Vol. 35, No. 1INFECTION AND IMMUNITY, Jan. 1982, p. 296-3040019-9567/82/010296-09$02.00/0

Cultivation and Partial Characterization of Spiroplasmas inCell Cultures

THEODOR STEINER,' GERARD J. McGARRITY,l* AND DAVID M. PHILLIPS2Department of Microbiology, Institute for Medical Research, Camden, New! Jersey 081031; and Population

Council, Rockefeller University, New York, New York 100212

Received 25 June 1981/Accepted 2 September 1981

Spiroplasmas were propagated in the Drosophila melanogaster cell line Dm-1.Spiroplasma citri and unidentified strains (corn stunt organism, 277F [tickisolate], powder puff, BNR-1, honey bee, and OBMG) grew to 108 to 109 colony-forming units per ml and could be passaged. Cytopathic effect (CPE) varied withthe infecting spiroplasma. The honey bee isolate killed Dm-1 within 2 to 4 daysand produced CPE in four mammalian cells tested. At 25°C, suckling mousecataract agent produced no CPE in Dm-1 cells. Dm-1 cells did not support growthof the spiroplasmal sex ratio organism. Spiroplasmas could be detected in the cellcultures by agar inoculation, dark-field microscopy, scanning electron microsco-py, and DNA fluorescent staining. The uridine phosphorylase test showedsignificant levels of conversion of [14C]uridine to [14C]uracil for all but some plantisolates: S. citri, corn stunt organism, lettuce, cactus, and powder puff strains, thefirst mycoplasmas to lack the enzyme. Primary isolations of corn stunt organismfrom infected corn plants were made in Dm-1 and I-XII cultures. The course ofcorn stunt organism infection of Dm-1 was monitored for three passages. The useof agarose and Dienes staining of the colonies improved growth and colonycounting of corn stunt organism. The number of viable infected Dm-1 cellsdecreased from 1.2 x 107 at passage 1 to 7.0 x 106 at passage 2 and 3 x 105 atpassage 3.

Procaryotic organisms in diseased plants re-sembling mycoplasmas with "sinusoidal" andhelical morphologies have been described previ-ously (10, 19). In 1971, the first plant mycoplas-mas were isolated in cell-free medium (14, 26),but the helical morphology of these isolates wasrecognized later (7, 27). The term spiroplasmafor helical mycoplasmas was proposed to de-scribe these organisms (9). Helical forms wererecognized earlier, but could not be cultured andwere thought to be spirochetes (24, 25). Spiro-plasmas have been isolated with increasing fre-quency from both diseased and apparentlyhealthy plants and insects. Isolates from ticksare also known (32). Only one organism has yetbeen identified to species: Spiroplasma citri(27).

Pathogenicity of several spiroplasmas hasbeen documented. S. citri and the "corn stunt"organism (CSO) cause "citrus stubborn" andcorn stunt disease, respectively (3, 7, 27, 39).CSO affects the longevity and fecundity of thevector Dalbulus elimatus (15). Spiroplasmaspathogenic for insects also include honey beeisolates (HB) that produce a rapidly lethal infec-tion (5) and a group of organisms that produces amale lethality in certain species of Drosophila(37). The suckling mouse cataract agent (SMCA)isolated from ticks produces cataracts and neu-

rological lesions upon appropriate intracerebralinoculation into suckling mice and other rodents(33, 34). Spiral structures of a size similar tospiroplasmas have been observed in brain biop-sies of patients with Creutzfeldt-Jacob disease,although cultural studies have not been reported(1). Other spiroplasmas have been isolated thatare either nonpathogenic or whose pathogenicityis unknown (8, 21, 35).Some spiroplasmas cannot yet be grown in

cell-free media (37; R. McCoy, Phytopathol.News 12:217, 1978). Few attempts to propagatespiroplasmas in cell or organ cultures have beenpublished. SMCA has been grown in embryonat-ed eggs and whole rabbit eye lens organ cultures(13); attempts by the same and other authorsusing primary tissue cultures were unsuccessful(4, 13). McBeath (Ph.D. Thesis, Rutgers Univer-sity, New Brunswick, N.J., 1974) was unable toinfect cells of the leafhopper Dalbulus elimatuswith CSO. The objective of the present studieswas to determine whether spiroplasmas could begrown in appropriate cell cultures and, if so, tocharacterize the spiroplasma-cell interactions.

