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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.
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ThesisItem Open Access Production and characterization of recombinant beta toxin of Clostridium perfringens(College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-03) Bharali, Arpita; Sharma, R KClostridium perfringens causes several forms of enteric disease in human and animals. Different toxinotypes of C. perfringens produce different combinations of lethal toxins. C. perfringens type C produces beta toxin which is a kind of lethal pore-forming toxin that is responsible for necrotic enteritis in animals. Although C. perfringens type C infects several livestock species, the juvenile pigs are most susceptible. In India, the north-eastern states have the highest pig population in the country but no indigenous vaccine against C. perfringens type C is currently available. Therefore, the present study was conducted with an aim to produce recombinant beta toxin protein in a heterologous host and to evaluate the immunogenicity of the recombinant toxin adjuvanted with calcium phosphate nanoparticles in the mice model. For this, the cpb gene of C. perfringens type C that encodes the beta toxin was cloned into a prokaryotic expression vector, pET28a(+). Then the recombinant clone was transformed into BL21-CodonPlus®(DE3)-RIL E. coli cells. Expression of the recombinant beta toxin was induced by 1 mM IPTG for 12 hours at 37C. The recombinant beta toxin protein was present as inclusion bodies in the insoluble fraction of the cell lysate which was further purified by Ni-NTA column affinity chromatography and confirmed by SDS-PAGE analysis. The specificity and the reactivity of the recombinant beta toxin protein were confirmed by western blotting using anti-sheep C. perfringens beta toxin serum. In-vitro and in-vivo toxicity of the recombinant beta toxin protein was evaluated in MDCK cell line and mice, respectively. The recombinant beta toxin protein did not show cytotoxicity in the concentrations from 3500-6.89 ng/ml as well as failed to produce clinical signs or death in mice when administered intravenously at a 100μg dose. The recombinant beta toxin protein was loaded into calcium phosphate nanoparticles (CaP-NPs) used as an adjuvant, and a calcium phosphate nanoparticles-recombinant beta toxin protein complex was produced which was characterized using a zetasizer. The immunogenicity of the recombinant beta toxin protein adjuvanted with CaP-NPs was evaluated and compared with the conventional Freund’s adjuvant in the mice model. Three groups of six mice each were inoculated separately with 30 μg of the recombinant beta toxin protein with Freund’s adjuvant (Group-I), 30 μg of the recombinant beta toxin adjuvanted with CaP-NPs (Group-II), and PBS in the control group (Group-III). The booster doses with corresponding inocula were given in all the groups 3 weeks apart and the level of antibody was estimated in serum samples collected at 7 days interval from day 0 to day 35 post-primary inoculation by indirect ELISA. The specific antibody response against the recombinant toxin protein was significantly higher (p<0.05) in Groups I and II from day 7 to day 35 compared to the control group. The level of antibody was at its peak on day 28 in the experimental groups (Groups I and II) and from day 28 to day 35, the level of antibody was significantly higher (p<0.05) in Group-II compared to Group-I. Hence, in the present study a non-toxic form of recombinant beta toxin of C. perfringens could be expressed in E. coli and the combination of the recombinant beta toxin protein with CaP-NPs as adjuvant could elicit better antibody response compared to its combination with the conventional oil adjuvant. However, its ability to produce neutralizing antitoxin and the protective efficacy against challenge infection are to be ascertained in future for considering it as a potential vaccine candidate against C. perfringens type C infection.ThesisItem Open Access DNA POLYMORPHISM IN MITOCHONDRIAL GENES ENCODING ND1, CO1 AND CYTB IN CANINE MALIGNANT TUMOURS(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2020-01) REHMAN, SHAKEEL-UL; Borah, ProbodhMalignant tumours in dogs