INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1988, p. 103-107 Vol. 38, No. I 0020-7713/88/010103-05$02.00/0 Copyright 0 1988, International Union of Microbiological Societies

Spiroplasma taiwanense sp. nov. from Culex tritaeniorhynchus Mosquitoes Collected in Taiwan M. L. ABALAIN-COLLOC,l* L. ROSEN,* J. G. TULLY,3 J. M. BOVE,4 C. CHASTEL,l AND D. L. WILLIAMSON' Laboratoire de Bact&riologie,Faculte' de Me'decine, 22, Avenue Camille Desmoulins, 29285 Brest, France'; University of Hawaii at Manoa, Pacijic Biomedical Research Center, Honolulu, Hawaii, 968222; Mycoplasma Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Center, Frederick, Maryland 21 7013; Laboratoire de Biologie Cellulaire et Mole'culaire, Institut National de la Recherche Agronomique et Universite' de Bordeaux 11, Domaine de la Grande Ferrade, 33140 Pont de la Maye, France4; and Department of Anatomical Sciences, Health Sciences Center, State University of New York, Stony Brook, New York I I 794'

Spirophsmu isolates recovered from female mosquitoes (Culex tritueniorhynchus) collected in Taiwan were found to be similar in their serological properties. Strain CT-lT (T = type strain) proved to be serologically unrelated to all currently recognized groups and subgroups. Strain CT-lT was studied by using criteria proposed by the International Committee on Systematic Bacteriology Subcommittee on of for the description of new mollicute species. The organisms were shown to belong to the class Mollicutes by the ultrastructure of their limiting membrane, their colonial morphology, and their filtration patterns and to the family Spiroplusmatuceue by their helical morphology and motility. Growth in SP-4, MlA, and M1D media occurred at 22 to 30°C. Cholesterol was required for growth. Glucose was fermented, but arginine was not hydrolyzed. The base composition (guanine-plus-cytosine content) of the deoxyribonucleic acid of strain CT-lTwas found to be 25 f 1 mol%. On the basis of these findings, we propose that spiroplasma strains with these characteristics should be recognized as a new species, Spiroplusmu tuiwunense. Strain CT-lT has been deposited in the American Type Culture Collection as strain ATCC 43302T.

In July 1981, female mosquitoes were collected in Taiwan Taiwan, Republic of China, on 2 July 1981. After capture, in a survey of a natural population of mosquitoes for the mosquitoes were held for 4 days to allow any blood present presence of Japanese encephalitis virus @a). In 1985, tritu- to be digested. They were then separated by species and rates of these mosquitoes, which had been stored at -7O"C, stored at -70°C until thawed for processing. were tested for the possible presence of by Culture media and cultivation procedures. A triturate was culture in SP-4 medium. Several isolates of helical motile prepared for viral assay by grinding a pool of 100 adult organisms were obtained in these trials. One of the spiro- female Culex tritaeniorhynchus mosquitoes in 1 to 2 ml of plasma isolates was designated strain CT-lT (T = type phosphate-buffered saline containing 30% calf serum, peni- strain) to reflect the name of the mosquito species (Culex cillin (750 U/ml), and streptomycin (750 pg/ml). After viral tritaeniorhynchus) from which it had been isolated. assay, the triturate was refrozen and stored at -70°C. In Strain CT-lT was shown to be distinct from all previously 1985, the frozen triturate was thawed and filtered through a described group I through XXI strains by one-way deforma- 450-nm membrane filter, and 0.1 ml was inoculated into 1.5 tion, metabolism inhibition, and growth inhibition tests and ml of SP-4 medium (29, 30). The culture was incubated at was designated the representative strain of a new group, 28°C. Color change was noted 12 days later, and motile group XXII, according to the classification originally pro- helical filaments were observed in samples of the cultures posed by Junca et