CULTIVATION OF CILIA-ASSOCIATED RESPIRATORY BACILLUS IN ARTIFICIAL MEDIUM AND DETERMINATION OF THE 16S RIBOSOMAL-RNA GENE SEQUENCE

Cilia-associated respiratory (CAR) bacillus, an unclassified gliding b acterium associated with respiratory disease in rats, mice, and rabbit s, has previously been cultivated only in embryonated chicken eggs, ce ll culture, or cell culture medium supplemented with conditioned mediu m from cultured tracheas. A reference strain of CAR bacillus, original ly isolated in eggs, grew in cell culture flasks as adherent individua l bacilli and ropy, whorled fascicles in cell culture media supplement ed only with fetal calf serum. Using Dulbecco's minimal essential medi um, we isolated CAR bacillus from naturally infected rats and a natura lly infected rabbit and from experimentally inoculated mice and rats. Isolates were maintained for up to 20 passages. Isolates from rats wer e similar in morphology to the reference strain, but most were more ac tively motile and formed pincushion-like aggregates. The rabbit bacill i were smaller and formed fewer aggregates. DNAs of rat isolates diffe red only slightly in restriction fragment patterns from that of the re ference strain, whereas that of the rabbit isolate was distinctly diff erent. Cultures of CAR bacilli of all strains from rats contained Myco plasma fermentans, Mycoplasma pulmonis, or both, and cultures of the C AR bacillus from the rabbit contained an unidentified arginine-utilizi ng mycoplasma. The sequence of the 16S rRNA gene of the reference stra in was determined by amplification by polymerase chain reaction, cloni ng of the product, and sequencing by the dideoxynucleotide chain termi nation method. Comparison of the sequence with sequences in the GenBan k data base indicated that CAR bacillus is a unique organism most clos ely related to Flavobacterium ferrugineum and Flexibacter sancti.