Small subunit ribosomal RNA gene sequence of the parasitic protozoan Haplosporidium nelsoni provides a molecular probe for the oyster MSX disease

109

The development of diagnostic assays more sensitive and specific than traditional histological techniques is important for the management of bonamiasis in flat oysters Ostrea edulis. A specific polymerase chain reaction (PCR) protocol was developed for the detection of very small amounts of Bonamia ostreae (Pichot et al. 1980) ribosomal DNA (rDNA) in bulk DNA from oyster gill and hemolymph. The presence of a 760 bp PCR amplification product corresponded with B. ostreae infections determined cytologically in 185 oysters from Ireland, Spain, and the USA. All (100%) 'heavily' and 'moderately' infected oysters, 86.7% of the 'lightly' infected oysters, and 66.7% of the 'scarcely' infected oysters were confirmed to be infected using the PCR. In addition, 37.9% of the oysters in which B. ostreae was not detected using cytology were positive using the PCR. Sampling error and the subjectivity of cytological diagnoses are the likely sources of disagreement between diagnostic methods in oysters with very light infections. The PCR assay developed here is more sensitive and less ambiguous than standard histological and cytological techniques. Phylogenetic analysis of DNA sequence data confirmed B. ostreae to be a member of the Haplosporidia. KEY WORDS: Bonamia ostreae · PCR · Haplosporidia · Ostrea edulisResale or republication not permitted without written consent of the publisher

Section: Introductionmentioning

confidence: 99%

Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia

Carnegie¹,

Barber²,

Culloty³

et al. 2000

109

“…This gene has been widely used in the taxonomic study of oyster pathogens including B. ostreae and B. exitiosus and Haplosporidium spp. (Fong et al 1993, Ko et al 1995, Stokes et al 1995, Flores et al 1996, Carnegie et al 2000, Cochennec et al 2000, Hine et al 2001a. Adlard & Lester (1995) proposed a M. roughleyi-diagnostic method based on amplification of a segment of the internal transcribed spacer region (ITS) within the ribosomal gene cluster (rRNA).…”

mentioning

confidence: 99%

Mikrocytos roughleyi taxonomic affiliation leads to the genus Bonamia (Haplosporidia)

Cochennec-Laureau¹,

Reece²,

Berthe³

et al. 2003

Microcell-type parasites of oysters are associated with a complex of diseases in different oyster species around the world. The etiological agents are protists of very small size that are very difficult to characterize taxonomically. Associated lesions may vary according to the host species, and their occurrence may be related to variations in tissue structure. Lesion morphology cannot be used to distinguish the different agents involved. Ultrastructural observations on Mikrocytos roughleyi revealed similarities with Bonamia spp., particularly in regard to the presence of electron-dense haplosporosomes and mitochondria, whose absence from M. mackini also indicate that M. roughleyi and M. mackini are not congeneric. A partial small subunit (ssu) rRNA gene sequence of M. roughleyi was determined. This partial sequence, 951 nucleotides in length, has 95.2 and 98.4% sequence similarities with B. ostreae and B. exitiosus ssu rDNA sequences, respectively. Polymorphisms among the ssu rDNA sequences of B. ostreae, B. exitiosus and M. roughleyi allowed identification of restriction enzyme digestion patterns diagnostic for each species. Phylogenetic analysis based on the ssu rDNA data suggested that M. roughleyi belongs in the phylum Haplosporidia and that it is closely related to Bonamia spp. On the basis of ultrastructural and molecular considerations, M. roughleyi should be considered a putative member of the genus Bonamia.KEY WORDS: Mikrocytos roughleyi · Saccostrea glomerata · Bonamiosis · Microcell · Taxonomy · Small subunit rDNA Resale or republication not permitted without written consent of the publisherDis Aquat Org 54: [209][210][211][212][213][214][215][216][217] 2003 Farley et al. 1988), and M. roughleyi causes winter mortality in the Sydney rock oyster Saccostrea glomerata in SE Australia (Roughley 1926, Farley et al. 1988). Experimentally, Bonamia spp. can infect Ostrea spp. (Grizel et al. 1983, Le Borgne & Le Pennec 1983, Bougrier et al. 1986, Pascual et al. 1991 and C. rivularis (Cochennec et al. 1998). M. mackini has been shown to infect C. gigas, C. virginica, O. edulis and O. concaphila (Bower et al. 1997) (present Table 1). Therefore, Bonamia spp. and Mikrocytos spp. cannot be distinguished solely by the hosts which they infect. However, to date the geographic distributions of the 2 Mikrocytos species appear to be distinct, with M. mackini having been reported only in western Canada and M. roughleyi only in eastern Australia.Several studies have included observations of the ultrastructure of Bonamia ostreae (Comps et al. 1980, Pichot et al. 1980, Bréhelin et al. 1982, Grizel et al. 1983, Grizel 1985, Chagot et al. 1992, Montes et al. 1994 and B. exitiosus (Dinamani et al. 1987, Hine 1991, Hine & Wesney 1994a,b, Hine et al. 2001b), but there are none on Bonamia sp. in Ostrea angasi in Australia or Tiostrea chilensis in Chile. A recent study on the ssu rDNA gene sequences of B. ostreae (Carnegie et al. 2000, Cochennec et al. 2000 and Bonamia sp. from New Zealand showed that they are co...

