Mycoplasma Mobile Sp. Nov., a New Species from Fish H

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1987, p. 192-197 Vol. 37, No. 3 0020-7713/87/030192-06$02.00/0 Copyright 0 1987, International Union of Microbiological Societies

Mycoplasma mobile sp. nov., a New Species from Fish H. KIRCHHOFF," P. BEYENE, M. FISCHER, J. FLOSSDORF, J. HEITMANN, B. KHATTAB, D. LOPATTA, R. ROSENGARTEN, G. SEIDEL, AND C. YOUSEF Institut fur Mikrobiologie und Tierseuchen, Tierarztliche Hochschule, D 3000 Hannover, Federal Republic of Germany

Mycoplasma strain 163K was isolated from the gills of a tench (Tinca tinca L.) with red disease. The cells are elongated, ovoid or flask shaped, consisting of a thicker body and a more slender part ending in a hemispherical terminal structure, that is apparently stabilized by a cytoskeleton. They are able to attach to inert surfaces and living cells. The most exciting property of the organisms is their fast gliding motion, which occurs on glass and plastic surfaces and which is not interrupted by resting periods. Growth occurs between 17 and 30°C, with an optimum at 25"C, in modified Hayflick medium with horse or bovine serum and in medium with swine serum. Acid is produced from several carbohydrates, but arginine and urea are not catabolized. The organism reduces tetrazolium chloride, produces film and spots and hydrogen peroxide, and is able to hemolyze and to adsorb to erythrocytes of several animal species. Indirect immunofluorescencetest, growth inhibition, and metabolism iqhibition tests indicate that the mycoplasma represents a new species for which the name Mycoplasma mobile is proposed. Strain 163K (ATCC 43663) is the type strain.

In a study of 85 fish of several species, mycoplasmalike colony count method (6) diluting the samples in microtiter colonies were obtained on agar plates directly inoculated plates (1).Growth curves were determined for strain 163K in with unfiltered organ material of 16 of the 85 fish examined several media and at several temperatures. (17). Despite immense efforts, cultivation in further passages Morphological studies. The colonies of strain 163K were was only possible in one case, with an isolate from the gills examined with a stereo microscope (Leitz). Cellular mor- of a tench (Tinca tinca L.). In a second study of 95 fish of phology was examined by dark-field microscopy, by scan- several species (unpublished data), again mycoplasmalike ning electron microscopy and by transmission electron mi- colonies were observed repeatedly on agar plates directly croscopy, using a negative staining technique and sectioning inoculated with materials from the gills, skin, spleens and of organism pellets. intestines, of the fish, but subcultivation did not succeed in For scanning electron microscopy, organisms were grown any case. Consequently, there was only one type of myco- on glass cover slips in petri dishes. Cover slips with attached plasma isolated from these fish. However, this mycoplasma cells were rinsed three times in 0.1 M sodium cacodylate distinguished itself by such unique properties that its de- buffer (PH 7.2), fixed in 2.5% (wt/vol) glutaraldehyde in the scription as a new species appears to be justified. same buffer for 2 h at 4"C, and postfixed in 1% (wtlvol) cacodylate-buffered osmium tetroxide for 2 h at room tem- MATERIALS AND METHODS perature. After washing in buffer and dehydration in a Mycoplasma. The mycoplasma isolate originated from the graded series of ethanol and double-distilled water, followed gdls of a tench showing symptoms of red disease. Growth by pure ethanol and amylacetate as intermediate fluid, the was obtained under aerobic conditions at an incubation preparations were critical point dried in carbon dioxide, temperature of 15°C on modified Hayflick