Development of a TaqMan PCR assay for the detection of Bonamia species

Abstract

The development of molecular diagnostic assays with increased sensitivity compared with conventional histological techniques is highly desirable for effective management of bonamiosis in cultured oyster stocks and wild populations. A real-time TaqMan PCR assay was developed for the specific detection of Bonamia species in infected oyster tissues. The TaqMan assay was shown to be significantly more sensitive than histopathology. Although a real-time TaqMan PCR assay is comparable with conventional PCR in terms of sensitivity, it offers the advantages that it is a rapid test and has a very low risk of sample cross-contamination. Furthermore, it can be optimised to quantify the parasite load in samples. The assay detected Bonamia isolates from Australia, New Zealand, Europe, Canada, Chile and the USA and therefore demonstrated genus specificity as tested in this study. KEY WORDS: Bonamia spp. · Real-time TaqMan PCR assay · Oyster Resale or republication not permitted without written consent of the publisherDis Aquat Org 71: [75][76][77][78][79][80] 2006 known to harbour Bonamia sp., an endemic isolate that has caused mortalities in the states of Victoria, Tasmania and Western Australia (Hine 1996, CochennecLaureau et al. 2003, Diggles 2003. Other isolates of Bonamia have been reported from O. chilensis in Chile (Campalans et al. 2000), Crassostrea ariakensis in North Carolina (Burreson et al. 2004) and O. puelchana in Argentina (Kroeck & Montes 2005). Taxonomic relationships between these isolates and described species within the genus need to be established.Although recent developments of PCR-based assays have partially addressed the limitations of histology (Carnegie & Cochennec-Laureau 2004), real-time PCR (polymerase chain reaction) has the potential to provide rapid and quantitative results. In order to establish an effective diagnostic capability that will help prevent the introduction of exotic Bonamia spp. and to avoid the spread of enzootic isolates, the development of a molecular-based diagnostic assay that allows rapid, reliable and sensitive detection of Bonamia spp. is required. This report describes the development of a real-time PCR assay capable of detecting Bonamia isolates with greater sensitivity than currently available methods. MATERIALS AND METHODSIsolates of Bonamia spp. Wild flat oysters Ostrea angasi, used as a source of Bonamia sp.-infected tissue, were collected and fixed in 95% ethanol, during the course of a survey on the health and genetics of stocks in 5 estuaries on the south coast of New South Wales, Australia (Heasman et al. 2004). European flat oyster O. edulis tissues, infected with isolates of B. ostreae and fixed in 95% ethanol, were obtained from France (6 samples) and the Netherlands (2 samples). Four bluff oyster O. chilensis tissues, infected with B. exitiosa and fixed in 95% ethanol, were obtained from New Zealand. In addition, DNA prepared from Bonamia-infected oysters O. edulis (Canada and USA) and O. chilensis (Chile) was included in the study. Furthermore,...

Isolation of Tasmanian Rickettsia-like organism (RLO) from farmed salmonids: identification of multiple serotypes and confirmation of pathogenicity

“…After injection, fish were closely monitored, and any fish displaying overt signs of disease were removed from their tanks, euthanized by overdose of anaesthetic (AQUI-S) and necropsied. During necropsy, samples of the liver, kidney and spleen were aseptically removed and the gDNA extracted for qPCR (Corbeil et al 2003) as described above. Samples were also obtained using sterile loops from the liver, kidney and spleen for direct inoculation onto BCG (Mauel et al 2008), CHAB (Mikalsen et al 2008) and the selectivelynegative sheep blood agar plates.…”

“…Samples were also obtained using sterile loops from the liver, kidney and spleen for direct inoculation onto BCG (Mauel et al 2008), CHAB (Mikalsen et al 2008) and the selectivelynegative sheep blood agar plates. Plates were incubated at 20°C, and qPCR (Corbeil et al 2003) was used to verify the identity of TRLO-suspected growth. After the first TRLO isolate was obtained, the isolation process was simplified: pooled liver, kidney and spleen samples were screened by TRLO PCR (Corbeil et al 2003) as described above, and then a sterile-loop sample from the liver of TRLO PCR-positive fish was directly inoculated onto BCG agar and the plates were incubated at 22°C in air.…”

“…At the conclusion of the first experiment, a standard volume of cell culture supernatant was harvested from each flask. Genomic DNA was extracted from the supernatant using a QIAamp DNA mini kit (Qiagen) and the gDNA was used as template in a TaqMan ® realtime PCR assay (Corbeil et al 2003), as a proxy indicator of TRLO proliferation in the CHSE-214 cell line. The remaining cell culture supernatant was discarded, the cells were fixed in 10% neutral-buffered formalin and then May-Grünwald-Giemsa stained.…”

“…Fresh mortalities or moribund fish were selected from net pens, and samples of the liver, kidney and spleen were aseptically removed and placed in sterile specimen containers and kept on ice until required. Approximately 5 mm 3 of each organ were pooled from each fish, the genomic DNA (gDNA) was extracted using a QIAamp DNA mini kit (Qiagen), and the gDNA was amplified using the TaqMan ® real-time qPCR method described by Corbeil et al (2003). The remaining portions of tissues that tested positive for TRLO gDNA by PCR were homogenised in 15 ml of minimum essential medium (Invitrogen) containing 10% (v/v) foetal bovine serum (Invitrogen) using a Dounce homogeniser.…”

Detection and characterization of Bonamia ostreae in Ostrea edulis imported to China

“…Considering the importance of the commercial exchange of live oyster stocks around the world and the associated risk of spreading the disease from infected to non-infected areas, many detection techniques have been developed. These techniques include microscopic observation, conventional PCR combined with restriction fragment length polymorphism (RFLP) analysis, real-time quantitative PCR (qPCR), in situ hybridization or fluorescent in situ hybridization (FISH) and electrochemical genosensors (Berthe et al 1999, Carnegie et al 2000, 2003, Cochennec et al 2000, da Silva & Villalba 2004, Corbeil et al 2006, Narcisi et al 2011. Among these methods, FISH is a powerful complement to morphological and PCR-based detection methods.…”