DAO 104:149-161 (2013)  -  DOI: https://doi.org/10.3354/dao02597

Species-specific diagnostic assays for Bonamia ostreae and B. exitiosa in European flat oyster Ostrea edulis: conventional, real-time and multiplex PCR

Andrea Ramilo1, J. Ignacio Navas2, Antonio Villalba1,*, Elvira Abollo

1Centro de Investigacións Mariñas, Consellería do Medio Rural e do Mar, Xunta de Galicia, 36620 Vilanova de Arousa, Spain
2Instituto de Investigación y Formación Agraria y Pesquera (IFAPA), Centro ‘Agua del Pino’, Junta de Andalucía, 21450 Cartaya, Spain
3Centro Tecnológico del Mar – Fundación CETMAR, 36208 Vigo, Spain
*Corresponding author. Email:

ABSTRACT: Bonamia ostreae and B. exitiosa have caused mass mortalities of various oyster species around the world and co-occur in some European areas. The World Organisation for Animal Health (OIE) has included infections with both species in the list of notifiable diseases. However, official methods for species-specific diagnosis of either parasite have certain limitations. In this study, new species-specific conventional PCR (cPCR) and real-time PCR techniques were developed to diagnose each parasite species. Moreover, a multiplex PCR method was designed to detect both parasites in a single assay. The analytical sensitivity and specificity of each new method were evaluated. These new procedures were compared with 2 OIE-recommended methods, viz. standard histology and PCR-RFLP. The new procedures showed higher sensitivity than the OIE recommended ones for the diagnosis of both species. The sensitivity of tests with the new primers was higher using oyster gills and gonad tissue, rather than gills alone. The lack of a ‘gold standard’ prevented accurate estimation of sensitivity and specificity of the new methods. The implementation of statistical tools (maximum likelihood method) for the comparison of the diagnostic tests showed the possibility of false positives with the new procedures, although the absence of a gold standard precluded certainty. Nevertheless, all procedures showed negative results when used for the analysis of oysters from a Bonamia-free area.


KEY WORDS: Bonamiosis · Bonamia exitiosa · Ostrea edulis · Conventional PCR · Real-time PCR · Multiplex PCR · Bivalve


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Cite this article as: Ramilo A, Navas JI, Villalba A, Abollo E (2013) Species-specific diagnostic assays for Bonamia ostreae and B. exitiosa in European flat oyster Ostrea edulis: conventional, real-time and multiplex PCR. Dis Aquat Org 104:149-161. https://doi.org/10.3354/dao02597

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