Occurrence of the haemocyte parasite Bonamia sp. in flat oysters Ostrea puelchana farmed in San Antonio Bay (Argentina)

The flat oyster Ostrea edulis L. is widespread along the Italian coasts. In particular, the Manfredonia Gulf (Adriatic Sea) represents an important site where natural beds subsist. Previous monitoring conducted in 1990 by light microscopy and ultrastructural studies revealed the presence of Bonamia-like microcell parasites in some flat oysters: following this observation, a new sampling of O. edulis was carried out at this location in 2007. Of 750 oysters collected, 3 showed the presence of uninucleated microcells (2 to 3 µm diameter) free or inside the haemocyte cytoplasm by cytology and histopathology. Molecular analysis confirmed that the microcells in 2 oysters were B. exitiosa, whereas in the third oyster the microcells were B. ostreae. Moreover, molecular studies were carried out to confirm the existence of Bonamia sp. in archived samples, confirming the presence of B. ostreae in the Manfredonia Gulf since 1990. KEY WORDS: Bonamia spp. · Ostrea edulis · Molecular analysis · Electron microscopy Resale or republication not permitted without written consent of the publisherDis Aquat Org 89: [79][80][81][82][83][84][85] 2010 Bonamia exitiosa infects Ostrea chilensis in New Zealand (Hine et al. 2001) and O. angasi in Australia (Corbeil et al. 2006) and has been recently reported in O. edulis in Spain (Abollo et al. 2008). B. roughleyi, previously called Mikrocytos roughleyi, infects Saccostrea glomerata in southeast Australia (Farley et al. 1988). B. perspora is a newly described protozoan species found in Ostreola equestris (North Carolina, USA) and represents the first Bonamia species producing a typical haplosporidian spore (Carnegie et al. 2006). Other B. exitiosa-like organisms have been described in O. chilensis from Chile (Kern 1993, Campalans et al. 2000, O. puelchana from Argentina (Kroeck & Montes 2005), and Crassostrea ariakensis from North Carolina ); these organisms have been subsequently declared B. exitiosa (Lopez-Flores et al. 2007).Previous studies have reported by microscopic and ultrastrustural means the occurrence in Italy of small parasites similar to Bonamia sp. in flat oysters collected from the southern (Tiscar et al. 1991) and northern Adriatic Sea (Tiscar et al. 2002). A small number of molluscs appeared infected by the parasites (1/161 from the southern and 8/600 from the northern Adriatic Sea, respectively). Nevertheless all the infected oysters showed high infection level of the haemocytes with the presence of numerous protozoans per cell (Tiscar et al. 1991(Tiscar et al. , 2002.Taking into account these previous reports, the aim of the present study was to investigate by cytology and histology the occurrence of microcell parasites in flat oysters Ostrea edulis newly collected from natural beds in the southern Adriatic Sea and to characterize the recovered parasites by molecular and ultrastructural analyses as well as to confirm and characterize the presence of Bonamia sp. in one positive sample collected during 1990 from the Manfredonia Gulf (Tiscar et al. 1991). MAT...

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Detection of Bonamia ostreae and B. exitiosa (Haplosporidia) in Ostrea edulis from the Adriatic Sea (Italy)

Narcisi¹,

Arzul²,

Cargini³

et al. 2010

“…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.…”

Section: Resale or Republication Not Permitted Without Written Consenmentioning

confidence: 96%

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

Corbeil

Arzul²,

Diggles³

et al. 2006

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,...

