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Mycoplasmal infection of insect cell cultures

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Summary

Twenty-five cell cultures of three insect orders from eight laboratories were tested for mycoplasmal infection.Acholeplasma laidlawii was detected in one culture, an incidence of 4.0%.A. laidlawii, Mycoplasma orale, M. arginini, but notM. hyorhinis, could establish infections of drosophila Dm-1 cell cultures at 25° C. In prospective studies, drosophila Dm-1 cultures were intentionally infected with broth-propagatedA. laidlawii andM. hyorhinis. M. hyorhinis did not grow and was eliminated from the Dm-1 cultures during consecutive passages.A. laidlawii grew without obvious cytopathic effects during six weekly passages; titers of over 107 CFU/ml were recorded at Passages 2 and 5 (p2 and p5). Minimal cell culture infectious doses were also determined during these studies. 0.1 milliliter cell samples were inoculated into Leighton tubes containing either fresh M1A culture medium or 3T6 indicator cells in McCoy’s 5a medium. After 4 d of incubation at 25 and 37° C, respectively, the cover slips were stained by DNA fluorochrome Hoechst 33258 (A. laidlawii) or by specific fluorescein-conjugated antiserum (M. hyorhinis). At p2 with both mycoplasma species, the procedure using M1A medium and incubation at 25° C without 3T6 cells was inferior to indicator cells. In five of six experiments at least a two-log higher titer of mycoplasmas was needed to be detected with M1A and 25°C. At p5 no difference could be found.

Uridine phosphorylase assays of Dm-1 cultures infected withA. laidlawii, M. hyorhinis, M. orale, andM. arginini gave clearly positive results only withA. laidlawii. The ratio of incorporated uridine to incorporated uracil method yielded false positives with two drosophila cell lines. Suggestions for assay of mycoplasmas in invertebrate cell cultures are given.

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Author notes

  1. Theodor Steiner

    Present address: Laboratorie de Biologie, Cellulaire et Moleculaire, I.N.R.A., 33140, Pont-de-la-Maye, France

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  1. Institute for Medical Research, Copewood Street, 08103, Camden, New Jersey

    Theodor Steiner & Gerard McGarrity

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This work was supported by Grant AI-15748 from the National Institute of Allergy and Infectious Diseases and by a grant from the U. S. Department of Agriculture.

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Steiner, T., McGarrity, G. Mycoplasmal infection of insect cell cultures. In Vitro 19, 672–682 (1983). https://doi.org/10.1007/BF02628958

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