Occurrence and characteristics of the haemocyte parasite Bonamia sp. in the New Zealand dredge oyster Tiostrea lutaria

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,...

Section: Diagnosis Of Infection Withmentioning

confidence: 99%

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

Corbeil

Arzul²,

Diggles³

et al. 2006

“…The cup-like nuclear indentations with associated Golgi complexes and MVBs (Dinamani et al 1987, Hine 1991b, 1992, Hine & Wesney 1992, and the vacuolated stage (Hine 1991b) have not been reported from Bonamia ostreae. The reticulated structure (Hine & Wesney 1992), which may be connected to anastomosing endoplasmic reticulum (Hine & Wesney 1994a), and the Golgi associated with lipoid body genesis (Hine & Wesney 1994a) have not been described in B. ostreae.…”

Section: Comparison With Bonamia Ostreaementioning

confidence: 99%

“…It resembled Bonamia ostreae Pichot et al 1979 in Ostrea edulis in Europe and was therefore called Bonamia sp. (Dinamani et al 1987, Hine 1991a,b, 1992, 1996, Hine & Wesney 1992, 1994a,b, Hine & Jones 1994. Subsequent investigations have shown that the parasite was present in Foveaux Strait oysters in 1964, and a longstanding association between host and parasite is suggested by a consistent annual pattern of infection (Hine 1991a,b).…”

Section: Introductionmentioning

confidence: 99%

Bonamia exitiosus n.sp. (Haplosporidia) infecting flat oysters Ostrea chilensis in New Zealand

Hine¹,

Cochennec-Laureau²,

Berthe³

2001

Self Cite

100

Bonamia sp. is a pathogenic parasite that occurs in the haemocytes of dredge oysters Ostrea chilensis Philippi in New Zealand. Ultrastructurally it resembles other haplosporidians in the possession of haplosporosomes, haplosporogenesis, persistence of mitotic microtubules during interphase and of the nuclear envelope during mitosis, and occurrence of a diplokaryotic or multi-nucleate plasmodial stage. Another stage containing a large vacuole derived from enlargement of 1 or more mitochondria has not previously been described from other haplosporidians. It most closely resembles B. ostreae Pichot et al., 1979, which parasitises and is pathogenic in haemocytes of European flat oysters, O. edulis. However, B. ostreae is smaller and denser, and has fewer lipoid bodies and haplosporosomes. We have nearly completely sequenced the small ribosomal gene of the organism from O. chilensis. Initial comparisons of these sequences with those of other protozoans showed similarities to B. ostreae. Polymorphism within Bonamia sp. was confirmed by restriction fragment length polymorphism analysis. On the basis of ultrastructural and molecular considerations it is proposed that this organism be named Bonamia exitiosus sp. nov. KEY WORDS: Bonamia exitiosus · Ostrea chilensis · Ultrastructure · 18S rDNA · New Zealand Resale or republication not permitted without written consent of the publisherDis Aquat Org 47: [63][64][65][66][67][68][69][70][71][72] 2001 MATERIALS AND METHODS Ultrastructural studies. Infected Ostrea chilensis (n = 237) from Foveaux Strait were opened one at a time, the heart was removed and cut in two, imprints were made with one half, and the other half was fixed for ultrastructural studies. Imprints were stained with Hemacolor™ (Merck) and microscopically examined, and heavily infected oysters were selected for further study. Hearts were fixed in 2.5% glutaraldehyde in 0.22 µm filtered seawater for 1 to 2 h, washed twice in filtered seawater, post-fixed in 1% OsO 4 for 1 h, stained en bloc with 5% uranyl acetate in 0.1 M sodium acetate buffer for 45 min, dehydrated in ascending (50 to 100%) ethyl alcohol, embedded in Araldite, sectioned, stained with 5% uranyl acetate for 10 min and in 5% lead citrate for 5 to 6 min, and examined on a Philips 420ST TEM.To compare Bonamia sp. with B. ostreae, quantitative data were taken from published studies on the ultrastructure of B. ostreae (Pichot et al. 1979, Brehélin et al. 1982, Balouet et al. 1983, Grizel 1985, Hervio et al. 1991 and from archived material held at the IFREMER laboratory at Ronce-les-Bains, La Tremblade, France. Samples were compared directly under the TEM from blocks of B. ostreae infected Ostrea edulis kindly lent by Steve Feist of the MAFF Fish Diseases Laboratory, Weymouth, England.Base sequence studies. DNA extraction: Genomic DNA was extracted from Bonamia sp. infected Ostrea chilensis using the following procedure. Ethanol fixed tissues were suspended in 1 ml of extraction buffer (NaCl 100 mM, EDTA 25 mM, pH 8, SDS 0.5%) with proteina...

“…edulis in California in the past (Katkansky et al 1969) and more recently (Elston et al 1986, Friedman et al 1989. Bonamia sp., the subject of this study, is highly pathogenic in Tiostrea chilensis in Foveaux Strait on the south coast of the South Island of New Zealand (Dinamani et al 1987). and since 1986 has destroyed ca 80 % of the oyster stocks.…”

Section: Introductionmentioning

confidence: 99%

Interrelationships of cytoplasmic structures in Bonamia sp. (Haplosporidia) infecting oysters Tiostrea chilensis: an interpretation

Hine¹,

Wesney²

1992

Self Cite

Reticular structures, confronting cisternae (CC), including cylindrical confronting cisternae (CCC), an unusual surface projection, formation of haplosporosome-like bodies (H-LB) and haplosporogenesis are described from Bonamia sp. infecting oysters Tiostrea chilensis from New Zealand. Formation of H-LB began when balls of putative nuclear material derived from indentations in the nuclear surface were encircled by cisternae of Golgi bearing a layer of dense material on their inner, concave surface. Also. H-LB were formed when CC split longitudinally and putative nuclear material filled the lumen of the resultant tubular structure, which subsequently pinched off H-LB. Haplosporogenesis began with the formation of cytoplasmic multi-vesicular bodies (MVB), which filled with dark osmiophilic material derived from nearby Golgi. The characteristic membranes of haplosporosomes appeared to form within these MVBs. It is suggested that H-LB are unusual structures in haplosporidans and that their core is formed from nuclear material. The CC from which they may form are also unusual, and in mammals CC occur in rapidly divid~ng or diseased cells. As CCC can only be induced in virally infected, transformed or neoplastic mammalian cells, it is considered that in Bonamia sp. CCC may be overt signs of an underlying disease. The significance of this is discussed in relation to the sudden appearance and pathogenicity of Bonamia sp. and, possibly, Haplosporidium nelsonj (MSX).