Covert infection of a mouse mammary tumor cell line withMycoplasma hyorhinis: Cosedimentation with mouse mammary tumor virus in sucrose density gradients

Summary

Supernatant fluids from cultures of a mouse mammary tumor (MT) cell line were found to produce a specific cell detachment effect when inoculated into HeLa cells. The cell detachment factor (CDF) responsible for this effect was examined. Repeated attempts to cultivate this CDF in bacteriologic, fungal, and mycoplasma media were unsuccessful. However, using the DNA fluorochrome staining technique and specific immunofluorescent staining procedures, the CDF was identified positively as a noncultivable strain ofMycoplasma hyorhinis. It was also noted that this CDF could be labeled with [³H] uridine in MT cell cultures, concentrated, and banded at a density of 1.18 g/cm³ when centrifuged to equilibrium in a 20 to 60% sucrose gradient. Using a multiple antibiotic treatment regimen, the MT cells were “cured” of theM. hyorhinis contaminant. Re-infection of these cells with an exogenous strain ofM. hyorhinis resulted in the same cell detachment effect, and this strain when labeled with [³H]uridine also sedimented at a density of 1.18 g/cm³. The salient feature of these studies is thatM. hyorhinis sediments at the same density of mouse mammary tumor virus (MMTV) in sucrose density gradients. This was demonstrated by sucrose density gradient analyses of a purified sample of MMTV, assaying for reverse transcriptase activity, and a [³H] uridine labeled sample of theM. hyorhinis present in the MT cell cultures.