sequencing of the products generated by RT-PCR was carried out by Sanger sequence method. The obtained sequences were aUgned with each other by using SeqScapc v 5.2.0 sequence analysis software. Sequences were compiled and analyzed using BioEdit software. Deduced amino acid sequences of IBV SI gene were obtained using ExPASy proteomics tools. Length of the nucleotide sequences for ANDGUJIBVl, ANDGUnBV2, ANDGUJIBV3, ANDGUnBV4, ANDGUHBVS, and ANDGUJmVvac were 444 bp, 440 bp, 447 bp, 439 bp, 441 bp, 442 bp and length of the amino acid sequences were 148, 146, 148, 146, 146 and 147 amino acids respectively. The GenBank accession numbers of the nucleotide sequences of Anand isolates obtained were KJ577258 (ANDGUJIBVl); KJ577259 (ANDGUJIBV2); KJ577260 (ANDGUJIBV3); KJ577261 (ANDGU.TIBV4); K.T577262 (ANDGUJIBV5) andKJ577263 (ANDGUnBVvac) obtained. The sequencing of five field samples (Anand isolates) revealed that all the five sequences were 99.09-100% similar among themselves, and 99.10-100% similar with the vaccine sample. The consensus sequence (length 437 bp) was obtained. The multiple sequence alignment of Anand isolates and vaccine sample was done with the three reference vaccine strains (Ma5, HI20 and Mass 41). The nucleotide sequences of Anand isolates were found to have >97% identity with M41,11120, Mass 41 and Ma5 strains of Massachusetts serotype of IB virus. The three reference vaccine strains i.e. Ma5, H120 and Mass41 showed SNVs at positions 708, 1143 and 1146 with ' C ,'T' and 'A' respectively, while the Anand isolates including the vaccine showed 'T', ' C and G at the same positions. Mass41 showed additional 7 SNVs at positions 737, 846, 861, 1017, 1104, 1119 and 1136 with T, T, T, C, T, T, and C nt respectively, while all other strains showed C, A, C, T, C, C and T at the respective positions. Multiple amino acid sequence aligimient revealed that all the strains under comparison had similar amino acid sequences except the reference vaccine virus strain Mass41, which showed I (isoleucine) and S (serine) at positions 246 and 379, while all the other strains including Anand isolates showed T (threonine) and L (leucine) at the same positions. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 6. Nucleotide sequences of 16 IBV isolates/strains with different pathogenicity were retrieved from the Genbank database and were aligned for sequence analysis and phylogenetic study using the Clustal W programme. The Phylogenetic analysis of nucleotide and aminoacid sequences of selected 16 IBV isolates/strains revealed that the Anand IBV isolates were less similar to Arkansas and Cormecticut strains of USA. The Anand isolates were found to have similarity with nephropathogenic strains 4-91 pathogenic (UK), Australia T (Australia) and CK/CH/Chongqing/0909 (China) with average identity of >78%, >85% and >78% respectively. In addition, the Anand isolates also showed maximum similarity (>99%) with the mass type strain M41 reported from Andra Pradesh, India. This indicates that the Anand isolate are of nephropathogenic Massachusetts serotype. The Anand isolates showed similarities in the range of 77.59% to 78.08% with earlier reported isolate of Gujarat and also lower similarities with isolates from Maharashtra, Orissa, Punjab and Assam. The sequence of Gujarat IBV isolate was reported in the year 2009 and the lower similarity as observed in the current study indicates further genetic changes in the circulating IBV in Gujarat. Phylogenetic analysis revealed that all the Anand IBV isolates were very similar and probably had a common origin. The results of present study revealed that different strain variants of IBV may be circulating among chicken populations in India and recent outbreaks of nephropathogenic IB were due to this reason. Continuous genotyping and phylogenetic analysis of IBV from field outbreaks would be required so as to update the IB vaccines for better protection of chicken populationsThesisItem Open Access ISOLATION, IDENTIFICATION AND PATHOTYPING OF NEWCASTLE DISEASE VIRUS FROM POULTRY(AAU, Anand, 2014) PATEL, HARDIK S.; Jhala, M. K.Newcastle disease (ND) is one of the most important infectious diseases of poultry and has the potential to cause large economic losses in the poultry industry. Several outbreaks of ND recorded in and around Anand area of Gujarat despite routine vaccination programs demanded a scientific investigation. Hence, the present research work was carried out to isolate, identify and pathotype Newcastle disease virus (NDV) by using Haemagglutination (HA), Haemagglutination Inhibition (HI) test. Mean Death Time (MDT), Intra Cerebral Pathogenicity Index (ICPI) and Reverse Transcription- Polymerase Chain Reaction (RT-PCR) assay. Total of 38 samples from 11 poultry farms in and around Anand city and during the post-mortem examination at the Department of Veterinary Pathology, College of Veterinary Science & Animal Husbandry, AAU, Anand were collected. Tissues (trachea, lung, liver, spleen, proventriculus, caecal tonsils and intestine) from each bird were pooled and processed as single sample. From 38 pooled tissue homogenates, 18 tissue suspensions representative of 11 poultry farms of different areas in and arovind Anand, along with NDV F vaccine sample were inoculated in 200µl volumes into the allantoic cavity of embryonated SPF eggs of 9-11 days incubation. Eggs were fiirther incubated until death or for upto 72 hrs and allantoic fluid was collected after overnight