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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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Subject: Veterinary Microbiology × End date: 2014 × Start date: 2012 × Type: Thesis × Thesis Type: Ph.D ×
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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
    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).