Loading...
Assam Agricultural University, Jorhat
Assam Agricultural University is the first institution of its kind in the whole of North-Eastern Region of India. The main goal of this institution is to produce globally competitive human resources in farm sectorand to carry out research in both conventional and frontier areas for production optimization as well as to disseminate the generated technologies as public good for benefitting the food growers/produces and traders involved in the sector while emphasizing on sustainability, equity and overall food security at household level.
Genesis of AAU -
The embryo of the agricultural research in the state of Assam was formed as early as 1897 with the establishment of the Upper Shillong Experimental Farm (now in Meghalaya) just after about a decade of creation of the agricultural department in 1882. However, the seeds of agricultural research in today’s Assam were sown in the dawn of the twentieth century with the establishment of two Rice Experimental Stations, one at Karimganj in Barak valley in 1913 and the other at Titabor in Brahmaputra valley in 1923. Subsequent to these research stations, a number of research stations were established to conduct research on important crops, more specifically, jute, pulses, oilseeds etc. The Assam Agricultural University was established on April 1, 1969 under The Assam Agricultural University Act, 1968’ with the mandate of imparting farm education, conduct research in agriculture and allied sciences and to effectively disseminate technologies so generated. Before establishment of the University, there were altogether 17 research schemes/projects in the state under the Department of Agriculture. By July 1973, all the research projects and 10 experimental farms were transferred by the Government of Assam to the AAU which already inherited the College of Agriculture and its farm at Barbheta, Jorhat and College of Veterinary Sciences at Khanapara, Guwahati.
Subsequently, College of Community Science at Jorhat (1969), College of Fisheries at Raha (1988), Biswanath College of Agriculture at Biswanath Chariali (1988) and Lakhimpur College of Veterinary Science at Joyhing, North Lakhimpur (1988) were established. Presently, the University has three more colleges under its jurisdiction, viz., Sarat Chandra Singha College of Agriculture, Chapar, College of Horticulture, Nalbari & College of Sericulture, Titabar. Similarly, few more regional research stations at Shillongani, Diphu, Gossaigaon, Lakhimpur; and commodity research stations at Kahikuchi, Buralikson, Tinsukia, Kharua, Burnihat and Mandira were added to generate location and crop specific agricultural production packages.
Browse
6 results
Search Results
ThesisItem Open Access MOLECULAR AND ANTIGENIC CHARACTERIZATION OF CLASSICAL SWINE FEVER VIRUS ISOLATED FROM NORTH EASTERN REGION OF INDIA(Assam Agricultural University, Khanapara, Guwahati, 2017-07) Roychoudhury, Parimal; Sarma, Dilip KumarThe present study “Molecular and antigenic characterization of classical swine fever virus isolated from North Eastern region of India” was undertaken to explore the genogroups and subgroups of CSF virus isolated from different geographical location of North Eastern region of India, their genetic relatedness within and when compared to other isolates from different areas from the available data. Antigenic relatedness of the isolates to vaccine virus and to a local isolate(Lab. No.852) were studied by serological tests such as liquid phase blocking ELISA (LPB-ELISA) and neutralization peroxidase linked assay(NPLA).Antisera for the serological tests were raised against vaccine virus and a local isolate(Lab. No.852) in pigs and rabbits. Tissue samples collected during January 2011 to October 2012 were screened for Classical swine fever(CSF) virus antigen by sandwich ELISA(sELISA) and subsequently confirmed nested reverse transcriptase polymerase chain reaction (RT-nPCR). Out of 32 positive samples, virus could be isolated from 26 samples in PK-15 cell line. Seventeen isolates were selected for molecular characterization and 20 isolates for antigenic characterization. Virus Infectivity titres are expressed as log value