medium. The sputter coated with gold, and then examined in a Jeol model details of primary isolation have been described previously JSM 35C scanning electron microscope operating at 25 kV. (17). The isolate was designated strain 163K. Pictures were taken at approximately 30°C to the plane of the Media and cultivation. Strain 163K was isolated and gen- specimen holder. erally cultivated in Hayflick medium (7), modified as de- For negative staining, organisms were grown in petri scribed before (17). For the preparation of antisera dishes on Pioloform films supported by electron microscope rnycoplasmas were cultivated in Friis medium (13), modified as described before (17). For the preparation of solid me- copper grids. The adherent cells on these films were directly dium, 0.7% (wthol) purified agar (Oxoid no. 1) and 0.01% stained with 2% (wt/vol) aqueous ammonium molybdate for dextran were added. Strain 163K was usually incubated 20 to 60 s without any preceding washing and fixation aerobically at 25°C. For determination of the temperature procedures. range enabling growth and reproduction, mycoplasmas were For thin sectioning, fluid cultures were initially fixed by incubated at 4, 17, 25, 30, and 37°C. Strain 163K was filter adding glutaraldehyde to a final concentration of 2.5% cloned three times according to the recommended proce- (vol/vol). After 4 h at room temperature or overnight at 4"C, dure, using a 220-nm-pore membrane filter (Millipore Cop) cells were pelleted by centrifugation at 2,500 x g, washed in (23). 0.1 M sodium cacodylate buffer (pH 7.2) or in veronal Growth curves. Broth medium (5 ml) was inoculated with acetate buffer (pH 7.1), and postfixed in 1% (wt/vol) cacodyl- 0.5 ml of a mycoplasma suspension containing 7.85 x lo6 ate or veronal acetate buffered Noble agar. Small cubes of organisms (stock inoculum, kept at -70°C). The number of the cell-containing agar were dehydrated in increasing con- viable and reproductive mycoplasmas was determined at centrations af ethanol, and embedded in Ducupan ACM by various intervals during incubation for 0,6,12,15,24,30,36, the method of Luft (19). Thin sections were made with an 48, 54, 60, 72, 96, 120, 144, 168, 192, 216, and 240 h by the LKB model 4800 ultramicrotome, using glass knives, and were stained with 2% (wthol) aqueous uranylacetate for 10 * Corresponding author. min (28) followed by Reynolds lead citrate for 6 min (21).

192 VOL. 37, 1987 MYCOPLASMA MOBILE SP. NOV. 193

TABLE 1. Mycoplasma and Acholeplasma strains and antisera TABLE l-Continued used in serological tests Source' Source' Strain Strain Mycoplasmas Antisera Mycoplasmas Antisera M. pneumoniae FHT NIH NIH, IMT M. agalactiae PG2T IRC IRC M. primatum HRC 292T NIH NIH M alkalescens DBS 803 NIH NIH M. pullorum CKKT IRC IRC M alvi IlsleyT IRC IRC M. pulmonis PG34T NIH NIH, IMT M anatis 1340T NIH NIH, IMT M. putrefaciens KSIT IRC IRC, IMT M arginini G230T NIH NIH, IMT M. salivarium PG20T NIH NIH M arthritidis PG6T NIH NIH, IMT M. spumans PG13T NIH NIH, IMT M bovigenitalium PGllT NIH NIH, IMT M. sualvi Mayfield BT GIRA GIRA M bovirhinis PG43T NIH NIH, IMT M. subdolum TBT MIT MIT M bovis DonettaT IRC IRC, IMT M. synoviae WVU 1853T IRC IRC M bovoculi M 165/69T IRC IRC M. testudinis 01008T MRC MRC M buccale CH20274T NIH NIH M. verecundum 107T IRC IRC, IMT M. californicum ST6T IRC IRC M. canadense 275CT IRC IRC, IMT A. axanthum H86N IRC IRC, IMT M. canis PG14T NIH NIH, IMT A. equifetale C112T IMT IMT M. capricolum California kidT IRC IRC A.florum LIT FCR FCR M. caviae G122T IRC NIH, IMT A. granularum BTS39T NIH NIH, IMT M. cavipharyngis 117CT FCR FCR A. hippikon CIT IMT IMT M. citelli RG-2CT IRC IRC A. laidlawii PG8* NIH NIH, IMT M. cloacale 383T FCR FCR A. modicum PG49T IRC IRC, IMT M. collis 58BT FCR FCR A. morum 72-043T