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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 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 Reagent® 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 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 Reagent® 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.ThesisItem Open Access CHARACTERIZATION OF PASTEURELLA MULTOCIDA ISOLATES BY THEIR OUTER MEMBRANE PROTEIN PROFILES, RAPD PATTERNS AND T0X A GENE DETECTION(AAU, Anand, 2004) JAIN, ANSHU; ROY, ASHISHPasteurella multocida is one of the most fascinating gram-negative bacteria and is a commensal of the upper respiratory tract of many animal species. However, under predisposing circumstances the organism is the etiological agent of a wide range of economically important diseases. Thus to understand this microbial diversity, characterization of the isolates was done by their outer membrane protein profdes, RAPD patterns and lox A gene detection. For characterization of the isolates by their outer membrane protein (OMP) profiles, the cultures were grown in BHl broth and then subjected to sarkosyl method of OMP extraction. The electrophoretic protein profiles of the capsular type B showed the presence of 9 to II protein fractions. Based on band intensity, two polypeptides with approx. MW of 31.7 kDa and 34.9 kDa were considered to be the major OMPs. The OMP patterns of buffalo isolates revealed a total number of 9 bands with MW ranging from 21.1 to 89.2 kDa. While the H.S.Vaccine (P52) strain revealed a total number of II bands with protein fractions of 23.5 to 97.4 kDa. The OMP profiles from the field isolates of capsular type A revealed about 8 to 10 discernible protein bands. Based on band intensity, two polypeptides with MW of 31.7 kDa and 34.9 kDa were considered to be the major OMPs in all the isolates except one isolate of sheep. The rabbit isolates demonstrate a total number of 9 to 10 bands with molecular weights of 21 to 102 kDa. The protein fractions of sheep isolate revealed a total number of 8-9 bands with MW ranging from 21 to 135 kDa. The SDS-PAGE profiles of poultry isolates revealed a total of 9 bands with molecular weights of 18.6 to 88 kDa. The present research work was also carried out to study the effect of iron restriction on the OMP composition of P. multocida isolates. Among the capsular type B isolates, buffalo isolates expressed an additional protein of molecular weight 102 kDa while the vaccine strain (P52) did not express any additional protein as compared to iron sufficient medium. In capsular type A isolates, the rabbit isolates expressed additional protein band of molecular weight 77 kDa on a medium supplemented with iron chelator. Sheep isolates expressed additional protein band of 44 kDa in iron restricted medium and the poultry isolates, revealed protein bands of 44 kDa and 98 kDa when grown in iron restricted medium. To determine the genetic differences between the isolates, two random primers OPA-11 and 0PG-13 were used. The random primer OPA-11 generated nine different profiles. All the isolates belonging to different species viz. buffalo, rabbit, sheep and poultry isolates showed distinct RAPD profiles specific to their species of origin. All the buffalo isolates showed similar profile. While H.S. vaccine strain showed different profile from the buffalo isolate. The two rabbit isolates showed similar profile. The two sheep isolates showed two distinct RAPD profiles. The eleven poultry isolates showed four different RAPD profiles. The random primer OPG-13 showed only three RAPD profiles. All the capsular type A isolates of rabbit, sheep and poultry origin had identical RAPD pattern except one sheep isolate PS-1. Capsular type A exhibited all the bands present in capsular type B except the heaviest band of 1438 bp. PS-1 isolate expressed eight bands, all seven of capsular type B and an additional band of 773 bp. By Popgen and Network analysis it was found that 100% similarity exists among all the buffalo and among the rabbit isolates. While dissimilarity exists among the sheep as well as among the poultry isolate. The H.S. vaccine strain shows similarity to one of the poultry isolate. The tox A gene was detected in only 3 isolates (2 from sheep and one from poultry) of capsular type A while capsular type B isolates did not show any amplification product.ThesisItem Open Access GENOMIC FINGERPRINTING OF ESCHERICHIA COLI