Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 3.1.3.

MARTEILIOSIS
(Marteilia refringens and M. sydneyi)

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GENERAL INFORMATION

Marteiliosis here refers only to the diseases caused by Marteilia refringens and M. sydneyi. Other Marteilia spp. infect oysters and other bivalves. Until more is known about the identity and biology of these other Marteilia spp., their presence in any bivalve should be regarded as potentially serious and the OIE Reference Laboratory should be consulted.
 
Marteiliosis is primarily caused by two protistan parasites of the genus Marteilia: M. refringens and M. sydneyi (phylum Paramyxea) (3, 11, 12, 26, 30). Marteiliosis is also known as Aber disease (M. refringens), and QX disease (M. sydneyi).
 
The type species of the genus, M. refringens, is a lethal parasite of the European flat oyster, Ostrea edulis (12). Marteilia sydneyi infects Saccostrea glomerata (= commercialis) and possibly also Saccostrea echinata (30, 31).
 
In addition, M. maurini (9) in Mytilus galloprovincialis and Mytilus edulis has been described from France, Italy and Spain (21). Because M. maurini is not easily distinguished from M. refringens, detection of the parasite in mussels would require confirmatory diagnosis as described in this chapter. Marteilia lengehi (7, 16) in Saccostrea cucullata from the Persian Gulf and Western Australia, and M. christenseni (8) in Scrobicularia plana from France can apparently be differentiated from other species by the characteristics of the cytoplasmic contents of sporangia and spore morphology. Marteilia spp. has also been described in the following species: Tiostrea chilensis (13), Ostrea angasi (5), O. puelchana (25), Cerastoderma (= Cardium) edule (10), Mytilus edulis (10, 22), Mytilus galloprovincialis (29), Crassostrea gigas (6) and C. virginica (27). A protistan similar to M. sydneyi was reported in giant clams Tridacna maxima (24). The scallop Argopecten gibbus is infected by a nonspecified Marteilia sp. in Florida, United States of America (USA) that has not been identified to species level (23).
 
The geographical distribution of M. refringens is: France, Greece, Italy, Morocco, Portugal, and Spain. Marteilia sydneyi is found in New South Wales, Queensland and Western Australia.
 
Marteilia refringens and M. sydneyi sporulate in the epithelia of the digestive gland, where infection is associated with poor condition index, emaciation of the oyster and exhaustion of its reserves of energy (glycogen), discoloration of the digestive gland, cessation of growth, and mortalities. Mortality appears to be related to the sporulation of the parasite (12). The initial site of infection of M. sydneyi is in the epithelia of the palps and gills and presporulating stages are found throughout the connective tissue and in digestive gland epithelia (18). Presporulating stages of M. refringens occur in the epithelia of the palps, stomach, digestive ducts and possibly the gills. Oysters infected with M. sydneyi and M. refringens are in poor condition with completely resorbed gonads (28). Massive invasion by M. sydneyi leads to complete disorganisation of the digestive gland epithelia. Death results from starvation in under 60 days after initial infection
 
The period of infection for M. refringens in Ostrea edulis is confined to spring and summer, when water temperature is greater than 17°C (1, 12, 14). Oysters may become infected with M. sydneyi in summer and early autumn. However, the disease is not seasonal, heavy mortality occurs and spores may be found all year round. High salinities limit the development of Marteilia spp.
 
The mode of infection and the life cycle outside the host are unknown. Because it has not been possible to transmit the disease experimentally in the laboratory, possible intermediate hosts are suspected (2, 4, 14, 22, 28).
 
For diagnosis, the recommended guidelines for sampling are those stated in Chapter 1.1.4 and Chapter I.2. of this Aquatic Manual.
 