FCR FCR M. columbinasale 694T IRC IRC A. oculi 19LT IRC IRC, IMT M. columbinum MMP-lT IRC IRC A. parvum H23MT FCR, VFV FCR, VFV M. columborale MMP-4T IRC IRC M. conjunctivae HRC 581T IRC NIH, IMT Bovine serogroup 7 PGSO IRC IRC M. cricetuli CHT FCR FCR M. cynos H831T IRC IRC, IMT ' Abbreviations: CVM (R. F. Ross), College of Veterinary Medicine, Iowa M. dispar 462/2T State University, Ames, Iowa; FCR (J. G. Tully), Mycoplasma Section, IRC IRC, IMT Frederick Cancer Research Facility, Frederick, Md.; GIRA (R. M. Gourlay), M. edwardii PG24T IRC IRC, IMT Institute for Research Animal Diseases, Compton Newbury, Berkshire, M. equigenitalium T37T IMT IMT England; IMT (H. Kirchhoff and J. Heitmann), Institut fur Mikrobiologie und M. equirhinis M 432/72T LIRA LIRA, IMT Tierseuchen, Tierarztliche Hochschule, Hannover, Federal Republic of Ger- M. fastidiosum 4822= IRC IRC many; IRC (E. A. Freundt), FAO/WHO International Reference Centre for M. faucium DC333T NIH NIH Animal Mycoplasmas, Aarhus Denmark; LIRA (R. Lemcke), Institut for M.felifaucium PVT MRC MRC Research Animal Diseases, Compton Newbury, Berkshire, England; MRC M.feliminutum BenT IRC IRC (A. Hill), Medical Research Council Laboratories, Carshalton, Surrey, En- M. felis gland; NIH (M. F. Barile and J. G. Tully), National Institutes of Health, COT IRC NIH, IMT Bethesda, Md. ; SVS (N. F. Friis), Statens Veterinaere Serum Laboratorium, M. fermentans PG18T NIH NIH, IMT Copenhagen, Denmark; VFV (M. Ogata), Department of Veterinary Public M. fEocculare M~42~ svs svs Health, Azabu University, Fuchinobe Sagamihara, Kanagawa, Japan. M. gallinaceum DDT NIH NIH, IMT M. gallinarum PG16T NIH NIH M. gallisepticum PG31T NIH NIH, IMT M. gallopavonis WRIT IRC IRC All specimens were examined with a Zeiss EM 10 electron M. gatae C1 IRC IRC, IMT microscope, using an accelerating voltage of 60 and 80 kV. M. genitalium G37T FCR FCR Filtrations studies. Filterability of strain 163K was deter- M. glycophilum 486T FCR FCR M. hominis PG21T NIH NIH mined by membrane filters (Millipore Corp.) with pore M. hyophatyngis H3-6BFT FCR FCR diameters of 450 and 220 nm. An unfiltered suspension was M. hyopneumoniae JT svs SVS, IMT used as a control, and this suspension and each filtrate were M. hyorhinis BTS7T NIH NIH, CVM diluted in 10-fold steps and spread (0.1 ml) on agar plates. M. hyosynoviae S16T CVM CVM, IMT After incubation for 5 days, the number of colonies was M. iners PG30T NIH NIH determined. M. iowae 69ST IRC IRC Reversion experiments. Strain 163K was passaged twelve M. lipofaciens R171T FCR FCR consecutive times on growth medium devoid of penicillin or M. lipophilum MaB yT NIH NIH any other antibiotics by pushing inverted agar blocks across M. maculosum PGIST NIH NIH M. meleagridis 17529T IRC IRC the agar surface. In each passage cell suspensions obtained M. moatsii MK405T IRC IRC by rinsing the agar plates were examined by dark-field M. molare H542T IRC IRC, IMT microscopy. M. muris RII14T FCR FCR Sterol requirement. Inhibition of growth by 5,10,and 20% M. mustelae MXST FCR FCR sodium polyanetholsulfonate (Liquoid; Roche Diagnostics) M. mycoides subsp. mycoides PGIT IRC IRC and by 1% digitonin was tested by the disk method (12). M. mycoides subsp. Capri PG3T IRC IRC Investigations for cholesterol requirement were performed M. neurolyticum type AT NIH NIH, IMT in eight 100-ml bottles of media, consisting of a bottle with M. opalescens MH5408T IRC IRC, IMT modified Hayflick medium containing 4% bovine serum M. orale CH19299T NIH NIH M. ovipneumoniae Y98T IRC IRC, IMT (growth control), three bottles of serum-free medium (rea- M. pirum HRC 70-159T FCR FCR gent controls), and four bottles of serum-free medium containing increasing amounts of cholesterol (26). Tween 80 Continued could not been added as recommended by