MATERIALS AND METHODS

Spiroplasmas. The following organisms were stud-ied: S. citri strains R8A2 (ATCC 27556), C189 (ATCC

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27665), and strains from lettuce (ATCC 29594) andcactus (F. Kondo, A. H. McIntosh, S. B. Padhi, andK. Maramorosch, Abstr. Proc. Soc. Gen. Microbiol.3:154, 1976); CSO isolate E (ATCC 27954); 277Fisolated from ticks (ATCC 29761); SMCA (ATCC29335): HB strain BC-3 (ATCC 33219); OBMG isolat-ed from magnolias (ATCC 33221); BNR-1 isolatedfrom tulip trees (ATCC 33220); powder puff (PP)isolate (21); and the sex ratio organism (SRO). SROwas derived from Drosophila willistoni and maintainedin Drosophila pseudoobscura supplied by D. William-son, State University of New York, Stony Brook.SRO does not grow in cell-free medium. The PP isolatewas supplied in cloned form by Randy McCoy, Uni-versity of Florida, Fort Lauderdale. Clones of all otherorganisms were generously supplied in lyophilizedform by J. G. Tully, National Institute of Allergy andInfectious Diseases, Bethesda, Md. Serogroup identi-fication (18) of organisms was confirmed at the end ofthe studies by the growth inhibition test (6), usingantisera supplied by J. G. Tully. Broth cultures weregrown in MlA medium (17) at 30°C, except HB, whichwas grown at 25°C. For cell culture inoculations, late-log-phase broth cultures were routinely used.

Cell cultures. Cell lines of D. melanogaster weregrown at 25°C. The Dm-1 line was obtained from I.Schneider, Walter Reed Army Institute, Washington,D.C., and grown in M1A (17) or Schneider's Drosophi-la medium (28). The I-XII line obtained from S. Faccio-Dolfini, Institute of Genetics, University of Milano,Italy, was also studied. The I-XII line was adapted toSchneider's medium. All cells were assayed for myco-plasmas by inoculation onto mycoplasmal and spiro-plasmal MlA agar (17) and SP4 (33) agar and by DNAfluorescent staining and immunofluorescence (11, 22)at 25 and 37°C.

Quantitative counts. Cell numbers were determinedin triplicate with a hemacytometer; viability was calcu-lated by using trypan blue staining. To determinespiroplasma viability, serial dilutions of late-log-phasecultures were made in MlA broth and plated ontoMlA agar in triplicate. For solid media, agarose (Sea-kem) was used for CSO, and Noble agar (Difco) wasused for other spiroplasmas. This choice was based onresults described below. Colonies were counted after10 to 14 days of incubation at 30°C with x100 magnifi-cation. To improve visualization of colonies, agarplates were stained with a 1% Dienes methylene blue-azure II solution for 10 min and rinsed with phosphate-buffered saline (12).

Microscopy. DNA fluorescent staining of spiro-plasma-infected cell cultures was performed by usingHoechst 33258 fluorochrome. The procedure was asdescribed previously (11) except that fixation was with2% glutaraldehyde in MlA medium without serum, pH7.3. After fixation, cover slips were washed threetimes in phosphate-buffered saline, pH 7.2, air dried,and stained. Slides were observed in a Leitz Orthoplanfluorescent microscope equipped with an H2 filtermodule.

Scanning electron microscopy (SEM) was per-formed on spiroplasma-infected Dm-1 cells grown inMlA in Leighton tubes containing cover slips. Dm-1cells (1 x 105 to 2 x 105) were seeded, and approxi-mately 106 to 107 colony-forming units (CFU) of theappropriate spiroplasma were inoculated into freshlypassaged cells. The cultures were incubated at 25°C

for 2 to 7 days, depending on the specific spiroplasmainoculated, and then fixed in 2% glutaraldehyde inMlA as described for DNA staining. The cover slipswere processed and scanned as described previously(23).

Transmission electron microscopy was also per-formed on Dm-1 cells infected with spiroplasmas. Themethod of plating, inoculation, and fixation was as forSEM except that the Dm-1 cells were grown in T25flasks. Cells were rinsed, postfixed, dehydrated, em-bedded, and viewed as described previously (23).

Cytadsorption was studied by means of dark-fieldmicroscopy, DNA fluorescent staining, and SEM.Criteria for dark-field microscopy was adsorption ofone end of the spiroplasma to Dm-1 cells, with theother end exhibiting normal motility. DNA stain andSEM preparations werejudged by the relative locationof Dm-1 cells and spiroplasmas.

