Detection of 'Candidatus Xenohaliotis californiensis' (Rickettsiales-like prokaryote) inclusions in tissue squashes of abalone (Haliotis spp.) gastrointestinal epithelium using a nucleic acid fluorochrome

Rickettsiales-like prokaryotes appear to be etiologic agents of a number of newly described diseases of fish and shellfish. 'Candidatus Xenohaliotis californiensis' is a Rickettsiales-like prokaryote responsible for withering syndrome, a fatal disease of wild and farmed Eastern Pacific abalone, Haliotis spp. The bacterium proliferates in gastrointestinal epithelial cells, forming large intracytoplasmic inclusions. We describe a method of rapidly detecting and assessing the intensity of 'Candidatus Xenohaliotis californiensis' infections in abalone gastrointestinal tissue using the nucleic acid-specific fluorochrome Hoechst 33258. In excised tissue pieces dried onto slides, rehydrated in the Hoechst stain and viewed with ultraviolet light, the large bacterial inclusions were strongly fluorescent and could be easily distinguished from smaller host cell nuclei, This provided a rapid, inexpensive alternative to paraffin section microscopy or molecular techniques, allowing detection of the pathogen within minutes of tissue excision. Comparison of the fluorochrome method with conventional histological analysis for the ability to detect inclusions in 109 samples was 90 % accurate, with discrepancies due to false negative diagnosis of low-level infections, An alternative nucleic acid-specific fluorochrome, propidium iodide, showed a staining pattern identical to that of Hoechst 33258. These methods should prove useful for the rapid detection of inclusion-forming Rickettsiales-like prokaryotes in tissues from many host species.