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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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2019 - 201912020 - 20222
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Subject: Animal Biotechnology × End date: 2023 × Type: Thesis × Thesis Type: Ph.D ×
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    ThesisItemOpen 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 K
    Clostridium 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 37C. 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.
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    ThesisItemOpen 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, Probodh
    Extended-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.
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    ThesisItemOpen 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, Probodh
    Mastitis 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.