Uridine phosphorylase. Uridine phosphorylase activ-ity was determined in broth-propagated spiroplasmasand in spiroplasma-free and spiroplasma-infected Dro-sophila cell cultures. The uridine phosphorylase assayhas been used to detect mycoplasmas in mammaliancell cultures. All mycoplasmas previously tested bythe procedure of Levine (20) contain uridine phosphor-ylase, whereas the overwhelming majority of mamma-lian cultures lack the activity. Uridine phosphorylaseactivity is suggestive of mycoplasmal infection. Themethod was essentially that of Levine (20) except thatthe temperature of incubation was varied as noted, andcentrifugation was at 10,000 x g for 30 min in aBeckman rotor type 30. For the SRO assay, hemo-lymph from 500 flies (D. pseudoobscura) was pooledand frozen at -70°C. The thawed suspension wascentrifuged at 10,000 x g for 30 min and washed threetimes with serum-free M1A before assay. The criterionfor a positive reaction was more than 50% conversionof [14C]uridine to [14C]uracil in 30 min or an increaseof 10%o or more from 30 to 180 min of incubation (20).Primary isolations. Extracts of CSO-infected corn

plants were inoculated into cell cultures. The infectedcorn plants were supplied by T. Chen, Rutgers Univer-sity, New Brunswick, N. J. Leaves were surfacedecontaminated by washing with 90% ethanol. In 3 to5 ml ofM1A medium, leaves were either cut into small1-mm2 pieces or pressed with the flat tip of a forceps tosqueeze fluid from the leaf. The suspension was fil-tered through a 0.45-p.m membrane filter. Aliquots (0.2ml) were immediately inoculated into Multi-Wellplates (Falcon Plastics, Oxnard, Calif.) containingeither MlA medium alone, Dm-1 cells (2 x 105) inMlA or Schneider's medium, or I-XII cells (2 x 105) inSchneider's medium.

RESULTSDm-1 cell cultures grew in MIA medium as

well as in Schneider's Drosophila medium. Cellcounts of Dm-1 grown in 25-cm2 flasks for 7 daysin MlA or Schneider's medium were 1.21 x 107or 0.85 x 107, respectively (three tests, each intriplicate; no significance). All further experi-ments with Dm-1 cells were performed in MiAmedium.

Table 1 lists the results of spiroplasma infec-tion of Dm-1. The spiroplasma peak titers

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achieved in Dm-1 cultures within 3 to 7 dayswere similar to those found and reported inbroth. However, despite similar spiroplasma ti-ters, the type and extent of cytopathic effect(CPE) observed in Dm-1 cells varied among

different spiroplasmas, but included detachmentfrom the monolayer surface, granularity,changes in membranes, and increase in culturedebris. CPE varied, but included a rapidly lethalinfection ofHB that killed the culture within 2 to4 days, ofCSO infection that killed the culture in3 weeks, and of SMCA that produced a chronicinfection which the Dm-1 cells survived. How-ever, at 30°C, SMCA was cytopathic to the cells.Contrary to uninfected cells at 25°C, mostSMCA-infected cells detached from the mono-

layer surface. Table 1 also shows that the spiro-plasmas cytadsorb. The adsorption is speciesdependent. Under the conditions of these ex-

periments, HB, 277F, CSO, and S. citri stronglycytadsorbed. SMCA cytadsorbed to a lesserdegree, and BNR-1 and OBMG cytadsorbedpoorly, if at all. BNR-1 and OBMG lost theirspiral in broth and Dm-1 cultures and becamespheroidal within 24 h. Therefore, it was difficultto determine whether these spheroids cytad-sorbed to Dm-1. Guinea pig and human group 0erythrocytes did not hemadsorb to colonies of277F and HB, the strains that exhibited thehighest degree of cytadsorption.HB grew in Dm-1 and I-XII Drosophila cell

cultures. HB produced CPE in these lines. In-oculation of 105 to 107 CFU of HB producedCPE within 4 to 7 days in the following mamma-lian cell lines: human IMR-90, mouse 3T6, Veromonkey kidney, and mouse A-9 cells. Studieswith mammalian cells were performed at 37°C in5% C02-air. CSO did not grow in the abovemammalian cell cultures because it was inacti-vated at 37°C.

Microscopy. The nuclei of spiroplasma-freeDm-1 cells exhibited a diffuse fluorescent stainwith Hoechst 33258, but still were readily dis-tinct from the cytoplasm. Fluorescent DNAstaining of spiroplasma-infected Dm-1 culturesmostly showed clusters of spiroplasmas at-tached to Dm-1 or to cover slips as well as singleorganisms. A representative DNA fluorescent-stained 277F-infected Dm-1 cell is shown in Fig.1. The large fluorescent bodies are cells withhigh numbers of cytadsorbed spiroplasmas. Thehelical morphology of 277F is apparent.

Helical morphology is also apparent in SEM.Figure 2 shows SEM of uninfected Dm-1 (Fig.2a) and Dm-1 infected with HB (Fig. 2b). HBmaintained tight coiling and cytadsorbed to theDm-1. Spiroplasmas adsorbed to Dm-1 cellsgenerally exhibited a minimum of five turns perorganism. Dark-field observation confirmed thatthe organisms attached to the cell. Figure 2b alsoshows a rough Dm-1 cell surface, suggestingcytopathology as a result of HB infection. SEMof Dm-1 infected with spiroplasmas other thanHB confirmed that the spiroplasma-cell cytad-sorption and CPE varied with the infecting spir-oplasmas, as shown in Table 1 (data not shown).

