Development and application of a quantitative virulence assay for Haemophilus ducreyi in an in vivo model of infection.

Abstract

A temperature-dependant rabbit model of Haemophilus ducreyi infection was used as a quantitative virulence assay to evaluate the effect of host factors upon ulcerative cutaneous disease production. New Zealand White rabbits underwent inoculation with H. ducreyi strain #35000 either after iron loading, dexamethasone immunosuppression, prior infection or immunization. Chancroid-like disease was scored for severity, time, culture of lesions, and serologic response. In primary infections, culture-positive ulcerative lesions were consistently produced at and above 105 colony forming units inocula. Iron and dexamethasone treatment increased lesion severity and duration of culture positivity. Infection of previously infected animals produced sterile lesions of greater size and higher cumulative disease score at 105 colony forming units inocula. Infection of immunized animals produced sterile lesions of lesser severity at 105 colony forming units, with protection from ulcer formation. Efficacy of ceftriaxone treatment was tested in naive control and iron loaded rabbits. Antibiotic was injected as a single intramuscular dose of 0.1 mg/Kg and 5 mg/Kg, four days following inoculation with Haemophilus ducreyi #35000. In naive control rabbits, antibiotic treatment at each dose sterilized the lesions within 24 hours with attenuation of disease effect. In naive iron loaded rabbits lesions were sterilized later on day 10 with 0.1 mg/Kg ceftriaxone and on day 5 with 5 mg/Kg ceftriaxone. Virulence scores corroborated the lessened microbiologic efficacy of antibiotic treatment. We conclude that quantitative assay of infection and disease in iron loaded animals, with relative prolongation of disease effect and culture positivity of lesions may be a sensitive model in which to comparatively measure virulence related to bacterial factors. As well, limitations of efficacy or synergy of antibiotic treatments may be evaluated. We conclude that prior antigenic exposure and immune response through infection or immunization attenuates subsequent homologous infection. Disease produced in re-infection is amplified, while disease produced in inoculation after immunization is attenuated at lower inocula. This suggests that while there is inducible immunity to H. ducreyi infection and disease, the disease effect may be in part related to host inflammatory response to bacterial antigen or effect of bacterial toxin. This system of measurement of virulence may be useful towards understanding pathogenesis, and identifying strategy for vaccine development in chancroid.