Cytotoxicity of Mycoplasma mycoides subsp. mycoides small colony type to bovine epithelial cells

Abstract

The cytotoxicities of various strains of Mycoplasma mycoides subsp. mycoides small colony type (SC), the agent of contagious bovine pleuropneumonia (CBPP), were measured in vitro using embryonic calf nasal epithelial (ECaNEp) cells. Strains isolated from acute cases of CBPP induced high cytotoxicity in the presence of glycerol, concomitant with the release of large amounts of toxic H2O2 that were found to be translocated into the cytoplasms of the host cells by close contact of the Mycoplasma strains with the host cells. Currently used vaccine strains also showed high cytotoxicity and high H2O2 release, indicating that they are attenuated in another virulence attribute. Strains isolated from recent European outbreaks of CBPP with mild clinical signs, which are characterized by a defect in the glycerol uptake system, released small amounts of H2O2 and showed low cytotoxicity to ECaNEp cells. M. mycoides subsp. mycoides SC strain PG1 released large amounts of H2O2 but was only slightly cytotoxic. PG1 was found to have a reduced capacity to bind to ECaNEp cells and was unable to translocate H2O2 into the bovine cells, in contrast to virulent strains that release large amounts of H2O2. Thus, an efficient translocation of H2O2 into host cells is a prerequisite for the cytotoxic effect and requires an intact adhesion mechanism to ensure a close contact between mycoplasmas and host cells.

FIG. 1.

Glycerol uptake. Numbers of disintegrations per minute (dpm) of ¹⁴C incorporated by mycoplasmas are shown as a measure for glycerol incorporation. Triangles, strain Afadé; squares, strain KH3J. Error bars indicate standard deviations.

FIG. 2.

Fluorescence micrographs showing detection of intracellular H₂O₂ in ECaNEp cells infected with M. mycoides subsp. mycoides SC at an MOI of 500 mycoplasmas per cell. Results are shown for ECaNEp cells 20 min after infection with strain Afadé in the absence (A) and in the presence (B) of glycerol, with strain PG1 in the absence (C) and in the presence (D) of glycerol, and with strain L2 in the absence (E) and in the presence (F) of glycerol.

FIG. 3.

Adherence assay. Shown is a graph representing the relative levels for adherence of strain Afadé and type strain PG1 to ECaNEp cells in culture at different MOIs after 2 h of incubation at 37°C. Triangles, strain Afadé; squares, strain PG1. Lines are trend lines. Error bars represent standard deviations for three measurements.