Identification of molecular markers for the diagnostic identification of the intracellular prokaryote associated with the appearance of withering syndrome in the abalone Haliotis midae
Withering syndrome is a severe disease of abalone, Haliotis species that has been associated with mortality ranging from 99% in black, H. cracherodii Leach and 30% in red abalone, H. rufescens Swainson. The disease was first observed in California, along the west coast of North America and is an economically important disease that has led to the closure of the black abalone fishery throughout the southern California State. The causative agent of withering syndrome is a gram-negative intracellular Rickettsiales-like prokaryote designated Candidatus xenohaliotis californiensis. The geographical range of C. xenohaliotis californiensis is broad, besides red and black abalone it has also been reported in yellow, H. corrugate and blue abalone, H. fulgens all caught in Baja California, Mexico. In 2000 a Rickettsiales-like prokaryote resembling the disease-causing agent was observed in the digestive gland of the South African abalone H. midae. In this study we aimed to determine the relationship of the bacterium observed in the local abalone species, H. midae to the disease-causing agent of withering syndrome. A specific PCR and in situ hybridization test using primers and probes specific for the C. xenohaliotis californiesis 16S rDNA gene were used to screen H. midae digestive gland tissues showing classical features of the Rickettsiales-like prokaryote. Both analyses indicated that C. xenohaliotis californiensis is not present in the local abalone species. We therefore aimed the identification of the organism parasiting the local abalone species by DNA sequence analysis of the 16S rDNA gene. The 16S rDNA gene was amplified by PCR, cloned and sequenced. Phylogenetic trees, constructed by maximum parsimony analysis revealed a diverse community comprised of α - Proteobacteria, Mollicutes and Spirochaetes. In the class α - Proteobacteria a novel group of sequences showing phylogenetic affinities to the order Rickettsiales was identified as likely candidate for forming the Rickettsiales-like inclusions in the digestive gland of H. midae. Oligonucleotide probes that bind to four variable regions of the novel group were used to confirm their presence in infected H. midae digestive gland tissue by in situ hybridization. Although these probes did not recognize the inclusions formed by the Rickettsiales-like organisms, they revealed the presence of a group of free-living bacteria abundant in the host tissue. We therefore conclude that (1) C.xenohaliotis californiensis is not present in the South- African abalone, H. midae; (2) the organisms isolated from the digestive gland of H. midae are part of the normal microflora and (3) the group of sequences showing phylogenetic affinities to the order Rickettsiales is not responsible for the Rickettsiales-like inclusions in infected digestive gland tissues but represent a novel group of organisms that are abundant in the host tissue.