are frequently reported. The types of malignancies commonly reported in canines include female breast cancers, lymphomas, adenomas and carcinomas of mast cells. Specific mutations and polymorphism in mitochondrial genes have been shown to be associated with different types of human malignancies. However, similar studies in respect to malignant tumours in dogs are very limited. Hence in the present study, an attempt was made to identify frequency of occurrence of mutation and polymorphism in gene sequences encoding NADH dehydrogenase subunit 1 (ND1), cytochrome b (CYTB) fragments of mtDNA and cytochrome c oxidase subunit 1 (CO1) in dogs, and to define the association of DNA polymorphic mutations with different tumour types. Based on histopathology, out of 10 tumours examined 5 (50%) were found to be of epithelial and the rest 5 (50%) of mesenchymal origin. Two of the five epithelial tumors were recognized as adenonocarcinoma and three as squamous cell carcinoma. Of the five mesenchymal tumors, four were identified as fibrosarcoma and one as liposarcoma. Of the 10 cases, 8 (80%) were recorded in local and 2 (20%) in crossbred dogs. While 7 (70%) cases were recorded in male, 3 (30%) were observed in females. Location-wise, two each of the tumours were observed in skin and mammary gland, while one each was observed in mouth, left flank, abdominal region, testicle, right elbow and left forelimb. The dogs suffering from the neoplastic growth in different parts of the body were within the range of 5 - 13 years of age. Analysis of three mtDNA gene fragments established a relatively low level of molecular genetic variation between the tissues (tumour tissue, normal tissue and blood) of the individuals examined. Majority of the mutational changes in the ND1, COI and CYTB gene fragments in the analyzed tissues in most of the dogs with tumours were insertions and deletions. Only a few polymorphisms were noted in the partial gene fragments of the analyzed tissues when compared to reference successions. Multiple substitutions and insertions have been noted in ND1 gene fragment; these included four substitutions (C218T, T455C, G498A and C666T) and three insertions (341InsC, 355InsC and 718InsT). However, no mutations were recorded in ND1 gene fragment from any of the three types of tissues examined in case of a dog affected with squamous cell carcinoma. Changes in CYTB gene fragment included two substitutions (C322T and T799C) and one insertion (303InsG) mutation. Polymorphism C322T in the CYTB fragment was noted in 40% of the samples analysed. No mutation was, however, detected in this gene fragment in one case of fibrosarcoma. In the COI gene fragment, A735G polymorphic mutation was recorded in all (100%) the 10 cases of malignant tumours investigated in the present study. In this gene fragment, instances of mutations recorded were comparatively lesser. Except for C218T mutation observed in ND1 gene fragment seven cases of canine malignant tumour that induced S (Serine) to Y (Tyrosine) variation in the amino acid sequence of the coded protein at position 72, no other substitution mutation recorded in this gene fragment could cause a variation at the level of amino acid sequence. On the other hand, none of the mutations detected in CYTB gene fragment could induce any change in the level of amino acid sequence of the coded protein. Similarly, the only substitution mutation in the CO1 gene fragment that induced a change at the amino acid level was A813T mutation observed in a case of fibrosarcoma, which caused a G (Glycine) to A (Alanine) variation at 71 position. Results of the present study showed the effect of two alleles (ND1: 218, CO1: 813) on the amino acid sequence of the coded proteins which suggested consequently their potential role in carcinogenesis. However, the sample size in the present study was too small to infer conclusively about the association of the mutations and polymorphisms identified in the present study with specific malignant tumours in dog.ThesisItem Open Access PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF EXTENDEDSPECTRUM β-LACTAMASE PRODUCING Escherichia coli AND Klebsiella ISOLATES FROM ANIMAL SOURCES(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2019-07) DAS, LEENA; Borah, ProbodhExtended-spectrum