Mycoplasma gallisepticum invades chicken erythrocytes during infection

Abstract

Recently, it was demonstrated using in vitro assays that the avian pathogen Mycoplasma gallisepticum is able to invade nonphagocytic cells. It was also shown that this mycoplasma can survive and multiply intracellularly for at least 48 h and that this cell invasion capacity contributes to the systemic spread of M. gallisepticum from the respiratory tract to the inner organs. Using the gentamicin invasion assay and a differential immunofluorescence technique combined with confocal laser scanning microscopy, we were able to demonstrate in in vitro experiments that M. gallisepticum is also capable of invading sheep and chicken erythrocytes. The frequencies of invasion of three well-defined M. gallisepticum strains were examined over a period of 24 h, and a significant increase in invasiveness occurred after 8 h of infection. In addition, blood samples derived from chickens experimentally infected via the aerosol route with the virulent strain M. gallisepticum R(low) were analyzed. Surprisingly, M. gallisepticum R(low) was detected in the bloodstream of infected chickens by nested PCR, as well as by differential immunofluorescence and interference contrast microscopy that showed that mycoplasmas were not only on the surface but also inside chicken erythrocytes. This finding provides novel insight into the pathomechanism of M. gallisepticum and may have implications for the development of preventive strategies.

FIG. 1.

Scanning electron micrographs of sheep (A) and chicken (B) erythrocytes after in vitro infection with a clonal derivative of M. gallisepticum strain R_low. The arrows indicate mycoplasmas or imprints of mycoplasmas appearing to sink into the erythrocyte surface.

FIG. 2.

Confocal z scan of a chicken RBC infected in vitro with M. gallisepticum strain R_low after DIF staining. The same area of a confocal microscopic image after DIF staining was analyzed for extracellular M. gallisepticum labeled with FITC (green fluorescence) (A) and for extra- and intracellular M. gallisepticum labeled with Alexa Fluor 405 (red fluorescence) (B). (C to F) Superimposition of the green and red fluorescence images (D to F) and a DIC micrograph (C) resulted in yellow fluorescence, indicating extracellular M. gallisepticum, while the red fluorescent focus indicates an intraerythrocytic mycoplasma cell. When the image was scanned from the top to the bottom (D to F) of the erythrocyte, first an extracellular mycoplasma cell located at the surface (yellow) was visible, which slowly faded out as the cross section layer was moved downward (E and F). At the same time the intracellular mycoplasma cell came to the fore (E and F), showing the difference in the localizations of the two mycoplasma cells.

FIG. 3.

Frequencies of invasion of chicken RBCs by M. gallisepticum strains R_low, R_high, and 6/85 at different times. The values are the means ± standard deviations of a minimum of five independent gentamicin invasion assays. The asterisks indicate statistically significant differences in RBC invasiveness between R_low (open bars) and R_high (gray bars) or 6/85 (black bars), and the plus sign indicates statistically significant differences between R_high and 6/85.

FIG. 4.

RBCs from an experimentally M. gallisepticum-infected chicken after DIF staining: superimposed image after FITC and Alexa Fluor 633 labeling, showing M. gallisepticum attached to an RBC's surface (yellow) and inside an RBC (red) after experimental in vivo infection. The erythrocytes were visualized by differential interference microscopy.

FIG. 5.

Agarose gel electrophoresis of nested PCR products from blood samples of experimentally infected chickens. Blood samples were taken before experimental infection of chickens (lane 0) and on day 6 p.i. (lanes 1 and 2), day 12 p.i. (lanes 3 to 5), and day 20 p.i. (lanes 6 to 8). A positive control containing M. gallisepticum-spiked chicken blood (lane +) and a PCR negative control (lane −) were included. Lane M contained a molecular size marker (1-kb ladder; Invitrogen).