Marteilia refringens

Clinical pathology

Infection with Marteilia refringens is a lethal disease of oysters. Death occurs during the second year after initial infection. As gross sign, we can usually observe a discolouration of the digestive gland.

Agent description

Marteilia refringens is a protistan parasite belonging to the phylum Paramyxea which is responsible for marteiliosis in the flat oyster Ostrea edulis and the mussels Mytilus edulis and M. galloprovincialis. The parasite has also been sporadically reported in other bivalve species such as clams Solen marginatus and Chamelea gallina (Lopes-Flores et al. 2008), and the mussel Xenostrobus securis (Pascual et al. 2010).

Two M. refringens types have been defined based on differences on the ITS region of the genome (Le Roux et al. 2001). : the type O, more often detected in flat oysters and the type M more often detected in mussels.

However, whether Marteiliosis in mussels and in flat oysters is attributed to the same parasite is not clear. Two distinct species were previously recognized: M. refringens infecting oysters and M. maurini infecting mussels. Further studies suggested that genomic differences between M. refringens and M. maurini were too low to consider them as separate species (Balseiro et al., 2007; Lopez-Flores et al., 2004), and both species were officially named M. refringens in 2007. More recently, a taxonomic study using high throughput sequencing suggested restoring the two separate species recognition. Authors proposed to rename the M-type as Marteilia pararefingens (Kerr et al., 2018).

Different stages of the parasite can be observed in infected oysters and mussels. Young plasmodia are mainly found in the epithelia of labial palps and stomach. Sporulation takes place in the digestive gland tubules and ducts (picture 1). Propagules are released into the lumen of the digestive tract and shed into the environment in faeces. They can survive from several days up to 2-3 weeks depending on the environmental conditions.

Mussels are usually not adversely affected by marteilioisis. However few important mussel mortalities (up to 100%) associated with heavy infection by Marteilia refringens were reported in the past in France. They occurred on mussels Mytilus edulis bought in Northern European countries for relaying in France. These mussels have never been in contact with M. refringens in their originating countries and this can explain their high susceptibility to the disease.

Picture 1 : Five plasmodia of Marteilia refringens seen in the digestive tubule epithelium of a flat oyster Ostrea edulis (H&E staining).

Other Marteilia species have been described associated with mortalities of marine molluscs such as M. sydneyi, the parasite responsible of the Queensland Unknown disease causing recurring mortalities of Sydney rock oysters Saccostrea glomerata in Australia (Perkins and Wolf 1976), or M. cochillia, a parasite associated with high mortalities of cockles in Spain since 2012, (Carrasco et al. 2013, Villalba et al. 2014).

Screening techniques for the pathogen

Imprints

Tissue imprints can be made by using digestive gland from live or gapping bivalves (picture 2). Marteilia refringens appears as cells ranging in size up to 30–40 µm. Using Wright, Wright-Giemsa or equivalent stain (e.g., Hemacolor, Merck; Diff-QuiK, Baxter) these parasites show a basophilic cytoplasm and an eosinophilic nucleus. Pale halo around large, strongly stained (refringent) granules and in larger cells, cell within cell arrangements are observed.

Picture 2 : Imprint of digestive gland from Ostrea edulis showing young stages (upper left) and sporulated cells (center) of Marteilia refringens.

Histopathology

Histopathology should be performed on tissue sections that include digestive gland (picture 1), gills and palps and stained with hematoxylin and eosin or equivalent staining. Marteilia cells have a size ranging from 4 µm up to 40 µm. Young plasmodia (uninucleate) are mainly found in the epithelium of labial palps and stomach. Sporulation involves divisions of cells within cells and takes place in the digestive gland tubules and ducts. Refringent granules appear in the course of sporulation, but are not observed in early stages. In late phases of infection, sporangia are observed free in the lumen of the digestive tract.

Values of sensitivity and specificity for histopathology were estimated at 0.7 and 0.99, respectively when co validated with in situ hybridization (Thébault et al. 2004). Tissue imprints appear less reliable than histopathology for the detection of the parasite in case of low level of infections. However, tissue imprints are more rapid and less expensive than histopathology (cost for one individual is estimated at about 5 € and 20 € -including personal cost- respectively).

PCR

Several conventional PCR have been developed. The conventional PCR targeting the ITS1 region, described by Le Roux et al. 2001 (Primers Pr4/Pr5, also named M2A/M3AS), is recommended because it is M. refringens specific. However, this PCR also amplifies M. cochillia. Two real-time Taqman® PCR assays have been developed to rapidly detect and type M. refringens (Carrasco et al 2017, EURL unpublished). Those assays target the ITS-1 region and were designed with two Taqman® probes for the specific detection of the type M and the type,O and do not amplify M. cochillia. The Taqman® PCR for the detection and typing of M. refringens developed by Carrasco et al.  was compared to histology on a set of 154 mussels and flat oysters, and results showed a good agreement between both methods (kappa value of 0.85) (Carrasco et al. 2017). The one developed by the EURL was evaluated in the context of an Inter Laboratory Comparison test on a set of 24 mussel and flat oyster samples by 7 laboratories in 2014. Results showed a good sensibility, specificity and reproducibility.

