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Subject: Veterinary Microbiology × End date: 2016 × Start date: 2010 × Type: Thesis ×
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    ThesisItemOpen Access
    IMMUNO PROTECTIVE POTENTIAL OF PARTIALLY PURIFIED TOXOIDS OF Clostridium difficile IN MICE
    (Assam Agricultural University, Khanapara,Guwahati, 2016-11) HAZARIKA, PARISHMITA; SHARMA, R. K.
    The present study was undertaken to characterize Clostridium difficile toxins, in respect to the influence of glucose and stages of growth (incubation period) on release of toxins, cytotoxic activities in Vero cells and the immune-protective potential of partially purified toxoids of C. difficile in mice. A total of 10 isolates of C. difficile from the repository of Department of Microbiology, College of Veterinary Science, Khanapara, Guwahati were revived and reconfirmed, based on morphological and staining characteristics, and molecular detection of gluD gene. Characterization of all the 10 isolates, in respect to certain virulence associated genes revealed presence of tcdA (toxin A) and tcdB (toxin B) genes in three strains of C. difficile each. Another three strains could reveal tcdA and tcdB together in the same isolate, while one strain was found to be negative for tcdA and tcdB gene.The protein concentration in the cell free supernatant of toxin A and toxin B positive isolate of C. difficile growth in nutrient media without addition of glucose was found to increase with advancement of growth phases and reached the highest conc. during the decline phase of 48 hr (5.24 µg/µl and 5.06 µg/µl, respectively). Similar trend of protein conc. was observed in the cell free supernatants of both the isolates, in presence of glucose in the nutrient media. However, the presence of glucose was found to suppress the protein conc. in the cell free supernatants of toxin A and toxin B of C. difficile (3.49 µg/µl and 3.99 µg/µl, respectively). The protein profile of toxin A positive C. difficile isolate, in presence of glucose could show 10 protein bands with mol. wt. ranging from 25 to 135 kDa, while the same isolates in absence of glucose in nutrient media revealed 16 protein bands within the range of 22.4 kDa and 100.0 kDa. Similarly, the isolate positive for toxin B revealed 8 protein bands of 35 to 135 kDa range in the cell free supernatant with addition of glucose, while the growth of the same isolate in nutrient media without glucose could exhibit 15 protein bands within the range of mol. wt. 20.0 to 135.0 kDa. Toxin A, B and AB of C. difficile were extracted in thioglycolate media without addition of glucose at 48 hr of incubation and were partially purified by ammonium sulphate precipitation. The partially purified toxins were found to be cytotoxic for Vero cells at two dilution (1:10 and 1:100). Among the three toxins, toxin B was found to be more prominent cytotoxic activities than the other two toxins, A and AB. Complete detoxification was confirmed by testing the monolayer of Vero cells for no cytopathic effect. The immune-protective efficacy of the three toxoid vaccine preparations were tested by immunization of groups of mice with challenge trial on 34th day of post immunization revealed variable protection level. The immunized groups of mice were found to have 100.0 percent protection against homologous challenge dose of 6.0x108CFU. However, the groups of mice, immunized with toxoid A and B could show 75.0 percent protection against challenge with 9.0x108CFU of homologous strains of C. difficile. On the other hand, the vaccine prepared from toxoid AB could confer only 25.0 percent protection in mice, following homologous challenge with 9.0x108CFU. The immunized affected mice, following challenge with 9.0x108CFU dose could show clinical symptoms, suggestive of intestinal disorder, with any mortality. All the affected immunized mice with clinical symptoms were found to recover by the end of the challenge study. The challenge trial with 6.0 x 108 and 9.0x108CFU / dose of homologous strain of C. difficile could produce 100.0 percent mortality in the mice of control group during 48 hr of post challenge observation. The affected mice of the control group revealed an initial development of clinical symptoms, suggesting intestinal infection during 24 hr of observation and all the clinically affected mice were died within 48 hr of challenge. Mortality in mice of control group due to inoculated strain of C. difficile was confirmed by re-isolation of the inoculated strains from the affected liver as well as haemorrhagic part of intestine and intestinal contents.
