Vol. 42, No. 3INFECTION AND IMMUNITY, Dec. 1983, p. 1168-11750019-9567/83/121168-08$02.00/0Copyright C) 1983, American Society for Microbiology

Characterization of Spiroplasma mirum (Suckling MouseCataract Agent) in a Rabbit Lens Cell Culture

FRANCIS MEGRAUD,t LINDSAY B. GAMON, AND GERARD J. McGARRITY*Department of Microbiology, Institlite for Medical Research, Caimden, Newt Jersey 08103

Received 27 June 1983/Accepted 19 September 1983

Spiroplasma mirum (suckling mouse cataract agent) was studied in an epithelialcell line AG-4676, derived from rabbit eye lens. Rabbit eye lens is a natural targettissue of S. mirum infection. The organism grew rapidly in this cell line, reachingtiters of 107 to 109 color change units per ml at 7 days after infection. This is thesame level as that achieved in SP-4 medium designed specifically for S. tnirlum.No lag period was apparent in growth in AG-4676. S. mirnlm did not grow inDulbecco minimal essential medium-10% fetal bovine serum, the medium for AG-4676, indicating the need for cells or a cellular product. S. mini-m-infected AG-4676 cells exhibited vacuolization and granulation and an increase in polynuclea-tion compared with uninfected controls (36/100 versus 14/100, P < 0.001).Infection significantly decreased the growth rate of AG-4676, especially late in thegrowth cycle. In a representative experiment, growth of AG-4676 at 11 days wasreduced from 9 x 105 to 2 x 104 cells by S. mirum infetion. S. mirilm grew to hightiters in conditioned medium of AG-4676, obtained from cell-free supernatants of1- to 5-day-old AG-4676 cultures. This growth promotion was not due to osmoticconditioning of the medium. Preliminary characterization of this growth promo-tion substance showed it to be active after 0.22-.m filtration, heating at 56°C for30 min, freezing and thawing, and dilution at 10-1 but not 10-2. AG-4676-propagated S. mirlum produced death or cataracts in suckling Wistar rats at thesame frequency (55/60, 91.7%) as SP-4-propagated organisms (60/65, 92.3%).

Spiroplasma mirum, formerly known as suck-ling mouse cataract agent (SMCA) was original-ly isolated from embryonated eggs inoculatedwith a rabbit tick pool (Haemaphysalis lepori-spalustris), as reported by Clark in 1964 (3).Clark and Rourke (5) have reviewed the charac-teristics of the SMCA strain and a second relat-ed strain (GT-48) recovered from rabbit ticks ayear after isolation of SMCA. A third strain of S.mirum (TP-2) was recovered directly in SP-4broth medium from a rabbit tick pool collectedin Maryland in 1977 (15). Several other spiro-plasmas, serologically distinct from S. mirium,have been recovered from ticks, including the277F strain from rabbit ticks (2) and the Y32group from Ixodes ticks (16). The spiroplasmalnature of SMCA and GT-48 strains was conclu-sively established in 1976 by Tully et al. (18),and organisms were cultivated for the first timein a cell-free medium, SP-4, in 1977 (17). S.mirum killed 7-day-old embryonated chickeneggs during an incubation period of 4 to 10 days.Intracerebral inoculation of S. mirum into suck-

t Present address: University of Bordeaux 11, Bordeaux,France.

ling mice, rats, rabbits, and hamsters producescataracts, encephalitis, and death (3, 5, 9, 10).The specific effect depends on the medium usedto propagate the organism, the animal speciesstrain, and the number of organisms injected (7).The GT-48 strain, antigenically related to S.mirum, produces more severe pathogenic effectswhich generally result in overwhelming enceph-alitis, killing suckling animals within 7 days.Strain 277F did not produce cataracts in intra-cerebrally inoculated rats. It did kill sucklingrats intracerebrally inoculated in high doses,>109 organisms. Strain 277F did not kill embry-onated eggs (2).

