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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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2012 - 201410
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Subject: Veterinary Microbiology × End date: 2014 × Start date: 2012 × Type: Thesis ×
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    ThesisItemOpen 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 populations
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    ThesisItemOpen 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.
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    ThesisItemOpen Access
    ISOLATION, IDENTIFICATION, MEMBRANE PROTEIN PROFILING AND MOLECULAR CHARACTERIZATION OF MYCOPLASMA AGALACTIAE ISOLATES FROM GOATS OF GUJARAT STATE
    (AAU, Anand, 2012) KUMAR, PRANAY; ROY, ASHISH
    The present study was undertaken to isolate and identify Mycoplasma agalactiae (genus Mycoplasma), a major cause of contagious agalactiae in goats, from different types of clinical samples by conventional methods and 16S rRNA based PCR and to characterize them by biochemical and molecular techniques like restriction enzyme (RE) analysis. Characterization of M. agalactiae isolates by membrane protein profiling by SDS-PAGE was also undertaken. Characterization of membrane protein coding genes viz. P30 and P48 was also done by PCR-RE analysis and sequencing. Comparative analysis of 16S-23S rRNA ITS region of different Mycoplasma spp. was also done to establish their importance in phylogenetic evolutionary studies. The thirteen isolates obtained from 748 samples were characterized biochemically as M. agalactiae. They were found sensitive to digitonin and negative to glucose fermentation, arginine hydrolysis and serum digestion whereas found positive for tetrazolium reduction, phosphatase activity and film and spot foimation. All the 13 isolates carried multiple drug resistance against Ampicillin, Erythromycin and Streptomycin. Four isolates (MAGE3, MAGT2, MAGT3, MAGM7) were additionally carrying resistance to Chloramphenicol, while one isolate (MAGT2) was resistant to Ampicillin, Erythromycin, Streptomycin, Chloramphenicol and Gentamicin and one isolate (MAGTl) was resistant to Ampicillin, Erythromycin, Streptomycin and Gentamicin. Membrane protein profile of four representative isolates (MAGEl, MAGTl, MAGMl and MAGM2) of the M. agalactiae was studied by SDS-PAGE of whole cell lysate and more than 20 bands of proteins of different intensities were visualized after the SDS-PAGE analysis. The separated bands revealed presence of protein bands of different molecular weights varying from 10 kDa to around 110 IcDa. The protein bands of 30, 40, 48 and 80 kDa were also clearly visualized after resolving on electrophoresis. All the thirteen isolates were screened by PCR using 16S rRNA based genus specific primers (Gpo-1 and MGSO) and species-specific primers (MagaF and MagaR) yielding 715 bp and 360 bp product respectively in case of M agalactiae. Four representative isolates of M. agalactiae (MAGEl, MAGTl, MAGMl and MAGM2) were subjected to RE analysis. Restriction enzyme analysis of the 360 bp long amplicon of 16S rRNA of M agalactiae with AluI yielded two products (121 bp and 239 bp) revealing a single cutting site. On RE analysis, the amplicon (1329 bp) of P48 gene yielded 2 products (683 bp and 646 bp) with TaqI and 3 fragments (1224 bp, 48 bp and 57 bp) with Sau3AI in accordance with the expected restriction map showing the presence of single and double cutting sites, respectively. On RE analysis, 730 bp amplicon of P30 gene yielded 2 fragments with RsaI (654 bp and 74 bp) and 3 fragments (111 bp, 375 bp and 244 bp) with MbolI in accordance with the expected restriction map. After digestion, Sau3AI yielded two fragments (461 bp and 269 bp) which were in contrast to the three expected products (461 bp, 105 bp and 164 bp) showing the presence of only one cutting site in contrast to the expected two sites. With AluI, 3 fragments (342 bp, 328 bp and 60 bp) were obtained revealing the presence of only two cutting sites in contrast to the expected three. Restriction patterns of P30 gene were