TY - JOUR
T1 - Development of a real-time PCR for detection of the oyster pathogen Nocardia crassostreae based on its homogeneous 16S-23S rRNA intergenic spacer region
AU - Carrasco, Noèlia
AU - Roozenburg, Ineke
AU - Voorbergen-Laarman, Michal
AU - Itoh, Naoki
AU - Engelsma, Marc Y.
N1 - Funding Information:
The authors are grateful to Harry Heidekamp, Flip Tanis and Jos Hensen for their help during the surveys on Lake Grevelingen, as well as Rob Zwart, Ad Bartese and Ralph Kok for histological processing of the samples, and Yvonne Dijkstra and Frido de Zwart for providing the N. farcinica , N. asteroides and N. cyriacigeorgica isolates. Furthermore, the authors would like to acknowledge the helpful advice of Olga Haenen and Steven van Beurden during the study, and Gary Meyer is gratefully acknowledged for his suggestions for textual improvement of the manuscript. The work described in this study was made possible by a postdoctoral fellowship for Noèlia Carrasco from Beatriu de Pinós, Generalitat de Catalunya, Spain and was further supported by funding from the Dutch Ministry of Economic Affairs, Agriculture and Innovation. We would also like to thank Chris Rodgers for the English revision of the manuscript.
PY - 2013/10
Y1 - 2013/10
N2 - Nocardia crassostreae, the causative agent of Pacific oyster nocardiosis (PON), is a Gram-positive actinomycete bacterium associated with Pacific oyster (Crassostrea gigas) mortalities. Oysters infected with this bacterium have been reported previously from the west coast of North America and Japan. More recently, N. crassostreae was reported in oyster culture areas in the Netherlands. In this study, a sensitive real-time PCR for specific detection of N. crassostreae was developed, and the intra-species divergence of N. crassostreae from different geographical locations was studied. The 16S-23S rRNA intergenic spacer (ITS) region of N. crassostreae was sequenced for a number of infected oysters originating from the Netherlands, Japan and Canada. The sequence analyses showed an absence of genetic variation in the ITS region between N. crassostreae from different geographical locations. Based on these ITS sequences a species-specific and highly sensitive SYBR Green real-time PCR assay was developed to facilitate detection of N. crassostreae in oyster tissue. To evaluate this new detection tool for N. crassostreae a preliminary validation was carried out and real-time PCR results were compared with other detection methods (histology, conventional PCR and bacterial isolation) using field samples from Lake Grevelingen, the Netherlands. The genetic homogeneity in the ITS region between N. crassostreae from different geographical locations might be explained by the recent spread of the organism via the international trade in Pacific oysters for aquaculture purposes. However, the lack of genetic variation could also suggest that N. crassostreae is a genetically monomorphic species.
AB - Nocardia crassostreae, the causative agent of Pacific oyster nocardiosis (PON), is a Gram-positive actinomycete bacterium associated with Pacific oyster (Crassostrea gigas) mortalities. Oysters infected with this bacterium have been reported previously from the west coast of North America and Japan. More recently, N. crassostreae was reported in oyster culture areas in the Netherlands. In this study, a sensitive real-time PCR for specific detection of N. crassostreae was developed, and the intra-species divergence of N. crassostreae from different geographical locations was studied. The 16S-23S rRNA intergenic spacer (ITS) region of N. crassostreae was sequenced for a number of infected oysters originating from the Netherlands, Japan and Canada. The sequence analyses showed an absence of genetic variation in the ITS region between N. crassostreae from different geographical locations. Based on these ITS sequences a species-specific and highly sensitive SYBR Green real-time PCR assay was developed to facilitate detection of N. crassostreae in oyster tissue. To evaluate this new detection tool for N. crassostreae a preliminary validation was carried out and real-time PCR results were compared with other detection methods (histology, conventional PCR and bacterial isolation) using field samples from Lake Grevelingen, the Netherlands. The genetic homogeneity in the ITS region between N. crassostreae from different geographical locations might be explained by the recent spread of the organism via the international trade in Pacific oysters for aquaculture purposes. However, the lack of genetic variation could also suggest that N. crassostreae is a genetically monomorphic species.
KW - Crassostrea gigas
KW - ITS
KW - Nocardia crassostreae
KW - Ostrea edulis
KW - Phylogeny
KW - Real-time PCR
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U2 - 10.1016/j.jip.2013.07.002
DO - 10.1016/j.jip.2013.07.002
M3 - Article
C2 - 23876658
AN - SCOPUS:84880979203
VL - 114
SP - 120
EP - 127
JO - Journal of Invertebrate Pathology
JF - Journal of Invertebrate Pathology
SN - 0022-2011
IS - 2
ER -