To facilitate the monitoring of regulated pathogens of flat oysters, the EURL also developed a real-time Taqman® PCR assay for the detection of both M. refringens and Bonamia sp.in one step targeting the 18S rDNA gene. This Taqman® PCR was shown to also detect M. cochillia and M. sydneyi. It was compared to the conventional PCR (Le Roux et al. 2001) on a set of 386 flat oysters and was shown to have a better sensitivity. Both methods had an equivalent specificity. (Estimated sensitivity of the new PCR was 95.7% against 65.1% for the PCR-RFLP, estimated specificity of the new PCR was 98.7% against 99.8% for the PCR-RFLP)

Because infection may be focal and also because infection targets different tissues in the early and late stages, the sensitivity of PCR detection may be lower than expected theoretical PCR performances.

The following standard operating procedures are available on the "SOPs and quality" page:

-          The conventional PCR-RFLP for Marteilia refringens detection and characterisation (La Roux et al. 2001)

-          The real-time Taqman® PCR for Marteilia refringens detection and typing (EURL)

-          The real-time Taqman® PCR for the detection of Marteilia refringens and Bonamia sp. (EURL)

Confirmatory techniques for diagnosis

PCR

PCR protocols previously described in the section “screening techniques” below can also be used as confirmatory technique. The conventional PCR (La Roux et al. 2001) can not differentiate Marteilia refringens type M and type O. A protocol of RFLP can be applied on PCR products to differentiate M. refringens type M and type O, however the M-type and M. cochillia shows the same HhaI restriction profil (Carrasco et al. 2015).

ISH

An in situ hybridization protocol has been developed based on the use of Smart2, a digoxygenin-labelled probe targeting the SSU rDNA (Le Roux et al. 1999, SS2/SAS1 primers). This ISH protocol has been validated against histology (estimated sensitivity 90%, and specificity 99%) (Thébault et al. 2004). However, the probe was shown to cross react with Marteilia sydneyi and Marteilioïdes chungmuensis (Kleeman et al. 2002). For a specific detection of M. refringens, it is recommended to use a probe targeting the ITS region. The PCR product from the conventional PCR Le Roux et al. 2001 (M2A/M3AS primers) can be used as a probe for ISH.

The SOP “Marteilia refringens detection and characterization by in situ hybridization (ISH)” is available on the "SOPs and quality" page.  

In situ hybridization can help to detect early infection which is more difficult to detect in traditional histological sections (picture 3).

Picture 3 : In situ hybridization test on connective tissue from mussel  Mytilus edulis confirming previous screening diagnosis of young stages of Marteilia sp (see inserted picture of a H&E stained slide from the same specimen tissue) .

TEM

Transmission electron microscopy is time consuming and cannot be applied in routine but can be recommended when Marteilia like parasites are described in a new host species. Ultrastructural criteria are not enough discriminant to differentiate Marteilia refringens type M and type O. Haplosporosomes in mature Marteilia from oysters and mussels appear similar in shape, although those from mussels seem to be marginally smaller in size, and spore wall morphology vary depending on the state of maturity of the parasite (Longshaw et al. 2001).

Sequencing

Sequencing is recommended as one of the final steps for confirmatory diagnostic. Targeted regions are SSU rDNA and ITS1. Obtained sequences should be compared with available ones in gene banks.

What should we do for diagnosis at suspicion?

When suspected, Marteilia refringens can be detected by digestive gland imprints. In parallel, pieces of digestive gland can be fixed in ethanol for PCR analysis and a section of oysters should be fixed in Davidson’s fixative for histological examination.

EU-legislation related to techniques

Marteilia refringens is listed by the EU legislation: in Directive 2006/88/EC as non exotic pathogen, and in the recent Commission implementing regulation 2018/1882 in categories C+D+E.

Commission implementing decision 2015/1554 lay down rules for the application of Directive 2006/88/EC as regards requirements for surveillance and diagnostic methods for M. refringens.

OIE recommendations related to techniques

Marteilia refringens is listed by the OIE Manual of Diagnostic Tests for Aquatic Animals (2019 version) and by the Aquatic Animal Health Code (2019 version).

  • Histopathology and PCR for surveillance
  • Tissue imprints and PCR for presumptive diagnostic
  • PCR and sequencing for confirmatory diagnostic

OIE Reference Laboratory

: Ifremer, Laboratoire Génétique et Pathologie, Av. de Mus de Loup, 17390 La Tremblade, FRANCE

: Dr Isabelle Arzul, e-mail: iarzul@ifremer.fr

Assessment

The tests are discussed at the annual European EURL/NRL meetings. Use the methods according to the table below for screening and confirmation.