Tully (26) because 394 KIRCHHOFF ET AL. INT. J. SYST.BACTERIOL. of its toxicity for strain 163K, and cholesterol was solubil- ized in ethanol. The bottles were inoculated with about lo9 CFU of strain 163K. Organisms used for inoculation were washed twice with phosphate-buffered saline (PBS) (to re- move the cholesterol originating from the culture medium) and resuspended in 2 ml of PBS. This suspension served as inoculum. After incubation for 6 days at 25”C, cells were harvested by centrifugation (20,000 x g for 45 min) and washed with PBS, and the protein content of the cell pellet determined by the biuret method (Merck, Darmstadt). Biochemical tests. All biochemical tests (see Table 3) were performed as described by Aluotto et al. (3). The experiments were performed 5 to 10 times. Hemolysis and hemadsorption. The overlay technique (3) was used to test for hemolysis. Hemadsorption was determined as described by Sobeslavsky et al. (22). Both tests were performed with human, equine, bovine, ovine, swine, rabbit, guinea pig, and fish erythrocytes. Preparation of antisera. For the preparation of antisera, organisms were passaged six to eight times in modified Friis medium to eliminate traces of horse serum. They were then washed twice in PBS, resuspended in 1% of the initial volume, emulsified in the same volume of Freund adjuvant (Difco Laboratories), and administered to rabbits (20). Serological tests. For the serological comparison of strain 1163K with the established mycoplasma species, the indirect immunofluorescence test (IFT) (9) the disk-growth inhibition test (GIT) (8) and the metabolism inhibition test (MIT) (25) were used. The sources of the mycoplasma strains and antisera are indicated in Table 1. For the IFT, rabbit antisera were used in dilutions of l:lO, 150, and 1:lOO and goat anti-rabbit fluorescein isothiocyanate-conjugated serum (Nordic) in the dilution of 150. For the GIT, plates were FIG. 1. Negative-stain (a) and scanning (b) electron microphoto- inoculated by the running drop method with mycoplasma graphs of single cells of strain 163K showing the pleomorphic cultures usually diluted and 1:lOO. When the media flask-shaped cell form, the headlike structure, and the neck part of 150 the organisms. appeared to be dry, paper discs saturated with 0.25 ml of undiluted antiserum were placed onto the surface and plates were incubated aerobically. Zones of inhibition were recorded after an incubation period of 5 to 12 days. The MIT this observation and revealed details. The flask shape exists was performed using microtiter plates. Beside undiluted in many variations ranging from coccoid to strong elongated broth cultures, dilutions of l:lO, 1:100, and 1:1,000 were forms (Fig. 1) with dimensions from 0.3 to 1.6 pm in length tested against the serum dilutions in modified Hayflick and 0.1 to 0.5 pm in width. In spite of this pleomorphism all medium containing glucose or arginine (1%). the cells are divided in a coccoid “body part” and a “head DNA. The guanine-plus-cytosine (G +C) content of deoxy- part.” The head part is hemispherical or triangular and ribonucleic acid (DNA) of strain 163K was determined by somewhat thickened (Fig. 1 and 2). On many cells, between isopycnic centrifugation on three DNA batches (11). As a the head part and body part there is a “neck part” showing control for the technique, we also evaluated the G+C a different length. Electron microscopy of ultrathin sections contents of DNAs from Acholeplasrna laidlawii and Myco- revealed that the organisms lack a cell wall and are bounded plasma pulmonis. by a single membrane (Fig. 2a and b). An electron-dense membrane-associated inner layer can be seen in the area of RESULTS the headlike structure (Fig. 2c) representing probably a cytoskeletal element. The organisms