Search

Mycoplasma fermentans incognitus has been isolated from human tissue in patients both with and without AIDS who died of systemic infection. M. fermentans incognitus and other strains of M. fermentans have been associated with rheumatoid arthritis. While cell extracts of M. fermentans incognitus can induce changes in murine and human cells of the monocytic lineage, little is known about interactions of viable organisms with such cells. Because of the central role of macrophages in chronic inflammation, we examined the effects of M. fermentans incognitus on surface markers and functions of THP-1 cells, a well-characterized human monocytic cell line. This cell line has been used extensively in studies of macrophage differentiation, especially following exposure to phorbol esters. Changes in cell morphology, phagocytosis, rate of cell division, and selected surface markers were evaluated in cultures of THP-1 cells exposed to phorbol myristate acetate (PMA), M. fermentans incognitus, or both. As reported by other investigators, PMA induced THP-1 cells to differentiate into cells resembling tissue macrophages. M. fermentans incognitus only minimally affected changes induced by PMA, slightly increasing the percentage of cells positive for FCgammaRI and major histocompatibility complex (MHC) class II antigens. M. fermentans incognitus alone induced an incomplete arrest in the cell cycle at G0 phase, increased phagocytic ability, and enhanced expression of FCgammaRI, CR3, CR4, and MHC class II antigens.

Studies were conducted to determine whether the production of various cytokines is associated with Mycoplasma pulmonis disease expression. Susceptible C3H/HeN and resistant C57BL/6N mice were inoculated intranasally with 10(7) CFU of virulent M. pulmonis UAB CT or avirulent M. pulmonis UAB T. Expression of genes for tumor necrosis factor alpha (TNF-alpha), interleukin 1 alpha (IL-1 alpha), IL-1 beta, IL-6, and gamma interferon (IFN-gamma) in whole lung tissue and TNF-alpha gene expression in bronchoalveolar lavage (BAL) cells was determined by reverse transcription-PCR using specific cytokine primers at various times postinoculation. In addition, concentrations of TNF-alpha, IL-1, IL-6, and IFN-gamma were determined in BAL fluid and serum samples at various times postinoculation. Our results showed that there was a sequential appearance of cytokines in the lungs of infected mice: TNF-alpha, produced primarily by BAL cells, appeared first, followed by IL-1 and IL-6, which were followed by IFN-gamma. Susceptible C3H/HeN mice had higher and more persistent concentrations of TNF-alpha and IL-6 in BAL fluid than did resistant C57BL/6N mice, indicating that TNF-alpha and possibly IL-6 are important factors in pathogenesis of acute M. pulmonis disease in mice. Serum concentrations of IL-6 were elevated in C3H/HeN mice, but not C57BL/6N mice, following infection with M. pulmonis, suggesting that IL-6 has both local and systemic effects in M. pulmonis disease.

Ammonia (NH3) from soiled cage bedding is known to enhance the progression and severity of murine respiratory mycoplasmosis in rats. To test the hypothesis that NH3 directly or indirectly enhances the growth of Mycoplasma pulmonis in vivo, pathogen-free F344 rats were inoculated intranasally with 1 x 10(4) to 4 x 10(4) or 4 x 10(6) to 5 x 10(6) colony-forming units of M. pulmonis and exposed to less than or equal to 1.5 or 76 microgram of NH3 per liter (less than or equal to 2 or 100 ppm, respectively). Nasal passages, larynges, tracheas, and lungs from rats killed at intervals up to 28 days after inoculation were quantitatively cultured. Growth of M. pulmonis was much greater in NH3-exposed rats than in controls, particularly in those inoculated with the lower dose. Increases in M. pulmonis populations were more rapid in proximal airways than in distal airways. Serum immunoglobulin G and M antibody responses to M. pulmonis as measured by an enzyme-linked immunosorbent assay were greater in NH3-exposed rats. In other experiments, the nasal passages absorbed virtually all NH3 when the rats were exposed to less than 380 micrograms of NH3 per liter (500 ppm), indicating that NH3 induced increases in the numbers of organisms in the distal respiratory tract, probably by a secondary, rather than a direct, effect. Also, NH3 exposure did not inhibit pulmonary antibacterial activity as measured by clearance of radiolabeled Staphylococcus epidermidis. The growth of M. pulmonis in vitro was inhibited by 1 mM NH4+ added to the medium as NH4OH but not by NH4+ concentrations of 0.5, 0.1, or 0.01 mM, suggesting that NH3 increases growth indirectly through effects on the host.

The proteins of Mycoplasma pulmonis were examined by two-dimensional polyacrylamide gel electrophoresis and immunoblotting. Comparison of profiles from silver-stained two-dimensional polyacrylamide gel electrophoresis of 18 strains of M. pulmonis allowed identification of five proteins that were strain variable. These variable proteins were not dependent on the medium used to grow the organisms and were shown by reaction with serum samples from naturally infected rats and mice to be produced in vivo and not to be medium components. Identification of strain-variable proteins may lead to an explanation of the differences in properties found among M. pulmonis strains.