Pathogen

Screening technique

(well established)

Confirmatory technique

(well established)

Evaluation

Marteilia refringens

Histology, imprints, PCR

PCR & DNA sequencing, ISH, TEM

References

Balseiro, P., Montes, A., Ceschia, G., Gestal, C., Novoa, B., Figueras Huerta, A., 2007. Molecular epizootiology of the European Marteilia spp., infecting mussels (Mytilus galloprovincialis and M. edulis) and oysters (Ostrea edulis): an update.

Carrasco, N., Hine, P.M., Durfort, M., Andree, K.B., Malchus, N., Lacuesta, B., Gonzalez, M., Roque, A., Rodgers, C., Furones, M.D., 2013. Marteilia cochillia sp nov., a new Marteilia species affecting the edible cockle Cerastoderma edule in European waters. Aquaculture 412, 223-230.

Carrasco, N., Voorbergen-Laarman, M., Lacuesta, B., Furones, D., Engelsma, M.Y., 2017. Application of a competitive real time PCR for detection of Marteilia refringens genotype “O” and “M” in two geographical locations: The Ebro Delta, Spain and the Rhine-Meuse Delta, the Netherlands. Journal of invertebrate pathology 149, 51-55.

Kerr, R., Ward, G.M., Stentiford, G.D., Alfjorden, A., Mortensen, S., Bignell, J.P., Feist, S.W., Villalba, A., Carballal, M.J., Cao, A., Arzul, I., Ryder, D., Bass, D., 2018. Marteilia refringens and Marteilia pararefringens sp nov are distinct parasites of bivalves and have different European distributions. Parasitology 145, 1483-1492.

Kleeman S.N., Le Roux F., Berthe F. & Adlard R.D. (2002). Specificity of PCR and in situ hybridisation assays designed for detection of Marteilia sydneyi and M. refringens. Parasitology 125: 131–141.

Le Roux F., Audemard C., Barnaud A. & Berthe F.C.J. (1999). DNA probes as potential tools for the detection of Marteilia refringens. Mar. Biotechnol., 1(6): 588–597.

Le Roux F., Lorenzo G., Peyret P., Audemard C., Figueras A., Vivarès C., Gouy M. & Berthe F.C.J. (2001). Molecular evidence for the existence of two species of Marteilia in Europe. J. Euk. Microbiol., 48 (4): 449–454.

Longshaw M., Feist S.W., Matthews R.A. and A. Figueras (2001). Ultrastructural characterization of Marteilia species (Paramyxea) from Ostrea edulis, Mytilus edulis and  Mytilus galloprovincialis in Europe. Diseases of Aquatic Organisms 44: 137-142.

Lopez-Flores, I., De La Herran, R., Garrido-Ramos, M.A., Navas, J.I., Ruiz-Rejon, C., Ruiz-Rejon, M., 2004. The molecular diagnosis of Marteilia refringens and differentiation between Marteilia strains infecting oysters and mussels based on the rDNA IGS sequence. Parasitology 129, 411-419.

Lopez-Flores, I., Garrido-Ramos, M.A., de la Herran, R., Ruiz-Rejon, C., Ruiz-Rejon, M., Navas, J.I., 2008a. Identification of Marteilia refringens infecting the razor clam Solen marginatus by PCR and in situ hybridization. Molecular and Cellular Probes 22, 151-155.

Lopez-Flores, I., Robles, F., Valencia, J.M., Grau, A., Villalba, A., de la Herran, R., Garrido-Ramos, M.A., Ruiz-Rejon, C., Ruiz-Rejon, M., Navas, J.I., 2008b. Detection of Marteilia refringens using nested PCR and in situ hybridisation in Chamelea gallina from the Balearic Islands (Spain). Diseases of Aquatic Organisms 82, 79-87.

Pascual, S., Villalba, A., Abollo, E., Garci, M., Gonzalez, A.F., Nombela, M., Posada, D., Guerra, A., 2010. The mussel Xenostrobus securis: a well-established alien invader in the Ria de Vigo (Spain, NE Atlantic). Biological Invasions 12, 2091-2103.

Perkins, F.O., Wolf, P.H., 1976. Fine-Structure of Marteilia-Sydneyi Sp-N - Haplosporidan Pathogen of Australian Oysters. Journal of Parasitology 62, 528-538.

Thebault A, Bergman S, Pouillot R, Le Roux F and Berthe FC. (2005). Validation of in situ hybridisation and histology assays for the detection of the oyster parasite Marteilia refringens. Diseases of Aquatic Organisms, 65 (1): 9-16.

Villalba, A., Iglesias, D., Ramilo, A., Darriba, S., Parada, J.M., No, E., Abollo, E., Molares, J., Carballal, M.J., 2014. Cockle Cerastoderma edule fishery collapse in the Ria de Arousa (Galicia, NW Spain) associated with the protistan parasite Marteilia cochillia. Diseases of Aquatic Organisms 109, 55-80.