Transmission electron microscopy of HB-in-fected Dm-1 is shown in Fig. 3a. The CPEproduced by HB is evident in several cells. Thehelical nature of HB can be seen (arrows).Figure 3b shows some characteristics of HBsuch as the lack of a cell wall and enlargedregions. These enlargements were also seen inSEM and are located at one end of the spiral.Thin extracellular filaments were often ob-served. These were also seen in sections of 277Fspiroplasmas (not shown).

Studies were performed on CSO infection ofDm-1 since these organisms grew to high con-

centrations but did not kill Dm-1 cultures in

TABLE 1. Growth of spiroplasmas in Dm-1 cellsa

Spiroplasma Growth Culture death Titer Adsorptionisolate (days) (CFU/ml) AsrtoCSO + 21 2.6x108 +S. citri + 14 1.0 x 109 +Cactus + 14 9.2 x 108 +Lettuce + 14 4.4 x 108 +PP + 7 2.5 x 109 +BNR-1 + 5 3.3 x 109 b

OBMG + 5 3.0 x 109 +HB + 2 1.1x109 +SMCA + 1.0 x 109c +277F + 5 5.5 x 109 +SRO - NDd ND

a Approximately 105 to 107 spiroplasmal CFU or cells (SRO) were inoculated into Dm-1 cultures. Culturedeath was determined by trypan blue stain, adsorption by dark-field microscopy, and DNA staining. The titerswere determined within 3 to 7 days, depending on the spiroplasma strain.

b +, Questionable results.cColor-changing units, checked by dark-field microscopy.d ND, Not done.

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FIG. 1. Strain 277F-infected Dm-1 cells stained with fluorescent DNA stain Hoechst 33258. The fluorescentbodies are Dm-1 cells with cytadsorbed helical 277F. Pictures were taken with an Orthomat-W automaticmicroscope camera (x1,600).

passage 1. CSO produced small colonies on agarthat were difficult to count and to distinguishfrom background artifacts. Counts were facili-tated and made more reproducible by use of

Dienes staining that has been used previouslywith animal mycoplasmas. Viability counts ofCSO, but not HB, 277F, and S. citri could beincreased by substitution of agarose, a washed

FIG. 2. SEM of uninfected (a) and HB-infected (b) Dm-1 cells (x2,300).

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a

I. :. :

I~~~~~~~~~~~..

r

FIG. 3. Transmission electron micrographs of HB-infected Dm-1 cells. a. Arrows depict longitudinal sectorsthrough individual spiroplasmas (x 3,800). b. Arrows point to enlarged regions of HB and to extracellularfilaments (x 72,000).

agar, for Noble agar (Table 2). All experimentswith CSO were performed with agarose-contain-ing medium.

Dm-1 cell cultures were infected on day 0, andcell counts of Dm-1 and spiroplasmas were madeover a 3-week period. The cultures were pas-saged every 7 days. This experiment was per-formed three times, each in triplicate. The re-sults of the effects of CSO on Dm-1 are plotted inFig. 4. In contrast to uninfected controls, theCSO-infected Dm-1 cultures died within 3weeks. Viability of the infected cultures de-creased from 98% the first week to 84 and 25% inthe second and third weeks, respectively. Unin-fected cultures had 98% viable cells throughout.CPE became apparent during the second weekof infection. The peak titer ofCSO propagated inDm-1 cells did not differ significantly from CSOgrown in MlA broth. Peak CSO titers in Dm-1cultures and in MlA broth were achieved 7 daysafter infection or passage. The concentrations ofCSO in Dm-1 did not differ significantly at theend of the three passages studied: 2.6 x 108CFU/ml, 4.7 x 108 CFU/ml, and 1.6 x 108 CFU/ml at passages 1, 2, and 3, respectively. Thesedata indicate that the CPE observed was not duesolely to the presence of large numbers of CSO.

Uridine phosphorylase. Results of uridinephosphorylase assays of broth-propagated spiro-plasmas are presented in Table 3. SRO wasobtained from Drosophila hemolymph. SMCA,277F, HB, OBMG, BNR-1, and SRO contained

uridine phosphorylase activity, whereas S. citri,CSO, PP, and primary isolates of CSO failed todemonstrate activity. Agreement was alwaysobtained within spiroplasmal serovars. Furtherinvestigations were performed on the organismsthat yielded negative results. Variables studiedincluded incubation of the substrate spiroplasmapreparation at temperatures of 25, 30, and 37°Cfor periods up to 5 h. These also failed todemonstrate enzyme activity. To eliminate thepossibility that the phosphorylase-negative or-ganisms contained a material that affected uracilmigration, tests were performed in which[14C]uracil was added to the preparation instead

TABLE 2. Effect of agar on growth ofspiroplasmasa

Spiroplasma CFU/mlisolate Noble agar Agarose

HB 2.5 x 108 2.5 x 108277F 2.3 x 109 1.8 x 109S. citriR8A2 1.1 x 108 0.6 x 108Lettuce 6.9 x 108 6.3 x 108

CSO 4.7 x 105 2.5 x 108a Spiroplasmas were inoculated onto MlA plates

with agarose or Noble agar. After 10 to 14 days ofaerobic incubation at 30°C, the plates were Dienesstained, and the colonies were counted at x 100 magni-fication. The CSO result was significant (P < 0.05).

