Molecular Characterization of Coccidia Associated with an Epizootic in Green Sea Turtles (Chelonia mydas) in South East Queensland, Australia

Abstract

In the spring of 2014, mass mortalities among wild green sea turtles occurred off the coast of south-east Queensland, Australia. The suspected causative agent was Caryospora cheloniae, an eimeriid coccidian implicated in previous epizootics. Necropsies were undertaken on a subset of 11 dead turtles, with subsequent histopathology and molecular analyses. All turtles returned positive PCR results for coccidial infection in various tissues; these included the brain, gastrointestinal tract, lung, kidney and thyroid. Granulomatous encephalitis was consistently observed, as well as enteritis and, less frequently, thyroiditis and nephritis. Sequencing and phylogenetic analyses indicated the presence of two distinct coccidian genotypes, presumably separate species-one associated with the brain, gastrointestinal tract and lung, and the second with the thyroid and kidney. Maximum likelihood and Bayesian inference analyses placed the first genotype closest to the lankesterellid genus Schellackia, rather than in the Eimeriidae, while the second was paraphyletic to the eimeriids. Presence of coccidial stages in extra-intestinal tissues of the primary host raises questions about the potential presence of intermediate or paratenic hosts within the life cycles, as well as their current placement relative to the genus Caryospora. This study represents the first genetic characterization of this emerging disease agent in green sea turtles, an endangered species, and has relevance for life-cycle elucidation and future development of diagnostics.

Fig 1. Maximum likelihood analysis of 18S partial sequences from coccidia of green sea turtles relative to a range of eimeriid, lankesterellid and cyst-forming coccidians.

Numbers at each node represent bootstrap support values expressed as a percentage rounded to the nearest whole number. Scale-bar indicates the number of nucleotide substitutions per site. Species names are preceded by Genbank accession numbers, and definitive hosts follow in parentheses. Clade 1 indicates the clade containing coccidians from Eimeriidae and Lankesterellidae, while Clade 2 contains the families Sarcocystidae, Barrouxiidae and Calyptosporiidae.

Fig 2. Bayesian inference analysis of 18S partial sequences from coccidia of green sea turtles relative to a range of eimeriid, lankesterellid and cyst-forming coccidians.

Numbers at each node represent posterior probability values as a percentage rounded to the nearest whole number. Scale-bar indicates the number of nucleotide substitutions per site. Species names are preceded by Genbank accession numbers, and definitive hosts follow in parentheses. Clade 1 indicates the clade containing coccidians from Eimeriidae and Lankesterellidae, while Clade 2 contains the families Sarcocystidae, Barrouxiidae and Calyptosporiidae.

Fig 3. Histological views of green sea turtle coccidia.

a) Typical meront arrangement (arrow) in the brain of a green turtle (56168) HE, bar = 65μ; b) granuloma (g) associated with meronts (arrow) in the brain (56168). HE, bar = 260μ; c) Aggregation of merozites (arrow) in the thyroid (56168). HE, bar = 175μ; d) meronts (black arrows) and macrogametes (white arrows) within the intestinal epithelium (56143). HE, bar = 65μ. Abbreviations: HE = Haematoxylin and eosin