Mikrocytos mackini

 

Clinical pathology

Gross signs are ususally abscesses or green pustules on palps and mantle, though these signs are not pathognomonic. The disease is usually present from February to June with the higher prevalence in April-May. Up to 40% mortalities of Japanese oysters Crassostrea gigas have been observed in the past on oysters population of more than three years. Many oysters recover from the disease but green pustules on the mantle can reduce the market value of oysters (Quayle, 1988).

Histologically, lesions occur in the connective tissue of the palps, mantle, and vesicular tissue (picture 1). Although the life cycle outside the host is unknown, it is possible to transmit the disease experimentally in the laboratory by cohabitation or by intamuscular inoculation of purified parasites. Cohabitation in the field gives the same result.

Picture 1: Numerous Mikrocytos mackini inside cells from the vesicular tissue of a Pacific oyster Crassostrea gigas (H&E staining).

Agent description

Mikrocytos mackini is an eukaryotic protistan of uncertain taxonomy. Recent phyilogenetic analysis (Carnegie et al., 2003) shows that M. mackini is not affiliated to the Haplosporidian group including Bonamia spp. M. mackini is a microcell responsible for the disease called Denman Island Disease, named after the location in British Columbia (Pacific coast of Canada) were it was first described in 1960.   It has been experimentally demonstrated in laboratory and in the field that M. mackini can be highly infective for several other species of oysters : Crassostreae virginica, Ostrea edulis and Ostrea conchaphila (Bower et al. 1997).

Screening techniques for the pathogen

Imprints

Tissue imprints (picture 2) can be made using adductor muscle, palps and mantle, particularly from yellow-green pustules. Mikrocytos mackini appears as small spherical or ovoid organisms (2-4 µm wide) free or inside vesicular cells or sometimes inside haemocytes. Using Wright, Wright-Giemsa or equivalent stain (e.g., Hemacolor, Merck; Diff-QuiK, Baxter) these parasites show a basophilic cytoplasm and an eosinophilic nucleus. Binucleated cells can be observed.

Picture 2 : Mantle imprint of Pacific oyster Crassostrea gigas showing numerous free cells of Mikrocytos mackini (Hemacolor® staining).

Histopathology

Histopathology should be performed on tissue sections that include palps, digestive gland and mantle. In infected oysters, parasites can be observed as very small cells of 2-4 µm wide, within the vesicular tissue cells (figure 1), sometines in haemocytes or freely in connective tissue or sinuses of palp, gill, gut and mantle epithelium, often associated with intense inflammatory reaction.

Comment: Tissue imprints appear less reliable than histopathology for the detection of the parasite in case of low level infections. However, tissue imprints are quicker and less expensive than histopathology (cost for one individual is estimated at about 5 € and 20 € -including personal cost- respectively).

PCR

A PCR technique has been described by Carnegie et al. (2003) after they design specific primers by aligning a presumptive M. mackini SSU rDNA with SSU rDNA gene sequences from numerous protists and a fungus. Primers are: MIKROCYTOS-F (AGATGGTTAATGAGCCTCC) and MIKROCYTOS-R (GCGAGGTGCCACAAGGC). Positive reaction gives a 546 bp product. PCR technique has been experimentally validated and appears to detect 15 times more M. mackini than histopathology and tissue imprint techniques (Carnegie et al. 2003).

Confirmatory techniques for diagnosis

PCR

The PCR technique described above can be use as confirmatory technique  when suspicious cells are observed in histology, because of its high specificity to M. mackini.

FISH and ISH

A fluorescent in situ hybridization (FISH) protocol using four Oregon Green labeled oligonucleotide probes (MACKINI-1-OG to MACKINI-4-OG) have been developed by Carnegie et al. (2003).

An ISH Digoxigenin labeled probe (5'-AGCCCACAGCCTTCAC-3'-DIG) based on the Carnegie probe MACKINI-1-0G has also been developped (Meyer et al. 2005) and used succesfully to validate the technique for specificity and sensitivity against histopathology diagnosis (see picture 3). The study showed that only 70% of the positive oysters diagnosed with ISH were found infected with histology. It demonstrated that M. mackini cells present in digestive gland, gut, heart, gills and kidney were hardly detectable with the histology technique.

