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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 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 PRISMĀ® 310 Genetic Analyzer (Applied Biosystems, USA), using BigDyeĀ® 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 ASSESSMENT OF NOVEL N-GENE TARGET FOR DETECTION OF PESTE DES PETITS RUMINANTS (PPR) VIRUS BY RT-PCR AND CHARACTERIZATION BY RFLP PROFILE(AAU, Anand, 2006) CHANDRA, VARTIKA; Jhala, M. K.Peste des Petits Ruminants (PPR) is an economically important viral disease of sheep and goats, characterized by high fever, ocular and nasal discharge, pneumonia, necrosis and ulceration of the mucous membrane and inflammation of gastrointestinal tract leading to severe diarrhea. The disease is caused by a Morbillivirus of the family Paramyxoviridae. The disease is highly contagious causing varying degree of morbidity and mortality in susceptible animals. In India, severity of the disease is more pronounced in goats than in sheep and with a combined susceptible population of about 200 million, it is one of the major threats to the small ruminant population of the country. The present study was aimed to study the incidence of Peste des petits ruminants virus (PPRV) in goats of selected areas of Gujarat by sandwich ELISA and to derive estimates of overall, locationwise, agewise and sexwise incidence and samplewise positivity rates. In addition, the study also involved standardization and application of novel Nucleocapsid (N) gene based RT-PCR, for diagnosis of PPR. Further, an attempt was also made to access possible genetic variation among the field and vaccine PPRVs by RFLP profile. A total of 46 clinical samples from 31 goats suspected of PPRV from selected areas of Gujarat were tested by sandwich-ELISA, of which 23 animals were found positive yielding an overall incidence rate of 74.19 percent. Out of 18 animals suspected of PPR at Clinical Service Complex, Veterinary College. Anand, 15 (83.33%) were found positive; whereas four (80%) out of five samples from Koth, two (66.67%)) out of three samples from Vasna and two (40%) out of five samples from Harsoli yielded positive results. Out of the 31 animals, 13, 11, 6 and one animals belonged to age groups of 0-12, 12-24, 24-48 and >48 months, respectively, showing the respective incidence of PPRV as 84.61, 63.63, 66.67 and 100 per cent. Incidence rate of PPR by sandwich-ELISA was 80%) and 71.43%) in males and females, respectively. The PPRV antigen could be detected slightly more in blood samples (73.33%) than in nasal swabs (66.67%o), in case where both the sample types were taken from the same animals. All the 46 clinical samples, a reference vaccine virus (Sungri isolate) and a blood sample from apparently healthy goat were processed for RNA extraction using TRI Reagent . RNA samples showing acceptable purity and concentration were reverse transcribed with random hexamers to generate cDNA templates, which were subjected to PCR amplification using in silico designed N-gene specific primer pair N1-N2. Reference vaccine virus as well as fifteen (32.61%) of the 46 clinical samples, including fourteen blood samples and one nasal swab, produced the desired amplion of approximately 463 bp with primer pair N1-N2. The amplicons from all the positive samples and the vaccine virus migrated similarly in the gel. Thirty one (67.39%)) samples including 12 blood samples, 17 nasal swabs and two tissue samples as well as the blood sample taken from apparently healthy goat failed to produce the targeted amplification with the primer pair N1-N2. Out of 46 clinical samples tested for PPRV, 31 and 15 samples were positive by sandwich-ELlSA and RT-PCR, respectively. "None of the samples negative in sandwich- ELISA yielded positive amplification in RT-PCR. Relative to sandwich-ELlSA, sensitivity and specificity of the RT-PCR was 48.39 and 100.00 per cent, respectively. Overall agreement between the two tests was 65.22 per cent. RT-PCR products (463 bp) amplified with primer pair N1-N2, were further digested with two restriction enzymes viz. Pst-I and Alu-I for RFLP analysis. RT-PCR products of the representative samples from each of the three locations and the reference vaccine virus produced similar RFLP patterns with each of the two REs, indicating absence of genetic variation at the corresponding restriction sites of these two REs. Digestion of RT-PCR products with Alu-l