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

<i>Mycoplasma bovis</i> is the most common mycoplasma associated with cattle diseases worldwide. However, other seemingly less virulent <i>Mycoplasma</i> spp. such as <i>M. bovigenitalium</i> and <i>M. bovirhinis</i> have also been associated with mycoplasmosis. The study objective was to compare the adhesion and cellular invasion characteristics of these bovine <i>Mycoplasma</i> spp. using Madin-Darby Bovine Kidney (MDBK) epithelial cells. MDBK cells were separately infected with 12 <i>M. bovis</i> strains and one strain each of <i>M. bovigenitalium</i> and <i>M. bovirhinis</i>. 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 <i>Mycoplasma</i> spp. and qPCR was used to estimate the average number of <i>Mycoplasma</i> bacterial cells that infected each MDBK cell (Myc/Cell ratio). Confocal and electron microscopy studies using <i>M. bovis</i> mNeonGreen strain were also performed. All 14 <i>Mycoplasma</i> strains multiplied within the MDBK cells, a finding confirmed by microscopy studies of the <i>M. bovis</i> mNeonGreen strain. Unexpectedly, the <i>M. bovis</i> strains, obtained from diseased and asymptomatic cattle and bison, had lower Myc/Cell ratios than <i>M. bovirhinis</i> and <i>M. bovigenitalium</i> 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.