Characterization of an intracellular bacterium infecting the digestive gland of the South African abalone Haliotis midae

A Rickettsiales-like prokaryote was observed in digestive gland epithelium of farmed South African abalone Haliotis midae sampled for routine surveillance. Histological examination showed basophilic inclusions typical of rickettsial infections in shellfish (Mouton, 2000). This study aimed to identify the Rickettsiales-like prokaryote (RLP) colonising H. midae. Total genomic DNA was extracted from infected digestive gland sampled from cultured H. midae. PCR performed with ". Candidatus Xenohaliotis californiensis" specific primers RA 5-1 and RA 3-6 did not yield positive results, indicating that the H. midae RLP is not the withering syndrome agent. The 16S rRNA gene sequence of the intracellular bacterium was determined following nested PCR using universal eubacterial primers fD1/Rp2 and the broad spectrum primers EHR16SD/EHR16SR which are specific for members of the Anaplasmataceae family within the Rickettsiales order. Phylogenetic and evolutionary distance analysis showed that the H. midae RLP is a unique member of the α-proteobacteria. The identity and location of the histologically observed intracellular bacterium was confirmed by in situ hybridization performed on sequential sections of infected H. midae digestive gland using a combination of three digoxigenin-labelled probes specific to various regions of the 16S rRNA gene of the RLP. Statement of relevanceThe manuscript shows that farmed abalone, showing no disease symptoms, can remain healthy despite the presence of intracellular bacteria colonising their digestive glands. The study shows that Rickettsiales-like prokaryotes detected in farmed abalone are not necessarily ". Candidatus Xenohaliotis californiensis", the causative agent of withering syndrome.