Search

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Neither naturally occurring chancroid nor experimental infection with H. ducreyi results in protective immunity. Likewise, a single inoculation of H. ducreyi does not protect pigs against subsequent infection. Accordingly, we used the swine model of chancroid infection to examine the impact of multiple inoculations on a host's immune response. After three successive inoculations with H. ducreyi, pigs developed a modestly protective immune response evidenced by the decreased recovery of viable bacteria from lesions. All lesions biopsied 2 days after the first and second inoculations contained viable H. ducreyi cells, yet only 55% of the lesions biopsied 2 days after the third inoculation did. Nearly 90% of the lesions biopsied 7 days after the first inoculation contained viable H. ducreyi cells, but this percentage dropped to only 16% after the third inoculation. Between the first and third inoculations, the average recovery of CFU from lesions decreased approximately 100-fold. The reduced recovery of bacteria corresponded directly with a fivefold increase in H. ducreyi-specific antibody titers and the emergence of bactericidal activity. These immune sera were protective when administered to naïve pigs prior to challenge with H. ducreyi. These data suggest that pigs mount an effective humoral immune response to H. ducreyi after multiple exposures to the organism.

We developed a new enzyme immunoassay (rpEIA) for use in determining the seroprevalence of chancroid. Three highly conserved outer membrane proteins from Haemophilus ducreyi strain 35000 were cloned, overexpressed, and purified from Escherichia coli for use as antigens in the rpEIA. Serum specimens from patients with and without chancroid were assayed to determine optimum sensitivity and specificity and to establish cutoff values. On the basis of these data, rpEIA was found to be both sensitive and specific when used to test a variety of serum specimens from patients with genital ulcers and urethritis and from healthy blood donors.

The response to heat shock in Haemophilus ducreyi has been investigated by labelling newly synthesized proteins with a pulse of 35S-methionine at a range of temperatures between 30-40 degrees C. Proteins were then separated by SDS-PAGE and analysed by autoradiography of the resultant profiles. Four heat shock proteins (HSP) were identified of apparent molecular weight < 14, 58.5, 74 and 78 kDa. The three larger proteins appeared to be independently controlled and inducible in a range of H. ducreyi strains. The protein profiles were also probed by Western blot with serum from an immunized rabbit, serum from a rabbit infected intradermally with H. ducreyi, mouse monoclonal antibodies (MAb) and a pool of human sera from patients with chancroid. The rabbit sera, the human serum pool and MAb (BB11) reacted with epitopes on the HSP of 58.5 kDa, whereas only the immunized rabbit serum and another MAb (CC11) reacted with epitopes on the HSP of 78 kDa. The HSP of 58.5 kDa appears to be immunogenic and may play a part in the host response to H. ducreyi and the pathogenesis of chancroid.

Haemophilus ducreyi cytotoxin-positive and -negative strains as well as bacterial sonicates and lipooligosaccharides (LOS) from such strains were evaluated for the capacity to produce dermonecrotic lesions, especially ulcers, after intradermal injections to rabbits and to different mouse strains, including nude mice. Dermonecrotic lesions of the ulcerous type were observed within 4 days and they were developed in both rabbits and mice with about 10(7) colony forming units (cfu) of H. ducreyi. Viable bacteria were isolated from the lesions up to 9 days after inoculation. All lesions healed spontaneously within 2-3 weeks. Bacterial sonicate (heated and unheated) and LOS preparations caused mainly abscess formation in rabbits, while in mice, a superficial, haemorrhagic ulceration was observed. To obtain ulceration at all injection sites, about 200 micrograms of LOS was required. Histological examination of acute, dermonecrotic lesions caused by viable bacteria showed deep necrosis, infiltrate of inflammatory cells, especially granulocytes and dilatation of blood vessels. The same type of inflammatory cells as seen in lesions caused by bacteria, were involved in the mouse lesions caused by bacterial sonicate and LOS preparations. The results indicate that LOS/endotoxin, probably in combination with other bacterial polysaccharides, can play a role in ulceration caused by H. ducreyi in animals; however, a relatively high amount of LOS preparation was necessary to cause dermal ulceration at all injection sites in the mouse model. The development of ulcers correlated with the endotoxin activity in bacterial sonicate and in LOS preparations. The model may therefore be useful to study the role of LOS components in development of ulceration. There was no significant difference in lesions caused by cytotoxin producing, respectively, non-producing H. ducreyi strains and cell-free preparations from such strains. The bacterial sonicates, cytotoxic for human cell lines, failed to kill animal cell lines, indicating that animal models do not adequately reflect the cytotoxin activity in experimental H. ducreyi infection. Antibodies to H. ducreyi sonicate and LOS, tested by means of ELISA, were found in pre-immune sera from both rabbits and mice. There was a significant antibody response to homologous cell sonicate and LOS, after primary and secondary infections with bacteria. Still, there was no clear difference between primary and secondary lesions in animals. Since animal lesions are mainly due to endotoxin activity, this may indicate that antibodies are of minor importance for protection in animal models.

In 1984 and 1985, outbreaks of genital ulcers occurred in Florida and New York. Initial investigations for syphilis, herpes simplex, Chlamydia trachomatis, and Haemophilus ducreyi did not implicate any of these organisms as etiologic agents. From the results of dot-immunobinding assays, we found that sera from the patients had higher levels of IgM (30 [47.6%] of 63 patients) and IgG (22 [34.9%] of 63 patients) reactivities with an outer-membrane preparation from H. ducreyi strain CIP542 than with outer-membrane preparations from Haemophilus influenzae ATCC 10211 or Haemophilus parainfluenzae ATCC 7901. In contrast, sera from 35 patients in the control group did not react with any of the outer-membrane preparations (P less than .01 for both IgM and and IgG reactivity), nor did sera from eight individuals with disease caused by H. influenzae (P = .051 for IgM reactivity, P = .02 for IgG reactivity). Indirect immunofluorescence assay using a monoclonal antibody reactive with outer-membrane preparations from H. ducreyi strain CIP542 stained organisms in smears of lesion material from genital ulcers from three of six patients. These results suggested that the cause of both outbreaks of genital ulcers was H. ducreyi which was subsequently isolated in both geographic areas.