of tissue culture infectious dose(logTCID50) per volume(0.1ml) of virus suspension, which varies in the range of 3.75 to 4.50 among the 20 isolates used for antigenic characterization. Cloning of three partial genomic region i.e.271nt fragment of 5′UTR, 271nt of E2 and 449nt of NS5B were carried out in pDrive vector and were subsequently sequenced by outsourcing. Phylogenetic analysis of the isolates using 150nt of UTR, 190nt of E2 and 409nt of NS5B revealed that 15 out of 17 isolates belonged to genogroup 1.1 and 2 isolates belong to genogroup 2.2. Nucleotide polymorphism at several locations were observed when compared to Alfort/187(GenBank Acc. No. X87939).Pair wise distance analysis of the E2 sequences of the 17 isolates showed 98.4% to 100% similarity within the same 1.1 genogroup While, two sequences of 2.2 genogroup showed 100% similarity with each other. Pair wise distance analysis of the 5′UTR sequences of the 17 isolates showed very close similarities, ranged between 99.3% to 100% While, two sequences of 2.2 genogroup showed 100% similarity with each other. Pair wise distance calculation of the NS5B sequence within the seventeen isolates from different parts of North Eastern India revealed overall similarity ranges from 85.1% to 100%. Homologous 2.2 group isolates showed 100% similarity with each other. Six representative isolates (ML-1, ML-2, ML-3, ML-4, AS-1 and AS-3) recovered during August and September 2011 within the genogroup 1.1 were compared among themselves as well as with available published sequences during 2005-2007. The results clearly indicate the close relation of the present isolates with the previously isolated virus from same North Eastern Region. However, the sequences are slightly diverge from each others, compared during 2005 to 2012 clearly indicates the endemic status of the region. Antigenic characterization of 20 isolates were carried out by comparing 50% inhibition log titre in LPB-ELISA and neutralization inhibition log titre in NPLA using hyperimmune serum against vaccine virus and a local isolate(Lab. No.852) raised in rabbit and pig.The 50% inhibition log titre of CSF virus isolates in LPB-ELISA obtained by using rabbit hyperimmune serum against the vaccine virus as well as against local isolate (Lab. No. 852) ranged from 1.505 to 2.107. The overall mean titre of CSF virus isolates in LPB-ELISA with the vaccine virus antiserum was 1.820±0.032 and with the local isolate (Lab. No.852) antiserum was 1.806±0.029 . The results of statistical analysis revealed no significant difference (’t’ value=0.373; P<0.05) between the mean of the 50% inhibition titre of CSF virus isolates with the vaccine and local isolate (Lab. No.852) antiserum in LPB-ELISA. The 50% inhibition log titre of the present isolates in LPB-ELISA obtained by using pig hyperimmune serum against the vaccine virus as well as against local isolate (Lab. No. 852) ranged from 1.505 to 2.107. The overall mean titre of CSF virus isolates in LPB-ELISA with the vaccine virus antiserum was 1.806±0.095 and with the local isolate antiserum was 1.818±0.025 . The results of statistical analysis revealed no significant difference (’t’ value=0.331; P<0.05) between the mean of the 50% inhibition titre of CSF virus isolates with the vaccine and local isolate antiserum in LPB-ELISA. Neutralization log titre of CSF virus isolates in NPLA using the rabbit hyperimmune serum raised against the vaccine virus as well as serum raised against a local isolate(Lab. No.852) ranged from 1.505 to 1.982. The overall mean titre of CSF virus in NPLA using rabbit hyperimmune serum, the vaccine virus antiserum was 1.754±0.028 and with the local isolate (Lab. No.852) antiserum was 1.789±0.026. The results of statistical analysis revealed no significant differences (‘t’ value,0.882; P<0.05) between the mean of the neutralization titre of CSF virus isolates with the vaccine virus and reference field virus antiserum in NPLA. The neutralization log titre of CSF virus isolates in NPLA using pig hyperimmune serum raised against the vaccine virus, ranged from 1.505 to 1.982 and against the local isolate(Lab. No.852), ranged from 1.505 to 1.806. The overall mean titre of CSF