The desert tortoise is listed by the United States government as a threatened species in part of its range. A major contributing factor in the decline of this animal has been the presence of an upper respiratory tract disease (URTD) which is characterized by a chronic disease which eventually leads to severe occlusion of the nares with viscous exudate and destruction of the respiratory epithelium. Electron microscopy of infected tissues demonstrated the presence of a mycoplasma-like organism attached to the respiratory surfaces. The mycoplasma was isolated and designated as a new species, with the proposed name Mycoplasma agassizii. The current study was designed to fulfill Koch's postulates and determine if M. agassizii was the etiologic agent of URTD. Clinically healthy animals with known antibody status were infused intranasally with pooled exudate (n = 8) from ill donor animals, with M. agassizii alone (n = 9) or in combination with PasteureUa testudinis (n = 8), with P. testudinis alone (n = 9), or with sterile broth (n = 12). The pooled exudate was culture positive for M. agassizii. Tortoises which received exudate or M. agassizii alone or in conjunction with P. testudinis were significantly more likely to develop clinical disease (P < 0.0004) than animals which received P. testudinis alone or the broth controls. Tortoises demonstrated a strong immune response to M. agassizii, and seroconversion was seen in all groups with clinical disease. M. agassizii was isolated from the upper respiratory tracts of clinically ill animals up to 6 months postinfection. On the basis of the results of these transmission studies, we conclude that M. agassizii is an etiologic agent of URTD in the desert tortoise.

Captive great egret (Ardea albus) nestlings were maintained as controls or were dosed with methylmercury chloride at low (0.5), and high doses (5 mg/kg, wet weight) in fish. Low dosed birds were given methylmercury at concentrations comparable to current exposure of wild birds in the Everglades (Florida, USA). When compared with controls, low dosed birds had lower packed cell volumes, dingy feathers, increased lymphocytic cuffing in a skin test, increased bone marrow cellularity, decreased bursal wall thickness, decreased thymic lobule size, fewer lymphoid aggregates in lung, increased perivascular edema in lung, and decreased phagocytized carbon in lung. High dosed birds became severely ataxic and had severe hematologic, neurologic, and histologic changes. The most severe lesions were in immune and nervous system tissues. By comparing responses in captive and wild birds, we found that sublethal effects of mercury were detected at lower levels in captive than in wild birds, probably due to the reduced sources of variation characteristic of the highly controlled laboratory study. Conversely, thresholds for more severe changes (death, disease) occurred at lower concentrations in wild birds than in captive birds, probably because wild birds were exposed to multiple stressors. Thus caution should be used in applying lowest observed effect levels between captive and wild studies.

Biochemical, serological and molecular genetic studies were performed on seven mycoplasma isolates that were recovered from the upper respiratory tract of clinically ill desert tortoises. The isolates were serologically related to each other but serologically distinct from previously described species. Unique mycoplasma species-specific 16S rRNA nucleotide sequences were found in the proposed type strain. The name Mycoplasma agassizii is proposed for these isolates. The type strain is PS6T (l ATCC 700616 T ) which caused upper respiratory tract disease (URTD) in experimentally infected tortoises.

