Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica.

The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

Strong seasonality of Bonamia sp. infection and induced Crassostrea ariakensis mortality in Bogue and Masonboro Sounds, North Carolina, USA.

Asian oyster Crassostrea ariakensis is being considered for introduction to Atlantic coastal waters of the USA. Successful aquaculture of this species will depend partly on mitigating impacts by Bonamia sp., a parasite that has caused high C. ariakensis mortality south of Virginia. To better understand the biology of this parasite and identify strategies for management, we evaluated its seasonal pattern of infection in C. ariakensis at two North Carolina, USA, locations in 2005. Small (<50 mm) triploid C. ariakensis were deployed to upwellers on Bogue Sound in late spring (May), summer (July), early fall (September), late fall (November), and early winter (December) 2005; and two field sites on Masonboro Sound in September 2005. Oyster growth and mortality were evaluated biweekly at Bogue Sound, and weekly at Masonboro, with Bonamia sp. prevalence evaluated using parasite-specific PCR. We used histology to confirm infections in PCR-positive oysters. Bonamia sp. appeared in the late spring Bogue Sound deployment when temperatures approached 25 degrees C, six weeks post-deployment. Summer- and early fall-deployed oysters displayed Bonamia sp. infections after 3-4 weeks. Bonamia sp. prevalences were 75% in Bogue Sound, and 60% in Masonboro. While oyster mortality reached 100% in late spring and summer deployments, early fall deployments showed reduced (17-82%) mortality. Late fall and early winter deployments, made at temperatures <20 degrees C, developed no Bonamia sp. infections at all. Seasonal Bonamia sp. cycling, therefore, is influenced greatly by temperature. Avoiding peak seasonal Bonamia sp. activity will be essential for culturing C. ariakensis in Bonamia sp.-enzootic waters.