al. (17) and subsequent revisions (27, 33, examined by dark-field microscopy. Tenfold dilutions of an 34). Two additional spiroplasma strains, CT-2 and CT-3, early pass (P3) were sent to D. L. Williamson, State Univer- isolated in the same year from the same species of mosquito sity of New York, Stony Brook. The isolate was first passed at the same site, had titers similar to those of strain CT-lT into M1D broth medium (35) and triply filter-cloned on M1D when they were tested against CT-lT antiserum in one-way agar medium, and then 1 to 2-ml samples of a batch culture deformation tests. were lyophilized. Strain C-intaiffeB in M1D In this report, we summarize evidence that strain CT-lT medium by passing a 1OYGnocu~umevery day into fresh and related strains fulfill the minimal species requirements media at 30°C. Agar colonies were obtained by plating for Mollicutes as proposed by the International Committee suspensions on 1.6% Noble agar (Difco Laboratories) in on Systematic Bacteriology Subcommittee on the Taxonomy M1D medium. To provide a uniform source of spiroplaSmas, of Mollicutes (16, 32). Therefore, we formally propose that a 25-ml batch culture was prepared and separated in mid- strain CT-lT and related strains be recognized as a new logarithmic phase into 0.5- and 1-ml volumes in microtubes, species in the genus Spiroplasma. which were stored at -70°C. Other spiroplasmas were compared with strain CT-lT. MATERIALS AND METHODS These included the following organisms: Spiroplasma citri Maroc R8A2T (= ATCC 27556T) (subgroup I-l), Spiro- Origin of strain. Female mosquitoes attracted to animal plasma melliferum BC-3T (= ATCC 33219T) (subgroup I-2), bait near rice fields were collected at Taishan (near Taipei), Spiroplasma kunkelii E275T (= ATCC 29320T) (subgroup I-3), tick spiroplasma strain 277F (= ATCC 29761) (subgroup * Corresponding author. I-4), strain LB-12 (= ATCC 33649) (subgroup I-5), Maryland

103 104 ABALAIN-COLLOC ET AL. INT.J. SYST.BACTERIOL. flower spiroplasma strain M55 (= ATCC 33502) (subgroup '[-6), Cocos spiroplasma strain N525 (= ATCC 33287) (sub- group I-7), Spiroplasrna phoeniceurn strain P40 (= ATCC 43115) (subgroup I-@, uncultivated strain WSRO of the Dro- sophila sex ratio organism (group 11), Spiroplasrna floricola OBMG (= ATCC 33221) (group 111), Spiroplasrna apis B31T I:= ATCC 33834T) (group IV), Spiroplasrna rnirurn SMUT I:= ATCC 29335T) (group V), Ixodes tick spiroplasma strain Y32 (= ATCC 33835) (group VI), Monobia spiroplasma strain MQ-1 (= ATCC 33825) (group VII), syrphid spiroplasma $strainEA-1 (= ATCC 33826) (group VIII), strain CN-5 (= ATCC 33827) (group IX), Spiroplasrna culicicola AES-1 (= ATCC 35112) (group X), strain MQ-4 (= ATCC 35262) (group XI), strain DU-1 (= ATCC 43210) (group XII), Spiroplasrna sabaudiense Ar-1343T (= ATCC 43303T) (group XIII), strain EC-1 (= ATCC 43212) (group XIV), strain 1-25 (= ATCC 43262) (group XV), strain CC-1 (= ATCC 43207) (group XVI), strain DF-1 (= ATCC 43209) (group XVII), strain TN-1 (= ATCC 43211) (group XVIII), strain PUP-1 (= ATCC 43206) '(group XIX), strain LD-1 (= ATCC 43213) (group XX), strain W115 (= ATCC 43260) (group XXI), and strain TG-1 (= ATCC 43525) (group XXIII). The origins and histories of these strains have been reviewed recently (1, 27). Filtration studies. An M1D broth culture of strain CT-lT FIG. 1. Electron micrograph of spiroplasma strain CT-lT. Thin- that had been incubated for 1day at 22°C was passed through section preparation of a cell pellet obtained from a 24-h broth culture a series of membrane filters with graded pore diameters (450, grown in M1D medium and stained with 2% aqueous uranyl acetate 300, 220, and 100 nm) by using a hypodermic syringe and and Reynold lead citrate. Arrows indicate the unit membrane on the minimum hand pressure. Each filtrate was diluted in a series cells. Bar = 100 nm. of 10-fold dilutions in M1D medium, and all tubes were incubated at 22°C. After 7 