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SPIROPLASMAS IN CELL CULTURES 301

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l 2 3 Weeks

FIG. 4. Viability cell counts of uninfected (*) and CSO-infected (O) Dm-1 cells. Cell counts were performedfor 3 weeks, using a hemacytometer and trypan blue stain. After 7 and 14 days, the cells were transferred intonew flasks at the same cell density as that on day 0.

of [14C]uridine. The presence of a possible en-zyme inhibitor was assayed by mixing in a 1:1ratio a phosphorylase-negative organism (CSO)with a positive organism (277F). These experi-ments showed that uracil migrated as expected,and there was no inhibitor of the phosphorylasereaction in the CSO-277F mixture.

Results of uridine phosphorylase assays ofspiroplasma-free and spiroplasma-infected Dro-sophila cell cultures are presented in Table 4.Dm-1 had no detectable activity, but I-XII hadsignificant enzyme levels. I-XII was free ofmycoplasmas as determined by plating on stan-dard mycoplasma, M1A, and SP4 agars, DNA

staining, and SEM. Inoculation of a 0.22-,umfiltrate of supernatant from I-XII into Dm-1failed to transfer phosphorylase activity, sug-gesting that uridine phosphorylase activity wasnot due to an infectious agent. Infection of Dm-1with a phosphorylase-containing spiroplasmaconferred activity to that culture. Infection witha phosphorylase-free spiroplasma had no effect.Primary isolations. To determine whether cell

cultures were appropriate substrates for primaryisolation of spiroplasmas, aliquots of fluids fromcorn plant leaves infected with CSO were inocu-lated into MIA medium with or without Dm-1and into Schneider's media with or without I-

TABLE 3. Uridine phosphorylase activity in spiroplasmasSerogroupa Spiroplasma isolate Strain No. of tests Activity (%)b Resultc

I-1 S. citri R8A2 12 4.9 (1-9)C189 3 10.7 (7-14) -

Cactus 3 7.1 (6-8)Lettuce 3 6.7 (4-9)

1-2 HB BC3 4 64.2 (41-83) +1-3 CSO E 10 9.4 (2-15)

Primary isolates 4 7.5 (3-11)1-4 Tick group 2 277F 11 91.2 (87-95) +II SRO SRO 1 36.0 +III Flower OBMG 4 86.3 (76-95) +

BNR-1 4 68.3 (22-95) +IV Flower PP 3 11.2 (8-18)V Tick group 1 SMCA 6 85.8 (72-96) +

a Serological groups or subgroups are as described by Junca et al. (18).b Conversion of ["4C]uridine to [14C]uracil in 180 min; range is shown within parentheses.c By Levine's criteria (20).

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TABLE 4. Uridine phosphorylase activity in cleanand infected cell cultures

Culture No. of Activity (%)a Resultbtests

Dm-1 8 4.3 (2-12)I-XII 3 69.5 (58-90) +Dm-1 + 277F 2 94.5 (93, 96) +Dm-1 + SMCA 1 49.0 +Dm-1 + CSO 1 5.0I-XII + CSO 2 40.9 (40.6, 41.1) +

a Conversion of [14C]uridine to [14C]uracil in 180min; range is shown within parentheses.

b By Levine's criteria (20).

XII cells. Results are shown in Table 5. In eachof three experiments, high concentrations ofCSO were definitely detected earlier in Dm-1 orI-XII cell cultures than in the correspondingmedium alone. These primary isolates of CSOcould be subcultured in both MlA andSchneider's medium. These isolates were identi-fied as CSO by D. Williamson, State Universityof New York, Stony Brook, by the combineddeformation-metabolism inhibition test (38).SRO. SRO failed to grow in the Dm-1 cells. It

also failed to grow in I-XII. In both cell lines, theSRO survived for several weeks. Modificationsof the MiA medium were also unsuccessful.

