Development of an In Vitro Clonal Culture and Characterization of the rRNA Gene Cluster of Perkinsus atlanticus, a Protistan Parasite of the Clam Tapes decussatus

Abstract

Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR-based species-specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5-kb in length including 5S, IGS, SSU, ITS1, 5.8S, ITS2, LSU, and an inter-cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3-100% identical to P. atlanticus sequences previously obtained from clam tissue (non-clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus "genus-specific" PCR-based assay complements the species-specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.

A Galectin of Unique Domain Organization from Hemocytes of the Eastern Oyster (Crassostrea virginica) Is a Receptor for the Protistan Parasite Perkinsus marinus

“…and Isochrysis sp., each species on alternate days). Oysters were confirmed as P. marinusfree by diagnostic PCR (24). Immune challenge, extraction of RNA and DNA, and cDNA synthesis Oysters were notched at the anterior side of the shell, close to the adductor muscle, allowed to acclimate for 3 days, and injected with 100 l of LPS (E. coli 0111:B4, 100 g/ml in artificial sea water (ASW)) into the adductor muscle sinus using a 30-gauge hypodermic needle.…”

Herbicides and Protozoan Parasite Growth Control: Implications for New Drug Development

“…Various in vitro cultures of the protozoan parasite of genus Perkinsus (Figure 1) have already been developed (Gauthier et al 1995;Robledo et al 2002;Casas et al 2008), and have the advantage of not requiring the presence of a host enhancing the capability of assessing the effects of drugs on parasite growth (Gauthier and Vasta 1994;Elandalloussi et al 2005;Leite et al 2008). The effects of a given drug can thus be determined by analyzing the survival of the parasites as a function of time in culture in the presence or absence of the drug to be tested ( Figure 2) and analyzing the data using specific software (Figure 3).…”

Perkinsus olseni detected in Vietnamese aquacultured reef clams Tridacna crocea imported to the USA, following a mortality event

“…DNA from frozen Tridacna crocea tissue (4 animals), fresh Crassostrea virginica tissue, and FTM cultures of C. virginica containing abundant hypnospores was subjected to PCR assays using published primers specific for Perkinsus marinus (forward: DERMO A1 5'-CACTTGTATTGTG AAGCACCC-3'; and reverse: DERMO D 5'-GTGACA TCTCCAAATGACC-3'), which were expected to yield a 304 base pair (bp) product (Penna et al 2001). PCR assays with the same tissue sets were also performed using published primers for amplification of partial inter-genic spacer (IGS) sequences (319 bp) of P. marinus, P. olseni and P. chesapeakii (= P. andrewsi) (forward: PER1 5'-TAGTACCCGCTCATY GTGG-3' and reverse: PER2 5'-TGCAATGCTTGC GAGCT-3') (Robledo et al 2002). PCR assays were performed with those tissue sets supplemented with an additional T. crocea using published primers for the NTS region (554 bp) of P. olseni (= P. atlanticus) (forward: P. olseni A 5'-ACCAGTCACAGGGCGTA AT-3'; and reverse: P. olseni B 5'-GTAGCGTGCTCT GATGATCACT-3').…”

“…All PCR reactions were performed in the Eppendorf Mastercycler gradient thermal cycler (Perkin-Elmer). Reactions containing the PER1 and PER2 primers were carried out using an initial denaturation step at 94°C for 4 min, followed by 35 cycles of 91°C for 1 min, 60°C for 1 min, then 72°C for 1 min, followed by final extension at 72°C for 7 min (Robledo et al 2002). Reactions containing the DERMO A1 and DERMO D primers were carried out using an initial denaturation step at 94°C for 12 min, followed by 35 cycles of 94°C for 45 s, 50°C for 1 min, then 72°C for 2.5 min, followed by final extension at 72°C for 7 min (Penna et al 2001).…”

“…Microscopically, the organisms in tissue are characterized by 5 to 15 µm spherical cells, with a prominent vacuole, an eccentric nucleus, and stimulate a host immune response characterized by hemocyte infiltration and a peripheral zone of eosinophilic material derived from hemocyte degranulation (Sagrista et al 1995, Montes et al 1997, Villalba et al 2004). Molecular diagnostic techniques are necessary to distinguish the many species and strains of Perkinsus, and development of these assays is an ongoing process (de la Herrán et al 2000, Penna et al 2001, Reece et al 2001, Robledo et al 2002, Brown et al 2004, Russell et al 2004, Burreson et al 2005, Park et al 2005. Molecular characterization involves comparison of nucleotide sequences of the internal transcribed spacer (ITS) region, large subunit (LSU), and nontranscribed spacer (NTS) region of the ribosomal RNA gene, and actin genes (Dungan & Reece 2006).…”