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-passage 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 ,Lm wide by 4 to 6 ,um 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.

Cilia‐Associated Respiratory (CAR) Bacillus Infection In Conventionally Reared Rabbits

“…In this study, as in most other reports, CAR bacillus was directly visualized in silverstained histological section, which represents the method of choice for the diagnosis of CAR bacillus infection ( CUNDIFF et al, 1994a). In vitro culture of CAR bacillus can be performed only in embryonated chicken eggs, mammalian cell cultures or cell culture medium supplemented with foetal calf serum ( GANAWAY et al, 1985; LA REGINA and UNDER, 1991;SCHOEB et al, 1993;CUNDIFF et al, 1994a). However, these techniques are expensive and time consuming, thus appearing not advisable as routine diagnostic methods.…”

“…In rats and mice, CAR bacillus infection has been related with chronic respiratory disease (CRD) characterized by histological lesions resembling those described for the murine respiratory mycoplasmosis (MRMJ These lesions consist of peribronchial cuffing with lymphocytes and plasma cells, accompanied by mucopurulent exudate filling the bronchial lumen and leukocytic infiltration in the lamina propria (GANAWAY et al, 1985;GRIFFITH et al, 1988;FRANCE. 1994;MEDINA et al, 1994).…”

“…1994;MEDINA et al, 1994). In addition, the pathogenic significance of CAR bacillus in rats and mice has been proved by experimental infection studies (GANAWAY et al, 1985; MATSUSHITA and JOSHIMA, 1989; MATsUSHlTA et d., 1989; SHOJI-DARKYE et d., 1991;SCHOEB et al, 1997).…”

Detection of Mycoplasma pulmonis in cilia-associated respiratory bacillus isolates and in respiratory tracts of rats by nested PCR

“…In addition to the positive results obtained with samples of all six strains known from culture results to contain M. pulmonis, M. pulmonis was detected in two additional strains from which M. pulmonis could not be isolated. One of these, CAR bacillus X2006C, was isolated from a domestic rabbit; the other, CAR bacillus NIH, was derived from the original rat isolate (6). Of samples from mycoplasma broth cultures in which growth was suspected due to weak pH changes but from which mycoplasmas could not be subcultured on solid media, nested PCR increased the number of samples giving positive results in the first PCR from 26 of 33 (79%) to 33 of 33 (100%).…”

“…CAR bacillus strains were designated according to their sources (16). CAR bacillus NIH, the reference strain, and CAR bacillus StL were derived from the original isolate obtained in embryonated eggs by Ganaway et al (6). CAR bacilli isolated in our laboratory were designated according to their accession numbers.…”