In the present study, we report for the first time the efficacy of recombinant Bm95 mid gut antigen isolated from an Argentinean strain of
Rhipicephalus microplus strain A in controlling the tick ...infestations in India. The synthetic gene for Bm95 optimized for expression in yeast was obtained and used to generate yeast transformants expressing Bm95 which was purified to apparent homogeneity. Liquid chromatography–mass spectrometry analysis of the purified protein confirmed its identity as Bm95. Vaccine was prepared by blending various concentrations of purified Bm95 with aluminium hydroxide as an adjuvant. Immunogenicity studies of the vaccine in rabbits and cattle indicated that the vaccine was highly immunogenic. The efficacy studies of the vaccine was done in cattle. Naïve
Bos indicus cattle were vaccinated with the recombinant vaccine and were challenged with the larval, nymphal and adult forms of
Rhiphicephalus haemaphysaloides. The vaccine protected the animals from larval, nymph and adult tick challenges with an efficacy of 98.7%, 84.6% and 78.9% respectively. The results obtained from the above studies clearly demonstrated the advantage and possibilities of the use of Bm95 in controlling
R. haemaphysaloides infestations in the field.
Bovine tuberculosis caused by Mycobacterium bovis is a zoonotic disease that is responsible for significant economic losses in many countries. The standard diagnostic method, the tuberculin test ...(TST) that is used in control programmes has serious shortcomings and, given the complex nature and the economic impact of the disease, a number of other diagnostic methods have been examined. The authors have attempted to characterise antibody response using the multi-antigen print immunoassay (MAPIA). A total of 511 serum samples were collected from farms in India on which bovine tuberculosis was prevalent and on farms with low incidence. These were tested using the MAPIA against a panel of five defined M. bovis recombinant antigens and two purified protein derivatives (bovine PPD and avian PPD) to study the seroprevalence of the disease on Indian cattle farms. Results indicated that the fusion protein of antigen CFP-10:MPB83 showed a positive response in 142 out of 298 serum samples from tuberculosis-prevalent farms, thereby indicating the serological dominance of the proteins post infection. The antigen selected could be used further in the development of a simple, rapid and accurate serological diagnostic test, paired with TST, for use in bovine tuberculosis control programmes.
Bovine tuberculosis (BTB) is a chronic bacterial disease, and a major animal health problem with zoonotic implications. Screening of mycobacterial infections in bovines is traditionally done using ...the single intradermal tuberculin test. Though the test is widely used, it has its own disadvantages and they include its inability to distinguish between pathogenic and non-pathogenic mycobacterial infections owing to its low specificity. Furthermore, the associated operative difficulties of this test have driven the quest for discovery of new antigens and diagnostic assays leading to the development of the interferon (IFN)- test. Presently, combinatorial testing using the skin test and the interferon gamma assays are being used in the diagnosis of BTB in various control and surveillance programs. In this study, we report the cloning, expression and purification of ESAT-6-CFP-10 fusion protein and its further use in the development of the IFN- gamma ELISPOT assay for accurate diagnosis of BTB in cattle. The BTB diagnosis employing the ELISPOT assay was evaluated using peripheral blood mononuclear cells from culture positive and culture negative cattle. The ELISPOT assay showed higher specificity and sensitivity in detecting BTB when a recombinant ESAT-6-CFP-10 fusion protein was used. The present study indicated that the usefulness of the fusion protein can replace the ESAT-6, CFP-10 or combination of both proteins for detecting BTB in IFN-gamma ELISPOT assay.
► Mycobacterium (TB) protein ESAT-6 and CFP-10 is cloned as a fusion ESAT-6-CFP-10 protein and purified from E. coli. ► Fusion protein has been evaluated in IFN-gamma ELISPOT assay. ► Fusion protein can replace ESAT-6, CFP-10 or a combination of both in ELISPOT assay for BTB diagnosis.
Phage display peptide libraries are widely used in various protein-protein interaction studies to determine the immunological binding of proteins and epitope mapping of different targets. In the ...present study, the peptide library is used to identify the Fab binding epitope site of Tetanus Toxoid (TT). The peptides were screened against Fab by biopanning, using a random peptide 12mer phage display library. Clones were selected based on the binding activity by phage ELISA and one of the peptide sequence (DTMSYTPNIHLL), which showed highest binding activity towards the Fab was cloned into pGEX-4T1 vector for expression and purified using glutathione agarose affinity chromatography. The binding activity of the purified peptide was demonstrated by ELISA and Immunoblot analysis. Further, analysis of the peptide sequence revealed homology of four amino acids (YTPN) to heavy chain sequence of TT. F13 Fab significantly binds to the YTPN epitope of the TT antigen as indicated by ELISA and may have potential in diagnostics.
In vitro production of bovine interferon gamma (BoIFN-γ) cytokine from bovine peripheral blood mononuclear cells (PBMCs) can be detected using the most sensitive enzyme-linked immunosorbent spot ...(ELISPOT) assay. ELISPOT assays are dependent on the quantity and quality of PBMC preparations and hence contribute significantly to the performance of this assay. In order to standardise the methods for isolation of PBMCs, we compared two methods for the processing of bovine blood which included aliquots of blood that were stored in a horizontal position without dilution or agitation and aliquots of blood that were immediately diluted 1:1 with complete Rosewell Park Memorial Institute (RPMI) 1640 medium and stored in a horizontal position with gentle agitation. PBMCs were isolated at 2, 4, 6, 8 and 24 h and at 4°C and at 22°C ± 2°C. They were stimulated using tuberculosis-specific antigens, after which the ELISPOT assay was performed. Quantities of spot-forming cells (SFC) created by the release of BoIFN-γ in ELISPOT assays were significantly greater in the samples stored at 22°C ± 2°C than those held +4°C and the intensity of the signals dropped following processing after 6 h. A further drop in SFC was observed in those samples that had been stored undiluted and without agitation. These findings demonstrated that optimisation of PBMC isolation procedures can lead to increased sensitivity in the detection of BoIFN-γ using the ELISPOT assay, thus contributing to an enhanced diagnosis of bovine tuberculosis.
Use of recombinant Bm95 isolated from an Argentinean strain of Rhiphicephalus microplus A as a vaccine candidate to control the tick infestation in India was investigated. Recombinant Bm95 expressed ...in yeast, Pichia pastoris. Purified protein blended into a vaccine using aluminium hydroxide as an adjuvant. An adverse effect on the reproduction of ticks was observed in vaccinated animals indicating reduced the environmental contamination and source of infestation indicating its use of Bm95 in the immunological control of ticks in India.
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