DISCUSSIONThe spiroplasmas constitute a recently recog-

nized and highly interesting group of organisms.Not all can presently be propagated in cell-freemedia, and new isolates are being made. Fre-quently, cell culture systems offer a potentiallyuseful methodology to aid in the development ofmedia and to study host-parasite relationships.In this study, cloned strains of spiroplasmas

multiplied in the Dm-1 cell line to high titers,and, more significantly, each produced a specif-ic type of CPE. These findings suggest that theCPE produced was not due to changes in themedia secondary to spiroplasmal growth, e.g.,

pH. Uninfected Dm-1 cells were resistant to anacid pH of 6.0 to 6.5 for more than 2 weeks inour laboratory. Strain 277F, which killed Dm-1cells in approximately 5 days, did not produce amarked acid shift in pH within this period.Further, different spiroplasmas killed Dm-1 cul-tures in periods ranging from 2 days to 3 weeksand from passages 1 to 3, although peak titers ofall organisms were approximately the same.SMCA did not kill the cultures at 25°C, althoughit grew to high concentrations. The precisecause of CPE by the various spiroplasmas wasnot determined in this study. Production of toxicmetabolites, membrane damage, and utilizationof media components are some possibilities.

Although the present studies were performedmainly on early passage-cloned laboratorystrains, the Dm-1 system was capable of cultur-ing primary isolates of CSO from diseased cornplants. In fact, in both Dm-1 and I-XII cell linesof Drosophila, high CSO titers were registeredmuch earlier than in MlA or Schneider's medi-um as determined by dark-field observation.These findings suggest that cell culture sub-strates may be useful in detection of spiroplas-mas from plants, insects, or the general environ-ment. Tully et al. also made primaryspiroplasma isolations from Ixodes ticks, usingtick cell cultures (32).The helical morphology was maintained in

Dm-1 cell cultures as shown by dark-field obser-vation, DNA staining, and SEM. The relativelylarge size of the organisms (4 to 5 ,um) allowseasy visualization in dark-field which then can

TABLE 5. Isolation of CSO from infected corn leavesaMlA Schneider's medium

Without Dm-1 With Dm-1 Without I-XII With I-XII

1 5-7 + + NDb +12-14 - + +++19-21 - ++ + +++26-28 + +++ +++ +++

2 5-7 + + - +12-14 + + + - +19-21 + +++ + ++26-28 ++ +++ + +++

3 5-7 + + ND +12-14 + +++ + +++19-21 ++ +++ +++ +++

a The cultures were classified by means of CSO concentration per dark-field: -, negative as judged bymorphology and motility; +, 1 to 20 CSO per field; + +, 20 to 80 CSO per field; + ++, too numerous to count.

b ND, Not done.

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serve as a basis for proper fixation methods.Curiously, long helices were observed attachedto Dm-1, with five or six turns or more beingusual. These were observed even during log-phase spiroplasmal growth. Bove and Saillard(2) reported that with S. citri, log-phase growthis accompanied with spirals of primarily two orthree turns.

Spiroplasmal cytadsorption varies in differentspecies; it was also found with mammalian cul-tures (unpublished data) such as IMR-90, 3T6,and Vero monkey kidney cells. Detailed elec-tron microscopic findings of spiroplasma-Dm-1cytadsorption and CPE will be published sepa-rately. It is unknown whether cytadsorption isrelated to pathogenicity. R. McCoy (personalcommunication) also found cytadsorption to he-mocytes in wax moth larvae (Galleria mellon-ella) with the HB spiroplasma AS576 and, to alesser extent, with the spiroplasma from Onco-metopia nigricans (R. McCoy, Phytopathol.News 12:217, 1978).Our transmission electron microscopy photo-

graph (Fig. 3) did not allow comparison of thethin extracellular filaments with other reportedspiroplasma features. Outer membrane projec-tions were found in 277F (29), in helical andnonhelical S. citri (7, 31), and in barred 5-nmstructures in helical and nonhelical S. citri (7,31). Striated 3.5-nm diameter fibrils were foundin lysed SRO (35), BC3 HB spiroplasma (30),and helical and nonhelical S. citri (31). Morpho-logically similar threadlike fibers were observedin 277F (29). The fibrils were sometimes report-ed to be associated with membrane fragments,supporting the hypothesis that they might beinvolved in maintaining the helical morphology(30, 31). Townsend et al. (30) purified and par-tially characterized these fibrils. Additionalstudies of these extracellular filaments are inprogress.

S. citri, CSO, and PP are the first mycoplas-mas reported to lack uridine phosphorjlase bythe Levine assay of [4C]uridine to [1 C]uracilconversion. Mycoplasma buccale lacked uridinephosphorylase as determined by another method(16). With the Levine method, we found thisstrain of M. buccale to be positive (86% conver-sion in 180 min, mean of three assays). Forspiroplasmas, findings were consistent for dif-ferent strains of the same organism and organ-

isms of the same serovar (18, 38). The distinc-tion between uridine phosphorylase-positive and-negative organisms does not apparently followany classification of spiroplasmas by pathoge-nicity, serological group, or host range, althoughall uridine phosphorylase-negative spiroplasmaswere plant isolates.Other cell and organ cultures from different

insects may be more useful for spiroplasma

studies than the Dm-1 system, depending on thespecific markers of the cell in culture, the mediaused, and the spiroplasma of interest. Proce-dures such as use of conditional medium orfeeder layers have been successful in cell culturesystems, and these may be appropriate in stud-ies on spiroplasmas.