virus in NPLA using pig hyperimmune serum, the vaccine virus antiserum was 1.758±0.03 and with the local isolate (Lab. No.852) antiserum was 1.746±0.021 . The results of statistical analysis revealed no significant differences (‘t’ value,0.319; P<0.05) between the mean of the neutralization titre of CSF virus isolates with the vaccine virus and local isolate(Lab. No.852) antiserum in NPLA. Comparison of neutralization pattern of the isolates compared to vaccine virus antiserum and local isolate(Lab. No.852) antiserum by LPB-ELISA and NPLA using rabbit and pig hyperimmune serum revealed that the 50% inhibition mean log titre obtained in LPB-ELISA was slightly higher than the mean neutralization log titre obtained in NPLA in all the cases, but the mean titre obtained in the tests did not differ significantly. Antigenic characterization of six selected isolates (MZ-1,AS-1,MN-1,TR-1,AR-1,ML-1) representing genogroup 1.1 and 2.2 were selected for LPBE and NPLA using monoclonal antibody specific for CSF virus E2 glycoprotein. In LPBE, 50% inhibition log titre of the five isolates (MZ-1,AS-1,MN-1,AR-1 and ML-1) along with the vaccine virus and field isolate was 0.903.The neutralization log titre of CSF virus isolates in NPLA using monoclonal antibody showed similar log titre of 0.779 in four isolates(MZ-1,AS-1,AR-1,ML-1) along with the vaccine virus and field isolate, while two isolates (MN-1 and TR-1) showed a titre of 0.602.The overall mean titre of six CSF virus isolates with the reference vaccine and field virus isolate using monoclonal antibody in LPB-ELISA was 0.865±0.037 and in NPLA was 0.734±0.028. Statistically, there was no significant difference (‘t’ value=0.015; P<0.05) between the mean of the 50% inhibition log titre and neutralization log titre of CSF virus isolates in LPB-ELISA and NPLA using monoclonal antibody. Comparison of 50% inhibition log titre and neutralization log titre of the isolates among the two geno groups and their subgroup 1.1 and 2.2 by LPB-ELISA and NPLA revealed no significant differences in the neutralization pattern using antisera against vaccine virus and a local isolate (Lab. No.852).ThesisItem Open Access CHARACTERIZATION OF OUTER MEMBRANE PROTEINS OF Pasteurella multocida OF PORCINE ORIGIN(Assam Agricultural University, Khanapara, Guwahati, 2016-07) Borah, Bornali; Saikia, G. K.The present study was undertaken with a view to isolate and identify Pasteurella multocida from apparently healthy, diseased and dead pigs by both conventional and molecular methods, to study the pathogenicity of the isolates in mice, to prepare partially purified outer membrane proteins (OMPs) from most local virulent porcine strains (capsular types A and D), to study the protein profile of OMPs extract by SDS-PAGE, to identify the immunogenic proteins of OMPs by western blotting, to purify these proteins by size exclusion chromatography and to study the immunogenic potential of oil-adjuvanted vaccine prepared from the purified immunodominant OMPs in mice challenged with virulent homologous and heterologous capsular types of P. multocida. In the present investigation, a total of 357 samples including nasal swabs (187), tracheal swabs (18), lung (125) and heart blood (27) from apparently healthy, diseased and dead pigs were examined for isolation of P. multocida. Of these, 17 (4.76%) samples yielded P. multocida. More isolates were obtained from nasal swabs (9) from apparently healthy and diseased pigs than that of tracheal swabs (4) and lung tissues (4) from apparently healthy and dead pigs. All the 17 isolates showed cultural, morphological, staining and biochemical characteristics typical of P. multocida. The isolates were further confirmed as P. multocida on the basis of detection of species-specific gene (KMT1) by P. multocida species-specific PCR (PM-PCR). Among the 17 isolates, 6 (35.29%) were identified as capsular type A and 11 (64.71%) were identified as capsular type D based on multiplex capsular PCR results targeting hyaD-hyaC and dcbF genes, respectively. Mouse pathogenicity trial of 19 isolates of P. multocida revealed that the isolates induced 33.33 to 100.00 per cent mortality within 72 hours of inoculation. Two