Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis

Abstract

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host-parasite relationship.

Bonamia in Ostrea angasi: Diagnostic performance, field prevalence and intensity

“…() found that heart smear had higher sensitivity than histology for B. exitiosa in New Zealand, and Lynch, Mulcahy, and Culloty () assessed heart smears as more sensitive than PCR or histology. PCR is usually regarded, however, as more sensitive than histology and/or heart smear (Carnegie, Barber, Culloty, Figueras, & Distel, ; Diggles et al., ; Marty et al., ; Michael, Forman, Hulston, Fu, & Maas, ; Ramilo, Navas, Villalba, & Abollo, ; Robert et al., ). Unrepresentative negative results will arise if DNA subsampled for qPCR does not contain target DNA (Lynch et al., ).…”

Moving from Histopathology to Molecular Tools in the Diagnosis of Molluscs Diseases of Concern under EU Legislation

“…For this reason, differentiation between species has been achieved using PCR-restriction fragment length polymorphism (PCR-RFLP) assays (Hine et al, 2001; Cochennec-Laureau et al, 2003; Abollo et al, 2008), sequencing the products obtained by PCR assays or by means of light microscopic techniques, thus causing the diagnostic process to be more expensive and slower. Recently species- specific quantitative real time PCR assays for B. ostreae have finally been developed targeting a region of the actin-1 gene (Robert et al, 2009) and the 18S-ITS 1 rDNA region (Ramilo et al, 2013). Other approaches has also been reported to face the problem of distinguishing among species based on DNA sensors.…”

“…The data thus generated is analyzed by computer software to detect and quantify the targeted DNA in samples to determine the presence and abundance of a particular pathogen. Different quantitative real time PCRs have already been described to detect molluscs pathogens (Corbeil et al, 2006b; Marty et al, 2006; Robert et al, 2009; Martenot et al, 2010; Umeda and Yoshinaga, 2012; Carrasco et al, 2013; Ramilo et al, 2013). There have also been attempts to develop multiplex PCRs to detect the presence of different molluscs pathogens in the same reaction in order to facilitate their diagnosis (Penna et al, 2001; Xie et al, 2010, 2013; Umeda and Yoshinaga, 2012).…”

Whole-genome amplification: a useful approach to characterize new genes in unculturable protozoan parasites such asBonamia exitiosa

“…detection. Following the criteria of Robert et al (2009), the most infected oysters were selected for parasite purification (Mialhe et al 1988). Bonamia exitiosa infection was confirmed by histological analysis performed on infected oysters sections.…”