10 – cell culture mycoplasmas

Purpose: To elucidate a previously unknown aetiology of rapidly progressive unilateral cataract in a premature baby associated with severe anterior uveitis. Methods: The lens and vitreous material were saved as part of a special protocol in a 4-month-old premature baby at the time of pars plana lensectomy with anterior vitrectomy. We performed (1) microbiological cultures to detect viable bacterial and fungal organisms; (2) PCR reaction to viral, bacterial and fungal agents; (3) transmission electron microscopy (TEM). In addition, serological examinations were performed for HSV-1 and -2, CMV, VZV and Mycoplasma infection. Results: PCR detected Spiroplasma sp.; TEM confirmed the presence of Spiroplasma within the lens fibres. Serological testing and microbiological cultures of the vitreous and lens were negative. Conclusion: Endogenous Spiroplasma infection in a premature baby may manifest as rapidly progressive acquired unilateral cataract with anterior uveitis. Beyond this, Spiroplasma infection has never been reported to occur naturally in vertebrates, although experimentally Spiroplasma mirum produces panophthalmitis associated with cataract in a wide range of rodents and in chicks. In acquired infantile cataract with inflammatory signs, PCR and TEM should be performed in the lensectomy/vitrectomy material to detect infectious agents not evident on routine laboratory and microbiological examinations.

First evidence of an endogenous Spiroplasma sp. infection in humans manifesting as unilateral cataract associated with anterior uveitis in a premature baby.

The Colorado potato beetle spiroplasma, which is not cultivable in conventional cell-free media, grew in tissue culture media in the presence of several coleopteran and lepidopteran insect cell lines. The cultured organisms attained titers of 1.2 x 10(9) spiroplasmas per milliliter of culture at the 100th passage and retained infectivity and a high capacity for translational motility at the 15th passage. Cell culture systems may facilitate the isolation of other presently uncultivable microorganisms and may be useful in the study of the role of microbial physiology and behavior in pathogenicity.

Cell-assisted growth of a fastidious spiroplasma.

A helical mycoplasma,Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T cells. The time of inoculation ofS. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation.

Malignant transformation of NIH-3T3 and CV-1 cells by a helical mycoplasma, Spiroplasma mirum, strain SMCA.

Objective Scrapie, a transmissible spongiform encephalopathy (TSE) occurring naturally in sheep, characteristically shows a severe retinopathy that is well developed in the terminal phases of the disease. In this study, we set out to demonstrate similar retinal changes in our ruminant spiroplasmosis TSE model.



Procedure  The eyes from deer, sheep, and goats that were inoculated intracranially with the laboratory strain of spiroplasma (suckling mouse cataract [SMCA] strain of Spiroplasma mirum) or with Spiroplasma sp. isolated from the brains affected with scrapie or with chronic wasting disease were examined by light microscopy for pathologic changes and by immunocytochemistry for distribution of spiroplasma antigen. The eyes were also obtained from a research flock of sheep with terminal scrapie, from which the intraocular tissues were submitted aseptically for culture assay in M1D broth or as explants on bovine corneal endothelia (BCE).



Results  The eyes from the spiroplasmosis ruminant models showed retinopathy remarkably similar to eye lesions seen in sheep with scrapie. The spiroplasma antigen accrued in the ruminant model eye tissues, particularly in the retina, the vitreous humor, and the corneal endothelia. A Spiroplasma sp. grew out of the scrapie-affected eyes both in the M1D broth and in the BCE cultures but did not expand. These new spiroplasma isolates differed immunologically from SMCA.



Conclusion  These data showed a clear association of spiroplasma with scrapie suggesting that these bacteria have a role in the pathogenesis of TSE and that the eye should be a research focus for future studies of TSE.

Spiroplasma found in the eyes of scrapie affected sheep

Spiroplasma mirum (suckling mouse cataract agent) was studied in an epithelial cell line AG-4676, derived from rabbit eye lens. Rabbit eye lens is a natural target tissue of S. mirum infection. The organism grew rapidly in this cell line, reaching titers of 10(7) to 10(9) color change units per ml at 7 days after infection. This is the same level as that achieved in SP-4 medium designed specifically for S. mirum. No lag period was apparent in growth in AG-4676. S. mirum did not grow in Dulbecco minimal essential medium-10% fetal bovine serum, the medium for AG-4676, indicating the need for cells or a cellular product. S. mirum-infected AG-4676 cells exhibited vacuolization and granulation and an increase in polynucleation compared with uninfected controls (36/100 versus 14/100, P less than 0.001). Infection significantly decreased the growth rate of AG-4676, especially late in the growth cycle. In a representative experiment, growth of AG-4676 at 11 days was reduced from 9 X 10(5) to 2 X 10(4) cells by S. mirum infection. S. mirum grew to high titers in conditioned medium of AG-4676, obtained from cell-free supernatants of 1- to 5-day-old AG-4676 cultures. This growth promotion was not due to osmotic conditioning of the medium. Preliminary characterization of this growth promotion substance showed it to be active after 0.22-micron filtration, heating at 56 degrees C for 30 min, freezing and thawing, and dilution at 10(-1) but not 10(-2). AG-4676-propagated S. mirum produced death or cataracts in suckling Wistar rats at the same frequency (55/60, 91.7%) as SP-4-propagated organisms (60/65, 92.3%).

https://iai.asm.org/content/iai/42/3/1168.full.pdf

Characterization of Spiroplasma mirum (suckling mouse cataract agent) in a rabbit lens cell culture.

Summary Spiroplasma mirum grew to high titres, 108 colour-changing units per ml of supernatant medium, and produced cytopathology which consisted of vacuolization, granulation and polynucleation. S. mirum did not grow in cell culture medium (Dulbecco's MEM+10% fœtal bovine serum), thereby indicating the need for cultured cells or a cell culture product. Growth was also obtained from cell-free supernatants from AG-4676 cultures. S. mirum propagated in AG-4676 produced cataracts and death in suckling Wistar rats.

Rabbit lens cell cultures in the characterization of Spiroplasma mirum pathogenicity

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Characterization of Spiroplasma mirum (suckling mouse cataract agent) in a rabbit lens cell culture.

Rabbit lens cell cultures in the characterization of Spiroplasma mirum pathogenicity