Shinji Takai

Expression of Virulence-Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH

We recently reported that there are two different virulence‐associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 106 cells expresses 15‐ to 17‐kDa antigens and intermediately virulent R. equi that kills mice with 107 cells expresses a 20‐kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence‐associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence‐associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20‐kDa antigen was found to be located on the cell surface, as were the 15‐ to 17‐kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence‐associated antigens of R. equi is dependent on the environmental conditions.

Immunohistochemical Detection of Virulence-associated Antigens of Rhodococcus equi in Pulmonary Lesions of Foals

Rhodococcus equi was isolated from the lungs of six foals with bronchopneumonia. All isolates expressed 15-17-kd antigens by immunoblot analysis and contained a virulence-associated plasmid of 85 or 90 kb. Immunohistochemically, R. equi from all pulmonary lesions showed the expression of 15-17-kd antigens mainly in the phagocytic cells. The specific monoclonal antibody to 15-17-kd antigens of R. equi (MAb 10G5) may be an aid in the diagnosis of R. equi-induced pneumonia.

Serum antibody responses of foals to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi.

Humoral immune responses in 16 foals to virulence-associated 15- to 17-kDa antigens of Rhodococcus equi were studied during the first fourteen weeks of life on two horse-breeding farms with a persistent incidence of R. equi infection. Serum antibody levels specific for 15- to 17-kDa antigens were measured by enzyme-linked immunosorbent assay and Western immunoblotting. Immunoglobulin G (IgG) antibodies specific to 15- to 17-kDa antigens were detected by all the foals. R. equi was found in the feces of foals during week 1 of life, and the number of fecal R. equi rapidly increased to the highest level. Virulent R. equi were isolated from the feces of the foals at a high frequency and from their environmental soil on the farms. Evidence that serum antibody response to 15- to 17-kDa antigens of virulent R. equi occurred naturally in every foal in correlation with the quantitative changes of fecal R. equi during the first 1 to 3 months of life suggests that intestinal virulent R. equi might be the most important source of antigenic stimulation in foals from contaminated farms.

Effect of growth temperature on maintenance of virulent Rhodococcus equi

Repeated passage of virulent Rhodococcus equi ATCC 33701 and L1 at 38 degrees C resulted in attenuation of the strains as a result of curing the virulence plasmid; at 30 degrees C, repeated passage had no such effect. At a temperature of 38 degrees C the plasmid-bearing cells replicated more slowly than their plasmid-cured derivatives and so were gradually replaced by cells lacking plasmids. In contrast, at a temperature of 30 degrees C the growth rate of either strain was not affected by the presence or absence of the plasmid. No plasmid-cured derivative was recovered from mouse organs at 48 h after inoculation of a mixture of equal numbers of bacteria with and without plasmids. It is concluded that under nonselective conditions growth temperature is an important factor in maintaining the virulence of R. equi.