STRAINS USING REPETITIVE SEQUENCE BASED POLYMERASE CHAIN REACTION(AAU, Anand, 2004) NIKAM, AMAR KISHOR; Purohit, J. H.Repetitive element PCR (rep-PCR) uses outward facing pnmers to amplify multiple segments of DNA located between conserved repeated sequences interspersed along the bacterial chromosome. Polymorphism of rep-PCR amplification products can serve as strain specific molecular fingerprints. In this study, capability of repetitive BOX elements, enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) was tested to detect genetic diversity among Escherichia coli strains recovered from autopsied poultry birds showing different pathological conditions. The DNAs from 52 avian E.coli isolates were extracted and used to amplify BOX, ERIC and REP sequences. DNA from avian strains produced 1-11 bands by BOX-PCR, 2- 13 bands by ERIC-PCR, where as 1-13 bands by REP-PCR. All these primers showed good discriminating power. BOX-PCR produced 23 different products where as ERIC-PCR and REP-PCR produced 26 and 25 products, respectively. All bands were showing 100 per cent polymorphism. Dendrograms based on different patterns revealed extensive genetic diversity among avian strains. Three major clusters were formed by BOXPCR and ERIC-PCR showing less than 25 per cent similarity where as two major clusters were formed by REP-RCR showing less than 25 per cent similarity. Network analysis of these patterns showed highly complex network suggesting intense heterogeneity among E.coli strains. Network obtained by BOX primer was most complex where as network obtained by REP-RCR was least complex among these three primers. The results obtained were compared with Serotypes, Biotypes and SSCP analysis. No correlation could be established but those isolates that could not be analyzed or discriminated by above typing techniques were easily analyzed and discriminated by rep-PCR analysis. These results suggest intense heterogeneity or genetic diversity among E.coli strains studied. Also, rep- PCR has discriminating capacity that could improve the studies needed to understand genetic makeup of E.co//strains.ThesisItem Open Access PREVALENCE OF PESTE DES PETITS RUMINANTS (PPR) VIRUS IN SMALL RUMINANTS OF GUJARAT AND ITS CHARACTERIZATION BY RT - PCR / RFLP AND SSCP PROFILES(AAU, Anand, 2004) TIWARI, ASHISH; JHALA, M. K.Peste des petits ruminants (PPR) is an acute viral disease of small ruminants caused by a Morbillmrus and characterized by fever, oculonasal discharges, stomatitis, diarrhoea and pneumonia. In India, PPR was first reported in 1987 from Tamil Nadu and for several years, the disease was thought to be restricted to southern India only, however in 1994, and after a series of PPR outbreaks was reported from many northern states as well as from West Bengal. The disease causes severe losses to small ruminant production and is presently considered as one of the major threats to about 200 million small ruminant population of the country. The present study was planned to carry out a seroprevalence study of Peste des petits ruminants virus (PPRV) in Gujarat state and to derive estimates of overall, zonewise, districtwise, yearwise and specieswise seroprevalence. The study was also aimed to detect PPRV in clinical samples using sandwich-ELISA and to derive estimates of overall, locationwise, specieswise and agewise prevalence of PPRV. Also, RT-PCR was standardized and applied for detection of PPRV from field samples and its sensitivity and specificity was compared with sandwich-ELISA. Further, an attempt was made to assess possible genetic variation among the field and vaccine PPRVs by RT-PCR /RFLP and SSCP profiles. A total of 520 Serum samples collected from 17 districts of the five different geographical zones viz. North, South, Central, Saurashtra and Kachchh of Gujarat state were screened for PPR specific antibodies using PPR c-ELISA kit developed by National Rinderpest Laboratory, Division of Virology, IVRI, Mukteswar. One hundred ninty one samples were positive for PPR antibodies yielding an overall seroprevalence of 36.70 per cent in the small ruminant population included in the study. Among the zones, the seroprevalence was 53.67, 33.33, 19.00, 43.18 and 15.00 per cent in North Gujarat, South Gujarat, Central Gujarat, Saurashtra and Kachchh zones, respectively. Among the districts sampled, highest seroprevalence of PPR was observed in goat population of Bharuch district (100%) and the lowest