chilling at 4°C and used for further biological as well as molecular characterization. All the 18 allantoic fluids from field samples along with F vaccine sample were screened for HA and HI activity using poultry RBCs as per standard method of OIE Terrestrial Manual 2012. Out of 18 field samples, 17 samples were found positive for HA activity, which was confirmed by HI using known NDV serum. The MDT of 6 representative samples was between 60.8 to 66.2 hrs indicating the mesogenic nature of the field strains. While, ICPI values of 2 for all the field samples were indicative of velogenic nature of the field NDV strains. As per OIE, ICPI is more reliable than MDT hence, we considered the samples as velogenic which was later also confirmed by RT-PCR. RT-PCR was used to amplify the F gene segment of all the isolates. Production of a 362 bp fragment specific for NDV in 15 out of 17 isolates using general primer pair NDV A+B confirmed the ND virus isolation. Amplification of a 254 bp product with both NDV A+C and A+D pans of primers specific for virulent strains in 14 samples indicated the high virulence of these isolates, while only one sample did not show amplification in A+C combination indicating non-virulent nature of the virus. Based on ICPI and RT-PCR, the NDV isolates could be placed in velogenic group. The study indicated prevalence of virulent NDV circulating in and around Anand area. Further study involving F gene sequencing and phylogenetic analysis is required to assess the genetic variation in the field NDV.ThesisItem Open Access F GENE SEQUENCING AND ANALYSIS OF NEWCASTLE DISEASE VIRUS CIRCULATING IN THE FIELD(AAU, Anand, 2014) RABARI, PANNABEN DHARAMSHIBHAI; JHALA, M. K.Newcastle disease (ND) is one of the most important infectious diseases of poultry. It is distributed world wide and has the potential to cause large economic losses in the poultry industry. ND is caused by Newcastle disease virus (NDV) which is a member of the family Paramyxoviridae, genus Avulavirus and is designated Avian paramyxovirus-I (APMV-1). The primary molecular determinant for tiie NDV pathogenicity is the fusion protein cleavage site amino acid sequences. Incidences of ND in vaccinated chicken population of Anand area of Gujarat demanded a study for pathotyping and genetic characterization of the field NDV isolates. Ten allantoic fluid samples from the SPF eggs inoculated with NDV suspected tissue suspension and preserved at the Department of veterinary Microbiology were used for the RNA isolation by QIAamp viral RNA mini kit, followed by one step RT-PCR using primers A and B to amplify F gene segments of NDV. All the ten isolates yielded expected 362bp product of F gene indicating them positive for NDV. Sequencing was done by ABI PRISM automated DNA sequencer. Length of the nucleotide sequences for NDVGUJl, NDVGUJ2, NDVGUJ3, NDVGUJ4, NDVGUJ5, NDVGUJ6, NDVGUJ7, NDVGUJ8, NDVGUJ9 and NDVGUJIO were 352nt, 352nt, 363nt, 352nt, 352nt, 353nt, 352nt, 352nt, 355nt and 354nt. The deduced amino acid sequences of F gene were obtained using ExPASy proteomics. Length of amino acid sequences were 115aa, 115aa, 119aa, 115aa, 115aa, 116aa, 115aa, 115aa, 116aaand 116aa respectively. The sequence were submitted to Genbank and the obtained Accession numbers are KJ754123, KJ754124, KJ754125, KJ754126, KJ754127, KJ754128, KJ754129, KJ754130, KJ754131 and KJ754132 respectively. The nucleotide sequences and deduced amino acid sequences of F gene of ten Anand isolates were aligned with the sequences of one Indian reference sequence Bareilly (Accession No. KF727980.1) and two reference vaccine strains viz. R2B and LaSota (Accession nos. JX316216.1 and JF950510.1 respectively) using ClustalW programme. Multiple sequence alignment of Anand isolates with reference Indian Bareilly strain and two vaccine strains R2B and LaSota revealed 7 unique mutations in Anand isolates viz. G222A, T231C, T300G, T420C, A426T, C432T and A438G. The amino acid sequences of all the ten Anand isolates showed RRQKRF between 112 and 117 positions indicating them to be of virulent type. All ten Anand isolates showed two unique amino acid changes i.e. I and E respectively at positions 69 and 104 with reference Bareilly sequence which showed M and G at these positions. Anand isolates differed with both the reference vaccine strains (R2B and LaSota) at six positions viz. 69, 82, 107, 121, 124 and 145 by showing I, E, S, A, S and N in place of L, D, T, I, G and K. The phylogenetic analysis based on F gene sequences of ten Anand isolates and 18 different NDV strains/isolates from different parts of the world available in GenBank was conducted using MEGA version 6. The analysis grouped all these 28 sequences into five different clusters. Cluster I included our ten isolates and strains Chicken/Sweden/97, Stema/Astr/2755/2001, and GDI003/2010. Our isolates showed maximum genetic similarity 94.32% with genotype XIII strain (Chicken/Sweden/97) indicating our isolates to be of genotype XIII. Our ten isolates showed least similarity 75.57% with Bl vaccine strain. The study thus emphasizes that the circulating NDV in the field are continuously evolving at genetic level and the variants are continuously emerging furthering the genetic distance with the other isolates as well the vaccine strains. This explains the vaccination failures observed in field condition and demands comprehensive studies to genotype large nimiber of NDV isolates so as to update the NDV vaccines.