Association of Three Avian Escherichia coli Strains with Chicken Macrophages
F.J. Azeredo; N.L. Barbieri; L.B. Matter; F. Horn
Departamento de Biofísica, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, Brasil.
Avian pathogenic Escherichia coli (APEC) strains, the etiological agent of colibacillosis in poultry, must resist the attack of incoming macrophages in order to cause disease. In this work, we quantified the association of two APEC strains (UEL31 and UEL13) to the HD11 cell line of chicken macrophages. An avian E. coli isolated from the intestinal flora of a healthy chicken (FN14) was used as control.
Monolayers of HD11 cells were infected with bacteria for 1 h; cells were then washed and reincubated in medium containing 50 mg/ml gentamicyn for another 3 h. Association of bacteria was assessed by looking at the Giemsa-stained samples under an optical microscope. When HD11 macrophages were infected at a multiplicity of infection (MOI) of 150 CFU/cell, strains UEL31 and UEL13 associated with ~96 % and ~35 % of HD11 cells, with ~16 and ~5 bacteria/infected cell, respectively, while the non-pathogenic strain (FN14) associated only with 9 % of HD11 (~2 bacteria/infected cell). Internalization of the associated bacteria was analysed by the gentamicyn resistant assay, in which, after the gentamicyn incubation, cells were lysed and extracts plated onto MacConkey-agar plates for bacterial growth. These assays revealed that the number of viable intracellular bacteria in HD11 is low. At a MOI of 150, for UEL31 we recovered an average of 0.67 CFU/cell, while for UEL13 and FN14 there was virtually no viable intracellular bacteria.
We also analysed cell membrane damage, through lactate dehydrogenase liberation, of HD11 cells after APEC infection. Compared to uninfected cells, the three strains were cytotoxic to HD11, UEL31 being the most cytotoxic.
Financial support: FAPERGS; BIC-Propesq UFRGS.
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