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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 PHENOTYPIC AND GENOTYPIC IDENTIFICATION AND METAGENOMIC ANALYSIS OF SUBCLINICAL MASTITIC PATHOGENS IN COWS(AAU, Anand, 2011) BHANDERI, BHARAT BABUBHAI; Jhala, M. K.Subclinical mastitis occurs with no visible changes in the appearance of the milk and/or the udder, but milk production decreases which leads to economic losses to the farmers and dairy industry. There are many microbial pathogens involved in causing subclinical mastitis in cows. The present study was undertaken to know incidences of subclinical mastitis in organized farms using Somatic Cell Count (SCC) and bacteriological examination (International Dairy Federation-IDF guidelines), California Mastitis Test (CMT) and impregnated pH strip test followed by characterization and PCR based detection of important mastitic pathogens. Metagenomic analysis of subclinical mastitis milk was also done to determine the complex microbial diversity in udder environment during subclinical mastitis. A total of 349 quarters of 89 lactating cows comprising 31 Triple cross (TP) (Kankrej x Jersey x Holstein Friesian), 29 Kankrej, 17 Gir and 12 Holstein Friesian (HF) affiliated with Anand Agricultural University, Anand were screened for subclinical mastitis. Overall 52.8 per cent (47/89) cows were found to be positive for subclinical mastitis infection in one or more quarters. The highest incidence of subclinical mastitis was found in Triple cross cows (74.19%), followed by Gir cows (58.82%), HF cows (50%) and Kankrej cows (27.58%). Overall quarter wise incidence for subclinical mastitis was found to be 30.66 per cent (107/349). The highest incidence was found in Gir cows (38.80%) followed by Triple cross cows (38.08), HF cows (33.33%) and Kankrej cows (15.04%). The highest incidence of subclinical mastitis was found in fore left quarter (28.03%), followed by hind left quarter (27.1%), fore right quarter (24.29%) and hind right quarter (20.56%). Of the 47/107 cows/quarters positive for subclinical mastitis, 39/47 (82.97%) cows and 82/107 (76.63%) quarters were also positive by CMT and 27/47 (57.44%) cows and 56/107 (52.33%) quarters were positive by impregnated pH strip test. Cultural isolation ft'om 107 subclinically positive quarter milk samples yielded 126 bacterial isolates. Staphylococci was the most predominant bacterial species accounting for 53.97 per cent (68/126) of all the isolates, followed by 21.43 per cent (27/126) CAMP (Christie-Atkins-Munch-Peterson) test positive Str. agalactiae, 18.25 per cent (23/126) Micrococci, 4.77 per cent (6/126) E. coli and 1.58 per cent (2/126) Bacillus species. Out of 68 Staphylococci isolates, 38 (55.89%) isolates showed fermentation on Mannitol Salt Agar (MSA), whereas 30 (44.11%) isolates were mannitol non fermentive. Of the total 30 S. aureus identified by PCR, 21 (70%) were mannitol fermentive and 9 (30%) mannitol non fermentive. Thirty one (45.58%)) Staphylococci were found to be positive for pigment production, whereas 37 (54.42%) isolates produced white colonies on nutrient agar. Forty eight (70.58%) isolates were found positive for coagulase reaction, whereas 20 (29.41%) were negative. Thirty one (45.58%)) isolates exhibited P haemolysin production, 4 (5.89%) a haemolysin and 33 (48.53%)) isolates were non-haemolytic on 5 per cent Sheep blood agar. Phage typing at National Staphylococci Phage typing Centre, Maulana Azad Medical College, New Delhi, using five phage group sets of International Basic Set of 23 phages revealed maximum number of the Staphylococci isolates lysed by group II 14 (82.35%), followed by groups III, Not alloted (NA), I and V with 12 (70.58%), 9 (52.94%), 5 (29.23%) and, 2 (11.76%) respectively. Maximum 11 (64.7%) isolates were lysed with phage number 47 with strong reaction, followed by 10 (58.82%)) isolates with phage numbers 42E and 81, while less effective phage numbers were 71 and 94, which lysed only one strain (5.89% each) and phage number 95 not giving strong reaction with any of the isolates. The methicillin and oxacillin antibiotic sensitivity pattern by disc diffusion method revealed that, all the 68 (100%)) Staphylococci isolates were sensitive. Serotyping of six E. coli isolates (at National Salmonella and Escherichia Centre, Kasauli, Himachal Pradesh for 'O' antigen) resulted in identifying 