Development of a real time PCR for detection of the oyster pathogen Nocardia crassostrea based on its homogeneous 16S-23S rRNA intergenic spacer region
Carrasco, N. ; Roozenburg, I. ; Voorbergen-Laarman, H.A. ; Engelsma, M.Y. - \ 2013
Journal of Invertebrate Pathology 114 (2013)2. - ISSN 0022-2011 - p. 120 - 127.
pacific oyster - summer mortality - gigas thunberg - gene-sequences - bacteria - identification - france
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.
First isolation of Nocardia crassostreae from Pacific oysters (Crassostrea gigas) in Europe
Engelsma, M.Y. ; Roozenburg, I. ; Joly, J.P. - \ 2008
Diseases of Aquatic Organisms 80 (2008)3. - ISSN 0177-5103 - p. 229 - 234.
summer mortality - thunberg - bay
In summer 2006 an extensive mortality of Pacific oysters Crassostrea gigas occurred in Lake Grevelingen, the Netherlands. A sample of Pacific oysters was investigated for the presence of shellfish pathogens as potential causes of the mortality. Yellow-green lesions were observed in several oysters upon clinical inspection. Histopathology showed that 6 out of 36 oysters had a suspected bacterial infection, including 4 Nocardia-like infections. Two bacterial species, Vibrio aestuarianus and Nocardia crassostreae, were isolated from haemolymph samples and identified using PCR and sequencing of the 16S rRNA gene. This is the first isolation of N. crassostreae from shellfish in European waters. The near full-length 16S rRNA sequence of this Dutch Nocardia sp. isolate was identical to other known N. crassostreae isolates from the west coast of North America. The primary cause of oyster mortality was thought to be the physiological stress from environmental conditions, including prolonged high water temperatures and low oxygen levels. The multiple bacterial species isolated from the diseased Pacific oysters may have been a secondary cause.