EXAMINATION PROCEDURES

1.	Screening Methods
	1.1.	Histology
		General histological procedures are detailed in Chapter I.2. of this Aquatic Manual. For histological examination, cut a section through the digestive gland and place the sample in a fixative liquid, such as Davidson's or Carson's solutions or 10% formalin in filtered seawater (these latter enables the sample to be reused for electron microscopy, if necessary). The ratio must be no more than 1 volume of tissue to 10 volumes of fixative.
		The samples are subsequently handled in accordance with classical histological methods (see chapter I.2.). Several nonspecific stains allow M. refringens to be observed, e.g. haematoxylin-eosin or Millot's trichrome. These examples are not exhaustive, and modified staining techniques (15) may enhance the detection of the parasite.
		The young stages of Marteilia refringens are present in the epithelia of the stomach, intestine and digestive ducts. Initial infective stages of Marteilia sydneyi are present in the palp and gill epithelia and presporulating stages are found in the connective tissues and the epithelia of the digestive tubules, digestive ducts, intestine and stomach. Sporulating stages of M. refringens and M. sydneyi can be found in the epithelia of the digestive tubules. Free sporangia can also be observed in the lumen of the intestine. The unique feature of internal cleavage to produce cells within cells during sporulation differentiates Marteilia spp. from all other protista.
2.	Presumptive Diagnostic Methods
	2.1.	Histology
		See Section 1.1. above.
	2.2.	Cytological examination: tissue imprints
		In order to prepare the smears, cut a section through the digestive gland, remove excess water by placing the sample on blotting paper, then imprint the sample corresponding to the section that passes through the digestive tract on to a slide. The slides are air-dried and then fixed in methanol (2-3 minutes).
		The samples are stained using a commercially available staining kit for blood cells in accordance with the manufacturer's instructions. After staining, rinse in tap water, allow to dry completely and mount with a cover-slip using an appropriate synthetic resin.
		The parasite is 5-8 µm in size in the early stages and may reach up to 40 µm during sporulation. The cytoplasm of the cells stains basophilic, the nucleus is eosinophilic. The secondary cells or sporoblasts are surrounded by a bright halo (colour may vary slightly with the stain used). An observation time of 10 minutes per slide is considered sufficient.
3.	Confirmatory Identification of the Pathogen
	3.1.	In-situ hybridisation examination of Marteilia spp.
		An in-situ hybridisation assay may be performed to assess the taxonomic affiliation of the observed organisms at the genus level (20). Cross reaction studies using this protocol demonstrated that both M. refringens and M. sydneyi may be detected by the probe (17, 19, 20). An M. sydneyi-specific DNA probe has been developed and determined to be specific to the pathogen in in-situ hybridisation assays (17, 19).
		The probe, Smart 2, is used to detect Marteilia spp. by in-situ hybridisation. For probe production, the probe is obtained by polymerase chain reaction (PCR) using the primers SS2 (5'-CCG-GTG-CCA-GGT-ATA-TCT-CG-3') and SAS1 (5'-TTC-GGG-TGG-TCT-TGA-AAG-GC-3'). The PCR reaction is performed as described in Section 3.2., from M. refringens purified from Ostrea edulis DNA, except that 1 µl of digoxigenin-dUTP (DIG-dUTP) 25 mM is added to the mix.
		The probe, M. sydneyi the ITS (internal transcribed spacer) 1, is used to specifically detect M. sydneyi among related species. For probe production, the probe is obtained by polymerase chain reaction (PCR) using the primers PRO2 (5'-TCA-AGG-GAC-ATC-CAA-CGG-TC-3') and LEG1 (5'-CGA-TCT-GTG-TAG-TCG-GAT-TCC-GA-3'). The PCR reaction is performed as described in Section 3.2., from M. sydneyi purified from Saccostrea glomerata DNA, except that 1 µl of digoxigenin-dUTP (DIG-dUTP) 25 mM is added to the mix.
		The in-situ hybridisation is conducted as follows. Positive and negative controls must be included in the procedure.
