Molecular detection of the oyster parasite Mikrocytos mackini, and a preliminary phylogenetic analysis

Abstract

The protistan parasite Mikrocytos mackini, the causative agent of Denman Island disease in the oyster Crassostrea gigas in British Columbia, Canada, is of wide concern because it can infect other oyster species and because its life cycle, mode of transmission, and origins are unknown. PCR and fluorescent in situ hybridization (FISH) assays were developed for M. mackini, the PCR assay was validated against standard histopathological diagnosis, and a preliminary phylogenetic analysis of the M. mackini small-subunit ribosomal RNA gene (SSU rDNA) was undertaken. A PCR designed specifically not to amplify host DNA generated a 544 bp SSU rDNA fragment from M. mackini-infected oysters and enriched M. mackini cell isolates, but not from uninfected control oysters. This fragment was confirmed by FISH to be M. mackini SSU rDNA. A M. mackini-specific PCR was then designed which detected 3 to 4 × more M. mackini infections in 1056 wild oysters from Denman Island, British Columbia, than standard histopathology. Mikrocytos mackini prevalence estimates based on both PCR and histopathology increased (PCR from 4.4 to 7.4%, histopathology from 1.2 to 2.1%) when gross lesions were processed in addition to standard samples (i.e. transverse sections for histopathology, left outer palp DNA for PCR). The use of histopathology and tissue imprints plus PCR, and standard samples plus observed gross lesions, represented a 'total evidence' approach that provided the most realistic estimates of the true prevalence of M. mackini. Maximum parsimony and evolutionary distance phylogenetic analyses suggested that M. mackini may be a basal eukaryote, although it is not closely related to other known protistan taxa. KEY WORDS: Denman Island disease · Mikrocytos mackini · PCR validation · Fluorescent in situ hybridization Resale or republication not permitted without written consent of the publisherDis Aquat Org 54: [219][220][221][222][223][224][225][226][227] 2003 (OIE 2000) because it is pathogenic to several oyster species and because its life cycle, mode of transmission, and origins are unknown.Mikrocytos mackini might be understood more thoroughly and managed more effectively if specific molecular diagnostic assays existed to complement conventional histopathological techniques. However, M. mackini small-subunit ribosomal DNA (SSU rDNA) eluded identification for a decade, presumably because 'universal' eukaryotic PCRs preferentially amplified the oyster DNA predominating in host-parasite mixtures, and because pure isolates of M. mackini are not available. We describe here: successful amplification of M. mackini 's SSU rDNA gene using a primer pair designed specifically not to amplify host DNA; proof of the identity of this gene using fluorescent in situ hybridization (FISH); design and validation of a PCR assay specific for M. mackini SSU rDNA; and a preliminary SSU rDNA phylogenetic examination of M. mackini 's affinities. MATERIALS AND METHODSAmplification of presumptive Mikrocytos mackini SSU rDNA. A rapid PCR cloning approa...

Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location

“…Deparaffinized 5 µm thick tissue sections were stained with Harris's modified hematoxylin and 0.5% alcoholic eosin (H&E) and also stained using a digoxigenin-labeled DNA probe in situ hybridization (DIG-ISH) technique . The probe was originally developed to be M. mackini specific (Carnegie et al 2003), but its sequence also matches perfectly with the 18S sequence of the Mikrocytos sp. found in the study area.…”

“…The tendency for outgroup taxa to form long branches creates a hazard towards misplacing long branched ingroup taxa as they are pulled towards the outgroup (Bergsten 2005). Although branch lengths are not shown in Carnegie et al (2003), inspection of 18S alignments clearly show M. mackini to be exceptionally divergent from other protistan taxa.…”

“…Among these are 2 genera of intracellular parasites called 'microcells', Bonamia and Mikrocytos, which have caused significant mortality to oyster populations around the world (Carnegie & Cochennec-Laureau 2004). Species of Bonamia are members of the eukaryotic phylum Haplosporidia (Carnegie et al 2000); however, multiple lines of evidence suggest against a close relationship between Mikrocytos and Bonamia (Carnegie et al 2003, Carnegie & Cochennec-Laureau 2004. The phylogenetic relationships of Mikrocytos are unknown.…”

“…The evolutionary history of Mikrocytos is entirely unknown. Phylogenetic analysis of 18S rDNA suggested that M. mackini is not closely related to any known protistan taxon and may be evolutionarily basal to other eukaryotes (Carnegie et al 2003). However, a long-branch attraction artifact may explain this basal positioning; it can cause taxa to group together due to high evolutionary rates and convergent evolution and/or unequal taxon sampling (see review by Bergsten 2005).…”

“…mentioned above crossreacts with the PCR reaction originally designed by Carnegie et al (2003) to be specific to M. mackini (Gagné et al 2008, Wang et al 2010. Although M. mackini was recently de-listed by the World Organisation for Animal Health's (OIE) Aquatic Animal Health Code, it continues to be subject to regulatory testing in Canada.…”