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  • Title: Replacement of the in vivo neutralisation test for efficacy demonstration of tetanus vaccines ad us. vet.
    Author: Rosskopf U, Noeske K, Werner E.
    Journal: ALTEX; 2005; 22(3):169-74. PubMed ID: 16186992.
    Abstract:
    The bacterium Clostridium (C.) tetani is an ubiquitous pathogen. This anaerobic, gram-positive bacterium can form spores and can be found in the whole environment. It enters the body via injuries of the skin and wounds where it releases the neurotoxin "tetanospasmin" (= tetanus toxin). The animals most susceptible to tetanus infection are horses and sheep. Only active immunisation by tetanus vaccine provides effective protection against tetanus intoxication. The marketing authorisation requirements stipulate that efficacy of tetanus vaccines ad us. vet. must be demonstrated in all target animal species via determination of neutralising tetanus serum antitoxin concentrations. The standard method used for this purpose is still the toxin neutralisation test (TNT), as it quantifies the tetanus toxin-neutralising effect of tetanus serum antibodies in vivo. In this test, tetanus toxin is added to dilutions of serum from vaccinated horse and sheep. The serum dilutions are then administered to mice or guinea pigs, which are observed for toxic symptoms. Against the background of animal protection, the goal of one project of the Paul-Ehrlich-Institut (Bundesministerium fuer Bildung und Forschung (Federal Ministry for Education and Research), 0312636) was to establish an alternative to the toxin neutralisation test, enabling the testing of efficacy of tetanus vaccines with serological in vitro methods. For this purpose, a so-called double antigen ELISA (DAE) was established which enables the testing of sera of different species in one assay. In addition, the sera were tested in an indirect ELISA for horses and sheep separately. Altogether, ten groups of horses and eight groups of sheep were immunised with ten animals per group each. The tetanus vaccines comprised almost all products authorised for the German market at the start of the project. 564 horse sera and 257 sheep sera were tested using the two ELISA methods. Some sera were also tested in vivo. The kinetics of antibody responses were recorded. The in vitro DAE method seems to be suitable to replace the mouse neutralisation test used for the detection of tetanus antitoxin in sera of target animal species. The comparison of some sera in the ELISA and the TNT showed good equivalence of results. Nevertheless, before an ELISA titre in horse and sheep sera indicating unambiguous protection against tetanus can be fixed, further comparative assays of low titre sera in the TNT and the DAE will have to be performed.
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