yielded a fragment of 395 bp, whereas the other fragment was not visible. Similarly, digestion with Pst-I yielded a single fragment of405bp.ThesisItem Open Access SEROEPIDEMIOLOGY AND ELECTROPHEROGRAM STUDIES OF BLUETONGUE VIRUS(AAU, Anand, 2000) Bhalodiya, M. B.; Jhala, M. K.Bluetongue (BT) is an insect transmitted viral disease of several species of domestic and wild ruminants. The disease is a cause for serious concern to livestock industry due to staggering direct and indirect economic losses. In many countries like India having considerable sheep population, the disease has become endemic. Severity of the infection depends upon the species and breed of animals, serotypes/strains of virus and prevalent ecosystem. The present study was aimed to find out prevalence of Bluetongue virus (BTV) antibodies in cattle and sheep of different regions of the Gujarat State. Avidin-Biotin enzyme-linked immunosorbent assay (AB-ELISA) and indirect enzyme-linked immunosorbent assay (I-ELISA) employed for antibody detection were also compared in terms of their sensitivity and specificity. An attempt was also made to standardize polyacrylamide gel electrophoresis (PAGE) for detecting BTV. Out of 527 cattle and sheep sera tested, 343 (65.08%) and 396 (75.14%) were found to be positive for BTV antibodies by AB-ELISA and IELISA respectively. Specieswise, 69.04 and 77.57 percent of cattle and 55.55 and 63.33 percent of sheep revealed antibodies to BTV by AB-ELISA and l-ELISA respectively. The highest prevalence rate was found in South Gujarat region (67.94 and 78.63 percent) by AB-ELISA and l-ELISA respectively and female animals (65.53 and 77.84 percent) showed more prevalence than male animals (64.35 and 70.79 percent). Districtwise seroprevalence study revealed highest incidence in Valasad district (68.51%), followed by Navsari (67.70%), Kutch (60.74%), Junagadh (57.69%) and Rajkot (51.72%) district by AB-ELISA, whereas IELISA yielded highest prevalence rate in Valasad (80.55%) followed by Navsari (77.82%), Rajkot (69.96%), Junagadh (69.23%)) and Kutch (66.96%>) district. In cattle, highest incidence was observed in Kankrej (75.52%)) by ABELISA, while HF cross-yielded highest positive samples (84.72%) by l-ELISA. Lowest incidence was seen in Gir breed by both the tests (51.51 and 66.66 percent), while in sheep, higher prevalence rate in native Patanwadi breed (64.28 and 73.21 percent) was observed than the crossbred (41.17 and 47.05 percent) by AB-ELISA and I-ELISA respectively. Comparison of l-ELISA and AB-ELISA for the detection of BTV antibodies, revealed that the former test was better in terms of sensitivity and specificity. Out of a total 527 serum samples tested 343 (65.08%) and (75.14%) reacted positively in AB-ELISA and l-ELISA respectively. l-ELISA detected BTV antibodies in 54 (10.25%) samples, which were negative by AB-ELISA. Relative sensitivity and specificity of AB-ELISA to l-ELISA were 86.63 and 99.23% respectively and overall agreement between both the tests was 89.56%. RNA-PAGE was employed for detecting BTV using a cell culture propagated BTV serotype 1. This was attempted essentially to standardize this highly sensitive technique, so as to use if routinely in future for the field samples. The study revealed 10 RNA segments with a migration pattern resembling to serotype 1 of BTV.ThesisItem Open Access CHARACTERIZATION OF Escherichia coli ISOLATES FROM DIARRHOEIC CALVES FOR TRANSFERABLE DRUG RESISTANCE, COLICINOGENY AND VIRULENCE ASSOCIATED GENES(AAU, Anand, 2006) CHOUDHARY, VANDANA; Roy, AshishThe present study was undertaken to analyze plasmid profile of E.coli isolates, to ascertain transferable nature of antibiotic resistance, colicinogeny and unusual biochemical characters among the isolates from calf diarrhoea cases. Further PCR based detection of virulence associated genes was also carried out. On conjugation 19 transconjugates were obtained which were tested for in vitro transferable nature of antibiotic resistance. In eighteen transconjugates en bloc transfer of Cephalexin, Cephalodrine and Kanamycin was seen. In 7 transconjugates en bloc transfer of Amikacin, Cephalexin, Cephaloridine, Kanamycin, Tetracycline and Enrofloxacin was observed. In 2 isolates, en bloc transfer of Amikacin, Cephalexin, Cephaloridine, Kanamycin and Tetracycline, to their respective transconjugates was achieved. In one transconjugate, Cephalexin, Cephaloridine, Enrofloxacin, Kanamycin