014, O20, 045, 055 and 0112 serotypes, while one isolate was untypeable (UT). Out of 68 Staphylococci isolates tested for identification of 5. aureus by PCR, 30 isolates were identified as S. aureus by obtaining amplification product of 1318bp using S. aureus specific primer for 23S rRNA. Out of 30 PCR positive S. aureus, 18 (60%)) were positive and rest were negative for coagulase test. All the 27 Streptococci isolates were identified as Str. agalactiae by amplifying 586bp product using Str. agalactiae specific primer for the 16S rRNA while, none were amplified for Str. dysgalactiae (401bp) and Str. uteris (94bp) based on primers specific for the 16S rRNA and 23 S rRNA respectively. All the six E. coli isolates yielded 232bp amplified product using E. coli specific primer targeting DNA sequence coding for the 23 S rRNA. Metagenomic analysis (using GS FLX 454 Life Sciences) of DNA of subclinical mastitis milk sample of TP, Kankrej and Gir cows yielded an out put of 274190 bp, 17,727 bp, 42,548 bp and 1,960, 170, 301 contigs respectively. Average fragment length obtained were 139.89, 104.28 and 141.36 bp for TP, Kankrej and Gir cows respectively. The longest sequence length was 560, 327 and 454 bp, while shortest sequence length was 40, 40, and 41 bp for TP, Kankrej and Gir cows respectively. A total of 54 (2.76%), 39 (22.94%) and 12 (3.99%) sequences for TP, Kankrej and Gir cows respectively could be matched to proteins in SEED subsystems of MG-RAST (Meta Genome Rapid Annotation with Subsystem Technology) (using an e-value cut-off of le-5). Metagenomic analysis of the three breeds identified bacterial organisms belonging to phyla (5), class (8), Subclass / order (15), Family (19), Genus (23) and species (28); of these, 19 genera and 26 species, many of which were fastidious/anaerobic organisms, were identified additionally than the cultural methods. Out of five genera Staphylococcus, Streptococcus, Micrococcus, Bacillus and Escherichia detected in the subclinical mastitis milk samples of TP, Gir and Kankrej breeds by culture based methods, four genera Staphylococcus, Streptococcus, Bacillus and Escherichia were also identified in the corresponding pyrosequencing data, while Micrococcus identified by culture based methods was not found in the pyrosequencing data. In pyrosequencing, over all 28 bacterial species were identified from all the three breeds of cows viz. Leifsonia xyli, Propionibacterium acnes, Streptomyces coelicolor, Chlamydophila abortus, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus acidophilus, Streptococcus mitis, Burkholderia cenocepacia, Burkholderia cepacia, Ralstonia solanacearum, Nitrosomonas europaea, Pseudoalteromonas atlantica. Salmonella Dublin, Serratia marcescens, Azotobacter vinelandii, Pseudomonas aeruginosa, Pseudomonas mendocina, Stenotrophomonas maltophilia. Bacillus subtilis, Lactobacillus delbrueckii. Aster yellows witches'-broom phytoplasma, Pannbaculum lavamentivorans, Thermosipho melanesiensis, Aeromonas hydrophila, Escherichia coli, Shigella hoydii and Pseudomonas fluorescens. Of these, except S. aureus and E. coli, all were additionally identified than the culture based method but, Str. agalactiae identified by cultural method was not found in the pyrosequencing data. The role of lesser known or less frequently involved organisms as identified by metagenomic analysis may be further explored in future so as to understand the complete etiopathology of subclinical mastitis in cows.ThesisItem Open Access ISOLATION AND CHARACTERISTICS OF Staphylococcus aureus FROM BOVINE MILK(AAU, Anand, 1990) Purohit, Jayantilal Hargovind; JHALA, V. M.The present study was undertaken with a view to know the occurrence of Staphylococcus aureus In bovine milk In relation to species, managemental conditions, breed, method of milking, parity, stage of lactation and Involvement of the quarters as well as to observe the relationships among the certain characteristics, Including enterotoxigenicIty, of S.aureus. The isolates were also phage typed to know the possible origin. The milk samples were collected from the animals maintained at six different farms comprising of four GAU farms and two private farms. The cows were maintained at four farms whereas buffaloes were maintained at remaining two farms. A total of 925 milk samples (758 from cows and 167 from buffaloes) from the individual quarters of 234 