![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Journal of Clinical Microbiology, December 2004, p. 5909-5912, Vol. 42, No. 12
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.12.5909-5912.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington,1 Center for Reproduction of Endangered Species, The Zoological Society of San Diego, San Diego, California,2 The Peregrine Fund, Boise, Idaho3
Received 7 October 2003/ Returned for modification 10 July 2004/ Accepted 31 July 2004
 |
ABSTRACT |
|---|
 Top Abstract TextReferences |
|---|
|
TEXT |
|---|
|
TopAbstractTextReferences |
|---|
Culture and biochemical characterization Gb-V33 was initially detected in primary chicken embryo fibroblasts (CEFs) on the third passage of a pool of lung and spleen samples from vulture 33, in which it caused detectable cytopathology characterized by pronounced vacuolation and occasional syncytium formation at approximately 6 days postinfection. The CEF cells were prepared as previously described (4) and maintained in Eagle's minimal essential medium supplemented with 10% (vol/vol) fetal bovine serum, 2 mM L-glutamine, 100 U of penicillin ml–1, 0.1 mg of streptomycin ml–1, and 2.5 µg of amphotericin B ml–1. To maintain and passage Gb-V33, it was harvested by mechanically scraping free infected cell monolayers, collecting aliquots of pooled supernatants and cells, and freezing them at –80°C until needed. In subsequent passages cytopathology was detectable at approximately 3 to 5 days postinfection.
Gb-V33 was initially classified as a bacterium based on biochemical and physical properties. The presence of a DNA genome was demonstrated by sensitivity to 5-bromo-2'-deoxyuridine, an inhibitor of DNA replication. Tenfold serial dilutions of Gb-V33 were made in media with and without 50 µg of 5-bromo-2'-deoxyuridine ml–1, inoculated onto confluent CEFs in 24-well plates, and observed daily for cytopathology. Gb-V33 was also shown to be sensitive to tetracycline in a similar fashion by using medium with tetracycline hydrochloride at a final concentration of 10 µg ml–1. Both treatments decreased the infectivity of the stock cultures by at least 1 log10 order of magnitude (data not shown). The size of Gb-V33 was shown to be between 0.2 and 0.45 µm based on the ability of 0.2 (but not 0.45)-µm-diameter-pore filters to remove infectivity from an infectious stock of organism. However, no bacterial growth was detected with standard aerobic or microaerophilic cultures by using nonselective agars and broths. Cell-free cultivation for mycoplasmas by using pleuropneumonialike organism broth and agar (Remel TI no. 20360-A and 20260-A; Remel Inc., Lenexa, Kans.) and SP4-glucose broth and agar (Remel TI no. 20376 and 20276) did not detect growth after 15 and 45 days of incubation, respectively.
Ultrastructural features Ultrastructural studies of Gb-V33 were performed on infected CEF cell monolayers fixed for 4 h with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, followed by rinsing with 0.1 M cacodylate buffer in 3.5% sucrose. The fixed cell pellet was embedded in Epon resin and thin sectioned for transmission electron microscopy. Cells infected with Gb-V33 revealed organisms within intracytoplasmic vacuoles (Fig. 1A). The salient ultrastructural features of Gb-V33 were a nonnucleated organism that lacked a cell wall, a coccoid to oval shape, and an approximate diameter of 0.5 µm. These features were characteristic of mycoplasmas (7, 8). Another feature was the organization of electron-dense granules, presumably ribosomes, on round or oval membrane-bound structures approximately 0.1 to 0.3 µm in diameter (Fig. 1B), resulting in structures similar in appearance to the rough endoplasmic reticulum in the cytoplasm of the CEFs. One or two of these structures per organism were found in approximately half of the organisms observed. Some Gb-V33 organisms demonstrated what appeared to be a membrane-bound structure that was compressed between the organism and the wall of the vacuole (Fig. 1B).
|
Comparison of a 1,429-nucleotide segment of the 16S rDNA sequence to the GenBank database (National Center for Biotechnology Information) with BLASTN.2.2.3 software (1) classified Gb-V33 as a mycoplasma. The sequence of Gb-V33 did not completely match any available sequences but was most closely related to mycoplasmas in the Mycoplasma neurolyticum cluster of the Mycoplasma hominis group (9) with an identity of approximately 90%. Similarity analysis (AlignX program, Vector NTI 8; Informax, Inc., Frederick, Md.) showed that Gb-V33 had 87% similarity to Mycoplasma collis, Mycoplasma cricetuli, and Mycoplasma hyorhinis and 86% similarity to Mycoplasma cloacale, Mycoplasma falconis, Mycoplasma lagogenitalium, Mycoplasma molare, and M. neurolyticum. Gb-V33 also had 86% similarity to Mycoplasma gypis, a mycoplasma that has been isolated from griffon vultures (Gyps fulvus) in Europe (15, 16). In contrast, Gb-V33 was only 73 to 84% similar to other pathogenic mycoplasmas of birds, including Mycoplasma gallisepticum, Mycoplasma meleagridis, and Mycoplasma sturni.
