Evaluation of Bacterial Burden and Neutrophil Response

The immunized mice were challenged intratracheally on day 42 with a lethal dose of 3 × 10⁸ CFU, mixed with 10% porcine mucin (type 3; Sigma-Aldrich, Taiwan) of mid-log phase E. anophelis C08. At 36 hpi (when the control mice were expected to begin dying), organs were harvested and homogenized in sterile PBS. The homogenized organs were quantitatively cultured for each separate mouse to determine the bacterial burden. The excised organs were placed in vials containing 4% formaldehyde and stained with hematoxylin and eosin and myeloperoxidase antibody (ab188211, Abcam, Cambridge, UK) for immunohistochemical analysis of neutrophilic granulocytes. Multiple tissue sections of each organ were scored by a blinded pathologist for injury severity, as previously described (45 (link), 46 (link)). MPO-positive areas were imaged using a Nikon 90i Eclipse widefield microscope (Nikon Instruments Inc., Melville, NY, USA) and quantified using NIS-Elements software.