A polymerase chain reaction assay for the detection of genomic DNA of a Rickettsiales-like prokaryote associated with withering syndrome in California abalone

The 16S rDNA from a Rickettsiales-like procaryote (RLP) infecting postesoph ageal tissues of black abalone Haliotis cracherodii Leach exhibiting signs of withering syndrome (WS) was amplified, cloned, and sequenced. The 16S rD NA sequence for the RLP was similar to that of species found in the genera Ehrlichia, Anaplasma, and Wolbachia. A polymerase chain reaction (PCR) test was developed that specifically amplifies a 160 bp segment of the 16S rDNA from the RLP associated with WS. Positive reactions were obtained for all black abalone samples of digestive gland or postesophagus known to be infec ted with the RLP by microscopic examinations of stained tissue sections. Th e PCR worked equally well for infected tissues of black and red abalone H. rufescens Leach. There was no amplification of genomic DNA from four other microbial species isolated from cultures of intestinal flora of abalone or from abalone deemed free of the RLP by microscopic examinations. This PCR t est greatly increases the ability to detect the bacterium, because to date no means to grow the organism from marine invertebrates on synthetic media or in cell Lines have been developed. This PCR test should allow detection of the RLP before the onset of clinical signs of withering syndrome in cult ured or wild abalone stocks. Furthermore, the test may be useful in identif ying reservoirs or other related RLPs in other marine invertebrates.