Initial attempts were made to cultivate S.mirum in cell culture but were unsuccessful forcytopathic effects as well as for multiplication ofthe organism (1, 3). Chick embryo chorioallanto-ic membrane and yolk sac fragment cultureswere unsuccessful (3). Organ culture system,using whole rabbit lens has been successful (6).In this study, Fabiyi et al. propagated unclonedS. mirum to obtain a 107 8 50% eye lens dose perlens culture. Steiner et al. (14) grew S. miruim inDrosophila Dm-1 cells and obtained a cytopathiceffect when incubation was made at 30°C but notwhen it was made at 250C. At 250C, S. mirllm

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established a chronic infection of Dm-1 cells.The infected culture could be passaged; concen-trations of ca. 109 color change units (CCU) of S.mirnii were obtained per ml within 7 days.The availability of a differentiated lens cell

line from the rabbit, a host susceptible to S.inirumn, encouraged us to examine S. miirtin inthis cell culture system to determine spiro-plasma-eukaryotic cell interactions. Since cata-racts are a major consequence of S. mirl(mininfection, an appropriate cell culture modelmight help explain mechanisms of pathogenesis.

MATERIALS AND METHODS

Organisms. S. iniruin strain SMCA was kindly pro-vided in lyophilized form by J. G. Tully of the Nation-al Institute of Allergy and Infectious Diseases, Bethes-da, Md. The strain was grown in SP-4 broth medium(17). All experiments were performed with a singlestock supply of 109 CCU/ml. Samples (1 ml) of thestock were frozen at -70°C. These samples werequick thawed for individual experiments. The passagenumber of the organism at the beginning of thesestudies was ca. 100. Strain GT-48 (CMRL P3) passage9 was also used and stored under the same conditions.

Cells. Two rabbit lens epithelial cell lines were used.These were established by Reddan et al. from 5-day-old rabbit (AG-4676) and 18-month-old rabbit (AG-4677) cell cultures. These have been characterizedpreviously (12). They were obtained from the AgingCell Repository, Institute for Medical Research, Cam-den, N.J.A rabbit fibroblast cell line (RAB 9) passage 20 was

obtained from the American Type Culture Collection(CCL 1414). A human fibroblast cell line (MRC-5) andthe continuous mouse cell line 3T6 were obtained fromdepartmental stocks. Medium for AG-4676 and AG-4677 was Dulbecco modified Eagle Earle medium(DMEM) 10% fetal bovine serum (FBS). RAB-9 andMRC-5 cells were grown in Ham F-12-10% FBS andMcCoy-20% FBS. respectively. The cells were as-sayed for mycoplasma at the beginning and end ofthese experiments by microbiological culture and fluo-rescent DNA staining to ensure absence of accidentalmycoplasmal infection (11).Spiroplasma growth and compatibility in cell cul-

tures. The growth curve of S. mirumn in cell cultureswas determined by inoculation of 10' CCU in 0.1-mlsamples in 1 ml of cell culture medium after cellattachment with incubation at 37°C in 5% CO,-air.Serial dilutions of this culture were made daily by theaddition of 0.5 ml of cell-spiroplasma suspension to 4.5ml of SP-4 medium. The number of viable organismswas made by determination of the number of CCUobserved weekly for 1 month. Growth was defined as

an increase of 2 logs in 6 days or less. Compatibility ofS. inirum in different cell culture media was deter-mined by dark-field microscopic examination for S.iirumin motility, growth. and helicity.

Cell culture growth curve. Cell culture growthcurves of AG-4676 or AG-4677 were performed in 25-cm2 flasks by inoculation of 105 cells in 6 ml ofmedium. Three flasks were counted daily in triplicatewith a hemacytometer: viability was calculated by

using trypan blue. All growth curves were performedat least twice.

Preparation of Spiroplasma extract. S. miirum wasgrown in SP-4 medium for 7 days. The suspension wascentrifuged for 30 min at 15.000 rpm in a Beckmantype 30 rotor. The supernatant was filtered through a0.22-,um filter and tested for toxic effects on cellcultures by inoculation of 0.1-ml samples onto mono-layers of AG-4676 cells. A sonicate was prepared fromthe centrifuged pellet to assay for a nondiffusible toxicmaterial as performed above for supernatant testing.Growth factor studies. AG-4676 was grown in

DMEM-10% FBS for 5 days or as described above.The supernatant medium was centrifuged at 500 x g inan International centrifuge for 15 min. The supernatantwas successively filtered through 0.45- and 0.22-p.mmembrane filters. This cell-free-conditioned mediumwas used to test growth promotion of S. nirumlo thathad been grown in SP-4 under various test conditions.All serial dilutions of S. imirum inocula were made inthe conditioned medium to minimize effects of SP-4nutrient carry-over. S. mirun (105 CCU) was used asinoculum into the conditioned medium.Animal studies. Newborn Wistar rats were injected

intracerebrally within 48 h of birth with 0.03 ml ofundiluted S. mnirum culture (ca. 107 CCU). Eachmother and her litter were kept together in a separatecage throughout the study. The number of CCU wasdetermined for each injection by inoculation of serialdilutions into SP-4 broth.

