ThesisItem Open AccessDEVELOPMENT OF A SUITABLE VACCINE FORMULATION AGAINST TYPE A Clostridium perfringens ASSOCIATED NECROTIC ENTERITIS IN BROILER CHICKEN(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2019-07) SARMAH, HIRAMONI; Sharma, Rajeev KumarNecrotic enteritis (NE) is one of the most clinically dramatic and important bacterial disease of poultry industry. It has a great negative impact on broiler industry due to production losses, increased mortality, increased feed conversion ratio. The cost of NE worldwide was estimated to 2 billion dollars per year with 1% daily mortality. Most common age of outbreaks of NE in broiler flocks raised on litter are between the second and fifth week of age. NE in broiler chicken is commonly associated with Clostridium perfringens toxin type A, while involvement of type C is very rare. The study was undertaken to develop a suitable vaccine preparation against C. perfringens type A associated NE for broiler chicken. During the study clinical samples. viz., intestinal content, intestinal scrapings from broilers died of suspected form of NE and faecal swabs from live affected birds with clinical symptoms suggestive of NE were screened for C. perfringens. A total of 26 repository isolates of C.perfringens maintained in Department of Microbiology, College of Veterinary Science, Khanapara were also considered for the present study. All the isolated C. perfringens recovered from NE affected broiler birds along with the repository were characterized with respect to the toxin types, detection of gene(s) associated with virulence and secretory protein, pathogenicity for mice, release of toxins and secretory proteins in cell free supernatant and resistance patterns towards antimicrobial agents. The detailed characterization was carried out with an idea to identify a suitable vaccine candidate for the development of vaccine preparations against NE in broiler chicken. Clinical samples, comprising of intestinal scrapings (42), intestinal contents (30) were collected from 72 dead broiler chickens with suspected form of necrotic enteritis. Another 23 faecal samples were collected from an equal no. of clinically affected live broiler birds by swabbing. A total of 41 isolates were identified as toxin type A, only 10 isolates isolates isolates isolates isolates isolates isolates isolates isolates exhibited additional virulent genes viz netB, tpeL and gapC genes either alone or in combination. All the eluted amplified PCR products of target genes with respective band sizes were confirmed by DNA sequencing. All total of 10 isolates of C. perfringens type A positive for netB alone (5), and netB with tpeL and gapC (5), were subjected to mouse pathogenicity trial. The mouse pathogenicity trial revealed variable pathogenicity, producing clinical symptoms in 21 inoculated mice within 72 hrs of observation, while 17 of the clinically affected mice were succumbed to death. The highest mortality was observed in group of mice inoculated with S8. On SDS-PAGE analysis cell free supernatant of S8 could exhibit highest 16 different visible bands with MW, ranging from 12 to 250 kDa. The four additional virulence associated proteins, NetB (33 kDa), GPD (40 kDa), α- toxin (43 kDa) and tpeL(180 kDa) were also distinctly visible. On immunoblotting clear immune dominant antigenic proteins identified as netB (33 kDa), GPD (40 kDa), alpha (43 kDa) and tpeL (180 kDa). were observed in cell free supernatant of S8 and other few strain. On antimicrobial resistance profiling highest resistance pattern was observed against ciprofloxacin (80.0%), followed by norfloxacin and tetracycline (60.0% each), gentamicin (30.0%) and levofloxacin (20.0%). Gatifloxacin, cefmetazole,clindamycin, metronidazole, and tigecycline were found to be effective against all the isolates. After selection of a suitable strain of C. perfringens type A, six different vaccine formulations, i.e., non-adjuvanted crude toxoid (I), non-adjuvanted crude toxoid with bacterin (II), non-adjuvanted crude toxoid with sonicated supernatant (SS) and bacterin (III), adjuvanted crude toxoid (IV), adjuvanted crude toxoid with bacterin (V) and adjuvanted crude toxoid with SS and bacterin (VI) were prepared. Comparative evaluation of the six vaccine formulations was carried out in respective groups of broiler birds, with respect to their serum antibody titer. Among the vaccine formulations, combination of crude toxoid, bacterin and SS was found to be superior in respect to the mean serum antibody titer in vaccinated bird (group VI), throughout the study period throughout study period. The passive mouse protection study could reveal that the pooled immunized serum samples of 21st, and 28th day could protect the mice with the challenge with homologous strain of C. perfringens. ThesisItem Open AccessEXPRESSION OF CERTAIN CYTOKINES IN RELATION TO PERSISTENCE OF FOOT AND MOUTH DISEASE VIRUS TYPE ‘O’ IN CATTLE(College of Veterinary Science Assam Agricultural University Khanapara, Guwahati-781022, 2017) Baro, Sangeeta; Sharma., K.The present study was undertaken to detect foot-and-mouth disease virus serotype ‘O’ in oro-pharyngeal fluid (OP fluid) and to quantify cytokines IL-1α, IL- 1β, IFN-α, TNF-α in blood of recovered animal by Real time PCR. Typing of infected clinical samples suspected for FMDV was done by sandwich ELISA followed by simultaneous detection of serotype by multiplex-PCR and detection of antibodies against NSP was done by DIVA ELISA in serum. The Relative Quantification (RQ) values for IL-1α gene during outbreak was 1.383 ±0.405 and after one month of post infection the RQ value was found to be 2.0223 ±0.592 which was found to be upregulated. Subsequently after three month of post infection the expression level of IL-1α was 23.8788±.993 which was upregulated. Later the expression level of IL-1α at 6month and nine month were 1.0223±0.299 and 1.9899±0.565 respectively. IL-1β gene expression was studied and the RQ values was found to be 0.0097±0.002 during one month of post infection which is down regulated and subsequently become undetectable during 3 month and in subsequent period of study period. The expression of IL-1β down regulation was observed in month 1 of post infection, whereas in subsequent period of the study the IL-1β was undetectable. Expression of IFN-α gene during outbreak was 1.0131±0.296. Up egulation of IFN-α in the 15 animals were found during 1, 3, 6 and 9 month respectively. The mRNA expression of TNF- α was studied and found to be upregulated during outbreak and during 1, 3, 6 and 9 month and the level of expression was 1.2361±0.362, 1.6346±0.478, 3.0521±0.893, 2.1447±0.628 and 1.3484±0.394 respectively. The present study thus supports the notion that real-time PCR is a powerful technique for reliable detection of persistent FMDV in recovered animals. The findings also indicated that IL-1α, IFN-α and TNF-α genes were gradually upregulated upto 3 months but IL-1β found to be down regulated with progression of recovery of the animals from the disease. Down regulation of the genes may be due to subside of the acute infection. ThesisItem Open AccessIMMUNO 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. ThesisItem Open AccessSERO-SURVEILLANCE AND MOLECULAR CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE FROM POULTRY OF ASSAM(Assam Agricultural University, Khanapara, Guwahati, 2016-01) MEDHI, MANISHA; Das, SutopaInfectious 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. ThesisItem Open AccessMOLECULAR 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. ThesisItem Open AccessMOLECULAR 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. ThesisItem Open AccessMOLECULAR 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 AccessCHARACTERIZATION 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 AccessDEVELOPMENT 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.