animals comprising of 191 cows and 43 buffaloes were collected and processed for isolation and identification of S.aureus. Of these, 94 quarters (10.16 per cent) of 67 animals (28.63 per cent) revealed the presence of S.aureus. The incidence of S.aureus was more commonly encountered amongst the cows on animal basis (31.94 per cent) as well as on quarter basis (11.35 per cent) than those of buffaloes (13.95 and 4.79 per cent, respectively).ThesisItem Open Access DETECTION AND DIFFERENTIATION OF MAREK'S DISEASE VIRUS SEROTYPES BY POLYMERASE CHAIN REACTION (PCR) AND CHARACTERIZATION BY DNA SEQUENCING OF THE PCR PRODUCT(AAU, Anand, 2006) KALYANI, IRSADULLAKHAN HABIBULLAKHAN; Purohut, J. H.Marek's disease (MD) is a lymphoproliferative disorder of chicken characterized by oncogenic transformation of T cells that infiltrate lymphoid tissues, peripheral nerves and visceral organs, resulting in a complex pathogenesis that usually leads to death of the affected chicken. The causative agent of MD, Marek's disease virus (MDV) a cell associated herpesvirus, is ubiquitous in almost all poultry population raised under commercial conditions. The present study was under taken to assess incidence of MD as per the farm records on the basis of clinical signs and post mortem lesions, detection of MDV from clinical cases exhibiting frank lesions of MD by PCR, comparing the efficacy of different primers in detecting the MDV as well as differentiating the serotypes in tissues and feather follicle and genetic characterization by sequencing of the PCR amplified fragment carrying 132 bp repeats. During June to November 2005 outbreaks of MD were suspected in flocks of egg type birds on farms situated in and around Anand district located of central Gujarat as well as from farms around Mahuva located in Saurastra region of Gujarat state. The strength of individual affected flocks varied from 4500 to 25000 birds with a total population of 4,26,991 birds. Of these, 14110 birds died due to MD (as per the farm records of history, symptoms and postmorterm lesions) with an over all mortality of 3.30 per cent during the months of June to November 2005. During the investigation, a total of thirty six commercial poultry farms and forty nine batches of layer birds suffered from mortality due to MD, in addition to Central Poultry Research Station (CPRS) Veterinary College Anand, where mortality occured in layer birds. Mortality in commercial poultry farms ranged between 0.6 to 7.3 per cent. A total of 34 clinical samples (17 feather follicles and 17 spleen ) from 27 birds suspected of MD were collected by making visits to the various commercial poultry farms and also from CPRS Veterinary College Anand. Suspected clinical samples and four MD vaccine strains, including three HVT (MDV-3) vaccines and one SB-I(MDV-2) vaccine were processed for DNA extraction. DNA was extracted from samples of feather follicle using proteinase K mixture, where as DNeasy Tissue Kit (QIAGEN Pvt. Ltd) was used for tissue samples. Sample showing acceptable purity and concentration were subjected to PCR amplification using six different primers. Total of six pairs of primers were used, which were selected to amplify different gene segments. The primer pair BamHl/BamH2 specifically amplifying a 434 bp segment from BamHI fragment (of MDV-1) containing 132 bp repeats located in TRL and IRL region of MDV genome, detected 32 samples positive indicating presence of double 132 bp repeats in the amplified segment. All the three HVT vaccines and one SB-1 vaccine failed to produce the amplification as the selected primer is specific for MDV-1 only. Primer pair AGAM1/AGAM2 and P1/P2, both targeting the antigen A gene (UL44) produced 314 bp and 199 bp products respectively in 30 out of 34 clinical samples tested, however, none of the HVT vaccine or SB-1 vaccine yielded positive result, suggesting the specificity of these primers for MDV-1. Primer set M1.1/M1.8 amplified a 318 bp product as against expected 247 bp product in 30 samples. To confirm the result, these primers were subjected to NCBI BLAST, and it was found that the primer specific segment of 318 bp does exist in pubhshed sequence of Md5, and Mdl IBAC. None of the HVT as well as SB-1 vaccine produce any amplification indicating the specificity of primer in detecting MDV-1. Only two field samples produced expected amplicon of 505 bp when