days, the tubes were examined for growth by identifying the last tube that showed growth of Genomic analysis. The techniques used for extraction and helical cells and a color change. The results were expressed determination of the guanine-plus-cytosine content (in moles in color-changing units (CCU) per milliliter. percent) of the deoxyribonucleic acid of strain CT-lr have Morphology. Cultures of strain CT-lT were routinely mon- been described elsewhere (3, 5, 6, 17). itored by dark-field microscopy (26). For electron micros- (copy, cells were fixed for 1 h in 2% glutaraldehyde in M1D RESULTS medium, postfixed for 1 h in 1% osmium tetroxide, dehy- Idrated in acetone, and embedded in Epon-Araldite; sections Morphological and cultural properties. A primary culture were stained with 2% aqueous uranyl acetate and Reynold of strain CT-lT was obtained after 12 days in SP-4 medium at lead citrate. 30°C. The subcultures grew rapidly, produced turbidity, and Temperature requirements. Temperature requirements acidified the medium within 24 h. Similar rapid growth was were determined by diluting the batch culture suspension in apparent when the organisms were subcultured in other M1D medium to give about lo6 CCU/ml. Six tubes, each spiroplasma broth media (M1A and M1D media and medium containing 2 ml of this dilution, were incubated at one of six containing 1% serum fraction). The presence of penicillin temperatures (22, 25, 30, 32, 35, and 37°C). At subsequent (500 U/ml) had no influence on growth rates or yields. Strain intervals, growth was assessed by removing 0.1 ml and CT-lT grew at 22,25, and 30"C, but not at 32,35, or 37°C. At preparing a 10-fold dilution series to determine the number 25 and 30"C, titers of lo1' CCUlml were achieved. Growth at of CCU per milliter. Each of the dilution series was evalu- 22°C was slower, and the peak titer was lo9 CCU/ml. ,ated by examining the dilutions near the endpoint by dark- Cultures examined by dark-field microscopy contained nu- field microscopy. merous motile helical cells. In stationary growth (cultures Sterol requirements. The growth response to cholesterol kept at 30°C more than 48 h), the organisms became de- .was determined by a technique modified slightly from the formed, but some evidence of helicity persisted for many irecommended method (20) and described elsewhere (1). days. Biochemical tests. Substrate tests were performed in 1% Electron microscopy of thin sections of plastic-embedded lbovine serum fraction broth by using procedures described pellets verified the presence of a unit membrane, typical of ]previously (1, 2). The film and spot reaction and the hemad- the genus (ll), surrounding each cell (Fig. 1). :sorption test were performed on solid medium (SP-4 broth On solid medium containing 1.6% Noble agar, classical containing 0.8% Noble agar) by using previously described "fried-egg" colonies, without satellites, appeared after 7 techniques (2, 4, 13, 19). days at 30°C (Fig. 2). Serological tests. Hyperimmune antiserum against strain Reversion studies. Cultures of strain CT-lT were main- CT-lT was prepared as described previously (28). Disk tained in antibiotic-free broth medium for five passages growth inhibition tests (10, 35) were performed with spiro- without reversion to bacterial forms. Cultures examined by plasma grown in M1D medium. The antigens grown in broth dark-field microscopy were characterized by typical helical 'were diluted to a concentration of about lo5 CCU/ml. The organisms with no evidence of walled forms. techniques used for the deformation and metabolism inhibi- Filtration studies. An 18-h broth culture of strain CT-lT tion tests have been described previously (36, 37). that contained lo1' CCU/ml was filtered through a series of VOL.38, 1988 SPIROPLASMA TAIWANENSE SP. NOV. 105