found almost similar in all the representative samples. The purified PCR products of membrane protein genes P48 and P30 of one representative isolate (MAGMl) were subjected to sequencing on ABI-PRISM automated DNA sequencer and raw data collected by data collection software were analyzed and curated on Sequence Analyser and SeqScape softwares. The consensus sequences were subjected to BLAST analysis, multiple sequence alignment by ClustalW with published sequences and phylogenetic analysis by Neighbour joining method by MEGA4 and BioEdit softwares. Nucleotide sequence alignment of P30 gene sequence with the published sequences showed homology varying from 92% to 97%. In the P30 gene sequence (MAGMl), 12 unique positions were found at position 57, 72, 80, 81, 90, 117, 139, 153, 270, 391, 432 and 448. Amino acid alignment of deduced amino acid sequence of P30 gene with eight other published sequences showed the homology ranging from 92% to 94%. Unique positions were found at 20, 24, 27, 30, 39, 46, 51, 90 and 144. The residue SN was found in the region from 63-65 in representative sequence whereas the three strains PG2, 7314, and 6833 possessed the residue FKY in this region. Phylogenetic analysis of the isolate on the basis of P30 gene sequence and pairwise distance analysis revealed the existence of two different clusters of the strains and the closer evolutionary relationship with 4021, 4210, 8750, 7375 and 5725 strains in cluster I. Amino acid sequence based phylogenetic analysis of P30 gene also revealed similar type of distribution of the strains under two clusters in the phylogram with the outrooting of the strain 7314. Alignment of the nucleotide sequence of P48 gene of representative isolate (MAGMl) with other published sequences showed the homology of 96% (EU000539.1/strain VP12/05, EU000537.1/strain RL14/95-96, NC_009497.1/ PG2 strain, FP671138.1/strain 5632) and 97% (EU0005 3 6.1/strain VP20-L15/02, EU000538.1/strain RL17). In the representative P48 sequence, unique bases were found at 141, 318, 339, 554, 626, 759 and 932. On amino acid aligrmient, the deduced amino acid sequence of P48 gene of representative (MAGMl) and published sequences showed the homology ranging from 96-98%. Unique positions were found at 5 positions viz. 94, 121, 185, 209 and 311. On phylogenetic analysis of P48 gene of representative isolate, the strains were found distributed in two different clusters and the representative isolate was found more closely related with the Indian strains EU000537.1 (strain RL14/95-96), EU000536.1 (strain VP20-L15/02) and EU000538.1 (strain RL17) in cluster I. Amino acid sequence based phylogenetic analysis of P48 gene of the representative (MAGMl) isolate revealed its place in cluster I alongwith strains EU000539.1 (strain VP12/05) and PG2 whereas cluster II was comprised of Indian strains EU000537.1 (strain RL14/95-96), EU000536.1 (strain VP20-L15/02) and EU000538.1 (strain RLl 7). Isolates of different Mycoplasma spp. viz. M. agalactiae, M. mycoides subsp. capri and M. capricolum subsp. capricolum were subjected to 16S-23S rRNA ITS region sequencing and its comparative analysis. After sequence analysis and alignment, the 95%, 98-99% and 97-99% homology were observed in case of M. agalactiae isolate (MAGMl), M. mycoides subsp. capri and M. capricolum subsp. capricolum, respectively with published sequences. After interspecies phylogenetic analysis, the strains of M. agalactiae were found as a distinct branch of the evolutionary tree and all the strains of M. agalactiae, M. mycoides subsp. capri and M capricolum subsp. capricolum were found distributed in three distinct clusters showing their evolutionary relationship and shown the similar relationship during interspecies and intraspecies analysis of the ITS sequences.
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    ThesisItemOpen 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.
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    ThesisItemOpen Access
    CULTURAL ISOLATION, IDENTIFICATION, BIOTYPING AND MOLECULAR CHARACTERIZATION OF CRYPTOCOCCUSS SPP. FROM VARIOUS AVIAN SPECIES
    (AAU, Anand, 2012) MATHAKIYA, RAFIYUDDIN A.; JHALA, M. K.