tested with MDV-3 specific HVT1/HVT2 to detect the vaccine virus (HVT). All the three HVT vaccines produced the amplicon of 505 bp, whereas SB-1 vaccine was negative, proving specificity of this primer pair for HVT. Out of the six primer pairs, BamHl/ BamH2 and AGA1/AGA2 detected MDV in maximum number (32) of field samples. Primers AGAM1/AGAM2, P1/P2 and Ml.l/M.1.8 detected the same number (30) of samples positive. Primer pair HVTl/ HVT-2 detected MDV-3 in two feather follicle samples indicating possible excretion of the vaccine virus. PCR products (434 bp) of MDV genome amplified by BamHl/BamH2 primers from two field samples (M3, M4) having good quality DNA (i.e. 1.7-1.9 OD at 260/280 nm) as detennined by spectrophotometry were used for sequencing by ABI PRISM® 310 Genetic Analyzer (Applied Biosystems, USA), using BigDye® Terminator v3.1 Cycle sequencing kit (Applied Biosystems, USA). The sequence obtained revealed two 132 bp repeats in the 378 bp stretch indicating virulent nature of the field MDV. The alignment of 378 bp sequence of M3 and M4 with previously published sequences of the MDV isolates Md5, Mdll, HPRS-16 and CDs of tumourogenicity associated m-RNA and CDs published from BamH gene family revealed that the field isolates of present study shared complete homology (100%). Results of sequencing along with history of vaccination in the affected birds and involvement of different organs on postmortem examination indicated about the increase in virulence of MDV circulating in field causing recent outbreaks.ThesisItem Open Access SEROEPIDEMIOLOGY, ISOLATION AND PATHOGENICITY OF BLUETONGUE VIRUS(AAU, Anand, 1996) Chandel, Bharat Singh; Kher, H. N.Bluetongue (BT) is an infectious, non contagious disease of domestic and wild ruminants. Bluetongue virus ( BTV ) causes severe disease in sheep, which i s transmitted by insect vectors (Culicoides spp.) . The ability of BTVs to inflict pathological changes in susceptible sheep depends on the virulence of a particular viral isolate , susceptibility of the host and a number of environmental factors related to climatic conditions. The present study was aimed at the seroepidemiology, prevalence of BTV serotypes in sheep, isolation, propagation and identification of local isolates and pathogenicity of BTV in natural and experimental cases of sheep. This study also covered the seroprevalence of epizootic haemorrhagic disease virus (EHDV) in cattle and buffaloes as it is related to orbivirus group. A seroepidentiological survey of BTV precipitating antibodies was carried out by agar gel immunodiffusicm ( AGID ) test in different species of livestock in Gujarat. Out of 1623 sera tested, 407 (25.07%) were found to be positive for BTV antibodies.ThesisItem Open Access ISOLATION, IDENTIFICATION, MEMBRANE PROTEIN PROFILING AND MOLECULAR CHARACTERIZATION OF MYCOPLASMA AGALACTIAE ISOLATES FROM GOATS OF GUJARAT STATE(AAU, Anand, 2012) KUMAR, PRANAY; ROY, ASHISHThe 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.ThesisItem Open Access ISOLATION OF INFECTIOUS BURSAL DISEASE VIRUS (IBDV), ITS COMPARISON WITH VACCINE STRAINS AND EFFICACY OF DIFFERENT VACCINATION REGIMENS IN PASSIVELY IMMUNE CHICKS(AAU, Anand, 1995) Hirpurkar, Sadananda D.; Kher, H. N.Infectious bursal disease (IBD) which was characterised by relatively less mortality but more immunosuppression was earlier prevalent in India. During a past couple of years, there is country-wide change in pattern of disease with high mortality rates. Some of the farms investigated in the present study have also experienced high mortality among both, layers (six to 14 weeks) and broilers (four to six weeks). Present study was undertaken on isolation of IBDV from field samples, study their pathogenicity and antigenicity in comparison with existing vaccine strains; and formulation of vaccination regimen effective enough to give protection to chicks in presence of maternal antibody. Before taking up virus isolation, tissue homogenates from clinical cases were examined by agar gel diffusion test (AGDT) against reference antiserum and their identity as IBDV was confirmed. Overall AGDT positive samples ranged between 40.0 and 70.5 per cent. Positive field samples were grouped under five groups (UNO, KAM, AND, HAL and NAV), according to different geographical regions. An attempt was made for isolation of IBDV from all the five representative samples