First molecular detection of Mycoplasma agassizii in captive tortoises in Portugal

The pathogen DNA was detected in 66.7% (52/78) of individuals. Phylogenetic analysis confirmed species identification, with all sequences forming a strongly supported monophyletic cluster together with <i>M. agassizii</i> reference sequences. A significant association was observed between tortoise genus and <i>M. agassizii</i> infection (<i>p</i> = 0.021), with <i>Chelonoidis</i> exhibiting a significantly lower infection frequency than Testudo (<i>p</i> = 0.029). No statistically significant associations were observed regarding geographic region, housing origin, or group size. These results reveal a high frequency of <i>M. agassizii</i> infection in apparently healthy captive tortoises in Portugal, emphasizing its potential for silent transmission in group or mixed-species settings. Our findings support the inclusion of this pathogen in the differential diagnosis of respiratory disease in tortoises, even when clinical signs are absent and underscore the need for routine molecular surveillance and strengthened biosecurity practices to mitigate transmission risks and foster chelonian conservation efforts.