The precarious status of desert (tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with 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 as antigens for ELISA and Western blot analyses. 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. Over the past two decades, disease has become an increasingly important issue for wildlife management. Disease surveillance is fundamental for disease prevention and control. Thus, the development of diagnostic assays will be critical to research and manage wildlife populations effectively. Diseases in free-ranging animal populations are often managed by isolation or culling, predominantly because treatment of individuals is impractical and vaccination programs can be instituted only in limited situations, if at all. As available habitat shrinks, translocation or relocation may be used as management tools, especially for at-risk species, and the use of diagnostic tools to minimize the risk of pathogen transmission will increase. It is critical that tests be appropriately validated and have quality control mechanisms established, as there may be adverse consequences for animals with positive diagnostic test results for infectious agents, as well as for na?ve animals that might be exposed to pathogens by the relocation of diseased animals. Mycoplasmal upper respiratory tract disease (URTD) (11, 13) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. Current diagnostic methods include culture, PCR, and enzyme-linked immunosorbent assay (ELISA) serology (9, 42, 43). Obtaining an adequate nasal flush or swab sample for culture and PCR is difficult, and both culture and PCR are significantly less sensitive when animals are not exhibiting overt clinical signs (9, 27, 42). The ELISA (43) has been extensively validated, using controlled experimental infection studies of both desert and gopher tortoises (total = 75) to establish sensitivity (0.983 to 0.985) and specificity (0.999 to 1 CP-673451 1) values. A sizeable serum bank from desert (= 4,830) FOS and gopher (= 1,124) tortoises was used to establish standard curves and quality control measures. Importantly, the presence of specific antibody has been highly correlated with the presence of histopathological lesions of the upper respiratory tract as well as the presence of clinical signs, especially CP-673451 a nasal discharge (8, 27, 36, 42). Recently it has been reported that the ELISA may misidentify true-negative tortoises as seropositive due to the presence of natural antibodies and that Western blot assays should be used as a confirmatory test for exposure (21). In that study, a single isolate of (PS6) was used as an antigen for immunoblotting. However, most mycoplasmas exhibit extensive intraspecies genotypic and phenotypic variability that can be manifested as antigenic variation in the context of immune recognition (2-5, 14, 17, 18, 22, 24, 30). This heterogeneity can confound analysis of mycoplasmal immunogen recognition CP-673451 when only a single mycoplasmal isolate is used as an antigen. Studies of swine, poultry, and ruminants (2-4, 18, 22, 24) document the necessity to use multiple strains of CP-673451 mycoplasmas as antigens in Western blots in order to avoid false negatives. Therefore, the objective of this study was to determine if strain variability impacted results obtained using Western blot analyses. Serum samples obtained from eight tortoises (gopher tortoise [antigen, (ii) be infected with as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of URTD were used to evaluate four distinct clinical isolates of as antigens for Western blot CP-673451 analyses. We also compared the reactivities of tortoise sera.