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Upper respiratory tract disease (URTD) in North American tortoises (Gopherus) has been the focus of numerous laboratory and field investigations, yet the prevalence and importance of this disease remains unclear across many tortoise populations. Furthermore, much research has been focused on understanding diagnostic biomarkers of two known agents of URTD, Mycoplasma agassizii and Mycoplasma testudineum, yet the reliability and importance of these diagnostic biomarkers across populations is unclear. Gopher Tortoises (Gopherus polyphemus) have experienced significant declines and are currently protected range wide. Geographically, Alabama represents an important connection for Gopher Tortoise populations between the core and periphery of this species’ distribution. Herein, we systematically sampled 197 Gopher Tortoises for URTD across seven sites in south-central and south-eastern Alabama. Plasma samples were assayed for antibodies to M. agassizii and M. testudineum; nasal lavage samples were assayed for the presence of viable pathogens as well as pathogen DNA. Lastly, animals were scored for the presence of external symptoms and nasal scarring consistent with URTD. External symptoms of URTD were present in G. polyphemus in all sites sampled in Alabama. There was no relationship between active symptoms of URTD and Mycoplasma antibodies, however the presence of URTD nasal scarring was positively related to M. agassizii antibodies (P = 0.032). For a single site that was sampled in three sequential years, seroprevalence to M. agassizii significantly varied among years (P < 0.0001). Mycoplasma agassizii DNA was isolated from four of the seven sites using quantitative PCR, yet none of the samples were culture positive for either of the pathogens. An analysis of disease status and condition indicated that there was a significant, positive relationship between the severity of URTD symptoms and relative body mass (P < 0.05). This study highlights the need for continued monitoring of disease in wild populations. Specifically, focus must be placed on identifying other likely pathogens and relevant biomarkers that may be important drivers of URTD in North American tortoises. Special consideration should be given to environmental contexts that may render wild populations more susceptible to disease. [ABSTRACT FROM AUTHOR]