beta-lactamase producing Enterobacteriaceae has become a major threat to both animals and human health globally. The present study was undertaken to isolate and identify ESBL producing Escherichia coli and Klebsiella pneumoniae from various sources, to study their resistant gene profile, to detect insertion sequences, to genogroup the isolates and to compare the efficacy of REP-PCR and PFGE to discriminate ESBL producing E. coli and K. pneumoniae isolates. Out of 385 samples from various sources, 31 (8.05%) were positive for ESBL producing E. coli. Such isolates could be isolated from 10.05, 8.33, 15.63, 6.67 and 4.35 per cent of cattle milk, curd, chicken, pork and cattle faeces samples, respectively. However, no ESBL producing E. coli could be isolated from goat milk, goat faeces and beef samples. A total of 59 (15.32%) samples were positive for ESBL producing K. pneumoniae, which could be isolated from 14.35, 6.25, 21.43 and 34.78 per cent samples of cattle milk, chicken, beef and cattle faeces, respectively. No ESBL producing K. pneumoniae isolates could, however, be isolated from goat milk and faeces, curd and pork. In-vitro drug susceptibility assay against 3rd and 4th generation cephalosporins showed resistance of all the 90 ESBL isolates to at least one antibiotic. In CDT, 93.55% of E. coli and 88.14% K. pneumoniae and in ESBL –E test, 96.77% E. coli and 88.14% K. pneumoniae showed positive results. Antibiogram of the ESBL producing E. coli and K. pneumoniae showed resistance of 74.19% and 69.49%, respectively to ceftizoxime, 25.81% and 23.73% to both co-trimoxazole and tetracycline, 19.35% and 25.42% to ciprofloxacin, 9.68% and 16.95% to chloramphenicol, 3.23% and 5.08% to pipercillin-tazobactam, and 3.23% and 3.39% to gentamicin. Resistance gene profiling showed blaCTX-M gene to be present in all the 90 (100%) ESBL isolates. The blaTEM gene was found in 54.84% and 55.93%, blaSHV gene in 90.32% and 77.97%, Sul 1 gene in 90.32% and 86.44% isolates. The Int1 gene was detected in 70.97% and 62.71% isolates, while qnrB gene was found in 3.23% and 10.17% of E. coli and K. pneumoniae isolates, respectively. Out of the insertion sequences under study, ISEcp1 was found to be present in all the 90 (100%) ESBL producing isolates, followed by IS26 (100% and 90.32%) and ISCR1 (80.65% and 45.76%) in E. coli and K. pneumoniae isolates, respectively. All the 90 ESBL producing isolates were subjected to PCR for detection of CTX-M genogroups. All the 90 (100%) ESBL producing isolates were found to be positive for group 1 gene. A total of 80.65% and 55.93% E. coli and K. pneumoniae isolates, respectively showed presence of group 2 genes. The corresponding percentages for group 25 gene were 27.27% and 67.8%. However, group 9 gene could be detected in 5.08% of K. pneumoniae isolates only. None of the E. coli isolates were found be positive for group 8 and 9 genes, while no isolate of K. pneumoniae was found to be positive for group 8 gene. The two molecular typing methods, REP-PCR and PFGE were found to show similar discriminatory power and could distinctly differentiate the ESBL producing E. coli and K. pneumoniae isolates. As both the methods were found equally competent, REP-PCR may be recommended as the preferred method of typing for epidemiological investigations owing to its advantages over PFGE in terms of rapidity, simplicity and ease of performance.ThesisItem Open Access MOLECULAR CHARACTERIZATION AND GENOTYPING OF BIOFILM-PRODUCING STAPHYLOCOCCI ASSOCIATED WITH BOVINE MASTITIS(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2022-03) DUTTA, MADHUSMITA; Borah, ProbodhMastitis is an inflammatory disease of dairy animals and is considered as one of the commonest problems of the dairy industry throughout the world. Though it is a multietiological disease, Staphylococcus aureus is considered to be a common and potent cause of mastitis due to its wide array of virulence factors and ability of forming biofilm. The present study aimed at isolation and identification of biofilm forming S. aureus from mastitic and apparently normal bovine milk samples, and molecular detection of genes related to adhesion and biofilm formation, antibiotic resistance and enterotoxin production. The study also