Detalle de: Hypnospores detected by RFTM in clam (Ruditapes decussatus) tissues belong to two different protozoan organisms, Perkinsus atlanticus and a Perkinsus-like organism

The development of hypnospores (large cells that stain dark blue with Lugol's iodine) after incubation of bivalves tissues in Ray's fluid thioglycollate medium (RFTM) has been used for Perkinsus diagnosis for the last 40 years. Polymerase chain reaction (PCR) was performed using conserved 18S rRNA gene primers on DNA from an enriched preparation of hypnospores obtained from carpet shell clam (Ruditapes decussatus) tissues incubated in RFTM. The restriction analysis of the cloned 18S rRNA gene PCR fragments showed different band profiles. The DNA sequencing analysis of the PCR products confirmed that they corresponded to Perkinsus atlanticus and Pseudoperkinsus tapetis, morphologically very similar to Perkinsus spp. All the cloned fragments of the 18S rRNA gene obtained from the in vitro cultured P. tapetis gave only one restriction profile, leading us to conclude that the culture was axenic containing only this organism. Hypnospores, obtained from clam tissues incubated in RFTM belonged at least, to these two morphologically similar although molecularly different organisms. The lack of specificity of RFTM makes its use questionable for the diagnosis of Perkinsus in Galicia and maybe in other parts of the world.