seroprevalence was recorded in goat populations of Panchmahal, Navasari and Surat districts (0.00%, each). The seroprevalence in other districts ranged from 15.00 to 68.18 per cent, of which Bhavnagar (68.18%), Mehsana and Sabarkantha (67.50%, each) showed higher seroprevalence rates. For the years 2003 and 2004, seroprevalence of PPRV in small ruminants was 34.04 and 38.25 per cent, respectively. In the year 2003, seroprevalence of PPRV in sheep and goat was 22.78 and 42.20 per cent, respectively. These values for the year 2004 were 54.02 and 32.65 percent, respectively. Specieswise seroprevalence of PPRV in sheep and goat was 39.16 and 35.59 per cent, respectively. A total of 30 clinical samples from 25 small ruminants (10 sheep and 15 goats) suspected of PPRV, were tested for PPRV antigen using PPR sandwich-ELlSA kit developed by National Rinderpest Laboratory, Division of Virology, IVRI, Mukteswar. Fifteen animals were found positive yielding an overall incidence rate of 60.00 per cent. Out of 20 animals (including 10 sheep and 10 goat) suspected of PPR at Sheep Breeding Farm, Patan, 11 (55.00%) were found positive; whereas, four (80.00%) out of five goats suspected of PPR at Veterinary Polyclinic, Vadodara, yielded positive results. Incidence rate of PPR by sandwich-ELISA was 30.00 and 80.00 per cent in sheep and goats, respectively. Out of the 25 animals, five, eight, nine and three animals belonged to age groups of 0-24, 24-48, 48-72, and >72 months, respectively, showing the respective incidence of PPRV as 100, 62.50, 44.44 and 33.33 per cent. All the 30 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 the F-gene specific primer pairs F1-F2 and Flb-F2d. Reference vaccine virus as well as eight (26.67%)) of the 30 clinical samples, including four each of blood and swab samples produced approximately 372 bp and 448 bp expected amplicons with primer pairs F1-F2 and Flb-F2d, respectively. The amplicons from all the positive samples migrated similarly in the gel at respective locations expected for the two primer pairs used. Twenty two (73.33%)) samples including 21 blood samples and one oral swab suspension as well as the blood sample taken from apparently healthy goat failed to produce the targeted amplification with both the primer pairs. Out of 30 clinical samples tested for PPRV, 15 and eight samples were positive by sandwich-ELISA and RT-PCR, respectively. None of the samples negative in sandwich-ELISA yielded positive amplification in RT-PCR. Relative to sandwich ELISA, sensitivity and specificity of the RT-PCR was 53.33 and 100.00 per cent, respectively. Overall agreement between the two tests was 76.67 per cent. RT-PCR products amplified with primer pair F1-F2, were ftirther digested with three restriction enzymes viz. Msp-l, Pst-l and EcoRl for RFLP analysis. RT-PCR products of all the eight positive samples and the reference vaccine produced similar RFLP patterns with each of the three REs. Digestion of RT-PCR products with Msp-l yielded two fragments of approximately 116 bp and 256 bp each. Digestion with Pst-l resulted in two fragments of 155 bp and 217 bp each. None of the sample was digested with EcoRl, thus showing the original RT-PCR product. To detect any possible mutation in the targeted sequence of PPRV F-gene, RTPCR products amplified with primer pair F1-F2, were subjected to SSCP analysis on 0.5X MDETM gel (with 10% glycerol) in 0.5X TBE buffer system. Seven out of the eight RT-PCR positive field samples and the reference vaccine virus (Sungri isolate) yielded similar SSCP profiles producing three bands each, one corresponding to faster migrating non-denatured PCR product and two relatively slow migrating bands corresponding to single stranded DNA. One sample (P/2) collected from a goat, generated a SSCP profile different fi-om all the other field samples as well as the vaccine virus. It yielded five bands, one of which corresponded to non denatured PCR product and the other four bands of single stranded DNA, possibly indicating a genetic variation in the targeted F-gene 372 bp amplicon.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 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, 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 RTPCR. 