and Tetracycline was transferred while in another one transfer of Amikacin, Cephalexin, Cephaloridine, Enrofloxacin and Kanamycin was seen. Transfer of Cephalexin, Cephaloridine, Kanamycin was seen in seven transconjugates. Cephalexin and Kanamycin were found to be transferred in JU13T. In 11 transconjugates Ampicillin resistance was not transferred, similarly in 2 transconjugates Co-trimoxazole resistance was not transferred. Sixteen transconjugates, whose wild strains exhibited colicin production, were tested for transfer of colicinogeny to the recipient strain. Out of 16, ten transconjugates (62.5percent) were detected for the ability to transfer colicinogeny. Out of 19 transconjugates obtained eleven were assayed for transferability of unusual biochemical character of the wild parent strain. The biochemical tests viz. Urea hydrolysis, production of H2S, Citrate utilization and Carbohydrate fermentation were assayed. Only three wild strains could transfer ability for raffmose fermentation to their respective transconjugates while no other unusual biochemical character could be transferred from wild to recipient strain. Plasmid profile analysis of wild isolates revealed the possession of single to multiple plasmids (lto4) and that of transconjugates revealed the plasmid profile of their parent strain indicating that all the plasmid carried by wild strain was transferable except for 2 transconjugates which did not reveal the transfer of 2 small sized plasmid of 5.5kbp and 5.8kbp from their respective wild strain. Eight wild strains contained large plasmid of molecular weight ranging from 54kbp to 56kbp. Medium sized plasmids ranging from 17kbp to 32kbp is found in eight strains. Small sized plasmids ranging from 3kbp to 8kbp is isolated from maximum of thirteen strains. Eight isolates showed the presence of one plasmid. In seven wild strains three plasmids were isolated. In three isolates two plasmids were isolated and in only one isolate four plasmids were found. Multiplex PCR based detection of virulence associated genes among 91 E.coli isolates showed the presence of amplified product size of 815bp in 15 (16.8%) isolates indicating the presence of intimin (eae) gene. Eighteen (19.78%) isolates possessed bfp gene of 597bp and 62 (68.13%) isolates harbored EASTl gene of lllbp and in 15 (16.48%) isolates amplification of 382bp product size indicated the presence of invE gene. Out of 91 isolates, 15 isolates which were positive for intimin gene were, typed for 6 intimin variant types by PCR using the total 6 sets of primer viz. eaeĪ²1(811bp), eaeĪ³l(804bp), eaeĪ³2(808bp), eaeĪ“ (833bp), eaeĪ¶, (206bp), and eaeĪ¾ (468bp). Eight out of 15 isolates could be typed for intimin variant types, however in seven isolates no intimin variant type was detected. Two out of 15 (13.3%) were positive for eaeĪ²1 (811bp) primer. One isolate (6.67%) was positive for eaeĪ³l primer (804bp). Five isolates (33.3%)) were positive for eaeĪ³2 (808bp) and no isolate was positive for other three primer viz., eaeĪ“(833bp), eaeĪ¶; (206bp), and eaeĪ¾ (468bp).ThesisItem Open Access BIOCHEMICAL CHARACTERIZATION, ANTIMICROBIAL SENSITIVITY, PCR - BASED DETECTION AND MOUSE PATHOGENICITY OF PASTEURELLA MULTOCIDA FIELD ISOLATES(AAU, Anand, 2004) KIRTIBHAI, PATEL HETALBEN; Purohit, J. H.Two isolates of Pasteurella multocida each from poultry, sheep, rabbit, buffalo and the vaccine strain (P52 strain) were studied for cultural, morphological and biochemical characterization, antimicrobial sensitivity, PCR-based detection and mouse pathogenicity of P. multocida field isolates. The heart blood and organs of the experimental mice were processed for reisolation of P. multocida. The heart blood and reisolated organisms from the experimental mice were subjected to PCRbased detection. All the test isolates were Gram negative, cocco-bacillary rods and produced non-haemolytic colonies on blood agar and failed to grow on MacConkey agar. All the isolates (100%) were positive for oxidase, catalase, indole production, nitrate reduction and fermentations of glucose, mannitol, sucrose and mannose while negative for citrate utilization and fermentation of maltose, arabinose, lactose, dulcitol, salicin and trehalose. The isolates (100%) were sensitive to enrofloxacin, flumequine, chloramphenicol and tetracycline, while nine isolates (90%) were sensitive to norfloxacin and cephalexin, two isolates (20%) were sensitive to