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1 Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA
2 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA
3 Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610, USA
4 Mycoplasma Section, National Institute of Allergy and Infectious Diseases, Frederick, MD 21702, USA
Correspondence
D. R. Brown
brownd{at}mail.vetmed.ufl.edu
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BH29T is AY366210.
Present address: 16400 Black Rock Road, Germantown, MD 20874, USA. 
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). Mycoplasma testudinis, a commensal mycoplasma first isolated from a captive tortoise in England (Hill, 1985), is easily distinguished from M. agassizii by restriction endonuclease analysis of the 16S rRNA gene (Brown et al., 1995) but has not been found in wild tortoises in North America. During routine screening of mycoplasmas isolated from nasal lavages of free-ranging desert tortoises with upper respiratory tract disease, the 16S rRNA gene sequence of isolate H3110 revealed the existence of a previously unrecognized species (Brown et al., 1995). Isolates cloned from single colonies obtained from seven tortoises appeared to be identical as determined by growth characteristics and biochemical tests. In this report, strain BH29T is fully characterized and compared with previously described mollicutes.
Descriptions of isolation procedures, host and culture medium formulation for primary isolation of the organism were presented earlier (Brown et al., 1994; Tully, 1995a). Seven triply cloned isolates of the organism (Tully, 1983), designated 86M, H3110, H3113, H3133, H3154, H3155 and BH29T, were selected for further characterization. Optimal growth in American Type Culture Collection (ATCC) medium 988 (SP4) broth occurred at 30 °C. The organisms grew slowly at 22 and 25 °C, but did not grow at 4, 34, 37 or 42 °C. The organisms grew on agar anaerobically at 30 °C but not at 34 °C. All isolates passed through 200 nm porosity membranes. Filtration (Tully, 1983) reduced the cell density of BH29T in the exponential phase of growth from 2·5x109 c.f.u. ml–1 in unfiltered broth to 1·7x109, 7·0x108 and 9·0x107 c.f.u. ml–1 in 800, 450 and 200 nm filtrates, respectively. Colonies had a typical fried-egg appearance when examined at x20 magnification after 1–2 weeks incubation on SP4 agar at 30 °C. Gram-stained cells were not visible by bright-field microscopy at x1000 magnification. No reversion to bacterial forms was detected during 10 passages of each isolate in broth without antibiotics. BH29T cells in the exponential phase of growth when viewed by transmission electron microscopy appeared as coccoid to pleomorphic forms, between 200 and 800 nm in size, surrounded by a trilaminar unit membrane. Some cells were observed to have a tip structure (Fig. 1). The 16S rRNA gene of isolate BH29T confirmed the sequence determined as previously described for isolate H3110 (GenBank/EMBL/DDBJ accession no. U19768; Brown et al., 1995).
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); hydrolysis of arginine, aesculin and urea (Aluotto et al., 1970); phosphatase (Bradbury, 1983); production of ‘film and spots' (Freundt, 1983); sterol requirement (Rose et al., 1993; Tully, 1995b). Appropriate control cultures of Acholeplasma laidlawii, M. agassizii, Mycoplasma alligatoris, Mycoplasma bovis, Mycoplasma hominis, Mycoplasma pulmonis and Ureaplasma urealyticum were obtained from the ATCC. All isolates fermented glucose, mannose, lactose and sucrose. They did not hydrolyse arginine, aesculin or urea, produce ‘film and spots' or display phosphatase activity. All isolates grew in SP4 broth containing 20 % (v/v) fetal bovine serum, but none grew in serum-free medium or in serum-free medium supplemented with 0·04 % polyoxyethylene sorbitan (Tween 80). In summary, there were no physical or metabolic features besides the 16S rRNA gene sequence that distinguished Mycoplasma testudineum sp. nov. from M. agassizii.