    Cryptococcus neoformans is an opportunistic basidiomycete yeast that causes life-threatening infections such as meningoencephalitis in human, primarily in immunocompromised hosts and in animals and birds. The source of this organism is mainly pigeon excreta; however, other avian species' excreta are implicated as a source of this yeast. C. neoformans is primarily associated with nests and soils containing avian droppings, especially those of pigeons. Domestic and wild birds are known to be possible carriers of fungi that can contaminate dwellings and public areas. Despite the fact that both Cryptococcus species, C. neofornans and C. gattii, are capable of growth on pigeon guano, only C. neoformans exhibit prolific mating, completing its life cycle. Bird guano may represent the ecological niche for C. neoformans. In the present study, a total of 695 samples comprising 633 zoo samples (607 avian droppings, 19 eucalyptus, 4 egg swabs, 2 soil and feather, and one nodular swab) and 62 samples collected from outside the zoo (31 pigeon droppings, 18 mastitic milk and 13 cat swabs) were included. A total of 638 avian droppings and 57 samples other than droppings were screened to know the prevalence status of Cryptococcus spp. Out of total 695 samples (638 avian droppings and 57 others) screened, per cent (39/695) samples were found to be positive for C. neoformans by cultural isolation and identification. Out of total 638 avian droppings screened, 5.80 per cent (37/638) samples from 11 avian species belonging to 5 different orders were found to be positive, while one sample each of soil and feather and nodular swab fi-om Vadodara zoo were positive for C. neoformans. A total of 607 avian droppings collected from four different zoos of Gujarat state were screened to know the prevalence of C neoformans. Zoo wise prevalence of C. neoformans fi^om avian droppings was found to be 7.25 per cent (14/193) in Vadodara Zoo, 5.34 per cent (14/262) in Ahmedabad Zoo, 3.61 per cent (3/83) in Junagadh Zoo and 2.90 per cent (2/69) in Surat Zoo. Out of 62 samples collected outside the zoo (31 pigeon droppings, 18 mastitic milk and 13 cat swabs), 4 samples (6.45%) of pigeon dropping were positive for C. neoformans. Prevalence of C. neoformans was observed in macaw (33.33%), cockatoo (16.67%), cockatiel (15.00%), budgerigar and kunj (each 12.50%), parakeet (11.11%), pigeon (9.78%), pheasant (8.00%), love bird and lory (each 7.14%) and duck (3.03%). Bird order wise prevalence of C. neoformans recorded, was in order Pscittaciformes (28%), followed by Columbiformes and Gruiformes (each 23%), Galliformes (19%) and Anseriformes (7%). C. neoformans could not be isolated from the birds of orders Casuariiformes (emus and cassowary), Ciconiiformes (egrets, flamingos, herons, ibises, spoonbills and storks), Falconiformes (vultures), Passeriformes (crows, finches, mynahs and sparrows), Peliconiformes (pelicans), Piciformes (hombills) and Strigiformes (owls). A total of 123 fiangal isolates were recovered by cultural isolation fi^om 695 samples. Of these isolates, 39 isolates showed cultural characters on different media viz. Sabouraud dextrose agar medium with chloramphenicol, Sunflower seed medium, Bird seed agar and Brain heart infusion (BHI) agar indicative of Cryptococcus spp. Other non-Cryptococcus spp. and other fungal isolates were not processed further. The isolates included 33 isolates from zoo avian droppings, 2 isolates from zoo other than avian droppings and 4 isolates from free living pigeons. All 39 isolates were further examined and identified as yeast by India ink preparation and Gram's staining. All C. neoformans isolates were positive for urea hydrolysis, negative for nitrate reduction, and positive for growth inhibition by cycloheximide (0.1%), and revealed a similar pattern for sugar utilization viz. positive for glucose (G), galactose (Ga), sucrose (Su), trehalose (Te), maltose (Ma), rhamnose (Rh), D-xylose (Xy), inositol (Is), marmitol (Mn), arabinose (Ar) and sorbitol (Sb), and negative for lactose (La) and melibiose (Mb) utilization. Two sugars raffinose (Rf) and cellobiose (Ce) showed variable results with 21 (53.85%) and 18 (46.15%) isolates, respectively, showing positive reactions. All the 39 isolates of Cryptococcus spp. were serotyped using L-canavanineglycine- bromothymol blue (CGB) agar for differentiation of C. neoformans and C. gattii. All the isolates on CGB agar showed no colour change of yellow coloured CGB medium indicating that all were C. neoformans i.e. (serotype A or D). Nested PCR of all 39 isolates of C. neoformans was done using oligonucleotide primers Fungus I and Fungus II and generated the expected 429 bp amplicons indicating them to be fungal isolates. These amplicons were PCR amplified using nested primers Cryp I and Cryp II, which were complementary to C. neoformans and C. gattii selective regions within the 18S rDNA target. All 39 isolates generated the expected products of 278 bp indicating them to be either C. neoformans or C. gattii. The results of nested PCR were further confirming by PCR using CN4 and CN5 primers targeting ITS rDNA gene, which generated expected products of 136 bp from all 39 C. neoformans isolates indicating them to be either C. neoformans or C. gattii. The mating type detection of all 39 C. neoformans isolates was done by PCR using primers STE12aF809/STE12aR1607 specific for both C. neoformans and C. gattii MATa strains, which generated the expected products of 760 bp from all 39 C. neoformans isolates indicating them to be more virulent MATa strains. Molecular typing of all 39 C. neoformans isolates was done using URA5- RFLP. The fast digest restriction enzymes viz. 5'aM96I and Hhal were used for digestion of PCR products of 700 bp generated after amplification of URA5 gene of C. neoformans. The RFLP profile of all 39 C neoformans isolates were compared with eight standard strains of Cryptococcus, neoformans and Cryptococcus gattii (obtained from Dr. Wieland Meyer, Australia) representing each molecular type. All 39 isolates revealed RFLP pattern similar to WM 148 (serotype A, VNI/AFLPl) as indicated by two bands of 490 bp and 210 bp. This confirmed that all 39 isolates were of C. neoformans var grubii serotype A (VNI).