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    ThesisItemOpen Access
    SERO-SURVEILLANCE AND MOLECULAR CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE FROM POULTRY OF ASSAM
    (Assam Agricultural University, Khanapara, Guwahati, 2016-01) MEDHI, MANISHA; Das, Sutopa
    Infectious Bursal disease (IBD) is a highly infectious and contagious disease that primarily affects chicks of 3-6 weeks of age causing immunosuppression by affecting the immune system of poultry where it damages the Bursa of Fabricius, thymus etc. The disease has great economic importance in both broiler and pullet growers as the affected birds are susceptible to minor environmental pathogens leading to high morbidity and mortality. The disease is caused by double stranded bisegmented IBD virus (IBDV) that comes under the genus Avibirnavirus of family Birinaviridae. The best way to prevent the disease is by vaccination and good managemental practices. However, there are frequent reports of the occurrence of the disease from different parts of India including Assam. So the present study was aimed to assess the infection by detecting presence of antibodies in the serum samples through indirect Enzyme linked immuno sorbent assay (ELISA) which were randomly collected from unvaccinated local birds from different parts of Assam and detection of virus from clinically affected tissue samples by direct Reverse Transcription- Polymerase Chain Reaction (RT-PCR) and by integrated cell culture PCR (ICC-PCR) after isolating it in the chicken embryo fibroblast (CEF) and Vero cell line. A total of 1093 sera samples were randomly collected from 19 different districts of Assam and screened for presence of antibodies against IBDV using commercial Indirect- ELISA kit. Out of which, 306 samples (27.99%) were found positive. Based on different age groups, collected serum samples were categorized out of which 2-4 weeks of age group meaning young chicks were found to contain antibodies against IBDV. Clinical samples were collected from different places of Assam which shown characteristic necropsy lesion for IBD infection. Total 23 numbers of clinical samples were collected for diagnosis of IBDV antigen through RT-PCR with specific set of primers. Out of which, 12 samples (52.173%) were confirmed for the presence of IBDV. Samples positive in PCR were stained with DNA loading dye and run under polyacrylamide gel electrophoresis along with DNA ladder. A distinct band was observed at 643 bp size region. Representative three PCR amplicon from Nalbari, Hajo and Bijoynagar were further sequenced via an out source and a phylogenetic tree was constructed by maximum likelihood method along with other IBDV isolates reported from various part of the world. Percent identities were analyzed within the isolates reported from our study, from different parts of India and other parts of the world. IBDV can be well adapted in CEF and Vero cells. Virus was passaged for five times in primary chicken embryo fibroblast cells before adaptation in Vero cells. Cytopathic effects (CPE) were observed from second passage onwards in both the cell line. IBDV was passaged in both Vero and CEF cells for at least five times and the cell lysates of each passage were checked by performing Integrated Cell Culture -PCR. All positive samples gave similar results to the pairs of primers which were used for the detection of IBDV nucleic acid in the tissue samples. The present study confirms the prevalence of the disease in poultry population of Assam.
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    ThesisItemOpen Access
    MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF WILD STRAIN OF DUCK PLAGUE VIRUS
    (Assam Agricultural University, Khanapara, Guwahati, 2016-07) SARMAH, HIRAMONI; DAS, S. K.