included molecular typing of representative isolates by three methods namely, multilocus sequence typing (MLST), surface protein A typing (spa typing) and accessory gene regulator typing (agr typing). The study was further extended to assessment of antibiofilm efficacy of three compounds- chitosan, EDTA and povidone iodine against selected S. aureus isolates in vitro. The study included a total of 136 milk samples comprising of 26 from mastitic and 110 from apparently healthy cows. In California Mastitis Test for detection of sub-clinical mastitis, 84 (76.40 %) of the 110 apparently normal milk samples tested positive. On bacteriological examination, 18 (69.23%) of 26 milk samples from mastitic cows and 64 (76.19%) of 84 apparently normal milk samples were to be positive for Staphylococcus with an overall positivity of 74.55%. Altogether 78 (95.12 %) of the 82 Staphylococcus isolates were found to be coagulase positive and confirmed as S. aureus based on detection of the species-specific aroA gene by polymerase chain reaction (PCR). Among the 78 S. aureus isolates, 54 (69.23 %) were identified as biofilm producers based on their characteristic growth on Congo red agar (CRA) plates. However, on PCR amplification, 58 (74.36%) of these isolates were found to carry icaA and icaD genes. All the isolates were found to have both fnbA and clfB genes, while 98.71, 61.50 and 11.53 per cent of the isolates were positive for clfA, cna and bap genes, respectively. On antimicrobial susceptibility testing, all the 78 isolates were found to be resistant to Oxacillin and Tetracycline. The isolates showed highest (25.64%) susceptibility to Cefepime followed by Ceftriaxone (23.08%) and Cefotaxime (20.51%). A very low susceptibility was shown to Gentamicin (10.26%), combination of Tricarcillin and Clavulanic acid (3.85%), Chloramphenicol (2.56%) and Co-Trimoxazole (2.56%). Among the 54 biofilm producing isolates, 48 (88.89%) and 45 (83.33%) were found to carry blaZ1 and blaZ2 genes, respectively, while not a single isolate carried the smr gene. On the other hand, among the 24 non-biofilm producing isolates, 22 (91.67%) possessed both blaZ1 and blaZ2 genes, while none carried the smr gene. Of the 78 isolates, 14 (17.94 %) were found to have at least one of the three staphylococcal enterotoxin genes (sea, seb and sed) included in the study. However, none of the isolates were found positive for see genes. Multi Locus Sequence typing (MLST) of 15 representatives biofilm-producing S. aureus isolates could detect three sequence types (STs) and one clonal complex (CC). Seven isolates belonged to ST672, five to ST1713 and three to ST2592 with slight variations in the allelic profile. ST672 had no CC while ST1713 and ST2592 belonged to CC1. Spa typing of the same isolates revealed three different spa types, t1309, t1611 and t267. Of the 15 isolates tested, two agr types were identified: agr I (60 %) and agr II (40 %). Strains belonging to agr types III and IV were not detected in this study. Antibiofilm efficacy of 5% povidone iodine (betadine®), 20 mM EDTA and 5 mg/ml of chitosan was tested in the present study based on determination of MIC of these compounds either alone or in combination against selected biofilm-producing S. aureus isolates. It was found that the combined effect of all the three compounds against biofilm-producing S. aureus was almost similar to that of the combination of chitosan and povidone iodine. Hence, the later combination was suggested as an alternative to using high concentration of an antiseptic for sanitization of the udder surface of milch cows to get rid of biofilm-producing S. aureus frequently associated with subclinical mastitis.ThesisItem Open Access EXPRESSION OF CAP PROTEIN OF PORCINE CIRCOVIRUS TYPE 2 (PCV2) AND EVALUATION OF ITS IMMUNOGENICITY IN MICE(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2022-03) BORAH, DEBARUN; Hazarika, GirinThe present study was conducted with an aim to express the Cap protein of Porcine Circovirus Type 2 (PCV2) in a prokaryotic host and to assess its immunogenicity in mice. The recombinant pET32a/CT-His plasmid containing a codon-optimised PCV2 cap gene was transformed into E. coli BL21(DE3) cells for expression of the Cap protein after 8 hours of IPTG induction