Intranuclear coccidiosis in tortoises - discovery of its causative agent and transmission.

Intranuclear coccidiosis of testudines (known as TINC) is an emerging disease in chelonians. Although endogenous stages were repeatedly detected in various tissues, attempts to find the oocysts in faeces failed, leaving the question of the transmission and classification of the causative agent of TINC unresolved. We recorded small spherical oocysts (∼6-7 µm in diameter) of an eimeriid coccidium in faeces of a leopard tortoise (Stigmochelys pardalis). Sporulated oocysts were used for the experimental oral inoculation of juvenile coccidia-free tortoises representing 5 species (S. pardalis, Testudo graeca, T. hermanni, T. horsfieldii, and Geochelone sulcata). The oocysts' association with TINC was confirmed based on clinical signs, histopathological findings of intranuclear endogenous stages of the coccidium in many organs (including intestine), and by the partial 18S rDNA sequence analysis of the DNA isolated from organs of the experimentally infected animals and from a single naturally infected as well as from all experimentally infected tortoises. Breeding colonies of chelonians should be screened for this pathogen in order to prevent its further spread and unwanted introduction into endangered free-ranging chelonian populations.