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

Abstract

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%).

Spiroplasma found in the eyes of scrapie affected sheep

“…Interestingly, the BCE cell cultures infected with the scrapie spiroplasma isolate from ID#301 showed cytopathogenic effect with nuclear enlargement and cytoplasmic vacuolization (Fig. 5a) similar to that described in mouse astrocytic cell culture inoculated with scrapie tissues 20 and SMCA cytopathology in rabbit lens culture 19 . We had previously established infection with a scrapie sheep brain isolate in mouse N2a neuroblastoma cultures showing similar cytopathology (Fig.…”

“…Vitreous humor from scrapie sheep ID#301 was inoculated onto a mouse N2a neuroblastoma cell culture (In Pro Biotechnology Inc. ICL‐N2AI, San Francisco, CA, USA) to enhance the growth of the spiroplasma isolate; and although a spiral form was seen in the spent cell‐free culture MEM media by DFM (data not shown), the culture did not passage. Spiroplasma are known to flourish in conditioned tissue culture media 19 . An aliquot of M1D culture inoculated with the cornea from sheep ID#301 (shown to be positive by DFM) was added to a monolayer of BCE cells (ATCC #CRL‐2048).…”

The genome and antigen proteome analysis of Spiroplasma mirum

“…Spiroplasma mirum , Suckling Mouse Cataract Agent (SMCA) ( Megraud et al, 1983 ), was originally isolated from rabbit ticks ( Haemaphysalis leporis-palustis ). We purchased SMCA [CIP 103925] from ATCC and cultured and cloned it with the same method ( Itoh et al, 1989 ).…”

“…Spiroplasma mirum originally isolated from a rabbit tick ( Haemaphysalis leporis-palustis ATCC 29335 = CIP 103925) ( Megraud et al, 1983 ). S. mirum and S. eriocheiris are close to each other in the phylogenetic tree and both can cause the cataract of neonatal rat ( Hou et al, 2017 ).…”

Spiroplasmas: evolutionary relationships and biodiversity

“…Only one characterized Spiroplasma sp., S. mirum, has been shown to experimentally infect immunocompromised vertebrates (chick embryos and suckling mice, rats, Syrian hamsters and rabbits) (28,147,148). In vitro growth of S. mirum has also been demonstrated in an epithelial cell line derived from the rabbit eye lens (149). Recent reports have suggested that Spiroplasma sp.…”