Not only is murine respiratory mycoplasmosis, due to Mycoplasma pulmonis, a complication of biomedical research, it provides excellent animal models to study the development of a naturally occurring respiratory disease induced by an infectious agent. The understanding of pathogenic mechanisms of disease can be greatly facilitated by studying genetic differences in susceptibility. Five strains of mice with various H-2K haplotypes were examined for their susceptibility to murine respiratory mycoplasmosis; of these, C57BL/6 and C3H/HeN mice were chosen for additional study. There were no significant differences in the incidence of infection in either the upper or lower respiratory tract or in the severity of upper respiratory tract lesions in the two strains as determined at 14 days postinfection. In striking contrast, the C57BL/6 mice were significantly more resistant to the development of gross and microscopic lung lesions and to death due to pneumonia as shown by an almost 100-fold difference in the 50% lethal dose, 50% gross pneumonia dose, and 50% microscopic lesion dose. The most apparent differences in lung lesions between the two strains were in the severity of acute lesions of the bronchial epithelium, the amount of mixed inflammatory response in the alveoli, and the amount of lymphoid infiltrates. All were significantly more severe in C3H/HeN mice. In addition, more C3H/HeN mice developed antibody responses to M. pulmonis. The amount of antibody correlated with lesion severity in both strains.

Pathogen-free C3H/HeN mice were exposed by aerosol to Mycoplasma pulmonis PG34(ASH), UAB 5782C, M1, UAB T, or UAB CT, and clearance of mycoplasmas from the nasal passages, trachea, and lungs was determined during the first 72 h postinoculation (PI). There were differences among strains of mycoplasmas in physical removal of organisms and in killing by nonspecific factors in the nasal passages and trachea. The avirulent strain, PG34(ASH), was quickly removed from the nasal passages and trachea. Physical removal of the other mycoplasmal strains occurred slowly, with 60 to 89% of the radioactive label remaining in the nasal passages and trachea even after 72 h. There were significant differences in killing among mycoplasmal strains by nonspecific host mechanisms in the nasal passages, trachea, and lungs. Strain UAB T was quickly killed at all levels of the respiratory tract. Strains UAB 5782C and M1 were killed at all three sites by 2 to 4 h PI. The most virulent strain, UAB CT, was killed much more slowly than the other strains. However, there was no statistical difference in the relative numbers of mycoplasmas present in the lungs at 72 h PI among strains UAB CT, UAB 5782C, and M1. These studies showed that the different mycoplasmal strains were cleared from the respiratory tract by different mechanisms and suggest that the differences in virulence among the mycoplasma strains can be explained, in part, by the differences in elimination of the organisms from the respiratory tract by nonspecific host defense mechanisms.

Colony opacity, hemadsorption and hemolysis of erythrocytes, and the ability of whole mycoplasmal cells to induce a blastogenic response when incubated with C3H/HeN or C57BL/6 mouse lymphocytes were examined for 18 strains of Mycoplasma pulmonis to determine if any of these characteristics could be associated with virulence in vivo. Although there were differences among strains in each of these characteristics, none of these parameters were associated with virulence.

The mouse model of acute murine respiratory mycoplasmosis was used to screen 18 strains of Mycoplasma pulmonis for their ability to establish respiratory infections and produce gross lung lesions in the susceptible C3H/HeN mouse strain. All experiments were designed to minimize host, environmental, and microbial differences to ensure that experimental results would reflect differences in mycoplasmal virulence. There were differences in the 50% infectious dose (range, 3 X 10(2) to greater than 10(7) CFU) and the 50% gross pneumonia dose (range, 10(3) to greater than 10(7) CFU) among the 18 mycoplasmal strains. Only 10 strains (UAB CT, M1, UAB 5782C, UAB 6510, 66, UAB T, UAB 8145D, Nelson C, Peter C, and Negroni) established respiratory infections, and only 2 of the 10 strains (UAB CT and M1) produced gross lung lesions. Strains UAB CT, UAB T, M1, UAB 5782C, and PG34(ASH) were chosen for qualitative and quantitative evaluation of lung lesions in C3H/HeN and C57BL/6N mice. Lesion incidence and severity was dependent on the mycoplasmal strain and the mouse strain. Microscopic lesions varied among mycoplasmal strains and mouse strains in the amount of lymphoid infiltrate, neutrophilic exudate, and consolidation, as well as overall severity. The most virulent strain, UAB CT, produced acute pneumonitis in the 10(7) CFU dosage group and required a threshold dose of 10(3) CFU to consistently produce microscopic lung lesions. These results suggest that M. pulmonis virulence is multifactorial and different strains of mycoplasmas yield disease expressions that differ both qualitatively and quantitatively.