at 37°C. Subsequent SDS-PAGE analysis revealed an intense band of 46 kDa. Following successful purification by affinity chromatography using Ni-NTA column under denaturing conditions, the recombinant protein was refolded by dialysis. In western blot analysis of the purified protein, a 46.0 kDa-sized intense band was detected in the PVDF membrane. In four groups of Swiss albino mice consisting of six mice in each group, the immunological response to the purified recombinant Cap protein was assessed and compared to that of a commercial vaccine. Group 1 served as the control group receiving PBS, while Group 2 received a commercial vaccine, Group 3 received the recombinant Cap protein, and Group 4 received the Cap protein combined with Freund's adjuvant. On the 14th day post-primary immunization, each group of mice received a booster dose of the corresponding inoculum. Serum was collected from all the experimental animals on 0, 7th, 14th, 21st, 28th, 35th, and 42nd days after immunization. As detected by indirect ELISA, the mean antibody level against PCV2 was significantly higher in Group 4 getting Cap protein with Freund's adjuvant, than in Group 2 receiving the commercial vaccine, and Group 3 receiving the Cap protein alone. The recombinant Cap protein was found to be immunogenic in mice and was capable of inducing a specific IgG response, as detected by indirect ELISA. Based on the results of the present study, it may be concluded that the recombinant Cap protein expressed in E. coli is a promising immunogen and may be further explored as a vaccine candidate against PCV2. Use of the recombinant protein as a potential diagnostic antigen may also be explored in future.ThesisItem Open Access Toxin gene profiling of Clostridium difficile in food and food products of animal origin(Assam Agricultural University, Khanapara,Guwahati, 2017-07) HAZARIKA, RITAM; SHARMA, R.K.The present work was undertaken with a view to investigate the presence of Clostridium difficile in food and food products of animal origin and profiling of toxin genes. A total of 235 samples were collected from different sources including raw meat (beef, pork, chicken and chevon), meat products (dry beef, dry pork, cooked chicken, chicken sausage and chicken salami), raw cow milk, milk product (Indian paneer) and fish products (dry fish and canned fish). Out of the total 235 samples, initially 56 (23.83%) samples revealed suspected colonies of C. difficile. All the suspected colonies exhibited the typical colony morphology with horse manure odour and typical Gram-positive rod shaped cell with sub-terminal spores. Among 56 tentative isolates, 17 (7.23%) could be confirmed as C. difficile, based on the detection of species specific gluD (GDH) and tpi (triose phosphate isomerase) genes. The 17 confirmed C. difficile isolates comprised of 15 (8.24%) from raw meat and meat product samples and remaining two (11.11%) from fish product samples. None of the samples from cooked chicken, raw chevon, chicken sausage, chicken salami, canned fish, cow milk and paneer yielded any C. difficile isolates. All the 17 C. difficile isolates were screened for detection of glutamate dehydrogenase (GDH) protein and toxin A and /or toxin B by enzyme immuno assay (EIA), out of which all isolates were found positive for GDH protein and six were found to be phenotypically positive for toxin production. The C. difficile isolates were characterized by PCR for detection of toxin genes (tcdA, tcd B and binary toxin) and PaLoc region. Antimicrobial resistance pattern was also tested against nine different antimicrobial agents by E-test. Altogether, six C. difficile isolates were found to be toxigenic. Out of which two were from chicken samples and four from pork samples. All the toxigenic isolates from chicken (2) samples possessed both tcdA and tcdB (A+ B+), while all the pork isolates (4) carried variant toxin genes (A-B+). All the (A-B+) isolates from pork were found to harbour the binary toxin genes (cdtA and cdtB). Based on the detection of PaLoc region comprising regulatory genes tcdC, tcdR and tcdE revealed that the two toxigenic chicken isolates (A+ B+) possessed tcdC and tcdE ,while the remaining four toxigenic pork isolates (A-B+) carry tcdR and tcdE, respectively. All