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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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2009 - 200912010 - 20121
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Subject: Veterinary Microbiology × Author: MATHAKIYA, RAFIYUDDIN A. ×
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION OF LISTERIA MONOCYTOGENES ISOLATES OBTAINED FROM DIFFERENT ANIMAL SPECIES
    (AAU, Anand, 2009) MATHAKIYA, RAFIYUDDIN A.; ROY, ASHISH
    Listeria monocytogenes, a facultative intracellular pathogen, is responsible for severe food-borne infections in humans and can also cause invasive disease in many different animal species, including farm ruminants cattle, sheep, and goats. Several animal-derived L. monocytogenes-contaminated food products, including raw milk, pasteurized milk, chocolate milk, butter, soft cheeses, processed meat and pouhry products are the main sources of human listeriosis cases and outbreaks. In human, it causes septicaemia, meningitis, encephalitis, abortion and death in some cases while in ruminants, it primarily causes encephalitis and uterine infections. The encephalitic form of animal listeriosis is characterized by neurological signs, including circling, excessive salivation, unilateral facial paralysis and uterine infections are characterized by late-term abortions or septicemia in neonates. The present study was undertaken to characterize all twenty-eight L. monocytogenes field isolates obtained from different animal species which were maintained at the Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand. The characterization was done on the basis of phenotypic character (CAMP test) and with an aid of new molecular techniques like PCR, PCR-RFLP. The quantitation by CFU counting and lowest detection limit by PCR was also performed to know the sensitivity level of PCR. Phenotypic characteration by CAMP test on all the 28 field isolates revealed positive reaction for twenty-three isolates with characteristics enhancement of haemolytic zone with S. aureus on Sheep Blood Agar (SBA) and five isolates (LM 01, LM 03, LM 05, LM 15 and LM 22) showed weak haemolytic zone. The template DNA was prepared from 28 field isolates and reference strain Listeria monocytogenes 4b (MTCC 1143) by boiling method as per standard protocol. L. monocytogenes inlA, inlB, plcB and m/J genes specific primers (inlA F: 5'-CCT AGC AGO TCT AAC CGC AC -3' and inlA R: 5'-TCG CTA ATT TGG TTA TGC CC-3'); (MB F: 5'-AAA GCA CGA TTT CAT GGG AG -3' and inlB R: 5'-ACA TAG CCT TGT TTG GTC GG-3'); (plcB F: 5'-GGG AAA TTT GAC ACA GCG TT -3' mdplcB R: 5'-ATT TTC GGG TAG TCC GCT TT-3') and (m//F: 5'-TGT AAC CCC GCT TAC ACA GTT-3' and inlJR: 5'-TTA CGG CTG GAT TGT CTG TG-3') were used to amplify virulence associated genes and virulent strain specific gene region. The PCR amplification carried out for the detection of virulence associated genes inlA (lmo0433), inlB (lmo0434), and plcB (lmo0205) gave a posifive result in all the 28 field isolates of of L. monocytogenes as well as all the reference strain Listeria monocytogenes 4b (MTCC 1143). The desired amplified product of approximately 255 bp, 146 bp and 261 bp was generated from virulence associated genes inlA, MB, and plcB, respectively, from all the 28 isolates of L. monocytogenes and the reference strain using the primer pairs for inlA, inlB, and plcB genes. Similarly, PCR amplification of inlJ (lmo282J) gene was carried out for the detection of virulent strain among all the 28 field isolates of L. monocytogenes. The desired amplified product of approximately 611 bp was generated from all the 28 isolates of L. monocytogenes and the reference strain Listeria monocytogenes 4b (MTCC 1143) using the primer pairs for m/J gene. PCR-RFLP analysis, which is commonly used for genotyping, was carried out for ten field isolates (LM 07, LM 13, LM 14, LM 20, LM 23, LM 24, LM 25, LM 26, LM 27 and LM 28) and reference strain Listeria monocytogenes 4b (MTCC 1143) having intense band in PCR product of inlA and inlJ genes was carried out for both inlA and inlJ genes by using four restriction endonucleases Rsal (Fast Digest), HindIII (Fast Digest) and DpnI, AluI, respectively. Digestion of 255 bp PCR products of inlA gene with Rsal yielded two fi-agments of approximately 151 bp and 104 bp, with Hindlll resulted in two fragments of 149 bp and 106 bp. On 2% agarose gel electrophoresis, all the 10 field isolates as well as the reference strain yielded similar RFLP profiles with each of the RE used. Similarly, Digesfion of 611 bp PCR products of inlJ gene with Alul yielded four fragments of approximately 274 bp, 161 bp, 150 bp and 26 bp digested. On 4% agarose gel electrophoresis, all the 10 field isolates as well as the reference strain yielded similar RFLP profiles with Alul while none of the samples was digested with DpnI. The quantitation by CFU counting and lowest detection limit by PCR was performed by ten fold serial dilution of the culture in BHI broth. For the sensitivity tests, the plate culture method was used to confirm the number of CFU and PCR was performed from each diluted culture and it showed the amplification up to 2 x10 to power 1 CFU/ml of broth using primer pair of inlA gene. This result indicates the lowest detection of Z. monocytogenes organism was 2 x10 to power 1 CFU/ml by PCR. Thus, in order to assess the analytical sensitivity, meaning the lowest number of L. monocytogenes detectable by PCR is a low-cost and rapid procedure that can be appropriated for the detection in real time of low L. monocytogenes levels in naturally contaminated food and is suitable to implement in the food industry.
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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).