		For the smart 2 probe, the visceral mass of molluscs is placed in Davidson's fixative AFA (glycerine [10%], formalin [20%], 95° ethanol [30%], dH2O [30%], and glacial acetic acid [10%]) or 10% buffered formalin for approximately 24 hours and then embedded in paraffin. Sections are cut 5 µm thick and placed on aminoalkylsilane-coated slides, which are then baked overnight in an oven at 40°C. The sections are dewaxed by immersing in xylene for 10 minutes. This step is repeated once and then the solvent is eliminated by immersion in two successive absolute ethanol baths for 10 minutes each. The sections are then rehydrated by immersion in an ethanol series. The sections are treated with proteinase K (100 µg/ml) in TE buffer (Tris [50 mM], ethylene diamine tetra-acetic acid [EDTA, 10 mM]), at 37°C for 30 minutes. Slides are dehydrated by immersion in an ethanol series and then air dried. Sections are incubated with 100 µl of hybridisation buffer (4 x SSC [standard saline citrate], 50% formamide, 1 x Denhardt's solution, 250 µg/ml yeast tRNA, 10% dextran sulfate) containing 10 ng (1 µl of the PCR reaction) of the digoxigenin-labelled probe. Sections are covered with in-situ plastic cover-slips and placed on a heating block at 95°C for 5 minutes. Slides are then cooled on ice for 1 minute before overnight hybridisation at 42°C in a humid chamber. Sections are washed twice for 5 minutes in 2 x SSC at room temperature, and once for 10 minutes in 0.4 x SSC at 42°C. The detection steps are performed according to manufacturer's instructions. The slides are then rinsed in dH2O. The sections are counterstained with Bismarck Brown Y, rinsed in dH2O, and cover-slips are applied using an aqueous mounting medium. The presence of Marteilia sp. is demonstrated by the purple-black labelling of the parasitic cells.
	3.2.	Polymerase chain reaction-restriction fragment length polymorphism for Marteilia refringens/M. sydneyi and M. refringens/M. maurini discrimination
		A PCR-restriction fragment length polymorphism (RFLP) assay was developed to differentiate M. refringens from M. maurini (21). A PCR assay was developed for the specific detection of M. sydneyi (17-19).
		For DNA extraction, infected animals are frozen at -80°C and the tissues are ground to powder. Around 10 volumes of extraction buffer (NaCl [100 mM], EDTA [25 mM], pH 8, sodium dodecyl sulfate [SDS, 0.5%]) are added with proteinase K (100 µg/ml). Following an overnight incubation at 50°C, DNA is extracted using a standard phenol/chloroform protocol, and precipitated with ethanol.
		PCR is carried out in 50 µl volume. After denaturation of DNA at 94°C for 5 minutes, 30 cycles are run as follows: denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and elongation at 72°C for 1 minute per kilo-base pair. A final elongation step of 10 minutes at 72°C is performed.
		For the detection of M. refringens, PCR is performed with primers (4 + 5) that target the ITS 1 (4: 5'-CCG-CAC-ACG-TTC-TTC-ACT-CC-3' and 5: 5'-CTC-GCG-AGT-TTC-GAC-AGA-CG-3').
		For the detection of M. sydneyi, PCR is performed with primers LEG1 (5'-CGA-TCT-GTG-TAG-TCG-GAT-TCC-GA-3') and PRO2 (5'-TCA-AGG-GAC-ATC-CAA-CGG-TC-3') that target the ITS.
		To differentiate M. refringens from M. maurini, polymorphism among the PCR products is identified by testing for cleavage with restriction enzyme Hha1. The resulting restriction fragments are analysed electrophoretically on 2% agarose gels. The profile corresponding to M. maurini gives three bands of 156 + 157 and 68 bp, respectively, whereas the profile corresponding to M. refringens gives two bands of 226 and 156 bp, respectively.
	3.3.	Electron microscopy examination
		Transmission electron microscopy procedures are outlined in Chapter I.2. of this Aquatic Manual.
		Marteilia sydneyi can be differentiated from M. refringens by a lack of striated inclusions in the plasmodia, formation of eight to sixteen sporangial primordia in each plasmodium (instead of eight for M. refringens), occurrence of two or three spores in each sporangium (rather than four in M. refringens), and the presence of a heavy layer of concentric membranes surrounding mature M. sydneyi spores.

REFERENCES

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