Isolation, propagation, and characterization of a newly recognized pathogen, cilia-associated respiratory bacillus of rats, an etiological agent of chronic respiratory disease

Abstract

A Gram-negative, filamentous, rod-shaped bacillus which failed to grow in cell-free media was isolated in apparently pure culture from the bronchial scraping and washing of a laboratory rat suffering from chronic respiratory disease by inoculating embryonated chicken eggs via the allantoic route. None of the embryos died during 20 serial passages at weekly intervals. The bacillus was reisolated in embryonated eggs from cesarean-derived barrier-maintained N:SD(SD) rats 8 and 12 weeks after intranasal inoculation with 10th-passsage allantoic fluid. The inoculated rats were housed in Horsfall-type units and remained free from other known respiratory pathogens, including mycoplasmas and murine viruses, throughout the study. The bacillus colonized the ciliated epithelial cells of the respiratory tract and caused a marked peribronchial infiltration and hyperplasia of mononuclear cells which progressed with time. The bacillus, ca. 0.2 μm wide by 4 to 6 μm long, stained very poorly with basic aniline dyes but was readily demonstrated with the Warthin-Starry silver technique. It was heat labile (56°C for 30 min); spore forms were not observed. It withstood freeze-thawing and was successfully stored at -70°C. Although no visible means of locomotion was observed with the electron microscope, a slow gliding motility, sometimes with bending and flexing of bacilli apparently adherent to the glass surface, was observed with phase microscopy. As an etiological agent of chronic respiratory disease of rats, this cilia-associated respiratory bacillus (tentatively designated the CAR bacillus) may be the first recognized gliding bacterium known to cause disease in a warm-blooded vertebrate.