stained pink in Giemsa Morphology and ultrastructure. Strain 163 K produced stain and were gram negative. typical fried-egg colonies on modified Hayflick medium Adherence. Organisms of strain 163K showed a remark- containing horse or bovine serum and on modified Friis able ability to adhere to glass and plastic surfaces as well as medium after incubation times of 2 to 6 days (17). The to erythrocytes and tissue cells. When broth cultures were diameter of the colonies varied from 10 to 500 km depending cultivated in plastic and glass petri dishes, the organisms on their density. Colonylike aggregates with a diameter of up attached to the bottom of the vessels, where they produced to 500 pm were observed in broth medium, as flocks in the colonies which could not be removed by repeated extensive fluid producing a granular turbidity or attached to the bottom washing with PBS (pH 7.2). The ability to adhere to glass of the culture vessel. appeared within a few minutes (to the slide as well as to the The cells of strain 163K distinguish themselves from most cover slip). The adherence to erythrocytes and tissue cells other mycoplasmas by their morphology: they are conical or was confirmed in dark-field preparations and in the adsorp- fllask shaped and have a distinct terminal structure. This tion of erythrocytes and tissue cells to colonies of strain could even be recognized in the dark-field microscope. 163K. Two types of hemadsorption depending on the age Scanning and transmission electron microscopy confirmed and the density of the colonies were observed (Fig. 3): the VOL. 37, 1987 MYCOPLASMA MOBILE SP. NOV. 195

FIG. 2. Ultrathin sections of strain 163K showing diffuse material around the cells, which probably represents a slime layer (a, sl), a trilaminar cytoplasm membrane (a and b, m), and a cytoskeletal element in the headlike structure (c, c).

erythrocytes adhered to the whole colony or they adhered showed a partial sensitivity to sodium polyanethol-sulfonate. only to the peripheral part of the colony. Around the saturated disks was a zone of 2 to 10 mm Gliding motility. The most exciting property of strain 163K (depending on the concentration of the polyanethol- is its ability to glide on glass and plastic surfaces. The gliding sulfonate) where colonies were much smaller and less dense motion of strain 163K is unique among mycoplasmas be- than on the remaining areas of the plate. The growth cause of its speed, which is 2 to 4 pds on average and 7.5 response of strain 163K to the cholesterol content of the pm/s maximum under adequate culture conditions. Gliding medium is shown in Table 2. No growth was apparent in motion can be observed in a simple dark-field preparation, in medium without serum and in serum-free medium enriched which the organisms are gliding on the slide as well as on the with 0.05% bovine serum albumin (fraction V) and 10 pg of lower side of the coverslip. The organisms move in straight palmitic acid per ml (base medium). However, there was lines, in arcs, or in circles, with their headlike structure increasing multiplication when 1 to 20 pg of cholesterol per always orientated in the direction of the movement, and ml was added to the base medium. show a tendency to aggregate. They form gliding chains and Biochemical characteristics. The results of the biochemical also lay together with their sides in close contact during investigations of strain 163K are given in Table 3. Variable gliding. The gliding is not interrupted by resting periods. results were obtained with some tests. Potassium tellurite Subcultivation of the organisms for more than 50 passages reduction was positive in about half of the experiments only on artificial medium did not have any effect on the ability to and depended on the size of the colonies, the