in different indicator systems. Among experimentally inoculated chicks, the clinical disease was reproduced in case of UND, KAM and AND isolate infected group. No clinical symptoms were observed in NAL and NAV isolates infected groups. The birds inoculated with each samples and sacrified 48 hours PI, had gross and histopathologic changes suggestive of IBD. Specific precipitation lines were demonstrated in bursal homogenates. Out of five field isolates, three isolates (UND, KAM and AND) were adapted to growth in chick embryos by chorioallantoic membrane (CAM) route. Predominant changes were observed in the embryos which included slow and stunted growth, cutaneous edema and hemorrhages in subcutis. UND isolate was serially passed onto chicken embryo fibroblast (CEF) cell culture for its adaptation to growth. Characteristic cytopathic effect (CPE) was observed earliest, at 30 hours PI, and maximum CPE showing degeneration of about 80 per cent cell monolayer, at 48 hours PI. Syncytia formation due to aggregation of large number of nuclei of dead cells was observed in later stages. Other field samples failed to induce CPE in cell culture upto five serial passages. Comparative pathogenicity studies on five field isolates, one reference isolate (BAN) and vaccine strain of intermediate (Vac-I) and mild (Vac-M) virulence each, were carried in chicks, chick enbryos and CEF cell culture. Birds infected with UHD and BAM isolates showed severe clinical synptons and also hemorrhagic lesions in the bursa of Fabricius (BF), skeletal muscles and proventriculus-gizzard junction. Concurrent histopathologic lesions in the BF, at 48 hours PI, includes marked edema, hyperemia, necrosis of lymphoid cells, and formation of vacuoles or cystic follicles. In case of birds inoculated with Vac-I strain, the enlargement and edema was more marked which persisted longer. Vaccine strain induced histopathological lesions in BF which were of mild degree and comparable to those found in birds infected with NAL and NAV isolates. As regard to the pathogenicity of field isolates in chick embryos inoculated by CAM route, UND and BAN isolates caused almost 80 per cent mortality. Among other isolates, AND and KAM showed relatively low mortality rates followed by HAL and NAV. Unlike any of the recent field isolates tested in the present study, the UND isolate grew well in CEF cells exhibiting CPE in infected monolayers. Thus, only the UND and BAN isolates, were able to maintain consistent pathogenic potential in every laboratory system tested. Slight variation as regard to period required for initiation of CPE was observed in UND and BAN isolates when compared with that of vaccine strains. Highest virus infectivity titre was recorded in Vac-I strain, while in Vac-M strain the titre was lowest. The virus titres in both field isolates (UND and BAN) were sane. Neutralization and cross neutralization tests were carried out to assess the antigenic relatedness of a field isolate, reference isolate and vaccine strains using homologus and heterologus innune sera. The results indicated close antigenic relationship of the isolates with vaccine strains. In the present study, efficacy of three vaccination regiuens was evaluated in the progeny obtained from breeder hens having high levels of maternal antibody (MA). The levels of precipitating antibodies of progeny chicks at hatch indicated, on an average, 50 to 55 per cent passive transfer of MA to progeny. After hatch, higher levels of MA sustained for one week and afterwards declined steadily at the half-life of approximately six to eight days. MA attained low levels between 21 and 28 days of age. While after 28 days of age, precipitating antibodies were undetected by quantitative AGDT (QAGDT), still these antibodies were detected by enzyme linked immunosorbent assay (ELISA). Live vaccine (mild) was used at day-old age for 'priming' of all the birds and subsequently, combination of live (intermediate) and/or inactivated vaccines was tested in three groups. One group served as control. The response of birds, vaccinated once or twice with live intermediate vaccine was not satisfactory as indicated by low levels of antibodies even after vaccination. On challenge, some birds showed clinical symptoms and bursal damage. The group in which birds received inactivated vaccine, at seven days of age and live vaccine at 14 and 28 days