Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 3.1.4.

MIKROCYTOSIS
(Mikrocytos mackini)

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GENERAL INFORMATION

Mikrocytosis here refers only to the disease in oysters caused by Mikrocytos mackini on the west coast of Canada. If detected outside the known range of M. mackini, electron microscopy or molecular probes, if available, must be used to identify and distinguish the detected organism from other microcell species (e.g. Mikrocytos roughleyi, Bonamia ostreae, B. exitiosus). The presence of these pathogens in any bivalve should be regarded as potentially serious and the OIE Reference Laboratory should be consulted.
 
Mikrocytos mackini has uncertain taxonomic affinities, but it is not closely related to other microcells (8, 10, 11). Mikrocytosis is also known as Denman Island disease (2, 13) and microcell disease of oysters. Mikrocytos mackini infects Crassostrea gigas, Ostrea edulis and O. conchaphila (= O. lurida); and infects experimentally Crassostrea virginica (1, 3, 4, 9). Crassostrea gigas seems to be more resistant to the disease than the other species.
 
Mikrocytos mackini occurs on the south-west coast of Canada. It is probably ubiquitous throughout the Strait of Georgia, including Henry Bay, Denman Island, and is confined to other localities around Vancouver Island (British Columbia).
 
Mikrocytos mackini produces a focal intracellular infection of muscle and vesicular connective tissue cells, which results in haemocyte infiltration and tissue necrosis (5). The parasite may induce yellow-greenish pustules, abscesses and ulcers, mainly on the mantle, with corresponding brown scars on the shell. Macroscopic lesions are not always present. Abscesses are composed of granular haemocytes and hyalinocytes, and may contain small cells of 1-3 µm in diameter. Severe infections appear to be restricted to oysters over 2 years of age.
 
The mortality rate has been recorded at around 40% for older oysters at low tide levels (13). The disease occurs most often in April and May, after a 3-4-month period when temperatures are less than 10°C. Harvesting or moving large oysters to locations high in the intertidal zone prior to March and not planting oysters at lower tide levels before June have been successfully implemented to control the disease (1).
 
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
		Cut a section through the body of the oyster to include the mantle and digestive gland. Include pustules, abscesses and ulcers, if they are present. Fix in the sample Davidson's or Carson's fixatives (the latter enables the samples to be re-used for electron microscopy, if necessary). The ratio must be no more than one volume of tissue to ten volumes of fixative.
		After processing for histology, stain sections with haematoxylin and eosin. It is recommended that two sections per oyster be examined at x100 magnification.
		Intracellular M. mackini, 1-3 µm in diameter, may be detected by examination of vesicular connective tissue cells immediately adjacent to abscess-like lesions. This parasite has also been observed in muscle cells and occasionally in haemocytes or free within the lesions, but it is much more difficult to detect in these situations.
		Microcytos mackini differs from all other microcells by its occurrence within the cytoplasm of vesicular connective tissue cells on the periphery of the lesions.
2.	Presumptive Diagnostic Methods
	2.1.	Cytological examination: tissue imprints
		For detection of the parasite, tissue imprints are better than histology (9). Cut a section through the abscesses or ulcers if present, blot on filter paper to remove excess water, then make imprints on a slide of the sample surface corresponding to the section that passed through the lesion. Air-dry the slides and fix in methanol (2-3 minutes).
		Stain the slides using a commercially available staining kit for blood cells in accordance with the manufacturer's instructions. After drying, the imprint can be examined directly using oil immersion at x100 magnification. However, if the slides are to be retained for future examination, they should be mounted with a cover-slip using a synthetic resin prior to applying immersion oil.
		The parasite, 1-3 µm in diameter, appears free of the host cell or, rarely, in haemocytes, and has blue (basophilic) cytoplasm and a small red (eosinophilic) nucleus (colours may vary with stain used). An observation time of 10 minutes per slide is sufficient at x100 magnification.
3.	Confirmatory Identification of the Pathogen
	3.1.	Histology
		Where infections have been observed within the known geographical range of M. mackini, visualisation of intracellular microcells within vesicular connective tissue cells, by histological examination, may be considered to be a confirmatory method (7).
	3.2.	Transmission electron microscopy examination
		Transmission electron microscopy procedures are described in Chapter I.2. of this Aquatic Manual.
		Ultrastructural morphology differentiates M. mackini from other microcells; M. mackini lacks mitochondria and haplosporosomes (10), and the nucleolus of M. mackini is located towards the centre of the nucleus while in Bonamia spp. it has an eccentric location.

REFERENCES

1.	Bower S.M. (1988). Circumvention of mortalities caused by Denman Island oyster disease during mariculture of Pacific oysters. Am. Fish. Soc. (Special Publication), 18, 246-248.
2.	Bower S.M. (2001). Synopsis of Infectious Diseases and Parasites of Commercially Exploited Shellfish: Mikrocytos mackini (Denman Island Disease) of Oysters.
	URL: http://www-sci.pac.dfo-mpo.gc.ca/sealane/aquac/pages/mikmacoy.htm
3.	Bower S.M., Hervio D. & McGladdery S.E. (1994). Potential for the Pacific oyster, Crassostrea gigas, to serve as a reservoir host and carrier of oyster pathogens. ICES Council Meeting Papers, Copenhagen, Denmark, ICES-CM-1994/F:30.
4.	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.
5.	Bower S.M., McGladdery S.E. & Price I.M. (1994). Synopsis of infectious diseases and parasites of commercially exploited shellfish. Ann. Rev. Fish Dis., 4, 1-199.
6.	Bower S.M. & Meyer G.R. (1999). Effects of cold water on limiting or exacerbating some oyster diseases. J. Shellfish Res., 18, 296 (Abstract).
7.	Cochennec N., Le Roux F., Berthe F. & A. Gerard (2000). Detection of Bonamia ostreae based on small subunit ribosomal probe. J. Invertebr. Pathol., 76, 26-32.
8.	Farley C.A., Wolf P.H. & Elston R.A. (1988). A long-term study of 'microcell' disease with a description of a new genus, Mikrocytos (g. n.), and two new species, Mikrocytos mackini (sp. n.) and Mikrocytos roughleyi (sp. n.). Fishery Bull., 86, 581-593.
9.	Hervio D., Bower S.M. & Meyer G.R. (1996). Detection, isolation and experimental transmission of Mikrocytos mackini, a microcell parasite of Pacific oysters Crassostrea gigas (Thunberg). J. Invertebr. Pathol., 67, 72-79.
10.	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.
11.	Hine P.M., Cochennec-Laureau N. & Berthe F.C.J. (2001). Bonamia exitiosus n. sp. (Haplosporidia) infecting flat oysters Ostrea chilensis (Philippi) in New Zealand. Dis. Aquat. Org., 47, 63-72.
12.	Joly J.-P., Bower S.M. & Meyer G.R. (2001). A simple technique to concentrate the protozoan Mikrocytos mackini, causative agent of Denman Island disease in oysters. J. Parasitol., 87, 432-434.
13.	Quayle D.B. (1982). Denman Island oyster disease 1960-1980. British Columbia Shellfish Mariculture Newsletter, 2, 1-5, (Victoria, Canada).

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