ACKNOWLEDGMENTS

We thank J. G. Tully for review of the manuscript andLindsay Gamon, Diane Meredith, Karen Pierce, and RileyHansom for technical assistance.

This work was supported by Public Health Service grant AI-15748 from the National Institute of Allergy and InfectiousDiseases.

LITERATURE CITED

1. Bastian, F. 0. 1979. Spiroplasma-like inclusions in Creutz-feldt-Jakob disease. Arch. Pathol. Lab. Med. 103:665-669.

2. Bove, J. M., and C. Saillard. 1979. Cell biology of spiro-plasmas, p. 83-153. In R. F. Whitcomb and J. G. Tully,(ed.), The mycoplasmas, vol. III. Plant and insect myco-plasmas. Academic Press, Inc., New York.

3. Chen, T. A., and C. H. Liao. 1975. Corn stunt spiro-plasma: isolation, cultivation and proof of pathogenicity.Science 188:1015-1017.

4. Clark, H F., and L. B. Rorke. 1979. Spiroplasmas of tickorigin and their pathogenicity, p. 155-174. In R. F. Whit-comb and J. G. Tully (ed.), The mycoplasmas, vol. III.Plant and insect mycoplasmas. Academic Press, Inc.,New York.

5. Clark, T. B. 1977. Spiroplasma sp., a new pathogen inhoney bees. J. Invert. Pathol. 29:112-113.

6. Clyde, W. A. 1964. Mycoplasma species identificationbased upon growth inhibition by specific antisera. J.Immunol. 92:958-965.

7. Cole, R. M., J. G. Tully, T. J. Popkin, and J. M. Bove.1973. Morphology, ultrastructure, and bacteriophage infec-tion of the helical mycoplasma-like organism (Spiroplasmacitri gen. nov., sp. nov.) cultured from "stubborn" dis-ease of citrus. J. Bacteriol. 115:367-386.

8. Davis, R. E. 1978. Spiroplasma associated with flowers ofthe tulip tree (Liriodendron tulipifera L.). Can. J. Micro-biol. 24:954-959.

9. Davis, R. E., and J. F. Worley. 1973. Spiroplasma: motile,helical microorganism associated with corn stunt disease.Phytopathology 63:403-408.

10. Davis, R. E., J. F. Worley, R. F. Whitcomb, T. Ishiima,and R. L. Steere. 1972. Helical filaments produced by amycoplasma-like organism associated with corn stuntdisease. Science 176:521-523.

11. DelGiudice, R. H., and H. E. Hopps. 1977. Microbiologicalmethods and fluorescent microscopy for the direct dem-onstration of mycoplasma infection of cell cultures, p. 57-69. In G. J. McGarrity, D. G. Murphy, and W. W. Nichols(ed.), Mycoplasma infection of cell cultures. PlenumPublishing Corp., New York.

12. Dienes, L., M. W. Ropes, W. E. Smith, S. Madoff, and W.Bauer. 1948. The role of pleuropneumonia-like organismsin genitourinary and joint diseases. N. Engil. J. Med.238:509-515, 563-567.

13. Fabiyi, A., T. S. Elizan, and J. E. Pounds. 1971. Sucklingmouse cataract agent (SMCA) in tissue culture (35200).Proc. Soc. Exp. Biol. Med. 136:88-91.

14. Fudl-Allah, A., E. C. Calavan, and E. C. K. Igwegbe.1972. Culture of a mycoplasma-like organism associatedwith stubborn disease of citrus. Phytopathology 62:729-731.

15. Granados, R. R., and D. J. Meehan. 1975. Pathogenicityof the com stunt agent to an insect vector, Dalbuluselimatus. J. Invert. Pathol. 26:313-320.

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16. Hamet, M., C. Bonissol, and P. Cartier. 1980. Enzymaticactivities on purine and pyrimidine metabolism in ninemycoplasma species contaminating cell cultures. Clin.Chim. Acta 103:15-22.

17. Jones, A. L., R. F. Whitcomb, D. L. Williamson, and M.E. Coan. 1977. Comparative growth and primary isolationof spiroplasmas in media based on insect tissue cultureformulations. Phytopathology 67:738-746.

