OPTIMIZATION OF THE PROCEDURE TO SEPARATE MEMBRANE BOUND VACUOLES OF Candidatus Xenohaliotis californiensis FROM HOST ABALONE TISSUE

Candidatus Xenohaliotis californiensis (CXc) is an obligated intracellular bacterial pathogen commonly found in Haliotis spp. form the west coast of North America and is the etiological agent of Withering Syndrome (WS) a chronic progressive wasting disease. This bacterium replicates within membrane bound vacuoles (MBVs) within the gastrointestinal epitheliums. The liberation of apparently intact MBVs from the host cells is generally observed in histological slides of red abalone (Haliotis rufescens) blue abalone (Haliotis fulgens) and yellow abalone (Haliotis corrugata). Recently we have developed a methodology for separation of the MBVs and modifications to this methodology have allowed detailed TEM analysis.

Separation of the MBVs was conducted from infected red abalone posterior esophagus (PE). The PE was excised, macerated and was passed though progressively smaller Falcon® Cell strainers (100, 70 and 40 μm) and handmade nylon cell strainers (28 and 10 μm).

Modification to this methodology consist of replacing the handmade 28 and 10 μm filters with 20 and 15 μm pluriStrainer® to retain a higher number of MBVs. Additionally filter content was fixed for TEM analysis.

The MBVs were effectively separated from host tissue and were visualized on the filters by staining with nucleic acid fluorochrome DAPI (Figure 1). The identity of the MBVs was confirmed by Laser Capture Microdissection and PCR. These results confirm a resistant nature of the MBVs formed by CXc and also suggest that the MBVs could maintain their integrity once liberated from the host cells. These findings also support that infection could occur without need a vector as is common for other bacteria of the order Rickettsiales. The methodology for separating the MBVs of CXc is simple, fast and straight forward and could further contribute to determining its taxonomic position and to the understanding of the biological cycle of this parasite. TEM analysis of filter content revealed the taxonomic position of the hyperphage.