mosquitoes (Aedes sollicitans) collected in New Jersey in 1981 (23). The geographic occurence of spiroplasmas in mosquitoes was extended when Chaste1 and colleagues discovered spiroplasmas in Aedes species collected in 1983 in the Northern Alps of France (7). One of the strains, strain Ar-1343T, from Aedes sticticuslvexans females, is the type strain of group XIII, recently described as S. sabaudiense (1). Another isolate, Ar-1357, from Aedes cantanslannulipes females, also from the French Alps, is a serovar of group XVI. Collections made at the same or nearby sites in the Northern Alps in 1984 and 1985 yielded additional spiro- plasma isolates from the same hosts represented in 1983, plus the following new mosquito species: Aedes cinereuslge- minus and Coquillettidia richiardii (6a). These latter isolates have not yet been serologically characterized. More re- cently, spiroplasmas have been isolated from mosquitoes collected in Alabama (22). Three species of mosquitoes (Aedes fulvuslpallens, Anopheles punctipennis, and Culex nigripafpus)collected in central Alabama in 1985 were found to carry spiroplasmas. These isolates have not yet been characterized. FIG. 2. Colonies of strain CT-lT on 1.6% agar in M1D medium Since both male and female mosquitoes frequently feed on after 7 days of incubation at 30°C. Bar = 100 pm. nectar (18), it is possible that spiroplasmas were acquired through flower-feeding activities. graded membrane filters. The numbers of CCU per milliliter Many spiroplasmas have been isolated from insect species after filtration through membrane filters having average pore (8), but the occurrence of spiroplasmas in blood-sucking diameters of 450, 300, and 220 nm were lo1', lo9, and lo', arthropods such as ticks, tabanid flies, and mosquitoes (6a, respectively. The organisms did not pass through membrane 7, 8a, 9, 23, 31) suggests the potential for transmission to a filters with an average pore diameter of 100 nm. variety of vertebrates, including humans. Whether spiropla- Biochemical properties. Strain CT-lT fermented glucose smas may have any association with vertebrate infections is but did not hydrolyze arginine and urea. Film and spots were still an unresolved matter, but this question is worthy of not formed by strain CT-lT. Colonies of strain CT-lT grown further attention and study. on soft agar did not hemadsorb guinea pig erythrocytes. Since they are wall-less, procaryotes are resistant to Sterol requirements. The results of the modified sterol test penicillin, and are filtrable through 220-nm membrane filters, are shown in Table 1. Significant growth of strain CT-lT was strain CT-lT spiroplasmas belong in the class Moflicutes (12, not observed in the basal medium without serum or when 14). This strain is placed in the family Spiropfasmataceae supplements of albumin, palmitic acid, Tween 80, or 1 pg of (24, 25) and in the genus Spiroplasma (21) because of its cholesterol per ml were added. However, the addition of 5 to helical morphology, growth characteristics, requirement for 20 pg of cholesterol per ml to the basal medium containing sterol, and genomic features. We propose that this organism fatty acid supplements did induce growth of the organisms. be designated Spiroplasma taiwanense sp. nov. The taxo- Serological tests. Strain CT-lT homologous antiserum pro- nomic description given below summarizes the properties of duced an 8-mm diameter of growth inhibition. Heterologous the organism. antisera directed against the representatives of serogroups I Spiroplasma taiwanense (tai.wan.en'se. L. neut. adj. tai- through XXI and XXIII failed to inhibit the growth of strain wanensis of Taiwan, referring to the geographic origin of the CT-lT. The results of reciprocal growth inhibition, deforma- isolate). Helical filaments 100 to 160 nm in diameter and 3.1 tion, and metabolism inhibition tests confirmed the unique to 3.8 pm long. The cells lack true cell walls and periplasmic serological status of spiroplasma strain CT-lT (Table 2). fibrils. Motile. Colonies on solid M1D medium containing