18. Junca, P., C. Saillard, J. G. Tully, 0. Garcia-Jurado, J.-R.Degorce-Dumas, C. Mouches, J.-C. Vignault, R. Vogel, R.McCoy, R. F. Whitcomb, D. L. Williamson, J. Latrille,and J. M. Bove. 1980. Caracterisation de spiroplasmesisoles d'insectes et de fleurs de France continentale, deCorse et du Maroc. Proposition pour une classificationdes spiroplasmes. C. R. Acad. Sci. 290:1209-1212.

19. Lafleche, D., and J. M. Bove. 1970. Mycoplasmes dans lesargumes atteints de "greening", de "stubborn" ou demaladies similaires. Fruits 25:455-465.

20. Levine, E. M. 1974. A simplified method for the detectionof mycoplasmas. Methods Cell Biol. 8:229-248.

21. McCoy, R. E., D. S. Williams, and D. L. Thomas. 1979.Isolation of mycoplasmas from flowers. Proc. U.S.-ChinaCoop. Sci. Progr. NSL Symp. Ser. 1:75-80.

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

23. Phillips, D. M. 1977. Detection of mycoplasma contamina-tion of cell cultures by electron microscopy, p. 105-118. InG. J. McGarrity, D. G. Murphy, and W. W. Nichols (ed.),Mycoplasma infection of cell cultures. Plenum PublishingCorp., New York.

24. Pickens, E. G., R. K. Gerloff, and W. Burgdorfer. 1968.Spirochete from the rabbit tick Haemaphysalis leporispa-lustris (Packard). J. Bacteriol. 95:291-299.

25. Poulson, D. F., and B. Sakaguchi. 1961. Nature of the"sex-ratio" agent in Drosophila. Science 133:1489-1490.

26. Saglio, P., D. Lafleche, C. Bonissol, and J. M. Bove. 1971.Culture in vitro des mycoplasmes associes au "stubborn"des agrumes et leur observation au microscope electroni-que. C. R. Acad. Sci. 272:1387-1390.

27. Saglio, P., M. L'Hospital, D. Lafleche, G. Dupont, J. M.Bove, J. G. Tully, and E. A. Freundt. 1973. Spiroplasmacitri gen. and sp. n.: a mycoplasma-like organism associat-ed with "stubborn" disease of citrus. Int. J. Syst. Bacter-iol. 23:191-204.

28. Schneider, I. 1972. Cell lines derived from late embryonic

stages of Drosophila melanogaster. J. Embryol. Exp.Morphol. 27:353-362.

29. Stahlheim, 0. H. V., A. E. Ritchie, and R. F. Whitcomb.1978. Cultivation, serology, ultrastructure, and virus-likeparticles of spiroplasma 277F. Curr. Microbiol. 1:365-367.

30. Townsend, R., D. B. Archer, and K. A. Plaskitt. 1980.Purification and preliminary characterization of spiro-plasma fibrils. J. Bacteriol. 142:694-700.

31. Townsend, R., J. Burgess, and K. A. Plaskitt. 1980.Morphology and ultrastructure of helical and non-helicalstrains of Spiroplasma citri. J. Bacteriol. 142:973-981.

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

33. Tully, J. G., R. F. Whitcomb, H. F. Clark, and D. L.Williamson. 1977. Pathogenic mycoplasmas: cultivationand vertebrate pathogenicity of a new spiroplasma. Sci-ence 195:892-894.

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

35. Vignault, J. C., J. M. Bov6, C. Saillard, R. Vogel, A.Farro, L. Venegas, W. Stemmer, S. Aoki, R. McCoy, A. S.Al-Beldawi, M. Larue, 0. Tuzcu, M. Ozsan, A. Nhami, M.Abassi, J. Bonfil, G. Moutous, A. Fos, F. Poutiers, and G.Viennot-Bourgin. Mise en culture de spiroplasmes a partirde materiel vegetal et d'insectes provenant de pays cir-cummediterraneens et du Proche-Orient. C. R. Acad. Sci.290:775-780.

36. Williamson, D. L. 1974. Unusual fibrils from spirochete-like sex ratio organism. J. Bacteriol. 117:904-906.

37. Williamson, D. L., and D. F. Poulson. 1979. Sex ratioorganisms (spiroplasmas) of Drosophila, p. 175-208. In R.F. Whitcomb and J. G. Tully (ed.), The mycoplasmas,vol. III. Plant and insect mycoplasmas. Academic Press,Inc., New York.

38. Williamson, D. L., J. G. Tully, and R. F. Whitcomb. 1979.Seriological relationships or spiroplasmas as shown bycombined deformation and metabolism inhibition tests.Int. J. Syst. Bacteriol. 29:345-351.

39. Williamson, D. L., and R. F. Vhitcomb. 1975. Plantmycoplasmas: a cultivable spiroplasma causes corn stuntdisease. Science 188:1018-1020.

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