duration of glide or velocity of movement. incubation and the concentration of the potassium tellurite. Growth characteristics. Strain 163K grows under aerobic The color reaction demonstrating phosphatase activity was and anaerobic conditions. The growth characteristics under weak in comparison to the reference species M. arthritidis aerobic conditions were recorded by growth curves during and present in only 25% of the experiments. Gas production growth at different temperatures (4, 17, 25, 30, and 37°C). Figure 4 shows good growth of the organisms from 17 to 30°C with an optimum at 25°C. Weak growth occurred even at 4"C, but organisms died within 10 h at 37°C. At all temperatures enabling growth, the maximum cell number was reached between 40 and 50 h of incubation. The media used in the experiments did not have any decisive influence on the growth of strain 163K. Thus, there were no significant differences in growth curves obtained by cultivation in Hayflick medium (with either bovine or horse serum) or in modified Friis medium. Fiterability and reversion. Organisms of strain 163K passed through membrane filters of 450-nm pore size with a slight reduction in the number of cells (from lo9 down to lo6 to lo7 CFU/ml) and through filters of 220-nm pore size with a strong reduction in the number of organisms (from lo9 down to lo2 to lo3 CFU/ml). No evidence of a change to bacterial forms was apparent during 12 consecutive passages on medium devoid of inhibiting substances, whether agar colonies or broth preparations, examined microscopically. FIG. 3. Adsorption of bovine erythrocytes to colonies of strain Sterol requirement. Strain 163K was strongly sensitive to 163K. (a) Hemadsorption is limited to the peripheral part of the 1% (wt/vol) digitonin (inhibiting zone, 12 to 15 mm) and colony. (b) The whole colony is covered with erythrocytes. 1196 KIRCHHOFF ET AL. INT. J. SYST.BACTERIOL.

(19 CFU/ml

I7 ;= 1 0 u r c5 -0 0 0

3 D------o 37oc ...... 0 50 100 150 200 (h) 250 Incubation lime - FIG. 4. Growth curves of strain 163K at 4, 17, 25, and 37°C obtained in modified Hayflick medium containing 20% bovine serum. after addition of hydrogen peroxide, demonstrating catalase attach to inert surfaces, erythrocytes, and tissue cells, and activity, was weak and could be observed only with young its extraordinarily fast gliding motion. colonies. The reactions for oxidase activity were positive in In its morphology, strain 163K has some similarities to about 50% of the experiments. A strong film and spot other Mycoplasma species which have terminal structures: production were observed on the modified Hay flick medium M. pneumoniae (4, 18), M. pulmonis (16, 18), M. genitaliurn containing horse serum within 3 to 5 days of incubation, (18, 27), M. gallisepticum (2, 18), M. alvi (14, 18), M. sualvi whereas no film and spots were produced on media contain- (15, 18), and M. pirum (10). These terminal structures have ing bovine or swine serum. been given different designations (bleb, stalk, tip, and pro- Serology. Strain 163K was compared with all of the tuberance) (18). Since it is always pointed toward the direc- established Mycoplasma and Acholeplasma species (Table tion of the gliding movement and has the appearance of a I) by the IFT, GIT, and MIT. The tests showed that strain head, we chose to call the terminal structure of strain 163K 1163K is serologically unrelated to all previously described Mycoplasma and Acholeplasma species. DNA. The G+C contents were 32.7 mol% for the DNA of TABLE 3. Biochemical characteristics of strain 163K A. laidlawii, 27.2 mol% for the DNA of M. pulmonis, and Reac- Characteristic 23.5 mol% (range, 22.4 to 24.6) for the DNA of strain 163K. tion