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-chloroform method (protocol 1) and Tri Reagent® 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 AGID, SRID and CIE with RT-PCR was 74.19, 77.42 and 87.10 per cent, while that of AGID 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 ISOLATION AND IDENTIFICATION OF PASTEURELLA MULTOCIDA OF ANIMAL AND AVIAN ORIGIN BY CULTURAL, BIOCHEMICAL AND MOLECULAR TECHNIQUES(AAU, Anand, 2004) JAVIA, BHAVESHKUMAR B .; ROY, ASHISHThe present study was undertaken with a view of isolation and identification of P. multocida from suspected cases of pasteurellosis in different animals and birds. A total of 89 samples were collected from suspected cases, of which 13 (14.6 %) P. multocida isolates (3 from buffalo, 2 from rabbit, 1 from sheep and 7 from poultry) were isolated using blood agar as primary culture medium. These isolates were studied for their biochemical behaviours, in vitro antibiotic sensitivity pattern, P. multocida species specific PCR (PM-PCR) and molecular characterization by PCR-SSCP and repetitive sequence based PCR. All the isolates produce non-haemolytic colonies on blood agar and failed to grow on MacConkey agar. On biochemical behaviours, all the 13 isolates (100%) were found positive for oxidase, catalase, indole production, nitrate reduction and fermentation of glucose, mannitol, sucrose and mannose. All the 13 isolates (100%) v'cre negetive for citrate utilization test and could not ferment maltube, arabinose, lactose, dulitol, salicin and trehalose. All the isolates revealed similar biochemical behaviour. All the isolates (100%) were sensitive to enrofloxacin, flumequine, chloramphenicol and cephalexin, while twelve isolates (92.31%) were sensitive to petloxacin, six isolates (46.15%) were sensitive to ciprofloxacin and four isolates (30.77%) were sensitive to amoxycillin. All the thirteen P. multocida isolates (100%) were found to be resistant to sulphadiazine. All the 13 field isolates along with one vaccine strain (P. multocida P52) and five other bacterial cultures were tested by PM-PCR, which revealed an amplified product of approximately 465 bp size for all the P. multocida isolates, while other bacterial culture did not. The PM-PCR product was subjected to SSCP analysis on 0.5X MDE gel with 10% glycerol. Total six different SSCP band patterns were obtained. These patterns contained a minimum one to maximum four ssDNA bands. Total numbers of bands across the patterns were found to be eight. In this study capability of rep-PCR [repetitive BOX elements, enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) polymerase chain reaction (PCR)] was tested for molecular characterization of P. multocida isolates. Fingerprinting with BOX-, ERIC- and REP-PCR generated 14, 16, and 13 different bands with molecular weight ranging from 310-1160 bp, 55-900 bp and 44- 500 bp, respectively. The number of bands amplified in different samples by BOX-, ERIC-, and REP-PCR were varied from 2 to 7, 6 to 11, and 1 to 10 with the band frequency ranging from 0.1429 to 0.7857. 0.0714 to 1.0000 and 0.1429 to 0.9286 respectively. The bands of BOX-, ERIC-, and REP-PCR were 100 %. 75 % and 100 % polymorphic, respectively. Dendrogram were constructed by POPGENE software using bands information from BOX-, ERIC-, and REP-PCR. Dendrogram of BOX-PCR data showed 61 % similarity among all buffalo isolates including vaccine strain and 91 % similarity among both rabbit isolates. Poultry isolates were formed two groups with no similarity. Dendrogram of ERIC-PCR data showed 100 % similarity among all buffalo isolates including vaccine strain and between both the rabbit isolates. All the poultry isolates showed 92 % similarity while sheep isolate showed 77 % similarity with all poultry isolates. Dendrogram of REP-PCR showed 100 % similarity among all buffalo isolates including vaccine strain, between both the rabbit isolates and also among all the poultry isolates. Sheep isolate was branched out separately. REP-PCR found most efficient to group isolates from different host origin. Dendrogram based on overall pooled data showed 89 % similarity among all buffalo isolates including vaccine strain, 80 % similarity between both rabbit isolates and 65 % similarity among all poultry isolates. Sheep isolate showed 36 % similarity to poultry isolates. Similar groups were also formed by NETWORK analysis. It is concluded that PM-PCR are useful for rapid and specific detection of P. multocida isolates. SSCP and rep-PCR are useful to identify and characterize P. multocida isolates which can not be classified and characterized by conventional ways.