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 c aliforniensis' is a Rickettsiales-like prokaryote responsible for withering syndrome, a fatal disease of wild and farmed Eastern Pacific abalone, Hali otis 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 californi ensis' infections in abalone gastrointestinal tissue using the nucleic acid -specific fluorochrome Hoechst 33258. In excised tissue pieces dried onto s lides, rehydrated in the Hoechst stain and viewed with ultraviolet light, t he large bacterial inclusions were strongly fluorescent and could be easily distinguished from smaller host cell nuclei, This provided a rapid, inexpe nsive alternative to paraffin section microscopy or molecular techniques, a llowing detection of the pathogen within minutes of tissue excision. Compar ison of the fluorochrome method with conventional histological analysis for the ability to detect inclusions in 109 samples was 90 % accurate, with di screpancies due to false negative diagnosis of low-level infections, An alt ernative nucleic acid-specific fluorochrome, propidium iodide, showed a sta ining pattern identical to that of Hoechst 33258. These methods should prov e useful for the rapid detection of inclusion-forming Rickettsiales-like pr okaryotes in tissues from many host species.