    Duck plague or duck viral enteritis is an acute and contagious viral disease of ducks, geese swan and other waterfowl. The disease is responsible for significant economic losses in duck husbandry due to decrease in egg production, condemnation and mortality in duck. The present study was undertaken to study the molecular and biological characterization of wild strains of duck plague virus. In the present study 6 wild strains of DPV (DP/As-Km/0010, DP/As-Nal/0012, DP/As-Km/0016, DP/As-Km/0019, DP/As-By/0022, DP/As-Km/0025) were revived in ducklings. All the inoculated ducklings developed distinct clinical signs like nasal discharge, lacrimation, pested eyelids, greenish watery diarrhea, soiled vents and sometimes sudden death etc. Post mortem examination revealed gross lesions in brain, oesophagus, liver, spleen, heart, bursa of Fabricious and in intestine. Presence of viral nucleic acid was detected by PCR and detection of duck plague virus antigen in post mortem samples was done with indirect FAT. All the isolates revived in ducklings were further propagated in DEF upto 5th serial passage. The clear CPE was observed from 1st passage onwards. On the basis of DID50 and TCID50, a VV strain of DPV was selected for further study. DID50 of DP/As-Km/0019 was found to be 10-2 and DID50 in case of DP/As-By/0022 and DP/As-Km/0025 was 10-1. Highest TCID50 was found to be 106.33 in case of DP/As-Km/0019. On the basis of these parameters (DID50, TCID50). The strain DP/As-Km/0019 was selected as VV strain of DPV. The pathodynamics of the VV strain was studied by using mean clinical and pathological scores and virus excretion pattern in blood and other clinical samples like tracheal swab and cloacal swab, nasal and ocular swab. Highest mean pathological score was observed in Liver and oesophagus (2.33±0.51) and lowest was observed in thymus and bursa (1.00±0.00).Molecular characterization of selected VV strain of DPV was done by sequencing two genes (UL30, US10) from different region of the virus. Phylogenetic analysis showed close relation with other isolates of DPV and vaccine strain. VNT50 titre of VV strain of DPV (DP/As-Km/0019) was found to be 1:223 which is similar to VNT50 of the vaccine strain and for other moderate virulent strains (DP/As-By/0022 and DP/As-Km/0025), VNT50 was 1:188 and 1:112 respectively. The selected VV strain of duck plague virus was adapted in 9-11 days old embryonated chicken eggs. Different changes like thickening of CAM with extensive haemorrhages, Haemorrhage and congestion throughout the body of infected embryos were observed from 3rd passage onwards. The chicken embryo adapted VV wild strain of DPV was again adapted and propagated in the CEF upto 10th serial passage. The most common CPEs were rounding of cell, vaculation in the cell, syncytia formation and finally detachment of cell monolayer which was observed from 3rd passage onwards.
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    ThesisItemOpen Access
    MOLECULAR CHARACTERISATION OF EXTENDED SPECTRUM BETA LACTAMASE (ESBL) PRODUCING Escherichia coli IN POULTRY
    (Assam Agricultural University, Khanapara, Guwahati, 2016-07) BASAIAWMOIT, ERNESTINE; Hazarika, A. K.
    The study was undertaken to isolate and identify Escherichia coli from poultry with or without the history of diarrhoea and to determine the occurrence of extended spectrum beta-lactamase (ESBL) producing Escherichia coli in Assam and Meghalaya, India. A total of 182 (67.40%) samples yielded E. coli which included 106 (67.08%) samples from Assam and 76 (67.85%) samples from Meghalaya. The samples were obtained from cloacal swabs, faecal samples and intestinal contents of diarrhoeic and non-diarrhoeic poultry birds. All the 182 strains of E. coli isolated from diarrhoeic and non-diarrhoeic birds were subjected to antibiotic susceptibility test and were phenotypically confirmed to be ESBL producers by DDST method. A total of 39 (21.42%) samples were confirmed as ESBL producers. Out of these, 19 (17.92%) samples were from Assam and 20 (26.31%) samples were from Meghalaya. Further, the extended-spectrum beta-lactamase genes viz., blaCTX-M, blaTEM, and blaSHV were detected by Polymerase Chain Reaction from the phenotypically confirmed isolates. 17 (9.34%) isolates were found to be positive for at least one of the two resistance genes, viz. blaTEM (686bp) and blaCTX-M (585bp). None of the isolates were found to contain the blaSHV gene. Of the 17 isolates, 5 (2.75%) were found to be positive for blaTEM gene, of which 3 (1.65%) were from Assam and 2 (1.09%) from Meghalaya. Similarly, 12 (6.59%) were found to be positive for blaCTX-M gene, of which 5 (2.75%) were from Assam and 7 (3.85%) were from Meghalaya. Prevalence of the resistant genes in poultry birds was found to be slightly higher in Meghalaya in comparison to Assam.
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    ThesisItemOpen 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.
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    ThesisItemOpen 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.
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    ThesisItemOpen 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 Kumar
    The 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.
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    ThesisItemOpen 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.
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    ThesisItemOpen 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.