Karyology was performed on uninfected and S.,nirumn-infected AG-4676 by standard methods (19).Karyotypes were performed on 5-day-old cultures.For infected cultures. 105 CCU of S. Inirutn was addedat the time of culture passage. and karyotypes wereperformed 5 days later.

RESULTS

Among the different cell culture media tested,the most effective for growth of S. iniriun in thecell cultures tested was DMEM. FBS which wasnot inactivated gave better results than inacti-vated FBS. Criteria for this evaluation wasabsence of cytopathology of cultured cells,maintenance of helical morphology of S. jni,llmfor a minimum of 24 h, and an increase in S.inirlmn numbers. Rabbit serum, inactivated andnot inactivated, resulted in poorer growth ofAG-4676.Growth of S. mirum in AG-4676 and AG-4677

and effects of growth. Rapid growth of S. iniiriinwas observed in AG-4676 cell cultures comparedto SP-4 broth medium. S. mirum did not grow incell-free DMEM-10% FBS medium, indicatingthe need for cultured cells for growth (Fig. 1).Serial dilutions of S. mirum showed that 10' to102 CCU could establish infection of AG-4676cell cultures, growing to 107 to 109 CCU/ml in 7days in different experiments.

Dark-field microscopy showed numerous spi-roplasmas in the supernatant, but few wereattached to the cells. This finding of few organ-isms attached to AG-4676 cells was confirmed

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1170 MEGRAUD, GAMON, AND McGARRITY

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miruxm in AG-4676 cell cultures. SP-4 spiroplasma medium and DMEM-10% FBS,

by DNA fluorescent staining. In many in-stances, it was difficult to distinguish S. mirumfrom fluorescent artifacts seen in uninfectedcells.

Light microscope observation of infected AG-4676 cells after 4 to 5 days showed numerousrounded cells floating in the medium and granu-lation and vacuolization of the cytoplasm.Giemsa staining showed an increase in size ofthe cells in relation to the control as well as anincrease in the number of bi- and polynuclearcells (36/100 versus 14/100, P < 0.001) (Fig. 2).Spiroplasma sp. GT-48 also grew to high titersand produced cytopathology in AG-4676 cells.No difference was noted in the karyology

between infected and noninfected AG-4676.However, both cultures exhibited an extra bandin chromosome 10 (Fig. 3). This was not ob-served in the original text describing this cell line(12).

Tests for a spiroplasmal toxin were negativewhen culture supernatants and sonicates wereused, indicating the lack of a demonstrable toxinof S. mirum for AG-4676 under these conditions.The effect of S. mirum on the growth of AG-

4676 cells is presented in Fig. 4. AG-4676 cellsgrew relatively slowly, reaching confluency af-ter ca. 7 days of incubation. Although S. mirumreached peak titers in 5 to 7 days in SP-4 brothmedium, it reached peak titers in AG-4676 cellsmuch faster, i.e., 2 to 3 days. S. mirum producedthe greatest inhibiting effect late in the growth ofAG-4676 cells. Growth of S. mirum-free AG-4676 cultures plateaued at 7 days; 7- to 11-day-old cultures had viabilities of 88 to 94%, similarto younger cultures. This was similar to thereport of Reddan et al. for this cell line (12).Growth curve of S. mirum in other cell lines. S.

