AVALIAÇÃO DO CRESCIMENTO BACTERIANO POR CITOMETRIA DE FLUXO E PRODUÇÃO DE ANTÍGENOS SECRETADOS DE DIFERENTES CEPAS DE Corynebacterium pseudotuberculosis

Abstract

Introduction: Corynebacterium pseudotuberculosis responsible for Caseous Lymphadenitis (LC), a chronic infectious-contagious disease, that usually affects small ruminants, compromising the economy of small producers and of the regions that count on agribusiness. The infection in human occurs by the handling of animals, consumption of its raw derivatives or in laboratories, with few cases described and only one study in Brazil. Animal diagnostics are relatively simple, but human has complications. Bacteriological culture derived from cheesy content, followed by biochemical tests is currently the gold standard test. Purpose: The present work aims to evaluate and compare bacterial growth and antigen production to evaluate the human seroreactivity of three strains of Corynebacterium pseudotuberculosis. Methods: For bacterial counts and cell viability study in FACSCalibur (BD) cultures of the VD57 strain for the three times (24, 48, 72 hours) and comparison with strains CAP3W (21) and CAPJ4 (76) were carried out in 24 and 48 hours. For the production of the antigen, the supernatant of the VD57, CAP3W (21) and CAPJ4 (76), semipurified by three phase partitioning (TPP) strains was used with 48 hours culture supernatant. Extract was produced in medium with and without Tween only for CAPJ4 strain (76). The electrophoretic profile of the four extracts was analyzed by SDS-PAGE in 12% gel and the Western blot analysis was performed with a pool of serum samples from the non-contacting group (composed of 4 participants with no history of contact with the bacteria or consumption of raw or (consisting of 4 individuals with contact history for activity in a microbiology laboratory) and the group contacting farm (composed of 4 individuals with a history of contact with infected animals). Results: FACs showed faster cell growth for VD57 strain with a 10-fold higher result than CAP3W (21) and CAPJ4 (76). The peak of the recorded cell viability was 48 hours, but no significant variation in cellular mortality was identified at 72 hours. The electrophoretic profile of the extracts produced revealed differences in protein expression among the three strains, as well as for cultures under different conditions for CAPJ4 (76). Human seroreactivity was recorded in the four extracts, confirming the presence of antigenic proteins in all strains, but it was possible to identify difference in the profile of recognition between the groups Buyers and Non Contacters only in the extract VD57. Conclusion: The VD57 strain shows growth apex and viability in the 48 hours of culture and in the comparison between the three strains, it shows growth and viability superior to the others. The electrophoretic profile of the extracts of Corynebacterium pseudotuberculosis strains cultivated under the same conditions, present similarity in the number of bands and difference in molecular weight. The use of Tween-80 in the culture medium generates a 50% increase in the number of bands for CAPJ4 (76). All the extracts produced present antigenicity for human serum, however it is possible to show a difference in the Profile of the Contacters of the Non Contact Only profile in VD57.