Upper respiratory tract disease (URTD) has been observed in a number of tortoise species, including the desert tortoise (Gopherus agassizii) and the gopher tortoise (Gopherus polyphemus). Clinical signs of URTD in gopher tortoises are similar to those in desert tortoises and include serous, mucoid, or purulent discharge from the nares, excessive tearing to purulent ocular discharge, conjunctivitis, and edema of the eyelids and ocular glands. The objectives of the present study were to determine ifMycoplasma agassizii was an etiologic agent of URTD in the gopher tortoise and to determine the clinical course of the experimental infection in a dose-response infection study. Tortoises were inoculated intranasally with 0.5 ml (0.25 ml/nostril) of either sterile SP4 broth (control group; n = 10) or 108 color-changing units (CCU) (total dose) of M. agassizii 723 (experimental infection group;n = 9). M. agassizii caused clinical signs compatible with those observed in tortoises with natural infections. Clinical signs of URTD were evident in seven of nine experimentally infected tortoises by 4 weeks postinfection (p.i.) and in eight of nine experimentally infected tortoises by 8 weeks p.i. In the dose-response experiments, tortoises were inoculated intranasally with a low (101 CCU;n = 6), medium (103 CCU;n = 6), or high (105 CCU;n = 5) dose of M. agassizii 723 or with sterile SP4 broth (n = 10). At all time points p.i. in both experiments, M. agassizii could be isolated from the nares of at least 50% of the tortoises. All of the experimentally infected tortoises seroconverted, and levels of antibody were statistically higher in infected animals than in control animals for all time points of >4 weeks p.i. (P < 0.0001). Control tortoises in both experiments did not show clinical signs, did not seroconvert, and did not have detectableM. agassizii by either culture or PCR at any point in the study. Histological lesions were compatible with those observed in tortoises with natural infections. The numbers of M. agassizii 723 did not influence the clinical expression of URTD or the antibody response, suggesting that the strain chosen for these studies was highly virulent. On the basis of the results of the transmission studies, we conclude that M. agassizii is an etiologic agent of URTD in the gopher tortoise.

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...

Several factors have combined with an upper respiratory tract disease (URTD) to produce declines on some population numbers of desert tortoises (Gopherus agassizii) in the western USA. This study was designed to determine the seroepidemiology of URTD in a population of wild adult tortoises at the Desert Tortoise Research Natural Area (DTNA) study site in Kern County (California, USA). Prior to initiation of the study, there was a dramatic decline in the number of individuals in this population. At each individual time point, samples were obtained from 12 to 20 tortoises with radiotransmitters during winter, spring, summer, and fall from 1992 through 1995. During the course of the study, 35 animals were sampled at one or more times. Only 10 animals were available for consistent monitoring throughout the 4 yr period. Specific antibody (Ab) levels to Mycoplasma agassizii were determined for individual tortoises by an enzyme-linked immunosorbent assay (ELISA) test. Specific Ab levels were not influenced by the gender of the tortoise. Levels of Ab and distribution of ELISAϩ, ELISAϪ and suspect animals were not consistently affected by season within a single year or for a season among the study years. Significantly more tortoises presented with clinical signs in 1992 and 1995. The profile of ELISAϩ animals with clinical signs shifted from 5% (1992) to 42% (1995). In 1992, 52% of tortoises lacked clinical signs and were ELISAϪ. In 1995, this category accounted for only 19% of tortoises. Based on the results of this study, we conclude that URTD was present in this population as evidenced by the presence of ELISAϩ individual animals, and that the infectious agent is still present as evidenced by seroconversion of previously ELISAϪ animals during the course of the study. There is evidence to suggest that animals may remain ELISAϩ without showing overt disease, a clinical pattern consistent with the chronic nature of most mycoplasmal infections. Further, there are trends suggesting that the clinical expression of disease may be cyclical. Continued monitoring of this population could provide valuable information concerning the spread of URTD in wild tortoise populations.

Mycoplasma agassizii (proposed species novum) is the etiologic agent of an upper respiratory tract disease in the desert tortoise (Gopherus agassizii), which is threatened in most of its range. An enzyme-linked immunosorbent assay (ELISA) for the detection of M. agassizii-specific antibodies in desert tortoises was developed with a monoclonal antibody with specificity for desert tortoise immunoglobulin light chain. Plasma samples from one group of tortoises were tested immediately before and 1 month after challenge either with nasal exudate containing M. agassizii or with a purified preparation of M. agassizii. Plasma samples from a second group of known healthy and sick tortoises were also tested. In the first group, the ELISA detected seroconversion in individual tortoises following challenge with M. agassizii. In the second group, ELISA results were positively correlated with the health status of the tortoises, as determined by clinical and pathologic

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