of age, showed significantly (P <0.05) higher levels of antibody. On challenge, the birds did not show clinical symptons and mortality, but still these birds were not prevented from infection because the bursal danage was apparent indicating inadequate response against virulent IBDV. Overall studies indicated the presence of highly virulent IBDV in sone pockets of Gujarat State. Therefore, it is important to locate such pockets at an earliest to prevent possible spread of highly virulent IBDV and carry out effective vaccination coupled with strict biosecurity measures, where immunization has failed probably because of increased virulence of IBDV. Serological monitoring of progeny flock is necessary to determine when MA in chicks decline to levels that the vaccine can overcome. Without this information, it is very difficult to control highly virulent IBDV.ThesisItem Open Access STUDIES ON IMMUNE RESPONSE TO NEWCASTLE DISEASE VIRUS VACCINES IN CHICKENS(AAU, Anand, 1989) PATEL, H. R.; JHALA, V. M.Studies on immune response to Newcastle disease virus (NDV) vaccines i n cockerel chicks, layers , broilers and pullets were undertaken experimentally at the Department of Bacteriology, Veterinary College, Anand. In the first experiment, 196 day-old White Leghorn (WLH) cockerel chicks from immunized parent flock were used to assess the levels of maternal antibodies at different ages and their effect on immune response to 'F' strain of NDV. The maternal antibody titres in chicks were ranging from log2 1.0 to 3.0 before vaccination. They were highest in day-old chicks and lowest in 15 day-old chicks which were significantly ( P < 0.05) different . In control chicks, the titre declined at a constant rate and was undetectable at 19th day of age, determining five days as half life time for maternal antibodies. Post-vaccination titres at 7th, 14th and 21st day in chicks vaccinated at 1st, 5th, 10th and 15th day of age were not significantly different in between the groups, though the difference was significant within the groups. This indicated that maternal antibody titres upto logp 3.0 did not interfere the immune response when chicks were vaccinated even at one day of age. The ohallenge study at three weeks post-vaccination revealed that the minimum HI titre of log2 3.0 was protective in cockerel chicks. In second experiment, 45 WLH birds of 18 week-old were used for comparative study of immune response to inactivated OE and live R2B vaccines and their effect on egg production. Peak antibody response was observed at 20 weeks of age in birds vaccinated at 18 weeks of age either with inactivated OE or live R2B vaccines. There was no significant difference in HI titres in between the two groups upto 26 weeks except 24th week. Twenty eighth week onward, the HI titres were significantly higher in birds vaccinated with inactivated OB vaccine (Group A) than those vaccinated with live R2B vaccine (Group B). The overall serological response was higher in birds of group A than birds of group B. During the period of 18 weeks of lay, the birds of group A averaged 99.3 eggs (71.09 per cent) per hen, whereas those of group B averaged 83.7 eggs (59.51 per oent) per hen. Control birds (Group C) produced 89.6 eggs (63.84 per oent) per hen. Thus comparing with control group, inactivated OE vaccine did not affect the egg production but rather boosted it , whereas live R2B vaccine suppressed the egg production.ThesisItem Open Access MICROBIOLOGICAL INVESTIGATIONS OF NEONATAL CALF DIARRHOEA WITH SPECIFIC REFERENCE TO DETECTION OF ROTAVIRUS AND ENTEROTOXIGENIC Escherichia coli(AAU, Anand, 1989) SHAH, N. M.; JHALA, V. M.The present study was undertaken with the objectives of detecting the presence of rotavirus, enteropathogenic bacteria and parasites, to study the enterotoxigenicity of E. coli isolates and to find out the in vitro antibiotic sensitivity patterns of bacterial isolates from the cases of neonatal calf diarrhoea. Fecal samples from 116 diarrhoeic calves of cattle (94) and buffaloes (22)were examined. There was no significant difference in occurence of various enteropathogens between cow calves and buffalo calves. Commomnly encountered enteropathogens were E. coli (61.70% cow calves and 59.09 % in buffalo calves) and rotavirus (20.21% in cow calves and 22.72 % in buffalo calves).ThesisItem Open 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).