Genomic analysis. The guanine-plus-cytosine content of 1.6% Noble agar frequently have a fried-egg morphology. the deoxyribonucleic acid of strain CT-lT was determined from its melting temperature (assayed spectrophotometri- TABLE 1. Sterol requirement test for strain CT-lT cally in three independent analyses) and from its buoyant Titer of spiroplasmas (CCU/ml) after density (obtained by equilibrium centrifugation in CsC1, in Basal medium incubation at 30°C on day: two independent analyses). The guanine-plus-cytosine con- supplement(s) 5 tent was 25.7 mol% as determined from the melting temper- 123 8111316 ature and 25.4 mol% as determined from the buoyant den- None N" N N N N N N N sity. Albumin (0.5%) + palmitic NNNN N N N N acid (10 pg/ml) DISCUSSION Albumin (0.5%) + palmitic NNNN N N N N acid (10 pg/ml) + Tween Serological analysis of strain CT-lT and other evidence 80 (0.01%) showed that this spiroplasma is unrelated to any previously Cholesterol (1 pg/ml) NNNN N N N N established spiroplasma group or subgroup and therefore Cholesterol (5 pg/ml) N lo2 lo2 103 103 103 103 103 Cholesterol (10 pglml) N 10' lo2 lo2 103 104 105 lo5 represents the type strain of a new group of spiroplasmas lo2 103 105 105 (group XXII). Cholesterol (20 pg/ml) N 10' lo2 lo4 Bovine serum fraction (1%) 102 103 105 lo8 lo8 lo8 lo8 lo8 The first mosquito-derived spiroplasma, S. culicicola (15) (group X), was obtained from a triturate of female salt marsh a N, No growth. 106 ABALAIN-COLLOC ET AL. INT.J. SYST.BACTERIOL.

TABLE 2. Serological tests with strain CT-lT Growth inhibition test: Deformation test: Metabolism inhibition test: Group Strain homologous reaction (mm)" homologous reaction tite? homologous reaction titef I- 1 S. citri Maroc R8A2T 16 2,560 >117,000 1-2 S. melliferum BC-3T 12 10,240 >117,000 1-3 S. kunkelii E275T 18 20,480 39,000 1-4 277F 14 5,120 39,000 1-5 LB-12 18 5,120 > 117,000 1-6 M55 13 1,280 13,000 1-7 N525 11 10,240 39,000 1-8 S. phoeniceum P40T 17 10,240 > 117,000 I1 WSRO ND~ 2,560 ND 111 S. jIoricoLa OBMG~ 6 10,240 13,000 IV S. apis B31T 5 1,280 13,000 V S. mirum SMCA~ 13 5,120 13,000 VI Y 32 16 2,560 4,374 VII MQ-1 6 1,280 >1 17,000 VIII EA-1 10 1,280 39,000 IX CN-5 10 640 4,374 X S. culicicofa AES-~~ 8 640 4,374 XI MQ-4 5 640 4,374 XI1 DU-1 5 2,560 13,000 XI11 S. sabaudiense Ar-1343T 8 2,560 13,000 XIV EC-1 5 1,280 13,000 xv 1-25 8 1,280 >117,000 XVI cc-1 5 1,280 39,000 XVII DF-1 10 5,120 39,000 XVIII TN-1 6 1,280 >117,000 XIX PUP-1 14 5,120 13,000 xx LD- 1 ND 5,120 ND XXI W115 15 1,280 4,374 XXII CT-IT 8 1,280 13,000 XXIII TG- 1 7 2,560 39,000

a When CT-lTantigen was tested against heterologous antisera, growth inhibition zones were not observed. Likewise, when CT-lT antiserum was tested against heterologous antigens, no growth inhibition zones were observed, except for group I1 (strain WSRO),which was not done. The homologous reactions represent zones of inhibition with the antigens tested versus type-specific antisera. The results are expressed as zones of inhibition around disks saturated with antiserum. In tests of CT-lT antigen against heterologous antisera, titers less than 1:20 were considered to be negative. Likewise, when CT-lT antiserum was tested against heterologous antigens, titers less than 1:20 were recorded as negative. The titers for the homologous tests of antigens against type-specific antisera are given as the reciprocal of the highest antiserum dilution in which one-half the spiroplasmas were free and motile and one-half were deformed. When CT-lT antigen was tested against heterologous antisera, titers less than 1:18 were considered to be negative. Likewise, when CT-lT antiserum was tested against heterologous antigens, titers less than 1: 18 were recorded as negative. The titers for the homologous tests of antigens against type-specific antisera are given as the recitxocal of the highestI antiserum dilution that inhibited metabolism. kD, Not done.