Picture 3 : ISH reaction with the digoxygenin-labeled probe used on an infected Pacific oyster Crassostrea gigas showing Mikrocytos mackini cells (in blue) inside the digestive gland and the surrounding connective tissue (Bismarck Brown counterstain). Slide courtesy of Gary Meyer, PBS, DFO, Canada.

In situ hybridization can help to detect early infection which is more difficult to detect in traditional histological sections (Bower et al. 2005).

TEM

Transmission electron microscopy is time consuming and cannot be applied in routine but is recommended when Mikrocytos like parasites are described in a new host species.

Different stages including uninucleate and binucleate microcells have been reported extra or intracellularly. Caracteristic intracellular structures include endoplasmic reticulum, Golgi apparatus and ribosomes but absence of mitochondria and haplosporosomes. Intracellular parasites are often observed closely associated with host mitochondria (and sometimes nuclei) suggesting the use of host organelles for energy production (Hines at al. 2001).

Sequencing

Sequencing of the SSU rDNA is recommended as one of the final steps for confirmatory diagnosis. Obtained sequences should be compared with available ones in gene banks (GenBank access n° AF477623).

Comments and recommendations on available techniques

Protocols for PCR and in situ hybridization are available in pre cited articles. PCR and in situ hybridization techniques developed by Carnegie et al (2003) and Meyer et al. (2005) have been submitted to validation tests against histological methods and found in both cases specific and more sensitive than histopathology.

What should we do for diagnosis at suspicion?

In some cases, highly infected oysters might present some gill indentations. When suspected, Bonamia ostreae can be detected by heart or gill imprints. In parallel, pieces of gill 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

Mikrocytos mackini is listed by the EU legislation (91/67/EEC Annex A), and also in the new EU Directive 2006/88/EC, as an exotic pathogen.

OIE recommendations related to techniques

Mikrocytos mackini was listed by the OIE Manual of Diagnostic Tests for Aquatic Animals (2006 version) but not by the Aquatic Animal Health Code (2007 version). It is not listed in the 2009 version of the Manual.

The OIE recommends:

• Histopathology and tissue imprints in some cases for surveillance 
• Histopathology, imprints, PCR and ISH for presumptive diagnostic
• PCR, ISH, sequencing and transmission electron microscopy for confirmatory diagnostic

OIE Reference Laboratory

: Fisheries and Oceans Canada, Pacific Biological Station, 3190 Hammond Bay Road, Nanaimo, BC, Canada V9T 6N7

: Gary Meyer, e-mail: MeyerG@pac.dfo-mpo.gc.ca 

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 techniques (well established)	Confirmatory techniques (well established)	Evaluation
Mikrocytos mackini	Histology, imprint, PCR	PCR, ISH, DNA sequencing, TEM	No special further test needed

References

Bower, S.M., Hervio, D., Meyer, G.R. (1997). Infectivity of Mikrocytos mackini, the causative agent of Denman Island disease in Pacific oysters Crassostrea gigas, to various species of oysters. Dis. Aquat. Org. 29: 111–116.

Bower, S.M., Bate, K. and Meyer, G. R., (2005). Susceptibility of juvenile Crassostrea gigas and resistance of Panope abrupta to Mikrocytos mackini . Journal of Invertebrate Pathology 88: 95-99.

Carnegie, R.B., Meyer, G.R., Blackbourn, J., Cochennec-Laureau, N., Berthe, F.C.J., Bower, S.M. (2003). Molecular detection of the oyster parasite Mikrocytos mackini and a preliminary phylogenetic analysis. Dis. Aquat. Org. 54, 219–227.

Hine, P.M., Bower, S.M., Meyer, G.R., Cochennec-Laureau, N., Berthe, F.C.J. (2001). Ultrastructure of Mikrocytos mackini, the cause of Denman Island disease in oysters Crassostrea spp. and Ostrea spp. in British Columbia, Canada. Dis. Aquat. Org. 45: 215–227.

Quayle, D.B. (1988). Pacific oyster culture in British Columbia. Can. Bull. Fish. Aquat. Sci. 218: 241.

Meyer, G. R., Bower, S.M. and Carnegie, R. B. (2005). Sensitivity of a digoxigenin-labelled DNA probe in detecting Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), in oysters. Journal of Invertebrate Pathology 88: 89-94.