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 and M. maurini are protistan parasites belonging to the phylum Paramyxea and which are responsible for marteiliosis in the flat oyster Ostrea edulis and the mussels Mytilus edulis and M. galloprovincialis respectively. 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.

Because taxonomic relationships between Marteilia refringens and M. maurini are still not clear, we consider in this chapter that Marteilia refringens infects flat oysters Ostrea edulis and Marteilia maurini infects mussels Mytilus edulis and M. galloprovincialis. Mussels are usually not adversely affected by marteilioisis. However few important mussel mortalities (up to 100%) associated with heavy infection by Marteilia maurini 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 or M. maurini 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).

Screening techniques for the pathogen

Imprints

Tissue imprints can be made by using digestive gland from live or gapping bivalves (picture 2). Marteilia refringens and M. maurini 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

A PCR protocol targeting the ITS1 has been developed for the detection of Marteilia refringens (Le Roux et al. 2001). No cross-reaction has occurred with tested samples and specificity is considered very high. This PCR is expected to detect both Marteilia refringens and Marteilia maurini. 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 However, this technique has not be validated against histology.

Confirmatory techniques for diagnosis

PCR

The PCR protocol previously described in the section “screening techniques” below can also be used as confirmatory technique. However, this assay can not differentiate Marteilia refringens and M. maurini. A protocol of RFLP applied on PCR products obtained using ITS-1 primers has been developed and allows differentiating Marteilia refringens and M. maurini (Le Roux et al. 2001). The PCR-RFLP standard operating procedure is available on the "SOPs and quality" page.

ISH

An in situ hybridization protocol has been developed and is based on the use of Smart2, a 266 bp digoxygenin-labelled probe targeting the SSU rDNA (Le Roux et al. 1999). Smart 2 is able to detect Marteilia species including Marteilia refringens, M. maurini and M. Sydneyi (Le Roux et al. 1999; Kleeman et al. 2002). Values of specificity and sensitivity for in situ hybridization were estimated at 0.9 and 0.99 respectively when co validated with histology (Thébault et al. 2004). 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 and M. maurini. 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.

Comments and recommendations on available techniques

Protocols for PCR and in situ hybridization are available in pre cited articles. In situ hybridization developed by Le Roux et al (1999) has been co validated with histology (Thébault et al. 2004). However, validation is still required for PCR.

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 (Directive 2006/88/EC) as non exotic pathogen.

OIE recommendations related to techniques

Marteilia refringens is listed by the OIE Manual of Diagnostic Tests for Aquatic Animals (2009 version) and by the Aquatic Animal Health Code (2009 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 CRL/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

ISH only genus specific

PCR needed for sequencing

References

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.

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.