isolates (LS-3 and NS-4) were found to be comparatively more pathogenic causing 100.00 per cent mortality in the inoculated mice within 24-48 hours post inoculation and were selected for extraction of OMPs. Two most pathogenic strains of P. multocida, one each of types A and D (LS-3 and NS-4) were selected for extraction of OMPs. Analysis of OMPs of P. multocida type A by SDS-PAGE revealed presence of 21 protein bands with MWs ranging from 192.1 to 20.0 kDa. Among these protein, 36.8 and 25.0 kDa proteins appeared to be the major OMPs followed by 20.0, 56.5, 83.4, 47.4, 76.1, 51.2, 35.0, 99.2, 67.5 and 105.5 kDa protein bands based on band intensity in SDS-PAGE. While, OMPs of P. multocida type D showed presence of 22 protein bands with MWs ranging from 134.0 to 15.0 kDa. Among these protein, 35.7 and 25.0 kDa proteins appeared to be the major OMPs followed by 15.0, 56.2, 99.2, 47.1, 44.4, 66.5, 50.8, 78.5, 40.8 and 73.7 kDa proteins. The comparative evaluation of protein profiles of OMPs of serotypes A and D of P. multocida of porcine origin revealed that both the types shared three proteins with MWs 134.0, 99.2 and 25.0 kDa, of which the 25.0 kDa protein was found to be a major OMPs based on band intensity. The western blot analysis of the partially purified OMPs of P. multocida type A showed five major immunogenic proteins of MWs 83.4, 56.5, 36.8, 25.0 and 20.0 kDa giving strong immunostaining reaction with hyperimmune serum raised in rabbits. On the other hand, the partially purified OMPs of P. multocida type D showed five major immunogenic proteins of MWs 99.2, 56.2, 35.7, 25.0 and 15.0 kDa. Both the types shared a major immunogenic protein with MW 25.0 kDa. Size exclusion chromatography showed 10 peaks in OMPs extract of P. multocida type A and 13 peaks in OMPs extract of type D. In P. multocida type A, the five peaks of OMPs contained the protein fractions with molecular masses 83.4, 56.5, 36.8, 25.0 and 20.0 kDa, while in type D, the five peaks contained the protein fractions with MWs 99.2, 56.2, 35.7, 25.0 and 15.0 kDa. Mice immunized with oil-adjuvanted purified OMPs vaccines of P. multocida types A and D were challenged with 1x102 cfu of live P. multocida types A and D through subcutaneous (s/c) route and were found to be fully protective (100%). The organisms could not be re-isolated from the inoculated mice sacrificed after 7 days. The control group of mice showed 100 per cent mortality and died of septicaemia within 48 to 72 hours after challenge with 1x102 cfu of live P. multocida types A and D. Re-isolation of the organisms used for challenge infection was possible from the heart blood and the internal organs of dead mice of the control group.ThesisItem Open Access DEVELOPMENT OF USER FRIENDLY DIAGNOSTICS AND CELL CULTURE ADAPTED VACCINE CANDIDATE FOR DUCK PLAGUE(Assam Agricultural University, Khanapara, Guwahati, 2016-05) NEHER, SAMSUN; Das, S. K.Duck plague or duck viral enteritis is an acute and contagious viral disease of ducks, geese swan and other species of the order Anseriformes. The disease is responsible for significant economic losses in duck husbandry due to heavy mortality, condemnation and decrease in egg production in duck. Besides clinical and postmortem findings, laboratory diagnosis is essential to confirm the disease in cases of outbreaks. Conventional diagnostic methods are labour intensive, time consuming and less sensitive. There is an urgent need for development of rapid, sensitive and cost effective in house as well as user friendly diagnostic test so as to confirm the disease at clinical phase in the field itself. Again, vaccination is the only available option for prevention and control of the disease. The present study was undertaken to develop user friendly diagnostics and potent cell culture adapted vaccine to control duck plague virus (DPV) infection. During the study period a total of 29 outbreaks of duck plague were attended. Various clinical and post mortem samples were processed for detection of viral DNA by PCR and viral antigen by S-ELISA. Serum samples collected from different districts were tested for presence of antibody by I-ELISA and Dot-ELISA. Duck plague virus was isolated