Morphologic, Immunohistochemical, and Molecular Characterization of a Novel Lankesterella Protozoan in Two White's Tree Frogs (Litoria caerulea)

Abstract

Two White's tree frogs (Litoria caerulea) housed at a zoological park died after a short period of lethargy, weight loss, and edema. Detailed postmortem examinations were performed on both frogs, including bacterial cultures and complete histologic examinations. Intracytoplasmatic as well as free protozoan parasites were identified in multiple organs from both frogs. The parasites were identified within erythrocytes, leukocytes, endothelial cells, and hepatocytes. Immunohistochemistry demonstrated a cross-reaction with Toxoplasma gondii antisera. Parasite ultrastructural analysis was performed by transmission electron microscopy. The parasites demonstrated an apical complex containing a conoid, rhoptries, and micronemes, demonstrating it was a member of the phylum Apicomplexa. In addition, the parasites had bipolar paranuclear bodies, organelles that are typical of coccidian sporozoites. The organisms were tentatively identified as members of the genus Lankesterella on the basis of histologic and ultrastructural morphology. A portion of the 18s ribosomal RNA (rRNA) gene was amplified via a polymerase chain reaction, sequenced, and used in a Basic Local Alignment Search Tool search of the GenBank database. The 18s rRNA gene sequence was found to be most similar to gene sequences isolated from Lankesterella organisms (88%). In aggregate, these data support the classification of these protozoa as a novel species of Lankesterella. A causal relationship between frog morbidity and protozoal parasitism was not determined. This is the first report of Lankesterella sp. in White's tree frogs.