Sequence alignment was performed with the CLUSTAL W (1.82) multiple sequence alignment program (European Bioinformatics Institute). The 1,429-nucleotide sequence of Gb-V33 was aligned with the GenBank sequences of 37 other species of mycoplasmas representing all of the major groups and clusters within the genus Mycoplasma (9, 15), and phylogenetic analysis was performed using 1,000 replicates of bootstrapping and 100 replicates of DNA parsimony with the PHYLIP package (5). A phylogenetic tree was constructed using strict majority rule consensus trees. The resulting tree (Fig. 2) had the same topology as previously published phylogenetic estimates based on 16S rDNA sequences (9, 15) and demonstrated that Gb-V33 is a member of the M. neurolyticum cluster in the M. hominis group and that it was also quite distinct from M. gypis and other avian mycoplasmas.
|
The PCR assay directly detected Gb-V33 in both lung and spleen, but not kidney, tissue of the original case (vulture 33). The amplicon was confirmed as identical to Gb-V33 by direct sequencing (data not shown), further indicating that Gb-V33 was not a cell culture contaminant. In addition, detection in the spleen suggested that Gb-V33 was causing a systemic infection. The strongest PCR amplification was from the lung (data not shown), indicating that this would be the optimal tissue to test in other vultures. Testing by PCR of DNA extracted from the lungs of 37 additional vultures indicated infection in eight of these birds and a crude estimated prevalence of 
24%. Histopathology of the other infected vultures did not detect lesions consistent with mycoplasmosis in the respiratory tract or other tissues.
Conclusions
Based on biochemical, ultrastructural, and genetic characteristics, we have identified Gb-V33 as a new mycoplasma taxon that infects Oriental white-backed vultures, and we propose that it be named "Mycoplasma vulturii." Preliminary PCR testing indicates a prevalence of 24% in the wild population of Oriental white-backed vultures. However, the data do not indicate that "M. vulturii" is an obligate pathogen. Although mild inflammation was noted in the respiratory tract of vulture 33, this vulture was ultimately shown to have died due to diclofenac poisoning (13). Respiratory or other lesions consistent with mycoplasmosis were not evident in the other infected birds examined (13). The requirement for nested PCR methods to consistently detect "M. vulturii" indicates that it was present in very low numbers in the infected birds, also suggesting that these infections were subclinical. Consequently, it is most likely that "M. vulturii" is a commensal organism of Oriental white-backed vultures and possibly an opportunistic pathogen.
"M. vulturii" is propagated in primary CEF cultures in which it causes vacuolating cytopathology and is present intracellularly. Intracellular replication has also been noted for other mycoplasmas including M. gallisepticum, Mycoplasma genitalium, Mycoplasma penetrans, and Mycoplasma pneumoniae (3, 11, 19). Initial attempts to cultivate "M. vulturii" on cell-free media were unsuccessful. Some mycoplasmas, such as the recently reclassified hemotrophic mycoplasmas formerly in the genera Haemobartonella and Eperythrozoon, appear to be obligate cellular parasites that cannot be propagated on cell-free media (12). Others, such as some strains of M. gallisepticum and Mycoplasma synoviae, appear to be uncultivable with present cell-free media (2). For "M. vulturii," more comprehensive attempts at cell-free cultivation will be required to determine if it is truly an obligate intracellular organism. The internal structures noted ultrastructurally in "M. vulturii" are unusual. Internal vacuoles have been described for M. genitalium, but no known function has been ascribed to them (8). The membrane-bound structures compressed between the organism and the wall of the host cell vacuole were another unusual feature of "M. vulturii." One possible explanation for this structure is that the organisms were elongated and folded over upon themselves. However, longitudinal sections showing elongated forms were not observed in the sections examined. Alternatively, this compressed structure could be a form of the electron-dense layer that has been previously described for the intracellular forms of Mycoplasma fermentans (11), a "nap" structure that has been described for M. genitalium (18), or part of the attachment organelle that has been described for M. pneumoniae (10) and the hemotrophic mycoplasmas (12).
Nucleotide sequence accession number The GenBank accession number for the 16S rDNA gene sequence of Gb-V33 is AY191226. Gb-V33 has also been deposited with the American Type Culture Collection (ATCC BAA-882).
|
ACKNOWLEDGMENTS |
|---|
This work was supported in part by The Peregrine Fund's Asian Vulture Crisis Project with grants from the Gordon and Betty Moore Foundation; the Disney Wildlife Conservation Fund; the Zoological Society of San Diego; and the United Nations, Ivorybill, and Summit Foundations.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractTextReferences |
|---|
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Antimicrob. Agents Chemother. | Clin. Microbiol. Rev. |
|---|---|
| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
|---|