mirum also grew in RAB 9 and MRC-5 fibro-

blasts, but no cytopathic effect was noticedduring the 1-week incubation period. S. mirumalso grew in the mouse 3T6 cells. No cytopathol-ogy was evident after four passages in 3T6, eventhough S. mirum concentrations in the superna-tant reached ca. 108 CCU/ml.Growth in AG-4676 cell culture-conditioned

medium. When 105 CCU of S. mirum wereinoculated into the cell-free conditioned medi-um, rapid growth was evidenced. As shown inFig. 5, growth was more rapid in the conditionedmedium than in the SP-4 medium, with a muchshorter lag period in the conditioned medium.No significant difference was noted in the peakconcentrations obtained in the conditioned me-dium compared with SP-4. Conditioned mediumprepared from RAB-9 and MRC-5 cells yieldedthe same results as that prepared from AG-4676cells, indicating that the growth promotion wasnot a specific characteristic of the rabbit lensculture. Initial attempts to isolate a specificgrowth factor were unsuccessful. Conditionedmedium from 1- and 5-day-old cultures yieldedsimilar results. Further characterization of theconditioned medium was performed with mediafrom 5-day-old AG-4676 cells. Initial character-ization of the conditioned medium is shown inTable 1.To determine whether the growth promotion

of the conditioned medium could be due toosmotic conditioning of the DMEM-10% FBSmedium, osmolarities were measured. No signif-icant differences in osmolarity were observedamong DMEM-10% FBS, conditioned mediumfrom AG-4676 culture, or spiroplasmal SP-4medium (330 mosM). In fact, adjustment ofDMEM-10% FBS to 322 mosM did not promotegrowth of S. miruim (Table 1).

AG-4676-conditioned medium was tested for

FIG. 1. Growth of S.medium for AG-4676.

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its ability to promote the growth of other spiro-plasmas. Spiroplasmni sp. GT-48, serologicallyand genetically related to S. minirtm, grew inconditioned medium, similar to S. miruim. Con-ditioned medium failed to promote the growth of

three other spiroplasmas tested, S. citri, cornstunt spiroplasma, and the sex ratio organism.

Pathogenicity of cell culture-propagated S.mirum for suckling rats. Intracerebral inocula-tion of suckling rats with S. miru-m grown in

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either SP-4 or AG-4676 showed that S. mirummaintained its pathogenicity for these animalsafter 10 passages in AG-4676 cells, still produc-ing death and cataracts. No difference in cata-ract formation or animal death was detectedbetween AG-4676-propagated (55/60, 91.7%) or

SP-4-propagated (60/65, 92.3%) S. mirum. Smallnumbers of animals died from each of threenegative control groups (8/95) inoculated withSP-4 medium, DMEM-10% FBS, or AG-4676cells in DMEM-10% FBS. Cataracts were pro-duced in five rats injected with AG-4676-pas-saged material and seven rats injected with SP-4-passaged S. inirutm.

DISCUSSION

The effects of S. mirim on rat and mouse eyetissues have been reported by Clark and Karzon(4) and Friedlander et al. (7), respectively. Simi-larities have been reported between rat andmouse S. mirum-induced eye disease (5). InSprague-Dawley rats, lens changes began ca. 5days after injection. Lens epithelium proliferat-ed, producing aberrant swollen and nucleatedfibers central to the proliferated equatorial epi-thelium. A continuous band of multilayered epi-thelium eventually surrounded the lens as inter-nal lens degeneration intensified (5).

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cell cultures. Symbols: 0, AG-4676 with S. mnirlo; *.AG-4676.

Clark and Karzon (4) reported similar findingsin C57BL/6Ha mice, the most susceptible strainof six tested. S. mirllm was first detected inretinal tissue 2 days after injection and then inthe lens (day 4), choriod and globe (day 7), andin the cornea and anterior uveat tissues (day 7 to10). Because infection and pathological changesin the retina consistently preceded lens effects,Clarke and Rourke have noted that S. mirimn-induced cataracts may be secondary cataracts(5).