Chemoorganotroph. Acid produced from glucose. Does LITERATURE CITED not hydrolyze arginine and urea. Agar colonies do not 1. Abalain-Colloc, M. L., C. Chastel, J. G. Tully, J. M. Bov4, R. F. hemadsorb guinea pig erythrocytes. Film and spot reaction Whitcomb, B. Gilot, and D. L. Williamson. 1987. Spiroplasma negative. sabaudiense, sp. nov., a new species from mosquitoes collected Cholesterol is required for growth. Facultative anaerobe. in France. Int. J. Syst. Bacteriol. 37:260-265. Temperature range, 22 to 30°C; optimum temperature, 25 2. Aluotto, B. B., R. G. Wittler, C. 0. Williams, and J. E. Faber. to 30°C. 1970. Standardized bacteriologic techniques for the characteri- Isolated from female mosquitoes, Culex tritaeniorhyn- zation of Mycoplasma species. Int. J. Syst. Bacteriol. 20:35-58. chus, at Taishan, Republic of China. 3. Bovd, J. M., and C. Saillard. 1979. Cell biology of spiroplasmas, The guanine-plus-cytosine content of the deoxyribonu- p. 83-153. In R. F. Whitcomb and J. G. Tully (ed.), The cleic acid is 25 k 1 mol%. mycoplasmas, vol. 3. Academic Press, Inc., New York. Serologically distinct from the type and representative 4. Bradbury, J. M. 1977. Rapid biochemical tests for characteri- zation of the Mycoplasmatales. J. Clin. Microbiol. 5531-534. strains of groups I through XXI and XXIII. 5. Carle, P., C. Saillard, and J. M. Bovd. 1983. DNA extraction The type strain is strain CT-1 (= ATCC 43302). and purification, p. 295-299. In S. Razin and J. G. Tully (ed.), Methods in mycoplasmology, vol. 1. Academic Press, Inc., ACKNOWLEDGMENTS New York. 6. Carle, P., C. Saillard, and J. M. Bovd. 1983. Determination of Part of this work was done in the Department of Anatomical guanine plus cytosine content of DNA, p. 301-308. In S. Razin Sciences, Health Sciences Center, State University of New York, and J. G. Tully (ed.), Methods in mycoplasmology, vol. 1. Stony Brook, while M.L.A. was a Visiting Assistant Professor in the Academic Press, Inc., New York. Laboratory of D.L.W. 6a.Chaste1, C., B. Devau, F. Le Goff, A. M. Simitzis-Le Flohic, R. We thank Pualani Kondo for her excellent technical assistance. Gruffaz, G. Kerdon, and B. Gilot. 1987. Mosquito spiroplasmas This work was supported in part by Public Health Service grant from France and their ecology. Isr. J. Med. Sci. 23:683-686. A1 17952 from the National Institutes of Health and by grant DCB 7. Chastel, C., B. Gilot, F. Le Goff, R. Gruffaz, and M. L. 8308482 from the National Science Foundation. Abalain-Colloc. 1985. Isolement de spiroplasmes en France VOL. 38, 1988 SPIROPLASMA TAIWANENSE SP. NOV. 107

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