in duckling, duck egg and DEF primary cell culture from the field tissue sample. Sequence and phylogenetic analysis of local DPV isolate and a vaccine strain was done to see the circulating virus in Assam. A cell culture adapted vaccine was developed, and safety and potency test was conducted to see the efficacy of the vaccine. In sero-epidemiological study, among the 445 serum samples tested by I-ELISA 348 (78.20%) were found positive for DPV antibody, however in Dot-ELISA 149 (33.48%) were found to be positive. A total of 380 samples were collected from clinically affected (107) and dead ducks (273). S-ELISA showing positive results in 25 (23.36%) in clinical samples and 188 (68.86%) post mortem samples, however in PCR a total of 231 (84.61%) post mortem samples and 68 (63.55%) clinical samples showed positive for duck plague virus specific nucleic acid. The present study showed that PCR is the suitable and reliable test for detection of duck plague virus. Among different tissue samples collected from dead birds, liver and spleen were found to be most suitable. In cases of clinical samples ducks whole blood was found to be preferred sample than the cloacal swabs and tracheal swab. However, due to simplicity of collection, cloacal swab may be the choice of sample from large flock. Reviving of field isolate in primary host followed by isolation in duck embryo and duck embryo fibroblast (DEF) cell culture made 100% recovery of virus. However, the DEF cell culture was found to be more suitable than embryonated duck egg for isolation. Sequence and phylogenetic analysis of the local isolates and a vaccine strain showed a close relationship among the local isolates with the vaccine strain. Local isolates also showed a significantly high degree of sequence identity with other DPV isolates from China, Vietnam, Korea and Germany. A highly virulent local strain was selected as vaccine candidate and adapted in CEF primary cell culture, whereas standard vaccines strain was adapted in CEF primary cell culture as well as in vero cell line. In safety and potency test of the CEF cell culture adapted DPV vaccine strain, ducklings were vaccinated with 0.5 ml of 103, 104 and 105 TCID50/ml dose of vaccine virus. All doses of vaccine were found to be safe and optimum for eliciting protective immunity in the vaccinated ducklings, and conferred 100% protection of ducklings challenged with 1 ml of 100 DID50 of virulent DPV. Thereby, the minimum dose containing 1 ml of 103 TCID50/ml of vaccine virus can be considered as optimum vaccine dose for providing protection, which can be further used for protection of ducks from duck plague. The present study clearly showed that duck plague is endemic in Assam causing high mortality in ducklings as well as in growing and adult ducks. Diagnostic tests I-ELISA, S-ELISA and Dot-ELISA along with molecular technique PCR could be companion diagnostic tools for confirmation of DPV as well as assessment of virus antibody. Significantly development of cell culture adapted vaccine and conferring of 100% protection can be an achievement of the study.ThesisItem Open Access CHARACTERIZATION OF BETA-2 TOXIN PRODUCING Clostridium perfringens ISOLATED FROM ANIMALS, FOODS OF ANIMAL ORIGIN AND HUMAN(AAU, Khanapara, 2016) Haque, Shahnaz; Bhattacharyya, Dilip KumarThe present work was undertaken with a view to study the prevalence of C. perfringens in animals, birds, foods of animal origin and human. A total of 661 samples were collected from different sources, viz. animal faecal samples (433), bird faecal samples (93), human stool samples (49) and food of animal origin (86), which revealed 119 isolates of C. perfringens. The isolates were confirmed by the detection of C. perfringens alpha toxin (cpa) gene and were characterized in respect to beta 2 toxin (cpb2) gene and beta 2 toxin (CPB2). Sample wise distribution of C. perfringens was 16.17, 27.96, 32.65 and 8.14 percent in animals, birds, human stool and foods of animal origin, respectively. Irrespective of health status, isolation of C. perfringens was done from different animal and bird species. However, none of the samples collected from buffalo and horse yielded any C. perfringens