Secreted sialidase activity of canine mycoplasmas

Abstract

Through a survey of the phylogenetic distribution of sialidase among mycoplasmas, we detected activity secreted by the type strains of 3 of 11 species frequently or first isolated from dogs. The specific activity of washed cells of the type strains of Mycoplasma canis, Mycoplasma cynos, and Mycoplasma molare ranged from 5.2+/-0.8 x 10(-6) to 1.1+/-0.3 x 10(-5) enzymatic units per colony-forming unit (U/CFU). Cells of M. molare strain H542(T) had twice the specific activity (P<0.05) of M. canis strain PG14(T) or M. cynos strain H831(T). Significant differences in sialidase activity existed among nine clinical isolates of M. canis, ranging from not detectable to 2.1+/-0.1 x 10(-5)U/CFU. The type strains of other species previously isolated from dogs (Mycoplasma arginini, Mycoplasma bovigenitalium, Mycoplasma edwardii, Mycoplasma felis, Mycoplasma gatae, Mycoplasma maculosum, Mycoplasma opalescens, and Mycoplasma spumans) did not exhibit either secreted or cell-associated sialidase activity. Neither specific nor degenerate PCR primers complementary to the three known mycoplasmal sialidase alleles were able to amplify orthologs in M. canis, M. cynos, or M. molare, further evidence that the secreted sialidase of those species is distinct from the strictly cell-associated sialidases of Mycoplasma alligatoris, Mycoplasma synoviae, and Mycoplasma gallisepticum. This is the first report of a well-known bacterial virulence factor whose expression varies among strains of certain Mycoplasma species that infect dogs.

Fig. 1

Intraspecific variation in sialidase activity of washed Mycoplasma canis cells. Bars depict mean ± standard error of enzyme units (U) per colony-forming unit (CFU) of clinical isolates and the type strain PG14^T. Means with a different letter are different (P < 0.05 to P < 0.001) by Fisher's Protected Least Significant Difference test (n = 3 independent replications each). The clinical isolate LV had no activity.

Fig. 2

Secreted sialidase activity of canine mycoplasmas. (A) Sialidase was detected by release of fluorescent 4-methylumbelliferone from labeled N-acetylneuraminic acid. Activity was present in washed cells (C) and cell-free conditioned supernatant SP-4 broth (S) from cultures of Mycoplasma cynos H831^T and Mycoplasma molare H542^T. No activity was detected in the type strains of other species isolated from dogs. Mycoplasma alligatoris A21JP2^T was the positive control for strictly cell-associated activity; Mycoplasma crocodyli MP145^T and fresh SP-4 broth were negative controls. (B) Sialidase activity was present in Mycoplasma canis PG14^T and eight clinical isolates of M. canis. Isolate LV had no activity.