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Rabbits developed severe keratoconjunctivitisleading to pannus and intense inflammatory re-sponse in the anterior chamber (3). Kirchhoff etal. (9) reported lens changes in the rabbits con-sisting of fiber degeneration, liquefaction, in-flammation, mineralization, fibrosis, and foreignbody granulomatous reactions.Lens epithelial cell culture is an appropriate in

vitro system since lenses in vivo contain noblood supply, are not innervated, are completelyencapsulated, and are derived entirely from ec-toderm (12). S. inirmn grew to high titers of 10iCCU/ml of supernatant medium from AG-4676cultures. Interestingly, the organism did notgrow in cell culture medium, indicating the needof cells or a cellular product for growth. Morestudies are required to determine the relativevalue of this and perhaps other lens cell lines asin vitro models for S. minrim. As shown in thisstudy, the organism grows to high titers in othercells which are not lens or rabbit cells. Clearly,however, these studies demonstrate the poten-tial value of cell and organ culture systems tostudy spiroplasmal pathogenicity. For years,attempts to propagate S. mirnim in cell-freemedium were unsuccessful until Tully et al.developed SP-4 medium (17). Interestingly, Fa-biyi et al. (6) previously clearly demonstratedthat S. mirirm consistently grew to high titers inrabbit eye lens organ cultures. For some reason,this study was not further developed, althoughthe value of the system was clearly apparent.

In a short study, Kirchhoff et al. (9) notedchanges in the lens epithelium of S. inim-itin-infected rabbits, namely degeneration of lensfibers, and liquefaction of lens substance. Theexpression of lens fibers and lens substance arenot precisely known in the AG-4676 cell line.Reddan et al. stated that the line expressed

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1174 MEGRAUD, GAMON, AND McGARRITY

TABLE 1. Characteristics of growth promotion ofS. miritmn by conditioned medium from AG-4676 cell

cultures

GrowthTi-eatment

Gotpromotion

DMEM-10% FBS (AG-4676 medium)(292 m0sM).

DMEM-10% FBS (322 mosM).

Conditioned mediumNone (control)........................ +Heat, 56°C, 30 min.................... +Freeze-thaw.......................... +Filtration, 0.220 pLm ................... +

Dilution1:10 ................................. +1:100 ................................

rabbit crystallins, but this was not completelyproven (12). More data are needed on the degreeof differentiation of this cell culture.One advantage of the AG-4676 cell culture

system is that this culture grows relatively slow-ly, reaching confluence in ca. 1 week. Thisgrowth parallels the growth curve of S. inirlrn inSP-4. As noted, S. miirunm grew more rapidly inAG-4676 than in SP-4. AG-4676 cells were stableand viable for periods after reaching confluency.In fact, this postconfluency period may repre-sent a more differentiated form of the cultureand be more important for the study of S.,ni,.I-in.Although no toxin could be demonstrated in

S. iniruin, cell-free conditioned medium fromAG-4676 cultures promoted growth to approxi-mately the same extent as SP-4 medium, butwith no appreciable lag period. The nature ofthis growth promotion is unknown and underinvestigation. It does not appear to be due toosmotic conditioning of the medium, since theconditioned medium had a similar osmolarity toSP-4 medium, and adjustment of DMEM-10%FBS to 322 mosM failed to promote S. miruim71growth.The effects of medium osmolarity on growth

of S. iniimin merits investigation. S. iniriim can

grow in SP-4 medium (332 mosM), conditionedmedium (292 mosM), Schneider Dr-osophila cellculture medium (318 mosM), and spiroplasmalMlA medium (534 mosM) (values obtained inthis laboratory). The growth of S. minirm over

such a wide range of osmolarities is of interest inview of its original isolation from a tick and itsability to produce disease under appropriateconditions in certain animals. Tick hemolymphhas a high osmolarity.

Apparently, a cellular product elaborated ear-

ly in the growth of AG-4676 cultures and other

mammalian cultures promoted S. miruin growth,even faster than SP-4 medium designed specifi-cally for S. mirum (17). Studies are in progressto attempt to characterize the nature of growthpromotion by the conditioned medium by usingstandard methods (8, 13). The substance may ormay not be a component of SP-4 medium. Ineither case, its identification may add to ourknowledge of S. miriin metabolism and result inthe development of new or modified media for S.mnirllmn.

ACKNOWLEDGMENTS

We thank Judi Sarama and Veronica Vanaman for technicalassistance and Riley Hansom for providing illustrations. Wealso thank Carole Bradt for performing the chromosomebanding.

This work was supported by Public Health Service grant Al-15748 from the National Institutes of Health.

LITERATURE CITED

1. Bastardo, J. N., D. Ou, and R. H. Russell. 1974. Biologicaland physical properties of the suckling mouse cataractagent grown in chicken embryos. Infect. Immun. 9:444-451.