“…As reported here, detailed analysis of these differences do not support this. Two haplosporidians (Haplosporidium nelsoni and H. costale) that inhabit the same area and host and have a similar morphology in the plasmodial stage, but different spore stages, were found to be clearly different when their 18S rDNA sequence were compared (Fong et al 1993, Ko et al 1995, Stokes et al 1995. Perkinsus olseni from Australia and P. atlanticus from Portugal are morphologically similar and were found to belong to a single species, but P. marinus (found in the USA) was well discriminated from these 2 species (Goggin 1994).…”

mentioning

confidence: 99%

Ultrastructural characterisation of Marteilia species (Paramyxea) from Ostrea edulis, Mytilus edulis and Mytilus galloprovincialis in Europe

Longshaw

Feist²,

Matthews³

et al. 2001

A focused ultrastructural study of Marteilia spp. found in cultured Ostrea edulis, Mytilus edulis and Mytilus galloprovincialis from France and Spain was conducted with emphasis placed on haplosporosomes, striated plate-like inclusions and spore wall morphology. Two types of haplosporosome were identified, sphaeroid and oblate, which were common to the parasite in all 3 host species. A total of 492 haplosporosomes were measured; those from the Marteilia sp. in Mytilus spp. were marginally smaller than those in Ostrea edulis. Spore wall morphology was found to vary depending on the state of maturity of the parasite -the more mature the parasite, the thicker the wall surrounding it. It is suggested that the current criteria used to distinguish M. maurini from M. refringens are invalid and that M. maurini was relegated to a junior synonym of M. refringens. KEY WORDS: Marteilia refringens · Marteilia maurini · Ultrastructure Resale or republication not permitted without written consent of the publisherDis Aquat Org 44: [137][138][139][140][141][142] 2001 healthy animals are transferred to areas where the diseases are known to be absent. This is the case with Marteilia refringens in Europe. The European Community Council Directive 91/67 (OJ No L46 19.2.1991) includes M. refringens in annex A, list II, as a serious pathogen of the susceptible species Ostrea edulis. The diagnosis of the 2 species of Marteilia found in Europe (M. refringens and M. maurini ) currently requires analysis of the ultrastructural characteristics and consideration of the host specificity (Grizel 1974, Comps et al. 1982. Indeed, the only ultrastructural features selected by Comps et al. (1982) to distinguish the 2 species were subtle differences in haplosporosome shape and the 'existence of a multimembranous envelope next to the spore wall'.The recent discovery of Marteilia refringens in Mytilus galloprovincialis (Villalba et al. 1993 has raised some doubt that Marteilia maurini, which was described as the only Marteilia species parasitizing mussels, is a distinct species from M. refringens. Experimental transmission of M. refringens between members of the same or differing host species has been unsuccessful, although it has been attempted in a number of experiments under differing conditions (Figueras & Robledo 1993, Berthe et al. 1998.Because flat oysters cultured in Europe suffer mortalities as a result of infections with Marteilia refringens and the questionable taxonomic status of the Marteilia sp. reported from mussels Mytilus edulis (described as M. maurini Comps et al., 1982) which has not been associated with mortalities, it is important to establish whether one or several European Marteilia species exist.The objective of the current study was to re-evaluate the taxonomic features used to discriminate the Marteilia species in Ostrea edulis, Mytilus galloprovincialis and Mytilus edulis. MATERIALS AND METHODSOstrea edulis and Mytilus edulis were collected from France in February and April 1995. These were screened for ...