Taxonomic Analysis of the Tortoise Mycoplasmas Mycoplasma agassizii and Mycoplasma testudinis by 16S rRNA Gene Sequence Comparison

Abstract

The nucleotide sequences of the 16s rRNA genes of two mycoplasmas, Mycoplusma agassizii (proposed sp. nov.) and Mycoplusma testudinis, isolated from tortoises were determined and used for taxonomic comparisons. Signature nucleotide sequence motifs and overall sequence similarities to other mollicutes positioned these mycoplasmas in the M. hyorhinis and M. pneumoniae phylogenetic groups, respectively. A third, previously unrecognized tortoise mycoplasma was detected by 16s rRNA gene amplification and sequence analysis and was positioned in the M. fernentans phylogenetic group. The 16s rRNA gene of Acholeplasma laidlawii was similarly detected in a tortoise isolate, showing that diverse mollicutes can share the same family of reptilian host.Two well-characterized mycoplasmal species have been isolated from tortoises: Mycoplasma agassizii (proposed sp. nov.), isolated from the upper respiratory tract of Gopherus agassizii and Gopherus polyphemus tortoises (4), and Mycoplasma testudinis, isolated from the cloaca but not the nasopharynx of a Testudo graeca tortoise (7). M. testudinis ferments glucose and grows readily in conventional mycoplasma media at 20 to 35°C. It is not a recognized tortoise pathogen. M. agassizii, a newly recognized species, ferments glucose and grows slowly (3 to 6 weeks for primary isolation) in SP4 broth (13) at an optimal temperature of 30°C. It is an etiological agent of a chronic upper respiratory tract disease of tortoises characterized by a nasal mucous discharge and degeneration of the airway epithelium (4). Upper respiratory tract disease is a significant health problem in captive and certain wild populations of tortoises (8). The causes and diagnosis of disease are important considerations in the management of legally protected populations of endangered tortoises.There was no cross-reaction of tortoise antimycoplasma antibodies with M. testudinis antigens in an M. agassizii-specific enzyme-linked immunosorbent assay (12). This suggested that, although they share the same family of reptilian host, the two mycoplasmas are not closely related. Taxonomies of mollicute species have been constructed recently on the basis of 16s rRNA gene nucleotide sequences, which reflect their phylogenetic relationships (6, 9, 15). We determined the 16s rRNA gene nucleotide sequences of M. agassizii and M. testudinis for comparison with previously published 16s rRNA sequences to position the tortoise mycoplasmas in the taxonomy of mollicutes. MATERIALS AND METHODSCulture. M. ugassizii PS6 was isolated from the nares of a G. ugussizii tortoise with upper respiratory tract disease (3). A pure culture was derived from a single colony. The M. testudinis strain used was ATCC 43263. Cells were cultured at 30°C in SP4 broth (13) ment Station, University of Florida, Gainesville.culture in the mid-log growth phase, as indicated by acidification of the medium, was the template for PCR amplification of the 16s rRNA gene. Amplification primers were complementary to terminal sequences (sense strand nucleotides[nt] 1...

Guidelines for the Field Evaluation of Desert Tortoise Health and Disease

“…Special precautions must be taken to prevent transmission of pathogens causing diseases such as mycoplasmosis Jacobson et al, 1995) within and between tortoise populations (Jacobson, , 1994aBerry, 1997b). The most likely sources of transmission of mycoplasmosis are direct contact, nasal exudate, and aerosols .…”

“…The tortoise was listed by the federal government as a threatened species under the Endangered Species Act (ESA) of 1973 (as amended) over approximately 30% of its geographic range in the arid southwestern USA and Mexico in 1990, because several populations were experiencing declines (Fish and Wildlife Service [FWS], 1994;Berry, 1997a). Two recently described diseases, upper respiratory tract disease (URTD) and cutaneous dyskeratosis, were associated with population declines in some areas Jacobson et al, 1995;Berry, 1997b). Upper respiratory tract disease is caused by Mycoplasma agassizii (Jacobson et al, 1991;Brown et al, 1994) and an as yet unnamed new mycoplasma organism (Brown et al, 1995).…”

“…Two recently described diseases, upper respiratory tract disease (URTD) and cutaneous dyskeratosis, were associated with population declines in some areas Jacobson et al, 1995;Berry, 1997b). Upper respiratory tract disease is caused by Mycoplasma agassizii (Jacobson et al, 1991;Brown et al, 1994) and an as yet unnamed new mycoplasma organism (Brown et al, 1995). An enzyme-linked immunosorbent assay (ELISA) test was developed to measure antibodies to Mycoplasma agassizii in tortoises .…”

“…An enzyme-linked immunosorbent assay (ELISA) test was developed to measure antibodies to Mycoplasma agassizii in tortoises . URTD and exposure to mycoplasma, as evidenced by positive ELISA tests and presence of mycoplasma in nasal secretions by cultures or polymerase chain reaction tests, have been documented in tortoises at multiple sites in the Mojave Desert (Jacobson et al, 1995;Dickinson et al, 1995;Homer et al, 1998;Brown et al, 1999). Upper respiratory tract disease is a transmissible disease, often subclinical and generally chronic Jacobson et al, 1995;Homer et al, 1998).…”

“…URTD and exposure to mycoplasma, as evidenced by positive ELISA tests and presence of mycoplasma in nasal secretions by cultures or polymerase chain reaction tests, have been documented in tortoises at multiple sites in the Mojave Desert (Jacobson et al, 1995;Dickinson et al, 1995;Homer et al, 1998;Brown et al, 1999). Upper respiratory tract disease is a transmissible disease, often subclinical and generally chronic Jacobson et al, 1995;Homer et al, 1998). Cutaneous dyskeratosis produces lesions on the shell and integument and is of unknown etiology , although environmental toxicants and nutritional deficiencies are suspected contributors (Homer et al, 1998).…”