the 17 C. difficile isolates showed higher resistance pattern to ciprofloxacin (70.59%), followed by cefotaxime (58.82%), clindamycin (29.41%) and tetracycline (17.64%) but 100 per cent sensitive to chloramphenicol, moxifloxacin, tigecycline, metronidazole and vancomycin. All the beef isolates were clindamycin- resistant with an MIC of 96 - 128 mg/ml. While, all C. difficile isolates from pork and dry fish were sensitive to the antimicrobials tested except ciprofloxacin and cefotaxime, the four chicken isolates were sensitive to the antimicrobials except ciprofloxacin.ThesisItem Open Access Toxin gene profiling of Clostridium difficile in food and food products of animal origin(Assam Agricultural University, Khanapara,Guwahati, 2017-07) HAZARIKA, RITAM; SHARMA, R. K.The present work was undertaken with a view to investigate the presence of Clostridium difficile in food and food products of animal origin and profiling of toxin genes. A total of 235 samples were collected from different sources including raw meat (beef, pork, chicken and chevon), meat products (dry beef, dry pork, cooked chicken, chicken sausage and chicken salami), raw cow milk, milk product (Indian paneer) and fish products (dry fish and canned fish). Out of the total 235 samples, initially 56 (23.83%) samples revealed suspected colonies of C. difficile. All the suspected colonies exhibited the typical colony morphology with horse manure odour and typical Gram-positive rod shaped cell with sub-terminal spores. Among 56 tentative isolates, 17 (7.23%) could be confirmed as C. difficile, based on the detection of species specific gluD (GDH) and tpi (triose phosphate isomerase) genes. The 17 confirmed C. difficile isolates comprised of 15 (8.24%) from raw meat and meat product samples and remaining two (11.11%) from fish product samples. None of the samples from cooked chicken, raw chevon, chicken sausage, chicken salami, canned fish, cow milk and paneer yielded any C. difficile isolates. All the 17 C. difficile isolates were screened for detection of glutamate dehydrogenase (GDH) protein and toxin A and /or toxin B by enzyme immuno assay (EIA), out of which all isolates were found positive for GDH protein and six were found to be phenotypically positive for toxin production. The C. difficile isolates were characterized by PCR for detection of toxin genes (tcdA, tcd B and binary toxin) and PaLoc region. Antimicrobial resistance pattern was also tested against nine different antimicrobial agents by E-test. Altogether, six C. difficile isolates were found to be toxigenic. Out of which two were from chicken samples and four from pork samples. All the toxigenic isolates from chicken (2) samples possessed both tcdA and tcdB (A+ B+), while all the pork isolates (4) carried variant toxin genes (A-B+). All the (A-B+) isolates from pork were found to harbour the binary toxin genes (cdtA and cdtB). Based on the detection of PaLoc region comprising regulatory genes tcdC, tcdR and tcdE revealed that the two toxigenic chicken isolates (A+ B+) possessed tcdC and tcdE ,while the remaining four toxigenic pork isolates (A-B+) carry tcdR and tcdE, respectively. All the 17 C. difficile isolates showed higher resistance pattern to ciprofloxacin (70.59%), followed by cefotaxime (58.82%), clindamycin (29.41%) and tetracycline (17.64%) but 100 per cent sensitive to chloramphenicol, moxifloxacin, tigecycline, metronidazole and vancomycin. All the beef isolates were clindamycin- resistant with an MIC of 96 - 128 mg/ml. While, all C. difficile isolates from pork and dry fish were sensitive to the antimicrobials tested except ciprofloxacin and cefotaxime, the four chicken isolates were sensitive to the antimicrobials except ciprofloxacin.ThesisItem Open Access DEVELOPMENT AND EVALUATION OF IMMUNOPOTENCY OF OUTER MEMBRANE VESICLES VACCINE OF Salmonella Typhimurium ADJUVANTED WITH CHITOSAN NANOPARTICLES(Assam Agricultural University, Khanapara,Guwahati, 2017-07) SARMAH, RAKESH KUMAR; Tamuly, ShantanuDuring the past two decades, Salmonella Typhimurium has emerged as a leading cause of human infections in many countries and the Salmonella spp has a broad host range and can infect broad array of animals causing diseases ranging from gastroenteritis to life threatening