Virulence Is More than Adhesion and Invasion Ability, an In Vitro Cell Infection Assay of Bovine Mycoplasma spp.

Abstract

Mycoplasma bovis is the most common mycoplasma associated with cattle diseases worldwide. However, other seemingly less virulent Mycoplasma spp. such as M. bovigenitalium and M. bovirhinis have also been associated with mycoplasmosis. The study objective was to compare the adhesion and cellular invasion characteristics of these bovine Mycoplasma spp. using Madin-Darby Bovine Kidney (MDBK) epithelial cells. MDBK cells were separately infected with 12 M. bovis strains and one strain each of M. bovigenitalium and M. bovirhinis. Following infection, a gentamicin protection assay was performed and the cells lysed at 6 and 54 h post-infection. The MDBK cell lysates were cultured for Mycoplasma spp. and qPCR was used to estimate the average number of Mycoplasma bacterial cells that infected each MDBK cell (Myc/Cell ratio). Confocal and electron microscopy studies using M. bovis mNeonGreen strain were also performed. All 14 Mycoplasma strains multiplied within the MDBK cells, a finding confirmed by microscopy studies of the M. bovis mNeonGreen strain. Unexpectedly, the M. bovis strains, obtained from diseased and asymptomatic cattle and bison, had lower Myc/Cell ratios than M. bovirhinis and M. bovigenitalium strains. These findings suggest that the ability for mycoplasmas to invade and replicate within host cells does not account for the differences in virulence between species.