Leukocyte counts in BALF were performed as previously described [42, 43] . Briefly, three mice were euthanized on days 4, 6, 8 and 10 p.i., and the lungs were lavaged twice in situ with the chest cavity opened by midline incision with a total volume of 1.0 ml saline (4uC) inserted through an endotracheal tube. The rate of recovery of BALF was not less than 90% for all animals tested. After the amount of fluid recovered was recorded, an aliquot of BALF was diluted 1:1 with 0.01% crystal violet and 2.7% acetic acid for leukocyte staining and erythrocyte hemolysis. The number of leukocytes in the BALF was counted with a hemacytometer under a microscope. For differential counts, the BALF samples from each mouse were stained with Wright stain, and the numbers of monocytes, neutrophil and lymphocytes were determined, on the basis of morphologic criteria, under a light microscope, with evaluation of at least 200 cells per slide. All slides were counted twice by different observers blinded to the status of the animal.