Growth inhibition tests (Clyde, 1983) were performed by using rabbit polyclonal antisera against BH29T and the following Mycoplasma strains: M. agalactiae PG2T, M. agassizii PS6T, M. agassizii Utah C, M. alvi IlsleyT, M. anatis 1340T, M. anseris 1219T, M. bovigenitalium PG11T, M. bovirhinis PG43T, M. bovis DonettaT, M. bovoculi M165/69T, M. buteonis Bb/T2gT, M. californicum ST-6T, M. canis PG14T, M. capricolum subsp. capricolum California kidT, M. capricolum subsp. capricolum GM13, M. capricolum subsp. capripneumoniae F38T, M. caviae G122T, M. cavipharyngis 117CT, M. citelli RG-2CT, M. collis 58BT, M. columborale MMP-4T, M. conjunctivae HRC581T, M. corogypsi BV1T, M. cottewii VIST, M. cricetuli CHT, M. crocodyli MP145T, M. cynos H831T, M. dispar 462/2T, M. edwardii PG24T, M. elephantis E42T, M. equigenitalium T37T, M. fastidiosum 4822T, M. feliminutum BenT, M. felis COT, M. fermentans PG18T, M. flocculare Ms42T, M. gallinaceum DDT, M. gallisepticum PG31T, M. gallopavonis WR1T, M. genitalium G37T, M. glycophilum 486T, M. hyopneumoniae JT, M. hyorhinis BTS7T, M. imitans 4229T, M. iowae 695T, M. iowae PPAV, M. leonicaptivi 3L2T, M. leopharyngis LL2T, M. lipofaciens R171T, M. moatsii MK405T, M. mobile 163KT, M. molare H542T, M. mustelae MX9T, M. mycoides subsp. mycoides PG1T, M. mycoides subsp. mycoides UM30847, M. mycoides subsp. capri PG3T, M. neurolyticum Type AT, M. ovipneumoniae Y98T, M. oxoniensis 128T, M. penetrans GTU-54-6A1T, M. pirum HRC 70-159T, M. pneumoniae FHT, M. pullorum CKKT, M. pulmonis PG34T, M. putrefaciens KS-1T, M. sturni UCMFT, M. sualvi Mayfield (clone B)T, M. synoviae WVU 1853T, M. testudinis 01008T, M. verecundum 107T, M. yeatsii GIHT. In addition, colonies of BH29T were probed with fluorescein-conjugated antisera to M. agassizii strains PS6T and Utah C in a direct epi-immunofluorescence test (Gardella et al., 1983). No growth inhibition by heterologous antisera was observed, and immunofluorescence tests clearly indicated that BH29T was distinct from M. agassizii. However, cross-reactivity was observed in an indirect ELISA of naturally infected tortoise plasma by utilizing M. agassizii PS6T and M. testudineum BH29T whole-cell lysates or lipid-associated membrane protein fractions as antigens (Schumacher et al., 1993).
The seven isolates described were obtained from desert tortoises with chronic upper respiratory tract disease characterized by rhinitis and conjunctivitis. The disease is common in some desert and gopher tortoise (Gopherus polyphemus) populations across North America, and is commonly observed in captive chelonians of many species. In an experimental inoculation study, three healthy adult seronegative (titres 1 : <10) gopher tortoises were inoculated with 108 c.f.u. M. testudineum isolate H3110 by instillation into the nares as described previously (Brown et al., 1994, 1999). Six control tortoises received sterile broth. The inoculated tortoises developed classic signs of upper respiratory tract disease and seroconverted within 8 weeks post-inoculation (Brown et al., 1999). Mycoplasma were re-isolated from nasal swabs following the onset of disease, and their identity as M. testudineum was confirmed by restriction endonuclease analysis of the PCR-amplified 16S rRNA gene (Brown et al., 1995). Control tortoises remained seronegative and free of mycoplasma at necropsy after 14 weeks. The Henle–Koch–Evans postulates were therefore fulfilled for M. testudineum as an aetiology of upper respiratory tract disease of tortoises (Evans, 1976). M. testudineum seems to have similar distribution, but lower prevalence than M. agassizii in desert and gopher tortoises across North America (our unpublished data).
The properties of strain BH29T described herein fulfil recently revised criteria (International Subcommittee on Systematic Bacteriology International Subcommittee on the Taxonomy of the Mollicutes, 1995) for new species descriptions in the class Mollicutes. Properties mandating assignment to this class include absence of a cell wall, filterability and penicillin resistance. The non-helical morphology of strain BH29T, optimum growth temperature of 30 °C, and its inability to hydrolyse urea place it within the order Mycoplasmatales, family Mycoplasmataceae. A sterol requirement and the presence of conserved sequences of the 16S rRNA gene indicate the organism belongs in the genus Mycoplasma. Finally, the lack of a serological relationship of strain BH29T to the type strains of recognized Mycoplasma species and a unique 16S rRNA gene sequence demonstrate its stature as a distinct species. Accordingly, we propose the designation of Mycoplasma testudineum for this organism, in consideration of the initial isolation from a desert tortoise.
Description of Mycoplasma testudineum sp. nov.
Mycoplasma testudineum (tes.tu.din'e.um. L. neut. adj. testudineum of or belonging to a tortoise).