2. Brinton, L. P., and W. Burgdorfer. 1976. Cellular andsubcellular organization of the 277F agent, a spiroplasmafrom the rabbit tick, Haenaplhysalis Ieporispli(Istris (Ac-ari: Ixodidae). J. Syst. Bacteriol. 26:554-560.

3. Clark, H F. 1964. Suckling mouse cataract agent. J.Infect. Dis. 114:476-487.

4. Clark, H F., and D. T. Karzon. 1969. Growth curvestudies of the suckling mouse cataract agent in individualcompartments of the eye. Proc. Soc. Exp. Biol. Med.131:693-696.

5. Clark, H F., and M. B. Rourke. 1979. Spiroplasmas oftick origin and their pathogenicity. p. 155-174. Iti R. F.Whitcomb and J. G. Tully (ed.), The spiroplasmas: vol.III. Academic Press. Inc.. New York.

6. Fabiyi, A., T. S. Elizan, and J. E. Pounds. 1971. Sucklingmouse cataract agent (SMCA) in tissue culture. Proc. Soc.Exp. Biol. Med. 136:88-91.

7. Friedlander, R. P., M. F. Barile, T. Kuwabara, and H F.Clark. 1976. Ocular pathology induced by the sucklingmouse cataract agent. Invest. Ophthalmol. 15:640-647.

8. Hayashi, I., J. Larner, and S. Sato. 1978. Hormonalgrowth control of cells in culture. In Vitro 14:23-30.

9. Kirchhoff, H., J. Heitmann, and G. Trautwein. 1981.Pathogenicity of spiroplasmas sp. strain SMCA in rabbits:clinical, microbiological and histological aspects. Infect.Immun. 33:292-296.

10. Kirchhoff, H., T. Kuwabara, and M. F. Barile. 1981.Pathogenicity of Spi-oplas.loio sp. strain SMCA in Syrianhamsters: clinical, microbiological, and histological as-pects. Infect. Immun. 31:445-452.

11. McGarrity, G. J., J. Sarama, and V. Vanaman. 1979.Factors influencing microbiological assay of cell culturemycoplasmas. In Vitro 15:73-81.

12. Reddan, J. R., T. B. Friedman, M. K. Mostafapour, S. H.Southerland, R. L. Bondy, S. J. McGee, and E. M.Goldenberg. 1981. Donor age influences the growth ofrabbit lens epithelial cells in vitro. Vision Res. 21:11-23.

13. Scher, C. D., J. I. Farhi, R. I. Handin, D. R. Kaplan, J. S.Lillquist, K. McGovern, and C. D. Stiles. 1980. Initiationof cell replication by cationic polypeptide hormones. p.39-53. In F. E. Bloom (ed.), Peptides: integrators of celland tissue function. Raven Press. New York.

14. Steiner, T., G. J. McGarrity, and D. M. Phillips. 1982.Cultivation and partial characterization of spiroplasmas incell cultures. Infect. Immun. 35:296-304.

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15. Stiller, D., R. F. Whitcomb, M. E. Coan, and J. G. Tully.1981. Direct isolation in cell-free medium of a spiroplasmafrom Haemaphysalis leporispalustris (Acari: Ixodidae) inMaryland. Curr. Microbiol. 5:349-352.

16. Tully, J. G., D. L. Rose, C. E. Yunker, J. Cory, R. F.Whitcomb, and D. L. Williamson. 1981. Helical mycoplas-ma (spiroplasma) from Ixodes ticks. Science 212:1043-1045.

17. Tully, J. G., R. F. Whitcomb. H F. Clark, and D. L.Williamson. 1977. Pathogenic mycoplasmas: cultivation

and vertebrate pathogenicity of a new spiroplasma. Sci-ence 195:892-894.

18. Tully, J. G., R. F. Whitcomb, D. L. Williamson, and H F.Clark. 1976. Suckling mouse cataract aigent is a helicalwall free prokaryote (spiroplaisma) pathogenic for verte-brates. Nature (London) 259:117-120.

19. Yu, R. L., M. M. Aronson, and W. W. Nichols. 1981. Highresolution bands in human fibroblast chromosomes in-duced by actinomycin D. Cytogenet. Cell Genet. 31:111-114.

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