“…This is particularly true since recent reports of Bonamia sp. Zealand and Australia have significantly broadened the recognised geographical distribution of this parasite (Campalans et al 2000, Burreson et al 2004, Kroeck & Montes 2005. With regards to those recent isolates from Chile, the USA and Argentina, it is believed that the sequence data provided in this paper will help to clarify taxonomic relationships within the genus Bonamia and, more particularly, the species B. exitiosa.…”

Section: Resultsmentioning

confidence: 97%

“…Subsequent molecular characterisation and sequencing of the small subunit (ssu) rRNA operon of Bonamia species confirmed the broad northern hemisphere distribution of B. ostreae (Carnegie et al 2000, Cochennec et al 2000. Conversely, since its redesignation as B. exitiosa , the exact distribution and geographical range of this antipodean Bonamia is unclear (Hine 1996, Campalans et al 2000, Burreson et al 2004, Kroeck & Montes 2005, but this information is of central importance to better address management of disease caused by this serious pathogen (OIE 2003). Sequence comparison of the 18S gene and Internal Transcribed Spacer 1 (ITS-1) region of the ssu rRNA operon has helped clarify the taxonomic position of these intra-haemocytic parasites and has led to the development of diagnostic methods capable of detecting and distinguishing different isolates -resources essential for the development of health surveillance, disease monitoring and oyster stock translocation programmes .…”

Section: Introductionmentioning

confidence: 99%

Molecular characterisation of an Australian isolate of Bonamia exitiosa

Corbeil¹,

Arzul²,

Robert³

et al. 2006

An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by sequencing the 18S-ITS-1 region of the small subunit rRNA operon obtained from flat oysters Ostrea angasi shown to be infected by histological examination. Sequence data alignment with homologous genes from 2 other isolates of Bonamia (New Zealand and France) revealed high levels of nucleotide identity with both isolates. However, the Australian Bonamia is shown to be more closely related to the New Zealand isolate, suggesting the existence of an oceanic subgroup of Bonamia.KEY WORDS: Bonamia species · Ostrea angasi · Flat oyster · Small subunit rRNA operon Resale or republication not permitted without written consent of the publisherDis Aquat Org 71: [81][82][83][84][85] 2006 nec- Laureau et al. 2003). A recent epidemiological study of bonamiosis carried out in southern New South Wales (NSW), Australia (Heasman et al. 2004), has allowed us to identify and characterise a Bonamia isolate at the molecular level and to establish its relatedness to other described Bonamia species. MATERIALS AND METHODSAustralian isolate of Bonamia sp. Ethanol (70%)-fixed, Bonamia-infected flat oysters Ostrea angasi, collected from the Merimbula estuary in NSW (during 2003), were provided by Dr. Symon Dworjanyn (NSW Fisheries, Port Stephens) (Heasman et al. 2004). A total of 200 oysters, ranging from 80 to 120 mm in diameter, were sampled from the site, and histological sections were examined by light microscopy. Of several samples confirmed positive, 3 were used for sequencing analysis.DNA extraction. Oyster tissues (mantle and gills) were rinsed in phosphate-buffered saline (PBS; pH 7.4), and then DNA was extracted using a QiaAmp DNA minikit (Qiagen) according to the manufacturer's instructions. DNA was eluted and resuspended in a final volume of 100 µl sterile deionised water.PCR amplification of the rRNA 18S gene and ITS-1 region. Taq DNA polymerase (Promega) was used for PCR assays. In all PCR assays, the primers (Table 1) were used at a final concentration of 1 µM. Aliquots (2 µl) of DNA samples were added to 23 µl of reaction mixture for amplification. Thermocycling conditions for the 18S gene PCR were: 95°C for 2 min (1 cycle), followed by 94°C for 30 s, 55°C for 45 s and 72°C for 45 s (40 cycles), followed by 72°C for 5 min (1 cycle). PCR cycling conditions for the ITS region were: 95°C for 2 min (1 cycle), 94°C for 30 s, 50°C for 45 s and 72°C for 45 s (40 cycles), followed by 72°C for 5 min (1 cycle). The resulting amplicons were resolved by electrophoresis in 2% agarose gels in 1 × TAE (0.004 M tris-acetate, 0.001 M EDTA) buffer. Gels were stained with ethidium bromide (0.5 µg ml -1 ). Amplicons were then extracted using a gel extraction kit (Qiagen) before sequencing.Sequence determination and comparison. The 18S rRNA gene and the ITS region (GenBank Accession Number DQ312295) of the Australian Bonamia isolate were determined by direct sequencing of the PCR product using an ABI PRISM Ready Reaction Big dye Termina...