isolate. All the human stool samples positive for C. perfringens were found to have a history of diarrhoea. Characterization of C. perfringens isolates in respect to the major toxin genes revealed presence of cpa (alpha), cpb (beta), etx (epsilon) and ιA (iota) toxin gene, either alone or with different combinations. Based on distribution of major toxin genes, a total of 25 isolates were identified to be of type A, one isolate as type B and 93 isolates to be of type C. Among them, type A was isolated from animal (16), birds (7) and human (2), while the only type B isolate belonged to animal. The remaining type C isolate of C. perfringens were recovered from animal (53), birds (19), human (14) and foods of animal origin (7). In addition to the major virulence genes, a total of 86 isolates of C. perfringens were found to be positive for beta2 (cpb2) toxin gene, while only 6 were found to bear the enterotoxin (cpe) gene. Out of the cpb2 positive isolates, 39 (55.71%) belonged to animals and 24 (92.31%) to birds. All the isolates of C. perfringens recovered from human and foods of animal origin were found to possess cpb2 gene. Among the cpe bearing isolates, 2 (2.86%) were of animal origin, 1 (3.85%) of birds, 2 (12.5) of human and 1 (14.29%) of foods of animal origin. Four randomly selected cpb2 positive C. perfringens isolates representing different sources were studied for their genetic diversity by PFGE, PCR-RFLP and gene sequencing. The PFGE of the cpb2 positive isolates of C. perfringens representing different sources revealed that the isolates of foods of animal origin and poultry faeces were closely related. On the other hand, the isolates of human stool and poultry faeces were found to be unrelated. Similar type of genetic diversity was observed between C. perfringens isolates of human stool and foods of animal origin. However, the human stool and animal faeces were found to be possibly related. The PCR-RFLP results revealed that isolates belonging to foods of animal origin and animal faeces were of same type. Similar restriction pattern was also exhibited by the cpb2 positive C. perfringens isolates recovered from poultry faeces and human stool. Gene sequencing results revealed that the cpb2 positive isolates of C. perfringens representing different sources were clustered into four main clusters. Cluster I contains cpb2 positive C. perfringens isolated from various sources. Similar is the case with the other two clusters. The fourth cluster contains only a faecal sample of poultry origin from NCBI. This indicates that C. perfringens have no specific host specificity and there may be interspecies transmission of the organisms. Protein profiling of the partially purified beta-2 toxin of randomly selected cpb2 positive strains of C. perfringens representing different sources was done to detect the presence of C. perfringens beta 2 toxin (CPB2) in the culture supernatant. The result revealed the presence of 28 kDa protein in all the isolates representing different sources besides the presence of various other protein bands. The presence of 43 kDa protein represents the alpha toxin and the presence of 35 kDa protein represents the beta toxin in all the isolates. Western blotting of the cpb2 positive C. perfringens isolates revealed that all the isolates were immunogenic.ThesisItem Open Access CHARACTERIZATION OF OUTER MEMBRANE PROTEINS OF Pasteurella multocida OF PORCINE ORIGIN(AAU, Khanapara, 2016-07) Borah, Bornali; Saikia, G.K.The present study was undertaken with a view to isolate and identify Pasteurella multocida from apparently healthy, diseased and dead pigs by both conventional and molecular methods, to study the pathogenicity of the isolates in mice, to prepare partially purified outer membrane proteins (OMPs) from most local virulent porcine strains (capsular types A and D), to study the protein profile of OMPs extract by SDS-PAGE, to identify the immunogenic proteins of OMPs by western blotting, to purify these proteins by size exclusion chromatography and to study the immunogenic potential of oil-adjuvanted vaccine prepared from the purified immunodominant OMPs in mice challenged with virulent homologous and