Cells predominantly coccoid in shape, varying from 200 to 800 nm in diameter. Cells devoid of cell wall and surrounded only by cytoplasmic membrane. Non-helical and non-motile. Cells filterable through 200 nm membranes. Colonies on solid medium with 0·8 % agar exhibit typical fried-egg forms. Chemo-organotrophic. Acid produced from glucose. Does not hydrolyse arginine or urea. Serum or sterol required for sustained growth. Growth temperature range 22–30 °C, optimum 30 °C. Serologically distinct from all other recognized Mycoplasma species. First isolated from the upper respiratory tract of a free-ranging desert tortoise (Gopherus agassizii) in the Mojave Desert. Pathogenic for gopher tortoises (Gopherus polyphemus).
The type strain is BH29T (=ATCC 700618T=MCCM 03231T). Its 16S rRNA gene sequence is unique (GenBank/EMBL/DDBJ accession no. AY366210).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bradbury, J. M. (1983). Phosphatase activity. In Methods in Mycoplasmology, vol. 1, pp. 363–366. Edited by S. Razin & J. G. Tully. New York: Academic Press.
Brown, M. B., Schumacher, I. M., Klein, P. A., Harris, K., Correll, T. & Jacobson, E. R. (1994). Mycoplasma agassizii causes upper respiratory tract disease in the desert tortoise. Infect Immun 62, 4580–4586.
Brown, D. R., Crenshaw, B. C., McLaughlin, G. S., Schumacher, I. M., McKenna, C. E., Klein, P. A., Jacobson, E. R. & Brown, M. B. (1995). Taxonomic analysis of the tortoise mycoplasmas Mycoplasma agassizii and Mycoplasma testudinis by 16S rRNA gene sequence comparison. Int J Syst Bacteriol 45, 348–350.
Brown, M. B., McLaughlin, G. S., Klein, P. A., Crenshaw, B. C., Schumacher, I. M., Brown, D. R. & Jacobson, E. R. (1999). Upper respiratory tract disease in the gopher tortoise is caused by Mycoplasma agassizii. J Clin Microbiol 37, 2262–2269.
Clyde, W. A. (1983). Growth inhibition tests. In Methods in Mycoplasmology, vol. 1, pp. 405–410. Edited by S. Razin & J. G. Tully. New York: Academic Press.
Evans, A. S. (1976). Causation and disease: the Henle-Koch postulates revisited. Yale J Biol Med 49, 175–195.[Medline]
Freundt, E. A. (1983). Film and spot production. In Methods in Mycoplasmology, vol. 1, pp. 373–374. Edited by S. Razin & J. G. Tully. New York: Academic Press.
Gardella, R. S., Del Giudice, R. A. & Tully, J. G. (1983). Immunofluorescence. In Methods in Mycoplasmology, vol. 1, pp. 411–439. Edited by S. Razin & J. G. Tully. New York: Academic Press.
Hill, A. C. (1985). Mycoplasma testudinis, a new species isolated from a tortoise. Int J Syst Bacteriol 35, 489–492.
International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes (1995). Revised minimum standards for the description of new species of the class Mollicutes (division Tenericutes). Int J Syst Bacteriol 45, 605–612.
Razin, S. & Cirillo, V. P. (1983). Sugar fermentation. In Methods in Mycoplasmology, vol. 1, pp. 337–344. Edited by S. Razin & J. G. Tully. New York: Academic Press.
Rose, D. L., Tully, J. G., Bové, J. M. & Whitcomb, R. F. (1993). A test for measuring growth responses of mollicutes to serum and polyoxyethylene sorbitan. Int J Syst Bacteriol 43, 527–532.
Schumacher, I. M., Brown, M. B., Jacobson, E. R., Collins, B. R. & Klein, P. A. (1993). Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease. J Clin Microbiol 31, 1454–1460.
Tully, J. G. (1983). Cloning and filtration techniques for mycoplasmas. In Methods in Mycoplasmology, vol. 1, pp. 173–177. Edited by S. Razin & J. G. Tully. New York: Academic Press.
Tully, J. G. (1995a). Culture medium formulation for primary isolation and maintenance of mollicutes. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 1, pp. 33–39. Edited by S. Razin & J. G. Tully. San Diego, CA: Academic Press.
Tully, J. G. (1995b). Determination of cholesterol and polyoxyethylene sorbitan growth requirements of mollicutes. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 1, pp. 381–389. Edited by S. Razin & J. G. Tully. San Diego, CA: Academic Press.
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