~~ ~ ~~ Fermentation of carbohydrates (glucose, lactose, mannose, + DISCUSSION maltose, galactose, fructose, saccharose, and arabinose) under aerobic and anaerobic conditions ...... Strain 163K is distinguished by its differentiated terminal Hydrolysis of arginine...... - structure containing cytoskeletal elements, its ability to Hydrolysis of urea...... - Reduction of 2,3,5-triphenyltetrazoliumchloride under aero- TABLE 2. Growth dependence of strain 163K on cholesterol bic and anaerobic conditions...... + Reduction of potassium tellurite ...... -+ content of the culture medium Reduction of methylene blue...... - Protein Medium composition Phosphatase activity ...... t (mg)" Hydrolysis of gelatin...... - - Modified Hayflick medium containing 5% bovine serum.. . 4.18 Digestion of serum and casein ...... Modified Hayflick medium without serum ...... 0.016 Catalase activity...... -t Base mediumb...... 0.284 Oxidase activity ...... t Oxidation of gluconate - Base medium plus 1 pg of cholesterol per ml ...... 2.44 ...... Deamination of phenylalanine - Base medium plus 5 kg of cholesterol per ml ...... 3.32 ...... €lase medium plus 10 pg of cholesterol per ml ...... 3.47 Production of film and spots ...... + €lase medium plus 20 pg of cholesterol per ml ...... 3.82 Production of hydrogen peroxide ...... + Hemolysis (human, equine, bovine, ovine, swine, rabbit, a Final protein level in a pellet from 100 ml. guinea pig, and fish erythrocytes)...... + Base medium consisted of modified Hayflick medium without serum, Hemadsorption (human, equine, bovine, ovine, swine, rab- enriched with 0.5% bovine serum albumin (fraction V) and 10 pg of palmitic acid per ml. bit, guinea pig, and fish erythrocytes)...... + VOL. 37, 1987 MYCOPLASMA MOBILE SP. NOV. 197 a headlike structure (17, 18). Like M. pneumoniae, M. nia and its identification as a pleuropneumonia-like organism. gallisepticum, M. genitalium, and M. alvi, strain 163K Proc. Natl. Acad. Sci. USA 48:41-48. possesses cytoskeletal elements. It is very likely that these 8. Clyde, W. A. 1964. Mycoplasma species identification based elements are the basis for the formation of the terminal upon growth inhibition by specific antisera. J. Immunol. 92: 958-965. structure and for the gliding motility which could be demon- 9. Del Giudice, R. A., F. Robillard, and T. R. Carski. 1967. strated also for M. pneumoniae, M. gallisepticum, M. Immunofluorescence identification of mycoplasma on agar by pulmonis, and M. genitalium (5, 24). However, the velocity use of incident illumination. J. Bacteriol. 93:1205-1209. of the movement of these mycoplasmas is very low. The 10. Del Giudice, R. A., J. G. Tully, D. L. Rose, and R. M. Cole. average speeds are as follows: M. pulmonis, 0.4 to 0.7 km/s; 1985. Mycoplasma pirum sp. nov., a terminal structured M. pneumoniae, 0.3 to 0.4 km/s; M. genitalium, 0.1 p,m/s; mollicute from cell cultures. Int. J. Syst. Bacteriol. 35285-291 and M. gallisepticum, only 0.03 to 0.05 pm/s (5, 24). The 11. Flossdorf, J. 1983. A rapid method for the determination of the velocity of movement of strain 163K, which is 2 to 4 pm/s on base composition of bacterial DNA. J. Microbiol. Methods 1: average and 7.6 pm/s maximum, is 10 to 100 times faster. In 305-311. 12. Freundt, E. A., B. E. Andrews, H. Ern@,M. Kunze, and F. T. addition, the motion of strain 163K is not interrupted by Black. 1973. The sensitivity of Mycoplasmatales to sodium resting periods, as is the motion of the other gliding polyanethol-sulfonate and digitonin. Zentralbl. Bakteriol. Abt. 1 mycoplasmas, and is easy to observe in a simple dark-field Orig. Reihe A 225104-112. preparation without time-lapse exposures, heated chambers, 13. Friis, F. 1975. Some recommendations concerning primary or increased viscosity, which are required for the demon- isolation of Mycoplasma suipneumoniae and Mycoplasma stration of movement of the other gliding mycoplasmas. flocculare. Nord. Vet. Med. 27:337-339. The evaluation of the properties of these organisms iso- 14. Gourlay, R. N., S. C. Wyld, and R. H. Leach. 