Application of alternative identification methods for live tularaemia and brucellosis vaccines

Abstract

SCIENTIFIC RELEVANCE. The sanctions currently imposed on the Russian Federation requireusing Russian diagnostic products in vaccine quality assessments, as well as searching for alternative testing methods, in particular, serological ones.AIM. This study aimed to demonstrate the possibility of using alternative serological methods, including immunoenzymatic, direct agglutination, and indirect haemagglutination methods, for the identification of tularaemia and brucellosis vaccines in studies and quality assessments.MATERIALS AND METHODS. This study used the established pharmacopoeial reference standard (RS) for the bacterial suspension opacity of 10 international opacity units, the established pharmacopoeial RS for and two commercial batches of the live brucellosis vaccine (Brucella abortus 19 BA), and the established and candidate pharmacopoeial RSs for the live tularaemia vaccine (Francisella tularensis 15 NIIEG). These pharmacopoeial RSs were certified by the Scientific Centre for Expert Evaluation of Medicinal Products. Serological testing used Russian commercial diagnostic products, including dry diagnostic sera (polyvalent brucellosis and tularaemia sera for agglutination tests), a liquid erythrocytic diagnostic preparation of tularaemia immunoglobulin, and enzyme immunoassay (ELISA) diagnostic kits for detecting the causative agents for brucellosis and tularaemia. Statistical analysis involved using Microsoft Excel (P=0.95) for ELISA results and qualitative evaluation for the results of direct agglutination and indirect haemagglutination tests.RESULTS. All the tested batches demonstrated positive results. Live brucellosis vaccine batches showed positive results in the slide agglutination tests, while live tularaemia vaccine batches yielded positive results in the tube agglutination tests. All indirect haemagglutination tests showed haemagglutination in live tularaemia vaccine samples at the same concentration as positive control samples (6.25×10⁶  cells/mL). ELISA tests showed similar optical density values (D) for the two vaccines and positive control samples. Live tularaemia and brucellosis vaccines (undiluted, 1.0×10⁹  cells/mL) had D=2.133±0.273 and D=0.127±0.013, whereas the corresponding control samples had D=1.942±0.056 and D=0.123±0.007, respectively. The results reflected the presence of brucellosis or tularaemia microbes in the samples, confirming the identity of the vaccines.CONCLUSIONS. Serological immunoenzymatic, direct agglutination, and indirect haemagglu tination methods with Russian diagnostic products can be used to identify live brucellosis and tularaemia vaccines during quality assessment. The agglutination method with Russian diagnostic sera can be recommended as an alternative quality assessment method for the identification of live brucellosis and tularaemia vaccines, as this method offers time efficiency, simple visual evaluation of results, and low costs and relatively long shelf lives of diagnostic products. However, ELISA and indirect haemagglutination methods cannot be recommended for this purpose because of their labour-intensive and uneconomical nature. The results of this study may support the introduction of the agglutination method in the regulatory documents for live brucellosis and tularaemia vaccines (as an alternative method).