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Filtrations were applied to separate vacuoles of Rickettsiales-like organism Candidatus Xenohaliotis californiensis. Vacuoles were visualized by staining with nucleic acid fluorochrome and their identity was confirmed by Laser Capture Microdissection and PCR. This methodology separates vacuoles and allows studies without the need of isolation in synthetic media or cell lines. [ABSTRACT FROM AUTHOR]

Candidatus Xenohaliotis californiensis (C Xc) is a Rickettsiales-like organism (RLO) that is considered the causal agent of withering syndrome (WS), a chronic and potentially fatal disease for several abalone species. C Xc can be infected by a bacteriophage (p C Xc), and both phage-infected and non-phage-infected C Xc can coexist with a presumable different species of RLO tentatively named stippled Rickettsiales-like organism (ST-RLO). Currently, the taxonomic affiliation of the ST-RLO is unknown, and no studies have been conducted to determine its presence in infected abalones in the Peninsula of Baja California, Mexico. To determine whether the ST-RLO is a histological variant of C Xc or a new species of RLO, we performed histology and species-specific in situ hybridization (ISH) for C Xc and p C Xc in red abalone, Haliotis rufescens , infected with C Xc and hyperparasitized by p C Xc, which also showed the presence of the ST-RLO. Additionally, a histological analysis of the presence of all of these RLOs from 2008 to 2019 from an aquaculture facility was performed. The results showed that all ST-RLOs were negative by ISH for C Xc and p C Xc, suggesting that the ST-RLO is a different species of bacterium and that it is not susceptible to infection by p C Xc. Histologically similar alterations to those produced by C Xc in the post esophagus were observed. The three RLOs regularly coexist in all the infected abalones, and there is a relatively constant proportion of each kind of RLO over time, suggesting that equilibrium is reached during coexistence; the largest percentage corresponded to C Xc non phage infected (81%), followed by C Xc infected by p C Xc (16%) and the ST-RLO (3%). In all cases, the ST-RLO infection remained at a subclinical level making it difficult to determine if it acts as pathogen or as a commensal. Therefore, the genetic characterization of the ST-RLO and pathogenicity studies are needed to further elucidate the role of this new RLO in the development of of WS in abalone species of North America. • Confirmation of the presence of an unidentified rickettsia in abalone. • This unidentified rickettsia produces similar damages to those attributed to Candidatus Xenohaliotis californiensis. • This rickettsia coexists with Candidatus Xenohaliotis californiensis non infected and infected by the bacteriophage pCXc. • The role of this rickettsia in the development of abalone Withering Syndrome is unknown. • This is the first study to perform ISH for pCXc and to confirm that the ST-RLO is not susceptible to infection by pCXc. [ABSTRACT FROM AUTHOR]

Formalin-fixed paraffin-embedded (FFPE) tissues stored in thousands of human and animal pathology laboratories around the globe represent mines of stored genetic information. In recent years, the use of FFPE tissues as a viable source of DNA for diverse genetic studies has attracted attention for interrogating microbiomes from this sample type. These studies have proven that 16S rRNA amplicon sequencing-based microbiome studies are possible from FFPE samples but present some particular challenges. In this review, we summarize all aspects of microbiome studies from FFPE tissues including the challenges associated with working highly degraded DNA, best practices for reducing environmental contamination, and we propose solutions to address these issues. Finally, we discuss how the combination of FFPE microbiome studies and Laser Capture Microdissection and/or Laser Microdissection could enable to determine the spatial heterogeneity underlying complex bacterial communities. [Display omitted] [ABSTRACT FROM AUTHOR]