Macrophage inflammatory response to Clostridium chauvoei infection

GALLO GIOVANNA LUCRECIA; VEGA ALBA EDITH; VILLA MARÍA CECILIA; CÁCERES CLAUDIA SOLEDAD; CORTIÑAS TERESA INÉS; STEFANINI ANA MARÍA

Carlos Paz, Córdoba

Congreso; VI Congreso Argentino de Microbiología General; 2009

Sociedad Argentina de Microbiología General

Clostridium chauvoei is an anaerobic bacillus, which is the causative agent of blackleg, a clostridial myonecrosis that has an important impact on the sheep and cattle industry worldwide. Blackleg is considered a gangrene gaseous, which is an acute disease with a poor prognosis and often fatal outcome. Initial trauma to host tissue damages muscle and impairs blood supply. Initial symptoms are generalized fever and pain in the infected tissue. As the clostridia multiply, various exotoxins (including hemolysins, collagenases, proteases, and lipases) are liberated into the surrounding tissue, causing more local tissue necrosis and systemic toxemia. Infected muscle is discolored (purple mottling) and edematous and produces a foul-smelling exudates with gas bubbles formed from the products of anaerobic fermentation. In many inflammatory diseases many immuno-mediators are involved in this process, amongst them, we can remark cytokines TNF-alfa, IL-6 and IL-1beta. IL-6 is a pleiotropic citokine of complex biological activity and TNF-alfa is a pro-inflammatory cytokine which main function is the recruitment and stimulation of neutrophils and monocytes and the induction of apoptosis, giving rise to inflammation and connective tissue destruction. We hypothesize that C. chauvoei may induce an inflammatory cascade which stimulates tissue destruction mediated by the host. Until now it is unknown which are the immune inflammatory response involved in C. chauvoei infection. The aim of this work was evaluate the capability of C. chauvoei to induce the expression of IL-1beta, IL-6 and TNF-alfa pro-inflammatory citokynes in peritoneal mouse macrophage. beta- actin was used as housekeeping gene. Macrophage (1 x 106 cell/well) were co-cultures with C. chauvoei cells obtained in log phase at different multiplicity of infections (MOI): 3:1, 10:1 y 20:1 and treated with culture supernatants at 5% and 10%, for 5, 12 and 21 h of incubation. Total RNA was obtained using TRIZOL reagent. For RT-PCR, RNA was reverse transcribed into cDNA and used for RNA amplification with specific primers. The bands were semi-quantitative analyzed by Scion image software. The results showed that C. chauvoei induces an increased expression of TNF-alfa between 3 to 10 times higher than the control at 21 h of postinfection. Meanwhile for IL-1beta expression the increase observed was between 3 times at 12 h to 10 times at 21h. The IL-6 showed a remarkable increased of 8 times higher than the control at 12 h. The expression was dose dependent in all cases and the lost of viability was observed in macrophages infected at higher MOI and treated with culture supernatants. Our results showed that the pro-inflammatory cytokines must play an important role in blackleg outcome.