systemic infections. Salmonella enterica serovar Typhimurium is most frequently isolated serovar causing global food born outbreaks. The control of Salmonellosis can be accomplished either by vaccination or medication. Antibiotic resistance and antibiotic residue is a major hurdle in medication. The present study was conducted to study the efficacy of the chitosan nanoparticle adjuvanted outer-membrane vesicle based vaccine candidate against Salmonella Typhimurium. The OMV was extracted from Salmonella Typhimurium (MTCC-98) strain and confirmed by SDS-PAGE. The (ChNP-OMV) vaccine was synthesized by standard method and nanometer size was confirmed by TEM studies. In the immunization study three vaccine formulation were compared and injected in the 7th day and followed by booster dose on the 14th day. The humoral immune response of the target vaccine was compared with OMV based vaccine with or without booster dose and oil adjuvanted bacterin vaccine with or without booster dose by indirect ELISA. Collection of serum was done on 0th day just before vaccination, and thereafter 7, 14, 28, 45 and 60 days post primary vaccination. Humoral immune response in mice of all the experimental groups determined by using indirect ELISA. The (ChNP-OMV) vaccine was able to elicit quick and higher antibody response in the 14th day followed by steady decline up to 60th day. All the vaccine formulation elicited similar IgG response on 7th day PPI, however chitosan nanoparticle adjuvanted outer-membrane vesicle based vaccine elicited highest IgG response on 14th days PPI (with or without booster dose) with statistical significance of 95 % confidence level. Serum enzymatic test revealed that the significantly lower serum creatine kinase level in 7th and 14th day in the group of mice that was administered chitosan nanoparticle adjuvanted outer-membrane vesicle based vaccine as compared to the group of mice that was injected oil adjuvanted bacterin vaccine on 14th days PPI. However a significant decrease in antibody response was observed for the groups containing (ChNP-OMV) complex. From this study, it can be concluded that the ChNP-OMV (S. Typhimurium) vaccine was indicative of quick immune response for immediate vaccination in outbreaks against salmonellosis.ThesisItem Open Access CHARACTERIZATION AND QUALITY EVALUATION OF PIG FIBROBLAST CELLS AT DIFFERENT SUBCULTURES(Assam Agricultural University, Khanapara,Guwahati, 2017-07) DHAR, PRIYA; Nath, N. Ch.Fibroblast cells are the most common cells of connective tissue. In the present study, porcine fibroblast cells were isolated, cultured and subcultured upto six passages from adult and fetal skin samples. The time required to attain 70% confluence in primary culture of fetal fibroblast cells was found to be 26.40±2.40 hours, which was significantly lower (p≤0.01) than 67±4.80 hours of adult fibroblasts culture. The cells were maintained upto six passages which was 38.8 days for adult and 26 days for fetal fibroblast cells. The fetal fibroblast cells (days) required significantly lower (p≤0.01) time to subculture than adult fibroblast cells. Morphologically the isolated cells had fibroblastic character like typical fusiform shape, turgor vitalis cytoplasm, centrally located nuclei and flame like migration pattern upto sixth passage. The subculture time and cell viability (%) was significantly (p≤0.01) better from third subculture onwards upto sixth subculture as compare to primary and secondary cultures. TUNEL assay revealed a decreasing trend of apoptotic index from primary culture onwards to sixth subculture for both adult and fetal fibroblast cells. Fetal fibroblast cells maintained a lower apoptotic index than adult fibroblast in each of the subcultures. Therefore, fibroblast cells were found to be better from third subculture onwards to sixth subculture in regards to subculture time, viability (%), apoptotic index and in maintaining its normal morphological characteristics. ALP is considered as important marker for identification of pluripotent embryonic stem cells as well as multipotent mesenchymal stem cells. Isolated fibroblast cells of all the subcultures showed activity for ALP test indicating their progressive and undifferentiated quality and may be of multipotent in nature.
