Localization of Haemophilus ducreyi at the pustular stage of disease in the human model of infection

To localize Haemophilus ducreyi in vivo, human subjects were experimentally infected with H. ducreyi until they developed a painful pustule or for 14 days. Lesions were biopsied, and biopsy samples were fixed in 4% paraformal dehyde, and cryosectioned, Sections were stained with polyclonal anti-H. du creyi antiserum or H. ducreyi-specific monoclonal antibodies (MAbs) and flu orescently tagged secondary antibodies and examined by confocal microscopy. We identified H. ducreyi in 16 of 18 pustules but did not detect bacteria in the one papule examined. H. ducreyi was observed as individual cells and in clumps or chains. Staining with MAbs 2D8, 5C9, 3B9, 2C7, and 9D12 demon strated that H. ducreyi expresses the major pilus subunit, FtpA, the 28-kDa outer membrane protein Hlp, the 18-kDa outer membrane protein PAL, and the major outer membrane protein (MOMP) or OmpA2 in vivo. By dual staining wit h polyclonal anti-II. ducreyi antiserum and MAbs that recognize human skin components, we observed bacteria within the neutrophilic infiltrates of all positively staining pustules and in the dermis of 10 of 16 pustules, We we re unable to detect bacteria associated with keratinocytes in the samples e xamined. The data suggest that II, ducreyi is found primarily in associatio n with neutrophils and in the dermis at the pustular stage of disease in th e human model of infection.