Detection of Mycoplasma agassizii in the Texas Tortoise (Gopherus berlandieri)

Forty-five Horsfield’s tortoises ( Testudo horsfieldii; syn. Agrionemys horfieldii, Russian tortoise) belonging to different owners had decreased appetite and respiratory issues. Twenty-nine tortoises had epiphora, dyspnea, and white necrotic diphtheroid oral plaques (group G1). Ten of the remaining 16 tortoises had serious dehydration, appetite disorder, and depression (G2). The last 6 tortoises had only decreased appetite and moderate conjunctival discharge (G3). During the physical examination of all 45 tortoises, a cytologic sample and an oral swab for herpesvirus and Mycoplasma agassizii PCR testing were taken. In 20 of 29 specimens from G1, in 8 of 16 from G2, and 0 of 6 from G3, the cytologic exam revealed intranuclear acidophilic inclusion bodies, multinucleate cellular syncytia, and further abnormalities caused by herpesviral infection. Moreover, all 45 tested subjects were found to be positive for testudinid herpesvirus 1; 2 were positive for M. agassizii. This prospective study suggests that Horsfield’s tortoises with such signs would benefit from this screening procedure, given that it was effective in a significant proportion of infected and symptomatic animals, and no negative effects were seen.

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Mycoplasma agassizii Strain Variation and Distinct Host Antibody Responses Explain Differences between Enzyme-Linked Immunosorbent Assays and Western Blot Assays

Clinical and Vaccine Immunology ◽

10.1128/cvi.00215-10 ◽

2010 ◽

Vol 17 (11) ◽

pp. 1739-1745 ◽

Cited By ~ 9

Author(s):

Lori D. Wendland ◽

Paul A. Klein ◽

Elliott R. Jacobson ◽

Mary B. Brown

Keyword(s):

Western Blot ◽

Goodness Of Fit ◽

False Negative ◽

Well Being ◽

Management Decision ◽

Enzyme Linked Immunosorbent Assay ◽

Upper Respiratory Tract ◽

Strain Variation ◽

Serum Samples ◽

Mycoplasma Agassizii

ABSTRACT The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A 405 values were significantly correlated (r 2 goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations.

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Improved Enzyme-Linked Immunosorbent Assay To Reveal Mycoplasma agassizii Exposure: a Valuable Tool in the Management of Environmentally Sensitive Tortoise Populations

Clinical and Vaccine Immunology ◽

10.1128/cvi.00108-07 ◽

2007 ◽

Vol 14 (9) ◽

pp. 1190-1195 ◽

Cited By ~ 31

Author(s):

Lori D. Wendland ◽

Laurie A. Zacher ◽

Paul A. Klein ◽

Daniel R. Brown ◽

Dina Demcovitz ◽

...

Keyword(s):

Immunosorbent Assay ◽

Wildlife Conservation ◽

Control Measures ◽

Youden Index ◽

Management Decision ◽

Enzyme Linked Immunosorbent Assay ◽

Upper Respiratory Tract ◽

Serum Samples ◽

Assay Sensitivity ◽

Mycoplasma Agassizii

ABSTRACT The precarious status of desert (Gopherus agassizii) and gopher (Gopherus polyphemus) tortoises has resulted in research and conservation efforts that include health assessments as a substantial component of management decision-making. Therefore, it is critical that available diagnostic tests for diseases impacting these species undergo rigorous standardization and validation. Since 1992, analysis of exposure of tortoises to Mycoplasma agassizii, an etiological agent of upper respiratory tract disease, has relied on the detection of specific M. agassizii antibody by enzyme-linked immunosorbent assay (ELISA). We report here substantive refinements in the diagnostic assay and discuss the implications of its use in wildlife conservation and management. The ELISA has been refined to include more stringent quality control measures and has been converted to a clinically more meaningful titer reporting system, consistent with other diagnostic serologic tests. The ELISA results for 5,954 desert and gopher tortoises were plotted, and a subset of these serum samples (n = 90) was used to determine end-point titers, to establish an optimum serum dilution for analyzing samples, and to construct a standard curve. The relationship between titer and A 405 was validated using 77 serum samples from known positive (n = 48) and negative (n = 29) control tortoises from prior transmission studies. The Youden index, J, and the optimal cut point, c, were estimated using ELISA results from the 77 control sera. Based on this evaluation, the refinement has substantially improved the performance of the assay (sensitivity of 0.98, specificity of 0.99, and J of 0.98), thus providing a clinically more reliable diagnostic test for this important infection of tortoises.

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