heterologous capsular types of P. multocida. In the present investigation, a total of 357 samples including nasal swabs (187), tracheal swabs (18), lung (125) and heart blood (27) from apparently healthy, diseased and dead pigs were examined for isolation of P. multocida. Of these, 17 (4.76%) samples yielded P. multocida. More isolates were obtained from nasal swabs (9) from apparently healthy and diseased pigs than that of tracheal swabs (4) and lung tissues (4) from apparently healthy and dead pigs. All the 17 isolates showed cultural, morphological, staining and biochemical characteristics typical of P. multocida. The isolates were further confirmed as P. multocida on the basis of detection of species-specific gene (KMT1) by P. multocida species-specific PCR (PM-PCR). Among the 17 isolates, 6 (35.29%) were identified as capsular type A and 11 (64.71%) were identified as capsular type D based on multiplex capsular PCR results targeting hyaD-hyaC and dcbF genes, respectively. Mouse pathogenicity trial of 19 isolates of P. multocida revealed that the isolates induced 33.33 to 100.00 per cent mortality within 72 hours of inoculation. Two isolates (LS-3 and NS-4) were found to be comparatively more pathogenic causing 100.00 per cent mortality in the inoculated mice within 24-48 hours post inoculation and were selected for extraction of OMPs. Two most pathogenic strains of P. multocida, one each of types A and D (LS-3 and NS-4) were selected for extraction of OMPs. Analysis of OMPs of P. multocida type A by SDS-PAGE revealed presence of 21 protein bands with MWs ranging from 192.1 to 20.0 kDa. Among these protein, 36.8 and 25.0 kDa proteins appeared to be the major OMPs followed by 20.0, 56.5, 83.4, 47.4, 76.1, 51.2, 35.0, 99.2, 67.5 and 105.5 kDa protein bands based on band intensity in SDS-PAGE. While, OMPs of P. multocida type D showed presence of 22 protein bands with MWs ranging from 134.0 to 15.0 kDa. Among these protein, 35.7 and 25.0 kDa proteins appeared to be the major OMPs followed by 15.0, 56.2, 99.2, 47.1, 44.4, 66.5, 50.8, 78.5, 40.8 and 73.7 kDa proteins. The comparative evaluation of protein profiles of OMPs of serotypes A and D of P. multocida of porcine origin revealed that both the types shared three proteins with MWs 134.0, 99.2 and 25.0 kDa, of which the 25.0 kDa protein was found to be a major OMPs based on band intensity. The western blot analysis of the partially purified OMPs of P. multocida type A showed five major immunogenic proteins of MWs 83.4, 56.5, 36.8, 25.0 and 20.0 kDa giving strong immunostaining reaction with hyperimmune serum raised in rabbits. On the other hand, the partially purified OMPs of P. multocida type D showed five major immunogenic proteins of MWs 99.2, 56.2, 35.7, 25.0 and 15.0 kDa. Both the types shared a major immunogenic protein with MW 25.0 kDa. Size exclusion chromatography showed 10 peaks in OMPs extract of P. multocida type A and 13 peaks in OMPs extract of type D. In P. multocida type A, the five peaks of OMPs contained the protein fractions with molecular masses 83.4, 56.5, 36.8, 25.0 and 20.0 kDa, while in type D, the five peaks contained the protein fractions with MWs 99.2, 56.2, 35.7, 25.0 and 15.0 kDa. Mice immunized with oil-adjuvanted purified OMPs vaccines of P. multocida types A and D were challenged with 1x102 cfu of live P. multocida types A and D through subcutaneous (s/c) route and were found to be fully protective (100%). The organisms could not be re-isolated from the inoculated mice sacrificed after 7 days. The control group of mice showed 100 per cent mortality and died of septicaemia within 48 to 72 hours after challenge with 1x102 cfu of live P. multocida types A and D. Re-isolation of the organisms used for challenge infection was possible from the heart blood and the internal organs of dead mice of the control group.ThesisItem Open Access DEVELOPMENT OF USER FRIENDLY DIAGNOSTICS AND CELL CULTURE ADAPTED VACCINE CANDIDATE FOR DUCK PLAGUE(AAU, 2016) Neher, Samsun; Das, S. K.Duck plague or duck viral enteritis is an acute and contagious viral disease of ducks, geese swan and other species of the order Anseriformes. The disease is responsible for significant economic