1977. Myco- lated from a tench fulfills the essential criteria for Mollicutes, plasma alvi, a new species from bovine intestinal and urogenital including typical fried-egg colony form, pleomorphic cell tracts. Int. J. Syst. Bacteriol. 27:89-96. 15. Gourlay, R. N., S. C. Wyld, and R. H. Leach. 1978. Myco- form, absence of cell wall, passage through 450- and 220-nm- plasma sualvi, a new species from the intestinal and urogenital pore filters, lack of reversion to bacteria, resistance to tracts of pigs. Int. J. Syst. Bacteriol. 28:289-292. penicillin, and a low G+C content. According to the sensi- 16. Hummeler, K., N. Tomassini, and L. Hayflick. 1965. Ultrastruc- tivity of the organism to digitonin and its cholesterol require- ture of mycoplasma (Negroni) isolated from human letlkemia. J. ment, it may be classified in the family Mycoplasmataceae, Bacteriol. 90517-523. although the cells multiply at temperatures from 17 to 30°C, 17. Kirchhoff, H., and R. Rosengarten. 1984. Isolation of a motile but not at 37°C. The inability to hydrolize urea indicates that mycoplasma from fish. J. Gen. Microbiol. 130:2439-2445. the organism does not belong to the genus Ureaplasma. 18. Kirchhoff, H., R. Rosengarten, W. Lotz, M. Fischer, and D. Strain was serologically distinct not only from other Lopatta. 1984. Flask-shaped mycoplasmas properties and 163K pathogenicity for man and animals. Isr. J. Med. Sci. 20:848-853. members of the genus Mycoplasma but also from the mem- 19. Luft, J. H. 1961. Improvements in epoxy resin embedding bers of the genus Acholeplasma. From these results it can be methods. J. Biophys. Biochem. Cytol. 9:409414. concluded that strain 163K represents a new species. For 20. Morton, H. E., and R. J. Roberts. 1966. Productiod of this species we propose the name Mycoplasma mobile (L. antimycoplasma (PPLO) antibodies in rabbits. Proc. SOC.Exp. adj, mobilis, motile; referring to the most striking property of Biol. Med. 125538-542. this organism). A cloned line of this strain is designated as 21. Reynolds, E. S. 1963. The use of lead citrate at high pH as an the type strain and has been deposited in the American Type electron-opaque stain in electron microscopy. J. Cell. Biol. 17: Culture Collection as strain ATCC 43663. 208-212. 22. Sobeslavsky, O., B. Prescott, and R. M. Chanock. 1968. Adsorp- tion of Mycoplasma pneumoniae to neuraminic acid receptors LITERATURE CITED of various cells and possible role in virulence. J. Bacteriol. 96: 695-705. 1. Albers, A. C., and R. D. Fletcher. 1982. Simple method for 23. Subcommittee on the Taxonomy of Mollicutes. 1979. Proposal of quantitation of viable mycoplasmas. Appl. Environ. Microbiol. minimal standards for description of new species of the class 43: 95 8-960. Mollicutes. Int. J. Syst. Bacteriol. 29:172-180. 2. Allen, T, C., J. 0. Stevens, E. R. Florance, and R. 0. Hampton. 24. Taylor-Robinson, D., and W. Bredt. 1983. Motility of Myco- 1970. Ultrastructure of Mycoplasma gallisepticum isolate 10.56. plasma strain G37. Yale J. Biol. Med. 56:91&911. J. Ultrastruct. Res. 33:31&331. 25. Taylor-Robinson, D., H. Purcell, D. C. Wong, and R. M. 3. Aluotto, B. B., R. G. Wittler, C. 0. Williams, and J. E. Faber. Chanock. 1966. A colour test for the measurement of antibody 70. Standardized bacteriologic techniques for the character- to certain Mycoplasma species based upon the inhibition of acid tion of Mycoplasma species. Int. J. Syst. Bacteriol. 20:35-58. production. J. Hyg. 64:91-104. 4. berfeld, G., and P. Biberfeld. 1970. Ultrastructural features of 26. Tully, J. G. 1983. 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