penicillin G. All the isolates were (100%) resistant against sulphadiazine while four isolates(40%) were intermediate to penicillin G. Sixty per cent of P. multocida isolates were sensitive to Allium sativum, 40% sensitive to Ocimum sanctum, 20% sensitive to Zingiber officinale, 10% sensitive to Azadirachta indica, 10% sensitive to Curcuma longa. All the isolates were lethal to mice. Significant difference was observed in death time between species and between dilutions. Poultry isolates were found highly pathogenic to mice. Gross changes in mice were characterized by congestion and haemorrhages in most of vital organs. Histopathological lesions were characterized by mild to sever congestion, haemorrhages, and bacterial emboli. PM-PCR from reisolated colonies of P. multocida and gemomic DNA of test isolates were tested by PCR-based characterization, which revaled amplified product of approximately 465 bp size of all the P. multocida isolates, E.coli did not show amplification. Direct PCR from mice blood could not show any amplification.ThesisItem Open Access SURVEILLANCE OF HAEMORRHAGIC SEPTICAEMIA IN GUJARAT STATE WITH ISOLATION, BIOCHEMICAL CHARACTERIZATION AND PCR BASED DETECTION OF PASTEURELLA MULTOCIDA FROM THE FIELD OUTBREAKS(AAU, Anand, 2004) NADODHA, J. V.; Purohit, J. H.The present research work was undertaken with a view to study the surveillance of Haemorrhagic Septicaemia in relation to agro-climatic zones of Guajrat State during the period of January 1998 to December 2003 with isolation and identification of Pasteurella multocida from the field outbreaks in cattle and buffaloes. The isolates were studied for their in vitro antibiotic sensitivity. The PM-PCR was also tried for detection of P. multocida. The data during the study period were collected from the respective sources. The data were compiled and distributed year, month and zonewise. Incidences of HS were correlated to meteorological parameters of different zones. A total of 226 clinical samples (109 each of blood and nasal swabs and eight morbid material) were collected from suspected cases of HS in cattle and buffaloes and processed for demonstration of bipolar organisms and isolation of P. multocida. The isolates were identified by cultural, morphological and biochemical characteristics. The isolates were subjected to in vitro antibiotic sensitivity. The colony PCR was carried out for detection of P.multocida. The highest number of HS outbreaks were recorded during the year 1998 and lowest were found in 2000. The outbreaks were observed throughout the year but more number of outbreaks were found during the rainy season, i.e., in the months of August and September. Amongst the agro-climatic zones, maximum number of outbreaks were recorded in Zone-IV, whereas minimum in Zone-VII. Positive correlation was observed between outbreaks of HS and rainfall, relative humidity and minimum temperature. While no correlation was observed with outbreaks of HS and maximum temperature. A total of four isolates of P.multocida were recovered only from buffaloes blood which showed the presence of bipolar organisms in smear. All the isolates produced non-haemolytic, round, grayish, smooth and mucoid colonies on blood agar but failed to grow on MacConkey agar. The isolates were found non-motile, Gram negative, coccobacillary rods. All the isolates produced oxidase, catalse, indole and reduced nitrate but did not utilise citrate. They fermented glucose, sucrose, mannitol and maimose but not fermented maltose, arabinose, lactose, dulcitol, salicin, inositol and trehalose. All the isolates were found sensitive to gentamicin, chloramphenicol and cephalexin, while two isolates were sensitive to tetracycline, ampicillin and nitrofurantoin. All the isolates were found to be resistant against penicillin-G and streptomycin. All the P. multocida isolates along with P52 vaccine strain amplified product of approximately 465 bp size while E.coli failed to amplify.ThesisItem Open Access DETECTION OF INFECTIOUS BURSAL DISEASE VIRUS (IBDV) FROM BURSAL TISSUE BY RT-PCR AND ITS COMPARATIVE EFFICACY WITH CONVENTIONAL PRECIPITATION ASSAYS(AAU, Anand, 2004) Makadiya, Nirajkumar Ratilal; Jhala, M. K.Infectious bursal disease (IBD), an economically important infectious viral disease of poultry, is caused by Infectious bursal disease virus (IBDV) belonging o to Avibirnavirus genus of Birnaviridae family. The disease causes considerable morbidity