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    ThesisItemOpen Access
    DETECTION OF BRUCELLOSIS AMONG SHEEP AND GOATS IN GUJARAT STATE BY CULTURAL ISOLATION, SEROLOGICAL TESTS AND MOLECULAR TECHNIQUES
    (Anand Agricultural University, Anand, 2012) SUTARIYA MOHIT BABUBHAI; Dr. Ashish Roy
    Brucellosis is a major bacterial zoonosis of global importance. The causative organism belongs to genus Brucella is a Gram-negative facultative intracellular pathogen and its various species may affect a range of different mammals including man, cattle, sheep, goats, swine, rodents and marine mammals. Brucellosis is caused by different species of Brucella and it causes significant economical losses to live stock industry. The disease is manifested by reproductive disorder, viz., infertility, retained placenta, abortion and endometritis in livestock species, hence it leads to loss in productivity of animals and consequently to economic losses. The present study was undertaken to detect Brucella antibodies in serum,
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    ThesisItemOpen Access
    “CULTURAL ISOLATION, IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF BACTERIAL PATHOGENS FROM CLINICAL AND SUB CLINICAL MASTITIC MILK OF GOATS
    (Anand Agricultural University, Anand, 2012) PATEL PRADIPKUMAR KANTIBHAI; Dr. Ashish Roy
    Small ruminants (sheep and goats) production is popular and they play vital role in rural economies through provision of meat, milk, hides, skin and manure ensuring household income. Other advantages include their low cost of maintenance and high reproductive rates.
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    ThesisItemOpen Access
    ISOLATION, IDENTIFICATION, MOLECULAR CHARACTERIZATION AND DETECTION OF VIRULENCE ASSOCIATED GENES OF PASTEURELLA MULTOCIDA OF AVIAN ORIGIN
    (Anand Agricultural University, Anand, 2013) MAYURKUMAR P. BHIMANI; Dr. Ashish Roy
    The present study was undertaken with a view of isolation and identification of P. multocida from suspected cases of fowl cholera in different avian species. A total of 312 samples were collected from suspected cases, of which 24 (7.69%) P. multocida isolates (22 from emu and 2 from chicken) were isolated using blood agar as primary culture medium. These isolates were studied for their biochemical behaviour, in vitro antibiotic sensitivity pattern, P. multocida species specific PCR (PM-PCR), molecular characterization by multiplex PCR assay for capsular typing and detection of various virulence associated genes by PCR.
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    ThesisItemOpen Access
    COMPARATIVE ANALYSIS OF INTRADERMAL TUBERCULIN TEST AND γ- INTERFERON ASSAY FOR DIAGNOSIS OF BOVINE TUBERCULOSIS
    (Anand Agricultural University, Anand, 2012) SAGAR SHANKAR VYAVAHARE; Dr. M. K. Jhala
    A comparative study on interferon-γ (IFN-γ) assay, single intradermal tuberculin skin test (TST) and Enzyme linked immunosorbant spot (ELISPOT) for the diagnosis of Bovine tuberculosis was conducted. A total of 252 cattle stationed at a cattle farm were tested for Bovine tuberculosis by TST, IFN-γ assay and 23 of them by ELISPOT analysis. TST was carried out as per OIE Terrestrial Manual (2009) using PPD- Tuberculin. The reaction was considered to be positive if there was an increase of 4 mm or more in skin-fold thickness along with the local signs. Out of 252 cattle tested for Bovine tuberculosis, 66 (26.19%) cattle were found positive by TST. Breedwise, 13 (16.45%) animals of Kankrej (n=79), 30 (38.96%) of Gir (n=77) and 23 (23.95%) of Triple cross (n=96) breed were positive by TST. Agewise, 47 (37.90%) adults (n=124), 11 (11.22%) heifers (n=98) and 8 (26.66%) calves (n= 30) were positive by TST. Sexwise, 18 (56.25%) males (n=32) and 48 (21.81%) females