Thumbnail Image

Theses

Browse

2020 - 20224
Reset filters
Subject: Animal Biotechnology × End date: 2022 × Start date: 2020 × Type: Thesis ×
Reset filters

Search Results

Now showing 1 - 4 of 4
  • Thumbnail Image
    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.
  • Thumbnail Image
    ThesisItemOpen 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, Probodh
    Malignant 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.
  • Thumbnail Image
    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.
  • Thumbnail Image
    ThesisItemOpen 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, Girin
    The 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.