and mortality mainiy by immunosuppression. Emergence of very virulent IBDV (vvIBDV) strains in different parts of the world including India during the last couple of decades, have demanded further research efforts in understanding the added complexicity of the disease process and the means to diagnose and control it. To establish the proper control procedures, it is important to characterize the antigenic and virulent properties of the viral strains prevalent in a geographic area, and hence, it is necessary to develop rapid and accurate methods suitable for identifying as well as characterizing the virus. The present study was aimed at screening IBD suspected bursal samples by Reverse transcription-polymerase chain reaction (RT-PCR). Conventional precipitation assays viz. Agar gel immunodiffusion (AGID), Single radial immunodiffusion (SRID) and Counter Immunoelectrophoresis (CIE) were also employed so as to compare their relative sensitivity with RT-PCR as well as among themselves. Two viral RNA extraction protocols viz. phenol-chloroform and Tri Reagent method were compared for their efficacy and suitability for RT-PCR. Incidence of IBDV in relation to epidemiological factors like age, breed and strain of the birds was also studied. Processing of the 147 collected samples by AGID, SRID and CIE revealed 70.07, 72.79, and 78.23 per cent positivity respectively. CIE gave the highest positivity among the precipitation assays and hence was used for comparison of incidences as well as of sensitivity and specificity between the tests. Thirty four samples were from layer birds and 113 from broiler birds, of which, 29 (85.29%) and 86 (76.11%) were positive for IBDV antigen respectively by CIE. The strainwise incidence revealed the maximum incidence in C&M chicks (100%), followed by BV-300 (85.29%), Cobb (78.26%) Hybro (68.75%), Hubchix (57.14%) and Croiler (50.00%). The highest incidence was found in the age group > 3 <= 4 weeks, followed by > 5 <=6 and > 2 <=3 weeks age group, while, the least incidence was found in > 4 < =5 weeks of age. Thirty seven bursal samples and two live IBD vaccines were used for viral RNA extraction by phenol-chlorofomi method (protocol 1) and Tri ReagentĀ® method (protocol 2). RNA extracted by protocol 1 was of poor quality and quantity, and failed to yield targeted amplification in all the samples and vaccines by RT-PCR, where as protocol 2 yielded RNA of good quality and quantity, and produced targeted amplicon of approximately 643 bp and 557 bp respectively for primer pairs P1, P2 and U2, L2, specific for hypervariable region of IBDV VP2 gene, for 31 (83.78%) samples and two vaccines. All the positive samples behaved similarly to the two pairs of primers, which were equally sensitive in detecting the IBDV. Sensitivity of AGIO, SRID and CIE with RT-PCR was 74.19, 77.42 and 87.10 per cent, while that of AGIO and SRID with CIE was 89.57 and 93.04 per cent respectively. Overall agreement of AGID, SRID and CIE with RT-PCR was 78.38, 81.08 and 89.19 per cent, while that of AGID and SRID with CIE was 91.84 and 94.56 per cent respectively. Specificity of all the precipitation assays used with RT-PCR was found 100 per cent.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF ESCHERICHIA COLI STRAINS USING PCR-SSCP AND HETERODUPLEX ANALYSIS(AAU, Anand, 2001) Anand, Sanjeev Kumar; Purohit, J. H.The present study was undertaken with a view to characterize and classify the various strains of E.coli on the basis of SSCP and heteroduplex analysis. DNA from 150 isolates of E.coli, collected earlier from autopsied birds, was extracted using either lysozyme or proteinase K method and V3 region of 168 rRNA gene was amplified by PCR using universal primer designed for that region. The PCR product was subjected to SSCP and heteroduplex analysis on different gel matrices. Five different gel matrices were tested to find out suitability for SSCP and heteroduplex analysis. Among the gels tested 0.5X MDE with 10% glycerol gave the best results. Twenty two different SSCP band patterns were obtained by electrophoresis of ssDNA on non-denaturing gel matrix. The bands of ssDNA in these patterns corresponded to 983 to 379bp equivalent mobility of dsDNA ladder. These patterns contained a minimum of two ssDNA bands to maximum seven. Total 25 bands were found. On heteroduplex analysis keeping the target DNA of Proteus vulgaris as constant, 13 distinct band patterns were