Development of a real time quantitative PCR assay for the hard clam pathogen Quahog Parasite Unknown (QPX)

Abstract

Quahog Parasite Unknown (QPX) is a thraustochytrid pathogen responsible for catastrophic mortalities of the northern quahog (hard clam) Mercenaria mercenaria. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to assist research efforts on QPX ecology and pathology. Sensitivity of the assay was evaluated with serial dilutions of QPX-cultured cells to determine the lowest concentration of DNA that remained detectable in both the presence and absence of extraneous environmental substances. QPX cells were quantified before DNA extraction to calibrate standard curves to cell counts. Based on our results, the qPCR assay is able to quantify QPX within the range of 1 to several thousand organisms per reaction. Specificity of the assay was assessed by testing 29 thraustochytrid-like protists isolated from suspension-feeding bivalves from China, Oregon, Maryland, and Virginia. Application of the assay was demonstrated with positive qPCR results from naturally contaminated environmental samples including marine aggregates (i.e. marine snow), clam pseudofeces, and inflammatory nodules from infected clams. This quantitative assay for QPX will provide a valuable tool for characterizing QPX parasite abundances in coastal environments and for improving clam disease diagnostics.KEY WORDS: Quahog Parasite Unknown · QPX · Thraustochytrids · Mercenaria mercenaria · Real-time PCR · Histology · Marine aggregates · Pseudofeces · Clams Resale or republication not permitted without written consent of the publisherDis Aquat Org 72: [45][46][47][48][49][50][51][52] 2006 recorded (Dove et al. 2004). These large-scale mortality events in both cultured and wild clam beds have devastated parts of the hard clam shellfishing industry. QPX remains a primary concern for regulators because there are several clam-growing areas within the range of QPX (i.e. Canada to Virginia) that have not experienced clam mortalities due to QPX.QPX is broadly classified with the thraustochytrids (Maas et al. 1999, Ragan et al. 2000, Stokes et al. 2002, a group of elusive marine protists common in coastal environments (Raghukumar 2002). They are facultative parasites recently detected embedded within marine aggregates (i.e. marine snow) collected from clam-growing coastal waters affected by QPX outbreaks (Lyons et al. 2005). In the environment, marine aggregates are aspirated through the inhalant siphon of the clam during normal feeding processes, and are thought to facilitate entry of this microscopic pathogen into its benthic host. Marine aggregates have been identified as a possible target for environmental surveillance of QPX (Lyons et al. 2005). However, ecological disease research is hindered because there is no quantitative detection technique to enumerate the number of QPX parasites in environmental samples.The primary diagnostic method for QPX infections in clams is histological analysis (Howard et al. 2004). This procedure cannot be used for environmental samples such as seawater, sediment, macrophytes, or marine a...

Quantitative Real-Time PCR Assay for QPX (Thraustochytriidae), a Parasite of the Hard Clam ( Mercenaria mercenaria )

“…This value is within the range reported for other heterokonts of similar sizes (19,31). With a conservative detection limit of 10 ITS region copies per reaction, our QPX qPCR assay can detect approximately 0.05 QPX cells per reaction, substantially lower than the 1 cell per reaction detection limit of the proteincoding gene qPCR assay for QPX developed by Lyons et al (15).…”

“…Thraustochytrids are abundant in coastal benthic habitats (21), and QPX has previously been detected in hard clam pseudofeces (15,16) and in sediment samples (11). The natural transmission mechanism of QPX disease is not yet known, but sediment, as the habitat of clams and a potential environmental reservoir for QPX, could play a role.…”

“…Better sensitivity might be achieved by collecting particulate matter on filters with a larger pore size than the 0.22-m-pore-size filters used here; for example, a 1-m-pore-size filter would still capture QPX cells (which typically range from 2 to 20 m in diameter [14]) while allowing larger volumes of water to be filtered and reducing the contribution of bacteria to the extracted DNA. Alternatively, if QPX is associated mainly with marine aggregates, where it has previously been detected (15,16), much larger particles might need to be collected from larger volumes of seawater in order to routinely detect QPX. The efficiency of QPX cell lysis may have been a factor limiting the recovery of QPX DNA.…”

Genus-specific quantitative PCR of thraustochytrid protists

“…qPCR-based quantification provides a highly sensitive and specific system for the identification of target organisms. This method is increasingly being used in marine microbiological studies, such as in the detection of dinoflagellates (Bowers et al 2000, Moorthi et al 2006, Yamashita et al 2011) and the thraustochytrid pathogen quahog parasite unknown (QPX) (Lyons et al 2006, Liu et al 2009). In a recent report, Bergmann et al (2011) developed a qPCR assay for detection of the labyrinthulid Labyrinthula zosterae (Labyrinthulomycetes), known as the causative agent of eelgrass wasting disease.…”

Development and Validation of a Real-Time Quantitative PCR Assay for the Detection and Quantification ofPerkinsus marinusin the Eastern Oyster,Crassostrea virginica

“…The presence of PCR inhibitors has long been a recognized issue in the use of PCR assays for the detection of parasites within samples (Audemard et al 2004, Audemard et al 2006, Lyons et al 2006. Organic and inorganic materials may be present in the sample, compromising the efficiency of DNA polymerases and ultimately producing a false negative.…”