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

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 cul...

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Bibliographic Details
Published in:The Journal of eukaryotic microbiology 2002-09, Vol.49 (5), p.414-422
Main Authors: ROBLEDO, JOSÉ A. F, NUNES, PATRÍCIA A, CANCELA, M. LEONOR, VASTA, GERARDO R
Format: Article
Language:English
Subjects:
Animal aquaculture
Animal productions
Animals
Apicomplexa - classification
Apicomplexa - genetics
Apicomplexa - growth & development
Aquaculture
Base Sequence
Biochemistry. Physiology. Immunology. Molecular biology
Biological and medical sciences
bivalves
Bivalvia - parasitology
clam farming
diagnostic PCR
DNA, Protozoan - analysis
DNA, Ribosomal Spacer - analysis
Fundamental and applied biological sciences. Psychology
Genes, rRNA
IGS
Invertebrate aquaculture
ITS
LSU
Molecular Sequence Data
Mollusca
Multigene Family
NTS
Polymerase Chain Reaction
Protozoa
RNA, Ribosomal - genetics
Sequence Analysis, DNA
Species Specificity
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Summary: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.
ISSN:1066-5234
1550-7408
DOI:10.1111/j.1550-7408.2002.tb00221.x