observed. The bands of DNA in these patterns corresponded to 900 to 331bp equivalent mobility of dsDNA ladder. These patterns contained a minimum of three to maximum seven heteroduplex bands. The total 17 bands were observed. No relationship could be observed amongst the serotypes, biotypes and SSCP band patterns of E.coli studied. Thus PCR-SSCP and heteroduplex analysis can be used independently for the analysis of the bacterial communities.ThesisItem Open Access DETECTION AND STRAIN VARIATION OF FIELD INFECTIOUS BURSAL DISEASE VIRUS (IBDV) BY RT - PCR - RELP PROFILES(AAU, Anand, 2004) CHOUDHARY, MADHU; Jhala, M. K.Infectious bursal, or Gumboro, disease is an acute, highly infectious viral disease of immature chickens, caused by Infectious bursal disease virus (IBDV) belonging to Avibirnavirus genus of Birnaviridae family. The disease causes considerable morbidity and mortality mainly by immunosuppression. Emergence of very virulent IBDV (vvIBDV) strains in different parts of the world including India, during the last couple of decades, has demanded further research efforts in understanding the added complexicity of the disease process and the means to characterize the genetic variation, so that proper control measures can be taken. The present study was aimed at screening IBD suspected field bursal samples by Agar gel immunodiffusion (AGID), followed by genetic variation study by molecular techniques i.e. Reverse transcription-polymerase chain reaction / restriction fragment length polymorphism (RT-PCR/RFLP). The genetic similarities among the IBDVs were estimated by phylogenetic relationship obtained by PopGene software analysis and Network analysis. Processing of the 37 field bursal samples by AGID revealed IBDV in 23 (62.16 %) samples. These samples and two live IBD vaccines were used for viral RNA extraction by Tri ReagentĀ® method, which yielded RNA of good quality and quantity, and produced targeted amplicons of approximately 643 bp by primer P1 & P2 specific for hypervariable region of IBDV VP2 gene, for 31 (83.78%) field samples and both the vaccines. RT-PCR products from 31 positive bursal and two vaccine samples were further digested with four restriction enzymes (RE) viz. Msp-I, Sac-I, Stu-I and Taq-I for RFLP analysis, which yielded two, one and zero restriction sites and eight different haplotypes (A, B, C, D, E, F, G & H). None of the samples was digested by Sac-I. Majority of the samples behaved identically to the individual RE, as 24, 28 and 28 field samples showed single RE site for Msp-I, Stu-I and Taq-I respectively. Both the vaccine samples behaved similar to the majority of the samples, except for Stu-I by showing an unique additional restriction site placing them exclusively in haplotype D. Locationwise, haplotype F was found at maximum four locations, and strainwise, haplotype A was only found in a layer strain (BV-300). Phylogenetic analysis was done with PopGene software, which clustered the 31 field IBDVs and two vaccine strains into six molecular groups (designated as IG1, IG2, IG3, IG4, IG5 and V), based on RE profiles among the IBDVs. These molecular groups were compared for the genetic similarities among them, as well as with 14 international IBDV strains (seven classical; two variant; three classical vaccine and two vvIBDV strains) taken in silico from GeneBank database and digested similarly with the same four REs by DNAsis software (Pharmacia, Uppsala, Sweden). Two field samples falling in molecular group IG5 differed maximally from majority of the field and vaccine samples, by possessing an additional Msp-I site. Taq-I digestion revealed a new restriction site in majority of the samples possibly indicating very virulent nature of the field IBDVs. Different degrees of similarity observed among the field samples, suggested circulation of variant IBDVs in the field. Network analysis further emphasized the heterogenicity among the field and vaccine IBDV strains to the reference IBDV strains taken from GeneBank. IG5 showed maximum seven and five mutations from the central cluster of the reference classical IBDV strains and vaccine strains (included in the study) respectively. IG4 showed only one mutation with V group and was thus maximally similar than the other molecular groups. Among the 14 reference strains, both the wIBDV (849VB and T1/TW) and variant (Variant A and MTA) strains showed higher number of mutations from the central cluster.