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Clinical and Vaccine Immunology, September 2007, p. 1190-1195, Vol. 14, No. 9
1071-412X/07/$08.00+0     doi:10.1128/CVI.00108-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Department of Infectious Diseases and Pathology, College of Veterinary Medicine,¹ Department of Pathology, Immunology and Laboratory Medicine, College of Medicine,² Department of Statistics, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida³

Received 26 October 2006/ Returned for modification 12 April 2007/ Accepted 8 June 2007

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 A₄₀₅ 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.

Published ahead of print on 11 July 2007.