losses in duck husbandry due to heavy mortality, condemnation and decrease in egg production in duck. Besides clinical and postmortem findings, laboratory diagnosis is essential to confirm the disease in cases of outbreaks. Conventional diagnostic methods are labour intensive, time consuming and less sensitive. There is an urgent need for development of rapid, sensitive and cost effective in house as well as user friendly diagnostic test so as to confirm the disease at clinical phase in the field itself. Again, vaccination is the only available option for prevention and control of the disease. The present study was undertaken to develop user friendly diagnostics and potent cell culture adapted vaccine to control duck plague virus (DPV) infection. During the study period a total of 29 outbreaks of duck plague were attended. Various clinical and post mortem samples were processed for detection of viral DNA by PCR and viral antigen by S-ELISA. Serum samples collected from different districts were tested for presence of antibody by I-ELISA and Dot-ELISA. Duck plague virus was isolated in duckling, duck egg and DEF primary cell culture from the field tissue sample. Sequence and phylogenetic analysis of local DPV isolate and a vaccine strain was done to see the circulating virus in Assam. A cell culture adapted vaccine was developed, and safety and potency test was conducted to see the efficacy of the vaccine. In sero-epidemiological study, among the 445 serum samples tested by I-ELISA 348 (78.20%) were found positive for DPV antibody, however in Dot-ELISA 149 (33.48%) were found to be positive. A total of 380 samples were collected from clinically affected (107) and dead ducks (273). S-ELISA showing positive results in 25 (23.36%) in clinical samples and 188 (68.86%) post mortem samples, however in PCR a total of 231 (84.61%) post mortem samples and 68 (63.55%) clinical samples showed positive for duck plague virus specific nucleic acid. The present study showed that PCR is the suitable and reliable test for detection of duck plague virus. Among different tissue samples collected from dead birds, liver and spleen were found to be most suitable. In cases of clinical samples ducks whole blood was found to be preferred sample than the cloacal swabs and tracheal swab. However, due to simplicity of collection, cloacal swab may be the choice of sample from large flock. Reviving of field isolate in primary host followed by isolation in duck embryo and duck embryo fibroblast (DEF) cell culture made 100% recovery of virus. However, the DEF cell culture was found to be more suitable than embryonated duck egg for isolation. Sequence and phylogenetic analysis of the local isolates and a vaccine strain showed a close relationship among the local isolates with the vaccine strain. Local isolates also showed a significantly high degree of sequence identity with other DPV isolates from China, Vietnam, Korea and Germany. A highly virulent local strain was selected as vaccine candidate and adapted in CEF primary cell culture, whereas standard vaccines strain was adapted in CEF primary cell culture as well as in vero cell line. In safety and potency test of the CEF cell culture adapted DPV vaccine strain, ducklings were vaccinated with 0.5 ml of 103, 104 and 105 TCID50/ml dose of vaccine virus. All doses of vaccine were found to be safe and optimum for eliciting protective immunity in the vaccinated ducklings, and conferred 100% protection of ducklings challenged with 1 ml of 100 DID50 of virulent DPV. Thereby, the minimum dose containing 1 ml of 103 TCID50/ml of vaccine virus can be considered as optimum vaccine dose for providing protection, which can be further used for protection of ducks from duck plague. The present study clearly showed that duck plague is endemic in Assam causing high mortality in ducklings as well as in growing and adult ducks. Diagnostic tests I-ELISA, S-ELISA and Dot-ELISA along with molecular technique PCR could be companion diagnostic tools for confirmation of DPV as well as assessment of virus antibody. Significantly development of cell culture adapted vaccine and conferring of 100% protection can be an achievement of the study.